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Plant organ size and shape are major agronomic traits that depend on cell division and expansion, which are both regulated by complex gene networks. In several eudicot species belonging to the rosid clade, organ growth is controlled by a repressor complex consisting of PEAPOD (PPD) and KINASE-INDUCIBLE DOMAIN INTERACTING (KIX) proteins. The role of these proteins in asterids, which together with the rosids constitute most of the core eudicot species, is unknown. We used Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 genome editing to target SlKIX8 and SlKIX9 in the asterid model species tomato (Solanum lycopersicum) and analyzed loss-of-function phenotypes. Loss-of-function of SlKIX8 and SlKIX9 led to the production of enlarged, dome-shaped leaves and these leaves exhibited increased expression of putative Solanum lycopersicum PPD (SlPPD target genes. Unexpectedly, kix8 kix9 mutants carried enlarged fruits with increased pericarp thickness due to cell expansion. At the molecular level, protein interaction assays indicated that SlKIX8 and SlKIX9 act as adaptors between the SlPPD and SlTOPLESS co-repressor proteins. Our results show that KIX8 and KIX9 are regulators of organ growth in asterids and can be used in strategies to improve important traits in produce such as thickness of the fruit flesh., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The understanding of plant organ-size determination represents an important challenge, especially because of the significant role of plants as food and renewable energy sources and the increasing need for plant-derived products. Most of the knowledge on the regulation of organ growth and the molecular network controlling cell division and cell expansion, the main drivers of growth, is derived from arabidopsis. The increasing use of crops such as tomato for research is now bringing essential information on the mechanisms underlying size control in agronomically important organs. This review describes our current knowledge, still very scarce, of the cellular and molecular mechanisms governing tomato fruit size and proposes future research to better understand the regulation of growth in this important crop., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The FW2.2 gene is associated with the major quantitative trait locus (QTL) governing fruit size in tomato, and acts by negatively controlling cell division during fruit development. FW2.2 belongs to a multigene family named the CELL NUMBER REGULATOR (CNR) family. CNR proteins harbour the uncharacterized PLAC8 motif made of two conserved cysteine-rich domains separated by a variable region that are predicted to be transmembrane segments, and indeed FW2.2 localizes to the plasma membrane. Although FW2.2 was cloned more than two decades ago, the molecular mechanisms of action remain unknown. In particular, how FW2.2 functions to regulate cell cycle and fruit growth, and thus fruit size, is as yet not understood. Here we review current knowledge on PLAC8-containing CNR/FWL proteins in plants, which are described to participate in organogenesis and the regulation of organ size, especially in fruits, and in cadmium resistance, ion homeostasis, and/or Ca2+ signalling. Within the plasma membrane FW2.2 and some CNR/FWLs are localized in microdomains, which is supported by recent data from interactomics studies. Hence FW2.2 and CNR/FWL could be involved in a transport function of signalling molecules across membranes, influencing organ growth via a cell to cell trafficking mechanism., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A key strategy to increase plant productivity is to improve intrinsic organ growth. Some of the regulatory networks underlying organ growth and development, as well as the interconnections between these networks, are highly conserved. An example of such a growth-regulatory module with a highly conserved role in final organ size and shape determination in eudicot species is the PEAPOD (PPD)/KINASE-INDUCIBLE DOMAIN INTERACTING (KIX)/STERILE APETALA (SAP) module. We review the proteins constituting the PPD pathway and their roles in different plant developmental processes, and explore options for future research. We also speculate on strategies to exploit knowledge about the PPD pathway for targeted yield improvement to engineer crop traits of agronomic interest, such as leaf, fruit, and seed size., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Small RNAs (sRNAs), mainly including miRNAs and siRNAs, are ubiquitous in eukaryotes. sRNAs mostly negatively regulate gene expression via (post-)transcriptional gene silencing through DNA methylation, mRNA cleavage, or translation inhibition. The mechanisms of sRNA biogenesis and function in diverse biological processes, as well as the interactions between sRNAs and environmental factors, like (a)biotic stress, have been deeply explored. Phytohormones are central in the plant's response to stress, and multiple recent studies highlight an emerging role for sRNAs in the direct response to, or the regulation of, plant hormonal pathways. In this review, we discuss recent progress on the unraveling of crossregulation between sRNAs and nine plant hormones., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Post-transcriptional gene silencing (PTGS) is a major mechanism regulating gene expression in higher eukaryotes. To identify novel players in PTGS, a forward genetics screen was performed on an Arabidopsis thaliana line overexpressing a strong growth-repressive gene, ETHYLENE RESPONSE FACTOR6 (ERF6). We identified six independent ethyl-methanesulfonate mutants rescuing the dwarfism of ERF6-overexpressing plants as a result of transgene silencing. Among the causative genes, ETHYLENE-INSENSITIVE5, SUPERKILLER2 and HASTY1 have previously been reported to inhibit PTGS. Notably, the three other causative genes have not, to date, been related to PTGS: UTP:RNA-URIDYLYLTRANSFERASE1 (URT1), C-TERMINAL DOMAIN PHOSPHATASE-LIKE3 (CPL3) and RESURRECTION1 (RST1). We show that these genes may participate in protecting the 3' end of transgene transcripts. We present a model in which URT1, CPL3 and RST1 are classified as PTGS suppressors, as compromisation of these genes provokes the accumulation of aberrant transcripts which, in turn, trigger the production of small interfering RNAs, initiating RNA silencing.

Key Message: Here, we used a hxk1 mutant in the Col-0 background. We demonstrated that HXK1 regulates cell proliferation and expansion early during leaf development, and that HXK1 is involved in sucrose-induced leaf growth stimulation independent of GPT2. Furthermore, we identified KINγ as a novel HXK1-interacting protein. In the last decade, extensive efforts have been made to unravel the underlying mechanisms of plant growth control through sugar availability. Signaling by the conserved glucose sensor HEXOKINASE1 (HXK1) has been shown to exert both growth-promoting and growth-inhibitory effects depending on the sugar levels, the environmental conditions and the plant species. Here, we used a hxk1 mutant in the Col-0 background to investigate the role of HXK1 during leaf growth in more detail and show that it is affected in both cell proliferation and cell expansion early during leaf development. Furthermore, the hxk1 mutant is less sensitive to sucrose-induced cell proliferation with no significant increase in final leaf growth after transfer to sucrose. Early during leaf development, transfer to sucrose stimulates expression of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSPORTER2 (GPT2) and represses chloroplast differentiation. However, in the hxk1 mutant GPT2 expression was still upregulated by transfer to sucrose although chloroplast differentiation was not affected, suggesting that GPT2 is not involved in HXK1-dependent regulation of leaf growth. Finally, using tandem affinity purification of protein complexes from cell cultures, we identified KINγ, a protein containing four cystathionine β-synthase domains, as an interacting protein of HXK1.

In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development., (© 2017 American Society of Plant Biologists. All rights reserved.)

The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins., (© 2017 Dong et al.; Published by Cold Spring Harbor Laboratory Press.)

Although phytohormones such as gibberellins are essential for many conserved aspects of plant physiology and development, plants vary greatly in their responses to these regulatory compounds. Here, we use genetic perturbation of endogenous gibberellin levels to probe the extent of intraspecific variation in gibberellin responses in natural accessions of Arabidopsis (Arabidopsis thaliana). We find that these accessions vary greatly in their ability to buffer the effects of overexpression of GA20ox1, encoding a rate-limiting enzyme for gibberellin biosynthesis, with substantial differences in bioactive gibberellin concentrations as well as transcriptomes and growth trajectories. These findings demonstrate a surprising level of flexibility in the wiring of regulatory networks underlying hormone metabolism and signaling., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Chloroplasts and mitochondria are indispensable for plant development. They not only provide energy and carbon sources to cells, but also have evolved to become major players in a variety of processes such as amino acid metabolism, hormone biosynthesis and cellular signalling. As semi-autonomous organelles, they contain a small genome that relies largely on nuclear factors for its maintenance and expression. An intensive crosstalk between the nucleus and the organelles is therefore essential to ensure proper functioning, and the nuclear genes encoding organellar proteins involved in photosynthesis and oxidative phosphorylation are obviously crucial for plant growth. Organ growth is determined by two main cellular processes: cell proliferation and cell expansion. Here, we review how plant growth is affected in mutants of organellar proteins that are differentially expressed during leaf and root development. Our findings indicate a clear role for organellar proteins in plant organ growth, primarily during cell proliferation. However, to date, the role of the nuclear-encoded organellar proteins in the cellular processes driving organ growth has not been investigated in much detail. We therefore encourage researchers to extend their phenotypic characterization beyond macroscopic features in order to get a better view on how chloroplasts and mitochondria regulate the basic processes of cell proliferation and cell expansion, essential to driving growth., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls., (© 2015 American Society of Plant Biologists. All rights reserved.)

Arabidopsis (Arabidopsis thaliana) leaf development relies on subsequent phases of cell proliferation and cell expansion. During the proliferation phase, chloroplasts need to divide extensively, and during the transition from cell proliferation to expansion, they differentiate into photosynthetically active chloroplasts, providing the plant with energy. The transcription factor GROWTH REGULATING FACTOR5 (GRF5) promotes the duration of the cell proliferation period during leaf development. Here, it is shown that GRF5 also stimulates chloroplast division, resulting in a higher chloroplast number per cell with a concomitant increase in chlorophyll levels in 35S:GRF5 leaves, which can sustain higher rates of photosynthesis. Moreover, 35S:GRF5 plants show delayed leaf senescence and are more tolerant for growth on nitrogen-depleted medium. Cytokinins also stimulate leaf growth in part by extending the cell proliferation phase, simultaneously delaying the onset of the cell expansion phase. In addition, cytokinins are known to be involved in chloroplast development, nitrogen signaling, and senescence. Evidence is provided that GRF5 and cytokinins synergistically enhance cell division and chlorophyll retention after dark-induced senescence, which suggests that they also cooperate to stimulate chloroplast division and nitrogen assimilation. Taken together with the increased leaf size, ectopic expression of GRF5 has great potential to improve plant productivity., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Although quantitative characterization of growth phenotypes is of key importance for the understanding of essential networks driving plant growth, the majority of growth-related genes are still being identified based on qualitative visual observations and/or single-endpoint quantitative measurements. We developed an in vitro growth imaging system (IGIS) to perform time-resolved analysis of rosette growth. In this system, Arabidopsis plants are grown in Petri dishes mounted on a rotating disk, and images of each plate are taken on an hourly basis. Automated image analysis was developed in order to obtain several growth-related parameters, such as projected rosette area, rosette relative growth rate, compactness and stockiness, over time. To illustrate the use of the platform and the resulting data, we present the results for the growth response of Col-0 plants subjected to three mild stress conditions. Although the reduction in rosette area was relatively similar at 19 days after stratification, the time-lapse analysis demonstrated that plants react differently to salt, osmotic and oxidative stress. The rosette area was altered at various time points during development, and leaf movement and shape parameters were also affected differently. We also used the IGIS to analyze in detail the growth behavior of mutants with enhanced leaf size. Analysis of several growth-related parameters over time in these mutants revealed several specificities in growth behavior, underlining the high complexity of leaf growth coordination. These results demonstrate that time-resolved imaging of in vitro rosette growth generates a better understanding of growth phenotypes than endpoint measurements., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

When plants develop, cell proliferation and cell expansion are tightly controlled in order to generate organs with a determinate final size such as leaves. Several studies have demonstrated the importance of the cell proliferation phase for leaf growth, illustrating that cell-cycle regulation is crucial for correct leaf development. A large and complex set of interacting proteins that constitute the cell-cycle interactome controls the transition from one cell-cycle phase to another. Here, we review the current knowledge on cell-cycle regulators from this interactome affecting final leaf size when their expression is altered, mainly in Arabidopsis. In addition to the description of mutants of CYCLIN-DEPENDENT KINASES (CDKs), CYCLINS (CYCs), and their transcriptional and post-translational regulators, a phenotypic analysis of gain- and loss-of-function mutants for 27 genes encoding proteins that interact with cell-cycle proteins is presented. This compilation of information shows that when cell-cycle-related genes are mis-expressed, leaf growth is often altered and that, seemingly, three main trends appear to be crucial in the regulation of final organ size by cell-cycle-related genes: (i) cellular compensation; (ii) gene dosage; and (iii) correct transition through the G2/M phase by ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C) activation. In conclusion, this meta-analysis shows that the cell-cycle interactome is enriched in leaf growth regulators, and illustrates the potential to identify new leaf growth regulators among putative new cell-cycle regulators., (© The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.

The transcriptional coactivator ANGUSTIFOLIA3 (AN3) stimulates cell proliferation during Arabidopsis thaliana leaf development, but the molecular mechanism is largely unknown. Here, we show that inducible nuclear localization of AN3 during initial leaf growth results in differential expression of important transcriptional regulators, including GROWTH REGULATING FACTORs (GRFs). Chromatin purification further revealed the presence of AN3 at the loci of GRF5, GRF6, CYTOKININ RESPONSE FACTOR2, CONSTANS-LIKE5 (COL5), HECATE1 (HEC1), and ARABIDOPSIS RESPONSE REGULATOR4 (ARR4). Tandem affinity purification of protein complexes using AN3 as bait identified plant SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodeling complexes formed around the ATPases BRAHMA (BRM) or SPLAYED. Moreover, SWI/SNF ASSOCIATED PROTEIN 73B (SWP73B) is recruited by AN3 to the promoters of GRF5, GRF3, COL5, and ARR4, and both SWP73B and BRM occupy the HEC1 promoter. Furthermore, we show that AN3 and BRM genetically interact. The data indicate that AN3 associates with chromatin remodelers to regulate transcription. In addition, modification of SWI3C expression levels increases leaf size, underlining the importance of chromatin dynamics for growth regulation. Our results place the SWI/SNF-AN3 module as a major player at the transition from cell proliferation to cell differentiation in a developing leaf.

Size control of multicellular organisms poses a longstanding biological question that has always fascinated scientists. Currently the question is far from being resolved because of the complexity of and interconnection between cell division and cell expansion, two different events necessary to form a mature organ. Because of the importance of plants for food and renewable energy sources, dissecting the genetic networks underlying plant growth and organ size is becoming a high priority in plant science worldwide. Here, we review the current understanding of the cellular and molecular mechanisms that govern leaf organ size and discuss future prospects on research aiming at understanding organ size regulation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Because seed yield is the major factor determining the commercial success of grain crop cultivars, there is a large interest to obtain more understanding of the genetic factors underlying this trait. Despite many studies, mainly in the model plant Arabidopsis thaliana, have reported transgenes and mutants with effects on seed number and/or seed size, knowledge about seed yield parameters remains fragmented. This study investigated the effect of 46 genes, either in gain- and/or loss-of-function situations, with a total of 64 Arabidopsis lines being examined for seed phenotypes such as seed size, seed number per silique, number of inflorescences, number of branches on the main inflorescence and number of siliques. Sixteen of the 46 genes, examined in 14 Arabidopsis lines, were reported earlier to directly affect in seed size and/or seed number or to indirectly affect seed yield by their involvement in biomass production. Other genes involved in vegetative growth, flower or inflorescence development or cell division were hypothesized to potentially affect the final seed size and seed number. Analysis of this comprehensive data set shows that of the 14 lines previously described to be affected in seed size or seed number, only nine showed a comparable effect. Overall, this study provides the community with a useful resource for identifying genes with effects on seed yield and candidate genes underlying seed QTL. In addition, this study highlights the need for more thorough analysis of genes affecting seed yield., (© 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Functionally distinct Arabidopsis (Arabidopsis thaliana) genes that positively affect root or shoot growth when ectopically expressed were combined to explore the feasibility of enhanced biomass production. Enhanced root growth resulting from cytokinin deficiency was obtained by overexpressing CYTOKININ OXIDASE/DEHYDROGENASE3 (CKX3) under the control of the root-specific PYK10 promoter. Plants harboring the PYK10-CKX3 construct were crossed with four different transgenic lines showing enhanced leaf growth. For all combinations, the phenotypic traits of the individual lines could be combined, resulting in an overall growth increase. Unexpectedly, three out of four combinations had more than additive effects. Both leaf and root growth were synergistically enhanced in plants ectopically expressing CKX3 and BRASSINOSTEROID INSENSITIVE1, indicating cross talk between cytokinins and brassinosteroids. In agreement, treatment of PYK10-CKX3 plants with brassinolide resulted in a dramatic increase in lateral root growth that could not be observed in wild-type plants. Coexpression of CKX3 and the GROWTH-REGULATING FACTOR5 (GRF5) antagonized the effects of GRF5 overexpression, revealing an interplay between cytokinins and GRF5 during leaf cell proliferation. The combined overexpression of CKX3 and GIBBERELLIN 20-OXIDASE1 led to a synergistic increase in leaf growth, suggesting an antagonistic growth control by cytokinins and gibberellins. Only additive effects on root and shoot growth were visible in plants ectopically expressing both CKX3 and ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1, hinting at an independent action mode. Our results show new interactions and contribute to the molecular and physiological understanding of biomass production at the whole plant level.

Leaf primordia are iteratively formed on the flanks of the shoot apical meristem (SAM) at the vegetative shoot apex of Arabidopsis thaliana. The youngest leaf primordia and the SAM are extensively covered by older proliferating leaves, making it difficult to obtain accurate volumetric data from these structures. Combination of serial histological sections combined with 3D reconstruction software allowed us to acquire such data. Here, we compared the SAMs of wild-type plants of the Columbia-0 and Landsberg erecta ecotypes with those of clavata3-2 (clv3-2) mutants, which produce an enlarged SAM. In addition, the SAM size and morphology of plants over-expressing the gibberellin-20 oxidase (GA20OX) gene was examined, and the effect of mild osmotic stress on primordium size was measured. Efficient 3D visualization of gene expression patterns is also possible with this method, as illustrated by the analysis of SHOOTMERISTEMLESS:GUS and WUSCHEL:GUS reporter lines., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

The final size of plant organs, such as leaves, is tightly controlled by environmental and genetic factors that must spatially and temporally coordinate cell expansion and cell cycle activity. However, this regulation of organ growth is still poorly understood. The aim of this study is to gain more insight into the genetic control of leaf size in Arabidopsis (Arabidopsis thaliana) by performing a comparative analysis of transgenic lines that produce enlarged leaves under standardized environmental conditions. To this end, we selected five genes belonging to different functional classes that all positively affect leaf size when overexpressed: AVP1, GRF5, JAW, BRI1, and GA20OX1. We show that the increase in leaf area in these lines depended on leaf position and growth conditions and that all five lines affected leaf size differently; however, in all cases, an increase in cell number was, entirely or predominantly, responsible for the leaf size enlargement. By analyzing hormone levels, transcriptome, and metabolome, we provide deeper insight into the molecular basis of the growth phenotype for the individual lines. A comparative analysis between these data sets indicates that enhanced organ growth is governed by different, seemingly independent pathways. The analysis of transgenic lines simultaneously overexpressing two growth-enhancing genes further supports the concept that multiple pathways independently converge on organ size control in Arabidopsis.

The number of cells in an organ is a major factor that specifies its size. However, the genetic basis of cell number determination is not well understood. To obtain insight into this genetic basis, three grandifolia-D (gra-D) mutants of Arabidopsis thaliana were characterized that developed huge leaves with two to three times more cells than the wild-type. Genetic and microarray analyses showed that a large segmental duplication had occurred in all the gra-D mutants, consisting of the lower part of chromosome 4. In the duplications, genes were found that encode AINTEGUMENTA (ANT), a factor that extends the duration of cell proliferation, and CYCD3;1, a G(1)/S cyclin. The expression levels of both genes increased and the duration of cell proliferation in the leaf primordia was extended in the gra-D mutants. Data obtained by RNAi-mediated knockdown of ANT expression suggested that ANT contributed to the huge-leaf phenotype, but that it was not the sole factor. Introduction of an extra genomic copy of CYCD3;1 into the wild-type partially mimicked the gra-D phenotype. Furthermore, combined elevated expression of ANT and CYCD3;1 enhanced cell proliferation in a cumulative fashion. These results indicate that the duration of cell proliferation in leaves is determined in part by the interaction of ANT and CYCD3;1, and also demonstrate the potential usefulness of duplication mutants in the elucidation of genetic relationships that are difficult to uncover by standard single-gene mutations or gain-of-function analysis. We also discuss the potential effect of chromosomal duplication on evolution of organ size.

Tomato fruit size results from the combination of cell number and cell size which are respectively determined by cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of pericarp and locular tissues is characterized by the concomitant arrest of mitotic activity, inhibition of cyclin-dependent kinase (CDK) activity, and numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. To decipher the molecular basis of the endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the WEE1 kinase (Solly;WEE1). We here report a functional analysis of Solly;WEE1 in tomato. Impairing the expression of Solly;WEE1 in transgenic tomato plants resulted in a reduction of plant size and fruit size. In the most altered phenotypes, fruits displayed a reduced number of seeds without embryo development. The reduction of plant-, fruit- and seed size originated from a reduction in cell size which could be correlated with a decrease of the DNA ploidy levels. At the molecular level downregulating Solly;WEE1 in planta resulted in the increase of CDKA activity levels originating from a decrease of the amount of Y15-phosphorylated CDKA, thus indicating a release of the negative regulation on CDK activity exerted by WEE1. Our data indicated that Solly;WEE1 participates in the control of cell size and/or the onset of the endoreduplication process putatively driving cell expansion.

We have previously reported that ATPgammaS, a slowly hydrolyzed analog of ATP, inhibits the activation of human CD4(+) T lymphocytes by anti-CD3 and anti-CD28 mAb. In this report we have partially characterized the signaling mechanisms involved in this immunosuppressive effect. ATPgammaS had no inhibitory effect on CD4(+) T-cell activation induced by PMA and anti-CD28, indicating that it acts proximally to the TCR. It had no effect on the calcium rise induced by CD3/CD28 stimulation, but inhibited the phosphorylation of three kinases, ERK2, p38 MAPK and PKB, that play a key role in the activation of T cells. The receptor involved in these actions remains unidentified.

Mechanisms controlling brain size include the regulation of neural progenitor cell proliferation, differentiation, survival and migration. Here we show that ephrin-A/EphA receptor signalling plays a key role in controlling the size of the mouse cerebral cortex by regulating cortical progenitor cell apoptosis. In vivo gain of EphA receptor function, achieved through ectopic expression of ephrin-A5 in early cortical progenitors expressing EphA7, caused a transient wave of neural progenitor cell apoptosis, resulting in premature depletion of progenitors and a subsequent dramatic decrease in cortical size. In vitro treatment with soluble ephrin-A ligands similarly induced the rapid death of cultured dissociated cortical progenitors in a caspase-3-dependent manner, thereby confirming a direct effect of ephrin/Eph signalling on apoptotic cascades. Conversely, in vivo loss of EphA function, achieved through EphA7 gene disruption, caused a reduction in apoptosis occurring normally in forebrain neural progenitors, resulting in an increase in cortical size and, in extreme cases, exencephalic forebrain overgrowth. Together, these results identify ephrin/Eph signalling as a physiological trigger for apoptosis that can alter brain size and shape by regulating the number of neural progenitors.

The release of nucleotides in extracellular fluids can result from cell necrosis, exocytosis of secretory granules (such as platelet dense granules), or efflux through membrane channels. In addition, recent evidence suggests that vesicular trafficking is an important pathway of nucleotide release. Once in the extracellular fluids, they are rapidly degraded by ectonucleotidases, such as CD39, that play a key role in neutralizing the platelet aggregatory action of adenosine diphosphate (ADP), and act on two families of receptors: the ionotropic P2X receptors and the G-protein-coupled P2Y receptors. The family of P2X receptors encompasses seven genes. Currently, there are eight genuine P2Y receptors that can be subdivided into two structurally distinct subfamilies. Whereas P2X receptors are receptors of ATP, the different P2Y receptors are activated by distinct nucleotides, diphosphates or triphosphates, or purines or pyrimidines, some of them being conjugated to sugars. The study of knockout mice has demonstrated that P2X receptors play important roles in the neurogenic control of smooth muscle contraction, in pain and visceral perception, and in macrophage functions. The phenotype of P2Y null mice so far is more restricted: inhibition of platelet aggregation to ADP and increased bleeding time in P2Y (1)(-/-) and P2Y (12)(-/-) mice and lack of epithelial responsiveness to nucleotides in airways (P2Y (2)(-/-)) and intestine (P2Y (4)(0/-)).

Several orphan G protein-coupled receptors are structurally close to the family of P2Y nucleotide receptors: GPR80/99 and GPR91 are close to P2Y(1/2/4/6/11) receptors, whereas GPR87, H963 and GPR34 are close to P2Y(12/13/14). Over the years, several laboratories have attempted without success to identify the ligands of those receptors. In early 2004, two papers have been published: One claiming that GPR80/99 is an AMP receptor, called P2Y(15), and the other one showing that GPR80/99 is a receptor for alpha-ketoglutarate, while GPR91 is a succinate receptor. The accompanying paper by Qi et al. entirely supports that GPR80/99 is an alpha-ketoglutarate receptor and not an AMP receptor. The closeness of dicarboxylic acid and P2Y nucleotide receptors might be linked to the negative charges of both types of ligands and the involvement of conserved Arg residues in their neutralization.

Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds in two distinct phases of growth that comprise cell division and cell expansion, respectively. In pericarp and the jelly like locular tissue of tomato fruit, the transition between cell division to cell expansion is characterized by the arrest of mitotic activity, numerous rounds of nuclear DNA endoreduplication and the inhibition of Cyclin-Dependent Kinase A (CDKA) activity. To investigate whether the WEE1 kinase may play a role during the endoreduplication process, we isolated and characterized the tomato homologue for WEE1. The LeWEE1 gene consisted of 10 exons with a predicted 510 amino acid-long protein. The accumulation of the corresponding transcripts was associated with mitotically active organs: developing fruits, seeds and roots. Interestingly, LeWEE1was expressed in the jelly like locular tissue concomitant with endoreduplication during fruit development. Using tobacco BY-2 synchronized cells, we showed that the WEE1 gene expression is cell-cycle regulated with a maximum transcript accumulation at S phase. Our data indicate the putative dual contribution of LeWEE1 in the classical cell cycle and the endocycle.