Size is important for cell function and is characteristic of different species and of different cell types in the same organism. How cell size is specified has intrigued researchers ever since cells became visible to biologists. Cell size determines the geometry of all intracellular compartments and sets the scale of biosynthetic processes. Biosynthesis increases in larger cells, which have proportionally more protein, total RNA, total mRNA, and mRNA for specific investigated genes.Plants are a good multicellular system for studying the regulation of cell size. As plant cells cannot migrate because they are encased in a cell wall matrix, their growth and division can be readily tracked and measured by live cell imaging and three-dimensional (3D) image analysis. Plants are also particularly relevant for studying how cell size relates to ploidy and to organ size because of the prominent role of polyploidy in plant evolution, plant development, and crop domestication.

1. CELL SIZE CONTROL IN PLANTS
TAMIL NADU AGRICULTURAL UNIVERSITY
Doctoral Seminar : II
BY
BHIMIREDDY SUKRUTHA
2020608008
II PhD
Department of Plant Breeding and Genetics

2. Advisory Committee
Advisory
Committee
Name
Designation
Chairperson
Dr. S. Rajeswari Professor & Head, Dept. of Cotton
CPBG, TNAU, Coimbatore
Member
Dr. N. Premalatha
Asst. Professor (PBG),Dept. of Cotton,
CPBG, TNAU, Coimbatore
Member Dr. N. Manikanda Boopathi
Professor (Biotech.), DPB,
CPMB&B, TNAU, Coimbatore
Member
Dr. K.Thirukumaran Associate Professor, Dept of Agronomy,
TNAU, Coimbatore.
Additional
Member
Dr. A. Manivannan
Senior Scientist (Genetics)
Central Institute of Cotton Research,
Regional station, Coimbatore

3. Cells
 Smallest living unit
 Most are microscopic

4.  Diffusion limits the cell size
 DNA limits cell size
 Surface area to volume ratio
Why are cells small??

5. Diffusion Limits Cell Size
• Diffusion is fast and efficient
over short distances, it
becomes slow and inefficient
over larger distances.
• Small cell - good diffusion
• Big cell - bad diffusion
Speed of Diffusion
• An organelle in the center of a
20μm diameter cell receives
supplies a fraction of a second
after it enters the cell.
• An organelle in the center of a
20 cm diameter cell would have
to wait months before receiving
supplies that enter the cell.

6. DNA Limits Cell Size
• The DNA tells the cell which
proteins to be made.
• Not enough protein - the cell will
die. Take time for protein to be
produced.
• As cells increase in size, no extra
DNA is made

7. Surface Area-Volume Ratio
• As cell size increases, volume
increases much faster than surface
area.
• A 1mm cube has a surface area of
6mm2 and a volume of 1mm3. A
2mm cube has a surface area of
24mm2 and a volume of 8mm3
• The larger the volume of the cell,
the smaller the surface area gets.

8. Cell Size

9. Skotheim et al.,2015

10. As an organism grows, can the cells get bigger?
 Before a cell gets too large, it
divides into two “daughter”
cells.
 Before cell division takes place,
the cell must replicate all of its
DNA.
 The cell then divides and
solves the problem of the cell
becoming too large.

11. 2 Reasons why cells divide
• As the cell grows larger the cells DNA will no longer be able to serve the
increasing needs of the growing cell.
• The larger the cell the more trouble it has moving nutrients such as food,
oxygen and water and wastes across the cell membrane.

12. Possible scenarios in which centrosome centering (a) and spindle
elongation (b) set the upper and lower limit of cell size
If the cell exceeds the upper limit of size,
the centrosome, and consequently the
mitotic spindle, cannot position at the cell
center, leading to nonsymmetrical cell
division.
If the cell falls below the lower limit, the
centrosome may not stably position at the
cell center due to the excess elastic forces
of the microtubules

13. If the cell exceeds the upper limit, astral
microtubules do not reach the cell
cortex, potentially leading to insufficient
spindle elongation.
If the cell falls below the lower limit,
there may not be sufficient space for
accurate chromosome segregation
compared to the size of the cell’s
chromosomes.
Marshall et al., 2012

14. • The genetic control of the characteristic cell sizes of different species and tissues is a long-
standing enigma. Plants are convenient for studying this question in a multicellular context,
as their cells do not move and are easily tracked and measured from organ initiation in the
meristems to subsequent morphogenesis and differentiation.
• Molecular mechanisms for cell size control have implications for how cell size responds to
changes in ploidy, which are particularly important in plant development and evolution.
• Plants are also particularly relevant for studying how cell size relates to ploidy and to organ
size because of the prominent role of polyploidy in plant evolution, plant development, and
crop domestication
Early experiments using sea urchins of different ploidy revealed that cell size is strongly
affected by nuclear contents, while experiments using protozoans showed that
progression through cell division depends on cytoplasmic volume
In multicellular organisms, cell size regulation is particularly complex, partly because cell
autonomous mechanisms for size regulation overlap with developmental control of
growth and cell division by intercellular signals

15. PHENOMENOLOGICAL MODELS FOR CELL SIZE CONTROL
3 models: Timer model
Sizer model
Adder model
Sablowski et al.,2019
Jones et al., 2019

16. No active control of cell size
Division triggered after certain time period
Division triggered after fixed growth increment of volume
Large cells divide faster than small cells
Cell division could be inhibited until a fixed threshold size is reached
Large cells divide faster than small cells
Division is triggered by a proxy that is positively or negatively correlated to cell size
Relation b/n cell size and proxy is dependent on multiple factors and it varies a/c
to conditions
Jones et al., 2019

17. Cell Size Control through Tissue- and Cell-Level Mechanisms
cell growth and division are
regulated by tissue-level
hormonal signals. Cells
with different birth sizes,
but receiving the same
tissue-level signals, are
expected to divide after the
same amount of time.
cell growth rate and division
rate are hypothesized to be
dependent on cell size as
well as being regulated by
tissue-level signals
cell-cycle length is inversely
dependent on cell size such
that large cells divide more
rapidly than small cells

18. RELATION BETWEEN PLOIDY AND CELL SIZE
The hypothesis that DNA could be used as a molecular ruler in cell size homeostasis also
suggests a link to the observation that cell size correlates with ploidy levels in a wide variety of
eukaryotes, including plants
Sugimoto-Shirasu and Roberts , 2003
different cell layers have
different ploidies, show a
clear relationship between
nuclear DNA content, nuclear
volume and cell size
Cell division and cell expansion in the different layers mutually adjust to avoid distortion of
meristem anatomy, indicating that cell–cell signaling is involved in overall control of organ size

19. Endoreduplication
• One common mechanism by which plants achieve this increase in cell size is through
increasing their ploidy level by successive rounds of DNA replication, a process called
endoreduplication.
• Although endoreduplication is widespread in eukaryotes, it is commonplace in plants . Not
only higher plants but also algal and fern cells undergo endoreduplication.
• The entire complement of chromosomes is usually re-replicated during endoreduplication
but, depending on the final configuration of chromosomes, two distinct processes can be
discerned.
• In one of these processes chromosomes go through condensation and de-condensation
stages after replication and sister chromatids separate, resulting in polyploidy; in the second,
the chromosomes replicate without undergoing such condensation stages and sister
chromatids remain closely associated, resulting in polyteny

20. How do cells switch from mitosis to endoreduplication?
• Although this process is still poorly understood, it is clear that it requires important changes
in the cell cycle machinery.
• For instance, cyclins that are active in G2 are no longer expressed, and others have modified
activities as was reported for E cyclins in Drosophila: E cyclin is present at constant levels in
mitotic cells, but fluctuating levels are required for multiple rounds of endocycles.
• One of the components necessary for the transition to endoreduplication is the FIZZY
RELATED protein, first identified in Drosophila. It was proposed that FIZZY RELATED
functions in the degradation and inactivation of mitotic cyclins during interphase, thus
allowing endoreduplication to occur.

21. Robinson et al.,2018

22. trichome (32C)
stomatal guard cells (2C) small epidermal cell (4C)
large epidermal cell (8C)
The brightly stained heterochromatic
centromeric regions are few in
number (ten or fewer), strongly
suggesting that the endoreduplicated
chromosomes are polytene in nature
in the 32C trichomes.
The volume of each nucleus is
directly proportional to its ploidy.
Endoreduplication in the Arabidopsis leaf epidermis

23. Ploidy level and cell size: how far does the correlation go?
found in the polyploid periclinal chimeras of Datura
meristems, in which each discrete cell layer has a different
ploidy with corresponding nuclear and cell-size changes
Endoreduplication is also associated
with a cell-size increase in Arabidopsis,
as has been seen in wildtype leaf
epidermal cells

24. Counter examples in which ploidy level does not coupled to final cell size
1) Arabidopsis root cells from different ecotypes have varied sizes, but no positive correlation
was found between their ploidy level and cell size
2) Many Arabidopsis dwarf mutants that have defects in hormone signaling or cell wall
biosynthesis have a similar distribution of cell ploidy as the wildtype but remarkably reduced
cell sizes
3) Several transgenic Arabidopsis plants that overexpress key cell cycle regulatory genes are
altered in their ploidy levels but do not necessarily show corresponding cell-size changes
These findings suggest that polyploidization is not the only mechanism that controls
cell size in plants and that other distinct pathways also contribute to this control.

26. The first of these processes is increased growth
coupled to endoreduplication, whereas the second is
cell expansion (i.e. increase in cell volume through
vacuolation). The two processes combine to
determine the final size of the differentiated cell.
Growth and cell-size control in plants
Increases in the size of proliferating cells are
largely dependent on cell growth, whereas most of
the size increase in postmitotic cells is achieved by
cell expansion.

27. A simple schematic model of some of the
key processes that regulate cell size.
The input signals are a combination of
endogenous signal molecules and
environmental cues, which determine the local
balance between cell proliferation, growth (i.e.
increase in macromolecular mass) and
expansion (i.e. vacuolation-driven increase in
volume).
Cell number and size often have higher-order
constraints that are imposed by feedback loops
that regulate total organ size.

28. MOLECULAR MODELS
1. Size-Dependent Transition to the DNA Replication Phase
In budding yeast cells, the G1 cyclin Cln3 phosphorylates Whi5
to derepress the SBF transcription factor that activates
transcription
The fixed number of SBFbound sites in the genome has
been proposed to provide the ruler to measure Cln3 levels
Increasing cell volume dilutes the Whi5 protein to a
threshold concentration that allows the G1-to-S transition
In this case, Whi5 can function as the ruler because its synthesis rate is limited by gene copy number,
not by protein synthesis capacity, which scales up with cell size, so the Whi5 dilution model indirectly
uses the genome as the internal ruler.
 One of the challenges in revealing molecular mechanisms for cell size control is to identify a structure or
molecule that could be used as a ruler to measure cell size.
 Most organelles and the total amount of most proteins scale up with cell size, so they would not be
appropriate rulers. One obvious exception would be DNA, or specific sites on the genome
 This idea has been a key part of models for the size-dependent progression from the gap 1 (G1) phase to the
DNA synthesis (S) phase of the cell cycle.
Sablowski et al.,2019

29. Progressive dilution of CDKG1 in relation to its targets on increasing
amounts of chromatin has been proposed to link the number of S/M
cycles to initial cell size because each round of DNA replication
dilutes CDKG1 until a threshold level is reached
Accordingly, cdkg1 mutants divide fewer times and produce larger
daughter cells, whereas rb mutants divide too many times, resulting
in small cells
 In Chlamydomonas reinhardtii, DNA replication is also linked to
cell size.
 In this alga, a vegetative cell maintained in the light grows
during a prolonged G1 phase to several times its initial size;
shifting the cells to darkness stops growth, but if a size
threshold has been passed, the cell undergoes a series of rapid
cycles through S and M phases .
The number of these cycles is adjusted to the starting cell
size through the retinoblastoma (RB) pathway
 In C. reinhardtii, the cyclin dependent kinase G1 (CDKG1)
inactivates RB to promote S-phase entry; CDKG1 reaches a size
dependent concentration in the intitial cell and is not produced
during the subsequent divisions.
Together with the budding yeast and C. reinhardtii models, these results highlight an intimate
connection between the Whi5/RB pathway and the size-dependent G1-to-S transition.
Sablowski et al.,2019

30. 2. Mechanisms Linking Mitosis to Cell Size
In contrast to budding yeast and mammalian cells, the main target
for cell size control in Schizosaccharomyces pombe is the
transition from the second gap (G2) phase of the cell cycle to
mitosis (M).
In this case, different sizing mechanisms have been proposed,
mostly converging on a series of kinases (Pom1, Cdr2,Wee1) that
function sequentially to control cyclin-dependent kinase 1 (Cdk1),
which triggers the G2-to-M transition
In this model - Cdr2 accumulates in the medial region of the cell in a way that reflects cell
surface area, eventually reaching a threshold that initiates the G2-to-M transition
Sablowski et al.,2019

31. Complications: Overlapping Mechanisms and Cell-Type-Specific Features
• Although supported by extensive evidence, the mechanisms described above remain debated because
mutation of key genes such as whi5 in budding yeast and pom1 or cdr2 in fission yeast does not
eliminate cell size homeostasis
• Robust cell size homeostasis has been suggested to result from the overlap of multiple size-sensing
mechanisms at different stages of the cell cycle.
• For example, cell size uniformity in S.cerevisiae depends not only on the well-studied G1/S sizer but
also on the less well-characterized size control at the G2-to-M transition. Similarly, S. pombe too has a
G1/S sizing mechanism, which becomes visible only when the G2/M size control is compromised in
wee mutants.
• In the S. pombe cdr2 mutant, the area-sensing mechanism appears to revert to a secondary, volume-
sensing mechanism. In plants, there is evidence that both the G1-to-S and the G2-to-M transitions are
responsive to cell size
• In summary, the molecular mechanisms for cell size regulation remain an area of active investigation in
all eukaryotic models. So, to understand cell size regulation in multicellular plants, we should consider
specific features of plant cell growth

32. SPECIFIC FEATURES OF PLANT CELLS FOR SIZE REGULATION
The increased volume created by wall extension is
occupied by enlargement of the cytoplasm and
nucleus or by water uptake and expansion of
vacuoles, with the balance of the two depending on
cell type and developmental stage
Cell expansion associated with vacuole enlargement
probably evolved as a metabolically low-cost
mechanism for rapid and extensive organ growth
under competition for light and other environmental
resources.
Leaf cell
Shoot meristem

34. Objective: To dissect the function of the four NMCP(Nuclear Matrix Constituent
Protein) family proteins in Arabidopsis encoded by the CRWN genes
Materials : Agrobacterium T-DNA insertion alleles to study the effects of inactivating
different combinations of CRWN genes
 FISH
 Flow cytometry – to calculate average ploidy level for each genotype
CASE STUDY 1

35. Phylogenetic relationships among CRWN proteins
2 clades – one clade includes
CRWN1,2,3 and other clade
has CRWN4
Dicots contain CRWN1 proteins
dicot CRWN4-like proteins are distinct
from their monocot counterparts in
lacking conserved amino acid motif at
the extreme C terminus

36. Whole plant phenotypes of crwn mutants at rosette stage
Plants carrying a mutation in any
single CRWN gene had
phenotypes similar to wild-type
Columbia plants, as did the
double crwn2 crwn3 and crwn3
crwn4 mutants.
Triple mutant plants carrying
only CRWN2 or CRWN3 were
extremely stunted, but still viable
that suggests CRWN2 or
CRWN3 alone can cover the
minimum requirements for the
entire CRWN protein family.
plants carrying only CRWN1 or only CRWN4 were not recovered, suggesting that CRWN1 and
CRWN4 are specialized and that neither protein alone can express the full range of functions of the
CRWN protein family

37. Nuclear phenotypes of crwn mutants in adult leaf tissue
deficiency of CRWN1 or CRWN4
reduced nuclear size, while loss of
CRWN2 or CRWN3 had no
effect
In contrast, combination of a crwn1 with a
crwn4 mutation had an additive effect on
nuclear size. These findings indicate that
CRWN1 and CRWN4 are the major
determinants of nuclear size among the
CRWN paralogs.
spherical
Combining a crwn1 mutation with a crwn2
or crwn3 mutation had a synergistic effect
on nuclear size, suggesting that CRWN1
function overlaps, at least partially, with
those of CRWN2 and CRWN3.
Double mutant combinations containing
crwn4 and either crwn2 or crwn3 did not
show additive phenotypes but rather
resembled crwn4.

38. Relationship between nuclear size and DNA
content - Average leaf guard cell nuclear
sizes in crwn mutants
Effects of crwn mutations on nuclear size and nuclear DNA density in leaf cells
crwn mutants showed a decrease in
endopolyploidy levels, particularly the crwn
triple mutants and the crwn1 crwn2 double
mutant
Consistent with their effects on nuclear size shown in
Figure 4A Neither the crwn2 nor crwn3 mutation
affected nuclear size in guard cells.
CRWN1 plays the major role in affecting nuclear size in the absence of changes in endopolyploidy
Direct correlation between endopolyploidy
and nuclear size in wild-type Arabidopsis
cells prompted them to examine this
relationship within the crwn mutants
With the exception of crwn2 and crwn3, the crwn
mutations caused a more pronounced reduction
in nuclear size than predicted from the observed
endopolyploidy level. As a consequence, crwn
mutants display a spectrum of nuclear
DNA densities
However, crwn2 crwn3, crwn2 crwn4, and crwn3
crwn4 double mutants had nuclei approximately
20% smaller than those seen in wild-type guard
cells, suggesting some functional redundancy
among CRWN2, CRWN3 and CRWN4 proteins

39. Chromocenter morphology changes in crwn mutants Role of CRWN proteins on the
internal organization of the nucleus
average chromocenter number in crwn1,
crwn2 and crwn3 leaf cell nuclei was
similar to that seen in wild-type leaf cell
nuclei
crwn4 nuclei exhibited a wide range of
chromocenter numbers (2–27)
To explore the chromocenter phenotype in
more detail, aggregation index (AI), to
characterize the distribution of visible
DAPI-bright spots within interphase nuclei
Chromocenter
in
cell
nuclei
Chromocenter number remains fairly constant over a
wide range of nuclear sizes and endopolyploidy levels
(2n to 16n), most likely as a result of lateral association
of sister chromatids after endoreduplication
AI index of wild-type nuclei averaged close to
0.1 nd was not affected significantly by nuclear
size. The absence of a significant correlation
between AI and nuclear size indicates that
chromocenter organization remains constant
across different endopolyploidy levels in wild-
type nuclei
The variability in chromocenter size and
number in crwn mutant nuclei suggests that
CRWN proteins are required for proper
organization of heterochromatin in interphase
nuclei.

40. spatial arrangement of Chromocenter organization is altered in crwn1 crwn2 and crwn4 mutants
It was common to find a decondensed centromere
signal at several chromocenters in wild-type nuclei;
however, decondensed centromeric repeat clusters
were infrequently observed in crwn1 crwn2 nuclei and
the total number of clusters was reduced
number of discrete centromere
repeat clusters visible in crwn4 nuclei
was more variable
the apparent dispersal of chromocenters in larger
crwn4 nuclei and the mis-positioning of
centromeric and 5S RNA repeats outside of the
chromocenter indicates that higher-order
organization of heterochromatin breaks down in
interphase in the absence of CRWN4.
Using FISH, we examined the spatial
organization of the major 180-bp centromeric
tandem repeat and the 5S RNA gene arrays in
both large and small nuclei from wild-type,
crwn1 crwn2 and crwn4 plants
These findings indicate that there is a
compaction of the centromere repeat arrays
within coalesced chromocenters in crwn1 crwn2
nuclei
CRWN4 - controlling distribution and
number of heterochromatic chromocenters

41. Conclusion
• This study addresses fundamental questions about how plant cells specify and control the morphology
of their nuclei and its relationship with internal chromatin organization.
• CRWN proteins are important architectural components of plant nuclei which play diverse roles in both
heterochromatin organization and the control of nuclear morphology.
• CRWN 1 and CRWN4 are major determinants of nuclear size and shape out of which CRWN1- in
controlling nuclear size
CRWN4 - controlling distribution and number of heterochromatic chromocenters.
• The specificity of the nuclear morphological and higher-order chromatin organization defects seen in
crwn mutants reveals the interplay between nuclear morphology and the three-dimensional packaging
of the genome.

42. CASE STUDY 2
Regulation of grain size is crucial for improving crop yield and is also a basic aspect in developmental
biology. However, the genetic and molecular mechanisms underlying grain size control in crops remain
largely unknown despite their central importance
Aim: To reveal a significant genetic and molecular mechanism of the GSK2-OML4 regulatory module
in grain size Control
Materials:
 wild type - japonica var Zhonghuajing (ZHJ)
 ꞃ-Rays to irradiate the grains of the wild-type ZHJ
 RNA Extraction and RT-qPCR Analysis

43. • As grain size is a key component of grain weight, regulation of grain size is a crucial strategy to
increase grain production. Grain growth is restricted by spikelet hulls, which influence final grain size in
rice. The growth of the spikelet hull is determined by cell proliferation and cell expansion processes.
• Several genes that regulate the grain size by influencing cell proliferation in the spikelet hull have been
described in rice- GW2, GW5, GW8, GS5 etc
• SHAGGY-LIKE KINASE2 (GSK2), a homologue of the Arabidopsis (Arabidopsis thaliana)
BRASSINOSTEROID INSENSITIVE2 (BIN2) kinase, has been reported to influence grain size and also
other growth processes in rice.
• Overexpression of GSK2 leads to small grains and short plants, whereas downregulation of GSK2
produces long grains.
• To further reveal the mechanisms of grain size determination, we have identified several grain size
genes whose loss and gain of function lead to opposite effects on grain size in rice - LARGE1, which
encodes MEI2-LIKE PROTEIN4 (OML4) with three RNA recognition motif (RRM) domains, regulates
grain size and weight by restricting cell expansion in spikelet hulls in rice
• The large1-1 mutant forms large and heavy grains, while overexpression of OML4 causes small and
light grains.

44. LARGE1 Influences Grain Size and Plant Morphology
large1-1 mutant displayed large grains and tall
plants. LARGE1 negatively regulates grain
size and weight in rice
LARGE1 also negatively influences
panicle length
The number of grains per panicle in large1-1
was decreased in comparison with that in
ZHJ. These results suggest that LARGE1
influences mainly the grain size in rice.

45. LARGE1 Regulates Cell Expansion in Spikelet Hulls
Scanning electron microscopy analysis of lemma
Indicate that the long and wide grain
phenotypes of large1-1 result from
the long and wide cells in spikelet
hulls. Thus, LARGE1 regulates
grain size by limiting cell
expansion in spikelet hulls.
Grain growth is limited by spikelet hulls,
and spikelet hull growth is determined by
cell proliferation and cell expansion. To
uncover the cellular mechanism for
LARGE1 in grain growth, investigation
carried in cells of ZHJ and large1-1
spikelet hulls.
outer epidermal cells in large1-1
lemmas were longer and wider cells
than those of ZHJ lemmas, while cell
numbers in large1-1 lemmas were
similar to that in the wild-type lemmas in
both longitudinal and transverse
directions

46. LARGE1 Encodes the Mei2-Like Protein OML4
INDEL contained a 4-bp
deletion in large1-1 in the
gene (LOC_Os02g31290)
which leads to a premature
stop codon
confirmed this deletion
in LOC_Os02g31290 by
developing a derived
CAPS marker
A genetic complementation test
was conducted to confirm whether
the deletion in LOC_Os02g31290
was responsible for the large1-1
phenotypes. The genomic
fragment of LOC_ Os02g31290
(gLARGE1) was transformed into
the large1-1 mutant, and 11
transgenic lines were generated.
The Glarge1 construct
complemented the large-grain
phenotypes of the large1-1 mutant
Complementation test
supported that the LARGE1
gene is LOC_Os02g31290
The mutation in large1-1
resulted in a premature
stop codon. The proteins
encoded by large1- 1
(OML4large1-1) lacked
RRM motifs, which
indicated that large1-1 is a
loss-of-function allele.
Expression of OML4 in developing
panicles using RT-qPCR analysis
Subcellular localization of OML4
in rice, they generated gLARGE1-
GFP transgenic plants. the
gLARGE1-GFP construct rescued
the phenotypes of the large1-1
mutant, indicating that the
LARGE1-GFP fusion protein is
functional.
GFP signal in gLARGE1-
GFP; large1-1 roots was
predominantly detected in
nuclei. This indicates that
OML4 is localized in nuclei
in rice

47. Overexpression of OML4
Results in Short Grains Due to
Short Cells in Spikelet Hulls
The average length of proActin:OML4
panicles was significantly decreased
compared with that of ZHJ
As proActin:OML4 transgenic lines
produced short grains, they tested
whether overexpression of OML4
could decrease cell length in spikelet
hulls
Revealed that OML4 affects grain
growth by limiting cell expansion
in spikelet hulls.

48. OML4 Physically Interacts with GSK2 in Vitro and in Vivo
To further understand the
molecular role of OML4 in grain
growth control, they identified its
interacting partners through a
yeast two hybrid Assay. The OML4
full-length protein was used as the
bait. Among several interacting
proteins, six different clones
corresponding to GSK2 were found
in this screen. GSK2 has been
reported to restrict grain growth in
rice, suggesting that GSK2 is a
candidate OML4-interacting
partner. We further confirmed the
interaction of OML4 with the full
length GSK2 in yeast cells
Interaction between OML4 and GSK2
in plant cells using the firefly luciferase
(LUC)
performed a bimolecular
fluorescence complementation
(BiFC) assay to test the
interaction between OML4 and
GSK2 in plant cells
strong YFP fluorescence in nuclei
when coexpressed OML4-cYFP
and GSK2-nYFP in N. benthamiana
leaves which indicate that OML4
associates with GSK2 in plant
cells.
For invitro asay, they expressed
maltose binding protein (MBP)–
fused OML4 (OML4- MBP) and
glutathione S-transferase (GST)
tag–fused GSK2 (GSK2-GST)
proteins in Escherichia coli cells.
The coimmunoprecipitation analyss
were used to examine the
association of GSK2 and OML4 in
N. benthamiana. We coexpressed
GSK2-GFP and OML4-MYC in N.
benthamiana leaves. Total proteins
were isolated and incubated with
MYC beads to immunoprecipitated
OML4-MYC.
GSK2-GFP proteins were detected
in the immunoprecipitated OML4-
MYC complexes, indicating that
GSK2 associated with OML4 in
vivo

49. GSK2 Acts Genetically with OML4 to Regulate Grain Size
GSK2-RNAi lines showed longer and
slightly wider grains than ZHJ,
indicating that GSK2 predominantly
regulates grain length in rice
GSK2-RNAi spikelet hulls
contained longer and slightly
wider epidermal cells than ZHJ
spikelet hulls
cell number in the grain-length and grain-width
directions in GSK2-RNAi lemmas was similar to
that in the wild-type lemmas demonstrates that
GSK2 controls grain growth by limiting cell
elongation in spikelet hulls.
crossed large1-1 with GSK2-RNAi and
isolated large1-1;GSK2-RNAi plants. As
shown in Figure, the length of large1-1
grains was increased by 16.24% in
comparison with that of ZHJ, while the
length of large1-1;GSK2-RNAi grains was
increased by 7.90% compared with GSK2-
RNAi.
To investigate in detail the role of GSK2 in
grain size control, they downregulated the
expression of GSK2 using RNA interference
(RNAi; GSK2-RNAi)
These results suggest that GSK2 acts, at least in part,
in a common genetic pathway with OML4 to control
grain length

50. Conclusion
• MEI2-LIKE PROTEIN4 (OML4) encoded by the LARGE1 gene is phosphorylated by
GLYCOGEN SYNTHASE KINASE2 (GSK2) and negatively controls grain size and weight in
rice.
• Loss of function of OML4 leads to large and heavy grains, while overexpression of
OML4 causes small and light grains
• OML4 regulates grain size by restricting cell expansion in the spikelet hull
• Biochemical analyses showed that the GSK2 physically interacts with OML4 and
phosphorylates it, thereby possibly influencing the stability of OML4. Genetic analyses
support that GSK2 and OML4 act, at least in part, in a common pathway to control grain size
in rice
• Revealed the genetic and molecular mechanism of a GSK2-OML4 regulatory module in grain
size control, suggesting that this pathway is a suitable target for improving seed size and
weight in crops.

51. • Cell-size control in animals appears to be fundamentally different from that of yeast, whereas
plants, with their own unique cellular structures and lifestyle
• Identifying further genetic variants with altered ploidy and/or cell size and characterising
them more systematically will lead to a better understanding of cell-size control in plants.
• A main challenge for the future will be to reveal the molecular mechanisms of cell size
regulation in plants, the extent to which they include conserved strategies and genetic
pathways, and how they relate to plant-specific features such as vacuolar growth and growth
constrained by interconnected cell walls

52. References
• Wang, H., Dittmer, T.A and Richards, E.J. 2013. Arabidopsis CROWDED NUCLEI (CRWN) proteins are
required for nuclear size control and heterochromatin organization. BMC Plant Biology. 13:200.
• Marco D’Ario and Robert Sablowski. 2019. Cell Size Control in Plants. Annual Review of Genetics.
https://doi.org/10.1146/annurev-genet-112618-043602.
• Jones et al., 2019. Double or Nothing? Cell Division and Cell Size Control. Trends in Plant Science.
https://doi.org/10.1016/j.tplants.2019.09.005
• Keiko Sugimoto-Shirasu and Keith Roberts. 2003. ‘‘Big it up’’: endoreduplication and cell-size control in
plants. Current Opinion in Plant Biology. 6:544–553.
• Kurt M.Schmolle and Jan M.Skotheim. 2015. The Biosynthetic Basis of Cell Size Control . Trends in
Cell Biology. http://dx.doi.org/10.1016/j.tcb.2015.10.006

53. • Lyu, J., Wang, D., Duan, P., Liu., Huang, K., Zeng, D., Zhang, L., Dong, G., Li, Y., Xu, R., Zhang, B.,
Huang, X., Li, N., Wang, Y., Qian, Q and Li, Y. 2020. Control of Grain Size and Weight by the GSK2-
LARGE1/OML4 Pathway in Rice. The Plant Cell. Vol. 32: 1905–1918
• Marshall et al., 2012. What determines cell size? BMC Biology. 10:101.
http://www.biomedcentral.com/1741-7007/10/101
• Kondorosi, E., Roudier, F and Gendreau, E. 2000. Plant cell-size control: growing by ploidy? Current
Opinion in Plant Biology. 3:488–492
• Traas, J., Hülskamp, M., Gendreau, E and Höfte,H. 1998. Endoreduplication and development: rule
without dividing? Current Opinion in Plant Biology. 1:498–503