Understanding megasporogenesis through model plants: contemporary evidence and future insights.

The megasporangium serves as a model system for understanding the concept of individual cell identity, and cell-to-cell communication in angiosperms. As development of the ovule progresses, three distinct layers, the epidermal (L1), the subepidermal or the hypodermal (L2) and the innermost layers (L3) are formed along the MMC (megaspore mother cell). The MMC, which is the primary female germline cell, is initiated as a single subepidermal cell amongst several somatic cells. MMC development is governed by various regulatory pathways involving intercellular signaling, small RNAs and DNA methylation. The programming and reprograming of a single nucellar cell to enter meiosis is governed by 'permissive' interacting processes and factors. Concomitantly, several nucellar sister cells are prevented from germline fate also by a set of 'repressive' factors. However, in certain angiosperms, anomalies in development of the female gametophyte have been observed. The sporophytic tissue surrounding the female gametophyte affects the gametophyte in multiple ways. The role of genes and transcription factors in the development of the MMC and in the regulation of various processes studied in selected model plants such as Arabidopsis is explained in detail in this paper. However, as angiosperms display enormous diversity, it is important to investigate early stages of megasporogenesis in other plant systems as well. Such studies provide valuable insights in understanding the regulation of megasporogenesis and the evolution of the female gametophyte from gymnosperms to flowering plants.

Peanut LEAFY COTYLEDON1-type genes participate in regulating the embryo development and the accumulation of storage lipids.

KEY MESSAGE: Two peanut LEC1-type genes exhibit partial functional redundancy. AhNFYB10 could complement almost all the defective phenotypes of lec1-2 in terms of embryonic morphology, while AhNF-YB1 could partially affect these phenotypes. LEAFY COTYLEDON1 (LEC1) is a member of the nuclear factor Y (NF-Y) family of transcription factors and has been identified as a key regulator of embryonic development. In the present study, two LEC1-type genes from Arachis hypogeae were identified and designated as AhNF-YB1 and AhNF-YB10; these genes belong to subgenome A and subgenome B, respectively. The functions of AhNF-YB1 and AhNF-YB10 were investigated by complementation analysis of their defective phenotypes of the Arabidopsis lec1-2 mutant and by ectopic expression in wild-type Arabidopsis. The results indicated that both AhNF-YB1 and AhNF-YB10 participate in regulating embryogenesis, embryo development, and reserve deposition in cotyledons and that they have partial functional redundancy. In contrast, AhNF-YB10 complemented almost all the defective phenotypes of lec1-2 in terms of embryonic morphology and hypocotyl length, while AhNF-YB1 had only a partial effect. In addition, 30-40% of the seeds of the AhNF-YB1 transformants exhibited a decreasing germination ratio and longevity. Therefore, appropriate spatiotemporal expression of these genes is necessary for embryo morphogenesis at the early development stage and is responsible for seed maturation at the mid-late development stage. On the other hand, overexpression of AhNF-YB1 or AhNF-YB10 at the middle to late stages of Arabidopsis seed development improved the weight, oil content, and fatty acid composition of the transgenic seeds. Moreover, the expression levels of several genes associated with fatty acid synthesis and embryogenesis were significantly greater in developing AhNF-YB10-overexpressing seeds than in control seeds. This study provides a theoretical basis for breeding oilseed crops with high yields and high oil content.

Regulation of adaptive growth decisions via phosphorylation of the TRAPPII complex in Arabidopsis.

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.

HD-Zip II transcription factors control distal stem cell fate in Arabidopsis roots by linking auxin signaling to the FEZ/SOMBRERO pathway.

In multicellular organisms, specialized tissues are generated by specific populations of stem cells through cycles of asymmetric cell divisions, where one daughter undergoes differentiation and the other maintains proliferative properties. In Arabidopsis thaliana roots, the columella - a gravity-sensing tissue that protects and defines the position of the stem cell niche - represents a typical example of a tissue whose organization is exclusively determined by the balance between proliferation and differentiation. The columella derives from a single layer of stem cells through a binary cell fate switch that is precisely controlled by multiple, independent regulatory inputs. Here, we show that the HD-Zip II transcription factors (TFs) HAT3, ATHB4 and AHTB2 redundantly regulate columella stem cell fate and patterning in the Arabidopsis root. The HD-Zip II TFs promote columella stem cell proliferation by acting as effectors of the FEZ/SMB circuit and, at the same time, by interfering with auxin signaling to counteract hormone-induced differentiation. Overall, our work shows that HD-Zip II TFs connect two opposing parallel inputs to fine-tune the balance between proliferation and differentiation in columella stem cells.

Association mechanism between Arabidopsis immune coreceptor BAK1 and Pseudomonas syringae effector HopF2.

Brassinosteroid activated kinase 1 (BAK1) is a cell-surface coreceptor which plays multiple roles in innate immunity of plants. HopF2 is an effector secreted by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 into Arabidopsis and suppresses host immune system through interaction with BAK1 as well as its downstream kinase MKK5. The association mechanism of HopF2 to BAK1 remains unclear, which prohibits our understanding and subsequent interfering of their interaction for pathogen management. Herein, we found the kinase domain of BAK1 (BAK1-KD) is sufficient for HopF2 association. With a combination of hydrogen/deuterium exchange mass spectrometry and mutational assays, we found a region of BAK1-KD N-lobe and a region of HopF2 head subdomain are critical for intermolecular interaction, which is also supported by unbiased protein-protein docking with ClusPro and kinase activity assay. Collectively, this research presents the interaction mechanism between Arabidopsis BAK1 and P. syringae HopF2, which could pave the way for bactericide development that blocking the functioning of HopF2 toward BAK1.

Transcriptional and post-transcriptional regulation of chloroplast development by nuclear-localized XAP5 CIRCADIAN TIMEKEEPER.

Chlorophyll biosynthesis and breakdown, important cellular processes for photosynthesis, occur in the chloroplast. As a semi-autonomous organelle, chloroplast development is mainly regulated by nuclear-encoded chloroplast proteins and proteins encoded by itself. However, the knowledge of chloroplast development regulated by other organelles is limited. Here, we report that the nuclear-localized XAP5 CIRCADIAN TIMEKEEPER (XCT) is essential for chloroplast development in Arabidopsis. In this study, significantly decreased chlorophyll content phenotypes of cotyledons and subsequently emerging organs from shoot apical meristem were observed in xct-2. XCT is constitutively expressed in various tissues and localized in the nuclear with speckle patterns. RNA-seq analysis identified 207 differently spliced genes and 1511 differently expressed genes, in which chloroplast development-, chlorophyll metabolism- and photosynthesis-related genes were enriched. Further biochemical assays suggested that XCT was co-purified with the well-known splicing factors and transcription machinery, suggesting dual functions of XCT in gene transcription and splicing. Interestingly, we also found that the chlorophyll contents in xct-2 significantly decreased under high temperature and high light condition, indicating XCT integrates temperature and light signals to fine-tune the chlorophyll metabolism in Arabidopsis. Therefore, our results provide new insights into chloroplast development regulation by XCT, a nuclear-localized protein, at the transcriptional and post-transcriptional level.

WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development.

Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.

Functional screening of the Arabidopsis 2C protein phosphatases family identifies PP2C15 as a negative regulator of plant immunity by targeting BRI1-associated receptor kinase 1.

Genetic engineering using negative regulators of plant immunity has the potential to provide a huge impetus in agricultural biotechnology to achieve a higher degree of disease resistance without reducing yield. Type 2C protein phosphatases (PP2Cs) represent the largest group of protein phosphatases in plants, with a high potential for negative regulatory functions by blocking the transmission of defence signals through dephosphorylation. Here, we established a PP2C functional protoplast screen using pFRK1::luciferase as a reporter and found that 14 of 56 PP2Cs significantly inhibited the immune response induced by flg22. To verify the reliability of the system, a previously reported MAPK3/4/6-interacting protein phosphatase, PP2C5, was used; it was confirmed to be a negative regulator of PAMP-triggered immunity (PTI). We further identified PP2C15 as an interacting partner of BRI1-associated receptor kinase 1 (BAK1), which is the most well-known co-receptor of plasma membrane-localized pattern recognition receptors (PRRs), and a central component of PTI. PP2C15 dephosphorylates BAK1 and negatively regulates BAK1-mediated PTI responses such as MAPK3/4/6 activation, defence gene expression, reactive oxygen species bursts, stomatal immunity, callose deposition, and pathogen resistance. Although plant growth and 1000-seed weight of pp2c15 mutants were reduced compared to those of wild-type plants, pp2c5 mutants did not show any adverse effects. Thus, our findings strengthen the understanding of the mechanism by which PP2C family members negatively regulate plant immunity at multiple levels and indicate a possible approach to enhance plant resistance by eliminating specific PP2Cs without affecting plant growth and yield.

Two orthogonal differentiation gradients locally coordinate fruit morphogenesis.

Morphogenesis requires the coordination of cellular behaviors along developmental axes. In plants, gradients of growth and differentiation are typically established along a single longitudinal primordium axis to control global organ shape. Yet, it remains unclear how these gradients are locally adjusted to regulate the formation of complex organs that consist of diverse tissue types. Here we combine quantitative live imaging at cellular resolution with genetics, and chemical treatments to understand the formation of Arabidopsis thaliana female reproductive organ (gynoecium). We show that, contrary to other aerial organs, gynoecium shape is determined by two orthogonal, time-shifted differentiation gradients. An early mediolateral gradient controls valve morphogenesis while a late, longitudinal gradient regulates style differentiation. Local, tissue-dependent action of these gradients serves to fine-tune the common developmental program governing organ morphogenesis to ensure the specialized function of the gynoecium.

Characterization and expression analysis of GLABRA3 (GL3) genes in cotton: insights into trichome development and hormonal regulation.

BACKGROUND: GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) genes encode a typical helix-loop-helix (bHLH) transcription factors that primarily regulate trichome branching and root hair development, DNA endoreduplication, trichoblast size, and stomatal formation. The functions of GL3 genes in cotton crop have been poorly characterized. In this study, we performed comprehensive genome-wide scans for GL3 and EGL3 homologs to enhance our comprehension of their potential roles in trichome and fiber development in cotton crop. METHODS AND RESULTS: Our findings paraded that Gossypium hirsutum and G. barbadense have 6 GL3s each, unevenly distributed on 4 chromosomes whereas, G. arboreum, and G. raimondii have 3 GL3s each, unevenly distributed on 2 chromosomes. Gh_A08G2088 and Gb_A09G2187, despite having the same bHLH domain as the other GL3 genes, were excluded due to remarkable short sequences and limited number of motifs, indicating a lack of potential functional activity. The phylogenetic analysis categorized remaining 16 GL3s into three subfamilies (Group I-III) closely related to A. thaliana. The 16 GL3s have complete bHLH domain, encompassing 590-631 amino acids, with molecular weights (MWs) ranging from 65.92 to 71.36 kDa. Within each subfamily GL3s depicted shared similar gene structures and motifs, indicating conserved characteristics within respective groups. Promoter region analysis revealed 27 cis-acting elements, these elements were responsive to salicylic acid, abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellin. The expression of GL3 genes was analyzed across 12 tissues in both G. barbadense and G. hirsutum using the publicly available RNA-seq data. Among GL3s, Gb_D11G0219, Gb_D11G0214, and Gb_D08G2182, were identified as relatively highly expressed across different tissues, consequently selected for hormone treatment and expression validation in G. barbadense. RT-qPCR results demonstrated significant alterations in the expression levels of Gb_D11G0219 and Gb_D11G0214 following MeJA, GA, and ABA treatment. Subcellular localization prediction revealed that most GL3 proteins were predominantly expressed in the nucleus, while a few were localized in the cytoplasm and chloroplasts. CONCLUSIONS: In summary, this study lays the foundation for subsequent functional validation of GL3 genes by identifying hormonal regulation patterns and probable sites of action in cotton trichome formation and fiber development. The results stipulate a rationale to elucidate the roles and regulatory mechanisms of GL3 genes in the intricate process of cotton fibre and trichome development.

Unveiling the TRAPP: The role of plant TRAPPII in adaptive growth decisions.

The regulation of intracellular membrane traffic is coupled with the cell's need to respond to environmental stimuli, which ultimately is critical for different processes such as cell growth and development. In this issue, Wiese et al. (https://www.doi.org/10.1083/jcb.202311125) explore the role of the trans-Golgi network (TGN) in stress response, exposing its role in mediating adaptive growth decisions.

Three new discovery effector proteins from Candidatus Liberibacter asiaticus psy62 inhibit plant defense through interaction with AtCAT3 and AtGAPA.

KEY MESSAGE: We identify three SDEs that inhibiting host defence from Candidatus Liberibacter asiaticus psy62, which is an important supplement to the pathogenesis of HLB. Candidatus Liberibacter asiaticus (CLas) is the main pathogen of citrus Huanglongbing (HLB). 38 new possible sec-dependent effectors (SDEs) of CLas psy62 were predicted by updated predictor SignalP 5.0, which 12 new SDEs were found using alkaline phosphate assay. Among them, SDE4310, SDE4435 and SDE4955 inhibited hypersensitivity reactions (HR) in Arabidopsis thaliana (Arabidopsis, At) and Nicotiana benthamiana leaves induced by pathogens, which lead to a decrease in cell death and reactive oxygen species (ROS) accumulation. And the expression levels of SDE4310, SDE4435, and SDE4955 genes elevated significantly in mild symptom citrus leaves. When SDE4310, SDE4435 and SDE4955 were overexpressed in Arabidopsis, HR pathway key genes pathogenesis-related 2 (PR2), PR5, nonexpressor of pathogenesis-related 1 (NPR1) and isochorismate synthase 1 (ICS1) expression significantly decreased and the growth of pathogen was greatly increased relative to control with Pst DC3000/AvrRps4 treatment. Our findings also indicated that SDE4310, SDE4435 and SDE4955 interacted with AtCAT3 (catalase 3) and AtGAPA (glyceraldehyde-3-phosphate dehydrogenase A). In conclusion, our results suggest that SDE4310, SDE4435 and SDE4955 are CLas psy62 effector proteins that may have redundant functions. They inhibit ROS burst and cell death by interacting with AtCAT3 and AtGAPA to negatively regulate host defense.

An Escherichia coli-Based Phosphorylation System for Efficient Screening of Kinase Substrates.

Posttranslational modifications (PTMs), particularly phosphorylation, play a pivotal role in expanding the complexity of the proteome and regulating diverse cellular processes. In this study, we present an efficient Escherichia coli phosphorylation system designed to streamline the evaluation of potential substrates for Arabidopsis thaliana plant kinases, although the technology is amenable to any. The methodology involves the use of IPTG-inducible vectors for co-expressing kinases and substrates, eliminating the need for radioactive isotopes and prior protein purification. We validated the system's efficacy by assessing the phosphorylation of well-established substrates of the plant kinase SnRK1, including the rat ACETYL-COA CARBOXYLASE 1 (ACC1) and FYVE1/FREE1 proteins. The results demonstrated the specificity and reliability of the system in studying kinase-substrate interactions. Furthermore, we applied the system to investigate the phosphorylation cascade involving the A. thaliana MKK3-MPK2 kinase module. The activation of MPK2 by MKK3 was demonstrated to phosphorylate the Myelin Basic Protein (MBP), confirming the system's ability to unravel sequential enzymatic steps in phosphorylation cascades. Overall, this E. coli phosphorylation system offers a rapid, cost-effective, and reliable approach for screening potential kinase substrates, presenting a valuable tool to complement the current portfolio of molecular techniques for advancing our understanding of kinase functions and their roles in cellular signaling pathways.

Post-translational modifications: emerging directors of cell-fate decisions during endoplasmic reticulum stress in Arabidopsis thaliana.

Homeostasis of the endoplasmic reticulum (ER) is critical for growth, development, and stress responses. Perturbations causing an imbalance in ER proteostasis lead to a potentially lethal condition known as ER stress. In ER stress situations, cell-fate decisions either activate pro-life pathways that reestablish homeostasis or initiate pro-death pathways to prevent further damage to the organism. Understanding the mechanisms underpinning cell-fate decisions in ER stress is critical for crop development and has the potential to enable translation of conserved components to ER stress-related diseases in metazoans. Post-translational modifications (PTMs) of proteins are emerging as key players in cell-fate decisions in situations of imbalanced ER proteostasis. In this review, we address PTMs orchestrating cell-fate decisions in ER stress in plants and provide evidence-based perspectives for where future studies may focus to identify additional PTMs involved in ER stress management.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Organ-delimited gene regulatory networks provide high accuracy in candidate transcription factor selection across diverse processes.

Organ-specific gene expression datasets that include hundreds to thousands of experiments allow the reconstruction of organ-level gene regulatory networks (GRNs). However, creating such datasets is greatly hampered by the requirements of extensive and tedious manual curation. Here, we trained a supervised classification model that can accurately classify the organ-of-origin for a plant transcriptome. This K-Nearest Neighbor-based multiclass classifier was used to create organ-specific gene expression datasets for the leaf, root, shoot, flower, and seed in Arabidopsis thaliana. A GRN inference approach was used to determine the: i. influential transcription factors (TFs) in each organ and, ii. most influential TFs for specific biological processes in that organ. These genome-wide, organ-delimited GRNs (OD-GRNs), recalled many known regulators of organ development and processes operating in those organs. Importantly, many previously unknown TF regulators were uncovered as potential regulators of these processes. As a proof-of-concept, we focused on experimentally validating the predicted TF regulators of lipid biosynthesis in seeds, an important food and biofuel trait. Of the top 20 predicted TFs, eight are known regulators of seed oil content, e.g., WRI1, LEC1, FUS3. Importantly, we validated our prediction of MybS2, TGA4, SPL12, AGL18, and DiV2 as regulators of seed lipid biosynthesis. We elucidated the molecular mechanism of MybS2 and show that it induces purple acid phosphatase family genes and lipid synthesis genes to enhance seed lipid content. This general approach has the potential to be extended to any species with sufficiently large gene expression datasets to find unique regulators of any trait-of-interest.

NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

The small iron-deficiency-induced protein OLIVIA and its relation to the bHLH transcription factor POPEYE.

Iron (Fe) is a crucial micronutrient needed in many metabolic processes. To balance needs and potential toxicity, plants control the amount of Fe they take up and allocate to leaves and seeds during their development. One important regulator of this process is POPEYE (PYE). PYE is a Fe deficiency-induced key bHLH transcription factor (TF) for allocation of internal Fe in plants. In the absence of PYE, there is altered Fe translocation and plants develop a leaf chlorosis. NICOTIANAMINE SYNTHASE4 (NAS4), FERRIC-REDUCTION OXIDASE3 (FRO3), and ZINC-INDUCED FACILITATOR1 (ZIF1) genes are expressed at higher level in pye-1 indicating that PYE represses these genes. PYE activity is controlled in a yet unknown manner. Here, we show that a small Fe deficiency-induced protein OLIVIA (OLV) can interact with PYE. OLV has a conserved C-terminal motif, that we named TGIYY. Through deletion mapping, we pinpointed that OLV TGIYY and several regions of PYE can be involved in the protein interaction. An OLV overexpressing (OX) mutant line exhibited an enhanced NAS4 gene expression. This was a mild Fe deficiency response phenotype that was related to PYE function. Leaf rosettes of olv mutants remained smaller than those of wild type, indicating that OLV promotes plant growth. Taken together, our study identified a small protein OLV as a candidate that may connect aspects of Fe homeostasis with regulation of leaf growth.

Expression of the Arabidopsis redox-related LEA protein, SAG21 is regulated by ERF, NAC and WRKY transcription factors.

SAG21/LEA5 is an unusual late embryogenesis abundant protein in Arabidopsis thaliana, that is primarily mitochondrially located and may be important in regulating translation in both chloroplasts and mitochondria. SAG21 expression is regulated by a plethora of abiotic and biotic stresses and plant growth regulators indicating a complex regulatory network. To identify key transcription factors regulating SAG21 expression, yeast-1-hybrid screens were used to identify transcription factors that bind the 1685 bp upstream of the SAG21 translational start site. Thirty-three transcription factors from nine different families bound to the SAG21 promoter, including members of the ERF, WRKY and NAC families. Key binding sites for both NAC and WRKY transcription factors were tested through site directed mutagenesis indicating the presence of cryptic binding sites for both these transcription factor families. Co-expression in protoplasts confirmed the activation of SAG21 by WRKY63/ABO3, and SAG21 upregulation elicited by oligogalacturonide elicitors was partially dependent on WRKY63, indicating its role in SAG21 pathogen responses. SAG21 upregulation by ethylene was abolished in the erf1 mutant, while wound-induced SAG21 expression was abolished in anac71 mutants, indicating SAG21 expression can be regulated by several distinct transcription factors depending on the stress condition.

Disrupting FKF1 homodimerization increases FT transcript levels in the evening by enhancing CO stabilization.

KEY MESSAGE: FKF1 dimerization is crucial for proper FT levels to fine-tune flowering time. Attenuating FKF1 homodimerization increased CO abundance by enhancing its COP1 binding, thereby accelerating flowering under long days. In Arabidopsis (Arabidopsis thaliana), the blue-light photoreceptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) plays a key role in inducing the expression of FLOWERING LOCUS T (FT), encoding the main florigenic signal in plants, in the late afternoon under long-day conditions (LDs) by forming dimers with FT regulators. Although structural studies have unveiled a variant of FKF1 (FKF1 I160R) that disrupts homodimer formation in vitro, the mechanism by which disrupted FKF1 homodimer formation regulates flowering time remains elusive. In this study, we determined that the attenuation of FKF1 homodimer formation enhances FT expression in the evening by promoting the increased stability of CONSTANS (CO), a primary activator of FT, in the afternoon, thereby contributing to early flowering. In contrast to wild-type FKF1, introducing the FKF1 I160R variant into the fkf1 mutant led to increased FT expression under LDs. In addition, the FKF1 I160R variant exhibited diminished dimerization with FKF1, while its interaction with GIGANTEA (GI), a modulator of FKF1 function, was enhanced under LDs. Furthermore, the FKF1 I160R variant increased the level of CO in the afternoon under LDs by enhancing its binding to COP1, an E3 ubiquitin ligase responsible for CO degradation. These findings suggest that the regulation of FKF1 homodimerization and heterodimerization allows plants to finely adjust FT expression levels around dusk by modulating its interactions with GI and COP1.