Vacuolar H+-ATPase and BZR1 form a feedback loop to regulate the homeostasis of BR signaling in Arabidopsis.

Brassinosteroid (BR) is a vital plant hormone that regulates plant growth and development. BRASSINAZOLE RESISTANT 1 (BZR1) is a key transcription factor in BR signaling, and its nucleocytoplasmic localization is crucial for BR signaling. However, the mechanisms that regulate BZR1 nucleocytoplasmic distribution and thus the homeostasis of BR signaling remain largely unclear. The vacuole is the largest organelle in mature plant cells and plays a key role in maintenance of cellular pH, storage of intracellular substances, and transport of ions. In this study, we uncovered a novel mechanism of BR signaling homeostasis regulated by the vacuolar H+-ATPase (V-ATPase) and BZR1 feedback loop. Our results revealed that the vha-a2 vha-a3 mutant (vha2, lacking V-ATPase activity) exhibits enhanced BR signaling with increased total amount of BZR1, nuclear-localized BZR1, and the ratio of BZR1/phosphorylated BZR1 in the nucleus. Further biochemical assays revealed that VHA-a2 and VHA-a3 of V-ATPase interact with the BZR1 protein through a domain that is conserved across multiple species. VHA-a2 and VHA-a3 negatively regulate BR signaling by interacting with BZR1 and promoting its retention in the tonoplast. Interestingly, a series of molecular analyses demonstrated that nuclear-localized BZR1 could bind directly to specific motifs in the promoters of VHA-a2 and VHA-a3 to promote their expression. Taken together, these results suggest that V-ATPase and BZR1 may form a feedback regulatory loop to maintain the homeostasis of BR signaling in Arabidopsis, providing new insights into vacuole-mediated regulation of hormone signaling.

Corrigendum: Tonoplast proton pumps regulate nuclear spacing of female gametophytes via mediating polar auxin transport in arabidopsis.

[This corrects the article DOI: 10.3389/fpls.2022.1006735.].

Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis.

In Arabidopsis thaliana, female gametophyte (FG) development is accompanied by the formation and expansion of the large vacuole in the FG; this is essential for FG expansion, nuclear polar localization, and cell fate determination. Arabidopsis VACUOLELESS GAMETOPHYTES (VLG) facilitates vesicular fusion to form large vacuole in the FG, but the regulation of VLG remains largely unknown. Here, we found that gain-of-function mutation of BRASSINOSTEROID INSENSITIVE2 (BIN2) (bin2-1) increases VLG abundance to induce the vacuole formation at stage FG1, and leads to abortion of FG. Loss-of-function mutation of BIN2 and its homologs (bin2-3 bil1 bil2) reduced VLG abundance and mimicked vlg/VLG phenotypes. Knocking down VLG in bin2-1 decreased the ratio of aberrant vacuole formation at stage FG1, whereas FG1-specific overexpression of VLG mimicked the bin2-1 phenotype. VLG partially rescued the bin2-3 bil1 bil2 phenotype, demonstrating that VLG acts downstream of BIN2. Mutation of VLG residues that are phosphorylated by BIN2 altered VLG stability and a phosphorylation mimic of VLG causes similar defects as did bin2-1. Therefore, BIN2 may function by interacting with and phosphorylating VLG in the FG to enhance its stability and abundance, thus facilitating vacuole formation. Our findings provide mechanistic insight into how the BIN2-VLG module regulates the spatiotemporal formation of the large vacuole in FG development.

Asprosin in the Paraventricular Nucleus Induces Sympathetic Activation and Pressor Responses via cAMP-Dependent ROS Production.

Asprosin is a newly discovered adipokine that is involved in regulating metabolism. Sympathetic overactivity contributes to the pathogenesis of several cardiovascular diseases. The paraventricular nucleus (PVN) of the hypothalamus plays a crucial role in the regulation of sympathetic outflow and blood pressure. This study was designed to determine the roles and underlying mechanisms of asprosin in the PVN in regulating sympathetic outflow and blood pressure. Experiments were carried out in male adult SD rats under anesthesia. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) were recorded, and PVN microinjections were performed bilaterally. Asprosin mRNA and protein expressions were high in the PVN. The high asprosin expression in the PVN was involved in both the parvocellular and magnocellular regions according to immunohistochemical analysis. Microinjection of asprosin into the PVN produced dose-related increases in RSNA, MAP, and HR, which were abolished by superoxide scavenger tempol, antioxidant N-acetylcysteine (NAC), and NADPH oxidase inhibitor apocynin. The asprosin promoted superoxide production and increased NADPH oxidase activity in the PVN. Furthermore, it increased the cAMP level, adenylyl cyclase (AC) activity, and protein kinase A (PKA) activity in the PVN. The roles of asprosin in increasing RSNA, MAP, and HR were prevented by pretreatment with AC inhibitor SQ22536 or PKA inhibitor H89 in the PVN. Microinjection of cAMP analog db-cAMP into the PVN played similar roles with asprosin in increasing the RSNA, MAP, and HR, but failed to further augment the effects of asprosin. Pretreatment with PVN microinjection of SQ22536 or H89 abolished the roles of asprosin in increasing superoxide production and NADPH oxidase activity in the PVN. These results indicated that asprosin in the PVN increased the sympathetic outflow, blood pressure, and heart rate via cAMP-PKA signaling-mediated NADPH oxidase activation and the subsequent superoxide production.

Tonoplast proton pumps regulate nuclear spacing of female gametophytes via mediating polar auxin transport in Arabidopsis.

The vacuole is an important organelle with multiple functions in plants, and the tonoplast that wraps the vacuole also plays essential roles in intracellular trafficking and ion homeostasis. Previous studies found that tonoplast proton pumps regulate embryo development and morphogenesis through their effects on vacuole biogenesis and distribution, as well as polar auxin transport and concomitant auxin gradient. However, the precise roles of the tonoplast proton pumps in gametophyte development remain unclear. Here we demonstrated that the lack of two types of tonoplast proton pumps or the absence of V-ATPase alone leads to abnormal development and nuclear localization of female gametophyte (FG), and slowed endosperm nuclei division after fertilization of the central cell. We further revealed that V-ATPase regulates auxin levels in ovules through coordinating the content and localization of PIN-FORMED 1 (PIN1) protein, hence influencing nuclear spacing between centra cell and egg cell, and subsequent endosperm development. Collectively, our findings revealed a crucial role of V-ATPase in auxin-mediated FG development in Arabidopsis and expanded our understanding of the functions of tonoplast proton pumps in seed plants reproductive development.

Ovule initiation: the essential step controlling offspring number in Arabidopsis.

Seed is the offspring of angiosperms. Plants produce large numbers of seeds to ensure effective reproduction and survival in varying environments. Ovule is a fundamentally important organ and is the precursor of the seed. In Arabidopsis and other plants characterized by multi-ovulate ovaries, ovule initiation determines the maximal ovule number, thus greatly affecting seed number per fruit and seed yield. Investigating the regulatory mechanism of ovule initiation has both scientific and economic significance. However, the genetic and molecular basis underlying ovule initiation remains unclear due to technological limitations. Very recently, rules governing the multiple ovules initiation from one placenta have been identified, the individual functions and crosstalk of phytohormones in regulating ovule initiation have been further characterized, and new regulators of ovule boundary are reported, therefore expanding the understanding of this field. In this review, we present an overview of current knowledge in ovule initiation and summarize the significance of ovule initiation in regulating the number of plant offspring, as well as raise insights for the future study in this field that provide potential routes for the improvement of crop yield.

PIN3 positively regulates the late initiation of ovule primordia in Arabidopsis thaliana.

Ovule initiation determines the maximum ovule number and has great impact on seed number and yield. However, the regulation of ovule initiation remains largely elusive. We previously reported that most of the ovule primordia initiate asynchronously at floral stage 9 and PINFORMED1 (PIN1) polarization and auxin distribution contributed to this process. Here, we further demonstrate that a small amount of ovule primordia initiate at floral stage 10 when the existing ovules initiated at floral stage 9 start to differentiate. Genetic analysis revealed that the absence of PIN3 function leads to the reduction in pistil size and the lack of late-initiated ovules, suggesting PIN3 promotes the late ovule initiation process and pistil growth. Physiological analysis illustrated that, unlike picloram, exogenous application of NAA can't restore these defective phenotypes, implying that PIN3-mediated polar auxin transport is required for the late ovule initiation and pistil length. qRT-PCR results indicated that the expression of SEEDSTICK (STK) is up-regulated under auxin analogues treatment while is down-regulated in pin3 mutants. Meanwhile, overexpressing STK rescues pin3 phenotypes, suggesting STK participates in PIN3-mediated late ovule initiation possibly by promoting pistil growth. Furthermore, brassinosteroid influences the late ovule initiation through positively regulating PIN3 expression. Collectively, this study demonstrates that PIN3 promotes the late ovule initiation and contributes to the extra ovule number. Our results give important clues for increasing seed number and yield of cruciferous and leguminous crops.

Phosphatidic acid suppresses autophagy through competitive inhibition by binding GAPC (glyceraldehyde-3-phosphate dehydrogenase) and PGK (phosphoglycerate kinase) proteins.

Macroautophagy/autophagy is a finely-regulated process in which cytoplasm encapsulated within transient organelles termed autophagosomes is delivered to lysosomes or vacuoles for degradation. Phospholipids, particularly phosphatidic acid (PA) that functions as a second messenger, play crucial and differential roles in autophagosome formation; however, the underlying mechanism remains largely unknown. Here we demonstrated that PA inhibits autophagy through competitive inhibition of the formation of ATG3 (autophagy-related)-ATG8e and ATG6-VPS34 (vacuolar protein sorting 34) complexes. PA bound to GAPC (glyceraldehyde-3-phosphate dehydrogenase) or PGK (phosphoglycerate kinase) and promoted their interaction with ATG3 or ATG6, which further attenuated the interactions of ATG3-ATG8e or ATG6-VPS34, respectively. Structural and mutational analyses revealed the mechanism of PA binding with GAPCs and PGK3, and that GAPCs or ATG8e competitively interacted with ATG3, and PGK3 or VPS34 competitively interacted with ATG6, at the same binding interface. These results elucidate the molecular mechanism of how PA inhibits autophagy through binding GAPC or PGK3 proteins and expand the understanding of the functional mode of PA, demonstrating the importance of phospholipids in plant autophagy and providing a new perspective for autophagy regulation by phospholipids.Abbreviation: ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; co-IP: co-immunoprecipitation; Con A: concanamycin A; ER: endoplasmic reticulum; EZ: elongation zone; FRET-FLIM: fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S-transferase; MDC: monodansylcadaverine; MZ: meristem zone; PA: phosphatidic acid; PAS: phagophore assembly site; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PGK3: phosphoglycerate kinase; PtdIns3K: phosphatidylinositol 3-kinase; PLD: phospholipase D; TEM: transmission electron microscopy; TOR: target of rapamycin; VPS34: vacuolar protein sorting 34; WT: wild type; Y2H: yeast two-hybrid.

Interaction of brassinosteroid and cytokinin promotes ovule initiation and increases seed number per silique in Arabidopsis.

Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild-type plants. Here, we crossed BR- and CK-related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK-mediated and -independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops.

Multiple functions of the vacuole in plant growth and fruit quality.

Vacuoles are organelles in plant cells that play pivotal roles in growth and developmental regulation. The main functions of vacuoles include maintaining cell acidity and turgor pressure, regulating the storage and transport of substances, controlling the transport and localization of key proteins through the endocytic and lysosomal-vacuolar transport pathways, and responding to biotic and abiotic stresses. Further, proteins localized either in the tonoplast (vacuolar membrane) or inside the vacuole lumen are critical for fruit quality. In this review, we summarize and discuss some of the emerging functions and regulatory mechanisms associated with plant vacuoles, including vacuole biogenesis, vacuole functions in plant growth and development, fruit quality, and plant-microbe interaction, as well as some innovative research technology that has driven advances in the field. Together, the functions of plant vacuoles are important for plant growth and fruit quality. The investigation of vacuole functions in plants is of great scientific significance and has potential applications in agriculture.

Asynchrony of ovule primordia initiation in Arabidopsis.

Plant ovule initiation determines the maximum of ovule number and has a great impact on the seed number per fruit. The detailed processes of ovule initiation have not been accurately described, although two connected processes, gynoecium and ovule development, have been investigated. Here, we report that ovules initiate asynchronously. The first group of ovule primordia grows out, the placenta elongates, the boundaries of existing ovules enlarge and a new group of primordia initiates from the boundaries. The expression pattern of different marker genes during ovule development illustrates that this asynchronicity continues throughout whole ovule development. PIN-FORMED1 polar distribution and auxin response maxima correlate with ovule primordia asynchronous initiation. We have established computational modeling to show how auxin dynamics influence ovule primordia initiation. Brassinosteroid signaling positively regulates ovule number by promoting placentae size and ovule primordia initiation through strengthening auxin response. Transcriptomic analysis demonstrates numerous known regulators of ovule development and hormone signaling, and many new genes are identified that are involved in ovule development. Taken together, our results illustrate that the ovule primordia initiate asynchronously and the hormone signals are involved in the asynchrony.

Genome-Wide Association Analysis Identifies Candidate Genes Regulating Seed Number Per Silique in Arabidopsis thaliana.

Seed weight and number ultimately determine seed yield. Arabidopsis seed number comprised of silique number and seed number per silique (SNS). Comparing seed development and weight, determinants of seed number remain largely uncharacterized. In this study, taking advantage of 107 available Arabidopsis accessions, genome-wide association analysis (GWAS) was employed to identify the candidate genes regulating SNS. GWAS-based genotype and phenotype association analysis identified 38 most significant SNPs marker sites that were mapped to specific chromosomal positions and allowed us to screen for dozens of candidate genes. One of them (PIN3) was selected for functional validation based on gene expression analysis. It is a positive regulator of Arabidopsis SNS. Although silique length of PIN3 loss of function mutant was not significantly changed, its SNS and seed density (SD) were significantly reduced as compared with the wild type. Notably, PIN3 overexpression lines driven by a placenta-specific promoter STK exhibited significantly shorter siliques, slightly reduced SNS, but significant increased SD compared with wild type, suggesting that PIN3 positively regulates SD through inducing ovule primordia initiation regardless of the placenta size. Ovule initiation determines the maximal possibility of SNS, and new genes and mechanism regulating SNS through modulating ovule initiation is worth further investigated.

Two tonoplast proton pumps function in Arabidopsis embryo development.

Two types of tonoplast proton pumps, H+ -pyrophosphatase (V-PPase) and the H+ -ATPase (V-ATPase), establish the proton gradient that powers molecular traffic across the tonoplast thereby facilitating turgor regulation and nutrient homeostasis. However, how proton pumps regulate development remains unclear. In this study, we investigated the function of two types of proton pumps in Arabidopsis embryo development and pattern formation. While disruption of either V-PPase or V-ATPase had no obvious effect on plant embryo development, knocking out both resulted in severe defects in embryo pattern formation from the early stage. While the first division in wild-type zygote was asymmetrical, a nearly symmetrical division occurred in the mutant, followed by abnormal pattern formation at all stages of embryo development. The embryonic defects were accompanied by dramatic differences in vacuole morphology and distribution, as well as disturbed localisation of PIN1. The development of mutant cotyledons and root, and the auxin response of mutant seedlings supported the hypothesis that mutants lacking tonoplast proton pumps were defective in auxin transport and distribution. Taking together, we proposed that two tonoplast proton pumps are required for vacuole morphology and PIN1 localisation, thereby controlling vacuole and auxin-related developmental processes in Arabidopsis embryos and seedlings.

Effective Modulating Brassinosteroids Signal to Study Their Specific Regulation of Reproductive Development and Enhance Yield.

Brassinosteroid (BR) is a family of bioactive steroid hormones that plays vital roles in plant growth and development. The BR-mediated regulation of plant growth and architecture has been well studied. However, relatively few studies have investigated the BR-related regulation of reproductive development because of the difficulties in excluding non-specific regulation and secondary responses from severe vegetative phenotypes and poor nutritional status. Furthermore, differentially regulating the BR signal in vegetative and reproductive organs is problematic. Thus, establishing a method for modulating the BR signal only in reproductive organs or during reproductive developmental stages will be beneficial. Additionally, the utility of BR applications for crop production is limited because of deleterious side-effects, including the associated decrease in the planting density and lodging resistance. Moreover, enhancing the BR signal may lead to feedback inhibition. In this study, we developed a transformation system for modulating the BR signal differentially during reproductive and vegetative developmental stages. This system involves transformations with different combinations of a reproductive tissue-specific promoter, coding sequences that increase or decrease the BR signal, and various genotypic backgrounds with enhanced or decreased BR signals. The enhanced BR signal generated in transformants was targeted to reproductive organs without affecting vegetative organs. This system may be useful for studying the BR-specific regulation of plant reproductive development and shows promise for optimizing seed yield.