The A subunit of vacuolar H+-ATPase gene (CmVHA-A) plays opposite roles in plant growth and drought tolerance of chrysanthemum under different growing conditions.

As the most prominent proton pumps in plants, vacuolar H+-ATPases (VHAs) comprise multiple subunits that are important for physiological processes and stress tolerance in plants. However, few studies on the roles of subunit genes of VHAs in chrysanthemum have been reported to date. In this study, the gene of A subunit of V-ATPase in chrysanthemum (CmVHA-A) was cloned and identified. CmVHA-A was conserved with VHA-A proteins from other plants. Expression analysis showed that CmVHA-A was highly expressed in most tissues of chrysanthemum except for the flower bud, and was readily induced by polyethylene glycol (PEG) treatment. Functional analysis demonstrated that CmVHA-A exerted a negative influence on the growth and development of shoot and root of chrysanthemum under normal conditions. RNA-sequencing (RNA-seq) analysis revealed the possible explanations for phenotypic differences between transgenic and wild-type (WT) plants. Under drought conditions, CmVHA-A positively affected the drought tolerance of chrysanthemum by enhancing antioxidase activity and alleviating photosynthetic disruption. Overall, CmVHA-A plays opposite roles in plant growth and drought tolerance of chrysanthemums under different growing conditions.

The conserved evolution of plant H+-ATPase family and the involvement of soybean H+-ATPases in sodium bicarbonate stress responses.

Plant plasma membrane (PM) H+-ATPases are essential pumps involved in multiple physiological processes. They play a significant role in regulating pH homeostasis and membrane potential by generating the electrochemical gradient of the proton across the plasma membrane. However, information on soybean PM H+-ATPase is still limited. In this study, we conducted the evolutionary analysis of PM H+-ATPases in land plants and investigated the subfamily classification and whole genome duplication of PM H+-ATPases in angiosperms. We further characterized the extremely high conservation of the soybean PM H+-ATPase family in terms of gene structure, domain architecture, and protein sequence identity. Using the yeast system, we confirmed the highly conserved biochemical characteristics (14-3-3 binding affinity and pump activity) of soybean PM H+-ATPases and their conserved function in enhancing tolerance to high pH and NaHCO3 stresses. Meanwhile, our results also revealed their divergence in the transcriptional expression in different tissues and under sodium bicarbonate stress. Finally, the function of soybean PM H+-ATPases in conferring sodium bicarbonate tolerance was validated using transgenic Arabidopsis. Together, these results conclude that the soybean PM H+-ATPase is evolutionarily conserved and positively regulates the response to sodium bicarbonate stress.

Determinants, maintenance, and function of organellar pH.

The protonation state of soluble and membrane-associated macromolecules dictates their charge, conformation, and functional activity. In addition, protons (H+ or their equivalents) partake in numerous metabolic reactions and serve as a source of electrochemical energy to drive the transmembrane transport of both organic and inorganic substrates. Stringent regulation of the intracellular pH is therefore paramount to homeostasis. Although the regulation of the cytosolic pH has been studied extensively, our understanding of the determinants of the H+ concentration ([H+]) of intracellular organelles has developed more slowly, limited by their small size and inaccessibility. Recently, however, targeting of molecular probes to the organellar lumen together with advances in genomic, proteomic, and electrophysiological techniques have led to the identification and characterization of unique pumps, channels, and transporters responsible for the establishment and maintenance of intraorganellar pH. These developments and their implications for cellular function in health and disease are the subject of this review.

The molecular mechanism of plasma membrane H+-ATPases in plant responses to abiotic stress.

Plasma membrane H+-ATPases (PM H+-ATPases) are critical proton pumps that export protons from the cytoplasm to the apoplast. The resulting proton gradient and difference in electrical potential energize various secondary active transport events. PM H+-ATPases play essential roles in plant growth, development, and stress responses. In this review, we focus on recent studies of the mechanism of PM H+-ATPases in response to abiotic stresses in plants, such as salt and high pH, temperature, drought, light, macronutrient deficiency, acidic soil and aluminum stress, as well as heavy metal toxicity. Moreover, we discuss remaining outstanding questions about how PM H+-ATPases contribute to abiotic stress responses.

Functional Analysis of the Plasma Membrane H+-ATPases of Ustilago maydis.

Plasma membrane H+-ATPases of fungi, yeasts, and plants act as proton pumps to generate an electrochemical gradient, which is essential for secondary transport and intracellular pH maintenance. Saccharomyces cerevisiae has two genes (PMA1 and PMA2) encoding H+-ATPases. In contrast, plants have a larger number of genes for H+-ATPases. In Ustilago maydis, a biotrophic basidiomycete that infects corn and teosinte, the presence of two H+-ATPase-encoding genes has been described, one with high identity to the fungal enzymes (pma1, UMAG_02851), and the other similar to the plant H+-ATPases (pma2, UMAG_01205). Unlike S. cerevisiae, these two genes are expressed jointly in U. maydis sporidia. In the present work, mutants lacking one of these genes (Δpma1 and Δpma2) were used to characterize the role of each one of these enzymes in U. maydis physiology and to obtain some of their kinetic parameters. To approach this goal, classical biochemical assays were performed. The absence of any of these H+-ATPases did not affect the growth or fungal basal metabolism. Membrane potential tests showed that the activity of a single H+-ATPase was enough to maintain the proton-motive force. Our results indicated that in U. maydis, both H+-ATPases work jointly in the generation of the electrochemical proton gradient, which is important for secondary transport of metabolites and regulation of intracellular pH.

The plasma membrane H+-ATPase is critical for cell growth and pathogenicity in Penicillium digitatum.

The plasma membrane H+-ATPase (PMA1) is a major cytosolic pH regulator and a potential candidate for antifungal drug discovery due to its fungal specificity and criticality. In this study, the function of Penicillum digitatum PMA1 was characterized through RNA interference (RNAi) and overexpression technology. The results showed that silencing the PMA1 gene reduces cell growth and pathogenicity, and increases susceptibility of P. digitatum to proton pump inhibitors (PPIs). Under scanning electron microscopy (SEM) and transmission electron microscopy (TEM) examination, cell morphology was significantly altered in the PMA1- silenced mutant (si57). When compared with wild type (WT) and the overexpressed mutant (oe9), the cell walls of the si57 mutant were thicker and their cell membrane damage manifested particularly at sites of polarized growth. Consistent with the morphological change on the cell wall, chitin and glucan content of the cell wall of si57 were significantly lower and accompanied with increased activities of chitinase and glucanase. The lower ergosterol content in the si57 mutant then increased cell membrane permeability, ultimately leading to leakage of cytoplasmic contents such as ions, reduced sugars and soluble proteins. Furthermore, significantly decreased activity of cell wall degrading enzymes of si57 during citrus fruit infections indicates a reduced pathogenicity in this mutant. We conclude that PMA1 in P. digitatum plays an important role in maintaining pathogenesis and PMA1 could be a candidate novel fungicidal drug discovery for citrus green mold. KEY POINTS: Silencing PMA1 gene decreased the growth and pathogenicity of P. digitatum. Silencing PMA1 gene damaged cell wall and cell membrane integrity of P. digitatum. PMA1 appears to be a suitable fungicidal target against citrus green mold.

Arabidopsis Plasma Membrane ATPase AHA5 Is Negatively Involved in PAMP-Triggered Immunity.

Plants evolve a prompt and robust immune system to defend themselves against pathogen infections. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is the first battle layer activated upon the PAMP's perception, which leads to multiple defense responses. The plasma membrane (PM) H+-ATPases are the primary ion pumps to create and maintain the cellular membrane potential that is critical for various essential biological processes, including plant growth, development, and defense. This study discovered that the PM H+-ATPase AHA5 is negatively involved in Arabidopsis PTI against the virulent pathogen Pseudomonas syringae pvr. tomato (Pto) DC3000 infection. The aha5 mutant plants caused the reduced stomata opening upon the Pto infection, which was associated with the salicylic acid (SA) pathway. In addition, the aha5 mutant plants caused the increased levels of callose deposition, defense-related gene expression, and SA accumulation. Our results also indicate that the PM H+-ATPase activity of AHA5 probably mediates the coupling of H2O2 generation and the apoplast alkalization in PTI responses. Moreover, AHA5 was found to interact with a vital defense regulator, RPM1-interacting protein 4 (RIN4), in vitro and in vivo, which might also be critical for its function in PTI. In summary, our studies show that AHA5 functions as a novel and critical component that is negatively involved in PTI by coordinating different defense responses during the Arabidopsis-Pto DC3000 interaction.

Acid or base? How do plants regulate the ecology of their phylloplane?

Plants interface with and modify the external environment across their surfaces, and in so doing, can control or mitigate the impacts of abiotic stresses and also mediate their interactions with other organisms. Botanically, it is known that plant roots have a multi-faceted ability to modify rhizosphere conditions like pH, a factor with a large effect on a plant's biotic interactions with microbes. But plants can also modify pH levels on the surfaces of their leaves. Plants can neutralize acid rain inputs in a period of hours, and either acidify or alkalinize the pH of neutral water droplets in minutes. The pH of the phylloplane-that is, the outermost surface of the leaf-varies across species, from incredibly acidic (carnivorous plants: as low as pH 1) to exceptionally alkaline (species in the plant family, Malvaceae, up to pH 11). However, most species mildly acidify droplets on the phylloplane by 1.5 orders of magnitude in pH. Just as rhizosphere pH helps shape the plant microbiome and is known to influence belowground interactions, so too can phylloplane pH influence aboveground interactions in plant canopies. In this review, we discuss phylloplane pH regulation from the physiological, molecular, evolutionary, and ecological perspectives and address knowledge gaps and identify future research directions.

Gibberellin biosynthesis inhibitor mepiquat chloride enhances root K+ uptake in cotton by modulating plasma membrane H+-ATPase.

Potassium deficiency causes severe losses in yield and quality in crops. Mepiquat chloride, a plant growth regulator, can increase K+ uptake in cotton (Gossypium hirsutum), but the underlying physiological mechanisms remain unclear. In this study, we used a non-invasive micro-test technique to measure K+ and H+ fluxes in the root apex with or without inhibitors of K+ channels, K+ transporters, non-selective cation channels, and plasma membrane H+-ATPases. We found that soaking seeds in mepiquat chloride solution increased the K+ influx mediated by K+ channels and reduced the K+ efflux mediated by non-selective cation channels in cotton seedlings. Mepiquat chloride also increased negative membrane potential (Em) and the activity of plasma membrane H+-ATPases in roots, due to higher levels of gene expression and protein accumulation of plasma membrane H+-ATPases as well as phosphorylation of H+-ATPase 11 (GhAHA11). Thus, plasma membrane hyperpolarization mediated by H+-ATPases was able to stimulate the activity of K+ channels in roots treated with mepiquat chloride. In addition, reduced K+ efflux under mepiquat chloride treatment was associated with reduced accumulation of H2O2 in roots. Our results provide important insights into the mechanisms of mepiquat chloride-induced K+ uptake in cotton and hence have the potential to help in improving K nutrition for enhancing cotton production.

Phosphorus and iron deficiencies induce a metabolic reprogramming and affect the exudation traits of the woody plant Fragaria×ananassa.

Strawberries are a very popular fruit among berries, for both their commercial and economic importance, but especially for their beneficial effects for human health. However, their bioactive compound content is strictly related to the nutritional status of the plant and might be affected if nutritional disorders (e.g. Fe or P shortage) occur. To overcome nutrient shortages, plants evolved different mechanisms, which often involve the release of root exudates. The biochemical and molecular mechanisms underlying root exudation and its regulation are as yet still poorly known, in particular in woody crop species. The aim of this work was therefore to characterize the pattern of root exudation of strawberry plants grown in either P or Fe deficiency, by investigating metabolomic changes of root tissues and the expression of genes putatively involved in exudate extrusion. Although P and Fe deficiencies differentially affected the total metabolism, some metabolites (e.g. raffinose and galactose) accumulated in roots similarly under both conditions. Moreover, P deficiency specifically affected the content of galactaric acid, malic acid, lysine, proline, and sorbitol-6-phosphate, whereas Fe deficiency specifically affected the content of sucrose, dehydroascorbic acid, galactonate, and ferulic acid. At the same time, the citrate content did not change in roots under both nutrient deficiencies with respect to the control. However, a strong release of citrate was observed, and it increased significantly with time, being +250% and +300% higher in Fe- and P-deficient plants, respectively, compared with the control. Moreover, concomitantly, a significant acidification of the growth medium was observed in both treatments. Gene expression analyses highlighted for the first time that at least two members of the multidrug and toxic compound extrusion (MATE) transporter family and one member of the plasma membrane H(+)-ATPase family are involved in the response to both P and Fe starvation in strawberry plants.

SAUR Proteins as Effectors of Hormonal and Environmental Signals in Plant Growth.

The plant hormone auxin regulates numerous aspects of plant growth and development. Early auxin response genes mediate its genomic effects on plant growth and development. Discovered in 1987, small auxin up RNAs (SAURs) are the largest family of early auxin response genes. SAUR functions have remained elusive, however, presumably due to extensive genetic redundancy. However, recent molecular, genetic, biochemical, and genomic studies have implicated SAURs in the regulation of a wide range of cellular, physiological, and developmental processes. Recently, crucial mechanistic insight into SAUR function was provided by the demonstration that SAURs inhibit PP2C.D phosphatases to activate plasma membrane (PM) H(+)-ATPases and promote cell expansion. In addition to auxin, several other hormones and environmental factors also regulate SAUR gene expression. We propose that SAURs are key effector outputs of hormonal and environmental signals that regulate plant growth and development.

14-3-3 Proteins in Guard Cell Signaling.

Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses.

Adição de ácido cítrico potencializa a ação de ácidos húmicos e altera o perfil protéico da membrana plasmática em raízes de milho / Citric acid addition improve humic acids action and change proteins profile from plasma membrane of maize roots

A promoção do crescimento vegetal pelos ácidos húmicos tem sido atribuída a ações similares a hormônios, devido à promoção do desenvolvimento e proliferação das raízes, resultando numa absorção mais eficiente de água e nutrientes. O objetivo deste trabalho foi analisar as mudanças na arquitetura radicular em plântulas de milho e no perfil de proteínas da membrana plasmática (MP) promovidas pelo tratamento com ácidos húmicos (AH) isolados de vermicomposto (20mg C L-1). O efeito da adição de ácido cítrico (AC), importante ácido orgânico presente nos exudados radiculares, sobre a bioatividade destes AH também foi investigada. Foram analisados o comprimento da raiz principal, o número de sítios de mitose, o número e comprimento de raízes laterais e a área radicular total. Para a análise do perfil protéico, vesículas da MP de células de raízes foram obtidas por fracionamento celular e as proteínas analisadas por eletroforese uni (1D) e bidimensional (2D). Observou-se que a adição de AC (0,005mM) aos AH estimularam a promoção do crescimento das raízes laterais (126 por cento), da área radicular (58 por cento) e do número de raízes laterais (55 por cento) em relação às plantas controle. A atividade da bomba de H+ da membrana plasmática, analisada como marcador bioquímico de indução do mecanismo do crescimento ácido, também foi significativamente estimulada (374 por cento) pela solução húmica suplementada com AC. O perfil protéico da MP revelou uma supressão da expressão das proteínas nesta membrana, induzida pelo tratamento com AH e, na presença de AC, esse efeito foi ainda mais evidente. Os resultados obtidos corroboram o mecanismo proposto para a bioatividade de AH no qual a ação de ácidos orgânicos exudados pelas plantas, tais como o AC, promove o rompimento da associação supramolecular dessas substâncias, tornando as moléculas bioativas presentes nos agregados húmicos mais acessíveis aos receptores celulares das raízes.

The plant growth stimulation by humic acids (HA) has been attributed to a hormone-like effect as promoting the root development and proliferation, resulting in a more efficient water and nutrient absorption. This research aims to investigate how the humic acids isolated from vermicompost (20mg L-1) can modify the root architecture and the plasma membrane (PM) protein patterns in maize roots. It was also analyzed the effect of the citric acid (CA), an organic acid present in root exudates. The changes induced in the corn root system were estimated by measuring the taproot length, the amount of root mitotic sites and lateral roots, and the total root area. Plasma membrane vesicles were purified by cell fractionation and the protein patterns were analyzed by uni (1D) and bidimensional (2D) electrophoresis. The results show that the HA in solution with CA (0.005mM) increases the lateral root growth promotion (126 percent), the root area (58 percent), and the number of lateral roots (55 percent). The activity of the plasma membrane H+ pump, analyzed as a marker of the induction of the acid growth mechanism, was also enhanced (374 percent) by the humic solution supplemented with CA. Expression of several plasma membrane proteins was inhibited when plants were treated with HA and this effect was more pronounced upon CA supplementation. The obtained results corroborate the proposed mechanism for the HA bioactivity, by which under the action of root-exuded organic acids, such as CA, a disruption of the HA macrostructure is promoted releasing bioactive molecules presented in the humic aggregates, which becomes more accessible to the root cell receptors.