







































Abstract: Little is understood regarding the development and evolution of leaf shape and vein pattern. In 
particular, many mechanisms of plant growth and adaptation remain unknown. By reviewing the effects 
of ICK1, an Inhibitor of Cyclin-Dependent Kinase1, on cell cycling in Arabidopsis thaliana leaf mutants, 
this study seeks to determine the relationship between leaf marginal serrations and secondary veins in A. 
thaliana. Analyzing the overexpression of ICK1 in transgenic plants and the genetic pathways that regulate 
cell division will provide insight into the relationship between cell proliferation and margin development 
in A. thaliana leaves. Research has found that the overexpression of ICK1 increased the depth of marginal 
serrations while decreasing the number of serrations, overall leaf areas, total cell numbers, and vein densities. 
These results suggest that the loss of serrations induced by the inhibition of cell division is directly related to 
the loss of secondary veins in A. thaliana leaves.

Aisthesis      Volume 8,  20179

The Genetic Relationship between Leaf Margin Regulation 
and Vascular Patterning in Arabidopsis thaliana

by Lulua Rawwas

Introduction
 During the process of embryogenesis, the 
primary shoot apical meristem (SAM), a region 
of tissue at the tip of the plant shoot consisting of 
pluripotent cells, is initiated. The SAM is comprised 
of the central zone (CZ), in which cells proliferate 
slowly and tend to remain undifferentiated, and 
the peripheral zone (PZ), in which cells proliferate 
rapidly and differentiate into various organs such as 
the leaves, stem, and flowers (Kalve et al. 2014). WUS 
(Wuschel), a homeodomain transcription factor that 
designates the stem cell niche, is expressed by some 
cells in the CZ. The slow rate of cell proliferation in 
the stem cell niche dislocates cells, enabling them to 
initiate active division. The PZ, on the other hand, 
gives rise to leaf primordia cells, whose locations are 
determined by local auxin maxima. The plant hormone 
auxin and its efflux carrier, PIN1 (Pin-Formed1), 
modulate the differentiation of the progenitor 
cells into either lateral appendices or principal 
axis cells. PIN1 directs the polarized transport of 
auxin through the epidermis to neighboring cells 
of high auxin concentrations (Rodriguez et al. 
2014). In Arabidopsis, the developing leaves are 
positioned in a spiral phyllotaxy at approximately 
137˚ apart from each other (Rodriguez et al. 2014). 
Expressed throughout the meristem, the STM (Shoot 
Meristemless) gene and other KNOX1 (Knotted-

like homeobox) transcription factors also regulate 
the formation of the leaf primordia by suppressing 
premature cell differentiation (Rodriguez et al. 
2014). Cytokinin (CK), a plant hormone whose 
biosynthesis is stimulated by KNOX1 genes, may also 
play an important role in facilitating the growth and 
preservation of the SAM, as high levels of CK inhibit 
cell differentiation to protect cell identity (Kalve et 
al. 2014).
 Separated from the meristem by a boundary 
zone, differentiating cells within leaf primordia form 
along the edges of the SAM following germination. 
The presence of the boundary zone, distinguished 
by gene expression patterns and few cell divisions, 
facilitates leaf maturation and preserves the 
meristem’s functions (Wang et al. 2015). Following 
the establishment of a polarity gradient, primordium 
outgrowth is activated by intensified cell division. 
The final shape of the leaf is in part determined by 
varying rates of cell growth along the developmental 
axes (Kalve et al. 2014). Leaf primordia may mature 
into compound leaves consisting of leaflets or 
simple leaves comprising undivided blades, as in 
Arabidopsis, depending on genetic expressions and 
environmental conditions (Wang et al. 2015). The 
establishment of secondary shoot apical meristems 
in leaf axils, the regions between the leaf stalks and 
the leaf stems, promote the initiation of new leaf 



The Genetic Relationship between Leaf Margin Regulation and Vascular Patterning in A. thaliana

Aisthesis      Volume 8,  201710

primordia after embryonic development; this new 
leaf primordia may then develop into a side-shoot 
or enter a resting phase. Through the regulation of 
both the secondary meristem and the side-shoot 
formations, a plant adapts to its environmental 
conditions (Wang et al. 2015).
   
Inhibitors of Cyclin-Dependent Kinases
 At its core, the plant cell cycle, along with many 
other eukaryotic systems, is regulated by cyclin-
dependent kinases (CDK), cyclins, and inhibitors 
of cyclin-dependent kinases (ICK). The activities of 
cyclin-dependent kinases are vital to the initiation of 
DNA replication during the G1/S phase transition of 
mitosis (Kalve et al. 2014). The CDK complex plays 
an integral role in plant development, and research 
has shown that its mutation results in altered organ 
sizes and shapes (Mizukami and Fischer 2000). In 
nature, abiotic stress conditions, such as treatment 
with abscisic acid or low temperatures, induce ICK 
expression (Wang et al. 2000). At low concentrations, 
ICKs inhibit the CDK complex to perpetuate the 
CDK oscillations necessary for DNA duplication, 
but at high concentrations, cell cycle arrest occurs 
(Kalve et al. 2014). Along with its specificity for 
plant kinases and its distinct sequences, this trait 
distinguishes ICK1 from animal CDK inhibitors, 
suggesting that ICK1 represents a divergence of 
plant cell cycle regulation from other eukaryotes. 
However, ICK1 does share some characteristics with 
CDK inhibitors found in other species, such as its 
inhibitory effects, conserved region, and interactions 
with both a CDK and cyclin (Wang et al. 2000). 
Seven ICK genes have been found to be expressed 
in Arabidopsis tissues. The structure of the plant ICK 
protein sequence consists of a C-terminal domain 
that interacts with CDKs and a region that interacts 
with D-type cyclins, indicating that ICK seek to 
predominantly interact with complexes containing 
CDKA or CYCD. The ICK proteins are restricted to 
the nucleus in Arabidopsis, some of which transfer 
CDKA or CYCD proteins to the nucleus. It has been 
postulated that ICK regulates both the plant mitotic 
cycle and the endocycle (Cheng et al. 2013).
 Studies in which researchers overexpressed ICK1 
in transgenic plants found that cell proliferation was 
inhibited throughout the developing plant, leading 
to transformed morphologies. The decreased cell 

numbers seen in 35S::ICK1 plants, transgenic plants 
expressing ICK1 driven by the 35S promoter, may 
be due to fewer cells engaging in mitosis in the 
meristems, prolonged cell division in the meristems, 
or a combination of these events (Wang et al. 2000). 
In addition, ICK1 expression was found to repress 
lobe outgrowth when targeted to leaf margins and 
generate organs with extreme lobing when targeted 
to sinuses (Malinowski et al. 2011).
 The decreased cell numbers in 35S::ICK1 plants 
affected not only the morphology of the leaves, but 
also all of the vein orders. The overexpression of 
ICK1 led to significantly reduced secondary and 
minor vein densities, the absence of intersecondary 
and many higher order veins, and the cessation of 
most third order veinlets as freely ending. Mesophyll 
cells expanded to compensate for the reduced cell 
proliferation, resulting in the early termination of 
minor vein formation and an abnormal vascular 
pattern (Kang et al. 2007). These results have 
significant implications regarding the relationship 
between marginal leaf serrations, vascular patterning, 
and the genes that regulate them.

Marginal Serration Formation
 The gene CUC2 (Cup-shaped cotyledon 2) 
patterns serrations and promotes the separation 
of adjacent organs by suppressing growth in 
the boundary domain (Kawamura et al. 2010). 
Specifically, CUC2 is expressed in a small group of 
cells at the boundary between the meristem and 
the emergent leaf primordia, and its expression is 
augmented by STM gene expression (Rodriguez et 
al. 2014). Double mutants exhibited an abnormal 
SAM and fused cotyledons. Acting early in plant 
development, CUC2 regulates tooth size through 
cell proliferation at the sinuses primarily as a result 
of transcriptional regulation, while levels of CUC2 
expression are modulated by MIR164A. CUC2 and 
MIR164 promoter activities were acutely inhibited in 
cuc2 mutants, suggesting that CUC2 plays a vital role 
in the regulation of tooth outgrowth (Hasson et al. 
2011).
 The plant hormone auxin also defines the location 
of progenitor cells (cells capable of differentiation 
and limited division), regulates endoreduplication, 
and mediates blade expansion at the adaxial-abaxial 
boundary (Kalve et al. 2014). Auxin maxima, high 



The Genetic Relationship between Leaf Margin Regulation and Vascular Patterning in A. thaliana

Aisthesis      Volume 8,  201711

concentrations of auxin convergence, are markers 
of future tooth initiation sites that repress CUC2 
to regulate lamina growth and vein patterning 
(Kawamura et al. 2010). The convergence point 
formation of PIN1 (Pin-Formed1) is promoted 
to produce auxin maxima. PIN1 is a major auxin 
transporter, and its pin1 mutant removes leaf 
serrations and reduces auxin accumulation at the 
base of leaves, exhibiting decreased polar transport 
of auxin (Bilsborough et al. 2010). PIN1 expression 
is regulated by CUC2, suggesting that a mechanism 
involving CUC2, auxin, and PIN1 is necessary for 
serration formation (Kawamura et al. 2010). Auxin 
has been found to down-regulate CUC2 to position 
both PIN1 convergence points and auxin maxima. 
It has been posited that PIN1 convergence points 
are also regulated by CUC2 during organogenesis 
(Bilsborough et al. 2010).

Polarizing Genes
 Among the genes that coordinate the formation 
of auxin maxima is AS2 (Asymmetric 2), a regulator 
of marginal outgrowth and polarity. Low auxin 
concentrations are found at the adaxial boundary of 
leaf primordia as PIN1 polarity reverses and points 
towards the SAM, expending auxin and establishing 
the boundary (Wang et al. 2015).  AS2 functions in 
the AS1-AS2 complex to temporally repress ARF3 
(Auxin response factor 3), an abaxial (lower leaf 
domain) determinant, and non-repressively regulate 
TAS3A, an adaxial (upper leaf domain) determinant. 
ARF3 is also repressed by a ta-siRNA (trans-acting 
small interference RNA) pathway, which influences 
several nodes of development and enables AS1-AS2 
to maintain the flat surfaces and simple margins of 
the A. thaliana leaf. As a result, AS1-AS2 preserves 
the separation of the adaxial and abaxial domains by 
binding the promoters of its polarity targets as the 
patterning of polarity shifts from an external to an 
internal process (Husbands et al. 2015). The AS1-
AS2 complex also suppresses STM and other KNOX1 
transcription factors in the leaf primordia through 
several pathways to separate the meristem from the 
leaf primordia (Rodriguez et al. 2014).
 Regulators of the abaxial domain also contribute 
to the maintenance of bilateral symmetry through 
the establishment of leaf development processes. The 
polarizing gene FIL (Filamentous flower) determines 

the identities of cells on the abaxial leaf side. FIL is 
one of six YABBY genes found in the Arabidopsis 
genome. YABBY genes promote laminar growth, 
establish communication between growing leaves 
and the shoot apical meristem, regulate embryo 
patterning, and convert organ polarity to lamina-
specific programs. Furthermore, YABBY establishes 
linear marginal auxin flow to shape modified shoot 
systems into flat leaves (Sorojam et al. 2010).

Vascular Patterning
 In addition to regulating cell cycling and 
differentiation, auxin coordinates the formation of 
procambial strand (the precursor to mature xylem and 
phloem, cells that conduct water and carbohydrates, 
respectively). The expression of AtHB8 (Arabidopsis 
thaliana homeobox gene 8), an early marker for 
vascularization that precedes vein differentiation, 
is modulated by auxin (Kang and Dengler 2002). 
During vascular formation, differential cell divisions 
promote procambial cell growth and arrange strands 
in a hierarchy of vein orders (Kang et al. 2007). 
Research has suggested that AtHB8 may convert 
auxin signals into procambial strands and heighten 
tissue responsiveness to auxin (Kang and Dengler 
2002). Furthermore, auxin may direct secondary 
vein formation and regulate serration formation by 
initiating localized growth (Kang and Dengler 2004).

Conclusion
 Research has found that the overexpression 
of ICK1 is directly related to the loss of secondary 
leaf veins in A. thaliana. The complex interactions 
between diverse genetic pathways of other 
regulators of cell proliferation have also been 
shown to influence the vascular patterning and 
morphological development of Arabidopsis. CUC2 
governs the patterning of marginal serrations, while 
polarizing genes such as AS2 and FIL coordinate 
the establishment of the adaxial-abaxial domain. 
Furthermore, plant hormones such as auxin play 
significant roles in early plant development and the 
formation of leaf shape and vein pattern.

Implications
 The implications of further studying these genetic 
pathways are both extensive and multifaceted. At the 
cellular level, targeted expression of CDK inhibitor 



The Genetic Relationship between Leaf Margin Regulation and Vascular Patterning in A. thaliana

Aisthesis      Volume 8,  201712

genes such as ICK1 may be applied in the fields of 
agriculture, horticulture, and arboriculture to alter 
individual organs. It is known that mechanisms of 
cell division are maintained among crop species 
such as broccoli, cabbage, cauliflower, and kale, 
which belong to the Brassicaceae family along with 
Arabidopsis (Wang et al. 2000). Thus, researching the 
regulation of plant growth and organ development is 
essential to achieving a better understanding of crop 
yield and plant adaptation to both environmental 
and experimental settings (Kalve et al. 2014). In 
addition, the results of studying leaf morphology 
and vasculature may be employed to modify 
photosynthetic capacities and nutrient transport 
within plants, respectively. Finally, gaining insight 
into pathways involving auxin may lead to an 
enhanced comprehension of leaf development, 
specifically plant size, root systemization, and apical 
dominance (Kawamura et al. 2010).

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