Elucidating the functional role of endoreduplication in tomato fruit development

Background

Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague,although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination.

Scope

Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described.

Conclusions

The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits.     

The specific overexpression of a cyclin-dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth

The size of tomato fruit results from the combination of cell number and cell size, which are respectively determined by the cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of fleshy pericarp tissue is characterized by numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. Although a clear relationship exists between endoreduplication and cell growth in plants, the exact role of endoreduplication has not been clearly elucidated. To decipher the molecular basis of endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the tomato cyclin-dependent kinase inhibitor SlKRP1. We studied the kinetics of pericarp development in tomato fruit at the morphological and cytological levels, and demonstrated that endoreduplication is directly proportional to cell and fruit diameter. We established a mathematical model for tissue growth according to the number of divisions and endocycles. This model was tested in fruits where we managed to decrease the extent of endoreduplication by over-expressing SlKRP1 under the control of a fruit-specific promoter expressed during early development. Despite the fact that endoreduplication was affected, we could not observe any morphological, cytological or metabolic phenotypes, indicating that determination of cell and fruit size can be, at least conditionally, uncoupled from endoreduplication.     

Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes

Growth of tomato fruits is determined by cell division and cell expansion, which are tightly controlled by factors that drive the core cell cycle. The cyclin-dependent kinases (CDKs) and their interacting partners, the cyclins, play a key role in the progression of the cell cycle. In this study the role of CDKA1, CDKB1, and CDKB2 in fruit development was characterized by fruit-specific overexpression and down-regulation. CDKA1 is expressed in the pericarp throughout development, but is strongly up-regulated in the outer pericarp cell layers at the end of the growth period, when CDKB gene expression has ceased. Overexpression of the CDKB genes at later stages of development and the down-regulation of CDKA1 result in a very similar fruit phenotype, showing a reduction in the number of cell layers in the pericarp and alterations in the desiccation of the fruits. Expression studies revealed that CDKA1 is down-regulated by the expression of CDKB1/2 in CDKB1 and CDKB2 overexpression mutants, suggesting opposite roles for these types of CDK proteins in tomato pericarp development.     

Analysis of the tomato fruit growth response to temperature and plant fruit load in relation to cell division, cell expansion and DNA endoreduplication

BACKGROUND AND AIMS: To better understand the regulation of fruit growth in response to environmental factors, the effects of temperature and plant fruit load on cell number, cell size and DNA endoreduplication were analysed. METHODS: Plants were grown at 20/20 degrees C, 25/25 degrees C and 25/20 degrees C day/night temperatures, and inflorescences were pruned to two ('2F') or five ('5F') flowers. KEY RESULTS AND CONCLUSIONS: Despite a lower fruit growth rate at 20/20 degrees C, temperature did not affect final fruit size because of the compensation between cell number and size. The higher cell number at 20/20 degrees C (9.0 x 10(6) against 7.9 x 10(6) at 25/25 degrees C and 7.7 x 10(6) at 25/20 degrees C) resulted from an extended period of cell division, and the smaller cell size was due to a shorter period of expansion rather than a lower expansion rate. By contrast, the lower fruit growth rate and size of 5F fruits compared with 2F fruits resulted from the slow down of cell expansion, whereas the number of cells was hardly affected in the proximal fruit. However, within the inflorescence the decreasing gradient of fruit size from proximal to distal fruits was due to a decrease in cell number with similar cell size. Fruit size variations within each treatment were always positively correlated to variations in cell number, but not in cell size. Negative correlations between cell size and cell number suggested that cells of tomato pericarp can be seen as a population of competing sinks. Mean ploidy was slightly delayed and reduced in 5F fruits compared with 2F fruits. It was highest at 25/25 degrees C and lowest at 25/20 degrees C. Treatments did not affect ploidy and cell size in similar ways, but within each treatment, positive correlations existed between mean ploidy and cell size, though significant only in the 2F-25/20 treatment.     

Expression of genomic AtCYCD2;1 in Arabidopsis induces cell division at smaller cell sizes: implications for the control of plant growth

The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.     

The Arabidopsis cyclin-dependent kinase-activating kinase CDKF;1 is a major regulator of cell proliferation and cell expansion but is dispensable for CDKA activation

Cyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and post-embryonic development of various organisms. Full activation of CDKs requires not only binding to cyclins but also phosphorylation of the T-loop domain. This phosphorylation is catalysed by CDK-activating kinases (CAKs). Plants have two distinct types of CAKs, namely CDKD and CDKF; in Arabidopsis, CDKF;1 exhibits the highest CDK kinase activity in vitro . We have previously shown that CDKF;1 also functions in the activation of CDKD;2 and CDKD;3 by T-loop phosphorylation. Here, we isolated the knockout mutants of CDKF;1 and showed that they had severe defects in cell division, cell elongation and endoreduplication. No defect was observed during embryogenesis, suggesting that CDKF;1 function is primarily required for post-embryonic development. In the cdkf;1 mutants, T-loop phosphorylation of CDKA;1, an orthologue of yeast Cdc2/Cdc28p, was comparable to that in wild-type plants, and its kinase activity did not decrease. In contrast, the protein level and kinase activity of CDKD;2 were significantly reduced in the mutants. Substitution of threonine-168 with a non-phosphorylatable alanine residue made CDKD;2 unstable in Arabidopsis tissues. These results indicate that CDKF;1 is dispensable for CDKA;1 activation but is essential for maintaining a steady-state level of CDKD;2, thereby suggesting the quantitative regulation of a vertebrate-type CAK in a plant-specific manner.     

Tissue dependent variations of DNA methylation and endoreduplication levels during tomato fruit development and ripening

Tomato fruit cells are characterized by a strong increase in nuclear ploidy during fruit development. Average ploidy levels increased to similar levels (above 50C) in two distinct fruit tissues, pericarp and locular tissue. However, ploidy profiles differed significantly between these two tissues suggesting a tissue-specific control of endoreduplication in tomato fruit. To determine possible relationships between endoreduplication and epigenetic mechanisms, the methylation status of genomic DNA from pericarp and locular tissue of tomato fruit was analysed. Pericarp genomic DNA was characterized by an increase of CG and/or CNG methylation at the 5S and 18S rDNA loci and at gyspsy-like retrotransposon sequences during fruit growth. A sharp decrease of the global DNA methylation level together with a reduction of methylation at the rDNA loci was also observed in pericarp during fruit ripening. Inversely, no major variation of DNA methylation either global or locus-specific, was observed in locular tissue. Thus, tissue-specific variations of DNA methylation are unlikely to be triggered by the induction of endoreduplication in fruit tissues, but may reflect tissue-specific ploidy profiles. Expression analysis of eight putative tomato DNA methyltransferases encoding genes showed that one chromomethylase (CMT) and two rearranged methyltransferases (DRMs) are preferentially expressed in the pericarp during fruit growth and could be involved in the locus-specific increase of methylation observed at this developmental phase in the pericarp.     

The cell size distribution of tomato fruit can be changed by overexpression of CDKA1

Tomato is one of the most cultivated vegetables in the world and an important ingredient of the human diet. Tomato breeders and growers face a continuous challenge of combining high quantity (production volume) with high quality (appearance, taste and perception for the consumers, processing quality for the processing industry). To improve the quality of tomato, it is important to understand the regulation of fruit development and of fruit cellular structure, which is in part determined by the sizes and numbers of cells within a tissue. The role of the cell cycle therein is poorly understood. Plant cyclin‐dependent kinases (CDKs) are homologues of yeast cdc2, an important cell cycle regulator conserved throughout all eukaryotes. CDKA1 is constitutively expressed during the cell cycle and has dual functions in S‐ and M‐phase progression. We have produced transgenic tomato plants with increased expression of CDKA1 under the control of the fruit‐specific TPRP promoter, which despite a reduced number of seeds and diminished amount of jelly, developed fruits with weight and shape comparable to that of wild‐type fruits. However, the phenotypic changes with regard to the pericarp thickness and placenta area were remarkable. Fruits of tomato plants with the highest expression of CDKA1 had larger septa and columella (placenta), compared with wild‐type fruits. Our data demonstrate the possibility of manipulating the ratio between cell division and expansion by changing the expression of a key cell cycle regulator and probably its activity with substantial effects on structural traits of the harvested fruit.     

Cell and leaf size plasticity in Arabidopsis: what is the role of endoreduplication?

Leaf area expansion is affected by environmental conditions because of differences in cell number and/or cell size. Increases in the DNA content (ploidy) of a cell by endoreduplication are related to its size. The aim of this work was to determine how cell ploidy interacts with the regulation of cell size and with leaf area expansion. The approach used was to grow Arabidopsis thaliana plants performing increased or decreased rounds of endoreduplication under shading and water deficit. The shading and water deficit treatments reduced final leaf area and cell number; however, cell area was increased and decreased, respectively. These differences in cell size were unrelated to alterations of the endocycle, which was reduced by these treatments. The genetic modification of the extent of endoreduplication altered leaf growth responses to shading and water deficit. An increase in the extent of endoreduplication in a leaf rendered it more sensitive to the shade treatment but less sensitive to water deficit conditions. The link between the control of whole organ and individual cell expansion under different environmental conditions was demonstrated by the correlation between the plasticity of cell size and the changes in the duration of leaf expansion.     

WEE1 激酶及其抑制剂在抗肿瘤治疗中的应用进展

WEE1激酶是一种细胞周期调节蛋白,能调控细胞周期蛋白依赖性激酶1(cyclin-dependent kinase 1,CDK1)的磷酸化状态,从而调节CDK1与细胞周期蛋白B(cyclin B)复合物的活性从而实现对细胞周期的调控,且对DNA损伤检查点具有重要的调节作用。WEE1是G2/M期阻滞的关键基因,起着重要的监测作用,在一些癌症中过表达,抑制或下调WEE1激酶均能引发有丝分裂灾难,因此WEE1激酶抑制剂可能在抗癌治疗中有关键作用。在癌症的治疗过程中,WEE1抑制剂与DNA损伤剂、化学药物等联合使用会得到比单独使用更为有效,且在p53缺失的癌细胞中能发挥更好的效果。目前WEE1已成为许多癌症治疗的关键靶点之一,其抑制剂MK-1775已处于临床研究阶段,且能增强一些DNA损伤剂对p53缺失的癌细胞的杀伤能力。本文就WEE1激酶及其抑制剂在抗癌治疗中的应用作一综述。     

Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis

For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T-loop is required. This phosphorylation is catalyzed by CDK-activating kinases (CAKs). In Arabidopsis three D-type CDK genes (CDKD;1-CDKD;3) encode vertebrate-type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP-binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK - CDKF;1 - exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T-loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.     

Cyclin-dependent kinase complexes in developing maize endosperm: evidence for differential expression and functional specialization

Endosperm development in maize (Zea mays L.) and related cereals comprises a cell proliferation stage followed by a period of rapid growth coupled to endoreduplication. Regulation of the cell cycle in developing endosperm is poorly understood. We have characterized various subunits of cyclin-dependent kinase (CDK) complexes, master cell cycle regulators in all eukaryotes. A-, B-, and D-type cyclins as well as A- and B-type cyclin-dependent kinases were characterized with respect to their RNA and protein expression profiles. Two main patterns were identified: one showing expression throughout endosperm development, and another characterized by a sharp down-regulation with the onset of endoreduplication. Cyclin CYCB1;3 and CYCD2;1 proteins were distributed in the cytoplasm and nucleus of cells throughout the endosperm, while cyclin CYCD5 protein was localized in the cytoplasm of peripheral cells. CDKB1;1 expression was strongly associated with cell proliferation. Expression and cyclin-binding patterns suggested that CDKA;1 and CDKA;3 are at least partially redundant. The kinase activity associated with the cyclin CYCA1 was highest during the mitotic stage of development, while that associated with CYCB1;3, CYCD2;1 and CYCD5 peaked at the mitosis-to-endoreduplication transition. A-, B- and D-type cyclins were more resistant to proteasome-dependent degradation in endoreduplicating than in mitotic endosperm extracts. These results indicated that endosperm development is characterized by differential expression and activity of specific cyclins and CDKs, and suggested that endoreduplication is associated with reduced cyclin proteolysis via the ubiquitin–proteasome pathway.     

The anaphase promoting complex activator CCS52A,a key factor for fruit growth and endoreduplication in tomato

Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endoreduplication in connection to cell expansion and final fruit size determination. Endoreduplication occurs as an impairment of mitosis, which can originate from the selective degradation of M-phase-specific cyclins via the ubiquitin-mediated proteolytic pathway, requiring the E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C). In plants CCS52A is the ortholog of CDH1/FZR proteins from yeast, drosophila and human, belonging to the WD40-repeat protein family. During fruit development, the SlCCS52A gene expression is specifically associated to endoreduplication in tomato. Altering SlCCS52A expression in either negative or positive manner impacts the extent of endoreduplication in fruit and affects fruit size. When SlCCS52A is down-expressed endoreduplication is impaired during fruit growth leading to reduced fruit growth. However when SlCCS52A is overexpressed, endoreduplication is initially delayed, accounting for the altered final fruit size, but resumes and is even enhanced leading to fruit growth recovery, pointing at the physiological role of endoreduplication in growth induction during tomato fruit development.Key words: anaphase promoting complex, CCS52A, endoreduplication, fruit development, growth, tomatoEndoreduplication represents the most common mode of cell endopolyploidization in plants and is estimated to occur in over 90% of Angiosperms.^1,2 This process is an endonuclear DNA stock duplication leading to the production of chromosomes with 2n chromatids without any change in chromosome number.^3,4 As a consequence hypertrophying nuclei arise from successive cycles of DNA replication.In fruits of Cucurbitaceae and Solanaceae, mesocarp cells commonly undergo six rounds of DNA duplication (endocycle), and the highest ploidy levels for these cells being reached in tomato fruits where eight endocycles (up to 512C) can be observed.⁵ This high level of endopolyploidy in tomato fruits and the numerous data reported on this process in this species,^6–9 makes it an outstanding model for studying endopolyploidization and its physiological role during fruit development.     

Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage.     

A model describing cell polyploidization in tissues of growing fruit as related to cessation of cell proliferation

Endoreduplication is a phenomenon, widespread among plants, which consists of an incomplete cell cycle without mitosis and leads to the increase of the nuclear DNA content. In this work, a model was developed describing cell proliferation and DNA endoreduplication over the whole fruit development, from the pre-anthesis period until maturation. In each mitotic cycle of duration tau, the proportion of cells proceeding through division depends on a constant parameter rho and on the progressive decline of the proliferating capacity . The non-dividing cells may either stop the reduplication fully, or switch to repeated syntheses of DNA without cell division, resulting in cell endoreduplication. A single constant parameter sigma describes the proportion of cells that moves from one to the next class of DNA content after each lapse of time tauE, considered to be the minimum time required for an endocycle. The model calculates the total number of cells and their distribution among eight classes of ploidy level. The dynamic patterns of cell proliferation and ploidy were compared with those obtained experimentally on two contrasting tomato genotypes. The approach developed in this model should allow the future integration of new knowledge concerning the genetic and environmental control of the switch from complete to incomplete cell cycle.