The potassium transporter OsHAK21 functions in the maintenance of ion homeostasis and tolerance to salt stress in rice

The intracellular potassium (K⁺) homeostasis, which is crucial for plant survival in saline environments, is modulated by K⁺ channels and transporters. Some members of the high‐affinity K⁺ transporter (HAK) family are believed to function in the regulation of plant salt tolerance, but the physiological mechanisms remain unclear. Here, we report a significant inducement of OsHAK21 expression by high‐salinity treatment and provide genetic evidence of the involvement of OsHAK21 in rice salt tolerance. Disruption of OsHAK21 rendered plants sensitive to salt stress. Compared with the wild type, oshak21 accumulated less K⁺ and considerably more Na⁺ in both shoots and roots, and had a significantly lower K⁺ net uptake rate but higher Na⁺ uptake rate. Our analyses of subcellular localizations and expression patterns showed that OsHAK21 was localized in the plasma membrane and expressed in xylem parenchyma and individual endodermal cells (putative passage cells). Further functional characterizations of OsHAK21 in K⁺ uptake‐deficient yeast and Arabidopsis revealed that OsHAK21 possesses K⁺ transporter activity. These results demonstrate that OsHAK21 may mediate K⁺ absorption by the plasma membrane and play crucial roles in the maintenance of the Na⁺/K⁺ homeostasis in rice under salt stress.     

Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges

Plants take up both nitrate and ammonium as main nitrogen (N) sources. Although ammonium is the predominant form in anaerobic-flooded paddy soil, it has been proposed that rice and other wetland plants may take up significant amounts of nitrate formed by nitrification of ammonium in the rhizosphere. A two-component system for nitrate transport including NRT2s with a partner protein (NAR2 or NRT3.1) has been identified in Arabidopsis. We report the physiological function of another member of the NAR2 family, OsNAR2.1 in rice (Oryza sativa, ssp. Japonica, cv. Nipponbare). OsNAR2.1 was mainly expressed in roots and induced by nitrate and suppressed by ammonium and some amino acids. Knockdown of OsNAR2.1 by RNA interference synchronously suppressed expression of OsNRT2.1, OsNRT2.2 and OsNRT2.3a in the osnar2.1mutants. Both high- and low-affinity nitrate transports were greatly impaired by OsNAR2.1 knockdown. Yeast two hybridization showed that OsNAR2.1 not only interacted with OsNRT2.1/OsNRT2.2, but also with OsNRT2.3a. Taken together, the data demonstrate that OsNAR2.1 plays a key role in enabling the plant to cope with variable nitrate supply.     

Rice LTG1 is involved in adaptive growth and fitness under low ambient temperature

Low temperature (LT) is one of the most prevalent factors limiting the productivity and geographical distribution of rice (Oryza sativa L.). Although significant progress has been made in elucidating the effect of LT on seed germination and reproductive development in rice, the genetic component affecting vegetative growth under LT remains poorly understood. Here, we report that rice cultivars harboring the dominant LTG1 (Low Temperature Growth 1) allele are more tolerant to LT (15–25°C, a temperature range prevalent in high‐altitude, temperate zones and high‐latitude areas), than those with the ltg1 allele. Using a map‐based cloning strategy, we show that LTG1 encodes a casein kinase I. A functional nucleotide polymorphism was identified in the coding region of LTG1, causing a single amino acid substitution (I357K) that is associated with the growth rate, heading date and yield of rice plants grown at LT. We present evidence that LTG1 affects rice growth at LT via an auxin‐dependent process(es). Furthermore, phylogenetic analysis of this locus suggests that the ltg1 haplotype arose before the domestication of rice in tropical climates. Together, our data demonstrate that LTG1 plays an important role in the adaptive growth and fitness of rice cultivars under conditions of low ambient temperature.     

SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood‐tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A‐genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance‐related SUB1A‐1 and the intolerance‐related SUB1A‐2 alleles were found in tolerant and sensitive accessions, respectively. Flooding‐tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C‐genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence‐induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A‐1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C‐genome group, which show instead a SUB1A‐independent response to submergence.     

OsMOGS is required for N‐glycan formation and auxin‐mediated root development in rice (Oryza sativa L.)

N‐glycosylation is a major modification of glycoproteins in eukaryotic cells. In Arabidopsis, great progress has been made in functional analysis of N‐glycan production, however there are few studies in monocotyledons. Here, we characterized a rice (Oryza sativa L.) osmogs mutant with shortened roots and isolated a gene that coded a putative mannosyl‐oligosaccharide glucosidase (OsMOGS), an ortholog of α‐glucosidase I in Arabidopsis, which trims the terminal glucosyl residue of the oligosaccharide chain of nascent peptides in the endoplasmic reticulum (ER). OsMOGS is strongly expressed in rapidly cell‐dividing tissues and OsMOGS protein is localized in the ER. Mutation of OsMOGS entirely blocked N‐glycan maturation and inhibited high‐mannose N‐glycan formation. The osmogs mutant exhibited severe defects in root cell division and elongation, resulting in a short‐root phenotype. In addition, osmogs plants had impaired root hair formation and elongation, and reduced root epidemic cell wall thickness due to decreased cellulose synthesis. Further analysis showed that auxin content and polar transport in osmogs roots were reduced due to incomplete N‐glycosylation of the B subfamily of ATP‐binding cassette transporter proteins (ABCBs). Our results demonstrate that involvement of OsMOGS in N‐glycan formation is required for auxin‐mediated root development in rice.     

A glutathione responsive rice glyoxalase II,OsGLYII‐2, functions in salinity adaptation by maintaining better photosynthesis efficiency and anti‐oxidant pool

Glyoxalase II (GLY II), the second enzyme of glyoxalase pathway that detoxifies cytotoxic metabolite methylglyoxal (MG), belongs to the superfamily of metallo‐β‐lactamases. Here, detailed analysis of one of the uncharacterized rice glyoxalase II family members, OsGLYII‐2 was conducted in terms of its metal content, enzyme kinetics and stress tolerance potential. Functional complementation of yeast GLY II mutant (?GLO2) and enzyme kinetics data suggested that OsGLYII‐2 possesses characteristic GLY II activity using S‐lactoylglutathione (SLG) as the substrate. Further, Inductively Coupled Plasma Atomic Emission spectroscopy and modelled structure revealed that OsGLYII‐2 contains a binuclear Zn/Fe centre in its active site and chelation studies indicated that these are essential for its activity. Interestingly, reconstitution of chelated enzyme with Zn²⁺, and/or Fe²⁺ could not reactivate the enzyme, while addition of Co²⁺ was able to do so. End product inhibition study provides insight into the kinetics of GLY II enzyme and assigns hitherto unknown function to reduced glutathione (GSH). Ectopic expression of OsGLYII‐2 in Escherichia coli and tobacco provides improved tolerance against salinity and dicarbonyl stress indicating towards its role in abiotic stress tolerance. Maintained levels of MG and GSH as well as better photosynthesis rate and reduced oxidative damage in transgenic plants under stress conditions seems to be the possible mechanism facilitating enhanced stress tolerance.     

PP7L is essential for MAIL1‐mediated transposable element silencing and primary root growth

The two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon‐related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericentromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7‐LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells.     

Nucleocytoplasmic trafficking is essential for BAK1‐ and BKK1‐mediated cell‐death control

BRI1‐ASSOCIATED KINASE 1 (BAK1) was initially identified as a co‐receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1‐LIKE 1 (BKK1) regulate a BR‐independent cell‐death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid‐ and light‐dependent cell‐death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1–1). Genetic analyses indicated that cell‐death symptoms in a weak double mutant, bak1–3 bkk1–1, were completely suppressed by the loss‐of‐function mutation in SBB1, which encodes a nucleoporin (NUP) 85‐like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1‐located sub‐complex was also able to rescue the cell‐death phenotype of bak1–3 bkk1–1. In addition, a DEAD‐box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell‐death symptoms of bak1–3 bkk1–1. Further analyses indicated that export of poly(A)⁺ RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over‐expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell‐death phenotype of bak1–3 bkk1–1. Mutants suppressing cell‐death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1‐ and BKK1‐mediated cell‐death control.     

Rice cellulose synthase‐like D4 is essential for normal cell‐wall biosynthesis and plant growth

Cellulose synthase‐like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell‐wall polymers. The CSL D sub‐family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in‐depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map‐based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub‐family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C‐ or N‐terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype‐rescued transgenic plants expressing OsCSLD4–GUS under the control of its own promoter. Two phenotype‐altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell‐wall formation and plant growth.     

The auxin transporter,OsAUX1, is involved in primary root and root hair elongation and in Cd stress responses in rice (Oryza sativa L.)

Auxin and cadmium (Cd) stress play critical roles during root development. There are only a few reports on the mechanisms by which Cd stress influences auxin homeostasis and affects primary root (PR) and lateral root (LR) development, and almost nothing is known about how auxin and Cd interfere with root hair (RH) development. Here, we characterize rice osaux1 mutants that have a longer PR and shorter RHs in hydroponic culture, and that are more sensitive to Cd stress compared to wild‐type (Dongjin). OsAUX1 expression in root hair cells is different from that of its paralogous gene, AtAUX1, which is expressed in non‐hair cells. However, OsAUX1, like AtAUX1, localizes at the plasma membrane and appears to function as an auxin tranporter. Decreased auxin distribution and contents in the osaux1 mutant result in reduction of OsCyCB1;1 expression and shortened PRs, LRs and RHs under Cd stress, but may be rescued by treatment with the membrane‐permeable auxin 1‐naphthalene acetic acid. Treatment with the auxin transport inhibitors 1‐naphthoxyacetic acid and N‐1‐naphthylphthalamic acid increased the Cd sensitivity of WT rice. Cd contents in the osaux1 mutant were not altered, but reactive oxygen species‐mediated damage was enhanced, further increasing the sensitivity of the osaux1 mutant to Cd stress. Taken together, our results indicate that OsAUX1 plays an important role in root development and in responses to Cd stress.     

The wheat TaGBF1 gene is involved in the blue‐light response and salt tolerance

Light and abiotic stress both strongly modulate plant growth and development. However, the effect of light‐responsive factors on growth and abiotic stress responses in wheat (Triticum aestivum) is unknown. G–box binding factors (GBFs) are blue light‐specific components, but their function in abiotic stress responses has not been studied. Here we identified a wheat GBF1 gene that mediated both the blue light‐ and abiotic stress‐responsive signaling pathways. TaGBF1 was inducible by blue light, salt and exposure to abscisic acid (ABA). TaGBF1 interacted with a G–box light‐responsive element in vitro and promoted a blue‐light response in wheat and Aradidopsis thaliana. Both TaGBF1 over‐expression in wheat and its heterologous expression in A. thaliana heighten sensitivity to salinity and ABA, but its knockdown in wheat conferred resistance to high salinity and ABA. The expression of AtABI5, a key component of the ABA signaling pathway in A. thaliana, and its homolog Wabi5 in wheat was increased by transgenic expression of TaGBF1. The hypersensitivity to salt and ABA caused by TaGBF1 was not observed in the abi5 mutant background, showing that ABI5 is the mediator in TaGBF1‐induced abiotic stress responses. However, the hypersensitivity to salt conferred by TaGBF1 is not dependent on light. This suggests that TaGBF1 is a common component of blue light‐ and abiotic stress‐responsive signaling pathways.     

N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.     

Expression of Caenorhabditis elegans PCS in the AtPCS1‐deficient Arabidopsis thaliana cad1‐3 mutant separates the metal tolerance and non‐host resistance functions of phytochelatin synthases

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.