ABSTRACT 
 
 
 
 
Title of Dissertation: MECHANISM OF DREAM COMPONENT 
TSO1 IN PLANT STEM CELL REGULATION     
  
 Fuxi Wang, Doctor of Philosophy, 2022 
  
Dissertation directed by: Professor Zhongchi Liu, CBMG 
 
 
Plants are important for human survival and the environment. They provide oxygen, food, 
medicine and fuel. Understanding the development of plants has been a fundamental research 
question. Among all the plant tissues, the most important ones are the meristems. Sitting at the 
tip of the shoot and the root are the shoot apical meristem (SAM) and the root apical meristem 
(RAM). The shoot apical meristem gives rise to the above-ground organs like leaves and flowers 
while the root apical meristem produces all the root tissues that help to anchor the plants and 
transport water and nutrients. As the meristem is capable of producing new organs throughout 
the lifespan of a plant, the study of meristem maintenance and development provides the key to 
the understanding of plant development.  
Arabidopsis transcription factor TSO1 plays an essential role for the proper development of shoot 
apical meristem and root apical meristem. TSO1 encodes a protein with a cysteine-rich repeats 
domain and TSO1 is a potential component of a cell cycle regulating complex, the DREAM 
complex. The tso1-1 mutant has fasciated SAM due to shoot meristem cell over-proliferation and 
complete sterility due to lack of differentiated female and male floral organs. Interestingly, the 
tso1-1 mutant also produces shorter root than the wild type, presumably caused by early 
 
 
differentiation of the cells in the RAM. A prior mutagenesis screen identified two major 
suppressors of tso1-1. Characterization of these tso1-1 suppressor mutations provides important 
insights to the understanding of TSO1-regulatory pathways. My dissertation project focuses on 
analyzing one of these suppressors that was shown to be a mutated type-A cyclin gene named 
CYCA3;4. Mutations in CYCA3;4 suppress the shoot phenotype but not the root phenotype of 
tso1-1. The suppressed plants can produce normal floral organs and become partially fertile. 
Using transgenic method, I showed that the expression of CYCA3;4 was increased in the tso1-1 
SAM, and overexpression of CYCA3;4 in the tso1-3 mutant enhanced the fertility defect, 
suggesting that overexpression of CYCA3;4 partially mediates the tso1-1 shoot phenotype. In 
addition, I provided evidence supporting that TSO1 likely represses CYCA3;4 gene expression 
indirectly through MYB3R1, whose mutations also suppress tso1-1 mutants. My dissertation 
provides an important link between TSO1, a potential cell cycle regulatory complex component 
and meristem regulator, and cyclin A, a protein directly involved in cell cycle regulation. This 
link provides an important mechanistic insight into how plant meristems maintain their identity 
by limiting their cell division activity.  
To further investigate the mechanism of TSO1 action in the root, I collaborated with two other 
scientists to profile the gene expression in the tso1-1 root at single cell level. I compared the 
single cell RNA sequencing data of tso1-1 and wild type roots and identified molecular defects 
in the tso1-1 root vasculature. Correspondingly, the known regulators of vasculature 
development, the HD-ZIP III genes, are ectopically expressed in some of the vascular cells in the 
tso1-1 root. It suggests that the defects of root vasculature may be attributed to mis-expressed 
HD-ZIP III genes in the tso1-1 mutant. The HD-ZIPIII function was previously linked to their 
regulation of cytokinin biosynthesis genes, which were ectopically expressed in tso1-1 roots as 
 
 
revealed by our scRNA-seq data. Together, our data suggest that the over-production of 
cytokinin might be the cause of tso1-1 short root phenotype. 
In summary, my dissertation research revealed previously unknown links between TSO1 and cell 
cycle regulation in the shoot and root meristems as well as the molecular mechanisms of TSO1 
function in the root vascular development at single cell level. These findings have furthered our 
understanding of how cell cycle regulation is integrated with plant development. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MECHANISM OF DREAM COMPONENT TSO1 IN PLANT STEM CELL 
REGULATION     
 
 
 
by 
 
 
Fuxi Wang 
 
 
 
 
 
Dissertation submitted to the Faculty of the Graduate School of the  
University of Maryland, College Park, in partial fulfillment 
of the requirements for the degree of 
Doctor of Philosophy 
2022 
 
 
 
 
 
 
 
 
 
 
Advisory Committee: 
Professor Zhongchi Liu, Chair 
Associate Professor Stephen Mount 
Associate Professor Antony Jose 
Associate Professor Jianhua Zhu 
Associate Professor Yiping Qi 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
? Copyright by 
Fuxi Wang 
2022 
 
 
 
Acknowledgements 
I would like to thank my advisor Dr. Zhongchi Liu for her advice on experiments, 
critical thinking and writing over the past few years. She has been very patient and 
encouraging. She has confidence in me even sometimes I don?t have that in myself. I 
can?t be more grateful for the support she gave me: both the support on research and 
the support when I had hard times in my life. I see her as my role model as a scientist, 
especially as a female scientist.  
I also want to thank my committee members. I bothered Dr. Steve Mount a lot with 
my questions. He has been a great help for me on research and how to deal with 
Covid situation. I enjoyed the afternoon teatime when we talked about science and 
shared the mooncakes. I benefit a lot from Dr. Antony Jose?s writing class and 
continue to apply what I learned to my scientific writing, like this dissertation. Dr. 
Yiping Qi and Dr. Jianhua Zhu have been very supportive to my project and always 
make sure that I have a feasible plan for it. 
I would like to acknowledge my collaborators Dr. Rachel Shahan in Dr. Philip 
Benfey?s lab and Muzi Li in the Liu lab. They made the single cell project possible. It 
was difficult to perform the single cell sequencing during the pandemic, but Rachel 
did it! Muzi is a very good friend of mine. I am grateful not only for the work she has 
done but also her company when I had to work late. 
I am so glad that I joined the Liu lab for my PhD study. Everyone I met in the Liu lab 
are so nice and caring. We talk about each other?s project a lot and we are always 
happy to help each other with troubleshooting. I enjoyed a lot of our annual spring 
ii 
 
 
 
trips and crab feast/fall hiking. I am also very grateful for the former lab members, 
especially Dr. Julie Caruana, Dr. Wanpeng Wang (George) and Dr. Junhui Zhou. 
Julie was one of the few people with whom I could share all my ?nerdy? stories. 
George has worked on the TSO1 project before me. All my work wouldn?t be 
possible without his solid groundwork. Junhui has been a great teacher on how to 
perform experiments. Whenever I had questions about any type of experiment, I 
always went to him, and I always got helpful suggestions. I also want to thank the 
undergraduate students who I had work with. They are Nicole Szeluga, Merixia 
Kunjal and Emmanuel Mgboji. 
I am thankful for my friends outside the Liu lab too. Cust?dio Nunes and Matthew 
Fischer joined BISI the same year as me. We have been very good friends since then. 
We supported each other throughout the PhD study. Bilian Qian, Bixuan Wang and 
Jiming Wu also gave me a lot of support, especially during the pandemic. I also 
would like to thank my writing friend, Xiaojing Mao, who always encouraged me to 
write better and faster. I also would like to thank my idol Seventeen. Their music and 
stories have encouraged me to go further. 
I would like to acknowledge the Dean?s fellowship and the Ann G. Wylie dissertation 
fellowship. 
I am grateful for the support from my boyfriend, Anxin Bai. He makes me feel like 
home when he is around.  
Finally, I want to thank my families. My uncle has been taking good care of me since 
I arrived in Maryland. My parents have been incredibly supportive for every decision 
iii 
 
 
 
I made. They gave me unconditional love and trust. I could not go this far without 
them.  
iv 
 
 
 
Table of Contents 
 
 
Acknowledgements ....................................................................................................... ii 
Table of Contents .......................................................................................................... v 
Chapter 1: Introduction ................................................................................................. 1 
1.1 Plant meristem: origin of plant organs ................................................................ 1 
1.1.1 Shoot apical meristem (SAM): structure and how it is maintained ............. 1 
1.1.2 Root apical meristem (RAM): structure and regulation of development .... 4 
1.2 Arabidopsis TSO1 and its role in meristem development .................................. 9 
1.2.1 TSO1 encodes a transcription factor with cysteine-rich repeats .................. 9 
1.2.2 tso1 mutants exhibit defects in meristem development and tso1 alleles can 
be categorized into two classes ........................................................................... 10 
1.2.3 The animal homologs of TSO1 are cell cycle regulatory proteins ............ 13 
1.3 Cyclin genes and their roles in meristem development .................................... 20 
1.3.1 The classification of plant cyclin genes ..................................................... 20 
1.3.2 Functions of Arabidopsis CYCA3s ............................................................ 21 
Chapter 2: Mutations in a A-type cyclin gene suppresses tso1-1 shoot phenotype .... 26 
2.1 Introduction ....................................................................................................... 26 
2.2 Results ............................................................................................................... 29 
2.2.1 A splice-site mutation in CYCA3;4 suppresses tso1-1 shoot phenotype ... 29 
2.2.2 CRISPR/Cas9 mediated knockouts of CYCA3;4 also suppress tso1-1 ...... 32 
2.2.3 CYCA3;4 is mis-regulated in tso1-1 mutants ............................................. 35 
2.2.4 Overexpression of CYCA3;4 weakly enhances the tso1-3 fertility defect . 37 
2.2.5 A TSO1-MYB3R-CYCA3;4 regulatory module in shoot meristem regulation
............................................................................................................................. 40 
2.3 Discussion ......................................................................................................... 41 
2.3.1 CYCA3;4 is a unique CYCA3 ..................................................................... 43 
2.3.2 Potential mechanisms of CYCA3;4 in meristem regulation ....................... 44 
2.4 Methods............................................................................................................. 45 
Chapter 3: Single cell RNA sequencing revealed possible causes of tso1-1 short root 
phenotype .................................................................................................................... 50 
3.1 Introduction ....................................................................................................... 50 
3.2 Results ............................................................................................................... 54 
3.2.1 Single cell transcriptomes revealed reduced cell number in the vasculature 
of tso1-1 mutant root ........................................................................................... 54 
3.2.2 TSO1 is ectopic and overexpressed in the tso1-1 mutant root ................... 59 
3.2.3 Both cell type and cell numbers that express HD-ZIP IIIs are altered in 
tso1-1 root cells ................................................................................................... 62 
3.2.4 Expression of cytokinin biosynthesis genes is increased in tso1-1 root .... 65 
3.3 Discussion ......................................................................................................... 66 
3.3.1 TSO1 represses HD-ZIP III genes to inhibit cytokinin synthesis, which 
prevents premature root cell differentiation ........................................................ 66 
3.3.2 Integration of MYB3R1 in the TSO1 network ............................................ 68 
3.3.3 The increased expression of the HD-ZIP III genes affects vasculature 
development ........................................................................................................ 68 
v 
 
 
 
3.3.4 Overexpression of LOG1 suggest TSO1 may regulate cytokinin synthesis 
in SAM ................................................................................................................ 69 
3.3.5 TSO1 is involved in regulation of itself and plant DREAM complex 
components ......................................................................................................... 70 
3.4 Methods............................................................................................................. 70 
3.4.1 Protoplast isolation and scRNA-seq .......................................................... 70 
Chapter 4: Conclusion and future direction ................................................................ 73 
4.1 Conclusion ........................................................................................................ 73 
4.2 Future directions ............................................................................................... 73 
Bibliography ............................................................................................................... 76 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
vi 
 
 
 
Chapter 1: Introduction 
 
1.1 Plant meristem: origin of plant organs 
1.1.1 Shoot apical meristem (SAM): structure and how it is maintained 
One of the amazing facts about plants is that they continue to produce new organs 
throughout their lifetime, no matter it is a few weeks or hundreds of years. This is 
made possible by maintaining a small pool of pluripotent cells called the meristem. 
Similar to the stem cell in animals, the stem cells in plant meristem have the ability to 
maintain pluripotent state, continuously self-renewing and giving rise to daughter 
cells that form new plant organs. Different from animals, the fate of each plant cell is 
determined by its position relative to other cells rather than by the cell lineage as in 
most of the animals (Poethig, 1989).  
There are three important meristems in plants: the shoot apical meristem (SAM), root 
apical meristem (RAM), and the vascular meristem (De Rybel et al., 2016). The SAM 
gives rise to the above-ground tissues, the RAM produces cells to form the root, while 
the vascular meristem generates vascular tissues like xylem and phloem. This 
dissertation will focus on understanding the development of SAM and RAM. 
The dome shaped Arabidopsis shoot apical meristem (SAM) consists of three 
functional domains: the stem cell bearing central zone (CZ), the rapidly dividing 
peripheral zone (PZ), and the rib zone (RZ) that provides new cells to form the 
internal tissues like the vasculature (Figure1.1 A and B). Sitting at the top of the RZ is 
a small group of cells named organizing center (OC), where a homeodomain 
1 
 
 
 
transcription factor named WUSCHEL (WUS) is expressed. WUS plays a vital role in 
maintaining the stem cells in Arabidopsis SAM: the wus mutants  
Figure 1.1: The structure of Arabidopsis SAM and the regulatory pathways for 
maintaining it. 
(A) Arabidopsis shoot apical meristem under a scanning electron microscope. m =? (B) A 
diagram illustrating different domains within the SAM. CZ: central zone (pink), OC: 
organizing center (blue), PZ: prepherial zone (green), RZ: rib zone (gray). (C) The WUS-
CLV negative feedback regulatory pathway maintains the pluripotent stem cells in the CZ 
(pink) but also restrict the size of the SAM. WUS is also involved in regulating cytokinin at 
the SAM. It promotes cytokinin (CK) responses by repressing Type A ARR (?), a negative 
response factor of cytokinin. At the same time, cytokinin also induces WUS expression in 
the OC (blue). However, WUS represses the expression of LOG4,  a cytokinin synthesis 
gene, in the CZ to inhibit cell division of these stem cells. 
(A) and (C) were adapted from (Kitagawa and Jackson, 2019). 
 
 
2 
 
 
 
 
bear small, disorganized SAM , suggesting WUS promotes stem cell fate (Mayer et 
al., 1998). WUS proteins can move from OC to the CZ to activate a gene named 
CLAVATA3 (CLV3), which has opposite function as WUS by promoting 
differentiation of the cells at the shoot apex (Clark, Running and Meyerowitz, 1995; 
Fletcher et al., 1999; Yadav et al., 2011; Daum et al., 2014). The clv3 mutants have 
enlarged SAM and produce club-shaped siliques. CLV3 encodes a small peptide that 
travels back to the OC, where it can be perceived by several receptors: the 
CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase (LRR-RLK); the 
CLAVATA2 (CLV2) LRR-receptor like protein and CORYNE (CRN), a CLV2 co-
receptor, to repress WUSCHEL expression (Figure1.1 C). This negative feedback 
regulation allows preservation of stem cell pool size but also limit cell division at the 
SAM (Clark, Williams and Meyerowitz, 1997; Jeong, Trotochaud and Clark, 1999; 
Schoof et al., 2000; M?ller, Bleckmann and Simon, 2008; Ogawa et al., 2008). 
Besides the WUS-CLV pathway, there are many other factors influencing SAM size. 
For instance, the plant hormones cytokinin. Exogenous cytokinin was found to 
increase shoot meristem size in corn embryos (Giulini, Wang and Jackson, 2004). 
Plants with reduced cytokinin showed slow shoot growth and triple mutants of 
cytokinin receptors have smaller SAMs (Kieber, 2002; Higuchi et al., 2004).  
Interestingly, cytokinin also forms a feedback loop with WUS: WUS expression can 
be activated by type-B cytokinin response regulators (RRs) while WUS promotes 
cytokinin responses at the shoot apex by repressing a type-A RR, ARR7, a negative 
regulator of cytokinin response (Figure 1.1C) (Leibfried et al., 2005; Zhang, May and 
3 
 
 
 
Irish, 2017). Cytokinin induces WUS expression in the OC through a cytokinin 
receptor, ARABIDOPSIS HISTIDINE KINASE4 (AHK4), which exhibits overlapping 
expression domains as WUS (Gordon et al., 2009). Though it seems that cytokinin 
forms a positive feedback loop with WUS, it was reported that WUS represses 
cytokinin biosynthesis genes (the LOG genes) in the cells sitting at the topmost layer 
of SAM (Chickarmane et al., 2012). The complex regulations of cytokinin and WUS 
are great examples of how plant SAM finetune gene expression to maintain and 
restrict the stem cell pool, allowing healthy growth to occur. 
1.1.2 Root apical meristem (RAM): structure and regulation of development 
While the SAM giving rise to all the above-ground tissues, growth is also happening 
at another apex buried under the ground, the root. Arabidopsis root can be divided 
into three zones: the meristematic zone (MZ), the elongation zone (EZ) and the 
differentiation zone (DZ) (Figure1.2 A). The new cells are produced in the MZ and 
enter the EZ to elongate and prepare themselves for differentiation at the DZ.  
Similar to the SAM, there are a group of cells near the tip of the root called the root 
apical meristem (RAM) or the MZ. However, the cells at the RAM are arranged 
differently from SAM. The quiescent center (QC) at the root tip contains a few slowly 
dividing stem cells (Figure1.2 A-C). These cells give birth to daughter cells that 
become initials (stem cells) for different parts or layers of the root. Together, the QC 
and the initials form the stem cell niche (Petricka, Winter and Benfey, 2012). These 
initials give rise to the cell files (circular layers) at the proximal part of the root: 
4 
 
 
 
Figure 1.2: The organization and genetic regulation of Arabidopsis RAM. 
(A) Arabidopsis root is divided into three major zones: meristematic zone, elongation 
zone and differentiation zone (labeled with brackets). (B) Diagrams showing cell 
organizations in the RAM. The cells are aligned along the length of the root. Different 
cell types are marked with different colors. (C) The maintenance of QC (white cells in 
magnified region) and initials (colored cells in magnified region) is regulated by the 
WOX5-CLE40 pathway, transcription factors SHR, SCR and PLTs. 
(A) was adapted from (Jin et al., 2013). 
(B) was adapted from (Vaughan-Hirsch, Goodall and Bishopp, 2018). 
(C) was adapted from (Petricka, Winter and Benfey, 2012). 
 
 epidermis, cortex, endodermis, pericycle and stele (vascular tissues), and the 
columella cells at the more distal part of the root (Figure1.2 B). 
Though the structures are distinct from the SAM, the regulations of stem cell 
maintenance in RAM share some similarities. WUSCHEL-RELATED HOMEOBOX5 
(WOX5), a WUS homolog, is expressed in the QC and helps the QC to maintain 
5 
 
 
 
quiescence. WOX5 suppresses cell division in QC by repressing the expression of a 
cyclin gene, CYCD3;3 (Forzani et al., 2014). WOX5 also maintains stem cell 
identities of the surrounding initials in a non-cell-autonomous way. In the wux5 
mutant, the columella stem cells seem to lose their stem cell identity and acquire 
differentiated-cell like phenotypes (Sarkar et al., 2007). The expression of WOX5 is 
restricted to the QC in a similar manner to WUS in the shoot OC (Figure1.2 C). 
CLAVATA3/ESR-RELATED 40 (CLE40), a gene encodes a peptide closely related to 
CLV3, represses WOX5 expression through the receptor-like kinase ARABIDOPSIS 
CRINKLY4 (ACR4) and confers WOX5 expression to the QC (Stahl et al., 2009).  
Besides the WOX5-CLE40 pathway, there are other factors that contribute to the 
maintenance of root stem cells. Loss of function at the SHORT ROOT (SHR) and 
SCARECROW (SCR) loci reduces the root length of Arabidopsis seedlings due to 
defects in QC (Benfey et al., 1993; Laurenzio et al., 1996; Wysocka-Diller et al., 
2000), indicating that these two GRAS family of transcription factors are necessary 
for the RAM functions. Interestingly, SHR is expressed in the stele and moves to the 
adjacent layer (QC, cortex/endodermis initials and daughter cells, endodermis) to 
induce SCARE CROW (SCR) expression (Helariutta et al., 2000; Nakajima et al., 
2001; Levesque et al., 2006). SHR then forms a complex with SCR to maintain the 
stem cell pools (Cui et al., 2007). 
In parallel with SHR and SCR, AP2-domain transcription factor PLETHORAs (PLTs) 
function in the meristematic zone to maintain QC identity and activate division in the 
initials in a dosage-dependent manner. In other words, PLTs form a maximum at the 
QC to promote stem cell identity and lower expression of PLTs in the initials allow 
6 
 
 
 
cell division to occur. Interestingly, ectopic expression of PLTs were able to induce 
root formation at the shoot, suggesting the critical role of PLTs in specifying root 
identity. The double mutant of plt1;plt2 has much shorter root and reduced RAM 
size. PLTs also regulate the expression of auxin transporter PIN-FORMED (PINs). 
The PIN genes are downregulated in the plt mutants. So PLT promotes root stem cells 
by promoting auxin maximum at the root tip, which is consistent with auxin forming 
a similar gradient as PLTs (Aida et al., 2004; Galinha et al., 2007). Application of 
auxin promotes the root initiation but inhibits the root elongation (Thimann, 1936). It 
was believed that auxin was synthesized in the shoot and transported to the root 
through vascular tissues. However, auxin synthesis genes are also found expressed in 
the root stem cell, suggesting local auxin synthesis in the root (G?lweiler et al., 1998; 
Blilou et al., 2005; Ljung et al., 2005; Stepanova et al., 2005, 2008; Grieneisen et al., 
2007). In addition, auxin appears to regulate PLT expression, forming a positive 
feedback loop. Specifically, PLTs are thought to be activated by auxin through the 
auxin response factors MONOPTEROS (MP) and NONPHOTOTROPIC 
HYPOCOTYL4 (NPH4).  help stabilizing the auxin maximum (Petricka, Winter and 
Benfey, 2012). Together, auxin and PLTs specify and maintain the stem cell niche at 
the root tip and allow cells to divide only upon their exiting the stem cell niche. 
Cytokinin is an important phytohormone in plant meristems. One interesting 
observation is the opposite roles cytokinin plays in root vs. shoot. Exogenous 
application of cytokinin reduces the meristem size in root but increases meristem size 
in shoot. In root, cytokinin and auxin work antagonistically. Cytokinin promotes cell 
7 
 
 
 
differentiation at the transition zone (TZ), which is located between the MZ and the 
EZ (Miyawaki, Matsumoto-Kitano and Kakimoto, 2004; Dello Ioio et al., 2007).  
Cytokinin biosynthesis in the root is regulated by a family of transcription factors, the 
class III homeodomain-leucine zipper (HD-ZIP III). In Arabidopsis, the HD-ZIP III 
family has five members: PHABULOSA (PHB), PHAVOLUTA (PHV), REVOLUTA 
(REV), CORONA (CRN)/ATHB15 and ATHB8. They are involved in adaxial/abaxial 
polarity determination of lateral organs and vasculature patterning in plants. High 
expression of HD-ZIP IIIs induces metaxylem formation while downregulation of 
HD-ZIP IIIs promotes protoxylem formation. All members of HD-ZIP IIIs are targets 
of miR165/166. (McConnell and Barton, 1998; Emery et al., 2003; Tang et al., 2003; 
Zhong and Ye, 2004; Kim et al., 2005; Zhou et al., 2007).  
The PHB gain-of-function mutant phb-1d showed short root and small RAM 
phenotypes. The expression of PHB is upregulated and broadened in the phb-1d 
mutant. Similar phenotypes have been found in the PHV gain-of-function phv-1d. 
These phenotypes are reminiscent of root treated with exogenous cytokinin 
(McConnell and Barton, 1998; Dello Ioio et al., 2007; Dello Ioio et al., 2012), 
suggesting that the phenotype of HD-ZIP III gain-of-function could be mediated by 
an increase of cytokinin. Indeed, it was demonstrated that PHB could activate the 
transcription of ISOPENTENYLTRANSFERASE 7 (IPT7), a rate-limiting enzyme for 
cytokinin synthesis, suggesting that PHB directly regulates cytokinin synthesis in 
root. However, cytokinin produced in the vasculature restricts the expression of PHB 
and the negative regulator of PHB, miR165 and such regulations may provide 
8 
 
 
 
robustness against cytokinin fluctuations in the root and help balance cell 
proliferation and differentiation (Dello Ioio et al., 2012).  
Although much is known about the WUS-CLV and HD-ZIP III transcription factors in 
regulating plant meristems, their relationship with cell cycle regulation is not well 
established. How did stem cells remain quiescent and how the existing regulators 
interact with cell cycle regulators are not well known. My work focused on this area 
using TSO1, a putative plant cell cycle regulatory complex component, as an entry 
point. 
1.2 Arabidopsis TSO1 and its role in meristem development 
1.2.1 TSO1 encodes a transcription factor with cysteine-rich repeats 
TSO1 was discovered from an EMS mutagenesis screen (Liu, Running and 
Meyerowitz, 1997; Hauser, Villanueva and Gasser, 1998). The strong allele tso1-1 
was isolated for its severe phenotypes at the shoot. The tso1-1 plants have highly 
fasciated shoot apical meristems and lack most of the floral organs, thus they are 
sterile. TSO1 was then cloned and subsequently proven to form a regulatory module 
Figure 1.3: Structure of TSO1 
A simplified presentation of TSO1 protein structure. The conserved sequence of TCR 
motif and Hinge motif are listed. The number marked the amino acid position. 
 
9 
 
 
 
with MYB3R1 to control cell cycle at the SAM and RAM (Hauser et al., 2000; Song 
et al., 2000; Wang et al., 2018).  
 
Arabidopsis TSO1 encodes a nuclear protein with two cysteine-rich repeat motifs 
(TSO1 Cysteine Rich motif or TCR motif) separated by a conserved hinge region 
(Figure 1.3) (Song et al., 2000). Together, they are named as cysteine-rich repeats 
domain. The TCR motif has 9 conserved cysteine with invariable spacing (Figure1.3), 
which binds zinc and then DNA (Andersen et al., 2007; Marceau et al., 2016). 
Members of the cysteine-rich repeats domain protein family are found in both plants 
and animals. There are 8 members in Arabidopsis thaliana, which can be grouped 
into two groups based on domains outside the cysteine-rich repeats domain. TSO1, 
SOL2 and SOL1 fall into the first group (type I) while the rest 5 (TCX4, TCX5, TCX6, 
TCX7 and TCX8) fall into the second group (type II).  
1.2.2 tso1 mutants exhibit defects in meristem development and tso1 alleles can be 
categorized into two classes 
All 9 alleles of tso1 mutants are recessive mutations and they can be grouped into 2 
classes. tso1-1 and tso1-2 are Class I mutations. Both are missense mutations that 
convert one conserved cysteine in the TCR motif to a tyrosine and both exhibit strong 
phenotypes (Sijacic, Wang and Liu, 2011). The tso1-1 mutants exhibit developmental 
defects in both floral meristems and inflorescence meristems (Figure1.4 A and B), 
leading to sterility. The inflorescence meristems of tso1-1 are frequently fasciated 
while the mutant floral meristems fail to give rise to normal floral organs except 
sepals and remain callus-like (undifferentiated). The fasciation observed at the SAM 
10 
 
 
 
is caused by the split of one meristem into two or multiple meristems rather than 
Figure 1.4: tso1-1 (Class I) plants exhibit shoot apical meristem fasciation and short 
root phenotypes while Class II mutations of TSO1 cause fertility defect. 
(A) A wild type (Landsberg erecta or Ler-0) plant showing normal shoot apical meristem 
and normal flowers. A fertile silique is indicated by the white arrow. (B) A tso1-1 (type I) 
plant showing highly fasciated shoot meristem and failure of floral organ formation. (C) 
A tso1-3 (type II) plant with normal SAM and flowers. Reduced fertility indicated by the 
short siliques (white arrow). (D) The roots of tso1-1 plants are shorter than wild type 
plants. The tso1-3 plants have no root phenotype Scale bar in A and B: 500 ?m 
All pictures were adapted from (Sijacic, Wang and Liu, 2011; Wang et al., 2018). 
 
 enlargement of a single meristem. Electron microscopy of tso1-1 floral meristem 
sections showed large and aberrant nuclei, elevated DNA content and abnormal cell 
plate formation, indicating defects in cell division (Liu et al 1997; Song et al 2000). 
tso1-1 plants have short root phenotype resulting from the reduction in meristem size 
and possibly early differentiation of the stem cells (Figure1.4 D) (Wang et al., 2018). 
Therefore, TSO1 seems to cause opposite defects at the SAM and RAM. 
The rest of the TSO1 mutant alleles (tso1-3 to tso1-9) fall into Class II. The Class II 
mutants, represented by tso1-3 plants, exhibit weaker phenotypes than the Class I 
plants (Figure1.4 C). tso1-3 is a nonsense mutation that generates truncated TSO1 
transcripts lacking the hinge motif and the second TCR motif (Song et al., 2000). 
11 
 
 
 
tso1-3 plants have normal inflorescence meristems and can develop normal flowers. 
However, these plants have reduced fertility. Closer examination of the reproductive 
organs revealed that tso1-3 plants produce ovules without the embryo sac (Hauser, 
Villanueva and Gasser, 1998). These results agree with the phenotypes observed in 
another Class II mutant, the tso1-5 mutant. In addition to similar defects in ovules as 
tso1-3 plants, the pollen grains in tso1-5 are enlarged and collapsed (Andersen et al., 
2007). Together, we conclude that Class II tso1 mutant plants are defective not only 
in the female gametes but also in the male gametes. Taken together, the analysis of 
two classes of tso1 mutants indicates that TSO1 is involved in regulating reproductive 
organ development in Arabidopsis.  
It was puzzling why the missense tso1-1 causes more severe phenotypes? The 
mystery has been resolved by knocking down tso1-1 transcript using artificial 
microRNA. The resulting tso1-1; amiRNA plants produce normal flowers with 
reduced fertility, which resembles tso1-3 (Class II) (Sijacic, Wang and Liu, 2011). In 
addition, 3 intragenic suppressors were isolated from a tso1-1 suppressor screen. 
These alleles were named as tso1-7, tso1-8 and tso1-9 (Wang et al., 2018). These 
findings suggest that tso1-1 is a recessive antimorphic allele, which not only loses its 
own function but also interferes with the function of its paralogs such as SOL2. 
Specifically, double mutants of sol2; tso1-3 showed strong phenotypes similar to 
those of tso1-1. Moreover, a BiFC experiment showed that SOL2 interacts with 
TSO1-1 mutant protein but not the wild type TSO1. In summary, tso1-1 is a recessive 
antimorphic allele which interferes with SOL2 function by physical interaction, 
leading to a stronger phenotype than tso1 null. 
12 
 
 
 
1.2.3 The animal homologs of TSO1 are cell cycle regulatory proteins 
The animal homolog of TSO1, LIN54, is a component of the DREAM complex 
(Sadasivam and DeCaprio, 2013a; Fischer and M?ller, 2017). LIN54 is homologous 
with TSO1 only within the cysteine-rich repeats domain. The animal DREAM 
complex is a critical cell cycle regulator, acting both as a repressor and an activator of 
cell cycle genes depending on its interactors. The core complex of the DREAM 
complex, named as the MuvB complex or MuvB core (Korenjak et al., 2004; 
Harrison et al., 2006), consists of the core subunits of LIN54, the human homolog of 
TSO1, LIN53/RBBP4, LIN9, LIN37, and LIN52 (Litovchick et al., 2007). In 
mammalian cells, the MuvB core can associate with the retinoblastoma (RB) family 
members (P107 and p130), the E2F transcription factors and their dimerization 
partners (DP1-3) to form the DREAM complex. This complex represses G1/S genes, 
thus preventing cell cycle entry. In G2/M phase, the MuvB binds to B-Myb 
transcription factor instead to form MMB complex to promote mitotic gene 
expression (Litovchick et al., 2007; Sadasivam, Duan and DeCaprio, 2012). 
MuvB is an evolutionarily conserved complex. It has been identified in fly, worm, 
and Arabidopsis in addition to mammalian cells (Lewis et al., 2004; Harrison et al., 
2006; Litovchick et al., 2007; Schmit et al., 2007; Kobayashi et al., 2015). All of the 
homologs of the MuvB core have been found in plants and several DREAM-like 
complexes have been identified in Arabidposis (Ning et al., 2020; Lang et al., 2021). 
Additionally, plants have more copies of each of the complex components (Table1.1). 
For example, there are eight Arabidopsis homologs of LIN54 and five homologs of 
B-Myb. The existence of multiple paralogous genes coding each of the DREAM 
13 
 
 
 
component could potentially contribute to different isoforms of the DREAM/MMB 
complex, acting in different developmental contexts. So far, the plant DREAM-like 
complexes have been found to regulate meristem development, reproduction, stomata 
development, DNA methylation and response to DNA damage (Liu, Running and 
Meyerowitz, 1997; Hauser, Villanueva and Gasser, 1998; Song et al., 2000; Sijacic, 
Wang and Liu, 2011; Wang et al., 2018; Simmons et al., 2019; Ning et al., 2020; 
Lang et al., 2021). These findings support the hypothesis that different forms of plant 
DREAM function in different biological processes. 
Another distinct feature of plant DREAM-like complexes is that they seem to have 
components not found in the animal cells. Previously, two plant DREAM-like 
complexes were found to contain a cyclin-dependent kinase (CDK) CDKA;1, and one 
plant DREAM-like complex contains an uncharacterized protein encodes by 
AT2G40630 (DRC2) (Kobayashi et al., 2015; Ning et al., 2020). The presence of 
CDKA;1 in the purified complex suggests that the plant DREAM-like complex may 
regulate cell proliferation through somewhat different mechanisms from the animal 
DREAM complex. For instance, the plant MuvB core may associate with various 
CDKs to directly control cell proliferation in a developmental stage-specific or tissue-
specific manner.  
Like plants, disruptions of the DREAM/MMB complex in animal cells usually lead to 
severe developmental defects or cancer. Mutating either homolog of LIN37 (Mip40), 
LIN54 (Mip120 
14 
 
 
 
) or LIN9 (Mip130) leads to sterility in flies (Beall et al., 2007). The loss of both 
retinoblastoma protein (pRb) and Rb-like (p107/p130) induces retinoblastoma in mice 
while overexpression of B-Myb was found in many cancers including breast cancer 
and colorectal cancer, and often associated with poor patient outcomes (Musa et al., 
2017; Wu et al., 2017). Understanding the function of the DREAM/MMB complex 
during tumorigenesis has become an interesting research field. A recent finding 
revealed novel mechanisms of how overexpression of B-Myb disrupts cell cycle 
Table 1.1: Arabidopsis homologs of the DREAM complex components 
This table presents the plant homologs of each DREAM components. The mammalian 
DREAM protein names are listed in the first column. AGI stands for Arabidopsis gene 
ID and Alias stands for the annotated name of the corresponding gene. The Muv B core 
genes are shaded in light green color. 
The table was adapted from (Lang et al., 2021). 
 
 15 
  
 
 
(Iness et al., 2019). The over-accumulated B-Myb can interfere with DREAM 
assembly by affecting phosphorylation of LIN52, an essential process for forming the 
DREAM complex, which may lead to the poor patient outcomes. 
LIN54, the TSO1 homolog, is the only component in the MuvB core that interacts 
with DNA. It specifically recognizes and binds to the consensus sequence TTYRAA 
in the CHR (cell cycle genes homology region) in the target promoters (Marceau et 
al., 2016). This binding enables DREAM/MMB complex to be recruited to a large 
numbers of cell cycle genes, for example, the G2/M cell cycle genes cdc2 in human 
cells (Schmit et al., 2007; M?ller et al., 2014, 2016). The phenotypes observed in 
tso1-1 plants are consistent with the phenotypes found in animal systems. For 
instance, flies with the TSO1 homolog (Mip120) knock-out are sterile; LIN54 
depletion cells went through prolonged G2 phases (Beall et al., 2007; Schmit, Cremer 
and Gaubatz, 2009). It seems that TSO1 may function in a similar manner as LIN54 
in plant. The discoveries of several potential plant DREAM complex support this 
hypothesis (Kobayashi et al., 2015; Wang et al., 2018; Ning et al., 2020; Lang et al., 
2021). Nevertheless, little is known about the precise functions of these MuvB core 
proteins in plants.  
1.2.4 A mutagenesis screen identified myb3r1 a suppressor of tso1-1  
To understand how TSO1, a member of the plant DREAM complex ,regulates cell 
division in the context of shoot meristem development, a former graduate student set 
out to identify genetic suppressors of tso1-1 through mutagenesis screens (Wang et 
al., 2018). Mutations in genes downstream of TSO1 could potentially suppress tso1-1 
phenotype. Since tso1-1 is sterile, an inducible copy of wild type TSO1 was 
16 
 
 
 
introduced in to plants homozygous for tso1-1 in order to obtain homozygous mutant 
seeds. The full length TSO1 cDNA driven by the 35S promoter was fused to a rat 
glucocorticoid receptor (GR) and transformed into tso1-1 heterozygous plants. The 
TSO1-GR fusion proteins are retained in the cytoplasm without dexamethasone 
(DEX) treatment. When supplied with DEX, the fusion proteins can enter the nucleus 
and rescue tso1-1 phenotype, generating thousands of tso1-1 homozygous mutant 
seeds. These seeds were then treated with Ethyl methanesulfonate (EMS) and 
screened for the restoration of normal meristem and fertility without DEX 
application.  
Isolated from the screen were 45 suppressors. 32 of the suppressors were mapped to 
MYB3R1, one of the R1R2R3-MYB (MYB3R) genes that shares homology with 
animal B-Myb transcription factors (Wang et al., 2018). MYB3R1 and its close 
Arabidopsis homolog MYB3R4 are activators for G2/M phase genes. For instance, 
MYB3R1 and MYB3R4 activate KNOLLE to promote cytokinesis (Haga et al., 2007, 
2011). Knock-out of MYB3R1 in tso1-1 can suppress both shoot and root phenotype 
and restore fertility (Figure1.5 A, B and C). It has been found that MYB3R1 is 
ectopically expressed and overexpressed at both SAM and RAM in tso1-1 plants 
(Figure1.5 D), indicating that TSO1 represses MYB3R1 transcription in the wild type 
plants. Thus, any mutations removing the excessive MYB3R1 can suppress tso1-1 
phenotypes. 
17 
 
 
 
Figure 1.5: Overexpression of MYB3R1 mediates tso1-1 phenotypes. 
(A) An inflorescence of a homozygous tso1-1 mutant plant. (B) An inflorescence of 
tso1-1; myb3r1 double mutant. The mutation in MYB3R1 suppressed the fasciation and 
sterility of tso1-1. (C) Quantification of the RAM size. Mutating MYB3R1 also 
suppressed the short root phenotype of tso1-1. (D) Staining of pMYB3R1-GUS in wild 
type (WT) and tso1-1 SAM. Intense blue staining in tso1-1 suggests overexpression of 
MYB3R1. (E) Staining of pMYB3R1-GUS in WT and tso1-1 RAM (7dpg). Upper arrow: 
transition zone; lower arrow: QC; brackets: intense blue stain accompanied with 
emerging root hairs. Scale bar: 200 ?m. 
All pictures were adapted from (Wang et al., 2018). 
  18 
  
 
 
To further investigate the suppression mechanism, 35S::MYB3R1 has been 
transformed into wild type plants and tso1-3 plants. Surprisingly, neither of the 
resulting plants resemble tso1-1 phenotype. It seems that tso1-1 phenotype is not 
solely mediated by misexpression of MYB3R1. Later, it was found that phosphomimic 
of MYB3R1 at S656 position enhanced tso1-3 fertility defects but failed to 
phenocopy the SAM fasciation of tso1-1 mutants (Wang et al., 2018). It seems that 
overexpression of MYB3R1 is necessary but not sufficient to cause the tso1-1 
phenotype. A coimmunoprecipitation (Co-IP) experiment showed that TSO1 could 
physically interact with MYB3R1 to form a regulatory module, which agrees with 
previous publications on the MYB3R1 a component of the plant DREAM-like 
complexes.  
The result of simply overexpressing MYB3R1 did not phenocopy the SAM fasciation 
of tso1-1 led to the hypothesis that there might be other components in the TSO1-
MYB3R1 regulatory module. Although several DREAM-like complexes were 
discovered in Arabidopsis seedlings and leaves, none has been reported in SAM or 
RAM where TSO1 and MYB3R1 are expressed. It suggests that plants may have 
multiple DREAM-like complexes present in different tissues to coordinate cell cycle 
with development. My work has been focused on exploring other components 
involved in the TSO1-MYB3R1 regulatory module. A144 was isolated from the 
previous suppressor screen and was later characterized as a unique tso1-1 suppressor, 
indicating its role in the TSO1 regulatory pathway. The work will further our 
understanding of how plant meristems balance cell proliferation and differentiation 
19 
 
 
 
through manipulating regulatory networks of transcription factors and cell cycle 
machineries to develop and maintain their stem cell identities at the same time.   
1.3 Cyclin genes and their roles in meristem development 
1.3.1 The classification of plant cyclin genes 
Cyclins, as the name indicates, has unique cycling expression patterns. The 
abundance of a cyclin protein oscillates throughout the cell cycle. Cyclins are 
important cell cycle regulators. They bind to CDKs to form cyclin-CDK complexes. 
This binding is critical for CDK activation and substrate recognition. Cyclin also 
helps to bring CDK into the nucleus as CDK usually lacks a nuclear localization 
signal (David-Pfeuty and Nouvian-Dooghe, 1996; Schafer, 1998).  Cyclins can be 
divided into following categories according to their expression patterns, which also 
are indicators for their functions. G1/S cyclins promote entry into the cell cycle, 
whereas S cyclins promote DNA synthesis, and M cyclins help cells to enter M phase. 
Cyclins with D-box domains are targeted for degradation after fulfilling their 
functions, making cell cycle irreversible.  
Cyclins can be further classified according to their sequences, though they share poor 
homology in general. Based on the literature, there are 19 cyclins identified in human. 
Each can form a different complex with CDKs (Truman et al., 2012). This number 
greatly expands in plants, such as Arabidopsis, that has 50 putative cyclin genes. 
These cyclins can be grouped into eight classes: CYCA1-3, CYCB1-3, CYCC, 
CYCD1-7, CYCH, CYCL, CYCP1-4 and CYCT and 3 additional cyclins not belonging 
to these groups: CYCJ18, SOLODANCERS and CYL1 (Figure1.6) (Menges et al., 
20 
 
 
 
2005). The large number of cyclin genes brought great challenges when it comes to 
the analysis of their specific functions. In other words, single mutant is unlikely to 
show any phenotypes, making it hard to interpret the regulatory pathways each cyclin 
is involved in. 
 
Figure 1.6: The phylogenetic tree of Arabidopsis cyclin genes.  
Full length sequences of 50 Arabidopsis cyclins were used in the alignment. 
The unrooted neighbor-joining tree was based on multiple sequence alignment. 
Picture was adapted from (Nieuwland, Menges and Murray, 2007). 
 
1.3 .2 Functions of Arabidopsis CYCA3s 
 
Plants are sessile organisms. They are not able to move from places to places to avoid 
environmental stresses. Therefore, plants need more flexible regulatory modules for 
its growth and reproduction. This may explain why the number of cyclins expanded 
in plants. Cyclins have been found to play important roles in plant development and 
stress responses.  
Cyclins in the same class usually share some similarities in their functions. Though it 
is still hard to know the molecular function of each member, previous studies have 
21 
 
 
 
revealed some common functions of different classes plant cyclins. For instance, the 
expression of B-type cyclins (CYCBs) peak in early M phase (Menges et al., 2005). 
They are most likely to regulate M phase. The expression of D-type cyclins is 
dependent on the presence of mitogens. Adding cytokinin or sucrose induces 
expression of CYCD3;1 in plant cell cultures (Soni et al., 1995). Does this indicate 
that D type cyclins mainly regulate G1/S cell cycle check point? In Arabidopsis, one 
of the CYCB genes was shown to function in male meiosis. Ectopic cell wall 
formation was found in the cycb3;1 mutants, indicating that it prevents cell wall 
formation in Arabidopsis pollen mother cells (Bulankova et al., 2013).  
CYCA3 genes can be further divided into three subclasses: CYCA1 (CYCA1;1 and 
CYCA1;2), CYCA2 (CYCA2;1, CYCA2;2, CYCA2;3 and CYCA2;4) and CYCA3 
(CYCA3;1, CYCA3;2, CYCA3;3 and CYCA3;4). CYCA1 and CYCA2 genes are mainly 
expressed in G2/M while CYCA3s have diverse expression patterns (Menges et al., 
2005). For instance, the expression of CYCA3;1 and CYCA3;2 peaks in S phase, but 
CYCA3;3 is exclusively expressed in the meiotic cells. These expression patterns 
suggest that CYCA3;1 and CYCA3;2 may play roles in the S phase while CYCA3;3 
might specifically function in the meiosis. Since CYCA3;3 lacks a D-box domain, its 
degradation is not mediated by the anaphase-promoting complex (APC/C) but 
alternative mechanisms. Finally, CYCA3;4 show different expression patterns in the 
Arabidopsis MM2d cell line and roots. CYCA3;4 was found to have a constant 
expression in the MM2d cells. However, CYCA3;4 proteins mainly accumulate in the 
G2/M phase in the root. 
22 
 
 
 
None of the single mutant of CYCA3 genes showed significant phenotypes 
(Takahashi et al., 2010; Bulankova et al., 2013; Willems et al., 2020). The cyca3;1-1; 
cyca3;2-1 double mutant was indistinguishable from the wild type plant. Thus, 
overexpression has been used to reveal the potential functions of CYCA3 genes. 
Overexpression of CYCA3;1 and CYCA3;2 caused early termination of the apical 
buds and the lateral branches grow better than the main shoots perhaps as a result of 
loss of apical dominance (Takahashi et al., 2010). Overexpression of CYCA3;4 
caused short roots, defects in leaf growth, and fewer stomata (Willems et al., 2020). 
One intriguing finding about these plant CYCA3s is that they seem to control the 
phosphorylation of RBR1, which is the homolog of animal Retinoblastdoma (RB) and 
a member of the DREAM complex . The plant CYCA3;1-CDKA;1 complex was 
shown to phosphorylate RBR1 in vitro and overexpression of CYCA3;4 resulted in 
hyperphosphorylation of RBR1 in vivo (Takahashi et al., 2010; Willems et al., 2020).  
In a recent publication, mutations in CYCA3;4 were shown to partially suppress 
ccs52a2-1?s phenotype in short root and reduced rosette leaf size (Willems et al., 
2020). The CCS52A2 encodes a activator of the anaphase-promoting complex (Liu et 
al., 2012). CYCA3;4 can be targeted to degradation by APC/CCCS52A2 and hence 
proper degradation of CYCA3;4 by APC/CCCS52A2 is necessary for normal meristem 
development. Meanwhile, mutating CYCA3;1 and CYCA3;2 was not able to suppress 
ccs52a2-1, indicating they may not be regulated by APC/CCCS52A2 in this pathway. 
Meanwhile, efforts have been made to identify specific paring of Arabidopsis cyclins 
with their CDKs through yeast two hybrid assays and BiFC (Boruc et al., 2010; Van 
Leene et al., 2010). BiFC data showed that CYCA3;4 could interact with CDKA;1 
23 
 
 
 
and CDKB2;1, suggesting that CYCA3;4 may form functioning complexes with these 
two CDKs (Figure1.7).  
Figure 1.7: The protein-protein interaction network of CYCA3;4. 
The protein-protein interaction network of CYCA3;4 was based on BiFC data. Each 
node represents a gene, and the lines represent interaction and correlation of expression 
(transcription level). Red: highly positively correlated; Blue: highly negatively 
correlated; Gray: interaction detected without any significant correlation. All interactions 
were detected in the nuclear, which are labeled with ?nuclear? on the lines.  
Picture was adapted from (Boruc et al., 2010) 
  
 
  
24 
 
 
 
 
 
My dissertation investigated a novel suppressor of tso1-1 and identified a G-to-A 
mutation in a cyclin A gene, CYCA3;4, as the causal mutation. Using transgenic 
technique and transient expression assay, I showed that CYCA3;4 was overexpressed 
in the tso1-1 SAM and TSO1 likely regulate the expression of CYCA3;4 through 
regulating another TSO1 target gene MYB3R1.  
I discovered potential TSO1 target genes through comparing the single cell RNA 
sequencing data of tso1-1 root and wild type root. I found that the expression of the 
HD-ZIP III transcription factors and several cytokinin biosynthesis genes are 
increased in the tso1-1 root. The HD-ZIP III genes are mainly overexpressed in the 
vascular cells of tso1-1 while the ectopic expression of cytokinin biosynthesis genes 
is seen in vascular cells, endodermis and QC. The scRNA-seq data of tso1-1 also 
provide rich resources to explore TSO1 functions in regulating other plant hormones 
and transcription factors, which is not included in this dissertation.  
Overall, my work demonstrates the molecular mechanisms of TSO1 regulating shoot 
and root development through regulating a specific cyclin gene and cytokinin 
biosynthesis. It established TSO1 as a critical regulator of plant meristem 
maintenance and development. The regulatory pathway revealed by my work 
furthered our understanding of how conserved cell cycle machineries are involved in 
plant development.  
 
25 
 
 
 
Chapter 2: Mutations in a A-type cyclin gene suppresses tso1-1 
shoot phenotype 
 
2.1 Introduction 
Plant meristems are responsible for generating all above and below ground tissues. 
Identifying gene regulatory circuitries that confer and maintain the ?stem cell? 
property in plant meristems is fundamentally important. In plant shoot apical 
meristem (SAM), the WUSCHEL (WUS)-CLAVATA (CLV) negative feedback loop 
maintains the stem cell pool and limits the meristem size. Besides the WUS-CLV3 
pathway, there has been limited studies of other regulatory circuitries for SAM 
regulation, and little is known about how the stem cell pool regulation is integrated 
with the cell cycle regulation at SAM.  
The DREAM/MMB complex is a master cell cycle regulator in both animals and 
plants (Sadasivam and DeCaprio, 2013b; Kobayashi et al., 2015). The animal 
DREAM complex consists of Retinoblastoma (RB)-like proteins (P107 and p130), 
E2Fs and their dimerization partners DP1-3 and the MuvB core (LIN9, LIN37, 
LIN52, LIN54 and RBBP4). In animal quiescent cells, the DREAM complex prevents 
cells from entering cell cycles while in dividing cells, the MuvB core associates with 
B-Myb (also called MYBL2) instead of Rb-like to form the MMB complex that 
promotes the G2/M phase (Fischer and M?ller, 2017). Recent studies revealed that 
the DREAM/MMB complex exists in plants as well. The Arabidopsis genome 
possesses essentially all the homologous genes of the DREAM/MMB complex 
components and the corresponding plant homologs can form similar complexes (Ning 
et al., 2020; Lang et al., 2021). Additionally, plants have more copies of each the 
26 
 
 
 
complex component. For example, there are eight Arabidopsis homologs of LIN54, 
named as TSO1, SOL1, SOL2, TCX4, 5, 6, 7 and 8, all of which encode two cysteine-
rich CXC motifs separated by a linker region (Hauser et al., 2000; Song et al., 2000; 
Andersen et al., 2007). The existence of multiple paralogous genes coding each of the 
DREAM component could potentially contribute to different isoforms of the 
DREAM/MMB complex, acting in different developmental contexts. Though 
biochemical characterizations have been performed on the possible combinations of 
the plant DREAM/MMB complexes, the functions of each complex and of each 
member of the complex remain largely unknown. 
In animals, disruptions of the DREAM/MMB complex usually lead to cancer. The 
loss of both retinoblastoma protein (pRb) and Rb-like (p107/p130) induces 
retinoblastoma in mice (Wu et al., 2017), while overexpression of B-Myb was found 
in many cancers including breast cancer and colorectal cancer, and often associated 
with poor patient outcomes (Musa et al., 2017). While plants do not suffer from 
cancer, plant mutants defective in the DREAM/MMB components exhibit shoot 
meristem fasciation, an over-proliferation of SAM. In Arabidopsis, the tso1-1, an 
antimorphic mutation in the Arabidopsis homolog of LIN54, shows fasciated shoot 
apical meristems, and tso1-1 mutant flowers fail to differentiate into floral organs and 
are sterile (Liu, Running and Meyerowitz, 1997; Sijacic, Wang and Liu, 2011; Wang 
et al., 2018). Meanwhile, the cells in the tso1-1 root apical meristem (RAM) exit cell 
cycle early, resulting in a short root phenotype, suggesting opposite effects of TSO1 
on shoot and root meristems. tso1-3, a loss-of-function allele, on the other hand, has 
weak phenotypes; it develops normal flowers and normal length root but exhibits 
27 
 
 
 
reduced fertility (Hauser et al., 2000; Sijacic, Wang and Liu, 2011; Wang et al., 
2018). These two different mutant alleles of TSO1 in Arabidopsis provide a useful 
tool to dissect DREAM/MMB function in the context of plant meristem regulation. 
To identify genes that may act in the same pathway as TSO1, we previously 
conducted a genetic screen for suppressors of tso1-1 (Wang et al., 2018). Seeds of 
tso1-1 containing an inducible TSO1 (35S::TSO1-GR) were mutagenized. When 
applied with dexamethasone (DEX), tso1-1; 35S::TSO1-GR M1 plants were able to 
overcome sterility and gave rise to M2 progeny, which were screened for suppressors. 
Thirty-two suppressors from the screen were found to reside in MYB3R1, which 
encodes one of the five Arabidopsis homologs of B-Myb. The work established that 
the wild type TSO1 activity is required to repress MYB3R1 expression to prevent 
SAM over-proliferation.  
In this study, we characterized a second suppressor locus of tso1-1 from the same 
genetic screen. We showed that mutations in an A-type cyclin named CYCA3;4 
suppressed tso1-1 shoot fasciation and sterility to certain degrees. Further, TSO1 and 
MYB3R1 were shown to regulate the expression of CYCA3;4, and CYCA3;4 
overexpression enhances tso1-3 phenotype. The work reveals the function of a 
specific cyclin in shoot meristem regulation and provides mechanistic insights into 
the function of the TSO1-MYB3R1 regulatory module in shoot meristem regulation.  
28 
 
 
 
2.2 Results 
2.2.1 A splice-site mutation in CYCA3;4 suppresses tso1-1 shoot phenotype 
In order to identify new components of the TSO1 regulatory pathway, a second 
suppressor locus, defined by a single allele A144, was analyzed. A144 suppresses the 
shoot fasciation of tso1-1 mutant and the fertility defects of both strong (tso1-1) and 
weak (tso1-3) alleles of tso1 (Figure2.1). However, the suppression is not complete as 
A144 only partially restores the fertility of tso1-1.  
 
 
29 
 
 
 
 
Figure 2.1 Homozygous A144 mutation suppresses defects of tso1-1 and tso1-3. 
(A) Comparison of inflorescences and siliques among tso1-1 plants heterozygous or homozygous 
for the A144 mutation. The top panel illustrates suppression of fertility defects by homozygous 
A144. Arrowheads point at sterile carpels, and arrows indicate fertile siliques. Bottom images 
show shoot meristem fasciation in tso1-1 and tso1-1; A144/+ with more floral buds and wild type-
like inflorescence in tso1-1; A144/A144. All plants in (A) contain the 35S::TSO1-GR transgene 
but are not treated with DEX. (B) Comparison of inflorescences and siliques of a weak tso1 allele, 
tso1-3, which has reduced fertility (top panel) but normal SAM (bottom).  Homozygous A144 
further improves tso1-3 fertility with longer and fuller siliques. Scale bars in A, B: 1cm in the 
upper panel and 1mm in the lower panel. 
 
 
  
 
To isolate the gene defined by the A144 suppressor, an F2 mapping population was 
created. Using the SIMPLE mapping pipeline (Wachsman et al., 2017), A144 was 
mapped to an A-type cyclin gene CYCA3;4 on chromosome 1 (Figure2.2). A G-to-A 
mutation occurs in the last nucleotide of the first exon of CYCA3;4, however it is a 
synonymous mutation (E-to-E) (Figure2.3 A).  
30 
 
 
 
Figure 2.2 Mapping-by-sequencing result showing CYCA3;4 the best candidate for 
A144.  
An output plot from the SIMPLE pipeline (Wachsman et al., 2017) showing the allele 
frequency comparison between the unsuppressed and suppressed plants. CYCA3;4 
(AT1G47230) is located at the peak on Chromosome 1.  
 
  
 
RT-PCR was used to examine if this mutation affects the splicing of CYCA3;4 
transcripts. A CYCA3;4 transcript of wild-type size was not detected in the A144 
plants, but two transcripts of aberrant sizes were detected (Figure2.3 B). Sequence 
analysis of the RT-PCR products shows that the longer aberrant transcript retains the 
first intron while the shorter aberrant transcript used a cryptic splice donor site in the 
first exon. As both the aberrant transcripts contained premature stop codons, A144 
likely causes a non-functional CYCA3;4. 
 
31 
 
 
 
Figure 2.3 The G-to-A mutation affect splicing of CYCA3;4 transcripts. 
(C) Gene model of CYCA3;4. The G-to-A mutation is highlighted in red. (D) RT-PCR 
showing two aberrant transcripts in tso1-1; A144.  
 
 
 2.2.2 CRISPR/Cas9 mediated knockouts of CYCA3;4 also suppress tso1-1 
To confirm that the mutated CYCA3;4 is indeed the causal mutation for A144, we 
conducted both complementation tests and CRISPR/Cas9 knockout of CYCA3;4. The 
genomic sequence of CYCA3;4 (gCYCA3;4; from ~1.4 kb upstream to the end of 
3?UTR) was transformed into the A144 plants containing the tso1-1 mutation. If the 
transgene rescues A144, the resulting transgenic plants should regain the tso1-1 
mutant phenotype, which was indeed observed including meristem fasciation and 
complete sterility (Figure2.4).  
 
32 
 
 
 
 
Figure 2.4 Complementation test confirming CYCA3;4 as A144.  
(A) Top panel shows a tso1-1; A144 shoot with a normal inflorescence and fertile siliques. 
Bottom panel shows a top view of a wild type-like SAM in tso1-1; A144. (B)-(C) are two 
independent transgenic lines showing a loss of suppression due to their harboring the 
gCYCA3;4 transgene. Note the sterile carpels (red arrowheads). The bottom panel shows 
top view of SAMs and reveals a severely fasciated SAM in C. Scale bars: 1cm in the top 
panes and 500?m in the lower panel.  
 
 
  
 Second, a CRISPR/Cas9 construct with a gRNA targeting the first exon of CYCA3;4 
was transformed into tso1-1; 35S::TSO1-GR plants. Several T1 plants showed 
suppressed phenotypes even in the absence of DEX application (Figure2.5 A). These 
plants did not exhibit shoot fasciation and developed somewhat elongated siliques 
containing a few seeds, which contrasts with complete sterility of tso1-1; 35S::TSO1-
GR  plants in the absence of DEX (Figure2.5 B). Sequencing of the CYCA3;4 locus in 
these plants showed either homozygous or biallelic mutations in CYCA3;4 (Figure2.5 
E). Together, these data strongly support that CYCA3;4 defines the second suppressor 
33 
 
 
 
locus of tso1-1, and A144 is renamed as cyca3;4-4 (cyca3;4-1, cyca3;4-2, and 
cyca3;4-3 were previously reported (Willems et al., 2020)).  
Figure 2.5 CRISPR/Cas9 knockouts of CYCA3;4 suppress tso1-1 shoot but not root defects.  
(A) Defects of tso1-1 in fertility (top) and meristem fasciation (bottom) are suppressed by a 
CRISPR knockout mutation in CYCA3;4. (B) Comparing siliques of tso1-1 with tso1-1; 
cyca3;4CR3. While tso1-1 carpels stay unfertilized and contain no seed, the tso1-1; cyca3;4CR3 form 
short siliques with a few seeds inside. (C) tso1-1 mutants develop short roots in comparison to wild 
type (Ler). A tso1-1; cyca3;4CR3 double mutant has a root length similarly to tso1-1. (D) 
Quantification of root length in different genotypes. *** stands for p<0.001 (one-way ANOVA 
and Tukey?s test). (E) CRISPR/CAS9-generated mutant alleles of CYCA3;4. Red font marks the 
seed RNA, green font highlights insertions, ?-? and ?+? indicate deletion and insertion 
respectively, and blue font marks PAM. Scale bars: 1cm in A (upper), B and C, 500?m in A 
(lower).  
 
  
 
 CRISPR/Cas9 directed knock-out of CYCA3;4 allowed us to investigate if the 
 cyca3;4 mutations could suppress the tso1-1 short root phenotype without worries of 
background mutations caused by the EMS mutagenesis. Both tso1-1 and tso1-1; 
cyca3;4CR3 plants had similar root length, which is shorter than that of wild-type Ler 
(Figure2.5 C and D), suggesting that mutations in CYCA3;4 do not suppress tso1-1?s 
short root phenotype. It remains to be determined if this tissue-specific suppression of 
34 
 
 
 
tso1-1 by the cyca3;4 mutations is due to tissue-specific function of different 
members of the CYCA3 family, which are known to exhibit different expression 
patterns (Takahashi et al., 2010; Willems et al., 2020). 
2.2.3 CYCA3;4 is mis-regulated in tso1-1 mutants 
To understand the mechanism of tso1 suppression by cyca3;4, we analyzed the 
expression of CYCA3;4 in wild-type and tso1-1. A translational reporter of CYCA3;4 
(pCYCA3;4-gCYCA3;4-GUS) was constructed and transformed into plants 
heterozygous for tso1-1. Seven independent transgenic lines were analyzed. Reporter 
GUS expression was compared among the T2 siblings from the same T1 parent; these 
T2 siblings are either tso1-1 or wild type (+/+ or tso1-1/+). In the T2 wild type 
inflorescences, light and even blue staining was observed in the young floral buds 
(Figure2.6 A and B). At floral stages 9-10, the locule of anthers show intense blue 
staining (white arrow in Figure2.6 A); at stages 10-12, intense staining occurs in 
ovules inside the gynoecium as well as stigma (see inset of Figure2.6 A). Therefore, 
the CYCA3;4 expression is at the highest in actively dividing germ cells. In tso1-1 
mutant inflorescences, young floral buds showed significantly stronger GUS staining 
in young floral organ primordia and floral meristems (Figure2.6 C and D). In tso1-1 
plants with a strong phenotype including meristem fasciation and a lack of floral 
organ differentiation, strong and punctate staining were observed (Figure2.6 E and F). 
Since tso1-1 mutants do not form well differentiated stamen or carpels, we do not see 
strong blue staining in gynoecium nor anthers. The sustained and stronger reporter 
GUS expression in the tso1-1 young floral organs and meristems suggest two possible 
and not mutually exclusive possibilities. First, TSO1 may repress the expression of 
35 
 
 
 
CYCA3;4 in young floral primordia to limit proliferation and encourage 
differentiation. A loss of TSO1 resulted in over- and ectopic expression of CYCA3;4 
Figure 2.6 pCYCA3;4::CYCA3;4-GUS reporter expression in wild type and tso1-1 inflorescences 
and roots.  
(A) GUS reporter expression (blue) in an inflorescence of a wild type transgenic plant harboring 
pCYCA3;4::CYCA3;4-GUS. Inset shows a stage 11 flower. Arrows point to locule (L) of anther, 
stigma (S), and ovule (O). (B) A longitudinal section of the same wild type inflorescence in A. Eosin 
Y counterstain the tissues in pink. (C). GUS expression in an inflorescence of a tso1-1 transgenic 
plant containing pCYCA3;4::CYCA3;4-GUS. Inset is a stage 11 flower. (D) A longitudinal section of 
the same tso1-1 inflorescence shown in C with strong and patchy GUS staining throughout the young 
floral meristems and organ primordia. (E). GUS staining in a fasciated tso1-1 inflorescence. 
Significantly more young floral meristems are formed and all strongly stained blue. (F). A 
longitudinal section of the same tso1-1 inflorescence shown in E. (G) GUS staining of a 5 DPG root 
tip in wild type and tso1-1. Arrowheads indicate the upper boundary of RAM and brackets indicate a 
stronger GUS staining band at the most distal region of the RAM. Scale bars: 200?m in A, C and E, 
100?m in B, D, F, and G.  
 
 
 and hence meristem over-proliferation and failure in floral organ differentiation. 
 
 Alternatively, loss of TSO1 may let to many cells that are unable to complete cell 
 cycle and arrest at the G2/M or other cell cycle phases that express CYCA3;4. The 
partial suppression of the tso1-1 phenotype by removing CYCA3;4 function via 
mutations supports the first possibility. 
36 
 
 
 
 
We also compared the CYCA3;4 reporter expression in the roots. Consistent with 
previous published data (Willems et al., 2020), the CYCA3;4-GUS fusion proteins 
are located in the meristematic zone and the transition zone in all cell layers 
(Figure2.6 G). In the elongation zone, CYCA3;4-GUS fusion is restricted in the stele. 
Similar to the wild-type, the CYCA3;4-GUS fusion proteins are also restricted to the 
same tissues in tso1-1 roots (Figure2.6 G). However, the meristem zone is 
compressed in tso1-1 root. Further, while the CYCA3;4-GUS proteins are more 
abundant near the transition zone in the wild-type root, CYCA3;4-GUS seems to 
more evenly distributed throughout the meristematic zone and lacks a strong staining 
band at the transition zone. In summary, TSO1 does not seem to repress the 
expression of CYCA3;4 in the root, but it may impact the spatial distribution of 
CYCA3;4 due to TSO1?s impact on root development (Wang et al., 2018). 
2.2.4 Overexpression of CYCA3;4 weakly enhances the tso1-3 fertility defect 
As we revealed CYCA3;4 mis-expression in tso1-1, we wondered about the 
possibility of over expression or ectopic expression of CYCA3;4 in mediating the 
tso1-1 mutant phenotype. We tested this possibility by overexpressing CYCA3;4 in 
weak tso1-3 mutants to see if over-expressed CYCA3;4 enhances tso1-3 phenotype. 
pUBQ10::CYCA3;4 was introduced into tso1-3/+ plants and the phenotype was 
compared among T2 sibling in three independent lines. There was no visible 
phenotypic difference between wild type plants with or without the 
pUBQ10::CYCA3;4 transgene (Figure2.7 A and B). However, tso1-3 plants 
containing the pUBQ10::CYCA3;4 transgene showed smaller and more abnormal 
37 
 
 
 
siliques (Figure 4A, B). RT-qPCR showed 7-16 fold higher expression of CYCA3;4 in 
three different pUBQ10::CYCA3;4 transgenic lines (Figure2.7 C).  We quantified and 
compared the number of seeds per silique in these three transgenic lines in 
comparison to tso1-3; two of the three tso1-3 transgenic lines made fewer seeds per 
silique than the tso1-3 control although the difference is small (Figure2.7 D). 
Therefore, overexpressing CYCA3;4 enhances the fertility defect of tso1-3 and 
overexpressed CYCA3;4 may have contributed to the tso1 mutant shoot phenotypes. 
 
38 
 
 
 
Figure 2.7 Overexpression of CYCA3;4 enhances tso1-3 fertility defects.  
(A) Overexpression of CYCA3;4 in tso1-3 didn?t significantly alter the morphology of SAM. (B) 
tso1-3 plants with CYCA3;4OE have more unfertilized carpels due to severely abnormal carpels 
(marked by *). (C) RT-qPCR results showing higher expression levels of CYCA3;4 in the three 
CYCA3;4OE transgenic lines. (D) CYCA3;4OE leads to poorer fertility in tso1-3. Y-axis indicates 
percentage of siliques with specific number of seeds. Numbers of siliques used for quantification are 
on top of the graph. **stands for p<0.05, ***stands for p<0.001 (One-way ANOVA and Tukey?s 
test). Scale bars: 1cm in A (upper) and B, 500?m in A (lower). 
  
  39 
  
  
 
 
 
 
2.2.5 A TSO1-MYB3R-CYCA3;4 regulatory module in shoot meristem regulation 
Previously, TSO1-MYB3R1 was found to encode a cell cycle regulatory module 
(Wang et al., 2018). In SAM, TSO1 represses MYB3R1 transcription to prevent G1-S 
transition and limit cell proliferation. At the G2-M cell cycle phases, however, TSO1 
and MYB3R1 likely form a complex to promote G2-M gene expression and 
cytokinesis (Wang et al., 2018). We are curious about the role of CYCA3;4 in the 
context of this TSO1-MYB3R1 regulatory module. One hypothesis is that TSO1 may 
repress CYCA3;4 expression indirectly by repressing MYB3R1 expression to prevent 
the G1-S transition. In tso1-1, ectopic MYB3R1 leads to ectopic CYCA3;4 expression 
which may cause over-proliferation of shoot meristem cells. In this hypothesis, 
MYB3R1 may directly bind and activate CYCA3;4. We searched the Plant Cistrome 
Database containing the DAP-seq/ampDAP-seq data of Arabidopsis transcription 
factors (O?Malley et al., 2016) and found a MYB3R1 binding peak in the promoter of 
CYCA3;4 in the ampDAP-seq data, which shows in vitro-expressed-TF binding to 
PCR-amplified DNA fragments. Therefore, MYB3R1 can bind naked promoter 
region of CYCA3;4 (Figure2.8 A). To further test this possibility, a transient dual-
luciferase assay was subsequently performed in tobacco leaves testing the activity of 
MYB3R1 on the CYCA3;4 promoter driven LUC (Figure2.8 B). Compared with the 
control effector YFP, MYB3R1 caused higher luciferase activities even without 
fusion to the VP64 activation domain (Figure2.8 C, D). Combining with the 
observation that the CYCA3;4 expression domain (Figure2.6) overlaps with that of 
MYB3R1 (Wang et al., 2018), our data support a direct and positive regulatory role of 
MYB3R1 for CYCA3;4 expression. 
40 
 
 
 
Figure 2.8 MYB3R1 can bind to the promoter of CYCA3;4 and activates its transcription 
(A). ampDAP-Seq read abundance highlighting an MYB3R1 binding peak at the promoter of 
CYCA3;4, which is based on Plant Cistrome (O?Malley et al., 2016). (B) Diagram of the 
plasmid constructs used in the dual-luciferase assay. (C) and (D) Dual-luciferase assay results 
showing relative reporter expression of LUC/REN (Y-axis) in tobacco leaves. *** stands for 
p<0.001 (T-test, two-tailed). 
 
  
  
 
 2.3 Discussion 
 
 The plant DREAM/MMB complexes have been demonstrated to have an important 
 
impact on development, DNA damage response, and maintenance of methylations 
(Wang et al., 2018; Simmons et al., 2019, p. 1; Ning et al., 2020; Lang et al., 2021). 
Among the eight Arabidopsis homologs of animal LIN54, Arabidopsis TCX5 and 
41 
 
 
 
TCX6 were shown to act in plant DREAM complexes to preclude DNA 
hypermethylation and prevent excessive cell proliferation (Ning et al., 2020). The 
function of SOL1 and SOL2 is required for efficient cell fate transition in stomata 
lineage, but they act oppositely to TSO1 in regulating the final division to produce the 
guard cells (Simmons et al., 2019).  Previously, we showed that the TSO1-MYB3R1 
module regulates proper development of SAM and RAM (Wang et al., 2018). Despite 
the discovery of MYB3R1 itself as a target of regulation by the TSO1-MYB3R1 
module, little is known about how this module, and by inference the DREAM/MMB, 
balances the cell division and differentiation at the shoot and root meristems. The 
finding of CYCA3;4 as a target of the TSO1-MYB3R1 regulatory module directly links 
this meristem regulatory module to cell cycle regulation and reveals the mechanism 
by which TSO1-MYB3R1 may specifically regulate a member of the cyclin A family 
to control cell division rate at the shoot meristem.  
Figure 2.9 A regulatory module consisting of TSO1, MYB3R1 and CYCA3;4 regulates 
stem cell proliferation in Arabidopsis SAM.  
TSO1 represses the expression of MYB3R1, and MYB3R1 in turn promotes CYCA3;4 
expression. CYCA3;4, together with CDK, may activate MYB3R1 through phosphorylation, 
forming a feed-forward regulatory loop. Together, MYB3R1 and CYCA3;4 promote cell 
division. In wild type SAM, TSO1 limits cell division activity by repressing the expression of 
MYB3R1 and indirectly CYCA3;4.  
 
 
  
 
 42 
  
  
 
 
 
2.3.1 CYCA3;4 is a unique CYCA3 
In Arabidopsis, there are 50 putative cyclin genes in the genome (Nieuwland, Menges 
and Murray, 2007), which can be classified into eight classes (CYCA1-3, CYCB1-3, 
CYCC, CYCD1-7, CYCH, CYCL, CYCP1-4 and CYCT) plus 3 additional unclassified 
cyclins (CYCJ18, SOLODANCERS and CYL1) (Menges et al., 2005). Among the A-
type cyclins, CYCA3s were believed to govern the G1-S transition, a function that 
resembles the E-type cyclin in animals (Yu et al., 2003). The expression profiles of 
the four CYCA3 cyclins in Arabidopsis vary greatly, suggesting potentially diverse 
functions. For instance, CYCA3;1 and CYCA3;2 peak in the S phase while CYCA3;4 
was found to have a constant expression across all cell cycle phases in the 
synchronized Arabidopsis MM2d cell line and roots (Takahashi et al., 2010). 
However, CYCA3;4 proteins accumulate mainly in the G2/M phase in the root 
(Willems et al., 2020). CYCA3;3 is a meiosis-specific cyclin and not expressed in 
somatic cells (Bulankova et al., 2013).  
A prior study aimed at identifying substrates of the Anaphase Promoting 
Complex/Cyclosome (APC/CCCS52A2) isolated genetic suppressors of ccs52a2-1, 
which is defective in the activator subunit CCS52A2 of APC/CCCS52A2. A mutation in 
CYCA3;4A (cyca3;4-1) was identified that partially suppressed ccs52a2-1?s short root 
phenotype. Interestingly, single knockouts of CYCA3;4 in Arabidopsis, including 
cyca3;4-2 and cyca3;4-3 caused by T-DNA insertions, caused no obvious phenotype. 
Therefore, functional redundancy may still exist among family members (Willems et 
al., 2020). However, mutations in CYCA3;1 and CYCA3;2 did not suppress ccs52a2-
1. Thus CYCA3;4 is the main degradation target of APC/CCCS52A2.  
43 
 
 
 
Our genetic screen also identified a single mutation in CYCA3;4 as the suppressor of 
tso1-1. As our mutagenesis screen of tso1-1 is likely saturated due to the isolation of 
32 myb3r1 mutant alleles as the suppressors of tso1-1 (Wang et al., 2018), a lack of 
suppressor mutations in other CYCA3 genes suggests that CYCA3;4 is likely the only 
CYCA3 regulated by the TSO1-MYB3R1 module. In support of this, CYCA3;4 is the 
only CYCA3s in the target gene list of MYB3R1 in the ampDAP-seq data base 
(O?Malley et al., 2016), and CYCA3;4 expression is distinct from CYCA3;1, 
CYCA3;2, and CYCA3;3 (Takahashi et al., 2010; Willems et al., 2020). Therefore, 
CYCA3;4 could be the only or the major CYCA3 gene involved in the TSO1-MYB3R1 
regulatory module in the shoot meristem.  
2.3.2 Potential mechanisms of CYCA3;4 in meristem regulation 
Cyclins normally function by binding and activating cyclin-dependent kinases 
(CDKs) as well as helping specify substrates (K?ivom?gi et al., 2011; Tank and 
Thaker, 2011; Harashima and Schnittger, 2012). Earlier research showed that the 
Arabidopsis CYCA3;4 can bind CDKA;1 (Van Leene et al., 2010) suggesting 
CDKA;1 a likely partner of CYCA3;4 in meristem regulation. Previously, when 
CYCA3;4 was overexpressed in the wild type Arabidopsis seedlings, several 
phosphorylation targets including RBR1 were shown to be hyperphosphorylated 
(Willems et al., 2020); MYB3R1 was not among the phosphorylation targets in the 
study that uses young seedlings. Nevertheless, MYB3R1 could be a phosphorylation 
target of CYCA3;4/CDKA;1 in shoot and root meristems as MYB3Rs need to be 
phosphorylated in order to be active (Araki et al., 2004; Haga et al., 2007). A 
phosphomimic MYB3R1 (presumably over-active MYB3R1) was previously shown 
44 
 
 
 
to enhance the tso1-3 shoot phenotype (Wang et al., 2018). The phenotype of the 
CYCA3;4 overexpression plants resembled the phenotype of the phosphomimic 
MYB3R1 plants, that could possibly result from the over-activation of MYB3R1 by 
the increased CYCA3;4/CDKA;1 complex. 
Based on our findings as well as prior reports, we propose a model (Fig. 6), in which 
TSO1 may act at the G1-S phase to prevent MYB3R1 expression and cell proliferation 
in the SAM. In tso1-1 mutants, when TSO1 activity is lost, MYB3R1 is over- and 
constitutively expressed leading to constitutive expression of CYCA3;4. This 
increased CYCA3;4 subsequently promotes cell division cycle by phosphorylating 
RBR1, MYB3R1 and other substrates, causing over-proliferation of stem cells in the 
tso1-1 SAM. The model suggests a possible positive feedback loop between 
MYB3R1 and CYCA3;4 through phosphorylation. Unfortunately, we failed to purify 
the stable CYCA3;4/CDKA;1 complex that could be used for the kinase assay of 
MYB3R1.  
Interestingly, the binding of MYB3R1 to the CYCA3;4 promoter is observed in the 
ampDAP-seq that tests TF binding to PCR-amplified DNA fragment, but not in the 
DAP-seq that tests binding to genomic DNA fragments retaining 5-methylcytosines. 
Hence, the binding of MYB3R1 to the CYCA3;4 promoter might be sensitive to 
cytosine methylation and likely tissue- and cell cycle phase-specific. 
2.4 Methods 
2.4.1 Plant materials and growth conditions 
Plants were grown on soil (Sungrow) under a 16-h light/8-h dark cycle at 20?. All 
mutants used are in Landsberg erecta (Ler) background. tso1-1, tso1-3, and plants 
45 
 
 
 
heterozygous for tso1-1 or tso1-3 (tso1-1 +/+ sup-5 and tso1-3 +/ + sup-5) were 
described previously (Liu, Running and Meyerowitz, 1997; Hauser, Villanueva and 
Gasser, 1998; Sijacic, Wang and Liu, 2011; Wang et al., 2018). cyca3;4-4 (A144) 
was isolated from an EMS mutagenesis screen of tso1-1; 35S::TSO1-GR (Wang et 
al., 2018).  
2.4.2 Constructs and transformation 
All constructs were transformed through floral dip using Agrobacterium strain 
GV3101. All sequences were cloned from Ler. All relevant primers are listed in 
Table S1.  
For the complementation test construct, genomic sequence of CYCA3;4 (from the 
start of the 5?UTR to the end of the 3?UTR plus ~1.4kb upstream of the 5?UTR) was 
PCR amplified with primers (Table S1), cloned into pCR8/GW/TOPO, and LR-
recombined into pMDC99 (Curtis and Grossniklaus, 2003).  
For the CRISPR/Cas9 construct targeting CYCA3;4, the crRNA was designed using 
the website crispr.dbcls.jp/. Two 19-nt guide sequences (Table S1) were chosen as 
they had no off-target sites. crRNA1 and crRNA2 respectively target the first few 
nucleotides and the last few nucleotides of the first exon. To introduce the two 
crRNAs into the pHEE401E vector (Wang et al., 2015), cloning PCR was carried out 
using the pCBT-DT1T2 vector as the template and the PCR product containing the 
two crRNAs was introduced into pHEE401E via Gibson assembly (Gibson et al., 
2009). The construct was transformed into tso1-1; 35S::TSO1-GR plants. Only 
crRNA2 was able to generate successful knock-out of CYCA3;4. No DEX was 
applied when analyzing the phenotype of the transgenic plants. 
46 
 
 
 
The pCYCA3;4 (~1.4kb) and the genomic sequence of CYCA3;4 (from the start of 
5?UTR to the end of the coding region) were cloned into pCR8/GW/TOPO and 
recombined into pMDC162 (Curtis and Grossniklaus, 2003) to make the 
pCYCA3;4::CYCA3;4-GUS translational fusion. The construct was transformed into 
tso1-1 +/+ sup-5 plants. Seven independent transgenic lines were analyzed. GUS 
expression pattern was compared between wild type and tso1-1 T2 sibling plants 
derived from the same T1 parent (tso1-1 +/+ sup5).  
To overexpress CYCA3;4, the Arabidopsis UBQ10 promoter was cloned from the 
JH23 vector (Zhou et al., 2021) and used to drive full length cDNA of CYCA3;4 
(pUBQ10::CYCA3;4). pUBQ10::CYCA3;4 was first cloned into pCR8/GW/TOPO 
through Gibson assembly and LR recombined into pEarleyGate301 (Earley et al., 
2006). pUBQ10::CYCA3;4 was introduced into agrobacterium GV3101 and used to 
floral dip tso1-3 +/+ sup-5 plants. T  2 plants from three independent transgenic lines 
were analyzed. Seeds in the 6th ? 15th siliques on the main shoot were quantified.  
For dual luciferase assay, pCYCA3;4 (~1.4kb) was cloned into LEI01, LEI02, LEI03 
and LEI04 (Zhou et al., 2021) at NotI and BamHI. The CDS of MYB3R1 was 
amplified and cloned into pCYCA3;4-containing LEI01 and LEI02 at EcoRI and 
AscI. These four constructs were then used in LR reactions to transfer the insertions 
into the vector pLAH-LARm (Taylor-Teeples et al., 2015) to make 35S::MYB3R1-
pCYCA3;4::LUC-35S::REN, 35S::MYB3R1-VP64-pCYCA3;4::LUC-35S::REN, 
35S::Citrine-pCYCA3;4::LUC-35S::REN, 35S::Citrine-VP64-and pCYCA3;4::LUC-
35S::REN.  
47 
 
 
 
We noticed a mis-annotation of CYCA3;4 based on Col-0 and Ler cDNA sequences, 
where the first 3 nucleotides in the 3rd exon should be in the intron.  
2.4.3 Mapping by sequencing 
The mapping population was created by crossing A144 (in the tso1-1; 35S::TSO1-GR 
background) with the parent plant (+/+, tso1-1; 35S::TSO1-GR). Leaf tissues were 
collected and pooled from 34 suppressed F2 plants and 50 unsuppressed F2 plants, 
respectively. Genomic DNAs were extracted using the NucleoSpin Plant II Midi Kit 
(Macherey-Nagel) and then sent for Illumina sequencing (PE-150). The sequencing 
depth of the suppressed plants was 202 folds. The sequencing depth of the 
unsuppressed group was 95 folds. The SIMPLE pipeline (Wachsman et al., 2017) 
was employed for mapping A144 with default settings.  
2.4.4 Root assay 
The seeds were sterilized with 70% ethanol and 10% bleach and then kept in water at 
4? in the dark for 2 days, after which the seeds were planted on ? MS (RPI) 
medium and allowed to germinate under dim light environment for 2 days. Once 
germinated, they were transferred to the growth chamber. 5-DPG (Days Post 
Germination) roots were used for quantification.  
2.4.5 GUS staining and Sectioning 
Inflorescences or 5-DPG seedlings were soaked in 90% acetone for 20min at room 
temperature, followed by three washes of staining buffer [0.2% Triton X-100, 50mM 
NaHPO4 Buffer (pH7.2), 2mM Potassium Ferrocyanide, 2mM Potassium 
Ferricyanide]. They were then stained in a buffer with 2mM X-Gluc for 3 to 3.5 
hours. Tissues were cleared with ethanol series (20%, 35%, 50%) and then stored in 
70% ethanol at 4? before imaging. Subsequently, tissues were embedded and 
48 
 
 
 
sectioned based on a published protocol (Hollender et al., 2012). Imaging was 
performed with Zeiss LSM980 and Zeiss Stemi SV 6. 
2.4.6 Dual-luciferase assay 
Dual-luciferase assay were carried out according to the protocol described previously 
(Taylor-Teeples et al., 2015; Zhan et al., 2018; Zhou et al., 2021).  
2.4.7 RNA extraction and RT-qPCR experiments 
Arabidopsis SAMs were collected for RNA extraction using RNeasy Mini Kit 
(Qiagen). RNA samples were cleaned with DNase I. cDNAs were synthesized using 
RevertAid First Strand cDNA Synthesis Kit (ThermoFisher). RT-qPCR experiments 
were performed on the Bio-rad CFX 96 machine with PowerUp? SYBR? Green 
Master Mix (Thermo Fisher). Three biological replicates were conducted for the RT-
qPCR with three technical replicates. The TIP41(AT4G34270) gene was used as the 
internal control.  
   
49 
 
 
 
Chapter 3: Single cell RNA sequencing revealed possible causes 
of tso1-1 short root phenotype 
 
3.1 Introduction 
As one of the most important organs of vascular plants, roots play essential roles in 
up-taking water and nutrients as well as anchoring the plants to the ground 
(Macdonald and Stevens, 2019). Sitting at the tip of the root is a tissue called the root 
apical meristem (RAM). The RAM is responsible for generating new cells and 
maintaining pluripotent stem cells at the same time. Sitting at the tip of the RAM are 
a small group of cells named the quiescent center (QC) (Figure1.2 B). The QC 
contains pluripotent stem cells that give birth to the initials/stem cells for each layer 
of the root (Figure1.2 B). The maintenance of QC is essential for root growth; the 
root growth and the RAM size are greatly reduced in the mutant of genes that specify 
QC identity (Aida et al., 2004).  
The Arabidopsis transcription factor TSO1 has been shown to function in the root 
development. The tso1-1 mutant produces much shorter root when compared to the 
wild type (Wang et al., 2018). Closer examination revealed that this phenotype was 
due to the shrinkage of the RAM. Mutations in the TSO1 target gene MYB3R1 
suppressed the short root phenotype of tso1-1, and the double mutant tso1-1; myb3r1 
showed normal root length. MYB3R1 was found overexpressed in the tso1-1 root. 
These data suggest that the overexpression of MYB3R1 mediates the short root 
phenotype in the tso1-1 mutant. Nevertheless, no other TSO1 targets have been 
identified in the root. Little is known about the molecular mechanisms of how TSO1 
regulates root development. 
50 
 
 
 
The single-cell RNA sequencing (scRNA-seq) technique is becoming a popular tool 
for analyzing plant gene expression. Before the scRNA-seq, the bulk RNA 
sequencing was often used to examine gene expression within a mixture of different 
cell types. With bulk RNA-seq, it was hard to detect changes in gene expression for 
genes with cell type-specific expression, in particular those expressed in tissues like 
root QC (pluripotent stem cell), which consists of only a few cells. The scRNA-seq, 
on the other hand, allows analysis of gene expression at single cell level, revealing 
new information on gene expression and regulation (Shaw, Tian and Xu, 2021). 
Though scRNA-seq is only in its early stages in plant sciences and there are still 
many challenges, it has already demonstrated its power in revealing gene expression 
heterogeneity between cells (Shaw, Tian and Xu, 2021). The scRNA-seq has been 
applied to investigating developmental trajectories, identifying new developmental 
regulators as well as comparing cell differentiation pathways of cell identity mutants 
(Ryu et al., 2019; Liu et al., 2020; Zhang, Chen and Wang, 2021; Shahan et al., 
2022). It has also been used in rice to identify transcription factor targets (Xie et al., 
2020). In addition, the early-stage single-cell research provides useful tissue-
references for others who are interested in conducting scRNA-seq. The construction 
of several organ-scale scRNA-seq references/gene expression atlases greatly 
simplifies the process of scRNA-seq data analysis (Zhang, Chen and Wang, 2021; 
Shahan et al., 2022). These references are based on both established experimental cell 
markers as well as cell markers identified through bioinformatic analysis of scRNA-
seq data. With these references, specific tissues and cells can be clearly identified 
based on their characteristic gene expression signatures. The information provided by 
51 
 
 
 
these references can be projected onto any scRNA-seq dataset to identify specific cell 
types or developmental stages within that dataset.  
The availability of scRNA-seq for plants provides a great opportunity for the study of 
TSO1, especially for understanding the short root phenotype of tso1-1. The only 
difference observed between tso1-1 root and wild type is the reduction of the RAM 
size, indicated by the reduced number of cortex cells (Wang et al., 2018). The limited 
knowledge about tso1-1 root makes it hard to learn the molecular mechanisms behind 
TSO1?s function. However, with the scRNA-seq, we can determine if there?s any 
change in the cell identity, the ratio of each cell type, and the gene expression pattern 
within specific cell types in the tso1-1 mutant root all at once. 
By comparing the scRNA-seq data of tso1-1 and wild type roots, changes in the 
percentage of specific cell types were observed and several potential TSO1 target 
genes were found. Reduction of the percentage of vascular cells were seen in the 
tso1-1 mutant, indicating defects in vasculature development. Meanwhile, the 
potential TSO1 target genes can be divided into three categories. The first category is 
the plant DREAM complex components, including TSO1 itself. Increase of TSO1, 
ALY1 and MSI2 expression was seen in the tso1-1 root, which suggests that TSO1 
regulates the expression of potential plant DREAM complex components to balance 
cell proliferation and cell differentiation in the root. The second category is the HD-
ZIP III family. There are five members of the HD-ZIP III family: PHABULOSA 
(PHB), PHAVOLUTA (PHV), REVOLUTA (REV), CORONA (CRN)/ATHB15 and 
ATHB8. These genes are known as regulators of vascular patterning in the root: high 
expression levels of the HD-ZIP III genes lead to metaxylem formation while low 
52 
 
 
 
expression levels of HD-ZIP IIIs lead to protoxylem formation (Carlsbecker et al., 
2010). More detailed descriptions of the HD-ZIP III family can be found in Chapter 1 
of this dissertation. We found that the expression of HD-ZIP III genes is increased 
mainly in the vasculature of tso1-1. Two of the HD-ZIP IIIs (PHB and PHV) are 
ectopically expressed in the tso1-1 metaxylem cells. Moreover, one of the PHB direct 
target, IPT7, was also found overexpressed in the metaxylem in tso1-1. IPT7 falls into 
the third category of potential TSO1 target genes, consisting of the cytokinin 
biosynthesis genes. Four cytokinin biosynthesis genes including IPT7, IPT9, LOG1 
and LOG8 showed increased expression in the tso1-1 root. Among them, the LOG1 
was specifically overexpressed in the endodermis cells while elevated expression of 
IPT9 and LOG8 were seen mainly in the QC. 
Overall, these findings suggest that TSO1 is involved in the negative regulation of 
cytokinin biosynthesis either by repressing the expression of HD-ZIP III genes in the 
vascular cells or by unknown mechanisms in other tissues like the endodermis and 
QC. It helps to explain the short root phenotype of tso1-1 since applying exogenous 
cytokinin to Arabidopsis causes similar short root phenotype (Dello Ioio et al., 2007). 
The increased production of cytokinin may mediate the tso1-1 short root phenotype.  
This project is a collaborative project between me and two other scientists. The 
sample preparation for scRNA-seq, Illumina sequencing and raw sequencing file 
processing (using Cell Ranger) were carried out by Dr. Rachel Shahan in Dr. Philip 
Benfey?s lab at the Duke University. The global data analysis (protoplasting-induced 
gene removal, label transfer from reference and data integration) was performed by 
Muzi Li, a graduate student in Dr. Zhongchi Liu?s lab at the University of Maryland ? 
53 
 
 
 
College Park. The figures shown in this dissertation were generated by the 
dissertation author using Muzi Li?s R script. 
3.2 Results 
3.2.1 Single cell transcriptomes revealed reduced cell number in the vasculature of tso1-
1 mutant root 
To understand how TSO1 regulates root development, comparative single cell 
transcriptome was conducted to identify differentially expressed genes in tso1-1 and 
wild type (Ler) roots. The roots of tso1-1 and wild type (0.5cm from the tip, 5DPG) 
were collected for scRNA-seq (Figure 3.1 A). Two biological replicates were 
harvested for each genotype. The roots were then treated with enzymes to isolate the 
protoplasts/single cells. This step is required for the droplet-based scRNA-seq (10X 
Genomics) used in this study. After protoplasting, the samples were loaded onto 
microfluid chips (10X Genomics) to capture either 5,000 or 10,000 cells/sample. 
Barcoding of the cells were carried out with a Chromium Controller (10X Genomics). 
After reverse transcription and Illumina library preparation, the samples were 
sequenced with a Novaseq 6000 instrument (Illumina). The processing and analysis 
for our scRNA-seq datasets were summarized in Figure3.1 B. The raw scRNA-seq 
data were first processed by Cell Ranger to generate FASTQ files. Then the 
protoplasting-induced genes were removed using Seurat (Satija et al., 2015). A root 
cell type reference (primary root gene expression atlas) was used to identify different 
cell types in our scRNA-seq datasets. This organ-scale reference was generated based 
on the combination of established experimental cell type markers, previous published 
54 
 
 
 
Arabidopsis root scRNA-seq datasets and an integration of 110,427 wild type 
Arabidopsis root scRNA-seq data. It includes 14 root cell types and 7 developmental 
stages (Denyer et al., 2019; Ryu et al., 2019; Shahan et al., 2022). Using the label 
transfer function in Seurat (Satija et al., 2015), the primary root gene expression atlas 
was projected to our scRNA-seq data to identify clusters of cells belonging to one of 
the fourteen cell types. 
Combining the two biological replicates, about 27,000 cells from the wild type roots 
and 10,000 cells from tso1-1 roots were captured (Table 3.1). The number of cells 
from the tso1-1 roots is about 1/3 of the wild type, which is not surprising given past 
experiences that different mutant roots always yielded fewer cells than WT (Shahan 
Figure 3.1 The pipeline of single cell data analysis. 
(A) Photo and diagram showing the samples taken for scRNA-seq. The roots were 
harvested on 5DPG and 0.5cm was taken from the tip for protoplasting. The length 
covered three zones of both WT roots and tso1-1 roots. MZ: meristematic zone, EZ: 
elongation zone, DZ: differentiation zone. (B) The analysis pipeline used in this study.  
 
 
 55 
 
 
 
et al., 2022). After the label transfer, all 14 cell types were identified in wild type and 
tso1-1 roots (Figure3.2 A), suggesting that the short root phenotype of tso1-1 is 
probably not caused by missing certain cell types.  
Though there was no change in cell identities observed in tso1-1, differences were 
observed in the percentage of a specific cell type among the total cells examined. In 
particular, the percentage of several vascular cell types were reduced in tso1-1 when 
compared with WT (Figure3.2 D). The affected vascular cell types include 
protoxylem, metaxylem, procambium, metaphloem and companion cell, phloem pole 
pericycle and xylem pole pericycle. The result suggests that the short root phenotype 
of tso1-1 might be caused by defects in vasculature development. 
In order to compare the gene expression patterns in wild type and tso1-1 mutant, the 
scRNA-seq data of wild type and tso1-1 need to be combined and normalized 
(integration) by Seurat (Satija et al., 2015). After the integration, known cell type 
markers were examined to evaluate the quality of the label transfer and integration 
(Figure3.2 C). All markers showed expression in the corresponding cell types, 
suggesting that the label transfer and integration were successful. The data can be 
used for further gene expression comparison. 
56 
 
 
 
 
Table3.1: Number of cells in each cell type and percentage of cells in a specific cell type 
over all cells in a sequenced sample. 
Two biological replicates were collected and sequenced for each genotype: WT_1, WT_2 for 
wild type; tso1-1_1 and tso1-1_2 for tso1-1 mutant. 
57 
 
 
 
Figure 3.2: The single cell transcriptome analysis suggests that the percentage of vasculature 
cells is reduced in tso1-1 root. 
(A) UMAP showing the cell types of tso1-1 and WT (Ler) roots. Each dot represents a single cell, 
and fourteen different cell types are marked by different colors. Cells belonging to the same cell type 
are clustered together. All fourteen cell types are found in the tso1-1 root despite fewer total cells in 
the tso1-1 root sample. (B) UMAP showing integrated wild type and tso1-1 root single cell 
sequencing data. Normalization and scale were carried out using Seurat. (C) Feature plots 
(integrated) showing expression of known cell type marker genes. The name of the gene and cell 
type were labeled on the top of each plot. The scale shows log (log2) transformed normalized 
expression for corresponding gene. (D) A heatmap showing the ratio of cell type percentage between 
tso1-1 and WT. The cell type percentage is the percentage of cells in a specific cell type over all cells 
in a genotype. The percentage of vascular tissues (protoxylem, metaxylem, protophloem, 
metaphloem and companion cell, phloem pole pericycle, xylem pole pericycle and procambium) is 
reduced in tso1-1 root.  
 58 
  
 
 
3.2.2 TSO1 is ectopic and overexpressed in the tso1-1 mutant root 
Differentially expressed genes between WT and tso1-1 were examined for all cell 
types. Surprisingly, increased TSO1 expression was observed in nearly all cell types 
(Figure3.3 A). Specifically, TSO1 is ectopically expressed in atrichoblast, columella, 
lateral root cap, cortex, endodermis, protoxylem, metaxylem, protophloem, 
metaphloem and companion cell, procambium, phleom pole pericycle, xylem pole 
pericycle and QC (Figure3.3 B). The increased expression of TSO1 suggests that 
TSO1 or the TSO1-containing plant DREAM complex represses the expression of 
TSO1 in almost all wild type root cell types. 
As a potential member of the plant DREAM complex, altered TSO1 expression 
revealed above may affect DREAM complex activity. As DREAM complex is also 
involved in regulating the expression of DREAM complex components (Wang et al., 
2018; Iness et al., 2019), we examined gene expression patterns of the plant DREAM 
components (Figure3.3). No significant difference was observed for most of the 
DREAM components (Figure3.3 A). Surprisingly, one of the previously characterized 
TSO1 regulatory targets, MYB3R1, did not show any change in its expression pattern 
(Figure 3.3 C), even though MYB3R1 was shown to be overexpressed in both tso1-1 
shoot and root (Wang et al., 2018). Among the single cells captured in our study, very 
few cells express MYB3R1 (Figure 3.3 C), which may explain why MYB3R1 did not 
show significant expression difference due to too small number of cells. Similarly, 
very few cells express another TSO1 target, CYCA3;4 (Figure 3.3 D) described in 
chapter 2. Due to the small number of cells expressing these genes, statistically 
significant changes of gene expression may be difficult to detect. Increasing the total 
59 
 
 
 
number of cells in scRNA-seq may better enable us to examine MYB3R1 and 
CYCA3;4 expression. 
Besides TSO1, two core DREAM components, ALY1 and MSI2, are ectopically 
expressed in the tso1-1 QC cells (Figure 3.3 E and F). ALY1 and MSI2 are homologs 
of the mammalian DREAM complex core component LIN9 and RBBP4 respectively 
(Table1.1). In addition, in tso1-1 root, more cells expressed MSI2 in metaxylem and 
xylem pole pericycle (Figure3.3 F), and more cells expressed ALY1 in columella. 
Therefore, TSO1 may regulate the expression of some potential plant DREAM 
60 
 
 
 
Figure 3.3: TSO1 is both ectopic and overexpressed in tso1-1 root cells. 
(A) A dot plot showing the expression patterns of the plant DREAM components. X-axis 
shows potential plant DREAM complex component. Y-axis shows different cell types in WT 
(grey shade) and tso1-1 (pink shade) samples. The size of a dot represents the percentage of 
the cells in a tissue expressing the of interest. The color of a dot represents the average 
expression level of a particular gene in log transformed expression scaled by Seurat. (B)-(D) 
Feature plots (left panel) showing the cells expressing TSO1 (B), MYB3R1 (C), and CYCA3;4 
(D) and violin plots (right panel) showing the tissue-specific expression in log transformed 
expression normalized by Seurat.  There are fewer cells expressing MYB3R1 or CYCA3;4 than 
those expressing TSO1. No significant changes in expression of MYB3R1 and CYCA3;4 are 
detected in the violin plot. (E)-(F) are violin plots of ALY1 and MSI2. 
 
complex components (TSO1, MYB3R1, ALY1 and MSI2) in specific cell types, in 
particular, QC to maintain stem cell identity. 
61 
 
 
 
3.2.3 Both cell type and cell numbers that express HD-ZIP IIIs are altered in tso1-1 root 
cells 
HD-ZIP III genes are involved in regulating vasculature development (Carlsbecker et 
al., 2010). The reduction of the percentage of vascular cells (Figure 3.2D) led us to 
examinate the expression of HD-ZIP III genes. In tso1-1, the expression of several 
HD-ZIP III members was increased in the vascular tissues (Figure 3.4 A). The violin 
plot shows more cells in the tso1-1 metaxylem express PHB than wild type, while the 
average expression level of PHB per cell changes only slightly in tso1-1 (Figure 3.4 
A and B). The REV and ATHB8 show ectopic expression in some of the tso1-1 
vascular cells. Cells in procambium and protophloem express REV in tso1-1, but 
hardly any corresponding cells in WT express REV; some cells in tso1-1 protoxylem 
express ATHB8, but almost none in WT protoxylem express ATHB8 (Figure 3.4B). 
On the other hand, while significantly more tso1-1 QC cells express REV, 
significantly fewer QC cells in WT express ATHB8. Overall, HD-ZIP III gene 
expression appears increased or ectopically expressed in the absence of WT TSO1, 
indicating a negative regulatory relationship between TSO1 and HD-ZIPIII. 
62 
 
 
 
 
Figure 3.4: The HD-ZIP III family of genes are overexpressed or ectopically expressed in 
tso1-1 root vasculature tissues. 
(A) A dot plot comparing the expression patterns of the HD-ZIP III genes in WT (green shade) 
and tso1-1 (pink shade). More vascular cells, in particular, metaxylem and procambium, are 
overexpressing the HD-ZIP IIIs in tso1-1 root. (B) Violin plots showing the tissue-specific 
expression patterns of the HD-ZIP III genes. REV and ATHB8 are ectopically expressed in 
some of the vascular tissues, while more metaxylem cells express PHB. Y-axis shows log 
transformed expression normalized by Seurat. 
 
 
 
63 
 
 
 
 
Figure 3.5: The expression of cytokinin biosynthesis genes is increased in specific tso1-1 
root cells. 
(A) A dotplot showing the comparison of cytokinin biosynthesis gene expression in wild type 
(green) and tso1-1 (pink) root. The expression of IPT7, IPT9, LOG1 and LOG8 are altered in 
tso1-1. (B) Violin plots showing tissue-specific expression of IPT7, IPT9, LOG1 and LOG8. A 
significant higher number of metaxylem cells express IPT7 in tso1-1, while a higher number of 
endodermal cells express LOG1 in tso1-1. A larger number of QC cells express IPT9 and 
LOG8 in tso1-1 when compared with WT. (C) RT-qPCR showing that LOG1 is overexpressed 
in tso1-1 shoot apical meristem. ** stands for p < 0.05, * stands for p < 0.1 (One-way 
ANOVA and Tukey?s test). 
 
 
 
64 
 
 
 
3.2.4 Expression of cytokinin biosynthesis genes is increased in tso1-1 root 
The finding of increased HD-ZIP III gene expression in tso1-1 is exciting, as PHB 
regulates cytokinin biosynthesis in the vascular tissues (Dello Ioio et al., 2012). It 
activates the expression of a gene named ISOPENTENYLTRANSFERASE 7 (IPT7), 
which encodes a rate-limiting enzyme for cytokinin biosynthesis. The previously 
characterized tso1-1 short root phenotype resembles the short root phenotype caused 
by exogenous application of cytokinin (Dello Ioio et al., 2007; Wang et al., 2018). 
The RAM size of Arabidopsis root was reduced when treated with cytokinin, 
probably due to the early cellular differentiation of RAM cells facilitated by increased 
cytokinin (Dello Ioio et al., 2007). This phenotype is also observed in the tso1-1 root 
(reduced RAM size and premature cell differentiation). Therefore, we hypothesize 
that the short root phenotype of tso1-1 could be mediated by elevated cytokinin.  
In our scRNA-seq data, several cytokinin biosynthesis genes, in particular IPT7, 
IPT9, LOG1, and LOG8, are found ectopically expressed in specific root tissues 
(Figure3.5 A). In the violin plots, both the number of metaxylem cells that express 
IPT7 and the expression level of IPT7 are increased in tso1-1 relative to WT root 
(Figure3.5 B). This is consistent with a similar expression change of PHB in the tso1-
1 metaxylem (Figure3.4 B), in support of PHB?s role in the activation of IPT7 in 
metaxylem. The IPT9 is increased in the QC, suggesting tissue-specific regulation of 
IPT genes (Figure 3.5 B). Another family of cytokinin biosynthetic enzyme is called 
the LONELY GUY (LOG) family. The expression of two members of the LOG family 
is elevated in the tso1-1 root: LOG1 is specifically overexpressed in the endodermis 
while LOG8 is overexpressed in both atrichoblast and QC. These observations 
65 
 
 
 
support our hypothesis that loss of TSO1 may result in increased cytokinin and earlier 
differentiation, leading to the short root phenotype.  
Figure 3.6: A regulatory pathway consisting of TSO1, MYB3R1, HD-ZIP III and 
cytokinin (CK) that controls cell differentiation in Arabidopsis root.  
Based on the comparison of single cell RNA profiles of wild type and tso1-1, two tissue-
specific regulatory modules of TSO1 are proposed. In wild type vascular cells, the HD-ZIP III 
proteins activate cytokinin synthesis to induce cell differentiation in the root. TSO1 represses 
the expression of the HD-ZIP III genes by inhibiting the expression of MYB3R1. Ectopic 
overexpression of HD-ZIPIII in tso1-1 mutant roots causes increased cytokinin biosynthesis 
that leads to precocious differentiation. In endodermis and QC, TSO1 represses cytokinin 
synthesis to help maintain stem cell identity. Arrows and bars do not imply direct regulation.  
 
3.3 Discussion 
3.3.1 TSO1 represses HD-ZIP III genes to inhibit cytokinin synthesis, which prevents 
premature root cell differentiation 
Previous studies have revealed that TSO1 plays important roles in regulating SAM 
and RAM size. The tso1-1 mutant has enlarged and fasciated shoot apical meristem 
due to over-proliferation (Liu, Running and Meyerowitz, 1997). Meanwhile, the tso1-
1 mutant also produces much shorter root compared to wild type (Wang et al., 2018). 
66 
 
 
 
The short root phenotype was caused by premature cell cycle exit and precocious 
cellular differentiation at RAM. The underlying molecular mechanism of TSO1 
function in RAM is still lacking, especially how a loss of TSO1 leads to premature 
cell cycle exit. 
The root length is regulated in part by how soon the cells begin to differentiate at the 
transition zone, where cells exit cell cycle permanently and start to elongate (Baluska, 
Volkmann and Barlow, 1996). The transition zone is an active site for hormone 
crosstalk (Kong et al., 2018). Cytokinin controls RAM size by positioning the 
transition zone (Dello Ioio et al., 2007). Specifically, it acts in the vascular tissues in 
the transition zone to activate a repressor of auxin signaling gene SHORT 
HYPOCOTYL2 (SHY2) to promote cell differentiation (Dello Ioio et al., 2008). In 
addition, exogenous application of cytokinin led to premature cell cycle exit and 
differentiation (Dello Ioio et al., 2007), causing short root phenotype. This led to the 
hypothesis that the short root of tso1-1 might be related to increased cytokinin.  
The comparison of the scRNA-seq data of tso1-1 and wild type roots provided 
unprecedented details of TSO1 regulatory networks. One important observation is that 
the expression HD-ZIP III family members in root is increased in the tso1-1 
metaxylem, a subtissue of vasculature. Since the gain-of-function HD-ZIP III 
mutants, phb-1d and phv-1d, have much shorter roots and significantly smaller RAMs 
than the wild type (Dello Ioio et al., 2012), the observed over-expression of PHB and 
PHV in the tso1-1 metaxylem may mediate the tso1-1 short root phenotype.  
Moreover, PHB and PHV have been demonstrated as positive regulators of a 
cytokinin biosynthesis gene IPT7. Among them, PHB is necessary and sufficient to 
67 
 
 
 
directly activate the expression of IPT7 in the vascular tissues, leading to the 
production of cytokinin (Dello Ioio et al., 2012). Therefore, increased IPT7 
expression in the tso1-1 metaxylem, as revealed my our scRNA-seq data, suggests 
that TSO1 regulates root development by regulating cytokinin biosynthesis in specific 
root cells and this regulation is likely achieved indirectly by repressing PHB and PHV 
expression (Figure 3.6). 
3.3.2 Integration of MYB3R1 in the TSO1 network 
Previous studies showed that the tso1-1 short root phenotype could be attributed to 
the overexpression of MYB3R1 as removing MYB3R1 by a loss-of-function mutation 
suppressed the short root phenotype. However, it was unclear why an overexpression 
of MYB3R1 induces early root cell differentiation and short root phenotype. Is 
MYB3R1 function related to the HD-ZIP III genes or do MYB3R1 and HD-ZIPIII act 
in parallel pathways? Recent studies revealed the potential genetic relationship 
between MYB3R1 and the HD-ZIP IIIs. A chromatin immunoprecipitation sequencing 
(ChIP-seq) that aims at identifying MYB3R1 binding sites revealed PHB, REV and 
ATHB8 as the potential regulatory targets of MYB3R1 (Yang et al., 2021). While the 
experiment was performed using shoot meristem cells, the regulatory relationship 
could be preserved in the root cells. Therefore, TSO1 may repress the expression of 
the HD-ZIP III genes through the inhibition of MY3BR1 expression (Figure3.6).  
3.3.3 The increased expression of the HD-ZIP III genes affects vasculature development 
In addition to controlling cytokinin biosynthesis, the HD-ZIP III genes are also 
known as regulators of vascular patterning in Arabidopsis (Zhou et al., 2007; 
68 
 
 
 
Carlsbecker et al., 2010). Losing all five members of the HD-ZIP III family leads to 
failure of xylem formation, however, the quadruple mutant athb8-11 cna-2 phb-13 
phv-11 generates more vascular cells (Carlsbecker et al., 2010). It seems that the HD-
ZIP III genes are necessary for xylem formation but restrict vascular cell proliferation 
at the same time. Therefore, upregulation of the HD-ZIP III genes may result in fewer 
vascular cells. The discovery of increased expression of multiple HD-ZIP III genes 
and reduction of the vascular cells in tso1-1 (Figure 3.2) support this hypothesis. It 
suggests that TSO1 regulate vasculature development through regulating HD-ZIP IIIs. 
It will be interesting to examine the vascular tissues of tso1-1 in the root as well as 
shoot to see if there?s any existing developmental defects. 
3.3.4 Overexpression of LOG1 suggest TSO1 may regulate cytokinin synthesis in SAM 
Besides the increased expression of IPT7 in the vasculature, increased expression of 
cytokinin biosynthesis genes was also detected in other tissues. The upregulation of 
LOG1, IPT9 and LOG8 suggests that TSO1 regulates cytokinin biosynthesis not only 
in the vascular tissues but also in the endodermis and QC through unknown 
mechanisms. Though no significant morphological change of these tissues was 
observed under the microscope, nor from the scRNA-seq data, the gene expression 
network might be altered in these tissues in tso1-1 mutant (Wang et al., 2018). 
Compared to the wild type, the expression of LOG1 increased about one-fold in tso1-
1 shoot (Figure 3.5C), indicating there might be over-production of cytokinin in the 
SAM. Therefore, there are potentially conserved regulatory relationships between 
TSO1 and cytokinin synthesis in root as well as shoot. In fact, the enlargement of the 
tso1-1 SAM resembles the phenotype of applying cytokinin to the Arabidopsis SAM 
69 
 
 
 
(Giulini, Wang and Jackson, 2004). It is possible that the enlargement of tso1-1 SAM 
can be attributed, at least partially, to the increased cytokinin at the shoot. 
3.3.5 TSO1 is involved in regulation of itself and plant DREAM complex components 
The scRNA transcriptomes of wild type and tso1-1 roots show that TSO1 is highly 
and ectopically expressed in nearly all cell types in the tso1-1 roots. Therefore, TSO1 
may repress the expression of itself in most root cell types. Besides TSO1, two other 
plant DREAM complex component, ALY1 and MSI2, are found overexpressed in 
tso1-1, suggesting that TSO1 regulates the spatial expression of potential plant 
DREAM complex components in root to balance cell division and cell differentiation 
in the RAM. In addition, it was reported that the tso1-1 mutant protein can interfere 
with the function of its close homolog SOL2 and knocking down of the tso1-1 
transcript partially rescue the fasciation phenotype, suggesting the tso1-1 protein 
might be poisonous for plant meristem development (Sijacic, Wang and Liu, 2011). It 
is possible that the over-accumulated tso1-1 disrupt the function of its homolog, 
resulting in defects of root cell development.  
3.4 Methods 
3.4.1 Protoplast isolation and scRNA-seq 
1,000-3,500 primary roots per sample were cut from the tip (~0.5cm, 5DPG) and used 
for protoplasting. Protoplast isolation followed a protocol described in a previous 
publication (Shahan et al., 2022). The protoplast suspension was then loaded onto 
microfluidic chips (10X Genomics) to capture 5,000 or 10,000 cells per sample. 
Barcoding of cells was carried out with a Chromium Controller (10X Genomics). The 
70 
 
 
 
mRNA isolation, reverse transcription and Illumina library preparation also followed 
the protocol in the same publication (Shahan et al., 2022). The samples were 
sequenced with a Novaseq 6000 (Illumina) to generate 100bp pair-end reads. 
3.4.2 scRNA-seq data pre-processing 
Cell Ranger (v4.0.0) was applied to generate the row count matrices from FASTQ 
files for each sample (Zheng et al., 2017). Original STAR within the Cell Ranger was 
replaced by STAR (v2.7.3a) because of system incompatibility. TAIR10_chr_all.fas 
(https://www.arabidopsis.org/download_files/Genes/TAIR10_genome_release/TAIR
10_chromosome_files/TAIR10_chr_all.fas) and 
Araport11_GFF3_genes_transposons.201606.gtf.gz 
(https://arabidopsis.org/download_files/Genes/Araport11_genome_release/archived/
Araport11_GFF3_genes_transposons.201606.gff.gz) were used as the Arabidopsis 
reference genome and annotation, respectively.  
3.4.3 Label transfer and integration 
Seurat (v3.2.3) was utilized to transfer the annotations from the published atlas to the 
WT and tso1-1 mutant samples (Satija et al., 2015; Shahan et al., 2022). The cells 
that had greater than 5% mitochondrial and chloroplast counts were filtered out. And 
the genes affected by protoplasting (https://github.com/Hsu-Che-
Wei/COPILOT/blob/master/supp_data/Protoplasting_DEgene_FC2_list.txt) were 
removed from the analyses. Additionally, data normalization was performed using 
SCTransform approach. The WT and mutant Seurat objects were further integrated, 
and the RNA assay was normalized and scaled for downstream visualization after 
integration.  
71 
 
 
 
3.4.4  RNA extraction and RT-qPCR experiments 
Arabidopsis SAMs were collected for RNA extraction using RNeasy Mini Kit 
(Qiagen). RNA samples were cleaned with DNase I. cDNAs were synthesized using 
RevertAid First Strand cDNA Synthesis Kit (ThermoFisher). RT-qPCR experiments 
were performed on the Bio-rad CFX 96 machine with PowerUp? SYBR? Green 
Master Mix (Thermo Fisher). Three biological replicates were conducted for the RT-
qPCR with three technical replicates. The TIP41(AT4G34270) gene was used as the 
internal control.  
 
 
72 
 
 
 
Chapter 4: Conclusion and future direction 
 
4.1 Conclusion 
In summary, I characterized a genetic suppressor of the tso1-1 mutant. It suppresses 
the SAM fasciation and partially restores the sterility of the tso1-1 mutant. The causal 
mutation was mapped to a G-to-A mutation in a A-type cyclin gene, CYCA3;4. I 
showed that the expression of CYCA3;4 was increased in the tso1-1 SAM, suggesting 
that the overexpression of CYCA3;4 mediates the tso1-1 shoot phenotype. I also 
found that TSO1 might regulate the expression of CYCA3;4 indirectly by repressing 
MYB3R1 transcription in wild type plants. 
In collaboration with two other scientists, I analyzed the tso1-1 root phenotype by 
applying single cell RNA sequencing. I found that the short root phenotype of tso1-1 
might be caused by defects in the vasculature development but not by the absence of 
certain cell types. By looking at the gene expression profiles, I discovered that the 
HD-ZIP III genes and several cytokinin biosynthesis genes are ectopically expressed 
in tso1-1 root, which indicates that the tso1-1 short root phenotype might be attributed 
to over-production of cytokinin. 
4.2 Future directions 
Why the mutations in CYCA3;4 only partially suppressed the shoot phenotype and did 
not suppress the root phenotype? It leads to the questions that whether there is 
redundancy among the CYCA3 genes. It is unclear whether TSO1 can regulate the 
expression of other CYCA3s and whether the regulation is achieved in a similar 
manner. 
73 
 
 
 
Generating double knock-out or triple knock-out of CYCA3s in the tso1-1 background 
and observing if there are better suppression effects than the single knock-out of 
CYCA3;4 can help us answer the redundancy question. It can also help understand the 
roles of these cyclins in the TSO1 regulatory pathway and how TSO1 participates in 
the cell cycle regulation at molecular level.  
In addition, the result from the scRNA-seq suggests that TSO1 may regulate the 
expression of the HD-ZIPIII genes and several cytokinin biosynthesis genes in the 
root. One simple question is that whether the regulation pathways are similar or even 
the same in the shoot? To answer this question, we have examined the shoot 
phenotype of the tso1-1; rev double mutant plants and found that knock-out of the 
REV gene suppressed the fasciation of the tso1-1 SAM. Therefore, we proposed that 
TSO1 might regulate the HD-ZIPIII genes in both Arabidopsis shoot and root. 
It will be interesting to test this hypothesis by looking at the phenotype of other TSO1 
and HD-ZIPIIIs double knockout mutants.  
One of the experiments could be done to validate our scRNA-seq results, 
transcriptional reporters like promoter fusing to GFP can be used to examine the 
expression of the HD-ZIP III genes as well as IPT7, IPT9, LOG1 and LOG8 in tso1-1 
root. It can help to determine if the expression domains of these genes expand, or the 
expression levels are increased compared to the wild type.  
The finding of increased cytokinin biosynthesis is also quite interesting. It provides 
insights of how conserved cell cycle machineries can regulate plant development 
through regulating the plant hormones. Similar to the proposed experiments for the 
HD-ZIPIII genes, generating the knockouts of the cytokinin genes found in the 
74 
 
 
 
scRNA-seq analysis and observing if they suppress the short root phenotype of tso1-1 
can further confirm the proposed TSO1 regulatory pathway. Besides the cytokinin 
biosynthesis synthesis, we are curious about the cytokinin response in tso1-1 root too. 
Existing synthetic cytokinin reporters (Liu and M?ller, 2017) can be used to compare 
the response in the tso1-1 and the wild type roots.  
 
 
75 
 
 
 
Bibliography 
Aida, M. et al. (2004) ?The PLETHORA Genes Mediate Patterning of the 
Arabidopsis Root Stem Cell Niche?, Cell, 119(1), pp. 109?120. Available at: 
https://doi.org/10.1016/j.cell.2004.09.018. 
Andersen, S.U. et al. (2007) ?The conserved cysteine-rich domain of a tesmin/TSO1-
like protein binds zinc in vitro and TSO1 is required for both male and female fertility 
in Arabidopsis thaliana?, Journal of Experimental Botany, 58(13), pp. 3657?3670. 
Available at: https://doi.org/10.1093/jxb/erm215. 
Araki, S. et al. (2004) ?Mitotic Cyclins Stimulate the Activity of c-Myb-like Factors 
for Transactivation of G2/M Phase-specific Genes in Tobacco *?, Journal of 
Biological Chemistry, 279(31), pp. 32979?32988. Available at: 
https://doi.org/10.1074/jbc.M403171200. 
Baluska, F., Volkmann, D. and Barlow, P.W. (1996) ?Specialized Zones of 
Development in Roots: View from the Cellular Level?, Plant Physiology, 112(1), pp. 
3?4. Available at: https://doi.org/10.1104/pp.112.1.3. 
Beall, E.L. et al. (2007) ?Discovery of tMAC: a Drosophila testis-specific meiotic 
arrest complex paralogous to Myb?Muv B?, Genes & Development, 21(8), pp. 904?
919. Available at: https://doi.org/10.1101/gad.1516607. 
Benfey, P.N. et al. (1993) ?Root development in Arabidopsis: four mutants with 
dramatically altered root morphogenesis?, Development, 119(1), pp. 57?70. Available 
at: https://doi.org/10.1242/dev.119.1.57. 
Blilou, I. et al. (2005) ?The PIN auxin efflux facilitator network controls growth and 
patterning in Arabidopsis roots?, Nature, 433(7021), pp. 39?44. Available at: 
https://doi.org/10.1038/nature03184. 
Boruc, J. et al. (2010) ?Functional Modules in the Arabidopsis Core Cell Cycle 
Binary Protein?Protein Interaction Network?, The Plant Cell, 22(4), pp. 1264?1280. 
Available at: https://doi.org/10.1105/tpc.109.073635. 
Bulankova, P. et al. (2013) ?Identification of Arabidopsis Meiotic Cyclins Reveals 
Functional Diversification among Plant Cyclin Genes?, PLOS Genetics, 9(5), p. 
e1003508. Available at: https://doi.org/10.1371/journal.pgen.1003508. 
Carlsbecker, A. et al. (2010) ?Cell signalling by microRNA165/6 directs gene dose-
dependent root cell fate?, Nature, 465(7296), pp. 316?321. Available at: 
https://doi.org/10.1038/nature08977. 
Chickarmane, V.S. et al. (2012) ?Cytokinin signaling as a positional cue for 
patterning the apical?basal axis of the growing Arabidopsis shoot meristem?, 
Proceedings of the National Academy of Sciences, 109(10), pp. 4002?4007. Available 
at: https://doi.org/10.1073/pnas.1200636109. 
76 
 
 
 
Clark, S.E., Running, M.P. and Meyerowitz, E.M. (1995) ?CLAVATA3 is a specific 
regulator of shoot and floral meristem development affecting the same processes as 
CLAVATA1?, Development, 121(7), pp. 2057?2067. Available at: 
https://doi.org/10.1242/dev.121.7.2057. 
Clark, S.E., Williams, R.W. and Meyerowitz, E.M. (1997) ?The CLAVATA1Gene 
Encodes a Putative Receptor Kinase That Controls Shoot and Floral Meristem Size in 
Arabidopsis?, Cell, 89(4), pp. 575?585. Available at: https://doi.org/10.1016/S0092-
8674(00)80239-1. 
Cui, H. et al. (2007) ?An Evolutionarily Conserved Mechanism Delimiting SHR 
Movement Defines a Single Layer of Endodermis in Plants?, Science, 316(5823), pp. 
421?425. Available at: https://doi.org/10.1126/science.1139531. 
Curtis, M.D. and Grossniklaus, U. (2003) ?A gateway cloning vector set for high-
throughput functional analysis of genes in planta?, Plant Physiology, 133(2), pp. 462?
469. Available at: https://doi.org/10.1104/pp.103.027979. 
Daum, G. et al. (2014) ?A mechanistic framework for noncell autonomous stem cell 
induction in Arabidopsis?, Proceedings of the National Academy of Sciences, 
111(40), pp. 14619?14624. Available at: https://doi.org/10.1073/pnas.1406446111. 
David-Pfeuty, T. and Nouvian-Dooghe, Y. (1996) ?Human cyclin B1 is targeted to 
the nucleus in G1 phase prior to its accumulation in the cytoplasm?, Oncogene, 13(7), 
pp. 1447?1460. 
De Rybel, B. et al. (2016) ?Plant vascular development: from early specification to 
differentiation?, Nature Reviews Molecular Cell Biology, 17(1), pp. 30?40. Available 
at: https://doi.org/10.1038/nrm.2015.6. 
Dello Ioio, R. et al. (2007) ?Cytokinins Determine Arabidopsis Root-Meristem Size 
by Controlling Cell Differentiation?, Current Biology, 17(8), pp. 678?682. Available 
at: https://doi.org/10.1016/j.cub.2007.02.047. 
Dello Ioio, R. et al. (2008) ?A Genetic Framework for the Control of Cell Division 
and Differentiation in the Root Meristem?, Science, 322(5906), pp. 1380?1384. 
Available at: https://doi.org/10.1126/science.1164147. 
Dello Ioio, R. et al. (2012) ?A PHABULOSA/Cytokinin Feedback Loop Controls 
Root Growth in Arabidopsis?, Current Biology, 22(18), pp. 1699?1704. Available at: 
https://doi.org/10.1016/j.cub.2012.07.005. 
Denyer, T. et al. (2019) ?Spatiotemporal Developmental Trajectories in the 
Arabidopsis Root Revealed Using High-Throughput Single-Cell RNA Sequencing?, 
Developmental Cell, 48(6), pp. 840-852.e5. Available at: 
https://doi.org/10.1016/j.devcel.2019.02.022. 
77 
 
 
 
Earley, K.W. et al. (2006) ?Gateway-compatible vectors for plant functional 
genomics and proteomics?, The Plant Journal, 45(4), pp. 616?629. Available at: 
https://doi.org/10.1111/j.1365-313X.2005.02617.x. 
Emery, J.F. et al. (2003) ?Radial Patterning of Arabidopsis Shoots by Class III HD-
ZIP and KANADI Genes?, Current Biology, 13(20), pp. 1768?1774. Available at: 
https://doi.org/10.1016/j.cub.2003.09.035. 
Fischer, M. and M?ller, G.A. (2017) ?Cell cycle transcription control: 
DREAM/MuvB and RB-E2F complexes?, Critical Reviews in Biochemistry and 
Molecular Biology, 52(6), pp. 638?662. Available at: 
https://doi.org/10.1080/10409238.2017.1360836. 
Fletcher, J.C. et al. (1999) ?Signaling of Cell Fate Decisions by CLAVATA3 in 
Arabidopsis Shoot Meristems?, Science [Preprint]. Available at: 
https://doi.org/10.1126/science.283.5409.1911. 
Forzani, C. et al. (2014) ?WOX5 Suppresses CYCLIN D Activity to Establish 
Quiescence at the Center of the Root Stem Cell Niche?, Current Biology, 24(16), pp. 
1939?1944. Available at: https://doi.org/10.1016/j.cub.2014.07.019. 
Galinha, C. et al. (2007) ?PLETHORA proteins as dose-dependent master regulators 
of Arabidopsis root development?, Nature, 449(7165), pp. 1053?1057. Available at: 
https://doi.org/10.1038/nature06206. 
G?lweiler, L. et al. (1998) ?Regulation of Polar Auxin Transport by AtPIN1 in 
Arabidopsis Vascular Tissue?, Science, 282(5397), pp. 2226?2230. Available at: 
https://doi.org/10.1126/science.282.5397.2226. 
Gibson, D.G. et al. (2009) ?Enzymatic assembly of DNA molecules up to several 
hundred kilobases?, Nature Methods, 6(5), pp. 343?345. Available at: 
https://doi.org/10.1038/nmeth.1318. 
Giulini, A., Wang, J. and Jackson, D. (2004) ?Control of phyllotaxy by the cytokinin-
inducible response regulator homologue ABPHYL1?, Nature, 430(7003), pp. 1031?
1034. Available at: https://doi.org/10.1038/nature02778. 
Gordon, S.P. et al. (2009) ?Multiple feedback loops through cytokinin signaling 
control stem cell number within the Arabidopsis shoot meristem?, Proceedings of the 
National Academy of Sciences, 106(38), pp. 16529?16534. Available at: 
https://doi.org/10.1073/pnas.0908122106. 
Grieneisen, V.A. et al. (2007) ?Auxin transport is sufficient to generate a maximum 
and gradient guiding root growth?, Nature, 449(7165), pp. 1008?1013. Available at: 
https://doi.org/10.1038/nature06215. 
78 
 
 
 
Haga, N. et al. (2007) ?R1R2R3-Myb proteins positively regulate cytokinesis through 
activation of KNOLLE transcription in Arabidopsis thaliana?, Development, 134(6), 
pp. 1101?1110. Available at: https://doi.org/10.1242/dev.02801. 
Haga, N. et al. (2011) ?Mutations in MYB3R1 and MYB3R4 Cause Pleiotropic 
Developmental Defects and Preferential Down-Regulation of Multiple G2/M-Specific 
Genes in Arabidopsis1[C][W]?, Plant Physiology, 157(2), pp. 706?717. Available at: 
https://doi.org/10.1104/pp.111.180836. 
Harashima, H. and Schnittger, A. (2012) ?Robust reconstitution of active cell-cycle 
control complexes from co-expressed proteins in bacteria?, Plant Methods, 8(1), p. 
23. Available at: https://doi.org/10.1186/1746-4811-8-23. 
Harrison, M.M. et al. (2006) ?Some C. elegans class B synthetic multivulva proteins 
encode a conserved LIN-35 Rb-containing complex distinct from a NuRD-like 
complex?, Proceedings of the National Academy of Sciences, 103(45), pp. 16782?
16787. Available at: https://doi.org/10.1073/pnas.0608461103. 
Hauser, B.A. et al. (2000) ?TSO1 is a novel protein that modulates cytokinesis and 
cell expansion in Arabidopsis?, Development, 127(10), pp. 2219?2226. Available at: 
https://doi.org/10.1242/dev.127.10.2219. 
Hauser, B.A., Villanueva, J.M. and Gasser, C.S. (1998) ?Arabidopsis TSO1 Regulates 
Directional Processes in Cells During Floral Organogenesis?, Genetics, 150(1), pp. 
411?423. 
Helariutta, Y. et al. (2000) ?The SHORT-ROOT Gene Controls Radial Patterning of 
the Arabidopsis Root through Radial Signaling?, Cell, 101(5), pp. 555?567. Available 
at: https://doi.org/10.1016/S0092-8674(00)80865-X. 
Higuchi, M. et al. (2004) ?In planta functions of the Arabidopsis cytokinin receptor 
family?, Proceedings of the National Academy of Sciences, 101(23), pp. 8821?8826. 
Available at: https://doi.org/10.1073/pnas.0402887101. 
Hollender, C.A. et al. (2012) ?Flower and early fruit development in a diploid 
strawberry, Fragaria vesca?, Planta, 235(6), pp. 1123?1139. Available at: 
https://doi.org/10.1007/s00425-011-1562-1. 
Iness, A.N. et al. (2019) ?The cell cycle regulatory DREAM complex is disrupted by 
high expression of oncogenic B-Myb?, Oncogene, 38(7), pp. 1080?1092. Available 
at: https://doi.org/10.1038/s41388-018-0490-y. 
Jeong, S., Trotochaud, A.E. and Clark, S.E. (1999) ?The Arabidopsis CLAVATA2 
Gene Encodes a Receptor-like Protein Required for the Stability of the CLAVATA1 
Receptor-like Kinase?, The Plant Cell, 11(10), pp. 1925?1933. Available at: 
https://doi.org/10.1105/tpc.11.10.1925. 
79 
 
 
 
Kieber, J.J. (2002) ?Cytokinins?, The Arabidopsis Book, 2002(1). Available at: 
https://doi.org/10.1199/tab.0063. 
Kim, J. et al. (2005) ?microRNA-directed cleavage of ATHB15 mRNA regulates 
vascular development in Arabidopsis inflorescence stems?, The Plant journal?: for 
cell and molecular biology, 42(1), pp. 84?94. Available at: 
https://doi.org/10.1111/j.1365-313X.2005.02354.x. 
Kobayashi, K. et al. (2015) ?Transcriptional repression by MYB3R proteins regulates 
plant organ growth?, The EMBO journal, 34(15), pp. 1992?2007. Available at: 
https://doi.org/10.15252/embj.201490899. 
K?ivom?gi, M. et al. (2011) ?Dynamics of Cdk1 substrate specificity during the cell 
cycle?, Molecular Cell, 42(5), pp. 610?623. Available at: 
https://doi.org/10.1016/j.molcel.2011.05.016. 
Kong, X. et al. (2018) ?The Root Transition Zone: A Hot Spot for Signal Crosstalk?, 
Trends in Plant Science, 23(5), pp. 403?409. Available at: 
https://doi.org/10.1016/j.tplants.2018.02.004. 
Korenjak, M. et al. (2004) ?Native E2F/RBF complexes contain Myb-interacting 
proteins and repress transcription of developmentally controlled E2F target genes?, 
Cell, 119(2), pp. 181?193. Available at: https://doi.org/10.1016/j.cell.2004.09.034. 
Lang, L. et al. (2021) ?The DREAM complex represses growth in response to DNA 
damage in Arabidopsis?, Life Science Alliance, 4(12). Available at: 
https://doi.org/10.26508/lsa.202101141. 
Laurenzio, L.D. et al. (1996) ?The SCARECROW Gene Regulates an Asymmetric 
Cell Division That Is Essential for Generating the Radial Organization of the 
Arabidopsis Root?, Cell, 86(3), pp. 423?433. Available at: 
https://doi.org/10.1016/S0092-8674(00)80115-4. 
Leibfried, A. et al. (2005) ?WUSCHEL controls meristem function by direct 
regulation of cytokinin-inducible response regulators?, Nature, 438(7071), pp. 1172?
1175. Available at: https://doi.org/10.1038/nature04270. 
Levesque, M.P. et al. (2006) ?Whole-Genome Analysis of the SHORT-ROOT 
Developmental Pathway in Arabidopsis?, PLOS Biology, 4(5), p. e143. Available at: 
https://doi.org/10.1371/journal.pbio.0040143. 
Lewis, P.W. et al. (2004) ?Identification of a Drosophila Myb-E2F2/RBF 
transcriptional repressor complex?, Genes & Development, 18(23), pp. 2929?2940. 
Available at: https://doi.org/10.1101/gad.1255204. 
Litovchick, L. et al. (2007) ?Evolutionarily Conserved Multisubunit RBL2/p130 and 
E2F4 Protein Complex Represses Human Cell Cycle-Dependent Genes in 
80 
 
 
 
Quiescence?, Molecular Cell, 26(4), pp. 539?551. Available at: 
https://doi.org/10.1016/j.molcel.2007.04.015. 
Liu, J. and M?ller, B. (2017) ?Imaging TCSn::GFP, a Synthetic Cytokinin Reporter, 
in Arabidopsis thaliana?, Methods in Molecular Biology (Clifton, N.J.), 1497, pp. 81?
90. Available at: https://doi.org/10.1007/978-1-4939-6469-7_9. 
Liu, Y. et al. (2012) ?CCS52A2/FZR1, a cell cycle regulator, is an essential factor for 
shoot apical meristem maintenance in Arabidopsis thaliana?, BMC Plant Biology, 12, 
p. 135. Available at: https://doi.org/10.1186/1471-2229-12-135. 
Liu, Z. et al. (2020) ?Global Dynamic Molecular Profiling of Stomatal Lineage Cell 
Development by Single-Cell RNA Sequencing?, Molecular Plant, 13(8), pp. 1178?
1193. Available at: https://doi.org/10.1016/j.molp.2020.06.010. 
Liu, Z., Running, M.P. and Meyerowitz, E.M. (1997) ?TSO1 functions in cell division 
during Arabidopsis flower development?, Development (Cambridge, England), 
124(3), pp. 665?672. 
Ljung, K. et al. (2005) ?Sites and Regulation of Auxin Biosynthesis in Arabidopsis 
Roots?, The Plant Cell, 17(4), pp. 1090?1104. Available at: 
https://doi.org/10.1105/tpc.104.029272. 
Macdonald, H. and Stevens, D. (2019) Biotechnology and Plant Biology. Scientific e-
Resources. 
Marceau, A.H. et al. (2016) ?Structural basis for LIN54 recognition of CHR elements 
in cell cycle-regulated promoters?, Nature Communications, 7(1), p. 12301. Available 
at: https://doi.org/10.1038/ncomms12301. 
Mayer, K.F.X. et al. (1998) ?Role of WUSCHEL in Regulating Stem Cell Fate in the 
Arabidopsis Shoot Meristem?, Cell, 95(6), pp. 805?815. Available at: 
https://doi.org/10.1016/S0092-8674(00)81703-1. 
McConnell, J.R. and Barton, M.K. (1998) ?Leaf polarity and meristem formation in 
Arabidopsis?, Development, 125(15), pp. 2935?2942. Available at: 
https://doi.org/10.1242/dev.125.15.2935. 
Menges, M. et al. (2005) ?Global analysis of the core cell cycle regulators of 
Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of 
expression and provides a coherent model for plant cell cycle control?, The Plant 
Journal, 41(4), pp. 546?566. Available at: https://doi.org/10.1111/j.1365-
313X.2004.02319.x. 
Miyawaki, K., Matsumoto-Kitano, M. and Kakimoto, T. (2004) ?Expression of 
cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity 
81 
 
 
 
and regulation by auxin, cytokinin, and nitrate?, The Plant Journal, 37(1), pp. 128?
138. Available at: https://doi.org/10.1046/j.1365-313X.2003.01945.x. 
M?ller, G.A. et al. (2014) ?The CHR site: definition and genome-wide identification 
of a cell cycle transcriptional element?, Nucleic Acids Research, 42(16), pp. 10331?
10350. Available at: https://doi.org/10.1093/nar/gku696. 
M?ller, G.A. et al. (2016) ?Timing of transcription during the cell cycle: Protein 
complexes binding to E2F, E2F/CLE, CDE/CHR, or CHR promoter elements define 
early and late cell cycle gene expression?, Oncotarget, 8(58), pp. 97736?97748. 
Available at: https://doi.org/10.18632/oncotarget.10888. 
M?ller, R., Bleckmann, A. and Simon, R. (2008) ?The Receptor Kinase CORYNE of 
Arabidopsis Transmits the Stem Cell?Limiting Signal CLAVATA3 Independently of 
CLAVATA1?, The Plant Cell, 20(4), pp. 934?946. Available at: 
https://doi.org/10.1105/tpc.107.057547. 
Musa, J. et al. (2017) ?MYBL2 (B-Myb): a central regulator of cell proliferation, cell 
survival and differentiation involved in tumorigenesis?, Cell Death & Disease, 8(6), 
pp. e2895?e2895. Available at: https://doi.org/10.1038/cddis.2017.244. 
Nakajima, K. et al. (2001) ?Intercellular movement of the putative transcription factor 
SHR in root patterning?, Nature, 413(6853), pp. 307?311. Available at: 
https://doi.org/10.1038/35095061. 
Nieuwland, J., Menges, M. and Murray, J.A.H. (2007) ?The Plant Cyclins?, in Annual 
Plant Reviews Volume 32: Cell Cycle Control and Plant Development. John Wiley & 
Sons, Ltd, pp. 31?61. Available at: https://doi.org/10.1002/9780470988923.ch2. 
Ning, Y.-Q. et al. (2020) ?DREAM complex suppresses DNA methylation 
maintenance genes and precludes DNA hypermethylation?, Nature Plants, 6(8), pp. 
942?956. Available at: https://doi.org/10.1038/s41477-020-0710-7. 
Ogawa, M. et al. (2008) ?Arabidopsis CLV3 Peptide Directly Binds CLV1 
Ectodomain?, Science [Preprint]. Available at: 
https://doi.org/10.1126/science.1150083. 
O?Malley, R.C. et al. (2016) ?Cistrome and Epicistrome Features Shape the 
Regulatory DNA Landscape?, Cell, 165(5), pp. 1280?1292. Available at: 
https://doi.org/10.1016/j.cell.2016.04.038. 
Petricka, J.J., Winter, C.M. and Benfey, P.N. (2012) ?Control of Arabidopsis Root 
Development?, Annual Review of Plant Biology, 63(1), pp. 563?590. Available at: 
https://doi.org/10.1146/annurev-arplant-042811-105501. 
82 
 
 
 
Poethig, S. (1989) ?Genetic mosaics and cell lineage analysis in plants?, Trends in 
genetics: TIG, 5(8), pp. 273?277. Available at: https://doi.org/10.1016/0168-
9525(89)90101-7. 
Ryu, K.H. et al. (2019) ?Single-Cell RNA Sequencing Resolves Molecular 
Relationships Among Individual Plant Cells?, Plant Physiology, 179(4), pp. 1444?
1456. Available at: https://doi.org/10.1104/pp.18.01482. 
Sadasivam, S. and DeCaprio, J.A. (2013a) ?The DREAM complex: master 
coordinator of cell cycle-dependent gene expression?, Nature Reviews Cancer, 13(8), 
pp. 585?595. Available at: https://doi.org/10.1038/nrc3556. 
Sadasivam, S. and DeCaprio, J.A. (2013b) ?The DREAM complex: master 
coordinator of cell cycle-dependent gene expression?, Nature Reviews Cancer, 13(8), 
pp. 585?595. Available at: https://doi.org/10.1038/nrc3556. 
Sadasivam, S., Duan, S. and DeCaprio, J.A. (2012) ?The MuvB complex sequentially 
recruits B-Myb and FoxM1 to promote mitotic gene expression?, Genes & 
Development, 26(5), pp. 474?489. Available at: 
https://doi.org/10.1101/gad.181933.111. 
Sarkar, A.K. et al. (2007) ?Conserved factors regulate signalling in Arabidopsis 
thaliana shoot and root stem cell organizers?, Nature, 446(7137), pp. 811?814. 
Available at: https://doi.org/10.1038/nature05703. 
Satija, R. et al. (2015) ?Spatial reconstruction of single-cell gene expression data?, 
Nature Biotechnology, 33(5), pp. 495?502. Available at: 
https://doi.org/10.1038/nbt.3192. 
Schafer, K.A. (1998) ?The Cell Cycle: A Review?, Veterinary Pathology, 35(6), pp. 
461?478. Available at: https://doi.org/10.1177/030098589803500601. 
Schmit, F. et al. (2007) ?LINC, a Human Complex That is Related to pRB-Containing 
Complexes in Invertebrates Regulates the Expression of G2/M Genes?, Cell Cycle, 
6(15), pp. 1903?1913. Available at: https://doi.org/10.4161/cc.6.15.4512. 
Schmit, F., Cremer, S. and Gaubatz, S. (2009) ?LIN54 is an essential core subunit of 
the DREAM/LINC complex that binds to the cdc2 promoter in a sequence-specific 
manner?, The FEBS Journal, 276(19), pp. 5703?5716. Available at: 
https://doi.org/10.1111/j.1742-4658.2009.07261.x. 
Schoof, H. et al. (2000) ?The Stem Cell Population of Arabidopsis Shoot Meristems 
Is Maintained by a Regulatory Loop between the CLAVATA and WUSCHEL 
Genes?, Cell, 100(6), pp. 635?644. Available at: https://doi.org/10.1016/S0092-
8674(00)80700-X. 
83 
 
 
 
Shahan, R. et al. (2022) ?A single-cell Arabidopsis root atlas reveals developmental 
trajectories in wild-type and cell identity mutants?, Developmental Cell, 57(4), pp. 
543-560.e9. Available at: https://doi.org/10.1016/j.devcel.2022.01.008. 
Shaw, R., Tian, X. and Xu, J. (2021) ?Single-Cell Transcriptome Analysis in Plants: 
Advances and Challenges?, Molecular Plant, 14(1), pp. 115?126. Available at: 
https://doi.org/10.1016/j.molp.2020.10.012. 
Sijacic, P., Wang, W. and Liu, Z. (2011) ?Recessive Antimorphic Alleles Overcome 
Functionally Redundant Loci to Reveal TSO1 Function in Arabidopsis Flowers and 
Meristems?, PLOS Genetics, 7(11), p. e1002352. Available at: 
https://doi.org/10.1371/journal.pgen.1002352. 
Simmons, A.R. et al. (2019) ?SOL1 and SOL2 regulate fate transition and cell 
divisions in the Arabidopsis stomatal lineage?, Development (Cambridge, England), 
146(3), p. dev171066. Available at: https://doi.org/10.1242/dev.171066. 
Song, J.Y. et al. (2000) ?Regulation of meristem organization and cell division by 
TSO1, an Arabidopsis gene with cysteine-rich repeats?, Development (Cambridge, 
England), 127(10), pp. 2207?2217. 
Soni, R. et al. (1995) ?A family of cyclin D homologs from plants differentially 
controlled by growth regulators and containing the conserved retinoblastoma protein 
interaction motif.?, The Plant Cell, 7(1), pp. 85?103. Available at: 
https://doi.org/10.1105/tpc.7.1.85. 
Stahl, Y. et al. (2009) ?A signaling module controlling the stem cell niche in 
Arabidopsis root meristems?, Current biology: CB, 19(11), pp. 909?914. Available at: 
https://doi.org/10.1016/j.cub.2009.03.060. 
Stepanova, A.N. et al. (2005) ?A Link between ethylene and auxin uncovered by the 
characterization of two root-specific ethylene-insensitive mutants in Arabidopsis?, 
The Plant Cell, 17(8), pp. 2230?2242. Available at: 
https://doi.org/10.1105/tpc.105.033365. 
Stepanova, A.N. et al. (2008) ?TAA1-Mediated Auxin Biosynthesis Is Essential for 
Hormone Crosstalk and Plant Development?, Cell, 133(1), pp. 177?191. Available at: 
https://doi.org/10.1016/j.cell.2008.01.047. 
Takahashi, I. et al. (2010) ?Two Arabidopsis cyclin A3s possess G1 cyclin-like 
features?, Plant Cell Reports, 29(4), pp. 307?315. Available at: 
https://doi.org/10.1007/s00299-010-0817-9. 
Tang, G. et al. (2003) ?A biochemical framework for RNA silencing in plants?, Genes 
& Development, 17(1), pp. 49?63. Available at: https://doi.org/10.1101/gad.1048103. 
84 
 
 
 
Tank, J.G. and Thaker, V.S. (2011) ?Cyclin dependent kinases and their role in 
regulation of plant cell cycle?, Biologia Plantarum, 55(2), p. 201. Available at: 
https://doi.org/10.1007/s10535-011-0031-9. 
Taylor-Teeples, M. et al. (2015) ?An Arabidopsis gene regulatory network for 
secondary cell wall synthesis?, Nature, 517(7536), pp. 571?575. Available at: 
https://doi.org/10.1038/nature14099. 
Thimann, K.V. (1936) ?Auxins and the Growth of Roots?, American Journal of 
Botany, 23(8), pp. 561?569. Available at: https://doi.org/10.2307/2436087. 
Truman, A.W. et al. (2012) ?Cell Cycle: Regulation by Cyclins?, in eLS. John Wiley 
& Sons, Ltd. Available at: https://doi.org/10.1002/9780470015902.a0001364.pub3. 
Van Leene, J. et al. (2010) ?Targeted interactomics reveals a complex core cell cycle 
machinery in Arabidopsis thaliana?, Molecular Systems Biology, 6, p. 397. Available 
at: https://doi.org/10.1038/msb.2010.53. 
Wachsman, G. et al. (2017) ?A SIMPLE Pipeline for Mapping Point Mutations?, 
Plant Physiology, 174(3), pp. 1307?1313. Available at: 
https://doi.org/10.1104/pp.17.00415. 
Wang, W. et al. (2018) ?Arabidopsis TSO1 and MYB3R1 form a regulatory module 
to coordinate cell proliferation with differentiation in shoot and root?, Proceedings of 
the National Academy of Sciences of the United States of America, 115(13), pp. 
E3045?E3054. Available at: https://doi.org/10.1073/pnas.1715903115. 
Wang, Z.-P. et al. (2015) ?Egg cell-specific promoter-controlled CRISPR/Cas9 
efficiently generates homozygous mutants for multiple target genes in Arabidopsis in 
a single generation?, Genome Biology, 16, p. 144. Available at: 
https://doi.org/10.1186/s13059-015-0715-0. 
Willems, A. et al. (2020) ?The Cyclin CYCA3;4 Is a Postprophase Target of the 
APC/CCCS52A2 E3-Ligase Controlling Formative Cell Divisions in Arabidopsis?, 
The Plant Cell, 32(9), pp. 2979?2996. Available at: 
https://doi.org/10.1105/tpc.20.00208. 
Wu, N. et al. (2017) ?A mouse model of MYCN-driven retinoblastoma reveals 
MYCN-independent tumor reemergence?, The Journal of Clinical Investigation, 
127(3), pp. 888?898. Available at: https://doi.org/10.1172/JCI88508. 
Wysocka-Diller, J. et al. (2000) ?Molecular analysis of SCARECROW function 
reveals a radial patterning mechanism common to root and shoot.?, Development 
[Preprint]. 
Xie, Y. et al. (2020) ?Single-Cell RNA Sequencing Efficiently Predicts Transcription 
Factor Targets in Plants?, Frontiers in Plant Science, 11. Available at: 
85 
 
 
 
https://www.frontiersin.org/article/10.3389/fpls.2020.603302 (Accessed: 9 March 
2022). 
Yadav, R.K. et al. (2011) ?WUSCHEL protein movement mediates stem cell 
homeostasis in the Arabidopsis shoot apex?, Genes & Development, 25(19), pp. 
2025?2030. Available at: https://doi.org/10.1101/gad.17258511. 
Yang, W. et al. (2021) ?Molecular mechanism of cytokinin-activated cell division in 
Arabidopsis?, Science (New York, N.Y.), 371(6536), pp. 1350?1355. Available at: 
https://doi.org/10.1126/science.abe2305. 
Yu, Y. et al. (2003) ?The Tobacco A-Type Cyclin, Nicta;CYCA3;2, at the Nexus of 
Cell Division and Differentiation?, The Plant Cell, 15(12), pp. 2763?2777. Available 
at: https://doi.org/10.1105/tpc.015990. 
Zhan, J. et al. (2018) ?Opaque-2 Regulates a Complex Gene Network Associated with 
Cell Differentiation and Storage Functions of Maize Endosperm?, The Plant Cell, 
30(10), pp. 2425?2446. Available at: https://doi.org/10.1105/tpc.18.00392. 
Zhang, F., May, A. and Irish, V.F. (2017) ?Type-B ARABIDOPSIS RESPONSE 
REGULATORs Directly Activate WUSCHEL?, Trends in Plant Science, 22(10), pp. 
815?817. Available at: https://doi.org/10.1016/j.tplants.2017.08.007. 
Zhang, T.-Q., Chen, Y. and Wang, J.-W. (2021) ?A single-cell analysis of the 
Arabidopsis vegetative shoot apex?, Developmental Cell, 56(7), pp. 1056-1074.e8. 
Available at: https://doi.org/10.1016/j.devcel.2021.02.021. 
Zheng, G.X.Y. et al. (2017) ?Massively parallel digital transcriptional profiling of 
single cells?, Nature Communications, 8(1), p. 14049. Available at: 
https://doi.org/10.1038/ncomms14049. 
Zhong, R. and Ye, Z.-H. (2004) ?amphivasal vascular bundle 1, a Gain-of-Function 
Mutation of the IFL1/REV Gene, Is Associated with Alterations in the Polarity of 
Leaves, Stems and Carpels?, Plant and Cell Physiology, 45(4), pp. 369?385. 
Available at: https://doi.org/10.1093/pcp/pch051. 
Zhou, G.-K. et al. (2007) ?Overexpression of miR165 Affects Apical Meristem 
Formation, Organ Polarity Establishment and Vascular Development in Arabidopsis?, 
Plant and Cell Physiology, 48(3), pp. 391?404. Available at: 
https://doi.org/10.1093/pcp/pcm008. 
Zhou, J. et al. (2021) ?Gibberellin and auxin signaling genes RGA1 and ARF8 repress 
accessory fruit initiation in diploid strawberry?, Plant Physiology, 185(3), pp. 1059?
1075. Available at: https://doi.org/10.1093/plphys/kiaa087. 
 
86