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The leaf is a determinate organ with a final size under genetic control. Numerous factors that regulate the final leaf size have been identified in Arabidopsis thaliana ; although most of these factors play their roles during the growth of leaf primordia, much less is known about leaf initiation and its effects on the final leaf size. In this study, we characterized oligocellula6-D (oli6-D), a semidominant mutant of A. thaliana with smaller leaves than the wild type (WT) due to its reduced leaf cell numbers. A time-course analysis showed that oli6-D had approximately 50% fewer leaf cells even immediately after leaf initiation; this difference was maintained throughout leaf development. Next-generation sequencing showed that oli6-D had chromosomal duplications involving 2-kb and 3-Mb regions of chromosomes 2 and 4, respectively. Several duplicated genes examined had approximately 2-fold higher expression levels, and at least one gene acquired a new intron/exon structure due to a chromosome fusion event. oli6-D showed reduced auxin responses in leaf primordia, primary roots and embryos, as well as reduced apical dominance and partial auxin-resistant root growth. CRISPR-associated protein-9-mediated genome editing enabled the removal of a 3-Mb duplicated segment, the largest targeted deletion in plants thus far. As a result, oli6-D restored the WT leaf phenotypes, demonstrating that oli6-D is a gain-of-function mutant. Our results suggest a new regulatory point of leaf size determination that functions at a very early stage of leaf development and is negatively regulated by one or more genes located in the duplicated chromosomal segments. [ABSTRACT FROM AUTHOR]

The resistance of Arabidopsis thaliana to clubroot, a major disease of Brassicaceae caused by the obligate protist Plasmodiophora brassicae, is controlled in part by epigenetic factors. The detection of some of these epigenetic quantitative trait loci (QTLepi) has been shown to depend on experimental conditions. The aim of the present study was to assess whether and how temperature and/or soil water availability influenced both the detection and the extent of the effect of response QTLepi. The epigenetic recombinant inbred line (epiRIL) population, derived from the cross between ddm1-2 and Col-0 (partially resistant and susceptible to clubroot, respectively), was phenotyped for response to P. brassicae under four abiotic conditions including standard conditions, a 5°C temperature increase, drought, and flooding. The abiotic constraints tested had a significant impact on both the leaf growth of the epiRIL population and the outcome of the epiRIL-pathogen interaction. Linkage analysis led to the detection of a total of 31 QTLepi, 18 of which were specific to one abiotic condition and 13 common to at least two environments. EpiRIL showed significant plasticity under epigenetic control, which appeared to be specific to the traits evaluated and to the abiotic conditions. These results highlight that the environment can affect the epigenetic architecture of plant growth and immune responses and advance our understanding of the epigenetic factors underlying plasticity in response to climate change. [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]

Premise: With the global atmospheric CO2 concentration on the rise, developing crops that can thrive in elevated CO2 has become paramount. We investigated the potential of hybridization as a strategy for creating crops with improved growth in predicted elevated atmospheric CO2. Methods: We grew parent accessions and their F1 hybrids of Arabidopsis thaliana in ambient and elevated atmospheric CO2 and analyzed numerous growth traits to assess their productivity and underlying mechanisms. Results: The heterotic increase in total dry mass, relative growth rate and leaf net assimilation rate was significantly greater in elevated CO2 than in ambient CO2. The CO2 response of net assimilation rate was positively correlated with the CO2 response of leaf nitrogen productivity and with that of leaf traits such as leaf size and thickness, suggesting that hybridization‐induced changes in leaf traits greatly affected the improved performance in elevated CO2. Conclusions: Vegetative growth of hybrids seems to be enhanced in elevated CO2 due to improved photosynthetic nitrogen‐use efficiency compared with parents. The results suggest that hybrid crops should be well‐suited for future conditions, but hybrid weeds may also be more competitive. [ABSTRACT FROM AUTHOR]

Premise: With the global atmospheric CO2 concentration on the rise, developing crops that can thrive in elevated CO2 has become paramount. We investigated the potential of hybridization as a strategy for creating crops with improved growth in predicted elevated atmospheric CO2. Methods: We grew parent accessions and their F1 hybrids of Arabidopsis thaliana in ambient and elevated atmospheric CO2 and analyzed numerous growth traits to assess their productivity and underlying mechanisms. Results: The heterotic increase in total dry mass, relative growth rate and leaf net assimilation rate was significantly greater in elevated CO2 than in ambient CO2. The CO2 response of net assimilation rate was positively correlated with the CO2 response of leaf nitrogen productivity and with that of leaf traits such as leaf size and thickness, suggesting that hybridization‐induced changes in leaf traits greatly affected the improved performance in elevated CO2. Conclusions: Vegetative growth of hybrids seems to be enhanced in elevated CO2 due to improved photosynthetic nitrogen‐use efficiency compared with parents. The results suggest that hybrid crops should be well‐suited for future conditions, but hybrid weeds may also be more competitive. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: To understand complex developmental processes such as leaf growth, information on individual gene functions as well as a broader system‐wide view of the process are crucial. Here, we summarize known information on several important leaf cell proliferation‐controlling pathways as well as their interconnections among each other in Arabidopsis thaliana and draw comparisons to other plant species to provide an overview of where current knowledge stands and how this field of study might move forward. [ABSTRACT FROM AUTHOR]

SUMMARY: Vegetative shade causes an array of morphological changes in plants called shade avoidance syndrome, which includes hypocotyl and petiole elongation, leaf hyponasty, reduced leaf growth, early flowering and rapid senescence. Here, we show that loss‐of‐function mutations in HISTONE DEACETYLASE 9 (HDA9) attenuated the shade‐induced hypocotyl elongation in Arabidopsis. However, the hda9 cotyledons and petioles under shade were not significantly different from those in wild‐type, suggesting a specific function of HDA9 in hypocotyl elongation in response to shade. HDA9 expression levels were stable under shade and its protein was ubiquitously detected in cotyledon, hypocotyl and root. Organ‐specific transcriptome analysis unraveled that shade induced a set of auxin‐responsive genes, such as SMALL AUXIN UPREGULATED RNAs (SAURs) and AUXIN/INDOLE‐3‐ACETIC ACIDs (AUX/IAAs) and their induction was impaired in hda9‐1 hypocotyls. In addition, HDA9 binding to loci of SAUR15/65, IAA5/6/19 and ACS4 was increased under shade. The genetic and organ‐specific gene expression analyses further revealed that HDA9 may cooperate with PHYTOCHROME‐INTERACTING FACTOR 4/7 in the regulation of shade‐induced hypocotyl elongation. Furthermore, HDA9 and PIF7 proteins were found to interact together and thus it is suggested that PIF7 may recruit HDA9 to regulate the shade/auxin responsive genes in response to shade. Overall, our study unravels that HDA9 can work as one component of a hypocotyl‐specific transcriptional regulatory machinery that activates the auxin response at the hypocotyl leading to the elongation of this organ under shade. Significance Statement: Loss‐of‐function mutations of HDA9 exhibited short hypocotyl phenotype under shade. The organ‐specific transcriptome analysis indicated that HDA9 is required for activation of auxin response in the hypocotyl upon shade treatment. HDA9 associated with multiple auxin‐responsive gene loci in vivo to regulate shade‐induced hypocotyl elongation, possibly together with PIF7. This study introduces organ‐specific transcriptional regulatory machinery as a potential topic for further research. [ABSTRACT FROM AUTHOR]

The microRNA miR319 and its target JAW-TCP transcription factors regulate the proliferation-to-differentiation transition of leaf pavement cells in diverse plant species. In young Arabidopsis leaf primordia, JAW-TCPs are detected towards the distal region whereas the major mRNA319-encoding gene MIR319C, is expressed at the base. Little is known about how this complementary expression pattern of MIR319C and JAW-TCPs is generated. Here, we show that MIR319C is initially expressed uniformly throughout the incipient primordia and is later abruptly down-regulated at the distal region, with concomitant distal appearance of JAW-TCPs, when leaves grow to ~100 μm long. Loss of JAW-TCPs causes distal extension of the MIR319C expression domain, whereas ectopic TCP activity restricts MIR319C more proximally. JAW-TCPs are recruited to and are capable of depositing histone H3K27me3 repressive marks on the MIR319C chromatin. JAW-TCPs fail to repress MIR319C in transgenic seedlings where the TCP-binding cis-elements on MIR319C are mutated, causing miR319 gain-of-function-like phenotype in the embryonic leaves. Based on these results, we propose a model for growth patterning in leaf primordia wherein MIR319C and JAW-TCPs repress each other and divide the uniformly growing primordia into distal differentiation zone and proximal proliferation domain. Author summary: Young leaves in Arabidopsis exhibit a characteristic polar growth pattern where cells at the base grow due to continued cell division, while the tip stops growing as the cells exit division and enter differentiation. The MIR319C and its target JAW-TCP genes are expressed in the proximal and the distal domain respectively and are important for maintaining the polar growth pattern. However, it is not known how the complementary expression pattern of MIR319C and JAW-TCP genes is generated when leaves are initiated. Here, we show that MIR319C is expressed ubiquitously during leaf inception and later downregulated at the distal region. This distal loss of MIR319C is marked by an onset of JAW-TCP expression in the same domain. JAW-TCP proteins directly bind to, and downregulate the transcription of, the MIR319C gene to restrict its expression domain towards the base. Disruption of JAW-TCP-binding sites on the MIR319C promoter leads to miR319-overexpression-like phenotype. Together with the knowledge of miR319c-mediated post-transcriptional regulation of JAW-TCPs, we have shown that JAW-TCPs and miR319c mutually repress each other and generate growth polarity in early leaf primordia. [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]

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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]

Plants undergo extended morphogenesis. The shoot apical meristem (SAM) allows for reiterative development and the formation of new structures throughout the life of the plant. Intriguingly, the SAM produces morphologically different leaves in an age-dependent manner, a phenomenon known as heteroblasty. In Arabidopsis thaliana , the SAM produces small orbicular leaves in the juvenile phase, but gives rise to large elliptical leaves in the adult phase. Previous studies have established that a developmental decline of microRNA156 (miR156) is necessary and sufficient to trigger this leaf shape switch, although the underlying mechanism is poorly understood. Here we show that the gradual increase in miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE transcription factors with age promotes cell growth anisotropy in the abaxial epidermis at the base of the leaf blade, evident by the formation of elongated giant cells. Time-lapse imaging and developmental genetics further revealed that the establishment of adult leaf shape is tightly associated with the longitudinal cell expansion of giant cells, accompanied by a prolonged cell proliferation phase in their vicinity. Our results thus provide a plausible cellular mechanism for heteroblasty in Arabidopsis, and contribute to our understanding of anisotropic growth in plants. [ABSTRACT FROM AUTHOR]

Plant trichomes formed by specialized epidermal cells play a role in protecting plants from biotic and abiotic stresses and can also influence the economic and ornamental value of plant products. Therefore, further studies on the molecular mechanisms of plant trichome growth and development are important for understanding trichome formation and agricultural production. SET Domain Group 26 (SDG26) is a histone lysine methyltransferase. Currently, the molecular mechanism by which SDG26 regulates the growth and development of Arabidopsis leaf trichomes is still unclear. We found that the mutant of Arabidopsis (sdg26) possessed more trichomes on its rosette leaves compared to the wild type (Col-0), and the trichome density per unit area of sdg26 is significantly higher than that of Col-0. The content of cytokinins and jasmonic acid was higher in sdg26 than in Col-0, while the content of salicylic acid was lower in sdg26 than in Col-0, which is conducive to trichome growth. By measuring the expression levels of trichome-related genes, we found that the expression of genes that positively regulate trichome growth and development were up-regulated, while the negatively regulated genes were down-regulated in sdg26. Through chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we found that SDG26 can directly regulate the expression of genes related to trichome growth and development such as ZFP1, ZFP5, ZFP6, GL3, MYB23, MYC1, TT8, GL1, GIS2, IPT1, IPT3, and IPT5 by increasing the accumulation of H3K27me3 on these genes, which further affects the growth and development of trichomes. This study reveals the mechanism by which SDG26 affects the growth and development of trichomes through histone methylation. The current study provides a theoretical basis for studying the molecular mechanism of histone methylation in regulating leaf trichome growth and development and perhaps guiding the development of new crop varieties. [ABSTRACT FROM AUTHOR]

SUMMARY: Natural antisense long non‐coding RNAs (lncNATs) are involved in the regulation of gene expression in plants, modulating different relevant developmental processes and responses to various stimuli. We have identified and characterized two lncNATs (NAT1UGT73C6 and NAT2UGT73C6, collectively NATsUGT73C6) from Arabidopsis thaliana that are transcribed from a gene fully overlapping UGT73C6, a member of the UGT73C subfamily of genes encoding UDP‐glycosyltransferases (UGTs). Expression of both NATsUGT73C6 is developmentally controlled and occurs independently of the transcription of UGT73C6 in cis. Downregulation of NATsUGT73C6 levels through artificial microRNAs results in a reduction of the rosette area, while constitutive overexpression of NAT1UGT73C6 or NAT2UGT73C6 leads to the opposite phenotype, an increase in rosette size. This activity of NATsUGT73C6 relies on its RNA sequence and, although modulation of UGT73C6 in cis cannot be excluded, the observed phenotypes are not a consequence of the regulation of UGT73C6 in trans. The NATsUGT73C6 levels were shown to affect cell proliferation and thus individual leaf size. Consistent with this concept, our data suggest that the NATsUGT73C6 influence the expression levels of key transcription factors involved in regulating leaf growth by modulating cell proliferation. These findings thus reveal an additional regulatory layer on the process of leaf growth. In this work, we characterized at the molecular level two long non‐coding RNAs (NATsUGT73C6) that are transcribed in the opposite direction to UGT73C6, a gene encoding a glucosyltransferase involved in brassinosteroid homeostasis in A. thaliana. Our results indicate that NATsUGT73C6 expression influences leaf growth by acting in trans and by modulating the levels of transcription factors that are involved in the regulation of cell proliferation. Significance Statement: In this work we characterized at the molecular level two long non‐coding RNAs (NATsUGT73C6) that are transcribed in the opposite direction to UGT73C6, a gene encoding a glucosyltransferase involved in brassinosteroid homeostasis in Arabidopsis thaliana. Our results indicate that NATsUGT73C6 expression influences leaf growth by acting in trans and by modulating the levels of transcription factors that are involved in the regulation of cell proliferation. [ABSTRACT FROM AUTHOR]

Life evolved in the presence of reactive oxygen species (ROS) which resulted in an early integration of ROS signals and properties for cellular physiology. Originally ROS were viewed merely as metabolic by-products, but now they are viewed as conserved regulators of physiological and developmental processes throughout all kingdoms of life. Plants monitor constantly the fluctuating environmental conditions in which they grow, for which ROS signaling is essential. However, in recent years it has become clear that plants also use ROS homeostasis to guide their development. Here, an overview is presented for the role of ROS gradients and homeostasis in the development of leaves from the shoot apical meristem based mainly on the model plant species Arabidopsis thaliana. Furthermore, this chapter summarizes current knowledge on the involvement of different ROS species and molecular components in the regulation of leaf development. Although our current understanding of how plants employ ROS to guide their development is far from complete, it is clear that ROS is utilized during many developmental phases of leaf 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]

Peppermint (Mentha x piperita) is a species with inhibitory allelopathic properties due to its high amounts of terpenes. Recent studies have disclosed dosage dependent growth promotion or defense reactions in plants when facing appropriate amounts of Mentha bouquet terpenes. These positive effects could be of interest for agricultural applications. To obtain more insights into leaf growth modulations, the expression of Arabidopsis and Brassica rapa TCP transcription factors were studied after fumigation with M. x piperita bouquets (Arabidopsis), with M. x piperita essential oil or with limonene (Arabidopsis and Chinese cabbage). According to qPCR studies, expression of TCP3, TCP24, and TCP20 were downregulated by all treatments in Arabidopsis, leading to altered leaf growth. Expressions of B. rapa TCPs after fumigation with the essential oil or limonene were less affected. Extensive greenhouse and polytunnel trials with white cabbage and Mentha plants showed that the developmental stage of the leaves, the dosage, and the fumigation time are of crucial importance for changed fresh and dry weights. Although further research is needed, the study may contribute to a more intensive utilization of ecologically friendly and species diversity conservation and positive allelopathic interactions in future agricultural systems. [ABSTRACT FROM AUTHOR]

The leaf microbiota plays a key role in plant development, but a detailed mechanism of microbe-plant relationships remains elusive. Many genome-wide association studies (GWAS) have begun to map leaf microbes, but few have systematically characterized the genetics of how microbes act and interact. Previously, we integrated behavioral ecology and game theory to define four types of microbial interactions -- mutualism, antagonism, aggression, and altruism, in a microbial community assembly. Here, we apply network mapping to identify specific plant genes that mediate the topological architecture of microbial networks. Analyzing leaf microbiome data from an Arabidopsis GWAS, we identify several heritable hub microbes for leaf microbial communities and detect 140-728 SNPs (Single nucleotide polymorphisms) responsible for emergent properties of microbial network. We reconstruct Bayesian genetic networks from which to identify 22-43 hub genes found to code molecular pathways related to leaf growth, abiotic stress responses, disease resistance and nutrition uptake. A further path analysis visualizes how genetic variants of Arabidopsis affect its fecundity through the internal workings of the leaf microbiome. We find that microbial networks and their genetic control vary along spatiotemporal gradients. Our study provides a new avenue to reveal the "endophenotype" role of microbial networks in linking genotype to end-point phenotypes in plants. Our integrative theory model provides a powerful tool to understand the mechanistic basis of structural-functional relationships within the leaf microbiome and supports the need for future research on plant breeding and synthetic microbial consortia with a specific function. [ABSTRACT FROM AUTHOR]

Leaf growth and development are primarily driven by cell proliferation and expansion. Among a number of genes involved in the regulation of cell proliferation, the GROWTH-REGULATING FACTOR (GRF) transcription factors have been established to act as positive regulators of cell proliferation and leaf growth in angiosperms. While the Arabidopsis thaliana GRF family comprises nine members, not all members of the family have been experimentally confirmed for the positive role, not only due to no or only slight changes in leaf size of corresponding single mutants, but also due to unavailability of multiple mutants to overcome the obstacle. Furthermore, some discrepancies and confusion in their roles have been disclosed in the literature. Here, we systemically prepared a series of such multiple mutants and confirmed that all GRF members, except for GRF8, acted as positive regulators of cell proliferation and leaf growth. We also systematically examined the spatio-temporal distribution patterns of all nine GRF proteins in the leaf organ, and found that their distribution patterns were highly reminiscent of the behavior of the cell cycle arrest front. We therefore propose that GRFs play an important role in shaping the arrest front and growth patterns of leaves. [ABSTRACT FROM AUTHOR]

Plant acyl-CoA dominated acyltransferases (named BAHD) comprise a large appointed protein superfamily and play varied roles in plant secondary metabolism like synthesis of modified anthocyanins, flavonoids, volatile esters, etc. Tea (Camellia sinensis) is an important non-alcoholic medicinal and fragrancy plant synthesizing different secondary metabolites, including flavonoids. In the tea (C.A sinensis) cultivar Longjing 43 (LJ43), eight samples were performed into three groups for transcriptome analysis under three biological replications. Among the BAHD acyltransferase genes in tea cultivars, the expression of TEA031065 was highest in buds and young leaves following the RNA sequencing data, which was coincident with the tissue rich in catechins and other flavonoids. We then transformed this gene into wild-type Arabidopsis as an over-expression (OX) line 1 and line 2 in ½ MS media to verify its function. In the wild types (WT), the primary root length, number of secondary roots, and total root weight were significantly higher at 24%, 15%, and 53.92%, respectively, compared to the transgenic lines (OX1 and OX2). By contrast, the leaves displayed larger rosettes (21.58%), with higher total leaf weight (32.64%) in the transgenic lines than in the wild type (WT). This result is consistent with DCR mutant At5g23940 gene in Arabidopsis thaliana. Here, anthocyanin content in transgenic lines was also increased (21.65%) as compared to WT. According to the RNA sequencing data, a total of 22 growth regulatory genes and 31 structural genes with TFs (transcription factors) that are correlative with plant growth and anthocyanin accumulation were identified to be differentially expressed in the transgenic lines. It was found that some key genes involved in IAA (Auxin) and GA (Gibberellin) biosynthesis were downregulated in the transgenic lines, which might be correlated with the phenotype changes in roots. Moreover, the upregulation of plant growth regulation genes, such as UGT73C4 (zeatin), ARR15, GH3.5, ETR2, ERS2, APH4, and SAG113 might be responsible for massive leaf growth. In addition, transgenic lines shown high anthocyanin accumulation due to the upregulation of the (1) 3AT1 and (3) GSTF, particularly, GSTF12 genes in the flavonoid biosynthesis pathway. However, the TFs such as, CCoAMT, bHLH, WRKY, CYP, and other MYBs were also significantly upregulated in transgenic lines, which increased the content of anthocyanins in A. thaliana seedlings. In conclusion, a BAHD acyltransferase (TEA031065) was identified, which might play a vital role in tea growth and secondary metabolites regulation. This study increases our knowledge concerning the combined functionality of the tea BAHD acyltransferase gene (TEA031065). [ABSTRACT FROM AUTHOR]

Phosphorus (P) is an indispensable mineral nutrient for plant growth and agricultural production. Plants acquire and redistribute inorganic phosphate (Pi) via Pi transporters (PHT1s/PTs). However, apart from MtPT4, functions of the M. truncatula (Medicago truncatula) PHT1s remain unclear. In this study, we evaluated the function of the PHT1 family transporter MtPT5 in M. truncatula. MtPT5 was closely related to AtPHT1; 1 in Arabidopsis (Arabidopsis thaliana) and GmPT7 in soybean (Glycine max). MtPT5 was highly expressed in leaves in addition to roots and nodules. Ectopic expression of MtPT5 complemented the Pi-uptake deficiency of Arabidopsis pht1;1Δ4Δ double mutant, demonstrating the Pi-transport activity of MtPT5 in plants. When overexpressing MtPT5 in M. truncatula, the transgenic plants showed larger leaves, accompanying with higher biomass and Pi enrichment compared with wild type. All these data demonstrate that MtPT5 is important for leaf growth and Pi accumulation of M. truncatula and provides a target for molecular breeding to improve forage productivity. [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]

Shaded plants challenged with herbivores or pathogens prioritize growth over defense. However, most experiments have focused on the effect of shading light cues on defense responses. To investigate the potential interaction between shade-avoidance and wounding-induced Jasmonate (JA)-mediated signaling on leaf growth and movement, we used repetitive mechanical wounding of leaf blades to mimic herbivore attacks. Phenotyping experiments with combined treatments on Arabidopsis thaliana rosettes revealed that shade strongly inhibits the wound effect on leaf elevation. By contrast, petiole length is reduced by wounding both in the sun and in the shade. Thus, the relationship between the shade and wounding/JA pathways varies depending on the physiological response, implying that leaf growth and movement can be uncoupled. Using RNA-sequencing, we identified genes with expression patterns matching the hyponastic response (opposite regulation by both stimuli, interaction between treatments with shade dominating the wound signal). Among them were genes from the PKS (Phytochrome Kinase Substrate) family, which was previously studied for its role in phototropism and leaf positioning. Interestingly, we observed reduced shade suppression of the wounding effect in pks2pks4 double mutants while a PKS4 overexpressing line showed constitutively elevated leaves and was less sensitive to wounding. Our results indicate a trait-specific interrelationship between shade and wounding cues on Arabidopsis leaf growth and positioning. Moreover, we identify PKS genes as integrators of external cues in the control of leaf hyponasty further emphasizing the role of these genes in aerial organ positioning. Author summary: Plants face different types of stressful situations without the ability to relocate to favorable environments. For example, increasing plant density reduces access to sunlight as plants start to shade each other. Foliar shading represents a stress that many plants cope with by changing their morphology. This includes elongation of stem-like structures and repositioning of leaves to favor access to unfiltered sunlight. Plants also defend themselves against various pathogens including herbivores. Defense mechanisms include the production of deterrent chemical and morphological adaptations such as stunted growth and downwards leaf repositioning. Here we studied the morphological response of plants when simultaneously facing shade and herbivore stress. When facing both stresses petiole growth was intermediate between the shade-enhanced and wound-repressed response. In contrast, the shade cue overrides the wounding cue leading to a similar upwards leaf repositioning in the combined treatments or in the response to shade alone. Using gene expression analyses and genetics we identified two members of the Phytochrome Kinase Substrate family as playing a signal integration role when plants simultaneously faced both stresses. This contributes to our understanding of the mechanisms underlying plant morphological adaptations when facing multiple stresses. [ABSTRACT FROM AUTHOR]

Background: Leaf senescence, the final stage of leaf growth and development, is regulated by numerous internal factors and environmental cues. Ethylene is one of the key senescence related hormones, but the underlying molecular mechanism of ethylene-induced leaf senescence remains poorly understood. Results: In this study, we identified one AT-hook like (AHL) protein, AHL9, as a positive regulator of leaf senescence in Arabidopsis thaliana. Overexpression of AHL9 significantly accelerates age-related leaf senescence and promotes dark-induced leaf chlorosis. The early senescence phenotype observed in AHL9 overexpressing lines is inhibited by the ethylene biosynthesis inhibitor aminooxyacetic acid suggesting the involvement of ethylene in the AHL9-associated senescence. RNA-seq and quantitative reverse transcription PCR (qRT-PCR) data identified numerous senescence-associated genes differentially expressed in leaves of AHL9 overexpressing transgenic plants. Conclusions: Our investigation demonstrates that AHL9 functions in accelerating the leaf senescence process via ethylene synthesis or signalling. [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]

In plant chloroplasts, photosystem II (PSII) complexes, together with light‐harvesting complex II (LHCII), form various PSII‐LHCII supercomplexes (SCs). This process likely involves immunophilins, but the underlying regulatory mechanisms are unclear. Here, by comparing Arabidopsis thaliana mutants lacking the chloroplast lumen‐localized immunophilin CYCLOPHILIN28 (CYP28) to wild‐type and transgenic complemented lines, we determined that CYP28 regulates the assembly and accumulation of PSII‐LHCII SCs. Compared to the wild type, cyp28 plants showed accelerated leaf growth, earlier flowering time, and enhanced accumulation of high molecular weight PSII‐LHCII SCs under normal light conditions. The lack of CYP28 also significantly affected the electron transport rate. Blue native‐polyacrylamide gel electrophoresis analysis revealed more Lhcb6 and less Lhcb4 in M‐LHCII‐Lhcb4‐Lhcb6 complexes in cyp28 versus wild‐type plants. Peptidyl‐prolyl cis/trans isomerase (PPIase) activity assays revealed that CYP28 exhibits weak PPIase activity and that its K113 and E187 residues are critical for this activity. Mutant analysis suggested that CYP28 may regulate PSII‐LHCII SC accumulation by altering the configuration of Lhcb6 via its PPIase activity. Furthermore, the Lhcb6‐P139 residue is critical for PSII‐LHCII SC assembly and accumulation. Therefore, our findings suggest that CYP28 likely regulates PSII‐LHCII SC assembly and accumulation by altering the configuration of P139 of Lhcb6 via its PPIase activity. [ABSTRACT FROM AUTHOR]

Peppermint (Mentha x piperita) is a species with inhibitory allelopathic properties due to its high amounts of terpenes. Recent studies have disclosed dosage dependent growth promotion or defense reactions in plants when facing appropriate amounts of Mentha bouquet terpenes. These positive effects could be of interest for agricultural applications. To obtain more insights into leaf growth modulations, the expression of Arabidopsis and Brassica rapa TCP transcription factors were studied after fumigation with M. x piperita bouquets (Arabidopsis), with M. x piperita essential oil or with limonene (Arabidopsis and Chinese cabbage). According to qPCR studies, expression of TCP3, TCP24, and TCP20 were downregulated by all treatments in Arabidopsis, leading to altered leaf growth. Expressions of B. rapa TCPs after fumigation with the essential oil or limonene were less affected. Extensive greenhouse and polytunnel trials with white cabbage and Mentha plants showed that the developmental stage of the leaves, the dosage, and the fumigation time are of crucial importance for changed fresh and dry weights. Although further research is needed, the study may contribute to a more intensive utilization of ecologically friendly and species diversity conservation and positive allelopathic interactions in future agricultural systems.

In eukaryotes, N‐ethylmaleimide‐sensitive factor (NSF) is a conserved AAA+ATPase and a key component of the membrane trafficking machinery that promotes the fusion of secretory vesicles with target membranes. Here, we demonstrate that the Arabidopsis thaliana genome contains a single copy of NSF, AtNSF, which plays an essential role in the regulation of leaf serration. The AtNSF knock‐down mutant, atnsf‐1, exhibited more serrations in the leaf margin. Moreover, polar localization of the PIN‐FORMED1 (PIN1) auxin efflux transporter was diffuse around the margins of atnsf‐1 leaves and root growth was inhibited in the atnsf‐1 mutant. More PIN1‐GFP accumulated in the intracellular compartments of atnsf‐1 plants, suggesting that AtNSF is required for intracellular trafficking of PIN between the endosome and plasma membrane. Furthermore, the serration phenotype was suppressed in the atnsf‐1 pin1‐8 double mutant, suggesting that AtNSF is required for PIN1‐mediated polar auxin transport to regulate leaf serration. The CUP‐SHAPED COTYLEDON2 (CUC2) transcription factor gene is up‐regulated in atnsf‐1 plants and the cuc2‐3 single mutant exhibits smooth leaf margins, demonstrating that AtNSF also functions in the CUC2 pathway. Our results reveal that AtNSF regulates the PIN1‐generated auxin maxima with a CUC2‐mediated feedback loop to control leaf serration. [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]

Summary: Shade avoidance syndrome (SAS) arises in densely growing plants that compete for light. In Arabidopsis thaliana, phytochrome interacting factor (PIF) proteins link the perception of shade to stem elongation via auxin production.Here, we report that PIFs inhibit the shade‐induced expression of AUXIN RESPONSE FACTOR 18 (ARF18), and ARF18 represses auxin signaling. Therefore, PIF‐mediated inhibition of ARF18 enhances auxin‐dependent hypocotyl elongation in simulated shade.Furthermore, we show that both PIFs and ARF18 directly repress qua‐quine starch (QQS), which controls the allocation of carbon and nitrogen. Shade‐repressed QQS attenuates the conversion of starch to protein and thus reduced leaf area.Our results suggest that PIF‐dependent gene regulation coordinates multiple SAS responses, including altered stem growth via ARF18, as well as altered leaf growth and metabolism via QQS. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio‐lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1‐1 and 1‐2). Both PpAN1 genes complemented the A. thaliana an‐1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1‐promoter– uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1‐1 and PpAN1‐2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip‐growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α‐tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1‐1/1‐2 double‐knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed. Significance Statement: ANGUSTIFOLIA (AN) regulates leaf width in Arabidopsis thaliana by controlling diffuse cell growth in the medio‐lateral direction. An analysis of two homologs of AN (PpAN1‐1 and PpAN1‐2) found in the Physcomitrella patens genome suggested that both PpAN1 genes in P. patens function in diffuse cell growth in the haploid and diploid generations but not in tip growth of haploid protonemata. [ABSTRACT FROM AUTHOR]

Summary: Leaf growth is a complex trait for which many similarities exist in different plant species, suggesting functional conservation of the underlying pathways. However, a global view of orthologous genes involved in leaf growth showing conserved expression in dicots and monocots is currently missing. Here, we present a genome‐wide comparative transcriptome analysis between Arabidopsis and maize, identifying conserved biological processes and gene functions active during leaf growth. Despite the orthology complexity between these distantly related plants, 926 orthologous gene groups including 2829 Arabidopsis and 2974 maize genes with similar expression during leaf growth were found, indicating conservation of the underlying molecular networks. We found 65% of these genes to be involved in one‐to‐one orthology, whereas only 28.7% of the groups with divergent expression had one‐to‐one orthology. Within the pool of genes with conserved expression, 19 transcription factor families were identified, demonstrating expression conservation of regulators active during leaf growth. Additionally, 25 Arabidopsis and 25 maize putative targets of the TCP transcription factors with conserved expression were determined based on the presence of enriched transcription factor binding sites. Based on large‐scale phenotypic data, we observed that genes with conserved expression have a higher probability to be involved in leaf growth and that leaf‐related phenotypes are more frequently present for genes having orthologues between dicots and monocots than clade‐specific genes. This study shows the power of integrating transcriptomic with orthology data to identify or select candidates for functional studies during leaf development in flowering plants. [ABSTRACT FROM AUTHOR]

Imazethapyr (IM) is an acetolactate synthase (ALS)-inhibiting herbicide that has been widely used in recent years. However, IM spraying can lead to the accumulation of herbicide residues in leaves. Here, we determined the effects of IM spraying on the plant growth and leaf surface microbial communities of Arabidopsis thaliana after 7 and 14 days of exposure. The results suggested that IM spraying inhibited plant growth. Fresh weight decreased to 48% and 26% of the control value after 7 and 14 days, respectively, of 0.035 kg/ha IM exposure. In addition, anthocyanin content increased 9.2-fold and 37.2-fold relative to the control content after 7 and 14 days of treatment, respectively. Furthermore, IM spraying destroyed the cell structures of the leaves, as evidenced by increases in the number of starch granules and the stomatal closure rate. Reductions in photosynthetic efficiency and antioxidant enzyme activity were observed after IM spraying, especially after 14 days of exposure. The diversity and evenness of the leaf microbiota were not affected by IM treatment, but the composition of community structure at the genus level was altered by IM spraying. Imazethapyr application increased the abundance of Pseudomonas , a genus that includes species pathogenic to plants and humans, indicating that IM potentially increased the abundance of pathogenic bacteria on leaves. Our findings increase our understanding of the relationships between herbicide application and the microbial community structures on plant leaves, and they provide a new perspective for studying the ecological safety of herbicide usage. Unlabelled Image [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]

• 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]

Summary: Although cell number generally correlates with organ size, the role of cell cycle control in growth regulation is still largely unsolved. We studied kip related protein (krp) 4, 6 and 7 single, double and triple mutants of Arabidopsis thaliana to understand the role of cell cycle inhibitory proteins in leaf development.We performed leaf growth and seed size analysis, kinematic analysis, flow cytometery, transcriptome analysis and mathematical modeling of G1/S and G2/M checkpoint progression of the mitotic and endoreplication cycle.Double and triple mutants progressively increased mature leaf size, because of elevated expression of cell cycle and DNA replication genes stimulating progression through the division and endoreplication cycle. However, cell number was also already increased before leaf emergence, as a result of an increased cell number in the embryo. We show that increased embryo and seed size in krp4/6/7 results from seed abortion, presumably reducing resource competition, and that seed size differences contribute to the phenotype of several large‐leaf mutants.Our results provide a new mechanistic understanding of the role of cell cycle regulation in leaf development and highlight the contribution of the embryo to the development of leaves after germination in general. [ABSTRACT FROM AUTHOR]

• Leaf adaxial–abaxial polar establishment is crucial for leaf morphogenesis. • A novel leaf morphological mutant crsl with curled rod-shaped leaves and strikingly altered leaf adaxial–abaxial development was identified in Chinese cabbage. • BraA02g016100.3C (BrCRSL) gene is homologous to Arabidopsis MAB1 and encodes a putative mitochondrial PDH E1β subunit protein, which is the most likely causal gene for crsl and might serve as a new regulator of leaf polarity specification. • It's the first to identify BrCRSL function in leaf morphogenesis. Leaf morphogenesis requires precise genetic control and hormone regulation to develop into ideal plant architectures. Leaf flattening is an adaptive trait to obtain efficient photosynthesis, for which the leaf adaxial–abaxial polar establishment is of great importance. Herein, a novel leaf morphological mutant, curled rod-shaped leaf (crsl), was isolated and characterized from Chinese cabbage (Brassica rapa L. ssp. pekinensis) EMS-induced population. The leaves in crsl were slender and rod-shaped, with the abaxial side folded upwards and the distal side curled downwards, the mesophyll cells were loosely arranged, and the epidermal morphology was opposite on the adaxial and abaxial surface. Genetic analysis revealed that the curled rod-shaped leaf mutant phenotype was controlled by a dominate nuclear gene, named BrCRSL. MutMap and Kompetitive Allele Specifc PCR (KASP) genotyping demonstrated that BraA02g016100.3C was the most likely candidate for BrCRSL. Compared with the wild-type, a non-synonymous SNP mutation (C to T) occurred in the 8th exon of BraA02g016100.3C in crsl. BraA02g016100.3C is orthologous of Arabidopsis thaliana MAB1 (AT5G50850), which encodes a putative mitochondrial PDH E1β subunit protein and contributes to PIN-dependent auxin transport in organ development. The amino acid substitution from proline (P) to leucine (L) is conserved among the homologous proteins of BraA02g016100.3C. ​The crsl mutant exhibited abnormal auxin metabolism, primarily manifested by high level of L-tryptophan (TRP) accumulation that is the precursor of IAA biosynthesis. Furthermore, the expression of a wide range of leaf adaxial/abaxial polarity-related genes was affected in crsl. Our results contribute to a better understanding of the genetic control mechanism underlying leaf morphogenesis in Chinese cabbage and provide new insights into the auxin-related leaf axial growth. [ABSTRACT FROM AUTHOR]

Plants adapt to the prevailing photoperiod by adjusting growth and flowering to the availability of energy. To understand the molecular changes involved in adaptation to a long-day condition we comprehensively profiled leaf six at the end of the day and the end of the night at four developmental stages on Arabidopsis thaliana plants grown in a 16 h photoperiod, and compared the profiles to those from leaf 6 of plants grown in a 8 h photoperiod. When Arabidopsis is grown in a long-day photoperiod individual leaf growth is accelerated but whole plant leaf area is decreased because total number of rosette leaves is restricted by the rapid transition to flowering. Carbohydrate measurements in long- and short-day photoperiods revealed that a long photoperiod decreases the extent of diurnal turnover of carbon reserves at all leaf stages. At the transcript level we found that the long-day condition has significantly reduced diurnal transcript level changes than in short-day condition, and that some transcripts shift their diurnal expression pattern. Functional categorisation of the transcripts with significantly different levels in short and long day conditions revealed photoperiod-dependent differences in RNA processing and light and hormone signalling, increased abundance of transcripts for biotic stress response and flavonoid metabolism in long photoperiods, and for photosynthesis and sugar transport in short photoperiods. Furthermore, we found transcript level changes consistent with an early release of flowering repression in the long-day condition. Differences in protein levels between long and short photoperiods mainly reflect an adjustment to the faster growth in long photoperiods. In summary, the observed differences in the molecular profiles of leaf six grown in long- and short-day photoperiods reveal changes in the regulation of metabolism that allow plants to adjust their metabolism to the available light. The data also suggest that energy management is in the two photoperiods fundamentally different as a consequence of photoperiod-dependent energy constraints.

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]

Abstract: It has been reported in several pathosystems that disease resistance can vary in leaves at different stages. However, how general this leaf stage‐associated resistance is, and the molecular mechanism(s) underlying it, remain largely unknown. Here, we investigated the effect of leaf stage on basal resistance, effector‐triggered immunity (ETI) and nonhost resistance, using eight pathosystems involving the hosts Arabidopsis thaliana, Nicotiana tabacum, and N. benthamiana and the pathogens Sclerotinia sclerotiorum, Pseudomonas syringae pv. tabaci, P. syringae pv. tomato DC3000, and Xanthomonas oryzae pv. oryzae (Xoo). We show evidence that leaf stage‐associated resistance exists ubiquitously in plants, but with varying intensity at different stages in diverse pathosystems. Microarray expression profiling assays demonstrated that hundreds of genes involved in defense responses, phytohormone biosynthesis and signaling, and calcium signaling, were differentially expressed between leaves at different stages. The Arabidopsis mutants sid1, sid2‐3, ein2, jar1‐1, aba1 and aao3 lost leaf stage‐associated resistance to S. sclerotiorum, and the mutants aba1 and sid2‐3 were affected in leaf stage‐associated RPS2/AvrRpt2+‐conferred ETI, whereas only the mutant sid2‐3 influenced leaf stage‐associated nonhost resistance to Xoo. Our results reveal that the phytohormones salicylic acid, ethylene, jasmonic acid and abscisic acid likely play an essential, but pathosystem‐dependent, role in leaf stage‐associated resistance. [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]

Organ size control is of particular importance for developmental biology and agriculture, but the mechanisms underlying organ size regulation remain elusive in plants. Meristemoids, which possess stem cell-like properties, have been recognized to play important roles in leaf growth. We have recently reported that the Arabidopsis F-box protein STERILE APETALA (SAP)/SUPPRESSOR OF DA1 (SOD3) promotes meristemoid proliferation and regulates organ size by influencing the stability of the transcriptional regulators PEAPODs (PPDs). Here we demonstrate that KIX8 and KIX9, which function as adaptors for the corepressor TOPLESS and PPD, are novel substrates of SAP. SAP interacts with KIX8/9 and modulates their protein stability. Further results show that SAP acts in a common pathway with KIX8/9 and PPD to control organ growth by regulating meristemoid cell proliferation. Thus, these findings reveal a molecular mechanism by which SAP targets the KIX-PPD repressor complex for degradation to regulate meristemoid cell proliferation and organ size. [ABSTRACT FROM AUTHOR]

Ribosome biogenesis is one of the most energy-consuming events in the cell and must therefore be coordinated with changes in cellular energy status. Here, we show that the sugar-inducible gene ARABIDOPSIS PUMILIO PROTEIN24 (APUM24) encodes a Pumilio homology domain-containing protein involved in pre-rRNA processing in Arabidopsis thaliana. Null mutation of APUM24 resulted in aborted embryos due to abnormal gametogenesis and embryogenesis, whereas reduced expression of APUM24 caused several phenotypes characteristic of ribosome biogenesis or function-related mutants. APUM24 interacted with other pre-rRNA processing factors and a putative endonuclease for the removal of the internal transcribed spacer 2 (ITS2) of pre-rRNA in the nucleolus. The APUM24-containing complex also interacted with ITS2, and reduced APUM24 expression caused the overaccumulation of processing intermediates containing ITS2. Thus, APUM24 likely functions as an ITS2 removal-associated factor. Most importantly, the apum24 knockdown mutant was hypersensitive to highly concentrated sugar, and the mutant showed sugar-dependent overaccumulation of processing intermediates and nucleolar stress (changes in nucleolar size). Furthermore, reduced APUM24 expression diminished sugar-induced promotion of leaf and root growth. Hence, a breakdown in the coordinated expression of ribosome biogenesis-related genes with energy status may induce nucleolar stress and disturb proper sugar responses in Arabidopsis. [ABSTRACT FROM AUTHOR]

Here we report the discovery through activation tagging and subsequent characterization of the BIG LEAF (BL) gene from poplar. In poplar, BL regulates leaf size via positively affecting cell proliferation. Up and downregulation of the gene led to increased and decreased leaf size, respectively, and these phenotypes corresponded to increased and decreased cell numbers. BL function encompasses the early stages of leaf development as native BL expression was specific to the shoot apical meristem and leaf primordia and was absent from the later stages of leaf development and other organs. Consistently, BL downregulation reduced leaf size at the earliest stages of leaf development. Ectopic expression in mature leaves resulted in continued growth most probably via sustained cell proliferation and thus the increased leaf size. In contrast to the positive effect on leaf growth, ectopic BL expression in stems interfered with and significantly reduced stem thickening, suggesting that BL is a highly specific activator of growth. In addition, stem cuttings from BL overexpressing plants developed roots, whereas the wild type was difficult to root, demonstrating that BL is a positive regulator of adventitious rooting. Large transcriptomic changes in plants that overexpressed BL indicated that BL may have a broad integrative role, encompassing many genes linked to organ growth. We conclude that BL plays a fundamental role in control of leaf size and thus may be a useful tool for modifying plant biomass productivity and adventitious rooting. [ABSTRACT FROM AUTHOR]

Leaves are flat determinate organs derived from indeterminate shoot apical meristems. The presence of a specific leaf meristem is debated, as anatomical features typical of meristems are not present in leaves. Here we demonstrate that multiple NGATHA (NGA) and CINCINNATA-class-TCP (CIN-TCP) transcription factors act redundantly, shortly after leaf initiation, to gradually restrict the activity of a leaf meristem in Arabidopsis thaliana to marginal and basal domains, and that their absence confers persistent marginal growth to leaves, cotyledons and floral organs. Following primordia initiation, the restriction of the broadly acting leaf meristem to the margins is mediated by the juxtaposition of adaxial and abaxial domains and maintained by WOX homeobox transcription factors, whereas other marginal elaboration genes are dispensable for its maintenance. This genetic framework parallels the morphogenetic program of shoot apical meristems and may represent a relic of an ancestral shoot system from which seed plant leaves evolved. [ABSTRACT FROM AUTHOR]

Leaves are the plant's powerhouses, providing energy for all organs through sugar production during photosynthesis. However, sugars serve not only as a metabolic energy source for sink tissues but also as signaling molecules, affecting gene expression through conserved signaling pathways to regulate plant growth and development. Here, we describe an in vitro experimental assay, allowing one to alter the sucrose (Suc) availability during early Arabidopsis (Arabidopsis thaliana) leaf development, with the aim to identify the affected cellular and molecular processes. The transfer of seedlings to Suc-containing medium showed a profound effect on leaf growth by stimulating cell proliferation and postponing the transition to cell expansion. Furthermore, rapidly after transfer to Suc, mesophyll cells contained fewer and smaller plastids, which are irregular in shape and contain fewer starch granules compared with control mesophyll cells. Short-term transcriptional responses after transfer to Suc revealed the repression of well-known sugar-responsive genes and multiple genes encoded by the plastid, on the one hand, and up-regulation of a GLUCOSE-6-PHOSPHATE TRANSPORTER (GPT2), on the other hand. Mutant gpt2 seedlings showed no stimulation of cell proliferation and no repression of chloroplast-encoded transcripts when transferred to Suc, suggesting that GPT2 plays a critical role in the Suc-mediated effects on early leaf growth. Our findings, therefore, suggest that induction of GPT2 expression by Suc increases the import of glucose-6-phosphate into the plastids that would repress chloroplast-encoded transcripts, restricting chloroplast differentiation. Retrograde signaling from the plastids would then delay the transition to cell expansion and stimulate cell proliferation. [ABSTRACT FROM AUTHOR]

How gene activities and biomechanics together direct organ shapes is poorly understood. Plant leaf and floral organs develop from highly similar initial structures and share similar gene expression patterns, yet they gain drastically different shapes later—flat and bilateral leaf primordia and radially symmetric floral primordia, respectively. We analyzed cellular growth patterns and gene expression in young leaves and flowers of Arabidopsis thaliana and found significant differences in cell growth rates, which correlate with convergence sites of phytohormone auxin that require polar auxin transport. In leaf primordia, the PRESSED - FLOWER -expressing middle domain grows faster than adjacent adaxial domain and coincides with auxin convergence. In contrast, in floral primordia, the LEAFY -expressing domain shows accelerated growth rates and pronounced auxin convergence. This distinct cell growth dynamics between leaf and flower requires changes in levels of cell-wall pectin de-methyl-esterification and mechanical properties of the cell wall. Data-driven computer model simulations at organ and cellular levels demonstrate that growth differences are central to obtaining distinct organ shape, corroborating in planta observations. Together, our study provides a mechanistic basis for the establishment of early aerial organ symmetries through local modulation of differential growth patterns with auxin and biomechanics. [Display omitted] • Leaf and floral primordia share similar gene expression and initial domain partition • Local growth-rate differences across domains explain primordia shape differences • Two primordia have different patterns of cell-wall rigidity and auxin convergence • When incorporating growth dynamics, models explain different organ shapes Peng et al. show that different growth dynamics determine the shape of plant aerial organs, leaf and floral primordia, which share similar initial geometry. Experiments and simulations are combined to link growth dynamics with biomechanics and polar auxin transport. [ABSTRACT FROM AUTHOR]

Lateral appendages often show allometric growth with a specific growth polarity along the proximo-distal axis. Studies on leaf growth in model plants have identified a basipetal growth direction with the highest growth rate at the proximal end and progressively lower rates toward the distal end. Although the molecular mechanisms governing such a growth pattern have been studied recently, variation in leaf growth polarity and, therefore, its evolutionary origin remain unknown. By surveying 75 eudicot species, here we report that leaf growth polarity is divergent. Leaf growth in the proximo-distal axis is polar, with more growth arising from either the proximal or the distal end; dispersed with no apparent polarity; or bidirectional, with more growth contributed by the central region and less growth at either end. We further demonstrate that the expression gradient of the miR396- GROWTH-REGULATING FACTOR module strongly correlates with the polarity of leaf growth. Altering the endogenous pattern of miR396 expression in transgenic Arabidopsis thaliana leaves only partially modified the spatial pattern of cell expansion, suggesting that the diverse growth polarities might have evolved via concerted changes in multiple gene regulatory networks. [ABSTRACT FROM AUTHOR]