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Understanding the underlying mechanisms of plant development is crucial to successfully steer or manipulate plant growth in a targeted manner. Leaves, the primary sites of photosynthesis, are vital organs for many plant species, and leaf growth is controlled by a tight temporal and spatial regulatory network. In this review, we focus on the genetic networks governing leaf cell proliferation, one major contributor to final leaf size. First, we provide an overview of six regulator families of leaf growth in Arabidopsis: DA1, PEAPODs, KLU, GRFs, the SWI/SNF complexes, and DELLAs, together with their surrounding genetic networks. Next, we discuss their evolutionary conservation to highlight similarities and differences among species, because knowledge transfer between species remains a big challenge. Finally, we focus on the increase in knowledge of the interconnectedness between these genetic pathways, the function of the cell cycle machinery as their central convergence point, and other internal and environmental cues., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

We investigated the interaction between osmotic stress and auxin signaling in leaf growth regulation. Therefore, we grew Arabidopsis thaliana seedlings on agar media supplemented with mannitol to impose osmotic stress and 1-naphthaleneacetic acid (NAA), a synthetic auxin. We performed kinematic analysis and flow-cytometry to quantify the effects on cell division and expansion in the first leaf pair, determined the effects on auxin homeostasis and response (DR5::β-glucuronidase), performed a next-generation sequencing transcriptome analysis and investigated the response of auxin-related mutants. Mannitol inhibited cell division and expansion. NAA increased the effect of mannitol on cell division, but ameliorated its effect on expansion. In proliferating cells, NAA and mannitol increased free IAA concentrations at the cost of conjugated IAA and stimulated DR5 promotor activity. Transcriptome analysis shows a large overlap between NAA and osmotic stress-induced changes, including upregulation of auxin synthesis, conjugation, transport and TRANSPORT INHIBITOR RESPONSE1 (TIR1) and AUXIN RESPONSE FACTOR (ARF) response genes, but downregulation of Aux/IAA response inhibitors. Consistently, arf7/19 double mutant lack the growth response to auxin and show a significantly reduced sensitivity to osmotic stress. Our results show that osmotic stress inhibits cell division during leaf growth of A. thaliana at least partly by inducing the auxin transcriptional response., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Plant leaf margins produce small outgrowths or teeth causing serration in a regular arrangement, which is specified by auxin maxima. In Arabidopsis , the spatiotemporal pattern of auxin dependents on both, the transcription factor CUC2 and the signal peptide EPFL2, a ligand of the growth-promoting receptor kinase ERECTA (ER). Ectopic expression of CUC2 can have contrary effects on leaf growth. Ubiquitous expressed CUC2 suppresses growth in the whole leaf, whereas cuc2-1D mutants have enlarged leaves, through ER -dependent cell proliferation in the teeth. Here we investigated the growth dynamics of cuc2-1D leaves and the growth restricting the function of CUC2 using the ubiquitous inducible CUC2-GR transgene. In time courses, we dissected the serration promoting the function of CUC2 in the leaf margin and ectopic growth inhibition by CUC2 in the leaf plate. We found that CUC2 limits growth rather by cell cycle inhibition than by cell size control. Furthermore, endogenous CUC2 was rapidly induced by CUC2-GR indicating a possible auto-inducible feedback. In contrast, EPFL2 was quickly decreased by transient CUC2 induction but increased in cuc2-3 mutant leaves suggesting that CUC2 can also counteract the EPFL2-ER pathway. Therefore, tooth growth promotion and growth inhibition by CUC2 involve partially the same mechanism but in contrary ways.

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.

Plant leaves undergo a series of developmental changes from leaf primordium initiation through growth and maturation to senescence throughout their life span. Although the mechanisms underlying leaf senescence have been intensively elucidated, our knowledge of the interrelationship between early leaf development and senescence is still fragmentary. We isolated the oresara15-1Dominant (ore15-1D) mutant, which had an extended leaf longevity and an enlarged leaf size, from activation-tagged lines of Arabidopsis. Plasmid rescue identified that ORE15 encodes a PLANT A/T-RICH SEQUENCE- AND ZINC-BINDING PROTEIN family transcription factor. Phenotypes of ore15-1D and ore15-2, a loss-of-function mutant, were evaluated through physiological and anatomical analyses. Microarray, quantitative reverse transcription polymerase chain reaction, and chromatin immunoprecipitation as well as genetic analysis were employed to reveal the molecular mechanism of ORE15 in the regulation of leaf growth and senescence. ORE15 enhanced leaf growth by promoting the rate and duration of cell proliferation in the earlier stage and suppressed leaf senescence in the later stage by modulating the GROWTH-REGULATING FACTOR (GRF)/GRF-INTERACTING FACTOR regulatory pathway. Our study highlighted a molecular conjunction through ORE15 between growth and senescence, which are two temporally separate developmental processes during leaf life span., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Strobilurins are an important class of agrochemical fungicides used throughout the world on a wide variety of crops as protection against fungal pathogens. In addition to this protective role, they are reported to also positively influence plant physiology. In this study, we analysed the effect of Stroby® WG, a commercially available fungicide consisting of 50% (w/w) kresoxim-methyl (KM) as active strobilurin compound, on Arabidopsis leaf growth. Treatment of seedlings with Stroby resulted in larger leaves due to an increase in cell number. Transcriptome analysis of Stroby-treated rosettes demonstrated an increased expression of genes involved in redox homeostasis, iron metabolism and sugar transport. Stroby treatment strongly induced the expression of the subgroup Ib basic helix-loop-helix (bHLH) transcription factors, which have a role in iron homeostasis under iron-limiting conditions. Single loss-of-function mutants of three bHLHs and their triple bhlh039, bhlh100 and bhlh101 mutant did not respond to Stroby treatment. Although iron and sucrose content was not affected, nitric oxide (NO) levels and nitrate reductase (NR) activity were significantly increased in Stroby-treated rosettes as compared with control plants. In conclusion, we suggest that the Stroby-mediated effects on growth depend on the increased expression of the subgroup Ib bHLHs and higher NO levels., (© 2017 John Wiley & Sons Ltd.)

N -acetyl-5-methoxytryptamine (Melatonin), as a crucial messenger in plants, functions in adjusting biological rhythms, stress tolerance, plant growth and development. Several studies have shown the retardation effect of exogenous melatonin treatment on plant growth and development. However, the in vivo role of melatonin in regulating plant leaf growth and the underlying mechanism are still unclear. In this study, we found that high concentration of melatonin suppressed leaf growth in Arabidopsis by reducing both cell size and cell number. Further kinetic analysis of the fifth leaves showed that melatonin remarkably inhibited cell division rate. Additionally, flow cytometic analysis indicated that melatonin negatively regulated endoreduplication during leaf development. Consistently, the expression analysis revealed that melatonin regulated the transcriptional levels of key genes of cell cycle and ribosome. Taken together, this study suggests that high concentration of melatonin negatively regulated the leaf growth and development in Arabidopsis , through modulation of endoreduplication and the transcripts of cell cycle and ribosomal key genes.

I show that Arabidopsis leaf growth can be described with good precision by a conformal map, where expansion is locally isotropic (the same in all directions) but the amount of expansion can vary with position. Data obtained by tracking leaf growth over time can be reproduced with almost 90% accuracy by such a map. The growth follows a Möbius transformation, which is a type of conformal map that would arise if there were an underlying linear gradient of growth rate. From the data one can derive the parameters that describe this linear gradient and show how it changes over time. Such a rule has the property of maintaining the flatness of a leaf., (Copyright © 2016 The Author. Published by Elsevier Ltd.. All rights reserved.)

Foliar shade triggers rapid growth of specific structures that facilitate access of the plant to direct sunlight. In leaves of many plant species, this growth response is complex because, although shade triggers the elongation of petioles, it reduces the growth of the lamina. How the same external cue leads to these contrasting growth responses in different parts of the leaf is not understood. Using mutant analysis, pharmacological treatment and gene expression analyses, we investigated the role of PHYTOCHROME INTERACTING FACTOR7 (PIF7) and the growth-promoting hormone auxin in these contrasting leaf growth responses. Both petiole elongation and lamina growth reduction are dependent on PIF7. The induction of auxin production is both necessary and sufficient to induce opposite growth responses in petioles vs lamina. However, these contrasting growth responses are not caused by different auxin concentrations in the two leaf parts. Our work suggests that a transient increase in auxin levels triggers tissue-specific growth responses in different leaf parts. We provide evidence suggesting that this may be caused by the different sensitivity to auxin in the petiole vs the blade and by tissue-specific gene expression., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Although abscisic acid (ABA) and gibberellins (GAs) play pivotal roles in many physiological processes in plants, their interaction in the control of leaf growth remains elusive. In this study, genetic analyses of ABA and GA interplay in leaf growth were performed in Arabidopsis thaliana. The results indicate that for the ABA and GA interaction, leaf growth of both the aba2/ga20ox1 and aba2/GA20ox1 plants, which were derived from the crosses of aba2×ga20ox1 and aba2×GA20ox1 overexpressor, respectively, exhibits partially additive effects but is similar to the aba2 mutant. Consistently, the transcriptome analysis suggests that a substantial proportion (45-65%) of the gene expression profile of aba2/ga20ox1 and aba2/GA20ox1 plants overlap and share a pattern similar to the aba2 mutant. Thus, these data suggest that ABA deficiency dominates leaf growth regardless of GA levels. Moreover, the gene ontology (GO) analysis indicates gene enrichment in the categories of hormone response, developmental and metabolic processes, and cell wall organization in these three genotypes. Leaf developmental genes are also involved in the ABA-GA interaction. Collectively, these data support that the genetic relationship of ABA and GA interaction involves multiple coordinated pathways rather than a simple linear pathway for the regulation of leaf growth., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Several genes positively influence final leaf size in Arabidopsis when mutated or overexpressed. The connections between these growth regulators are still poorly understood although such knowledge would further contribute to understand the processes driving leaf growth. In this study, we performed a combinatorial screen with 13 transgenic Arabidopsis lines with an increased leaf size. We found that from 61 analyzed combinations, 39% showed an additional increase in leaf size and most resulted from a positive epistasis on growth. Similar to what is found in other organisms in which such an epistasis assay was performed, only few genes were highly connected in synergistic combinations as we observed a positive epistasis in the majority of the combinations with samba, BRI1(OE) or SAUR19(OE). Furthermore, positive epistasis was found with combinations of genes with a similar mode of action, but also with genes which affect distinct processes, such as cell proliferation and cell expansion.DOI: http://dx.doi.org/10.7554/eLife.02252.001., (Copyright © 2014, Vanhaeren et al.)

The REIL1 and REIL2 proteins of Arabidopsis thaliana are evolutionarily conserved homologs of the cytosolic 60S ribosomal maturation factors Rei1 and its paralog Reh1 of Saccharomyces cerevisiae. We previously demonstrated that the REIL proteins like the yeast homologs are required for the growth of both organisms at suboptimal temperatures. In addition, the cold sensitivity of the yeast Δrei1 mutant was almost fully rescued by heterologous expression of the REIL1 protein. These phenomena and conservation of co-expressed genes linked the function of REIL proteins to the maturation of the eukaryotic ribosome in A. thaliana. Here we demonstrate that REIL proteins interact in yeast-2-hybrid assays with A. thaliana homologs of the yeast proteins, Rlp24, Rpl24A, Rlp24B, Arx1, and Jjj1. These proteins take part in the cytosolic 60S ribosomal maturation process within yeast and physically interact with Rei1. Our study does not provide proof but is consistent with a conserved role of the A. thaliana REIL proteins in ribosomal maturation and demonstrates the potential of future investigations that aim to unravel the protein interactions of REIL proteins in planta.

Developmental biologists have been fascinated with the long-standing mystery of how multicellular organisms, such as plants and animals, sense and control their organ size. In plants, leaves are a suitable experimental system for elucidation of the mystery, because they, like animal organs, inherently exhibit a determinate growth pattern, meaning that they possess genetic information for the control of their final size. The cell proliferation and expansion processes are prerequisites for growth, so that the genetic controls should converge on the 2 cellular processes and decide their rate or duration during leaf growth. Plant scientists have found dozens of genes involved in the control of the cellular processes, including the Arabidopsis thaliana GRF-INTERACTING FACTOR (GIF) family. The GIF family consists of 3 members, GIF1 to GIF3, and encodes a class of transcription co-activators. Although the GIF family genes have been shown to play an essential role in the control of cell proliferation of the leaf organ, understanding of the spatio-temporal behaviors of GIF expression, in both aspects of their promoters and proteins, has been limited to GIF1 (also known as ANGUSTIFOLIA3, AN3). Here, we define kinematic growth properties of wild-type and gif leaf organs and present spatio-temporal expression patterns of all GIF genes, thus providing comprehensive insights into biological roles and expression behaviors of the whole GIF family members during leaf growth.

The polar auxin transport is required for proper plant growth and development. D6 PROTEIN KINASE (D6PK) is required for the phosphorylation of PIN‐FORMED (PIN) auxin efflux carriers to regulate auxin transport, while the regulation of D6PK stabilization is still poorly understood. Here, we found that Cytosolic ABA Receptor Kinases (CARKs) redundantly interact with D6PK, and the interactions are dependent on CARKs' kinase activities. Similarly, CARK3 also could interact with paralogs of D6PK, including D6PKL1, D6PKL2, and D6PKL3. The genetic analysis shows that D6PK acts the downstream of CARKs to regulate Arabidopsis growth, including hypocotyl, leaf area, vein formation, and the length of silique. Loss‐of‐function of CARK3 in overexpressing GFP‐D6PK plants leads to reduce the level of D6PK protein, thereby rescues plant growth. In addition, the cell‐free degradation assays indicate that D6PK is degraded through 26 S proteasome pathway, while the phosphorylation by CARK3 represses this process in cells. In summary, D6PK stabilization by the CARK family is required for auxin‐mediated plant growth and development. Summary statement: The AGC VIII kinase D6 PROTEIN KINASE (D6PK) phosphorylates PIN‐FORMED (PIN) auxin efflux carriers, which is essential for auxin transport in plant cells. This study uncovers a new mechanism for D6PK stability through Cytosolic ABA Receptor Kinases‐mediated phosphorylation to regulate auxin‐controlled Arabidopsis growth and development. [ABSTRACT FROM AUTHOR]

To cope with phosphate (Pi) starvation, plants trigger an array of adaptive responses to sustain their growth and development. These responses are largely controlled at transcriptional levels. In Arabidopsis (Arabidopsis thaliana), PHOSPHATE RESPONSE 1 (PHR1) is a key regulator of plant physiological and transcriptional responses to Pi starvation. PHR1 belongs to a MYB-CC-type transcription factor family which contains 15 members. In this PHR1 family, PHR1/PHR1-like 1(PHL1) and PHL2/PHL3 form two distinct modules in regulating plant development and transcriptional responses to Pi starvation. PHL4 is the most closely related member to PHR1. Previously, using the phr1phl4 mutant, we showed that PHL4 is also involved in regulating plant Pi responses. However, the precise roles of PHL1 and PHL4 in regulating plant Pi responses and their functional relationshipswith PHR1 have not been clearly defined. In this work, we further used the phl1phl4 and phr1phl1phl4 mutants to perform comparative phenotypic and transcriptomic analyses with phr1, phr1phl1, and phr1phl4. The results showed that both PHL1 and PHL4 act redundantly and equally with PHR1 to regulate leaf senescence, Pi starvation induced-inhibition of primary root growth, and accumulation of anthocyanins in shoots. Unlike PHR1 and PHL1, however, the role of PHL4 in maintaining Pi homeostasis is negligible. In regulating transcriptional responses to Pi starvation at genomic levels, both PHL1 and PHL4 play minor roles when acts alone, however, they act synergistically with PHR1. In regulating Pi starvation-responsive genes, PHL4 also function less than PHL1 in terms of the number of the genes it regulates and the magnitude of gene transcription it affects. Furthermore, no synergistic interaction was found between PHL1 and PHL4 in regulating plant response to Pi starvation. Therefore, our results clarified the roles of PHL1 and PHL4 in regulating plant responses to Pi starvation. In addition, this work revealed a new function of these three transcription factors in regulating flowering time. [ABSTRACT FROM AUTHOR]

A number of studies have demonstrated that epigenetic factors regulate plant developmental timing in response to environmental changes. However, we still have an incomplete view of how epigenetic factors can regulate developmental events such as organogenesis, and the transition from cell division to cell expansion, in plants. The small number of cell types and the relatively simple developmental progression required to form the Arabidopsis petal makes it a good model to investigate the molecular mechanisms driving plant organogenesis. In this study, we investigated how the RABBIT EARS (RBE) transcriptional repressor maintains the downregulation of its downstream direct target, TCP5, long after RBE expression dissipates. We showed that RBE recruits the Groucho/Tup1-like corepressor TOPLESS (TPL) to repress TCP5 transcription in petal primordia. This process involves multiple layers of changes such as remodeling of chromatin accessibility, alteration of RNA polymerase activity, and histone modifications, resulting in an epigenetic memory that is maintained through multiple cell divisions. This memory functions to maintain cell divisions during the early phase of petal development, and its attenuation in a cell division-dependent fashion later in development enables the transition from cell division to cell expansion. Overall, this study unveils a novel mechanism by which the memory of an epigenetic state, and its cell-cycle regulated decay, acts as a timer to precisely control organogenesis. Author summary: Epigenetic changes, such as histone modifications or DNA methylation, can modify gene expression without affecting the genetic code. Such alterations can persist for many cell generations, and often are induced by various environmental inputs in both plants and animals. By contrast, little is known of how intrinsic epigenetic changes can act to regulate the temporal progression of development. Here we showed that the expression of RABBIT EARS (RBE) represses the transcription of its target, TCP5, through inducing a variety of epigenetic changes at the TCP5 locus. These changes dissipated over a period of days, allowing for eventual activation of TCP5 expression and a concomitant shift from cell division to cell expansion during Arabidopsis petal development. The gradual loss of repressive epigenetic marks at the TCP5 locus represents an internal timing mechanism to temporally control the development of the Arabidopsis petal. [ABSTRACT FROM AUTHOR]

Plant growth is a complex process affected by a multitude of genetic and environmental factors and their interactions. To identify genetic factors influencing plant performance under different environmental conditions, vegetative growth was assessed in Arabidopsis thaliana cultivated under constant or fluctuating light intensities, using high-throughput phenotyping and genome-wide association studies. Daily automated non-invasive phenotyping of a collection of 382 Arabidopsis accessions provided growth data during developmental progression under different light regimes at high temporal resolution. Quantitative trait loci (QTL) for projected leaf area, relative growth rate, and PSII operating efficiency detected under the two light regimes were predominantly condition-specific and displayed distinct temporal activity patterns, with active phases ranging from 2 d to 9 d. Eighteen protein-coding genes and one miRNA gene were identified as potential candidate genes at 10 QTL regions consistently found under both light regimes. Expression patterns of three candidate genes affecting projected leaf area were analysed in time-series experiments in accessions with contrasting vegetative leaf growth. These observations highlight the importance of considering both environmental and temporal patterns of QTL/allele actions and emphasize the need for detailed time-resolved analyses under diverse well-defined environmental conditions to effectively unravel the complex and stage-specific contributions of genes affecting plant growth processes. [ABSTRACT FROM AUTHOR]

Protein carbonylation is an irreversible form of post-translational modification triggered by reactive oxygen species in animal and plant cells. It occurs either through the metal-catalyzed oxidation of Lys, Arg, Pro, and Thr side chains or the addition of α, β-unsaturated aldehydes and ketones to the side chains of Cys, Lys, and His. Recent genetic studies concerning plants pointed to an implication of protein carbonylation in gene regulation through phytohormones. However, for protein carbonylation to stand out as a signal transduction mechanism, such as phosphorylation and ubiquitination, it must be controlled in time and space by a still unknown trigger. In this study, we tested the hypothesis that the profile and extent of protein carbonylation are influenced by iron homeostasis in vivo. For this, we compared the profile and the contents of the carbonylated proteins in the Arabidopsis thaliana wild-type and mutant-deficient in three ferritin genes under normal and stress conditions. Additionally, we examined the proteins specifically carbonylated in wild-type seedlings exposed to iron-deficient conditions. Our results indicated that proteins were differentially carbonylated between the wild type and the triple ferritin mutant Fer1-3-4 in the leaves, stems, and flowers under normal growth conditions. The profile of the carbonylated proteins was also different between the wild type and the ferritin triple mutant exposed to heat stress, thus pointing to the influence of iron on the carbonylation of proteins. Consistent with this, the exposure of the seedlings to iron deficiency and iron excess greatly influenced the carbonylation of certain proteins involved in intracellular signal transduction, translation, and iron deficiency response. Overall, the study underlined the importance of iron homeostasis in the occurrence of protein carbonylation in vivo. [ABSTRACT FROM AUTHOR]

SEPALLATA (SEP) genes encode E-class floral homeotic transcriptional factors involved in regulation of flowering. In the present work, artificial microRNA (amiRNA) technology was adopted to downregulate the expression levels of SEP3-2, SEP3-3 and SEP4. Silencing of SEP3-2 and SEP3-3 did not generate obvious phenotypic changes in the four types of floral organs. However, knockdown of SEP3-2 and SEP3-3 resulted in suppression of FLOWERING LOCUS T (FT) and GIGANTEA (GI), accompanied by a delay in flowering time. SEP3-2 can directly bind to CArG-boxes existing in the promoter of AGL17, AGL20, AGL42, CCA1, FT, TCP3, TCP12, while SEP3-3 can directly interact with CArG-boxes existing in the promoter of AGL15, AGL24, CUC2, MAF5 and TCP2, indicating the encoded products of two alternative splicing variants of SEP3 can affect distinct target genes. These results suggest that SEP3-2 and SEP3-3 are probably involved in controlling different developmental processes. Compared to wild-type plants, the vegetative growth of amiRNA-SEP3-2 and amiRNA-SEP3-3 transgenic plants was more vigorous, and the morphology of leaves was changed significantly, including a widening the blade, increased surface area, longer petiole and serrated margin. In amiRNA-SEP4 transgenic plants, sepals were converted into petaloid structures, and the number of floral organs in the outside three whorls was reduced. In addition, amiRNA-SEP4 transgenic plants presented a late-flowering phenotype, illustrating the important roles of SEP4 in floral transition. These results showed that SEP3-2 and SEP3-3, two splicing variants of SEP3, involved in morphogenesis of leaves and floral organ development by conferring transcriptional activity. Moreover, SEP4 genes participate in development of sepals, petals, stamens and carpels. All these results indicated that SEPALLATA genes of Arabidopsis possessed diversified functions. In addition to the roles in reproductive growth, these genes can also influence the vegetative growth. [ABSTRACT FROM AUTHOR]

Chloroplast-localized NAD kinase (NADK2) is responsible for the production of NADP+, which is an electron acceptor in the linear electron flow of photosynthesis. The Arabidopsis T-DNA-inserted mutant of NADK2 (nadk2) showed delayed growth and pale-green leaves under continuous light conditions. Under short-day conditions (8 h light / 16 h dark), the nadk2 mutant showed more severe growth inhibition.The genomic fragment containing the promoter and coding region of NADK2 complemented the phenotypes of nadk2 obtained under continuous light and short-day conditions. The nadk2 mutant produced higher amounts of H2O2 and O2−, which were reduced in the complementary line. Under short-day conditions, the nadk2 mutant accumulated more H2O2 than under continuous light conditions. The accumulation of ascorbate and up-regulation of the PDF1.2 and PR1 genes indicated that the nadk2 mutant is under ROS stress and responding to keep its living activities. [ABSTRACT FROM AUTHOR]

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‐RESPONSIVE ELEMENT BINDING 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 ERF4 promotes 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. [ABSTRACT FROM AUTHOR]

Background: Salicylic acid (SA) is one of the plant hormones, which plays crucial roles in signaling transduction in plant growth, disease resistance, and leaf senescence. Arabidopsis (Arabidopsis thaliana) SA 3-hydroxylase (S3H) and 5-hydroxylase (S5H) are key enzymes which maintain SA homeostasis by catalyzing SA to 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA, respectively. Results: SA deficient transgenic Arabidopsis lines were generated by introducing two binary vectors S5Hpro::EGFP-S3H and 35Spro::EGFP-S3H respectively, in which the expression of S3H is under the control of the S5H promoter or CaMV 35S promoter. Compared with the constitutive expression of S3H gene under the control of 35S promoter, the S3H gene under the native S5H promoter is activated by endogenous SA and results in a dynamic control of SA catabolism in a feedback mode. The SA accumulation, growth, leaf senescence, and pathogen resistance of the S5Hpro::GFP-S3H transgenic plants were investigated in parallel with NahG transgenic plants. The SA levels in the S5Hpro::EGFP-S3H transgenic plants were similar to or slightly lower than those of NahG transgenic Arabidopsis and resulted in SA deficient phenotypes. The low-SA trait of the S5Hpro::EGFP-S3H transgenic lines was inherited stably in the later generations. Conclusions: Compared with NahG transgenic lines producing by-product catechol, S5Hpro::EGFP-S3H transgenic lines reduce SA levels by converting SA to a native product 2,3-DHBA for catabolism. Together, we provide new SA-deficient germplasms for the investigations of SA signaling in plant development, leaf senescence, and disease resistance. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is an essential nutrient for plants. Membrane lipid remodeling is an adaptive mechanism for P-starved plants that replaces membrane phospholipids with non-P galactolipids, presumably to retrieve scarce P sources and maintain membrane integrity. Whereas metabolic pathways to convert phospholipids to galactolipids are well-established, the mechanism by which phospholipid biosynthesis is involved in this process remains elusive. Here, we report that phospho-base N -methyltransferases 1 and 2 (PMT1 and PMT2), which convert phosphoethanolamine to phosphocholine (PCho), are transcriptionally induced by P starvation. Shoots of seedlings of pmt1 pmt2 double mutant showed defective growth upon P starvation; however, membrane lipid profiles were unaffected. We found that P-starved pmt1 pmt2 with defective leaf growth had reduced PCho content, and the growth defect was rescued by exogenous supplementation of PCho. We propose that PMT1 and PMT2 are induced by P starvation to produce PCho mainly for leaf growth maintenance, rather than for phosphatidylcholine biosynthesis, in membrane lipid remodeling. [ABSTRACT FROM AUTHOR]

Leaf laminar growth and adaxial–abaxial boundary formation are fundamental outcomes of plant development. Boundary and laminar growth coordinate the further patterning and growth of the leaf, directing the differentiation of cell types within the top and bottom domains and promoting initiation of lateral organs along their adaxial or abaxial axis. Leaf adaxial–abaxial polarity specification and laminar outgrowth are regulated by two transcription factors, REVOLUTA (REV) and KANADI (KAN). ABA INSENSITIVE TO GROWTH 1 (ABIG1) encodes a HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) class II transcription factor and is a direct target of the adaxial–abaxial regulators REV and KAN. To investigate the role of ABIG1 in leaf development and in the establishment of polarity, we examined the phenotypes of both gain-of-function and loss-of-function mutants. Through genetic interaction analysis with REV and KAN mutants, we determined that ABIG1 plays a role in leaf laminar growth as well as in adaxial–abaxial polarity establishment. Genetic and physical interaction assays showed that ABIG1 interacts with the transcriptional TOPLESS corepressor. This study provides new evidence that ABIG1, another HD-ZIP II, facilitates growth through the corepressor TOPLESS. [ABSTRACT FROM AUTHOR]

Background: Cymbidium is an economically important genus of flowering orchids cultivated in China because of showing graceful leaf shapes and elegant flower coloration. However, the deterioration of the ecological environment and the difficulty of conservation management have become increasing challenges for maintaining its germplasm resources. ARFs are critical transcription factors in the auxin signaling pathway and have been found to play pivotal roles in leaf growth and development in previous studies. However, their functions and mechanisms in Cymbidium goeringii remain to be clarified. Methods: The sequence of the CgARF1 gene was analyzed by bioinformatics. The expression of this gene in different tissues and under IAA treatment was detected by quantitative real-time PCR analysis. The CgARF1 gene was overexpressed in wild-type Arabidopsis and Nicotiana benthamiana via the Agrobacterium infection method. Acetone-ethanol solvent extraction was applied for the determination of chlorophyll contents, and the contents of endogenous hormones were determined using the enzyme-linked immunosorbent assay technique. Results: CgARF1 cloned from C. goeringii 'Songmei' was 2,049 bp in length and encoded 682 amino acids containing three typical domains: a B3 DNA binding domain, an Aux_resp domain and an AUX/IXX family domain. The expression pattern of CgARF1 in different tissues of C. goeringii showed that its expression was highest in the leaves and changed greatly under IAA treatment. Subcellular localization studies showed that the protein was mainly localized in the cell nucleus. CgARF1-overexpressing lines exhibited leaf senescence and a decreased chlorophyll content. Under IAA treatment, CgARF1 regulates the rooting length, rooting number and rooting rate from detached leaves. The levels of endogenous hormones in transgenic leaves were also significantly changed. Conclusion: These results indicated that CgARF1 overexpression is responsive to IAA treatment during leaf development. This study provides a foundation for future research on the function of the ARF gene family in C. goeringii. [ABSTRACT FROM AUTHOR]

Purine degradation products have been shown to play roles in plant response to stresses such as drought, salinity, extended dark, nitrogen deficiency, and pathogen infection. In this study, we used Arabidopsis wild-type (WT) and an Atxdh1 -knockout mutant defective in xanthine dehydrogenase1 (XDH1) to examine the role of degraded purine metabolites in the responses to wounding or UV-C stress applied to the middle leaves of the plant. Wounding or UV-C stress in the mutant resulted in lower fresh-weight, increased senescence symptoms, and increased cell death compared to WT plants. In addition, WT plants exhibited lower levels of oxidative stress indicators, reactive oxygen species, and malondialdehyde in their leaves than the mutant. Notably, transcripts and proteins functioning in the purine degradation pathway were regulated in such a way that it led to enhanced ureide levels in WT leaves 24h after applying the UV-C or wound stress. However, different remobilization of the accumulated ureides was observed after 72h of stress. In plants treated with UV-C, the concentration of allantoin was highest in young leaves, whereas in wounded plants it was lowest in these leaves and instead accumulated mainly in the middle leaves that had been wounded. These results indicated that in WT plants treated with UV-C, ureides were remobilized from the lower older and damaged leaves to support young leaf growth during the recovery period from stress. After wounding, however, whilst some ureides were remobilized to the young leaves, more remained in the wounded middle leaves to function as antioxidants and/or healing agents. [ABSTRACT FROM AUTHOR]

Arabidopsis non-host resistance against non-adapted fungal pathogens including Colletotrichum fungi consists of pre-invasive and post-invasive immune responses. Here we report that non-host resistance against non-adapted Colletotrichum spp. in Arabidopsis leaves requires CURLY LEAF (CLF), which is critical for leaf development, flowering and growth. Microscopic analysis of pathogen behavior revealed a requirement for CLF in both pre- and post-invasive non-host resistance. The loss of a functional SEPALLATA3 (SEP3) gene, ectopically expressed in clf mutant leaves, suppressed not only the defect of the clf plants in growth and leaf development but also a defect in non-host resistance against the non-adapted Colletotrichum tropicale. However, the ectopic overexpression of SEP3 in Arabidopsis wild-type leaves did not disrupt the non-host resistance. The expression of multiple plant defensin (PDF) genes that are involved in non-host resistance against C. tropicale was repressed in clf leaves. Moreover, the Octadecanoid-responsive Arabidopsis 59 (ORA59) gene, which is required for PDF expression, was also repressed in clf leaves. Notably, when SEP3 was overexpressed in the ora59 mutant background, C. tropicale produced clear lesions in the inoculated leaves, indicating an impairment in non-host resistance. Furthermore, ora59 plants overexpressing SEP3 exhibited a defect in leaf immunity to the adapted Colletotrichum higginsianum. Since the ora59 plants overexpressing SEP3 did not display obvious leaf curling or reduced growth, in contrast to the clf mutants, these results strongly suggest that concomitant SEP3 repression and ORA59 induction via CLF are required for Arabidopsis leaf immunity to Colletotrichum fungi, uncoupled from CLF's function in growth and leaf development. [ABSTRACT FROM AUTHOR]

The epidermal cell layer of plants has important functions in regulating plant growth and development. We have studied the impact of an altered epidermal cytokinin metabolism on Arabidopsis shoot development. Increased epidermal cytokinin synthesis or breakdown was achieved through expression of the cytokinin synthesis gene LOG4 and the cytokinin-degrading CKX1 gene, respectively, under the control of the epidermis-specific AtML1 promoter. During vegetative growth, increased epidermal cytokinin production caused an increased size of the shoot apical meristem and promoted earlier flowering. Leaves became larger and the shoots showed an earlier juvenile-to-adult transition. An increased cytokinin breakdown had the opposite effect on these phenotypic traits indicating that epidermal cytokinin metabolism can be a factor regulating these aspects of shoot development. The phenotypic consequences of abbreviated cytokinin signaling in the epidermis achieved through expression of the ARR1-SRDX repressor were generally milder or even absent indicating that the epidermal cytokinin acts, at least in part, cell non-autonomously. Enhanced epidermal cytokinin synthesis delayed cell differentiation during leaf development leading to an increased cell proliferation and leaf growth. Genetic analysis showed that this cytokinin activity was mediated mainly by the AHK3 receptor and the transcription factor ARR1. We also demonstrate that epidermal cytokinin promotes leaf growth in a largely cell-autonomous fashion. Increased cytokinin synthesis in the outer layer of reproductive tissues and in the placenta enhanced ovule formation by the placenta and caused the formation of larger siliques. This led to a higher number of seeds in larger pods resulting in an increased seed yield per plant. Collectively, the results provide evidence that the cytokinin metabolism in the epidermis is a relevant parameter determining vegetative and reproductive plant growth and development. [ABSTRACT FROM AUTHOR]

Light is a key limiting factor of plant growth and development under the canopy. Specific light signals, such as a low ratio of red : far‐red (R:FR) light, trigger the shade avoidance response, which affects hypocotyl, stem, and leaf growth. Although multiple components mediating shade avoidance responses have been identified in the past few decades, the underlying regulatory mechanism remains unclear. In this study, we found that the far‐red elongated hypocotyls 3 (fhy3) mutant exhibited longer hypocotyls and increased expression levels of core shade avoidance response genes under low R:FR shade conditions compared with the wild type No‐0, suggesting that FHY3 negatively regulates shade avoidance responses. Yeast one‐hybrid, chromatin immunoprecipitation, and RT‐qPCR assays revealed that FHY3 directly binds to the promoters and gene body of PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2 and activates their expression to inhibit shade responses. Furthermore, the overexpression of PAR1 or PAR2 rescued the enhanced shade avoidance responses of fhy3, indicating that both genes are direct downstream targets of FHY3 that mediate shade avoidance responses. Our findings demonstrate that the light‐signalling protein FHY3 positively regulates the transcription of PAR1 and PAR2, which encode two key negative regulators of shade avoidance responses, thus repressing plant responses to shade signals. Our findings demonstrate that the light‐signalling protein FHY3 positively regulates the transcription of PAR1 and PAR2, which encode two key negative regulators of shade avoidance responses, thus repressing plant responses to shade signals. [ABSTRACT FROM AUTHOR]

In this study, we investigated the regulatory function of miR396 in the phase transition in Arabidopsis thaliana. Using AtMIR396a/b knockout mutants generated through clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-directed genome editing, we showed that miR396 negatively regulates the leaf size and vegetative phase transition, and the first leaf with abaxial trichomes appeared earlier in the mir396ab double mutant than in the wild type (WT) and was significantly delayed in miR396 overexpression lines. Moreover, mir396ab exhibited early flowering, whereas 35S:MIR396a/b and cib4-1 delayed flowering, and the flowering time was negatively correlated with FT gene expression. Furthermore, in arp6 and pie1 mutants, which are deficient in the ATP-dependent chromatin remodeling complex (SWR1-C), miR396 expression was significantly repressed. Compared with the WT, reduced H2A.Z deposit and stronger relative nucleosome occupancy in the promoter region of MIR396a was found in the arp6 mutant, indicating that SWR1-C contributes to the transcriptional activation of MIR396a via nucleosome dynamics. In addition, miR396 displayed specific spatio-temporal expression patterns in the leaf, which was altered in arp6 and pie1 , and therefore affected the transcript levels of CIB4 and FT in these mutants. We propose that miR396 is not only a marker of cell differentiation, but also an age signal for leaf development and phase change. Meanwhile, SWR1-C-mediated epigenetic regulation contributes to the age-dependent enhancement of miR396 expression and differential miR396 accumulation among leaves. [ABSTRACT FROM AUTHOR]

FtsH is an essential ATP-dependent metalloprotease for protein quality control in the thylakoid membrane of Arabidopsis thaliana chloroplasts. It is required for chloroplast development during leaf growth, and particularly for the specific degradation of photo-damaged D1 protein in the photosystem II (PSII) complex to maintain photosynthesis activity. In the thylakoid membrane, the reversible phosphorylation of proteins is known to control the activity and remodeling of photosynthetic complexes, and previous studies implicate that FtsH is also phosphorylated. We therefore assessed the phosphorylation status of FtsH and its possible role in the regulatory mechanism in this study. The phosphorylation level of FtsHs that compose the FtsH heterohexameric complex was investigated by phosphate-affinity gel electrophoresis using a Phos-Tag molecule. Phos-tag SDS-PAGE of thylakoid proteins and subsequent immunoblot analysis showed that both type A (FtsH1/5) and type B (FtsH2/8) subunits were separable into phosphorylated and non-phosphorylated forms. Neither different light conditions nor the lack of two major thylakoid kinases, STN7 and STN8, resulted in any clear difference in FtsH phosphorylation, suggesting that this process is independent of the light-dependent regulation of photosynthesis-related proteins. Site-directed mutagenesis of putatively phosphorylated Ser or Thr residues into Ala demonstrated that Ser-212 may play a role in FtsH stability in the thylakoid membranes. Different phosphorylation status of FtsH oligomers analyzed by two-dimensional clear-native/Phos-tag SDS-PAGE implied that phosphorylation partially affects FtsH complex formation or its stability. [ABSTRACT FROM AUTHOR]

The mutants Atnoa1 and Atnia1nia2noa1-2 having a defective chloroplast developmental process, showed enhanced chlorophyll levels when they were grown on Murashige and Skoog (MS) medium and on exposure with uranium (U) on Hoagland medium. Thus we hypothesized that these mutants probably produced NO in MS medium and on exposure with U. Wild-type Col-0, Atnoa1 , Atnia1nia2noa1-2 plants were cultured on modified Hoagland and 1/10 MS media and NO generation in the roots of these mutants was monitored using NO selective fluorescent dyes, DAF-2DA and Fl2E. Both Atnoa1 and Atnia1nia2noa1-2 triple mutants produced NO as observed by increases in DAF-2T and Fl2E fluorescence when these mutants were grown on MS medium but not on Hoagland medium. In presence of NO scavenger, methylene blue (MB, 200 μM), DAF-2T and Fl2E fluorescence was completely abolished. On the other hand treatment of the plants with 25 μM U triggered NO generation. U-treated Atnoa1 and Atnia1nia2noa1-2 plants upregulated genes (POR B, POR D, CHL D) involved in the chlorophyll biosynthesis. From these results it was concluded that Atnoa1 and Atnia1nia2noa1-2 are conditional NO producers and it appears that NO generation in plants substantially depends on growth medium and NIA1, NIA2 or NOA1 does not appear to be really involved in NO generation in MS medium or after U exposure. • Alleged NO-suppressed mutants (Atnoa1 and Atnia1,2noa1-2) exhibit pale phenotype. • Atnoa1 and Atnia1,2noa1-2 exhibited greening in their leaves on MS growth medium. • Atnoa1 and Atnia1,2noa1-2 produced NO on MS growth medium. • Atnoa1 and Atnia1,2noa1-2 produced NO on U exposure in Hoagland medium. [ABSTRACT FROM AUTHOR]

• AtSPX1 might function as positive regulator of age-triggered leaf senescence in Arabidopsis thaliana. • During leaf ageing, AtSPX1 expression level is dramatically increased, leading to the upregulation of several key senescence-related transcription factors, such as WRKYs and NACs. • AtSPX1 plays a key role in the crosstalk among leaf senescence, Pi starvation and SA signal transduction. Leaf senescence is the final stage of leaf growth, a highly coordinated and complicated process. Phosphorus as an essential macronutrient for plant growth is remobilized from senescing leaves to other vigorous parts of the plant. In this study, through data mining, we found some phosphate starvation induced genes such as AtSPX1 , were significantly induced in aging leaves in Arabidopsis. We applied a reverse genetics approach to investigate the phenotypes of transgenic plants and mutant plants, and the results showed that the overexpression of AtSPX1 accelerated leaf senescence, suppressed Pi accumulation, promoted SA production and H 2 O 2 levels in leaves, while the mutant lines of AtSPX1 showed slightly delayed leaf senescence. We conducted RNA-seq-based transcriptome analysis together with GO and GSEA enrichment analyses for transgenic vs. wild-type plants to elucidate the possible underlying regulatory mechanism. The 558 genes that were up-regulated in the overexpression plants 35S::AtSPX1/WT, were significantly enriched in the process of leaf senescence, Pi starvation responses and SA signaling pathways, as were the target genes of some transcription factors such as WRKYs and NACs. In a word, we characterized AtSPX1 as a key regulator, which mediated the crosstalks among leaf senescence, Pi starvation and SA signaling pathways in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

The phenylpropanoid pathway, the source of a large array of compounds with diverse functions, starts with the synthesis of trans-cinnamic acid (t-CA) that is converted by cinnamate-4-hydroxylase (C4H) into p-coumaric acid. We have recently shown that in Arabidopsis, exogenous t-CA promotes leaf growth by increasing cell expansion and that this response requires auxin signaling. We have also shown that cell expansion is increased in C4H loss-of-function mutants. Here we provide further evidence that leaf growth is enhanced by either t-CA or a t-CA derivative that accumulates upstream of C4H. We also show that this growth response pathway has two components: one that requires auxin signaling and another which employs a currently unknown mechanism. [ABSTRACT FROM AUTHOR]

Developing leaves undergo sequential coordinated cell proliferation and cell expansion to determine their final size and shape. Although several important regulators of cell proliferation have been reported, the gene network regulating leaf developmental processes remains unclear. Previously, we showed that ORESARA15 (ORE15) positively regulates the rate and duration of cell proliferation by promoting the expression of direct targets, GROWTH-REGULATING FACTOR (GRF) transcription factors, during leaf growth. In the current study, we examined the spatiotemporal patterns of ORE15 expression and determined that ORE15 expression partially overlapped with AN3/GIF1 and ANT expression along the midvein in the proximal region of the leaf blade in young leaves. Genetic analysis revealed that ORE15 may function synergistically with AN3 to control leaf growth as a positive regulator of cell proliferation. Our molecular and genetic studies are the first to suggest the importance of functional redundancies between ORE15 and AN3, and between AN3 and ANT in cell proliferation regulatory pathway during leaf growth.

The information that connects genotypes and phenotypes is essentially embedded in research articles written in natural language. To facilitate access to this knowledge, we constructed a framework for the curation of the scientific literature studying the molecular mechanisms that control leaf growth and development in Arabidopsis thaliana (Arabidopsis). Standard structured statements, called relations, were designed to capture diverse data types, including phenotypes and gene expression linked to genotype description, growth conditions, genetic and molecular interactions, and details about molecular entities. Relations were then annotated from the literature, defining the relevant terms according to standard biomedical ontologies. This curation process was supported by a dedicated graphical user interface, called Leaf Knowtator. A total of 283 primary research articles were curated by a community of annotators, yielding 9947 relations monitored for consistency and over 12,500 references to Arabidopsis genes. This information was converted into a relational database (KnownLeaf) and merged with other public Arabidopsis resources relative to transcriptional networks, protein–protein interaction, gene co-expression, and additional molecular annotations. Within KnownLeaf, leaf phenotype data can be searched together with molecular data originating either from this curation initiative or from external public resources. Finally, we built a network (LeafNet) with a portion of the KnownLeaf database content to graphically represent the leaf phenotype relations in a molecular context, offering an intuitive starting point for knowledge mining. Literature curation efforts such as ours provide high quality structured information accessible to computational analysis, and thereby to a wide range of applications. DATA: The presented work was performed in the framework of the AGRON-OMICS project (Arabidopsis GRO wth Network integrating OMICS technologies) supported by European Commission 6th Framework Programme project (Grant number LSHG-CT-2006-037704). This is a data integration and data sharing portal collecting all the all the major results from the consortium. All data presented in our paper is available here. https://agronomics.ethz.ch/.

Leaf growth is a complex, quantitative trait, controlled by a plethora of regulatory mechanisms. Diverse environmental stimuli inhibit leaf growth to cope with the perceived stress. In plant research, mannitol is often used to impose osmotic stress and study the underlying growth-repressing mechanisms. In growing leaf tissue of plants briefly exposed to mannitol-induced stress, a highly interconnected gene regulatory network is induced. However, early signalling and associated protein phosphorylation events that probably precede part of these transcriptional changes and that potentially act at the onset of mannitol-induced leaf size reduction are largely unknown. Here, we performed a proteome and phosphoproteome analysis on growing leaf tissue of Arabidopsis thaliana plants exposed to mild mannitol-induced stress and captured the fast (within the first half hour) events associated with this stress. Based on this in-depth data analysis, 167 and 172 differentially regulated proteins and phosphorylated sites were found. We provide these data sets as a community resource and we flag differentially phosphorylated proteins with described growth-regulatory functions, but we also illustrate potential novel regulators of shoot growth. [ABSTRACT FROM AUTHOR]

Leaf growth is a complex process that involves the action of diverse transcription factors (TFs) and their downstream gene regulatory networks. In this study, we focus on the functional characterization of the Arabidopsis thaliana TF GROWTH-REGULATING FACTOR9 (GRF9) and demonstrate that it exerts its negative effect on leaf growth by activating expression of the bZIP TF OBP3-RESPONSIVE GENE 3 (ORG3). While grf9 knockout mutants produce bigger incipient leaf primordia at the shoot apex, rosette leaves and petals than the wild type, the sizes of those organs are reduced in plants overexpressing GRF9 (GRF9ox). Cell measurements demonstrate that changes in leaf size result from alterations in cell numbers rather than cell sizes. Kinematic analysis and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay revealed that GRF9 restricts cell proliferation in the early developing leaf. Performing in vitro binding site selection, we identified the 6-base motif 5'-CTGACA-3' as the core binding site of GRF9. By global transcriptome profiling, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) we identified ORG3 as a direct downstream, and positively regulated target of GRF9. Genetic analysis of grf9 org3 and GRF9ox org3 double mutants reveals that both transcription factors act in a regulatory cascade to control the final leaf dimensions by restricting cell number in the developing leaf. [ABSTRACT FROM AUTHOR]

N-acetyl-5-methoxytryptamine (Melatonin), as a crucial messenger in plants, functions in adjusting biological rhythms, stress tolerance, plant growth and development. Several studies have shown the retardation effect of exogenous melatonin treatment on plant growth and development. However, the in vivo role of melatonin in regulating plant leaf growth and the underlying mechanism are still unclear. In this study, we found that high concentration of melatonin suppressed leaf growth in Arabidopsis by reducing both cell size and cell number. Further kinetic analysis of the fifth leaves showed that melatonin remarkably inhibited cell division rate. Additionally, flow cytometic analysis indicated that melatonin negatively regulated endoreduplication during leaf development. Consistently, the expression analysis revealed that melatonin regulated the transcriptional levels of key genes of cell cycle and ribosome. Taken together, this study suggests that high concentration of melatonin negatively regulated the leaf growth and development in Arabidopsis, through modulation of endoreduplication and the transcripts of cell cycle and ribosomal key genes. [ABSTRACT FROM AUTHOR]

Simulation has become an important tool for studying plant physiology. An important aspect of this is discovering the processes that influence leaf growth at a cellular level. To this end, we have extended an existing, morphogen-based model for the growth of Arabidopsis leaves. We have fitted parameters to match important leaf growth properties reported in experimental data. A sensitivity analysis was performed, which allowed us to estimate the effect of these different parameters on leaf growth, and identify viable strategies for increasing leaf size. [ABSTRACT FROM AUTHOR]

AtLSG1-2 is a circularly permuted GTPase required for ribosome biogenesis and recently shown to be involved in early leaf development, although it was unclear how AtLSG1-2 affects leaf growth. Here, we found that atlsg1-2 mutants had reduced leaf size as a result of decreased cell size and cell number. Leaf kinematic analysis and CYCB1;1::GUS expression pattern in atlsg1-2 mutant indicated that loss of function of AtLSG1-2 delays the transition from cell division to cell expansion. Decreases in ploidy levels and trichome branch number suggest that AtLSG1-2 deficiency suppresses endoreduplication. Real-time PCR analysis showed that genes specifically expressed in the proliferation stage were highly expressed and those involved in endoreduplication were differentially regulated. LSG1 is known to mediate the recruitment of nucleocytoplasmic shuttling protein NMD3 back to the nucleus in yeast, yet their relationship was unclear in plants. Our genetic analysis revealed that the atlsg1 atnmd3 double mutant displayed enhanced phenotypes as compared with the respective single mutant and that AtLSG1-2 and AtNMD3 synergistically affect the cell proliferation process. [ABSTRACT FROM AUTHOR]

The developmental plasticity of leaf size and shape is important for leaf function and plant survival. However, the mechanisms by which plants form diverse leaves in response to environmental conditions are not well understood. Here, we identified TIE1-ASSOCIATED RING-TYPE E3 LIGASE1 (TEAR1) and found that it regulates leaf development by promoting the degradation of TCP INTERACTOR-CONTAINING EAR MOTIF PROTEIN1 (TIE1), an important repressor of CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, which are key for leaf development. TEAR1 contains a typical C3H2C3-type RING domain and has E3 ligase activity. We show that TEAR1 interacts with the TCP repressor TIE1, which is ubiquitinated in vivo and degraded by the 26S proteasome system. We demonstrate that TEAR1 is colocalized with TIE1 in nuclei and negatively regulates TIE1 protein levels. Overexpression of TEAR1 rescued leaf defects caused by TIE1 overexpression, whereas disruption of TEAR1 resulted in leaf phenotypes resembling those caused by TIE1 overexpression or TCP dysfunction. Deficiency in TEAR partially rescued the leaf defects of TCP4 overexpression line and enhanced the wavy leaf phenotypes of jaw-5D. We propose that TEAR1 positively regulates CIN-like TCP activity to promote leaf development by mediating the degradation of the TCP repressor TIE1. [ABSTRACT FROM AUTHOR]

Precise floral transition from vegetative growth phase to reproductive growth phase is very important in plant life cycle. In flowering genetic pathways, DELLA proteins are master transcriptional regulators of gibberelic acid ( GA) pathway, and FLOWERING LOCUS C ( FLC) is a core repressor of vernalization pathway as well as downstream of DELLAs. As a crucial messenger in plants, the possible involvement of melatonin ( N-acetyl-5-methoxytryptamine) in flowering and underlying molecular mechanism are unknown in Arabidopsis. In this study, we found that exogenous melatonin treatment delayed floral transition in Arabidopsis. Exogenous melatonin treatment conferred protein stabilizations of DELLAs [ REPRESSOR of ga1-3 ( RGA) and RGA- LIKE3 ( RGL3)], without regulating the transcripts of DELLAs and endogenous GA level. Notably, exogenous melatonin delayed plant flowering and DELLA-activated transcripts of FLC were alleviated in della mutants, and those were exacerbated in DELLA overexpressing plants. Taken together, this study provides direct link between melatonin and floral transition, and indicates the novel involvement of DELLAs-activated FLC in melatonin-mediated flowering in Arabidopsis. [ABSTRACT FROM AUTHOR]

Foliar shade triggers rapid growth of specific structures that facilitate access of the plant to direct sunlight. In leaves of many plant species, this growth response is complex because, although shade triggers the elongation of petioles, it reduces the growth of the lamina. How the same external cue leads to these contrasting growth responses in different parts of the leaf is not understood., Using mutant analysis, pharmacological treatment and gene expression analyses, we investigated the role of PHYTOCHROME INTERACTING FACTOR7 ( PIF7) and the growth-promoting hormone auxin in these contrasting leaf growth responses., Both petiole elongation and lamina growth reduction are dependent on PIF7. The induction of auxin production is both necessary and sufficient to induce opposite growth responses in petioles vs lamina. However, these contrasting growth responses are not caused by different auxin concentrations in the two leaf parts., Our work suggests that a transient increase in auxin levels triggers tissue-specific growth responses in different leaf parts. We provide evidence suggesting that this may be caused by the different sensitivity to auxin in the petiole vs the blade and by tissue-specific gene expression. [ABSTRACT FROM AUTHOR]

Leaf growth is a tightly regulated and complex process, which responds in a dynamic manner to changing environmental conditions, but the mechanisms that reduce growth under adverse conditions are rather poorly understood. We previously identified a growth inhibitory pathway regulating leaf growth upon exposure to a low concentration of mannitol and characterized the ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 transcription factor ERF6 as a central activator of both leaf growth inhibition and induction of stress tolerance genes. Here, we describe the role of the transcriptional repressor ERF11 in relation to the ERF6-mediated stress response in Arabidopsis (Arabidopsis thaliana). Using inducible overexpression lines, we show that ERF6 induces the expression of ERF11. ERF11 in turn molecularly counteracts the action of ERF6 and represses at least some of the ERF6-induced genes by directly competing for the target gene promoters. As a phenotypical consequence of the ERF6-ERF11 antagonism, the extreme dwarfism caused by ERF6 overexpression is suppressed by overexpression of ERF11. Together, our data demonstrate that dynamic mechanisms exist to fine-tune the stress response and that ERF11 counteracts ERF6 to maintain a balance between plant growth and stress defense. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]