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\begin{document}

\title{Tomato UVI4 and CCS52B play important roles in hypocotyl elongation via
modulation of cell cycle progression}



\author[1]{Shengqiang Di}%
\author[1]{Peng Zhang}%
\author[1]{Jiu-Cheng Zhang}%
\author[1]{Genzhong Liu}%
\author[1]{Fangfang Ma}%
\author[1]{Zhilong Bao}%
\affil[1]{Shandong Agricultural University}%


\vspace{-1em}



  
  \date{May 28, 2024}


\begingroup
\let\center\flushleft
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\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Tomato hypocotyl elongation is a consequence of active cell division and
expansion, both of which require precisely regulated cell cycle
progression. Little is known about the function of APC/C regulators in
cell cycle progression during hypocotyl growth. Here, we isolated and
characterized the positive and negative APC/C regulators, SlCCS52 and
SlUVI4 genes in tomato. We probed SlUVI4 and SlCCS52B roles in tomato
hypocotyl elongation via modulating cell cycle progression. Light
especially blue light represses the transcription of SlUVI4 and SlCCS52B
to inhibit the hypocotyl elongation through the suppression of
endoreduplication. MS basal salts and sugar both improve SlUVI4 and
SlCCS52B transcription to enhance hypocotyl length via the promotion of
endoreduplication. Hypocotyl elongation enhanced by heat might require
auxin-induced repression of SlUVI4 transcription. Salt inhibits
hypocotyl elongation possibly through ethylene-mediated modulation of
SlUVI4 and SlCCS52B transcription. Genetic studies reveal that tomato
deletion mutant of SlUVI4 and overexpression plants of SlCCS52B in
Arabidopsis both exhibit shorter hypocotyl with enhanced
endoreduplication. Thus, our results suggest that APC/C activities
stimulated by SlCCS52 genes requires SlUVI4-meidated inhibitory
machinery to reorchestrate cell cycle progression and facilitate
hypocotyl elongation.%
\end{abstract}%



\sloppy


\textbf{Tomato \emph{UVI4} and\emph{CCS52B} play important roles in
hypocotyl elongation via modulation of cell cycle progression}

Shengqiang Di\textsuperscript{\#}, Peng Zhang \textsuperscript{\#},
Jiucheng Zhang, Genzhong Liu, Fangfang Ma and Zhilong
Bao\textsuperscript{*}

State Key Laboratory of Crop Biology, College of Horticulture Science
and Engineering, Shandong Agricultural University, Tai-An, 271018,
Shandong, China

\textsuperscript{\#}These authors contributed equally.

\textsuperscript{*}Author for contact: zbao@sdau.edu.cn

Footnotes:

Author contributions: F.M. and Z.B. conceived and designed the
experiments. S.D., P.Z. and J.Z. performed most of experiments, and G.L.
provided technical assistance. S.D., P.Z., J.Z., F.M. and Z.B. analyzed
the data. S.D., F.M. and Z.B. wrote the manuscript.

Funding information: This work was supported by the National Natural
Science Foundation of China (31872951).

\textbf{Abstract}

Tomato hypocotyl elongation is a consequence of active cell division and
expansion, both of which require precisely regulated cell cycle
progression. Little is known about the function of APC/C regulators in
cell cycle progression during hypocotyl growth. Here, we isolated and
characterized the positive and negative APC/C regulators,
\emph{SlCCS52}and \emph{SlUVI4} genes in tomato. We probed \emph{SlUVI4}
and\emph{SlCCS52B} roles in tomato hypocotyl elongation via modulating
cell cycle progression. Light especially blue light represses the
transcription of \emph{SlUVI4} and \emph{SlCCS52B} to inhibit the
hypocotyl elongation through the suppression of endoreduplication. MS
basal salts and sugar both improve \emph{SlUVI4} and
\emph{SlCCS52B}transcription to enhance hypocotyl length via the
promotion of endoreduplication. Hypocotyl elongation enhanced by heat
might require auxin-induced repression of \emph{SlUVI4}
transcription\emph{.} Salt inhibits hypocotyl elongation possibly
through ethylene-mediated modulation of \emph{SlUVI4} and
\emph{SlCCS52B} transcription. Genetic studies reveal that tomato
deletion mutant of \emph{SlUVI4} and overexpression plants of
\emph{SlCCS52B} in Arabidopsis both exhibit shorter hypocotyl with
enhanced endoreduplication. Thus, our results suggest that APC/C
activities stimulated by \emph{SlCCS52} genes requires \emph{SlUVI4}
-meidated inhibitory machinery to reorchestrate cell cycle progression
and facilitate hypocotyl elongation.

\textbf{Keywords:} Tomato, hypocotyl elongation, UVI4, CCS52, APC/C,
cell cycle, light quality, heat, salt, plant hormones.



\textbf{Introduction}

Hypocotyl growth including elongation and thickening is important for
the emergence of tomato seedlings from soil and resistance to lodging.
In the dark, seedlings undergo skotomorphogenic development including
closed cotyledons, formation of an apical hook and elongated hypocotyls.
In the light, photomorphogenic development results in cotyledon
expansion, leaf development, photosynthesis initiation and reduced
hypocotyl elongation. Many mutants of light signaling components exhibit
defective hypocotyl growth. Loss-of-function mutant of
\emph{phyB}(\emph{Phytochrome B} ), the red-light receptor, has
elongated hypocotyls (de Lucas et al., 2008; Kim et al., 2016).
\emph{Phytochrome interacting factors} (\emph{PIFs} ) are transcription
factors downstream of light signaling to positively regulate hypocotyl
elongation, and monogenic\emph{PIF} mutants have shorter hypocotyls than
wild-type plants under light condition (Huq \& Quail, 2002; Kim et al.,
2003; Zhong et al., 2012). Ambient temperature also regulates hypocotyl
elongation. High temperature promotes hypocotyl elongation through the
increase of free IAA in Arabidopsis seedlings (Gray, Ostin, Sandberg,
Romano, \& Estelle, 1998). PIF4 promotes IAA biosynthesis, and is also
required for high temperature induced hypocotyl elongation (Franklin et
al., 2011; Koini et al., 2009; Stavang et al., 2009). PIF4 specifically
binds to the biologically active Pfr form of phyB, which results in the
degradation of PIF4 and prevents its transcription activity in the
hypocotyl elongation (Huq \& Quail, 2002; Park et al., 2012). PhyB-PIF
signaling module also regulates red light-induced hypocotyl elongation
under the warm temperature (Johansson et al., 2014). Blue light inhibits
Arabidopsis hypocotyl elongation requiring the function
of\emph{cryptochrome 1} (\emph{CRY1} ) and \emph{cryptochrome
2}(\emph{CRY2} ) (Ahmad \& Cashmore, 1993). Under blue light, CRY1
directly interacts with PIF4 to inhibit its transcription activity and
represses the hypocotyl elongation induced by high temperature (Ma et
al., 2016).

Plant hormones have diverse and strong impacts on hypocotyl growth.
Overproduction of auxin in plants promotes hypocotyl elongation (Boerjan
et al., 1995; Romano, Robson, Smith, Estelle, \& Klee, 1995; Zhao et
al., 2001). Chemical screens identify a new auxin analog named pro-2,4-D
which significantly promotes hypocotyl elongation (Savaldi-Goldstein et
al., 2008). Exogenous application of gibberellins promotes Arabidopsis
hypocotyl elongation under light via regulation of cellular elongation
(Cowling \& Harberd, 1999). Ethylene prevents the hypocotyl elongation
in the dark, while promotes hypocotyl elongation in the light (Bleecker,
Estelle, Somerville, \& Kende, 1988; Smalle, Haegman, Kurepa, Van
Montagu, \& Straeten, 1997). Brassinosteroid (BR) stimulates hypocotyl
elongation of pakchoi (\emph{Brassica chinensis} cv Lei-Choi) (T. W.
Wang, Cosgrove, \& Arteca, 1993) and BR signaling is required for
GA-induced hypocotyl elongation (Bai et al., 2012). Appropriate
endogenous level of Abscisic acid (ABA) is essential for tomato
hypocotyl elongation in the dark, which promotes the endoreduplication
in hypocotyl cells and inhibits cytokinin biosynthesis (Humplik et al.,
2015). Cytokinins inhibit hypocotyl elongation of Arabidopsis seedlings
in the dark, while induce hypocotyl elongation in light-grown
Arabidopsis plants via suppression of ethylene action or auxin transport
(Smets, Le, Prinsen, Verbelen, \& Van Onckelen, 2005).

Hypocotyl growth is species- and growth condition-dependent, resulting
from cell division and expansion (Gendreau et al., 1997; Raz \&
Koornneef, 2001; H. Wang \& Shang, 2020). In Arabidopsis, hypocotyl
growth is exclusively attributed to cell expansion (Boron \& Vissenberg,
2014), while both cell division and expansion exist in hypocotyls
of\emph{Helianthus annuus} (Kutschera \& Niklas, 2013). Accurate cell
cycle progression including mitotic cycle and endoreduplication results
in proper cell division and expansion during plant organ growth and
development (De Veylder, Beeckman, \& Inze, 2007; Sugimoto-Shirasu \&
Roberts, 2003). Cell cycle consists of four major phases, G1, S, G2 and
M, and the progression is governed by the activity of cyclin-dependent
kinases (CDKs)/cyclin protein complex, in which CDK is the catalytic
subunit and cyclin is the regulatory subunit (Inze \& De Veylder, 2006).
CDK inhibitors (CKIs) or Kip-related proteins (KRPs) directly bind to
the CDK/cyclin complexes to inhibit CDK activity (De Veylder et al.,
2001; Lui et al., 2000; Sherr \& Roberts, 1999; H. Wang, Fowke, \&
Crosby, 1997). SIAMESE(SIM)/SIAMESE RELATED (SMR) family proteins are
another type of inhibitors of CDK activity controlling endocycle onset
in Arabidopsis (Churchman et al., 2006; Kasili et al., 2010; Walker,
Oppenheimer, Concienne, \& Larkin, 2000). Cell cycle from G2 to M phase
requires E3 ubiquitin ligase complexes Anaphase-promoting
complex/cyclosome (APC/C) to degrade cyclins and inhibit CDK activity
(Fulop et al., 2005; Marrocco, Bergdoll, Achard, Criqui, \& Genschik,
2010; Peters, 2006; Sullivan \& Morgan, 2007). APC/C is a large protein
complex containing at least 11 different subunits referred as APC1 to
APC11, in which APC2 and APC11 are core components with catalytic
activities. APC/C has two types of activators, \emph{cell division cycle
20} (\emph{CDC20} ) and \emph{CELL CYCLE SWITCH52} (\emph{CCS52} )
(Fulop et al., 2005). Five \emph{CDC20} homologs (\emph{CDC20.1}
-\emph{CDC20.5} ) and three \emph{CCS52} homologs (\emph{CCS52A1} ,
\emph{CCS52A2} and \emph{CCS52B} ) were identified in Arabidopsis
genome. \emph{ULTRAVIOLET-B-INSENSITIVE4} (\emph{UVI4} )
and\emph{OMISSION OF SECOND DIVISION1} (\emph{OSD1} ) are two negative
regulators of APC/C, which have functional redundancy in the regulation
of female gametophyte development (Bao \& Hua, 2014; Heyman et al.,
2011; Iwata et al., 2011). Either \emph{UVI4} or
\emph{OSD1}overexpression leads to reduced plant size, lateral shoots
and enhanced disease resistance (Bao, Yang, \& Hua, 2013; Iwata et al.,
2011).

Studies on APC/C activities in tomato hypocotyl growth were seldom
reported. Here we accessed roles of the negative APC/C
regulator\emph{SlUVI4} and the positive regulator \emph{SlCCS52B} in
hypocotyl elongation via the modulation of cell cycle progression. High
expression levels of both genes correlated with the robust hypocotyl
elongation when seedlings grow under different lights or on the growth
medium supplemented with MS salts and sugar. Salt, heat and plant
hormones auxin and ethylene treatments affect their transcription
distinctly to modulate hypocotyl elongation via the perturbation of cell
cycle progression. Genetic studies revealed that
\emph{SlCCS52B}overexpression Arabidopsis plants and \emph{SlUVI4}
deletion mutants had defective hypocotyl elongation with enhanced
endoreduplication. Together, our study establishes cellular and genetic
connections between APC/C regulators, cell cycle progression and tomato
hypocotyl elongation.

\textbf{Materials and Methods}

\textbf{Plant materials and growth conditions}

Tomato cultivar `Heinz 1706', `Moneymaker' and `Ailsa Craig' seeds were
obtained from Tomato Genetics Resource Center
(\emph{https://tgrc.ucdavis.edu/}) and US National Plant Germplasm
System (\emph{www.ars-grin.gov/}) respectively, and propagated in the
greenhouse. Seeds were sterilized in 75\% alcohol for 1 minute and 8\%
NaClO solution for 15 minutes, and washed with autoclaved water for five
times, and planted on Murashige and Skoog (MS), non-MS (NMS) and MS with
sugar (MSS) medium plates. Dry seeds were directedly sown at the
different depth of soil mix containing 40\% organic matters. All
seedlings were grown in growth chamber at 25\selectlanguage{ngerman}°C with 12-hour dark and
12-hour light condition.

\textbf{Treatments with different lights, salt, heat and plant hormones}

For light treatment, sterilized tomato seeds planted on MS growth medium
were grown in red and blue light with the intensity of 100 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{-2} s\textsuperscript{-1} for 7 days, respectively.
For salt stress and plant hormone treatment, sterilized tomato seeds
were planted on MS growth medium supplemented with 150 mM NaCl or plant
hormones of different concentrations and grown for 7 days. For heat
treatment, sterilized tomato seeds were planted on MS growth medium and
grown at 33\selectlanguage{ngerman}°C for 7 days.

\textbf{Hypocotyl growth measurement}

Tomato seedlings grown under different conditions were photographed
first, and their hypocotyl length and diameter in the middle of
hypocotyl were measured by ImageJ software
(\emph{http://imagej.nih.gov/ij/}). Every experiment was independently
repeated at least twice.

\textbf{Cell Cycle analysis with flow cytometry}

Hypocotyls of more than 3 seedlings were collected for cell cycle
analysis. Samples were chopped in `Aru' buffer and filtered by the metal
mesh with 30\selectlanguage{greek}μ\selectlanguage{english}m pore size (Arumuganathan \& Earle, 1991; Bao, Zhang, \&
Hua, 2014). More than 5,000 nuclei in each sample were stained with
propidium iodide and measured in BD flow cytometer. Three biological
replicates were analyzed for each tomato variety. Endocycle index or the
cycle value were calculated to quantify the extent of endocycle using
the following formula (Bainard, Bainard, Henry, Fazekas, \& Newmaster,
2012; Barow \& Meister, 2003): EI = (\%4C nuclei x 1) + (\%8C nuclei x
2) + (\%16C nuclei x 3) + (\%32C nuclei x 4).

\textbf{Gene expression analysis}

Total RNA from hypocotyls was extracted by RNAisoPlus (TaKaRa, Otsu,
Japan, cat. \#) following the manufacturer's instruction. The first
strand complementary DNA (cDNA) synthesis using 2\selectlanguage{greek}μ\selectlanguage{english}g total RNA was
performed following instructions provided with HiScript III RT SuperMix
kit with gDNA wiper (Vazyme, cat. \#R323-01) for qPCR analysis and
HiScript III 1st Strand cDNA Synthesis Kit with gDNA wiper (Vazyme, cat.
\#R312-01) for full-length gene cloning. One microliter and 0.2\selectlanguage{greek}μ\selectlanguage{english}l of
cDNA templates were used the amplification of \emph{SlCBL1} gene by
RT-PCR to determine the quality of cDNA and qPCR analysis,
respectively.\emph{SlCBL1} was used as a reference gene for qRT-PCR data
normalization (Pombo et al., 2014). All primers were designed by on-line
tools (\emph{https://www.ncbi.nlm.nih.gov/tools/primer-blast/}) and
listed in Supplementary Table 1.

\textbf{Phylogenetic analysis}

Amino acid sequences of \emph{UVI4} and \emph{CCS52} genes in
Arabidopsis were obtained from the website (\emph{www.arabidopsis.org}),
and used for identification of their homologous genes in solanaceae
species by BLAST in the website (\emph{www.solgenomics.net}).
Phylogenetic analysis using amino acid sequences were performed in the
software MEGA X (Kumar, Stecher, Li, Knyaz, \& Tamura, 2018).

\textbf{Plasmid construction and plant transformation}

The coding sequence of each gene was first cloned into pDONR221 vector
by BP reaction following the manufacture's instruction (Life
Technologies, cat. \#11789-020). These entry vectors were recombined
into destination vectors by LR reactions (Life Technologies, cat.
\#11791-020). For Crispr-cas9 constructs, two guide RNAs, target 1 and
2, or Target 3 and 4 were designed following the instruction in the
website \emph{www.skl.scau.edu.cn}and included in primers for PCR
amplification (Xie et al., 2017). DNA fragments amplified using the
plasmid pCBC-DT1T2.2 as a template were ligated into pG3K-U6SC vector
using Golden Gate Assembly method, and all constructs were transformed
into \emph{Agrobacterium} strains LBA4404 harboring the helper plasmids
pVS1-VIR2 (Zhang et al., 2019). Transgenic Arabidopsis seedlings with
overexpression constructs were generated through\emph{Agrobaterium}
-mediated floral dipping method (Clough \& Bent, 1998). Transgenic
tomato plants using cultivar `Heinz 1706' were generated
through\emph{Agrobaterium} -mediated transformation as previously
described (H. J. Sun, Uchii, Watanabe, \& Ezura, 2006).

\textbf{Yeast two hybrid and bimolecular fluorescence complementation
assay}

For Y2H analysis, all cDNAs of \emph{SlUVI4} and \emph{SlCCS52s} were
cloned into pDEST-GADT7 and pDEST-GBKT7 (Rossignol, Collier, Bush, Shaw,
\& Doonan, 2007) and assays were performed following the user manual of
Matchmaker Gold Yeast Two-Hybrid System (Clontech Laboratories, Inc.).
For BiFC analysis, \emph{SlUVI4} cDNA was cloned into the destination
vector pSPYNE-35S GW and cDNAs of \emph{SlCCS52} genes were cloned into
the destination vector pSPYCE-35S GW (Walter et al., 2004). These
vectors were transiently transformed into onion cells via Agrobacterium
tumefacien strain GV3101 together with gene silencing inhibitor P19
(Schutze, Harter, \& Chaban, 2009; W. Sun, Cao, Li, Zhao, \& Zhang,
2007). GFP signals were detected using Nikon fluorescence microscope
ECLIPSE Ni-U.

\textbf{Data analysis}

Data were recorded and analyzed in Excel, and statistical analyses were
performed in Data Processing System (DPS) software (Tang \& Zhang,
2013). Means, standard errors and standard deviations of different
replicates were calculated in Excel. Least significance difference test
(LSD) was performed in DPS software to determine the statistical
difference with \emph{p} value less than 0.05.

\textbf{Results}

\textbf{Proper sowing depth is required for successful emergence of
tomato seedlings from soil.}

We investigated effects of the sowing depth on tomato seed germination
and hypocotyl elongation, both of which are important for tomato
seedling production. We observed that the seedling emergence from soil
was significantly reduced by 38\% and 50\% at the sowing depth of 4 and
5 cm compared to 2 and 3 cm, respectively (Figure 1A, B). We analyzed
effects of the sowing depth on the seed germination. Germination rates
of seeds sown at 4 and 5 cm deep soil reduced by 22\% and 33\% compared
to that at 2 and 3 cm deep soil (Figure 1C), suggesting that reduction
of seed germination in deep soil is one of factors influencing the
seedling emergence from soil. We further analyzed hypocotyl elongation
defects of tomato seedlings sown at 4 cm deep soil. Hypocotyls elongated
to various directions due to large resistance, some of which may not be
able to reach the surface of soil (Figure 1D). Taken together, these
data indicated that both seed germination and hypocotyl elongation are
significantly affected by the sowing depth, which results in dramatical
reduction of seedling emergence from soil and the ultimate yield of
plants.

\textbf{Identification of APC/C regulators in tomato}

We isolated tomato homologs of APC/C regulators through BLAST analysis
in the website (www.solgenomics.net). Tomato has only one
\emph{UVI4}homologous gene \emph{Solyc10g080400} in the genome referred
as\emph{SlUVI4} , which is different with other plant species such as
Arabidopsis, rice, maize, strawberry, etc. We further
identified\emph{UVI4} homologous genes in other Solanaceae species, and
revealed that pepper, potato and eggplant have one \emph{UVI4} homolog,
and tobacco and petunia have four and three homologs in the draft
genome, respectively (Supplementary Figure S1A). Similar to Arabidopsis,
tomato has three \emph{CCS52} homologous genes referred as
\emph{SlCCS52s} including \emph{Solyc08g080080}(\emph{SlCCS52A1} ,
previously named as \emph{SlCCS52A} ),\emph{Solyc12g056490}
(\emph{SlCCS52A2} ) and \emph{Solyc06g043150}(\emph{SlCCS52B} ) and four
\emph{CDC20} homologous genes referred as\emph{SlCDC20s} including
\emph{Solyc03g096870} (\emph{SlCDC20.1} ),\emph{Solyc08g005420}
(\emph{SlCDC20.2} ), \emph{Solyc06g072830}(\emph{SlCDC20.3} ) and
\emph{Solyc03g095210} (\emph{SlCDC20.4} ) (Supplementary Figure S1B).
\emph{SlCCS52A2} is newly isolated in tomato genome, and its amino acid
sequence has 83.05\% and 57.75\% identity with SlCCS52A1 and SlCCS52B,
respectively (Figure S2). We analyzed subcellular localizations of
SlUVI4 and SlCCS52 proteins, and revealed that all of them have nuclear
localization (Figure S3). SlCCS52A1 also has strong plasma membrane
localization, and SlCCS52B has weak cytoplasmatic localization. We
further tested the interactions between SlUVI4 and SlCCS52 proteins, and
both yeast-two hybrid and bimolecular fluorescence complementation assay
(BIFC) revealed that SlUVI4 could interact with SlCCS52A1 and SlCCS52A2,
but not with SlCCS52B (Figure 2). All these data suggest that
\emph{SlUVI4} and \emph{SlCCS52} genes might have similar functions as
those in Arabidopsis.

\textbf{Spatial and temporal expression of \emph{SlUVI4}
and\emph{SlCCS52} genes in tomato hypocotyls}

To understand the function of APC/C in tomato growth and development, we
measured transcript abundance of \emph{SlUVI4} and \emph{SlCCS52s} using
quantitative reverse transcription--polymerase chain reaction
(qRT-PCR).\emph{SlUVI4} had the highest expression level in stem and
young fruit, and moderate expression in fully expanded leaf and ovary
(Figure 3A).\emph{SlCCS52A1} had high expression levels in most organs
except root and stamen, and \emph{SlCCS52A2} had relatively low
expression levels in root, petal, sepal and young fruit (Figure 3B, C).
\emph{SlCCS52B} had similar tissue-specific expression patterns to
\emph{SlUVI4} (Figure 3D). These data suggested that \emph{SlCCS52}
genes are functionally redundant in the regulation of tomato organ
growth and development. We then analyzed the transcription of
\emph{SlUVI4} and \emph{SlCCS52}genes in hypocotyls of day 1, 3, 5 and 7
after seed germination. All genes were highly expressed at day 1
followed by dramatically reduced transcripts afterwards (Figure 3E). The
transcription of\emph{SlCCS52A1} and \emph{SlCCS52A2} did not change at
day 3 and 5, and had slight reduction and increase at day 7 (Figure 3E).
The transcription of \emph{SlUVI4} and \emph{SlCCS52B} had significant
reductions at day 5 and 7 compared to day 3. Meanwhile, we measured the
transcript abundances of APC/C core genes, \emph{SlAPC2}
and\emph{SlAPC11} . Both \emph{SlAPC2} and \emph{SlAPC11} had dramatic
reduction of transcript abundances at day 3, and restored the
transcription afterwards (Figure S4). Taken together, these data
indicate that APC/C activity is differentially regulated by\emph{SlUVI4}
and \emph{SlCCS52} genes in various tissues, and\emph{SlUVI4} and
\emph{SlCCS52B} may have more specific roles in the regulation of
hypocotyl elongation.

\textbf{Lights repress hypocotyl elongation through}\emph{SlUVI4}
\textbf{and\emph{SlCCS52B} mediated cell cycle progression.}

To investigate light effects on tomato hypocotyl growth, we measured
hypocotyl length and diameter, and cell cycle progression in hypocotyl
cells of cultivar `Heinz 1706' under dark and different light
conditions. The hypocotyl length was significantly longer under dark
condition than white light condition, while the hypocotyl diameter was
significantly thicker under white light condition (Figure S5A-C, E-G).
Cell cycle progression in hypocotyl cells was consistent in the dark,
resulting in relatively steady endoreduplication indices (EIs) (Figure
S5D). Under white light condition, cell cycle progression in hypocotyl
cells varied at different days, in which the proportion of 2C nuclei
increased gradually after day 1 resulting in reduction of EI values
(Figure S5H). Overall, EI values in the white light were smaller than
that in the dark, which correlated with hypocotyl elongation. We then
measured transcript abundances of \emph{SlUVI4} and \emph{SlCCS52} , and
all of them had highest transcription at day 1 under both dark and light
conditions (Figure S6). The transcription of \emph{SlUVI4}
and\emph{SlCCS52B} gradually reduced during the hypocotyl elongation
under light condition, which correlated with cell cycle progression. We
further investigated effects of light qualities on hypocotyl growth,
cell cycle progression and transcription of \emph{SlUVI4}
and\emph{SlCCS52} genes. The hypocotyls under red light were
significantly longer than that under blue light whereas diameters of
hypocotyls gradually increased under both light conditions (Figure 4A-C,
E-G). The proportion of 2C nuclei gradually increased under both light
conditions resulting in reduction of EI values, which was more
pronounced under red light condition (Figure 4D, H). qRT-PCR analyses
revealed that the transcription of \emph{SlUVI4} and \emph{SlCCS52B}
gradually reduced under both light conditions, which was parallel to
changes in cell cycle progression (Figure 4I). Their transcript
abundance accumulated more under red light than blue light condition,
which correlated with longer hypocotyls under red light condition. We
observed the reduction of\emph{SlCCA52A1} not \emph{SlCCS52A2}
transcription during the hypocotyl growth, and \emph{SlCCS52A1}
expression was slightly higher under red light, suggesting weak roles of
\emph{SlCCS52A1} in hypocotyl elongation under different light
conditions. All these data above suggest that\emph{SlUVI4} and
\emph{SlCCS52B} play important roles in hypocotyl elongation through the
regulation of cell cycle progression.

\textbf{MS salts and sugar} \textbf{enhance hypocotyl elongation
through\emph{SlUVI4} and \emph{SlCCS52B} mediated cell cycle
progression.}

To further understand roles of \emph{SlUVI4} and \emph{SlCCS52B} in
tomato hypocotyl elongation, we checked whether their expressions were
correlated with hypocotyl elongation under Murashige and Skoog (MS)
salts and sugar treatments. Without MS salts in the growth medium
(non-MS, NMS), the average hypocotyl lengths were 8.5, 18.6, 21.9 and
25.4 mm, and the average diameters of hypocotyls were 0.62, 0.76, 0.69
and 0.80 mm at day 1, 3, 5 and 7 (Figure 5A, B, C). With MS salts in the
growth medium (MS), the average hypocotyl lengths were 9.5, 31.3, 35.9
and 39.2 mm, and the average diameters of hypocotyls were 0.76, 0.92,
0.81 and 0.89 mm at day 1, 3, 5 and 7 (Figure 5E, F, G). With MS salts
and sugar both in the growth medium (MSS), the average hypocotyl lengths
were 5.3, 28.1, 37.7 and 49.2 cm, and the average diameters of
hypocotyls were 0.72, 0.89, 0.87 and 1.06 mm at day 1, 3, 5 and 7
(Figure 5I, J, K). These data above indicated that addition of MS salts
with or without sugar significantly promoted hypocotyl elongation and
thickening. Cell cycle analyses revealed that 16C nuclei only appeared
in seedlings grown on MS and MS with sugar growth medium (Figure 5H, L),
which resulted in the increase of EI values compared to that on non-MS
medium (Figure 5D). In fact, addition of MS salts with sugar
significantly increased abundances of 8C and 16C nuclei leading to
higher EI values. qRT-PCR analyses of transcription of \emph{SlUVI4} and
\emph{SlCCS52} genes in hypocotyls at day 7 showed that MS with or
without sugar had highest and moderate transcriptions of\emph{SlUVI4}
and \emph{SlCCS52B} , and only MS with sugar could enhance the
transcription of \emph{SlCCS52A1} and \emph{SlCCS52A2} (Figure 5M). All
these data suggested that MS salts with or without sugar promoted
hypocotyl elongation possibly through \emph{SlUVI4} and
\emph{SlCCS52B}coordinated cell cycle progression.

\textbf{Heat promotes hypocotyl elongation through auxin-mediated
transcriptional modulation of \emph{SlUVI4} and \emph{SlCCS52B}.}

We increased incubation temperature during the emergence of tomato
seedlings, and observed that 33\selectlanguage{ngerman}°C treatment significantly promoted
hypocotyl elongation of cultivar `Heinz 1706' by 1.6- and 1.3-fold at
NMS and MS growth medium compared to the normal growth temperature 25°C,
but did not affect the hypocotyl thickness (Figure 6A, B, C). Cell cycle
analysis revealed that 33°C treatment improved endoreduplication in
hypocotyl cells compared to 25°C resulting in increases of EI values
from 0.85 and 0.94 at 25°C to 0.99 and 1.00 at 33°C on NMS and MS growth
medium, respectively (Figure 6D). Heat induced promotion of
endoreduplication was more pronounced at NMS medium than MS medium,
which was in accord with the rate of improvement on hypocotyl
elongation. We also measured hypocotyl growth and cell cycle progression
of tomato cultivars `Moneymaker' and `Alisa Craig', and observed the
same phenomena on hypocotyl elongation and endoreduplication after high
temperature treatment except a slight decrease of hypocotyl diameter of
`Alisa Craig' grown on MS medium at 33°C comparted to that at 25°C
(Figure S7). qRT-PCR analysis revealed that 33°C treatment significantly
inhibited the transcription of\emph{SlUVI4} , but did not affect
\emph{SlCCS52B} transcription. The transcription of \emph{SlCCS52A1} was
not affected by 33°C treatment, while the transcript abundance of
\emph{SlCCS52A2} had a slight increase suggesting a weak role in the
regulation of endoreduplication (Figure 6E).

To investigate functions of plant hormones in heat treatment, we firstly
applied different concentrations of auxin (IAA), brassinosteroids (BRs),
gibberellins (GAs) and ethylene precursor
1-aminocyclopropane-1-carboxylic acid (ACC) in growth medium to analyze
hypocotyl growth. We observed slight enhancement of hypocotyl elongation
responding to low concentration of IAA (Figure 7A-C; Figure S8A, B). BR
and GA did not have consistent effects on hypocotyl growth (Figure
S8C-F). Higher concentration of ACC had more significant effects on
hypocotyl elongation and thickening (Figure S8G, H). Cell cycle analysis
in hypocotyls revealed no significant difference between the control and
0.1nM IAA treatment (Figure 7D). qRT-PCR analysis showed
that\emph{SlUVI4} transcriptions were significantly reduced,
and\emph{SlCCS52B} transcription had a slight reduction without
statistical significance (Figure 7E), suggesting that reduced
\emph{SlUVI4}transcription and steady \emph{SlCCS52B} transcription
might be required to maintain endoreduplication in auxin induced
hypocotyl elongation. Both \emph{SlCCS52A1} and \emph{SlCCS52A2}
transcription were significantly reduced suggesting that they might not
be as important as\emph{SlCCS52B} in hypocotyl elongation. All the data
above suggested that elevated temperature promoted hypocotyl elongation
possibly due to auxin-mediated repression of \emph{SlUVI4} transcription
and maintenance of \emph{SlCCS52B} transcription.

\textbf{Salt stress represses hypocotyl elongation possibly through
ethylene-mediated modulation of \emph{SlUVI4} and
\emph{SlCCS52B}transcription}

We investigated tomato hypocotyl growth under salt stress. NaCl with the
concentration of 150 mM in the MS medium significantly inhibited the
hypocotyl elongation by 39.3\% compared to the control, but did not
affect the hypocotyl thickening (Figure 8A-C). Cell cycle analysis
revealed that the proportion of 2C and 4C nuclei reduced, and 8C and 16C
dramatically increased which led to the increase of EI value from 1.01
to 1.19 (Figure 8D). Salt stress significantly promoted \emph{SlUVI4}
,\emph{SlCCS52A1} and \emph{SlCCS52A2} transcription, and
inhibited\emph{SlCCS52B} transcription (Figure 8E). To determine the
role of ethylene under salt stress condition, we applied 10\selectlanguage{greek}μ\selectlanguage{english}M ACC in the
growth medium and measured hypocotyl growth, cell cycle progression, and
the transcription of \emph{SlUVI4} and \emph{SlCCS52B} . The hypocotyl
length was reduced by 38.2\% and diameter was increased by 17.0\% after
10\selectlanguage{greek}μ\selectlanguage{english}M ACC treatment (Figure 9A-C). The proportion of 2C and 4C nuclei
decreased but 8C and 16C dramatically increased in ACC treatment, which
resulted in a higher EI value (Figure 9D). qRT-PCR analysis revealed
that the \emph{SlUVI4} transcript abundance increased by 32.3\%
and\emph{SlCCS52B} transcript abundance decreased by 36.3\% in ACC
treatment, whereas \emph{SlCCS52A1} and \emph{SlCCS52A2} transcription
were not altered (Figure 9E). Collectively, these data above suggested
that salt stress might trigger ethylene production which disrupted the
balance between \emph{SlUVI4} and \emph{SlCCS52B} to reprogram cell
cycle progression and eventually lead to the alteration of hypocotyl
elongation.

\textbf{Mis-regulation of \emph{SlUVI4} and \emph{SlCCS52B} leads to
repressed hypocotyl elongation.}

To further determine the role of \emph{SlUVI4} and \emph{SlCCS52B}played
in hypocotyl elongation, we generated \emph{sluvi4} mutants using
Crispr-Cas9 technology and \emph{SlCCS52B} overexpression lines in
Arabidopsis. We obtained several mutants with different mutation types.
Tomato plants with mutations in the second exon of \emph{SlUVI4} at T1
generation could not set up fruits, and we conducted reciprocal crosses
with wild type plants `Heinz 1706' and did not get success suggesting
that the second exon is pivotal for \emph{SlUVI4} function.
Interestingly, all of mutants with the capability of fruiting only had
mutations in the first exon, and the number of missing nucleotides is a
multiple of 3, resulting in truncated proteins of SlUVI4 (Figure S9).
Mutants of \emph{sluvi4 cr-1} and \emph{sluvi4 cr-2} lost 6 and 42
nucleotides encoding 2 and 14 amino acids, respectively. Both mutants
exhibited significantly shorter hypocotyls than wild type,
and\emph{sluvi4 cr-1} phenotype was weaker than that of \emph{sluvi4
cr-2}(Figure 10 A, B). Both mutants had thicker hypocotyls than wild
type (Figure 10C). Cell cycle analysis revealed that \emph{sluvi4}
mutants had enhanced endoreduplication indicated by more 8C and 16C and
a higher EI (Figure 10D). We further investigated three independent
overexpression lines of \emph{SlCCS52B} in Arabidopsis, which had
significantly shorter hypocotyls compared to wild type (Figure 11). Cell
cycle analysis revealed that overexpression of \emph{SlCCS52B} also
promoted endoreduplication in hypocotyl cells compared to wild type.
Meanwhile, we obtained overexpression lines of \emph{SlCCS52A2} in
tomato, and three independent lines exhibited shorter hypocotyls without
the alteration of cell cycle progression (Figure S10). All these data
indicated that mis-regulation of \emph{SlUVI4} and
\emph{SlCCS52B}affects tomato hypocotyl elongation through the
modulation of cell cycle progression.

\textbf{Discussion}

Accurate cell cycle progression is important for hypocotyl elongation
and thickening, which requires precisely mediated APC/C activities.
Several positive regulators of APC/C, \emph{SlCDC20} and
\emph{SlCCS52}genes were previously identified based on their homologous
sequences to Arabidopsis, Medicago and human homologs (Mathieu-Rivet,
Gevaudant, Sicard, et al., 2010). In this study, we isolated the
negative regulator\emph{SlUVI4} and new positive regulators,
\emph{SlCDC20.3} ,\emph{SlCDC20.4} and \emph{SlCCS52A2} using published
tomato genome sequence in solgenomics website (Tomato Genome, 2012).
Both\emph{SlUVI4} and \emph{SlCCS52B} had specifically high expressions
in stems. Further analyses revealed that their transcript abundances
correlated with cell cycle progression and hypocotyl elongation under
different light conditions, and in MS salts and sugar treatments. They
responded differently to exogenous hormones and environmental stresses
to modulate cell cycle progression and hypocotyl elongation. Their
overexpression lines in Arabidopsis and \emph{sluvi4} mutants had
altered cell cycle progression and hypocotyl elongation. Therefore, we
draw the conclusion that \emph{SlUVI4} and \emph{SlCCS52B} coordinated
cell cycle progression play important roles in the regulation of
hypocotyl elongation.

UVI4 proteins are unique and conserved in the plant kingdom (d'Erfurth
et al., 2009). Like most diploid Solanaceae species, tomato has only
one\emph{UVI4} gene in the genome indicating its central role in plant
growth and development. In Brassicaceae species, a genome duplication
brings about two homologous genes, \emph{UVI4} and \emph{OSD1} , which
could be clearly distinguished via sequence similarity analysis (Mieulet
et al., 2016). \emph{UVI4} and \emph{OSD1} mainly regulate mitosis and
meiosis respectively, and knockout of both genes leads to the female
gametophyte lethality (Bao \& Hua, 2014; Hase, Trung, Matsunaga, \&
Tanaka, 2006; Heyman et al., 2011; Iwata et al., 2011). Here we
presented results on tomato homologue \emph{SlUVI4} in the regulation of
cell cycle progression during hypocotyl elongation, while its roles in
the gametophyte development require further studies. \emph{CCS52} genes
are activators of APC/C to promote the onset and progression of
endoreduplication in plant organs (Cebolla et al., 1999; Vanstraelen et
al., 2009). In tomato, only \emph{SlCCS52A1} (previously as\emph{CCS52A}
) was reported to play important roles in fruit development through the
regulation of endoreduplication (Mathieu-Rivet, Gevaudant, Sicard, et
al., 2010). In Arabidopsis, UVI4 negatively regulates APC/C activity
through the interaction with CDC20 and CCS52 proteins. SlUVI4 can
interact with SlCCS52A1 and SlCCS52A2, but not with SlCCS52B. Whether or
not SlUVI4 can interact with the conserved domain of SlCCS52B remains to
be further explored. And here we want to emphasize that the similar
spatial and temporal expression patterns of \emph{SlUVI4}
and\emph{SlCCS52B} imply important roles of their coordination in the
regulation of tomato organ growth and development.

Hypocotyl elongation is based on cell division and expansion, which are
maintained by APC/C activities. Based on our observations, the highest
expression of \emph{SlCCS52} genes right after the seed gemination
results in the most robust APC/C activities to promote hypocotyl
elongation, which consequently ensures the successful emergence from
soil. Down-regulation of \emph{SlCCS52A1} transcription led to the
reduced plant heights and fruit size with decreased endoreduplication
(Mathieu-Rivet, Gevaudant, Sicard, et al., 2010). Overexpression
of\emph{SlCCS52A1} in tomato resulted in the promotion of
endoreduplication and abnormal growth phenotypes including impaired root
and leaf growth (Mathieu-Rivet, Gevaudant, Cheniclet, Hernould, \&
Chevalier, 2010; Mathieu-Rivet, Gevaudant, Sicard, et al., 2010).
Interestingly, overexpression lines of \emph{SlCCS52B} in Arabidopsis
generated in this study also had shorter hypocotyls with enhanced
endoreduplication (Figure 11). Therefore, excessive APC/C activities
triggered by overexpression of \emph{SlCCS52} genes
requires\emph{SlUVI4} -mediated inhibition machinery to fine-tune the
APC/C activities and facilitate plant growth and development. In this
case, tomato hypocotyl might grow faster when both \emph{SlUVI4}
and\emph{SlCCS52B} have higher transcript abundances, which proved to be
true when tomato seedlings grew under different lights and in MS salts
and sugar treatments. Light promotes cell division by activating
photoreceptors to suppress cell division inhibitors (Okello, de Visser,
Heuvelink, Marcelis, \& Struik, 2016). Light inhibits the transcription
of \emph{KRP1} , a CDK inhibitor, and activates cell cycle genes such
as\emph{CDKB1;1} , \emph{CDKB1;2} and \emph{CYCA2;2} (Lopez-Juez et al.,
2008). Under light conditions, the transcription of \emph{SlUVI4}
and\emph{SlCCS52B} is growth-dependent, and higher in younger
hypocotyls. Their reduced transcription during the hypocotyl growth
correlated with enhanced cell division suggesting their coordination in
endoreduplication.

Exogenous application of ethylene precursor ACC stimulates cambial cell
division in \emph{Populus} (Love et al., 2009), which may be the reason
of increased tomato hypocotyl thickness after ACC treatment (Figure 9C).
Application of ACC promotes \emph{SlUVI4} transcription, whereas
represses \emph{SlCCS52B} transcription, which collectively results in
the active cell division. However, we observed promotion of
endoreduplication in hypocotyl cells, which is due to ethylene-induced
DNA synthesis without cytokinesis through unknown mechanisms (Dan,
Imaseki, Wasteneys, \& Kazama, 2003). We observed the similar phenomena
of hypocotyl elongation, cell cycle progression and transcriptions
of\emph{SlUVI4} and \emph{SlCCS52B} under salt stress condition,
suggesting that ethylene signaling is involved in salt responses.
Ethylene drastically promotes hypocotyl elongation in nutrient-starved
Arabidopsis seedlings under light condition, and might be a mediator of
auxin-induced hypocotyl elongation (Smalle et al., 1997). High
temperature promotes Arabidopsis hypocotyl elongation due to enhanced
auxin synthesis or catabolism (Gray et al., 1998), which results in
enhanced tomato hypocotyl elongation as well. High temperature induced
promotion of endoreduplication in hypocotyl cells might be attributed to
enhanced ethylene biosynthesis mediated by auxin (Kang, Newcomb, \&
Burg, 1971), which correlates with the repressed and promoted
transcription of \emph{SlUVI4} and \emph{SlCCS52A2} in our study,
respectively. However, the regulation machinery of \emph{SlUVI4}
and\emph{SlCCS52A2} transcription by high temperature remains to be
further studied.

The deletion mutant of \emph{SlUVI4} and overexpression lines
of\emph{SlCCS52B} both have promoted endoreduplication in hypocotyl
cells, indicating their antagonistic roles in cell cycle progression as
the function of their homologues previously reported in Arabidopsis (Bao
\& Hua, 2014; Heyman et al., 2011; Iwata et al., 2011).
\emph{SlCCS52B}function remains to be further studied using tomato
mutants. Hypocotyl elongation is dependent on the active cell division
and consequent cell expansion, which are regulated by \emph{SlUVI4} and
\emph{SlCCS52B} , respectively. Based on our study, we proposed a
possible model of\emph{SlUVI4} and \emph{SlCCS52B} coordinated cell
cycle progression (Figure S11). Under normal growth condition, light
suppresses\emph{SlUVI4} and \emph{SlCCS52B} coordinated cell cycle
progression to inhibit hypocotyl elongation. MS salts and sugar promote
\emph{SlUVI4}and \emph{SlCCS52B} coordinated cell cycle progression to
improve hypocotyl elongation. Under stress condition, salt stress
triggers ethylene-mediated repression of \emph{SlUVI4} and
\emph{SlCCS52B} to inhibit hypocotyl elongation. Heat stimulates auxin
and its mediated ethylene production to modulate \emph{SlUVI4} and
\emph{SlCCS52B}function to promote hypocotyl elongation.

\textbf{Acknowledgements}

This research was supported by Startup funding from Shandong
Agricultural University for Dr. Zhilong Bao and National Natural Science
Foundation of China (31872951). We thank Tomato Genetics Resource Center
(TGRC) to provide tomato seeds. We thank Tsuyoshi Nakagawa from Shimane
University, Sumie Ishiguro from Nagoya University and Dr. Qijun Chen
from China Agricultural University to provide gateway destination
vectors and CRISPR-Cas9 gene editing system respectively. We thank plant
growth facility members in the state key laboratory of crop biology, and
the technical assistance on the measurement of plant hypocotyl growth
under different light qualities from Professor Kun Xu's Lab at Shandong
Agricultural University.

\textbf{Conflict of interest}

The authors declare no potential conflict of interest.

\textbf{References}

Ahmad, M., \& Cashmore, A. R. (1993). HY4 gene of A. thaliana encodes a
protein with characteristics of a blue-light photoreceptor.\emph{Nature,
366} (6451), 162-166. doi:10.1038/366162a0

Arumuganathan, K., \& Earle, E. D. (1991). Estimation of nuclear DNA
content of plants by flow cytometry. \emph{Plant Molecular Biology
Reporter, 9} (3), 229-241. doi:10.1007/bf02672073

Bai, M. Y., Shang, J. X., Oh, E., Fan, M., Bai, Y., Zentella, R., . . .
Wang, Z. Y. (2012). Brassinosteroid, gibberellin and phytochrome impinge
on a common transcription module in Arabidopsis. \emph{Nat Cell Biol,
14} (8), 810-817. doi:10.1038/ncb2546

Bainard, J. D., Bainard, L. D., Henry, T. A., Fazekas, A. J., \&
Newmaster, S. G. (2012). A multivariate analysis of variation in genome
size and endoreduplication in angiosperms reveals strong phylogenetic
signal and association with phenotypic traits. \emph{New Phytol, 196}
(4), 1240-1250. doi:10.1111/j.1469-8137.2012.04370.x

Bao, Z., \& Hua, J. (2014). Interaction of CPR5 with cell cycle
regulators UVI4 and OSD1 in Arabidopsis. \emph{PLoS One, 9} (6),
e100347. doi:10.1371/journal.pone.0100347

Bao, Z., Yang, H., \& Hua, J. (2013). Perturbation of cell cycle
regulation triggers plant immune response via activation of disease
resistance genes. \emph{Proc Natl Acad Sci U S A, 110} (6), 2407-2412.
doi:10.1073/pnas.1217024110

Bao, Z., Zhang, N., \& Hua, J. (2014). Endopolyploidization and
flowering time are antagonistically regulated by checkpoint component
MAD1 and immunity modulator MOS1. \emph{Nat Commun, 5} , 5628.
doi:10.1038/ncomms6628

Barow, M., \& Meister, A. (2003). Endopolyploidy in seed plants is
differently correlated to systematics, organ, life strategy and genome
size. \emph{Plant Cell Environ, 26} , 571-584.
doi:10.1046/j.1365-3040.2003.00988.x

Bleecker, A. B., Estelle, M. A., Somerville, C., \& Kende, H. (1988).
Insensitivity to Ethylene Conferred by a Dominant Mutation in
Arabidopsis thaliana. \emph{Science, 241} (4869), 1086-1089.
doi:10.1126/science.241.4869.1086

Boerjan, W., Cervera, M. T., Delarue, M., Beeckman, T., Dewitte, W.,
Bellini, C., . . . Inze, D. (1995). Superroot, a recessive mutation in
Arabidopsis, confers auxin overproduction. \emph{Plant Cell, 7} (9),
1405-1419. doi:10.1105/tpc.7.9.1405

Boron, A. K., \& Vissenberg, K. (2014). The Arabidopsis thaliana
hypocotyl, a model to identify and study control mechanisms of cellular
expansion. \emph{Plant Cell Rep, 33} (5), 697-706.
doi:10.1007/s00299-014-1591-x

Cebolla, A., Vinardell, J. M., Kiss, E., Olah, B., Roudier, F.,
Kondorosi, A., \& Kondorosi, E. (1999). The mitotic inhibitor ccs52 is
required for endoreduplication and ploidy-dependent cell enlargement in
plants. \emph{EMBO J, 18} (16), 4476-4484. doi:10.1093/emboj/18.16.4476

Churchman, M. L., Brown, M. L., Kato, N., Kirik, V., Hulskamp, M., Inze,
D., . . . Larkin, J. C. (2006). SIAMESE, a plant-specific cell cycle
regulator, controls endoreplication onset in Arabidopsis
thaliana.\emph{Plant Cell, 18} (11), 3145-3157.
doi:10.1105/tpc.106.044834

Clough, S. J., \& Bent, A. F. (1998). Floral dip: a simplified method
for Agrobacterium-mediated transformation of Arabidopsis
thaliana.\emph{Plant J, 16} (6), 735-743.
doi:10.1046/j.1365-313x.1998.00343.x

Cowling, R. J., \& Harberd, N. P. (1999). Gibberellins control
Arabidopsis hypocotyl growth via regulation of cellular
elongation.\emph{Journal of Experimental Botany, 50} (337), 1351-1357.
doi:10.1093/jxb/50.337.1351

d'Erfurth, I., Jolivet, S., Froger, N., Catrice, O., Novatchkova, M., \&
Mercier, R. (2009). Turning Meiosis into Mitosis. \emph{PLOS Biology, 7}
(6), e1000124. doi:10.1371/journal.pbio.1000124

Dan, H., Imaseki, H., Wasteneys, G. O., \& Kazama, H. (2003). Ethylene
stimulates endoreduplication but inhibits cytokinesis in cucumber
hypocotyl epidermis. \emph{Plant Physiol, 133} (4), 1726-1731.
doi:10.1104/pp.103.025783

de Lucas, M., Daviere, J. M., Rodriguez-Falcon, M., Pontin, M.,
Iglesias-Pedraz, J. M., Lorrain, S., . . . Prat, S. (2008). A molecular
framework for light and gibberellin control of cell
elongation.\emph{Nature, 451} (7177), 480-484. doi:10.1038/nature06520

De Veylder, L., Beeckman, T., Beemster, G. T., Krols, L., Terras, F.,
Landrieu, I., . . . Inze, D. (2001). Functional analysis of
cyclin-dependent kinase inhibitors of Arabidopsis. \emph{Plant Cell, 13}
(7), 1653-1668. doi:10.1105/tpc.010087

De Veylder, L., Beeckman, T., \& Inze, D. (2007). The ins and outs of
the plant cell cycle. \emph{Nat Rev Mol Cell Biol, 8} (8), 655-665.
doi:10.1038/nrm2227

Franklin, K. A., Lee, S. H., Patel, D., Kumar, S. V., Spartz, A. K., Gu,
C., . . . Gray, W. M. (2011). Phytochrome-interacting factor 4 (PIF4)
regulates auxin biosynthesis at high temperature. \emph{Proc Natl Acad
Sci U S A, 108} (50), 20231-20235. doi:10.1073/pnas.1110682108

Fulop, K., Tarayre, S., Kelemen, Z., Horvath, G., Kevei, Z., Nikovics,
K., . . . Kondorosi, E. (2005). Arabidopsis anaphase-promoting
complexes: multiple activators and wide range of substrates might keep
APC perpetually busy. \emph{Cell Cycle, 4} (8), 1084-1092.

Gendreau, E., Traas, J., Desnos, T., Grandjean, O., Caboche, M., \&
Hofte, H. (1997). Cellular basis of hypocotyl growth in Arabidopsis
thaliana. \emph{Plant Physiol, 114} (1), 295-305.
doi:10.1104/pp.114.1.295

Gray, W. M., Ostin, A., Sandberg, G., Romano, C. P., \& Estelle, M.
(1998). High temperature promotes auxin-mediated hypocotyl elongation in
Arabidopsis. \emph{Proc Natl Acad Sci U S A, 95} (12), 7197-7202.
doi:10.1073/pnas.95.12.7197

Hase, Y., Trung, K. H., Matsunaga, T., \& Tanaka, A. (2006). A mutation
in the uvi4 gene promotes progression of endo-reduplication and confers
increased tolerance towards ultraviolet B light. \emph{Plant J, 46} (2),
317-326. doi:10.1111/j.1365-313X.2006.02696.x

Heyman, J., Van den Daele, H., De Wit, K., Boudolf, V., Berckmans, B.,
Verkest, A., . . . De Veylder, L. (2011). Arabidopsis
ULTRAVIOLET-B-INSENSITIVE4 maintains cell division activity by temporal
inhibition of the anaphase-promoting complex/cyclosome. \emph{Plant
Cell, 23} (12), 4394-4410. doi:10.1105/tpc.111.091793

Humplik, J. F., Bergougnoux, V., Jandova, M., Simura, J., Pencik, A.,
Tomanec, O., . . . Fellner, M. (2015). Endogenous abscisic acid promotes
hypocotyl growth and affects endoreduplication during dark-induced
growth in tomato (Solanum lycopersicum L.). \emph{PLoS One, 10} (2),
e0117793. doi:10.1371/journal.pone.0117793

Huq, E., \& Quail, P. H. (2002). PIF4, a phytochrome-interacting bHLH
factor, functions as a negative regulator of phytochrome B signaling in
Arabidopsis. \emph{EMBO J, 21} (10), 2441-2450.
doi:10.1093/emboj/21.10.2441

Inze, D., \& De Veylder, L. (2006). Cell cycle regulation in plant
development. \emph{Annu Rev Genet, 40} , 77-105.
doi:10.1146/annurev.genet.40.110405.090431

Iwata, E., Ikeda, S., Matsunaga, S., Kurata, M., Yoshioka, Y., Criqui,
M. C., . . . Ito, M. (2011). GIGAS CELL1, a novel negative regulator of
the anaphase-promoting complex/cyclosome, is required for proper mitotic
progression and cell fate determination in Arabidopsis. \emph{Plant
Cell, 23} (12), 4382-4393. doi:10.1105/tpc.111.092049

Johansson, H., Jones, H. J., Foreman, J., Hemsted, J. R., Stewart, K.,
Grima, R., \& Halliday, K. J. (2014). Arabidopsis cell expansion is
controlled by a photothermal switch. \emph{Nat Commun, 5} , 4848.
doi:10.1038/ncomms5848

Kang, B. G., Newcomb, W., \& Burg, S. P. (1971). Mechanism of
Auxin-induced Ethylene Production. \emph{Plant Physiol, 47} (4),
504-509. doi:10.1104/pp.47.4.504

Kasili, R., Walker, J. D., Simmons, L. A., Zhou, J., De Veylder, L., \&
Larkin, J. C. (2010). SIAMESE cooperates with the CDH1-like protein
CCS52A1 to establish endoreplication in Arabidopsis thaliana
trichomes.\emph{Genetics, 185} (1), 257-268.
doi:10.1534/genetics.109.113274

Kim, J., Song, K., Park, E., Kim, K., Bae, G., \& Choi, G. (2016).
Epidermal Phytochrome B Inhibits Hypocotyl Negative Gravitropism
Non-Cell-Autonomously. \emph{Plant Cell, 28} (11), 2770-2785.
doi:10.1105/tpc.16.00487

Kim, J., Yi, H., Choi, G., Shin, B., Song, P. S., \& Choi, G. (2003).
Functional characterization of phytochrome interacting factor 3 in
phytochrome-mediated light signal transduction. \emph{Plant Cell, 15}
(10), 2399-2407. doi:10.1105/tpc.014498

Koini, M. A., Alvey, L., Allen, T., Tilley, C. A., Harberd, N. P.,
Whitelam, G. C., \& Franklin, K. A. (2009). High temperature-mediated
adaptations in plant architecture require the bHLH transcription factor
PIF4. \emph{Curr Biol, 19} (5), 408-413. doi:10.1016/j.cub.2009.01.046

Kumar, S., Stecher, G., Li, M., Knyaz, C., \& Tamura, K. (2018). MEGA X:
Molecular Evolutionary Genetics Analysis across Computing
Platforms.\emph{Mol Biol Evol, 35} (6), 1547-1549.
doi:10.1093/molbev/msy096

Kutschera, U., \& Niklas, K. J. (2013). Cell division and turgor-driven
stem elongation in juvenile plants: a synthesis. \emph{Plant Sci, 207} ,
45-56. doi:10.1016/j.plantsci.2013.02.004

Lopez-Juez, E., Dillon, E., Magyar, Z., Khan, S., Hazeldine, S., de
Jager, S. M., . . . Shanahan, H. (2008). Distinct light-initiated gene
expression and cell cycle programs in the shoot apex and cotyledons of
Arabidopsis. \emph{Plant Cell, 20} (4), 947-968.
doi:10.1105/tpc.107.057075

Love, J., Bjorklund, S., Vahala, J., Hertzberg, M., Kangasjarvi, J., \&
Sundberg, B. (2009). Ethylene is an endogenous stimulator of cell
division in the cambial meristem of Populus. \emph{Proc Natl Acad Sci U
S A, 106} (14), 5984-5989. doi:10.1073/pnas.0811660106

Lui, H., Wang, H., Delong, C., Fowke, L. C., Crosby, W. L., \& Fobert,
P. R. (2000). The Arabidopsis Cdc2a-interacting protein ICK2 is
structurally related to ICK1 and is a potent inhibitor of
cyclin-dependent kinase activity in vitro. \emph{Plant J, 21} (4),
379-385. doi:10.1046/j.1365-313x.2000.00688.x

Ma, D., Li, X., Guo, Y., Chu, J., Fang, S., Yan, C., . . . Liu, H.
(2016). Cryptochrome 1 interacts with PIF4 to regulate high
temperature-mediated hypocotyl elongation in response to blue
light.\emph{Proc Natl Acad Sci U S A, 113} (1), 224-229.
doi:10.1073/pnas.1511437113

Marrocco, K., Bergdoll, M., Achard, P., Criqui, M. C., \& Genschik, P.
(2010). Selective proteolysis sets the tempo of the cell
cycle.\emph{Curr Opin Plant Biol, 13} (6), 631-639.
doi:10.1016/j.pbi.2010.07.004

Mathieu-Rivet, E., Gevaudant, F., Cheniclet, C., Hernould, M., \&
Chevalier, C. (2010). The Anaphase Promoting Complex activator CCS52A, a
key factor for fruit growth and endoreduplication in Tomato. \emph{Plant
Signal Behav, 5} (8), 985-987. doi:10.4161/psb.5.8.12222

Mathieu-Rivet, E., Gevaudant, F., Sicard, A., Salar, S., Do, P. T.,
Mouras, A., . . . Hernould, M. (2010). Functional analysis of the
anaphase promoting complex activator CCS52A highlights the crucial role
of endo-reduplication for fruit growth in tomato. \emph{Plant J, 62}
(5), 727-741. doi:10.1111/j.1365-313X.2010.04198.x

Mieulet, D., Jolivet, S., Rivard, M., Cromer, L., Vernet, A., Mayonove,
P., . . . Mercier, R. (2016). Turning rice meiosis into
mitosis.\emph{Cell Res, 26} (11), 1242-1254. doi:10.1038/cr.2016.117

Okello, R. C. O., de Visser, P. H. B., Heuvelink, E., Marcelis, L. F.
M., \& Struik, P. C. (2016). Light mediated regulation of cell division,
endoreduplication and cell expansion. \emph{Environmental and
Experimental Botany, 121} , 39-47.
doi:\emph{https://doi.org/10.1016/j.envexpbot.2015.04.003}

Park, E., Park, J., Kim, J., Nagatani, A., Lagarias, J. C., \& Choi, G.
(2012). Phytochrome B inhibits binding of phytochrome-interacting
factors to their target promoters. \emph{Plant J, 72} (4), 537-546.
doi:10.1111/j.1365-313X.2012.05114.x

Peters, J. M. (2006). The anaphase promoting complex/cyclosome: a
machine designed to destroy. \emph{Nat Rev Mol Cell Biol, 7} (9),
644-656. doi:10.1038/nrm1988

Pombo, M. A., Zheng, Y., Fernandez-Pozo, N., Dunham, D. M., Fei, Z., \&
Martin, G. B. (2014). Transcriptomic analysis reveals tomato genes whose
expression is induced specifically during effector-triggered immunity
and identifies the Epk1 protein kinase which is required for the host
response to three bacterial effector proteins. \emph{Genome Biol, 15}
(10), 492. doi:10.1186/s13059-014-0492-1

Raz, V., \& Koornneef, M. (2001). Cell division activity during apical
hook development. \emph{Plant Physiol, 125} (1), 219-226.
doi:10.1104/pp.125.1.219

Romano, C. P., Robson, P. R., Smith, H., Estelle, M., \& Klee, H.
(1995). Transgene-mediated auxin overproduction in Arabidopsis:
hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl
elongation and axr1 auxin-resistant mutants. \emph{Plant Mol Biol, 27}
(6), 1071-1083. doi:10.1007/BF00020881

Rossignol, P., Collier, S., Bush, M., Shaw, P., \& Doonan, J. H. (2007).
Arabidopsis POT1A interacts with TERT-V(I8), an N-terminal splicing
variant of telomerase. \emph{J Cell Sci, 120} (Pt 20), 3678-3687.
doi:10.1242/jcs.004119

Savaldi-Goldstein, S., Baiga, T. J., Pojer, F., Dabi, T., Butterfield,
C., Parry, G., . . . Chory, J. (2008). New auxin analogs with
growth-promoting effects in intact plants reveal a chemical strategy to
improve hormone delivery. \emph{Proc Natl Acad Sci U S A, 105} (39),
15190-15195. doi:10.1073/pnas.0806324105

Schutze, K., Harter, K., \& Chaban, C. (2009). Bimolecular fluorescence
complementation (BiFC) to study protein-protein interactions in living
plant cells. \emph{Methods Mol Biol, 479} , 189-202.
doi:10.1007/978-1-59745-289-2\_12

Sherr, C. J., \& Roberts, J. M. (1999). CDK inhibitors: positive and
negative regulators of G1-phase progression. \emph{Genes Dev, 13} (12),
1501-1512. doi:10.1101/gad.13.12.1501

Smalle, J., Haegman, M., Kurepa, J., Van Montagu, M., \& Straeten, D. V.
(1997). Ethylene can stimulate Arabidopsis hypocotyl elongation in the
light. \emph{Proc Natl Acad Sci U S A, 94} (6), 2756-2761.
doi:10.1073/pnas.94.6.2756

Smets, R., Le, J., Prinsen, E., Verbelen, J. P., \& Van Onckelen, H. A.
(2005). Cytokinin-induced hypocotyl elongation in light-grown
Arabidopsis plants with inhibited ethylene action or indole-3-acetic
acid transport. \emph{Planta, 221} (1), 39-47.
doi:10.1007/s00425-004-1421-4

Stavang, J. A., Gallego-Bartolome, J., Gomez, M. D., Yoshida, S., Asami,
T., Olsen, J. E., . . . Blazquez, M. A. (2009). Hormonal regulation of
temperature-induced growth in Arabidopsis. \emph{Plant J, 60} (4),
589-601. doi:10.1111/j.1365-313X.2009.03983.x

Sugimoto-Shirasu, K., \& Roberts, K. (2003). ''Big it up'':
endoreduplication and cell-size control in plants. \emph{Curr Opin Plant
Biol, 6} (6), 544-553. doi:10.1016/j.pbi.2003.09.009

Sullivan, M., \& Morgan, D. O. (2007). Finishing mitosis, one step at a
time. \emph{Nat Rev Mol Cell Biol, 8} (11), 894-903. doi:10.1038/nrm2276

Sun, H. J., Uchii, S., Watanabe, S., \& Ezura, H. (2006). A highly
efficient transformation protocol for Micro-Tom, a model cultivar for
tomato functional genomics. \emph{Plant Cell Physiol, 47} (3), 426-431.
doi:10.1093/pcp/pci251

Sun, W., Cao, Z., Li, Y., Zhao, Y., \& Zhang, H. (2007). A simple and
effective method for protein subcellular localization using
Agrobacterium-mediated transformation of onion epidermal
cells.\emph{Biologia, 62} (5), 529-532. doi:10.2478/s11756-007-0104-6

Tang, Q. Y., \& Zhang, C. X. (2013). Data Processing System (DPS)
software with experimental design, statistical analysis and data mining
developed for use in entomological research. \emph{Insect Sci, 20} (2),
254-260. doi:10.1111/j.1744-7917.2012.01519.x

Tomato Genome, C. (2012). The tomato genome sequence provides insights
into fleshy fruit evolution. \emph{Nature, 485} (7400), 635-641.
doi:10.1038/nature11119

Vanstraelen, M., Baloban, M., Da Ines, O., Cultrone, A., Lammens, T.,
Boudolf, V., . . . Kondorosi, E. (2009). APC/C-CCS52A complexes control
meristem maintenance in the Arabidopsis root. \emph{Proc Natl Acad Sci U
S A, 106} (28), 11806-11811. doi:10.1073/pnas.0901193106

Walker, J. D., Oppenheimer, D. G., Concienne, J., \& Larkin, J. C.
(2000). SIAMESE, a gene controlling the endoreduplication cell cycle in
Arabidopsis thaliana trichomes. \emph{Development, 127} (18), 3931-3940.

Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake,
C., . . . Kudla, J. (2004). Visualization of protein interactions in
living plant cells using bimolecular fluorescence
complementation.\emph{Plant J, 40} (3), 428-438.
doi:10.1111/j.1365-313X.2004.02219.x

Wang, H., Fowke, L. C., \& Crosby, W. L. (1997). A plant
cyclin-dependent kinase inhibitor gene. \emph{Nature, 386} (6624),
451-452. doi:10.1038/386451a0

Wang, H., \& Shang, Q. (2020). The combined effects of light intensity,
temperature, and water potential on wall deposition in regulating
hypocotyl elongation of Brassica rapa. \emph{PeerJ, 8} , e9106.
doi:10.7717/peerj.9106

Wang, T. W., Cosgrove, D. J., \& Arteca, R. N. (1993). Brassinosteroid
Stimulation of Hypocotyl Elongation and Wall Relaxation in Pakchoi
(Brassica chinensis cv Lei-Choi). \emph{Plant Physiol, 101} (3),
965-968. doi:10.1104/pp.101.3.965

Xie, X., Ma, X., Zhu, Q., Zeng, D., Li, G., \& Liu, Y. G. (2017).
CRISPR-GE: A Convenient Software Toolkit for CRISPR-Based Genome
Editing. \emph{Mol Plant, 10} (9), 1246-1249.
doi:10.1016/j.molp.2017.06.004

Zhang, Q., Zhang, Y., Lu, M. H., Chai, Y. P., Jiang, Y. Y., Zhou, Y., .
. . Chen, Q. J. (2019). A Novel Ternary Vector System United with
Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize. \emph{Plant
Physiol, 181} (4), 1441-1448. doi:10.1104/pp.19.00767

Zhao, Y., Christensen, S. K., Fankhauser, C., Cashman, J. R., Cohen, J.
D., Weigel, D., \& Chory, J. (2001). A role for flavin
monooxygenase-like enzymes in auxin biosynthesis. \emph{Science, 291}
(5502), 306-309. doi:10.1126/science.291.5502.306

Zhong, S., Shi, H., Xue, C., Wang, L., Xi, Y., Li, J., . . . Guo, H.
(2012). A molecular framework of light-controlled phytohormone action in
Arabidopsis. \emph{Curr Biol, 22} (16), 1530-1535.
doi:10.1016/j.cub.2012.06.039



\textbf{Figure legends}

Figure 1. Sowing depth significantly affects both tomato seed
germination and hypocotyl elongation. (A) Morphology of 10-day tomato
cultivar `Heinz 1706' seedlings after seeds were sown at different depth
of soil. Dotted lines crossing the pots indicated the positions of
seeds. Scale bar, 1cm. (B) Percentage of tomato seedlings emerged from
soil. (C) Germination rate of tomato seeds at day 5 after sowing. (D)
Morphology of 7-day tomato seedlings after sown at 4 cm depth of soil.
Data are presented by means \selectlanguage{ngerman}± SE. Different letters indicate statistical
significance (\emph{p} \textless{} 0.05) determined by LSD Test in DPS
Software.

Figure 2. Interactions between SlUVI4 and SlCCS52 proteins. (A)
Yeast-two hybrid assay. Serial dilutions of yeast cells containing
SlUVI4 fused with the activation domain (AD) and SlCCS52 fused with the
DNA binding domain (BD) were plated on amino acid deficient yeast growth
medium. (B) Confocal microscopy images from BiFC assay in onion
epidermal cells. SlUVI4:YFP\textsuperscript{N} together with
SlCCS52A1:YFP\textsuperscript{C} or SlCCS52A2:YFP\textsuperscript{C}
were transiently expressed in onion epidermal cells. Images were taken
at 48\selectlanguage{english} h after incubation. Green signals are from YFP. Scale bar, 50\selectlanguage{english} \selectlanguage{greek}μ\selectlanguage{english}m.

Figure 3. Gene expression of \emph{SlUVI4} and \emph{SlCCS52s} in
different tissues and hypocotyls at different growth stages. The
transcript abundance of \emph{SlUVI4} (A), \emph{SlCCS52A1}
(B),\emph{SlCCS52A2} (C) and \emph{SlCCS52B} (D) in different tissues of
tomato cultivar `Heinz 1706' and hypocotyls at day 1, 3, 5 and 7 after
seed germination (1d, 3d, 5d, 7d) were analyzed by quantitative reverse
transcription polymerase chain reaction (qRT-PCR).
\emph{SlCBL1}(Calcineurin B-like protein, Solyc12g015870) transcript
abundance was used for data normalization. The transcript abundance of
each gene in the root was used for all comparisons to calculate the
relative expression. Data shown are means \selectlanguage{ngerman}± SE calculated from three
independent experiments. Letters on the top of each column indicate
statistically significant differences (\emph{p} \textless{} 0.05)
determined by LSD test in DPS software.

Figure 4. Different lights have distinct impacts on hypocotyl elongation
and gene expression of \emph{SlUVI4} and \emph{SlCCS52s} . Tomato
seedlings of cultivar `Heinz 1706' were grown on MS medium under red
light (A-D) and blue light (E-H) conditions. (A, E) Morphology of tomato
seedlings. (B, F) Hypocotyl length. (C, G) Diameter in the middle of
hypocotyl. Data represents means ± SE (n \textgreater{} 15). Different
letters indicate statistical significance (\emph{p} \textless{} 0.05)
determined by LSD test in DPS Software. (D, H) Nuclear DNA distribution
in hypocotyls. Data are presented by means ± SD. EI values are on the
top of columns. (I) qRT-PCR analysis of \emph{SlUVI4} , \emph{SlCCS52B}
,\emph{SlCCS52A1} and \emph{SlCCS52A2} in hypocotyls using
\emph{SlCBL1}as a reference gene. Data shown are means ± SE calculated
from three independent experiments.

Figure 5. MS salts and sugar enhance hypocotyl elongation through the
promotion of \emph{SlUVI4} and \emph{SlCCS52B} transcription. Tomato
seedlings were grown on non-MS agar (NMS, A-D), MS agar (MS, E-H) and MS
agar with sugar (MSS, I-L) for 1, 3, 5, and 7 days after seeds were
sown. (A, E, I) Morphology of tomato seedlings. (B, F, J) Hypocotyl
length. (C, G, K) Diameter in the middle of hypocotyl. Data represents
means ± SE. Different letters indicate statistical significance
(\emph{p} \textless{} 0.05) determined by LSD test in DPS Software. (D,
H, L) Nuclear DNA ploidy distribution in hypocotyls. Data represents
means ± SD. The number on the top of each column indicates EI. (M)
qRT-PCR analysis of \emph{SlUVI4} ,\emph{SlCCS52B} , \emph{SlCCS52A1} ,
and \emph{SlCCS52A2} in hypocotyls using \emph{SlCBL1} as a reference
gene. Data shown are means ± SE calculated from three independent
experiments. Letters on the top of each column indicate statistically
significant differences (\emph{p}\textless{} 0.05) determined by LSD
test in DPS software.

Figure 6. Heat enhances hypocotyl elongation through the transcriptional
modulation\emph{SlUVI4} and \emph{SlCCS52} genes. (A) Morphology of day
seedlings grown on NMS and MS agar plates at 25°C and 33°C. (B)
Hypocotyl length. (C) Diameter in the middle of hypocotyl. (D) Nuclear
DNA ploidy distribution in hypocotyls. (E) qRT-PCR analysis of
\emph{SlUVI4} ,\emph{SlCCS52B} , \emph{SlCCS52A1} and \emph{SlCCS52A2}
in hypocotyls using \emph{SlCBL1} as a reference gene\emph{.} The
transcript abundance of each gene at 25°C was used to calculate the
relative expression. Data shown are means ± SE calculated from three
independent experiments. The asterisk on the top of column indicates
statistically significant differences (\emph{p} \textless{} 0.05)
determined by student\emph{t} -test.

Figure 7. Auxin promotes the hypocotyl elongation via the
transcriptional modulation of \emph{SlUVI4} and \emph{SlCCS52} genes.
(A) Morphology of tomato cultivar `Heinz 1706' seedlings after 0.1 nM
IAA treatment for 7 days. (B) Hypocotyl length. (C) Diameter in the
middle of hypocotyl. (D) Nuclear DNA ploidy distribution in hypocotyls.
(E) qRT-PCR analysis of \emph{SlUVI4} and \emph{SlCCS52s} in hypocotyls
using \emph{SlCBL1} as a reference gene. Data shown are means ± SE
calculated from two independent experiments. The asterisk on the top of
column indicates statistically significant differences
(\emph{p}\textless{} 0.05) determined by student \emph{t} -test.

Figure 8. Salt stress inhibits hypocotyl elongation through the
transcriptional modulation of \emph{SlUVI4} and \emph{SlCCS52} genes.
(A) Morphology of tomato cultivar `Heinz 1706' seedlings after 150 mM
NaCl treatment for 7 days. (B) Hypocotyl length. (C) Diameter in the
middle of hypocotyl. (D) Nuclear DNA ploidy distribution in hypocotyls.
(E) qRT-PCR analysis of \emph{SlUVI4} and \emph{SlCCS52B} in hypocotyls
using \emph{SlCBL1} as a reference gene. Data shown are means ± SE
calculated from three independent experiments. The asterisk on the top
of column indicates statistically significant differences
(\emph{p}\textless{} 0.05) determined by student \emph{t} -test.

Figure 9. Ethylene affects the hypocotyl elongation via the
transcriptional modulation of \emph{SlUVI4} and \emph{SlCCS52} genes.
(A) Morphology of tomato cultivar `Heinz 1706' seedlings after 10\selectlanguage{greek}μ\selectlanguage{english}M ACC
treatment for 7 days. (B) Hypocotyl length. (C) Diameter in the middle
of hypocotyl. (D) Nuclear DNA ploidy distribution in hypocotyls. (E)
qRT-PCR analysis of \emph{SlUVI4}and \emph{SlCCS52B} in hypocotyls using
\emph{SlCBL1} as a reference gene. Data shown are means \selectlanguage{ngerman}± SE calculated
from three independent experiments. The asterisk on the top of column
indicates statistically significant differences (\emph{p} \textless{}
0.05) determined by student \emph{t} -test.

Figure 10. Deletion mutants of \emph{SlUVI4} exhibit defective hypocotyl
elongation. (A) Morphology of 7-day seedlings grown on MS medium. (B)
Hypocotyl length. (C) Diameter in the middle of hypocotyl. Data
represents means ± SE (n \textgreater{} 19). Different letters indicate
statistical significance (\emph{p} \textless{} 0.05) determined by LSD
test in DPS Software. (D) Nuclear DNA ploidy distribution in hypocotyls.
Data are presented by means ± SD. EI values are on the top of columns.

Figure 11. Overexpression of \emph{SlCCS52B} suppresses hypocotyl
elongation. (A) Morphology of 6-day seedlings grown on 1/2 MS medium
under 10\selectlanguage{greek}μ\selectlanguage{english}mol m\textsuperscript{-2} s\textsuperscript{-1} light. (B)
Hypocotyl length. Data represents means \selectlanguage{ngerman}± SE (n \textgreater{} 50).
Different letters indicate statistical significance (\emph{p}\textless{}
0.05) determined by LSD test in DPS Software. (c) Nuclear DNA ploidy
distribution in hypocotyls. Data are presented by means ± SD. EI values
are on the top of columns.

\textbf{Supporting Information}

The following supplemental materials are available.

Table S1. Primer sequences used in this study.

Figure S1. Phylogenetic analysis of \emph{UVI4} and
\emph{CCS52}homologous genes in \emph{Solanaceae} species. The
full-length amino acid sequences of UVI4 and CSS52 were isolated through
BLAST analysis in the website (https://solgenomics.net/) and analyzed in
the software MEGA X to determine their relationships. The bootstrap
values are shown at the nodes. (A) Phylogenetic relationship among
\emph{UVI4} homologous genes. (B) Phylogenetic relationship among
\emph{CCS52} homologous genes.

Figure S2. Amino acid sequence alignment of tomato CCS52 homologs.
Tomato \emph{CCS52} homologous genes were isolated from the
website\emph{www.solgenomics.net}. Alignment of amino acid sequences was
performed using online multiple sequence alignment program Clustal Omega
(https://www.ebi.ac.uk/Tools/msa/clustalo/).

Figure S3. Subcellular protein localization. Fully expanded leaves of
\emph{N. benthamiana} were infiltrated with \emph{A. tumefaciens} strain
GV3101 containing different constructs with 35S promoter-driven GFP or
\emph{SlCCS52}genes fused with GFP.

Figure S4. Gene expression of APC/C core genes in hypocotyls at
different growth stages after seed germinated. Hypocotyls of tomato
cultivar `Heinz1706' grown on agar with MS salts were harvested at day
1, 3, 5 and 7 (1d, 3d, 5d and 7d) after seeds germinated. qRT-PCR
analysis of \emph{SlAPC2} and \emph{SlAPC11} in hypocotyls using
\emph{SlCBL1} as a reference gene. The transcript abundance of each gene
at day 1 was used for all comparisons to calculate the relative
expression. Data shown are means ± SE calculated from three independent
experiments. Letters on the top of each column indicate statistically
significant differences (\emph{p} \textless{} 0.05) determined by least
significance difference test (LSD) in DPS software.

Figure S5. White light represses hypocotyl elongation through the
suppression of endoreduplication. Tomato seedlings of cultivar `Heinz
1706' were grown on MS medium in the dark (A-D) and white light (E-H)
conditions. (A, E) Morphology of tomato seedlings. (B, F) Hypocotyl
length. (C, G) Diameter in the middle of hypocotyl. Data represents
means ± SE (n \textgreater{} 15). Different letters indicate statistical
significance (\emph{p} \textless{} 0.05) determined by LSD test in DPS
Software. (D, H) Nuclear DNA distribution in hypocotyls. Data are
presented by means ± SD. EI values are on the top of columns.

Figure S6. Gene expression of\emph{SlUVI4} and \emph{SlCCS52s} in
hypocotyls at different growth stages in the dark and white light
condition. Transcript abundance of\emph{SlUVI4} (A), \emph{SlCCS52B}
(B), \emph{SlCCS52A1} (C), and\emph{SlCCS52A2} (D) in hypocotyls was
analyzed by qRT-PCR\emph{. SlCBL1} transcript abundance was used for
data normalization. Data shown are means ± SE calculated from three
independent experiments.

Figure S7. Heat promotes the endoreduplication to improve hypocotyl
elongation of different tomato cultivars. Two tomato cultivars `Alisa
Craig' (A-C) and `Moneymaker' (D-F) were grown on NMS and MS agar plates
at 25°C and 33°C for days. (A, D) Hypocotyl length. (B, E) Diameter in
the middle of hypocotyl. Data represents means ± SE (n \textgreater{}
15). Different letters indicate statistical significance (\emph{p}
\textless{} 0.05) determined by LSD test in Data Processing System (DPS)
Software. (C, F) Nuclear DNA ploidy distribution in hypocotyls. Data
represents means ± SD. EI values are on the top of columns.

Figure S8. Exogenous application of plant hormones affects hypocotyl
elongation. Tomato seedlings of cultivar `Heinz 1706' were grown on MS
medium supplemented with different concentration of plant hormones IAA
(A, B), BR (C, D), GA3 (E, F) and ACC (G, H). (A, C, E, G) Hypocotyl
length. (B, D, F, H) Diameter in the middle of hypocotyls. Data
represents means ± SE (n \textgreater{} 15). Different letters indicate
statistical significance (\emph{p} \textless{} 0.05) determined by LSD
test in DPS Software.

Figure S9. Deletion mutations of\emph{SlUVI4} generated using
Crispr-Cas9 technology. (A) Target sites of the guide RNAs in
Crispr-Cas9 gene editing system. (B) DNA sequence information of
\emph{SlUVI4 cr-1} and \emph{cr-2} mutations. Nucleotides in blue are
target sequences. The red hidden lines indicate missing nucleotides.
Black lines indicate the protospacer-adjacent motif (PAM).

Figure S10. Overexpression of\emph{SlCCS52A2} inhibits tomato hypocotyl
elongation. (A) Morphology of 7-day seedlings grown on MS medium. (B)
Hypocotyl length. (C) Diameter in the middle of hypocotyl. Data
represents means ± SE (n \textgreater{} 15). Different letters indicate
statistical significance (\emph{p}\textless{} 0.05) determined by LSD
test in DPS Software. (D) Nuclear DNA ploidy distribution in hypocotyls.
Data are presented by means ± SD. EI values are on the top of columns.

Figure S11. A possible model for\emph{SlUVI4} and \emph{SlCCS52B}
coordinated hypocotyl elongation under normal and stress conditions.
Under normal growth condition, light represses \emph{SlUVI4}
and\emph{SlCCS52B} coordinated endoreduplication to inhibit hypocotyl
elongation. MS salts and sugar promote \emph{SlUVI4} and
\emph{SlCCS52B}coordinated endoreduplication to enhance hypocotyl
elongation. Under stress condition, salt triggers ethylene production
which represses\emph{SlUVI4} and \emph{SlCCS52B} coordinated
endoreduplication to inhibit hypocotyl elongation, while ethylene itself
promotes endoreduplication via unknown cell cycle machinery.
Heat-stimulated auxin production and auxin-mediated ethylene production
reorchestrate\emph{SlUVI4} and \emph{SlCCS52B} coordinated
endoreduplication to promote hypocotyl elongation.\selectlanguage{english}
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