Plasma membrane H+-ATPases in mineral nutrition and crop improvement.

Plasma membrane H+-ATPases (PMAs) pump H+ out of the cytoplasm by consuming ATP to generate a membrane potential and proton motive force for the transmembrane transport of nutrients into and out of plant cells. PMAs are involved in nutrient acquisition by regulating root growth, nutrient uptake, and translocation, as well as the establishment of symbiosis with arbuscular mycorrhizas. Under nutrient stresses, PMAs are activated to pump more H+ and promote organic anion excretion, thus improving nutrient availability in the rhizosphere. Herein we review recent progress in the physiological functions and the underlying molecular mechanisms of PMAs in the efficient acquisition and utilization of various nutrients in plants. We also discuss perspectives for the application of PMAs in improving crop production and quality.

OsPHR2-mediated recruitment of Pseudomondaceae enhances rice phosphorus uptake.

Plant can shape their root microbiome for growth and nutrients uptake. PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) is a central regulator of phosphate signaling in rice, but whether OsPHR2 can shape the root microbiome for phosphorus uptake is unclear. Here, we investigate the roles of OsPHR2 in recruiting microbiota for phosphorus uptake using high-throughput sequencing and metabolites analysis. OsPHR2-overexpressing (OsPHR2 OE) rice exhibited 69.8% greater shoot P uptake in natural soil compared to sterilized soil under high phosphorus (HP) conditions; while there was only a 54.8% increase in wildtype (WT). Next, the abundance of the family Pseudomondaceae was significantly enriched in the OsPHR2 OE root, relative to WT rice. Compared to the WT, OsPHR2 OE had different root exudates (succinic acid and methylmalonic acid), which were associated with distinct changes in the root microbiome. After inoculation with Pseudomonas sp (P6), rice phosphorus uptake in WT and OsPHR2 OE rice was higher than that of uninoculated rice plants under low phosphorus (LP) conditions. Together, our results suggest that OsPHR2 can increase rice phosphorus use through root exudate-mediated recruitment of Pseudomonas. This finding reveals a cooperative contribution of OsPHR2-modulated root microbiome, which is important for improving phosphorus use in agriculture.

Auxin-producing bacteria promote barley rhizosheath formation.

The rhizosheath, or the layer of soil closely adhering to roots, can help plants to tolerate drought under moderate soil drying conditions. Rhizosheath formation is the result of poorly understood interactions between root exudates, microbes, and soil conditions. Here, we study the roles played by the soil microbiota in rhizosheath formation in barley (a dry crop). We show that barley rhizosheath formation is greater in acid soil than in alkaline soil, and inoculation with microbiota from acid soil enhances rhizosheath formation in alkaline soil. The rhizosheath-promoting activity is associated with the presence of Flavobacteriaceae and Paenibacillaceae bacteria that express genes for biosynthesis of indole-3-acetic acid (IAA, a common auxin), as determined by metagenomics and metatranscriptomics. Two bacterial strains isolated from rhizosheath (Chryseobacterium culicis and Paenibacillus polymyxa) produce IAA and enhance barley rhizosheath formation, while their IAA-defective mutants are unable to promote rhizosheath formation. Co-inoculation with the IAA-producing strains enhances barley grain yield in field experiments through an increase in spike number. Our findings contribute to our understanding of barley rhizosheath formation, and suggest potential strategies for crop improvement.

Rice ubiquitin-conjugating enzyme OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

Ubc13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity still remain largely unknown. Here, we used molecular biological, pathological, biochemical, and genetic approaches to evaluate the roles of rice OsUbc13 in response to pathogens. The OsUbc13-RNA interference (RNAi) lines with lesion mimic phenotypes displayed a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in defence-related genes expression or hormones as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. Strikingly, OsUbc13 directly interacts with OsSnRK1a, which is the α catalytic subunit of SnRK1 (sucrose non-fermenting-1-related protein kinase-1) and acts as a positive regulator of broad-spectrum disease resistance in rice. In the OsUbc13-RNAi plants, although the protein level of OsSnRK1a did not change, its activity and ABA sensitivity were obviously enhanced, and the K63-linked polyubiquitination was weaker than that of wild-type Dongjin (DJ). Overexpression of the deubiquitinase-encoding gene OsOTUB1.1 produced similar effects with inhibition of OsUbc13 in affecting immunity responses, M. oryzae resistance, OsSnRK1a ubiquitination, and OsSnRK1a activity. Furthermore, re-interfering with OsSnRK1a in one OsUbc13-RNAi line (Ri-3) partially restored its M. oryzae resistance to a level between those of Ri-3 and DJ. Our data demonstrate OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.

Soilborne bacterium Klebsiella pneumoniae promotes cluster root formation in white lupin through ethylene mediation.

Cluster roots of white lupin are induced by low phosphorus (LP) to efficiently access unavailable P, but how soilborne microbes are associated with cluster root formation (CRF) is unclear. We investigated the roles of soilborne bacteria in CRF response to LP by high-throughput sequencing and root-bacteria interactions. Cluster root number was significantly decreased in plants grown in sterilized soil compared with nonsterilized soil. Proteobacteria was enriched in CR, as shown by microbiome analysis of soil (bulk, rhizosphere, and rhizosheath) and roots (main, lateral, and CR). Large-scale gene expression level implicated ethylene mediation in CRF. Klebsiella pneumoniae (P7), a soilborne bacterium belonging to Proteobacteria, was isolated from CR. Among 11 isolated strains, P7 exhibited the highest 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity; this enzyme inhibits the biosynthesis of ethylene in plants by the cleavage of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and promotes CRF under LP. We constructed an ACCD-deficit mutant accd in the P7 genetic background. The loss-of-function mutation failed to promote CRF under LP conditions. Also, auxin responses may be involved in K. pneumoniae-ethylene-mediated CRF. Overall, we propose that the soilborne bacterium K. pneumoniae promotes CRF of white lupin in response to LP by ethylene mediation.

H+-ATPases in Plant Growth and Stress Responses.

H+-ATPases, including the phosphorylated intermediate-type (P-type) and vacuolar-type (V-type) H+-ATPases, are important ATP-driven proton pumps that generate membrane potential and provide proton motive force for secondary active transport. P- and V-type H+-ATPases have distinct structures and subcellular localizations and play various roles in growth and stress responses. A P-type H+-ATPase is mainly regulated at the posttranslational level by phosphorylation and dephosphorylation of residues in its autoinhibitory C terminus. The expression and activity of both P- and V-type H+-ATPases are highly regulated by hormones and environmental cues. In this review, we summarize the recent advances in understanding of the evolution, regulation, and physiological roles of P- and V-type H+-ATPases, which coordinate and are involved in plant growth and stress adaptation. Understanding the different roles and the regulatory mechanisms of P- and V-type H+-ATPases provides a new perspective for improving plant growth and stress tolerance by modulating the activity of H+-ATPases, which will mitigate the increasing environmental stress conditions associated with ongoing global climate change.

ABA regulation of root growth during soil drying and recovery can involve auxin response.

Abscisic acid (ABA) plays an important role in plant adaptation to water deficits, but its role in regulating root growth (primary root elongation and lateral root number) during different drought-phases remains unclear. Here, we exposed wild-type (WT) and ABA-deficient (not) tomato plants to three continuous drought-phases (moderate drying: day 0-21; severe drying: day 22-47 and re-watering: day 48-51). It was found that WT increased primary root growth during moderate drying; maintained more lateral roots, and greater primary root and total root length under severe drying; and produced more roots after re-watering. After RNA-Seq analysis, we found that the auxin-related genes in root showed different expression patterns between WT and not under drying or re-watering. Further, exogenous supply of IAA partially recovered the root growth of ABA-deficient not plants under three continuous drought-phases. Our results suggested that ABA regulation of tomato root growth during soil drying and recovery can involve auxin response.

Coordination of root auxin with the fungus Piriformospora indica and bacterium Bacillus cereus enhances rice rhizosheath formation under soil drying.

Moderate soil drying (MSD) is a promising agricultural technique that can reduce water consumption and enhance rhizosheath formation promoting drought resistance in plants. The endophytic fungus Piriformospora indica (P. indica) with high auxin production may be beneficial for rhizosheath formation. However, the integrated role of P. indica with native soil microbiome in rhizosheath formation is unclear. Here, we investigated the roles of P. indica and native bacteria on rice rhizosheath formation under MSD using high-throughput sequencing and rice mutants. Under MSD, rice rhizosheath formation was significantly increased by around 30% with P. indica inoculation. Auxins in rice roots and P. indica were responsible for the rhizosheath formation under MSD. Next, the abundance of the genus Bacillus, known as plant growth-promoting rhizobacteria, was enriched in the rice rhizosheath and root endosphere with P. indica inoculation under MSD. Moreover, the abundance of Bacillus cereus (B. cereus) with high auxin production was further increased by P. indica inoculation. After inoculation with both P. indica and B. cereus, rhizosheath formation in wild-type or auxin efflux carrier OsPIN2 complemented line rice was higher than that of the ospin2 mutant. Together, our results suggest that the interaction of the endophytic fungus P. indica with the native soil bacterium B. cereus favors rice rhizosheath formation by auxins modulation in rice and microbes under MSD. This finding reveals a cooperative contribution of P. indica and native microbiota in rice rhizosheath formation under moderate soil drying, which is important for improving water use in agriculture.

Abscisic acid is required for root elongation associated with Ca2+ influx in response to water stress.

Abscisic acid (ABA) is a critical hormone for plant survival upon water stress. In this study, a large-scale mutants of Arabidopsis ecotype Columbia-0 (Col-0) by ethyl methanesulfonate (EMS)-mutagenesis were generated, and an improved root elongation under water-stress 1 (irew1) mutant showing significantly enhanced root growth was isolated upon a water potential gradient assay. Then, irew1 and ABA-related mutants in Arabidopsis or tomato plants were observed under water potential gradient assay or water-deficient condition. ABA pathway, Ca2+ response and primary root (PR) elongation rate were monitored in addition to DNA- and RNA-Seq analyses. We found that based on phenotyping and transcriptional analyses, irew1 exhibited the enhanced PR growth, ABA and Ca2+ responses compared to wild-type subjected to water stress. Interestingly, exogenous Ca2+ application enhanced PR growth of irew1, ABA-biosynthesis deficient mutants in Arabidopsis and tomato plants in response to water potential gradients or water-deficient condition. In combination with other ABA-related mutants and pharmacological study, our results suggest that ABA is required for root elongation associated with Ca2+ influx in response to water stress.

G protein γ subunit qPE9-1 is involved in rice adaptation under elevated CO2 concentration by regulating leaf photosynthesis.

G protein γ subunit qPE9-1 plays multiple roles in rice growth and development. However, the role of qPE9-1 in rice exposed to elevated carbon dioxide concentration (eCO2) is unknown. Here, we investigated its role in the regulation of rice growth under eCO2 conditions using qPE9-1 overexpression (OE) lines, RNAi lines and corresponding WT rice. Compared to atmospheric carbon dioxide concentration (aCO2), relative expression of qPE9-1 in rice leaf was approximately tenfold higher under eCO2. Under eCO2, the growth of WT and qPE9-1-overexpressing rice was significantly higher than under aCO2. Moreover, there was no significant effect of eCO2 on the growth of qPE9-1 RNAi lines. Furthermore, WT and qPE9-1-overexpressing rice showed higher net photosynthetic rate and carbohydrate content under eCO2 than under aCO2. Moreover, the relative expression of some photosynthesis related genes in WT, but not in RNAi3 line, showed significant difference under eCO2 in RNA-seq analysis. Compared to WT and RNAi lines, the rbcL gene expression and Rubisco content of rice leaves in qPE9-1-overexpressors were higher under eCO2. Overall, these results suggest that qPE9-1 is involved in rice adaptation under elevated CO2 concentration by regulating leaf photosynthesis via moderating rice photosynthetic light reaction and Rubisco content.

Rice G protein γ subunit qPE9-1 modulates root elongation for phosphorus uptake by involving 14-3-3 protein OsGF14b and plasma membrane H+ -ATPase.

Heterotrimeric G protein is involved in plant growth and development, while the role of rice (Oryza sativa) G protein γ subunit qPE9-1 in response to low-phosphorus (LP) conditions remains unclear. The gene expression of qPE9-1 was significantly induced in rice roots under LP conditions. Rice varieties carrying the qPE9-1 allele showed a stronger primary root response to LP than the varieties carrying the qpe9-1 allele (mutant of the qPE9-1 allele). Transgenic rice plants with the qPE9-1 allele had longer primary roots and higher P concentrations than those with the qpe9-1 allele under LP conditions. The plasma membrane (PM) H+ -ATPase was important for the qPE9-1-mediated response to LP. Furthermore, OsGF14b, a 14-3-3 protein that acts as a key component in activating PM H+ -ATPase for root elongation, is also involved in the qPE9-1 mediation. Moreover, the overexpression of OsGF14b in WYJ8 (carrying the qpe9-1 allele) partially increased primary root length under LP conditions. Experiments using R18 peptide (a 14-3-3 protein inhibitor) showed that qPE9-1 is important for primary root elongation and H+ efflux under LP conditions by involving the 14-3-3 protein. In addition, rhizosheath weight, total P content, and the rhizosheath soil Olsen-P concentration of qPE9-1 lines were higher than those of qpe9-1 lines under soil drying and LP conditions. These results suggest that the G protein γ subunit qPE9-1 in rice plants modulates root elongation for phosphorus uptake by involving the 14-3-3 protein OsGF14b and PM H+ -ATPase, which is required for rice P use.

Abscisic acid mediates barley rhizosheath formation under mild soil drying by promoting root hair growth and auxin response.

Soil drying enhances root ABA accumulation and rhizosheath formation, but whether ABA mediates rhizosheath formation is unclear. Here, we used the ABA-deficient mutant Az34 to investigate molecular and morphological changes by which ABA could affect rhizosheath formation. Mild soil drying with intermittent watering increased rhizosheath formation by promoting root and root hair elongation. Attenuated root ABA accumulation in Az34 barley constrained the promotion of root length and root hair length by drying soil, such that Az34 had a smaller rhizosheath. Pharmacological experiments of adding fluridone (an ABA biosynthesis inhibitor) and ABA to drying soil restricted and enhanced rhizosheath formation respectively in Az34 and wild-type Steptoe barley. RNA sequencing suggested that ABA accumulation mediates auxin synthesis and responses and root and root hair elongation in drying soil. In addition, adding indole-3-acetic acid (IAA) to drying soil increased rhizosheath formation by promoting root and root hair elongation in Steptoe and Az34 barley. Together, these results show that ABA accumulation induced by mild soil drying enhance barley rhizosheath formation, which may be achieved through promoting auxin response.

Plasma membrane H+-ATPase overexpression increases rice yield via simultaneous enhancement of nutrient uptake and photosynthesis.

Nitrogen (N) and carbon (C) are essential elements for plant growth and crop yield. Thus, improved N and C utilisation contributes to agricultural productivity and reduces the need for fertilisation. In the present study, we find that overexpression of a single rice gene, Oryza sativa plasma membrane (PM) H+-ATPase 1 (OSA1), facilitates ammonium absorption and assimilation in roots and enhanced light-induced stomatal opening with higher photosynthesis rate in leaves. As a result, OSA1 overexpression in rice plants causes a 33% increase in grain yield and a 46% increase in N use efficiency overall. As PM H+-ATPase is highly conserved in plants, these findings indicate that the manipulation of PM H+-ATPase could cooperatively improve N and C utilisation, potentially providing a vital tool for food security and sustainable agriculture.

Abscisic Acid Is Required for Root Elongation Associated With Ca2+ Influx in Response to Water Stress.

Abscisic acid (ABA) is a critical hormone for plant survival under water stress. In this study, large-scale mutants of the Arabidopsis ecotype Columbia-0 (Col-0) were generated by ethyl methanesulfonate (EMS)-mutagenesis, and an improved root elongation under water-stress 1 (irew1) mutant showing significantly enhanced root growth was isolated under a water potential gradient assay. Then, irew1 and ABA-related mutants in Arabidopsis or tomato plants were observed under water potential gradient assay or water-deficient conditions. ABA pathway, Ca2+ response, and primary root (PR) elongation rate were monitored in addition to DNA- and RNA-Seq analyses. We found that based on phenotyping and transcriptional analyses, irew1 exhibited enhanced PR growth, ABA, and Ca2+ responses, compared to wild type subjected to water stress. Interestingly, exogenous Ca2+ application enhanced PR growth of irew1, ABA-biosynthesis deficient mutants in Arabidopsis, and tomato plants, in response to water potential gradients or water-deficient conditions. In combination with other ABA-related mutants and pharmacological studies, our results suggest that ABA is required for root elongation associated with Ca2+ influx in response to water stress.

Potassium alleviates ammonium toxicity in rice by reducing its uptake through activation of plasma membrane H+-ATPase to enhance proton extrusion.

Potassium (K+) has been reported to alleviate ammonium (NH4+) toxicity in rice through some underlying mechanisms, but it still not clear. In addition, K+ is an important cation for activation of plasma membrane (PM) H+-ATPase activity. Here, we hypothesized that K+ alleviated NH4+ toxicity by mediating PM H+-ATPase function in rice root. In this study, rice plants were cultivated in hydroponic solution with various concentrations of K+ and NH4+. By concurrently supplying K+ with NH4+ or re-supplying K+ after NH4+ toxicity, we found that high K+ concentration reduced the NH4+ uptake rate, enhanced the H+ extrusion rate by the roots, and alleviated rice NH4+ toxicity. The gene expression levels of PM H+-ATPase members (OsA1, 3, 7, 8, and 9) were upregulated by application of increasing concentrations of K+ under NH4+ toxicity. The PM H+-ATPase activity and protein expression in rice roots were also enhanced. Furthermore, the enhancement of PM H+-ATPase activity by a specific stimulator (fusicoccin) rescued rice seedlings from NH4+ toxicity. Taken together, these results indicate that K+ can alleviate NH4+ toxicity, possibly by activating PM H+-ATPase to extrude more H+ and inhibit NH4+ uptake by root. Our results may enhance understanding of the strategy of applying K+ fertilizer to mitigate crop NH4+ toxicity in agriculture.

In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes.

The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, 1/3 M519, and » M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L-1 + NAA 0.1 mg L-1) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L-1 + NAA 0.1 mg L-1 + BAP 1.67 mg L-1), and (KT 2.0 mg L-1 + NAA 0.1 mg L-1) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L-1 activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L-1 + KT 0.1 mg L-1 (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes.

Root-Bacteria Associations Boost Rhizosheath Formation in Moderately Dry Soil through Ethylene Responses.

The rhizosheath is a layer of soil around the root that provides a favorable environment for soil microbe enrichment and root growth. Rice (Oryza sativa) roots form rhizosheaths under moderate soil drying (MSD) conditions, but how the rhizosheath forms associations with microbes is unclear. To investigate rice rhizosheath formation under MSD, we employed a multiphasic approach, integrating data from high-throughput sequencing and root-bacteria interactions. Rice roots formed a pronounced rhizosheath under MSD, but not under continuous flooding regimens. Plant growth-promoting rhizobacteria of the Enterobacteriaceae were enriched in rhizosheaths of two different rice varieties, 'Gaoshan 1' (drought tolerant) and 'Nipponbare' (drought sensitive). RNA-sequencing analysis revealed that the ethylene pathway was induced in the rhizosheath-root system under MSD. Enterobacter aerogenes, a bacterium isolated from the rhizosheath, degrades the ethylene precursor 1-aminocyclopropane-1-carboxylate, thereby increasing rhizosheath formation. Furthermore, a 1-aminocyclopropane-1-carboxylate deaminase-deficient mutant of E. aerogenes failed to enhance rice rhizosheath formation. Our results suggest that root-bacteria associations substantially contribute to rhizosheath formation in rice under MSD conditions by mechanisms that involve the ethylene response. These data inform strategies to reduce water consumption in rice production, one of the most water-intensive human activities.

The genome evolution and low-phosphorus adaptation in white lupin.

White lupin (Lupinus albus) is a legume crop that develops cluster roots and has high phosphorus (P)-use efficiency (PUE) in low-P soils. Here, we assemble the genome of white lupin and find that it has evolved from a whole-genome triplication (WGT) event. We then decipher its diploid ancestral genome and reconstruct the three sub-genomes. Based on the results, we further reveal the sub-genome dominance and the genic expression of the different sub-genomes varying in relation to their transposable element (TE) density. The PUE genes in white lupin have been expanded through WGT as well as tandem and dispersed duplications. Furthermore, we characterize four main pathways for high PUE, which include carbon fixation, cluster root formation, soil-P remobilization, and cellular-P reuse. Among these, auxin modulation may be important for cluster root formation through involvement of potential genes LaABCG36s and LaABCG37s. These findings provide insights into the genome evolution and low-P adaptation of white lupin.

Moderate water stress in rice induces rhizosheath formation associated with abscisic acid and auxin responses.

The rhizosheath is known to be beneficial for drought resistance in many plants, but the regulation of rhizosheath formation in rice plants is unclear. Here, we investigate rhizosheath formation in different rice varieties and root hair mutants. Our results showed that moderate water stress in rice induced rhizosheath formation. The soil porosity and water content were higher in the rice rhizosheath than in the rice bulk soil under moderate water stress. Additionally, rhizosheath formation in short root hair mutants was lower than in wild-type rice under moderate water stress. Moreover, transcriptomic results indicated that abscisic acid (ABA) and auxin were involved in root and root hair responses in rhizosheath formation. Further, blocking ABA and auxin pathways in wild type and in rhl1-1, the shortest root hair mutant, rhizosheath formation and root hair length were significantly decreased under moderate water stress. However, wild type plants maintained a higher root ABA content, root basipetal auxin transport, root hair length, and amount of rhizosheath than did rhl1-1. Our results suggest that moderate water stress in rice induces rhizosheath formation by modulating the ABA and auxin responses to regulate root and root hair growth, which may be used to breed rice varieties resistant to drought.