Ectopic expression of VvFUS3,B3-domain transcription factor,in tomato influences seed development via affecting endoreduplication and hormones

FUSCA3(FUS3)is a member of B3-domain transcription factor family and master regulator of seed development.It has potential roles in hormone biosynthesis and signaling pathways and therefore plays diverse roles in plant life cycle,especially in seed germination,dormancy,embryo formation,seed and fruit development,and maturation.However,there is limited information about its functions in seed and fruit development of grapevine.In this study,we expressed VvFUS3 in tomato for its functional characterization.Overexpression of VvFUS3 in tomato led to a reduction in seed number and seed weight without affecting the fruit size.Histological analysis found that both cell expansion and cell division in transgenic seed and fruit pericarp have been affected.However,there were no obvious differences in pollen size,shape,and viability,suggesting that VvFUS3 affects seed development but not the pollen grains.Moreover,the expression of several genes with presumed roles in seed development and hormone signaling pathways was also influenced by VvFUS3.These results suggest that VvFUS3 is involved in hormonal signaling pathways that regulate seed number and size.In conclusion,our study provides novel preliminary information about the pivotal roles of VvFUS3 in seed and fruit development and these findings can potentially serve as a reference for molecular breeding of seedless grapes.

The Arabidopsis Transcription Factor AtTCP15 Regulates Endoreduplication by Modulating Expression of Key Cell-cycle Genes

Plant cells frequently undergo endoreduplication, a modified cell cycle in which genome is repeatedly rep- licated without cytokinesis. As the key step to achieve final size and function for cells, endoreduplication is prevalent during plant development. However, mechanisms to control the balance between endoreduplication and mitotic cell di- vision are still poorly understood. Here, we show that the Arabidopsis TCP （CINCINNATA-like TEOSINTE BRANCHED1~ CYCLOIDEA-_PCF）-family transcription factor gene AtTCP15 is expressed in trichomes, as well as in rapidly dividing and vascular tissues. Expression of AtTCP15SRDX, AtTCP15 fused with a SRDX repressor domain, induces extra endoredupli- cation in trichomes and cotyledon cells in transgenic Arabidopsis. On the contrary, overexpression of AtTCP15 suppresses endoreduplication in trichomes and other examined cells. Misregulation of AtTCP15 affects the expression of several im- portant genes involved in cell-cycle regulation. AtTCP15 protein binds directly to the promoter regions of CYCA2;3 and RETINOBLASTOMA-RELATED （RBR） genes, which play key roles in endoreduplication. Taken together, AtTCP15 plays an important role in regulating endoreduplication during Arabidopsis development.

ERF4 interacts with and antagonizes TCP15 in regulating endoreduplication and cell growth in Arabidopsis

Endoreduplication is prevalent during plant growth and development,and is often correlated with large cell and organ size.Despite its prevalence,the transcriptional regulatory mechanisms underlying the transition from mitotic cell division to endoreduplication remain elusive.Here,we characterize ETHYLENE-RESPONSIVEELEMENTBINDING FACTOR 4(ERF4)as a positive regulator of endoreduplication through its function as a transcriptional repressor.ERF4 was specifically expressed in mature tissues in which the cells were undergoing expansion,but was rarely expressed in young organs.Plants overexpressing ERF4 exhibited much larger cells and organs,while plants that lacked functional ERF4 displayed smaller organs than the wild-type.ERF4 was further shown to regulate cell size by controlling the endopolyploidy level in the nuclei.Moreover,ERF4 physically associates with the class I TEOSINTE BRANCHED 1/CYCLOIDEA/PCF(TCP)protein TCP15,a transcription factor that inhibits endoreduplication by activating the expression of a key cell-cycle gene,CYCLIN A2;3(CYCA2;3).A molecular and genetic analysis revealed that ERF4promotes endoreduplication by directly suppressing the expression of CYCA2;3.Together,this study demonstrates that ERF4 and TCP15 function as a module to antagonistically regulate each other?s activity in regulating downstream genes,thereby controlling the switch from the mitotic cell cycle to endoreduplication during leaf development.These findings expand our understanding of how the control of the cell cycle is fine-tuned by an ERF4–TCP15 transcriptional complex.

Observation of two amitotic ways in the epidermal cells of plant Pelargonium zonale ‘Kleiner Liebling＇-Amitotic nuclear division for endoreduplicated cells

Amitosis, different from mitosis, is a rare form of the proliferation of the cells. Most amitosis were observed in animal cells; only few of them were reported in plant cells. The cytological investigation in this study demonstrated first time in Pelargonium zonale two different amitotic nuclear division ways： cleavage and constriction, in which no appearance of the visible chromosomes and no spindles were observed. After amitotic nuclear division the cytokinesis was also observed, in which the cytoplasm divided directly into two or more parts accompany with formation of two or more daughter cells. This study showed the genetic material may sometimes be unequally distributed between the daughter cells in amitosis, and the amitosis could lead to bi-, tri- and multinucleated cells. These new findings are discussed in regard to the nuclear amitotic process in polyploid cells which gives rise to smaller, viable nuclei in multinucleated cells with reduced numbers of genomes. It was suggested that amitosis could be a way of the division of the endoreduplicated cells.

The trxG protein ULT1 regulates Arabidopsis organ size by interacting with TCP14/15 to antagonize the LIM peptidase DA1 for H3K4me3 on target genes

Plant organ size is an important agronomic trait that makes a significant contribution to plant yield.Despite its central importance,the genetic and molecular mechanisms underlying organ size control remain to be fully clarified.Here,we report that the trithorax group protein ULTRAPETALA1(ULT1)interacts with the TEOSINTE BRANCHED1/CYCLOIDEA/PCF14/15(TCP14/15)transcription factors by antagonizing the LIN-11,ISL-1,and MEC-3(LIM)peptidase DA1,thereby regulating organ size in Arabidopsis.Loss of ULT1 function significantly increases rosette leaf,petal,silique,and seed size,whereas overexpression of ULT1 results in reduced organ size.ULT1 associates with TCP14 and TCP15 to co-regulate cell size by affecting cellular endoreduplication.Transcriptome analysis revealed that ULT1 and TCP14/15 regulate common target genes involved in endoreduplication and leaf development.ULT1 can be recruited by TCP14/15 to promote lysine 4 of histone H3 trimethylation at target genes,activating their expression to determinefinal cell size.Furthermore,we found that ULT1 influences the interaction of DA1 and TCP14/15 and antagonizes the effect of DA1 on TCP14/15 degradation.Collectively,ourfindings reveal a novel epigenetic mechanism underlying the regulation of organ size in Arabidopsis.

The Anaphase-Promoting Complex/Cyclosome in Control of Plant Development

Temporal controlled degradation of key cell division proteins ensures a correct onset of the different cell cycle phases and exit from the cell division program. In light of the cell cycle, the Anaphase-Promoting Complex/Cyclosome （APC/C） is an important conserved multi-subunit ubiquitin ligase, marking targets for degradation by the 26S protea- some. However, whereas the APC/C has been studied extensively in yeast and mammals, only in the last decade has the plant APC/C started to unveil its secrets. Research results have shown the importance of the APC/C core complex and its activators during gametogenesis, growth, hormone signaling, symbiotic interactions, and endoreduplication onset. In addition, recently, the first plant APC/C inhibitors have been reported, allowing a fine-tuning of APC/C activity during the cell cycle. Together with the identification of the first APC/C targets, a picture emerges of APC/C activity being essential for many different developmental processes.

GLABROUS INFLORESCENCE STEMS regulates trichome branching by genetically interacting with SIM in Arabidopsis

Arabidopsis trichomes are large branched single cells that protrude from the epidermis. The first mor- phological indication of trichome development is an increase in nuclear content resulting from an initial cycle of endoreduplication. Our previous study has shown that the C2H2 zinc finger protein GLABROUS INFLORESCENCE STEMS (GIS) is required for trichome initiation in the inflorescence organ and for trichome branching in response to gibberellic acid signaling, although GIS gene does not play a direct role in regulating trichome cell division. Here, we describe a novel role of GIS, controlling trichome cell division indirectly by interacting genetically with a key endoreduplication regulator SIAMESE (SIM). Our molecular and genetic studies have shown that GIS might indireclty control cell division and trichome branching by acting downstream of SIM. A loss of function mutation of SIM signficantly reduced the expression of GIS. Futhermore, the overexpression of GIS rescued the trichome cluster cell phenotypes of sim mutant. The gain or loss of function of GIS had no significant effect on the expression of SIM. These results suggest that GIS may play an indirect role in regulating trichome cell division by genetically interacting with SIM.

Fruit size control by a zinc finger protein regulating pericarp cell size in tomato

Fruit size is largely defined by the number and size of cells in the fruit.Endoreduplication–a specialized cell cycle–is highly associated with cell expansion during tomato fruit growth.However,how endoreduplication coupled with cell size is regulated remains poorly understood.In this study,we identified a zinc finger gene SlPZF1(Solanum lycopersicum PERICARP-ASSOCIATED ZINC FINGER PROTEIN 1)that was highly expressed in the pericarp of developing fruits.Plants with altered SlPZF1 expression produced smaller fruits due to the reduction in cell size associated with weakened endoreduplication.Overexpressing SlPZF1 delayed cell division phase by enhancing early expression of several key cell cycle regulators including SlCYCD3;1 and two plant specific mitotic cyclin-dependent protein kinase(SlCDKB1 and SlCDKB2)in the pericarp tissue.Furthermore,we identified 14 putative SlPZF1 interacting proteins(PZFIs)via yeast two hybrid screening.Several PZFIs,including Pre-mRNA-splicing factor(SlSMP1/PZFI4),PAPA-1-like conserved region family protein(PZFI6),Fanconi anemia complex components(PZFI3 and PZFI10)and bHLH transcription factor LONESOME HIGHWAY(SILHW/PZFI14),are putatively involved in cell cycle regulation.Our results demonstrate that fruit growth in tomato requires balanced expression of the novel cell size regulator SlPZF1.

SD3, an Arabidopsis thaliana Homolog of TIM21, Affects Intracellular ATP Levels and Seedling Development

It is poorly understood how plants control their growth by cell division, elongation, and differentiation. We have characterized a seedling-lethal mutant segregation distortion 3 （sd3） that showed a very dwarf phenotype when grown in the light and, in the dark, had short hypocotyls with reduced ploidy levels. The corresponding gene of SD3 encodes a protein with high similarity to yeast translocase on the inner mitochondrial membrane 21 （TIM21）, which is a component of the TIM23 complex. Indeed, SD3 protein fused to GFP localized in the mitochondria. SD3 overexpression increased cotyledon size in the light and hypocotyl thickness in the dark. The expression of genes for several subunits of the respiratory-chain complexes III and IV was up-regulated in SD3-overexpressing plants. Furthermore, these plants showed high levels of ATP whereas those of sd3 were low. These results suggested that SD3 induced an increase in cell size by raising the expression of the respiratory-chain subunit genes and hence increased the intracellular ATP levels, We propose that intracellular ATP levels regulated by mitochondria control plant organ size.