RES EARCH

PLANT SCIENCE Equal inheritance and dilution of KRP4

Dilution of the retinoblastoma (RB) protein or
Cell size controlled in plants using DNA content the functionally equivalent Whi5 protein links
the G1/S transition to cell size in mammals
as an internal scale and yeast, respectively (6, 14). In plants, RB is
conserved (17, 18) and its function in delaying
S-phase is promoted by Kip-related proteins
Marco D'Ario1, Rafael Tavares1, Katharina Schiessl1†, Bénédicte Desvoyes3, Crisanto Gutierrez3,
(KRPs), which inhibit D-type cyclins (19). Un-
Martin Howard2, Robert Sablowski1*
like in mammals and yeast, an Arabidopsis RB
reporter (RBR1-GFP) (20) was not diluted by
How eukaryotic cells assess and maintain sizes specific for their species and cell type remains unclear. growth of meristem cells; instead, we saw that
We show that in the Arabidopsis shoot stem cell niche, cell size variability caused by asymmetric concentration of a functional KRP4-GFP fusion
divisions is corrected by adjusting the growth period before DNA synthesis. KIP-related protein 4 (KRP4) (21) was negatively correlated with cell size (fig.
inhibits progression to DNA synthesis and associates with mitotic chromosomes. The F BOX-LIKE 17 S2). Moreover, a comparable KRP4-mCherry
(FBL17) protein removes excess KRP4. Consequently, daughter cells are born with comparable amounts reporter (fig. S3) was present in similar amounts
of KRP4. Inhibitor dilution models predicted that KRP4 inherited through chromatin would robustly in sister cells of equal size but was more con-
regulate size, whereas inheritance of excess free KRP4 would disrupt size homeostasis, as confirmed by centrated in the small sister cells of asymmetric
mutant analyses. We propose that a cell cycle regulator, stabilized by association with mitotic pairs (Fig. 2, A and C). Similar results were seen
chromosomes, reads DNA content as a cell size–independent scale. using KRP4-GFP and sister cell pairs assigned
based on cell geometry (fig. S4, A to F). Thus, we

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C
ell size affects cell physiology, signaling,
and tissue mechanics (1, 2). During pro-
liferation, cell size depends on the balance
between cell growth and cell division.
From bacteria to humans, feedback be-
tween these two processes enforces predicta-
ble and uniform cell sizes, counteracting the
individual variability introduced, for example,
by imprecise divisions. The molecular basis for
these feedbacks remains uncertain (2, 3).
Most proteins and cellular components ac-
cumulate in proportion to cell size and there-
fore cannot be used as internal standards to
measure growth (4–6). Several models for cell
size control are based on cell cycle regulators
that break this rule and do not scale linearly
with cell size, such as Whi5 in budding yeast
(6), CYCLIN-DEPENDENT KINASE G1 (CDKG1)
in Chlamydomonas (7), and Cell Division Cycle
25 (Cdc25) in fission yeast (8). Other models
suggest accumulation of regulators in structures
that do not scale with cell volume, as proposed
for binding of Cyclin 3 (Cln3) to promoters in
budding yeast (9) and accumulation of Cell
Division Response 2 (Cdr2) in the medial cortex
of fission yeast (10).
The plant cell cycle includes DNA synthesis
(S) and mitotic (M) phases separated by two gap
phases (G1 and G2), typical of eukaryotic cells.
Progression through the cell cycle is adjusted to
cell size in the shoot meristem, a self-renewing
structure that originates and patterns all new
aerial organs (11–13). As in budding yeast and
mammals (6, 14), meristem cell size is coordinated
with the G1/S transition (15), although G2 length
1
Cell and Developmental Biology, John Innes Centre, Norwich
NR4 7UH, UK. 2Computational and Systems Biology, John
Innes Centre, Norwich NR4 7UH, UK. 3Centro de Biología
Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, 28049
Madrid, Spain.
*Corresponding author. Email: robert.sablowski@jic.ac.uk
†Present address: Sainsbury Laboratory, Cambridge University,
Cambridge CB2 1LR, UK.
also responds to meristem cell size (13). Here, we
show that meristem cells link their size to the G1/S
progression using chromosomes as a template to
allow inheritance of a cell cycle regulator in a
quantity independent of cell size.
Size asymmetry is corrected in G1
Frequent asymmetric divisions introduce cell
size variability in the meristem (11, 12). If the
progression from G1 to S phase were linked to
cell size, then asymmetrically sized sister cells
should enter the S phase at different times. To
test this prediction, we monitored G1 progres-
sion in relation to cell size using a reporter based
on the prereplication factor CDC10 Target1
(CDT1)-like protein a (CDT1a), which increases
during G1 and drops at the G1/S transition (16)
(Fig. 1 and fig. S1D). Cell growth and prolifer-
ation were not adversely affected by imaging
over 48 hours (fig. S1, A to C). Live tracking of
CDT1a-CFP expression during this time showed
that larger sister cells went more rapidly through
G1, both in individual cell pairs (Fig. 1, E to H)
and at the population level (Fig. 1I). Mitosis
increased the coefficient of variation of cell
volumes (11), but by the G1/S transition, var-
iability returned to that seen just before mitosis
(Fig. 1J). Correction of variability might result
from faster growth of smaller sister cells, which
was seen after normalization to cancel local
growth heterogeneity (12), but we could not
detect a correlation between relative growth
rates and either birth size asymmetry or cell
size in general (fig. S1, F and G). Faster growth
of small sister cells could have been obscured
by noise in our data but would still have
gradually reduced size differences. However,
the volume coefficient of variation remained
constant over time after mitosis (Fig. 1K). We
conclude that meristem cells counteract size
variability caused by asymmetric divisions
primarily by linking the timing of progres-
sion through G1 to birth size.
hypothesized that asynchronous G1/S progres-
sion in sister cells could be mediated by an
asymmetric concentration of KRP4, which delays
progression to the G1/S transition.
KRP4 concentration would reflect size at
birth if sister cells had comparable numbers of
KRP4 proteins. A mechanism for equal distribu-
tion was apparent from the association of KRP4
with chromosomes during mitosis (Fig. 2B and
fig. S7) (22). Accordingly, total KRP4 did not
correlate strongly with the asymmetry of sis-
ter cell volumes (Fig. 2D and fig. S4G). Time
courses aligned at cell division showed that
the overall levels of KRP4-mCherry rose in the
last few hours before mitosis, continued to in-
crease in early G1, then declined to a minimum
at the G1/S transition (Fig. 2, B, E, and F).
Together, these results suggested a mechanism
in which equal inheritance of KRP4 associated
with chromosomes results in a higher KRP4
concentration at birth in smaller cells. Consid-
ering that nuclear volume increases in propor-
tion to cell size (fig. S5) (12), smaller cells would
then require a longer period of growth to di-
lute KRP4 sufficiently to overcome its inhibi-
tory effect on G1 progression.
To ensure equal inheritance, the accumu-
lation of KRP4 by the end of G2 should not
exceed the amount that can be carried on the
replicated chromatin. The levels of KRP pro-
teins are regulated by targeted proteolysis as
mediated by the F-box protein FBL17, which
physically interacts with KRP4 (23). A func-
tional FBL17-GFP fusion driven by the FBL17
promoter (23) was seen in nuclei with low
levels of KRP4-mCherry in cells within the size
range expected for G2 (Fig. 3, A and B). To test
whether FBL17 lowers KRP4 levels, we used
plants with FBL17 function inhibited by an
inducible microRNA (miRNA) (24) (fig. S6)
because homozygous fbl17 mutants can only
be recovered at a very low frequency (25). In
those that we could recover, the KRP4 reporters
appeared to be silenced beyond the seedling

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Fig. 1. Size-dependent G1/S pro-

gression in the shoot meristem.
(A to D) Tracking of cell growth and
division: Three-dimensional (3D)
confocal images of a representative
inflorescence meristem expressing
the plasma membrane marker
pUBQ10::acyl-YFP (12) at 0 (A) and
48 (C) hours, with corresponding
segmented images [(B) and (D)];
selected cells within 40 mm of
the meristem center in (D) are
shown in the same color as their
progenitors in (B). Scale bars,
50 mm. (E to H) Size-dependent
G1/S progression shown by
CDT1a-CFP expression in
symmetrical [(E) and (F)] and
asymmetrical [(G) and (H)] sister
cells. In (E) and (G), top panels

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show confocal images of
CDT1a-CFP expression (cyan
signal) in sister cells (circled) at
different time points, with the
corresponding segmented
images below. Scale bars, 50 mm.
In (F) and (H), volume (green
triangles) and CDT1a concentra-
tion (magenta circles) over
time for the sisters shown in (E)
and (G), respectively; arrowheads
indicate mitosis (M) and the
start of S-phase (G1-S). (I) Differ-
ence in CDT1a signal density
(nuclear signal divided by nuclear
volume) between larger and
smaller sister cells plotted against
asymmetry in sister volumes,
measured within 6 hours of mitosis
(271 sister pairs from four
meristems); green bars indicate
the 95% confidence interval for
the mean difference in each
interval. (J) Distribution of the
relative deviation from mean
volume and overall coefficient of
variance (CV) for cells just before
mitosis (blue), at birth (green),
and at G1/S (magenta); dark colors
correspond to the subset of cells
that could be tracked from M to
G1/S. (K) Boxplots and individual
CV values (four biological replicates) for cells just before mitosis (blue), at progressive times after birth (green), and at G1/S (magenta).

stage. Meristems with inhibited FBL17 had ab-
normally large cells (as seen in the fbl17 mutant;
Figs. 3, D and E, and 4) with excess KRP4-GFP
more evident in larger cells (Fig. 3, D and E).
miRNA inhibition of FBL17 also increased
KRP4-GFP in cells <150 mm3, which were ex-
pected to be in G1 and therefore not to express
FBL17, suggesting that excess KRP4 was inherited
from mother cells. The KRP4-GFP signal re-
mained comparable in mitotic chromosomes of
the wild-type and miRNA lines, implying that
excess KRP4 inherited through mitosis would
be diluted in the cytoplasm, although the expected
increase in concentration would be difficult to
detect (fig. S7). We conclude that FBL17 prevents
excessive accumulation of KRP4 during G2.
Quantitative models of KRP4 function
As described above, KRP4 inherited in equal
amounts on chromatin is expected to constrain
cell size variability at G1/S. Conversely, free
KRP4 would be transmitted to daughter cells
in proportion to their volume and should am-
plify size differences during exponential cell
growth, which occurs in the meristem (fig. S1E)

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(12). To predict the outcome of these opposing
effects, we initially used numerical simulations
with values for division asymmetry, cell growth
rates, and variation in KRP4 accumulation
sampled from experimental data (Fig. 4A,
fig. S8, and dataset S3). The simulations made
three predictions (Fig. 4B). First, if all inherited
KRP4 were chromatin bound, then population
cell sizes would be stable and proportional to
total KRP4 levels. Second, if cells inherited only
free KRP4, then cell sizes would be unstable
and spiral either up or down to ever smaller
cells. Third, if cells inherited both bound and
free KRP4, then bound KRP4 would buffer cell
size variability but could be overwhelmed by
excess free KRP4. These predictions follow
from the summarizing equation in Fig. 4A:
KRP4 bound to chromatin (magenta term)
made a constant contribution to cell volume at
G1/S (VS), whereas the contribution of KRP4
inherited in the nucleoplasm (green term) was
proportional to volume at birth (V0) and thus
could not correct size variation. Production of
KRP4 in early G1 did not scale up with cell size
and was comparable between sister cells (fig.
S4, H and I), so on average, the black term was
also independent of V0. The reason that size-
independent accumulation of KRP4 extended
from late G2 into early G1 remains unknown.
However, because most of the KRP4 pres-
ent in G1 was inherited from mother cells,
we consider that production of KRP4 in G2
has the primary role in linking G1 duration
to cell size.

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An assumption of our model is that KRP4
accumulates on chromosomes in late G2, whereas
the steady-state levels of free KRP4 are kept
low. This might happen if association with con-
densing chromosomes renders KRP4 inaccessible
to FBL17-promoted degradation. Another as-
sumption is that some of the chromatin-bound
KRP4 is released to the nucleoplasm during G1.
To understand how changes in the dynamics of
KRP4 accumulation and binding to chromatin
could generate the behavior described in Fig.
4A, we developed a mathematical model based
on differential equations for the association,
dissociation, production, and degradation of
KRP4 (Fig. 4C and fig. S8). Assuming that the
pool of chromatin-bound KRP4 is in quasi–
steady state with respect to the concentration
of free KRP4 and ignoring the contribution of
KRP4 synthesis and degradation in early G1
(which can be assumed to add a constant
value to VS; see the supplementary materials),
the model yields an equation for VS compa-
rable to the one used in the numerical sim-
ulations (fig. S8). Thus, our conceptual model
(Fig. 4A) can be derived from changes in KRP4
turnover and affinity for chromatin during the
cell cycle. The mathematical model also illus-
trated (Fig. 4C) how decreased dissociation
from chromatin could result in a rapid increase
in KRP4 at the end of the cell cycle (as seen
in Fig. 2E) and how a reduction of the KRP4
Fig. 2. Equal inheritance and subsequent dilution of a KRP4 reporter. (A and B) Optical sections degradation rate could approximately double
through meristems expressing KRP4-mCherry (magenta), CDT1a-CFP (green), and pUBQ10::acyl-YFP (white). the total amount of KRP4, with little effect on
In (A), note the higher intensity in the smaller sister of asymmetrical pairs (arrows) and equal signal in the chromatin-bound pool, as seen after in-
symmetrical pairs (asterisks). In the 4-hour time course in (B), arrows and dotted circles mark cells that hibition of FBL17 (fig. S7F).
went through mitosis and G1/S, respectively. Scale bars, 10 mm. (C and D) Difference between larger and
smaller sisters for KRP4-mCherry signal density (C) and total amount of signal (D) within 6 hours of mitosis Model predictions matched mutant
plotted against asymmetry in sister volumes (101 sister pairs from three meristems); green bars indicate phenotypes
95% confidence interval for the mean difference. (E) Volume (gray), KRP4-mCherry signal density (dark Both the numerical simulations and the math-
magenta), and total KRP4-mCherry signal (light magenta) for cells with their time courses aligned at ematical model made specific predictions
mitosis (101 sister pairs from three meristems); note the increase in total KRP4-mCherry during the last about the cumulative effects of altered KRP4
6 hours and first 6 hours of the cell cycle and the dilution of KRP4-mCherry starting around 16 hours after and FBL17 levels over multiple cell cycles. First,
birth. (F) Signal density for CDT1a-CFP (green) and KRP4-mCherry (magenta) for cells with their time a change in overall KRP4 levels should affect
courses aligned at G1/S (36 cells from five meristems). the average, but not the distribution, of cell

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sizes in the meristem (Fig. 4B, top panel). Ac-

cordingly, the loss-of-function krp4-2 mutant
had a slight reduction in cell sizes, whereas
overexpression resulted in very large cells;
however, in both cases, there was no obvious
increase in size variability (11) (fig. S9). The
modest effect of krp4-2 suggested genetic re-
dundancy; likely candidates were the closest
homologs KRP3 and KRP5, which are expressed
in the meristem and also bind to mitotic chro-
mosomes (22). To test this, we generated a krp3
loss-of-function allele by CRISPR-Cas9 muta-
genesis and saw that the krp4-2 krp3-11 double
mutant had a stronger reduction in cell size
but a size distribution similar to the wild type
(fig. S9), as predicted by the model. For krp5,
we could only obtain in-frame deletions, sug-
gesting that null mutants are lethal. These
results confirmed that KRP activity is limit-
ing for meristem cell size. Furthermore, if an

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additional, independent mechanism links G2/M
progression to cell size (13), then this mecha-
nism must not be based on a size threshold
because if it was, it would have masked the
effect of krp mutants.
The second prediction was that an increase
in nucleoplasmic KRP4 relative to chromatin-
bound KRP4 at the end of the cell cycle should
disrupt size homeostasis, with a distribution
skewed toward large sizes; this was confirmed
in the fbl17 mutant (Fig. 4, D to F). A caveat of
this experiment was that the fbl17 mutant has
pleiotropic effects on gametophyte develop-
ment and shoot growth (23, 25), and genetic
confirmation that the fbl17 cell size defects
required KRP function was precluded by the
likely lethality of full loss of function. However,
we could exclude indirect effects of FBL17 loss
of function. First, the abnormal, very large
cells in the fbl17 meristem were not quiescent
or differentiated; they were cytoplasmically
dense, as expected for meristematic cells, and
continued to divide (fig. S10). Second, large
cells did not arise from polyploidy caused
by defects in G1/S control: 4′,6-diamidino-2-
phenylindole (DAPI) staining showed that the
large cells had proportionately large nuclei but
dilute DNA, unlike control polyploid cells
(fig. S10).

Wider relevance of mechanism

Our results support a three-component model
in which KRP links cell cycle progression to
DNA content, whereas FBL17 ensures that KRP4
is transmitted to newborn cells predominantly
in association with chromosomes. Cellular DNA
content, discrete and predictable, has been
proposed as a molecular standard against
which chromatin-binding proteins are titrated,
for example, to set the number of cell cycles be-
fore the mid-blastula transition in animals
(26) or the number of mitoses during dark
conditions in Chlamydomonas (7). Here, we
show how the relevant molecules are loaded
Fig. 3. FBL17 is expressed during G2 and inhibits accumulation of a KRP4 reporter. (A) Optical section
through meristem showing nuclei expressing FBL17-GFP (top left), KRP4-mCherry (bottom right), and a
merged image (bottom left). The top right panel shows background signal from a meristem imaged with
FBL17-GFP settings but lacking the reporter. Scale bars, 10 mm. (B) Expression in relation to cell volume for
FBL17-GFP (green) (104 cells from five meristems) compared with CDT1a-CFP (magenta) (217 cells from one
meristem). (C and D) 3D reconstructions from confocal image stacks showing, respectively, KRP4-GFP
expression (green) in a control meristem and in a meristem with miRNA-inhibited FBL17 (see fig. S6). Cell
outlines were stained with FM4-64 (magenta signal) and images were taken with the same settings. Note the
stronger signal and larger cells in (D). Scale bars, 50 mm. (E) Boxplots showing total KRP4-GFP signal in cells
within different volume ranges from control plants (blue; 618 cells from three meristems) or after miRNA
inhibition of FBL17 (orange; 598 cells from three meristems).

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Fig. 4. Model for cell size control using chromatin contents as a ruler.
(A) Conceptual model. Each sister cell inherits half the amount of KRP bound
to chromatin in the mother cell (Nkc2) and the same concentration of free
KRP in nucleoplasm (rF). During G1, KRP is released from chromatin and
diluted by growth until a threshold concentration (rks) triggers the S phase
(indicated by duplicated chromatin). In G2, KRP is made again and associates
with chromatin, whereas FBL17-mediated proteolysis maintains rF low. The
equation describes how volume in the S phase (VS) depends on volume at
birth (V0) and on the variables above; the additional term Nk1/rks accounts
for the size-independent KRP4 synthesis in early G1. (B) Cell size distribution
in numerical simulations compared with the experimental (exp) size
distribution (984 cells from four meristems). Nkc2 is relative to the median
wild-type value and rF is relative to rkS. Cell division ratios and growth rates
were sampled from experimental data (details in fig. S8). Magenta: with low
rF, cell sizes are stable and proportional to Nkc2. Green: without Nkc2, size is
unstable and critically dependent on rF. Orange: with Nkc2 and rF combined,
Nkc2 stabilized cell sizes but could be overwhelmed by increasing rF.
(C) Simulation of KRP4 dynamics in G2. The diagram represents KRP4
binding and unbinding to chromatin, production, and degradation, with rate
constants b, g, f, and m, respectively; graphs show the number of chromatin-
bound (Nkc) and free (Nk) KRP4 molecules and cell volume (V) during G2,
calculated from the mathematical model explained in fig. S8. Starting conditions
were Nk2 – Nkc2 = Nc2 = 100 and V2 = 250 mm3. To simulate the wild type (top)
and fbl17 mutant (bottom), m (min−1) was set as indicated. In both, g (min−1) was
decreased at 120 min to simulate the effects of higher chromosome-bound
KRP4; the remaining parameter values were b = 1.0 mm3 min−1, f = 0.1 mm−3
min−1, and relative growth rate = 1.00025 min−1. (D) Distribution of the relative
deviation from mean volume and volume CV in wild type (blue) and fbl17
mutants (orange) measured in meristems (exp; 263 cells from three wild-type
meristems; 406 cells from seven fbl17 meristems) or from simulations (sim)
based on the mathematical model (see the supplementary materials) and run for
10 cell cycles with parameters set as in (C). (E and F) Segmented 3D images
of wild-type (E) and fbl17 meristems (F), with cells colored by volume (color bar
on the left). Scale bars, 50 mm.

in the cell in the right amount to correctly
read the DNA content: Regulated by FBL17,
KRP uses DNA as a template to accumulate to
a predictable amount, which then affects cell
cycle progression.
The plant-specific KRP proteins are com-
ponents of the widely conserved RB pathway
(17, 19). Equal inheritance of RB might link the
G1/S transition to mammalian cell size (14). Thus,
diverse organisms may have converged on anal-
ogous mechanisms for cell size control, albeit in-
volving different components of a conserved
pathway. How KRP proteins associate with
chromosomes and are protected from turnover
remains unclear; one possibility is that these
proteins could be sequestered within a chro-
mosome periphery domain such as that formed
by the Ki67 protein in late G2 and mitosis (27).
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ACKN OWLED GMEN TS
We thank D. Twell for FBL17-GFP and fbl17 seeds, H. Jönsson for
pUBQ10::acyl-YFP seeds, K. Tamura for NUP136-GFP seeds,
G. Calder and E. Wegel for microscopy support, and members of
the Sablowski laboratory for critical comments. Funding: M.D. was
supported by a John Innes Foundation studentship. R.S. and M.H.
were supported by a UK Biotechnology and Biological Sciences
Research Council Institute Strategic Programme Grant (BB/
J004588/1). R.T. received a European Commission Marie-Curie
Fellowship (838718). C.G. received grants from the European
Commission (ERC-2018-AdG_833617) and the Ministry of
Science and Innovation (RTI2018-094793-B-I00) and
institutional grants from Fundación Ramón Areces and Banco
de Santander to the Centro de Biologia Molecular Severo Ochoa.
Author contributions: M.D. designed and performed
experiments, analyzed data, and developed the mathematical
model. R.T. performed experiments. K.S., B.D., and C.G.
provided materials. M.H. supervised development of the
mathematical model. R.S. conceived and supervised the project,
performed experiments, analyzed data, and wrote the
manuscript with contributions from all authors. Competing
interests: The authors declare no competing interests. Data
and materials availability: All data are available in the main
text or in the supplementary materials. Source images (28),
image analysis (29), and simulation scripts (30) are available on
FigShare, along with instructions and supporting files. Materials
are available from R.S. upon request.
SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/372/6547/1176/suppl/DC1
Materials and Methods
Figs. S1 to S10
References (31–41)
Datasets S1 to S3
MDAR Reproducibility Checklist
View/request a protocol for this paper from Bio-protocol.
2 May 2020; resubmitted 16 March 2021
Accepted 6 May 2021
10.1126/science.abb4348
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Cell size controlled in plants using DNA content as an internal scale
Marco D'Ario, Rafael Tavares, Katharina Schiessl, Bénédicte Desvoyes, Crisanto Gutierrez, Martin Howard and Robert
Sablowski

Science 372 (6547), 1176-1181.

DOI: 10.1126/science.abb4348

Cell size set by cell cycle regulation

In the Arabidopsis meristem, cell sizes are regularized despite asymmetric cell divisions. D'Ario et al. describe a
balanced regulatory system that controls the duration of the growth phase of the cell cycle preceding DNA synthesis.
KIP-related protein 4 (KRP4) inhibits progression to DNA synthesis. Because the amount of KRP4, which binds to mitotic

Downloaded from http://science.sciencemag.org/ on August 23, 2021

chromosomes, is titrated to the amount of chromosomal DNA, daughter cells begin with similar amounts of KRP4 despite
possible asymmetric cell divisions. Deviations are adjusted as excess KRP4 is degraded and the cell size is normalized.
Science, abb4348, this issue p. 1176

ARTICLE TOOLS http://science.sciencemag.org/content/372/6547/1176

SUPPLEMENTARY http://science.sciencemag.org/content/suppl/2021/06/09/372.6547.1176.DC1
MATERIALS

REFERENCES This article cites 38 articles, 10 of which you can access for free
http://science.sciencemag.org/content/372/6547/1176#BIBL

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