Regulation of Cytosolic pH: The Contributions of Plant Plasma Membrane H+-ATPases and Multiple Transporters.

Cytosolic pH homeostasis is a precondition for the normal growth and stress responses in plants, and H+ flux across the plasma membrane is essential for cytoplasmic pH control. Hence, this review focuses on seven types of proteins that possess direct H+ transport activity, namely, H+-ATPase, NHX, CHX, AMT, NRT, PHT, and KT/HAK/KUP, to summarize their plasma-membrane-located family members, the effect of corresponding gene knockout and/or overexpression on cytosolic pH, the H+ transport pathway, and their functional regulation by the extracellular/cytosolic pH. In general, H+-ATPases mediate H+ extrusion, whereas most members of other six proteins mediate H+ influx, thus contributing to cytosolic pH homeostasis by directly modulating H+ flux across the plasma membrane. The fact that some AMTs/NRTs mediate H+-coupled substrate influx, whereas other intra-family members facilitate H+-uncoupled substrate transport, demonstrates that not all plasma membrane transporters possess H+-coupled substrate transport mechanisms, and using the transport mechanism of a protein to represent the case of the entire family is not suitable. The transport activity of these proteins is regulated by extracellular and/or cytosolic pH, with different structural bases for H+ transfer among these seven types of proteins. Notably, intra-family members possess distinct pH regulatory characterization and underlying residues for H+ transfer. This review is anticipated to facilitate the understanding of the molecular basis for cytosolic pH homeostasis. Despite this progress, the strategy of their cooperation for cytosolic pH homeostasis needs further investigation.

Overexpression of OsMed16 Inhibits the Growth of Rice and Causes Spontaneous Cell Death.

The Mediator complex transduces information from the DNA-bound transcription factors to the RNA polymerase II transcriptional machinery. Research on plant Mediator subunits has primarily been performed in Arabidopsis, while very few of them have been functionally characterized in rice. In this study, the rice Mediator subunit 16, OsMed16, was examined. OsMed16 encodes a putative protein of 1301 amino acids, which is longer than the version previously reported. It was expressed in various rice organs and localized to the nucleus. The knockout of OsMed16 resulted in rice seedling lethality. Its overexpression led to the retardation of rice growth, low yield, and spontaneous cell death in the leaf blade and sheath. RNA sequencing suggested that the overexpression of OsMed16 altered the expression of a large number of genes. Among them, the upregulation of some defense-related genes was verified. OsMed16 can regulate the expression of a wealth of genes, and alterations in its expression have a profound impact on plant growth, development, and defense responses in rice.

The tonoplast-localized transporter OsABCC9 is involved in cadmium tolerance and accumulation in rice.

Cadmium (Cd) is a highly toxic element to living organisms, and its accumulation in the edible portions of crops poses a potential threat for human health. The molecular mechanisms underlying Cd detoxification and accumulation are not fully understood in plants. In this study, the involvement of a C-type ABC transporter, OsABCC9, in Cd tolerance and accumulation in rice was investigated. The expression of OsABCC9 was rapidly induced by Cd treatment in a concentration-dependent manner in the root. The transporter, localized on the tonoplast, was mainly expressed in the root stele under Cd stress. OsABCC9 knockout mutants were more sensitive to Cd and accumulated more Cd in both the root and shoot compared to the wild-type. Moreover, the Cd concentrations in the xylem sap and grain were also significantly increased in the knockout lines, suggesting that more Cd was distributed from root to shoot and grain in the mutants. Heterologous expression of OsABCC9 in yeast enhanced Cd tolerance along with an increase of intracellular Cd content. Taken together, these results indicated that OsABCC9 mediates Cd tolerance and accumulation through sequestration of Cd into the root vacuoles in rice.

Amino Acid Transporters in Plant Cells: A Brief Review.

Amino acids are not only a nitrogen source that can be directly absorbed by plants, but also the major transport form of organic nitrogen in plants. A large number of amino acid transporters have been identified in different plant species. Despite belonging to different families, these amino acid transporters usually exhibit some general features, such as broad expression pattern and substrate selectivity. This review mainly focuses on transporters involved in amino acid uptake, phloem loading and unloading, xylem-phloem transfer, import into seed and intracellular transport in plants. We summarize the other physiological roles mediated by amino acid transporters, including development regulation, abiotic stress tolerance and defense response. Finally, we discuss the potential applications of amino acid transporters for crop genetic improvement.

OsCASP1 Is Required for Casparian Strip Formation at Endodermal Cells of Rice Roots for Selective Uptake of Mineral Elements.

In response to diverse environmental conditions, rice (Oryza sativa) roots have developed one Casparian strip (CS) at the exodermis and one CS at the endodermis. Here, we functionally characterized OsCASP1 (Casparian strip domain protein 1) in rice. OsCASP1 was mainly expressed in the root elongation zone, and the protein encoded was first localized to all sides of the plasma membrane of endodermal cells without CS, followed by the middle of the anticlinal side of endodermal cells with CS. Knockout of OsCASP1 resulted in a defect of CS formation at the endodermis and decreased growth under both soil and hydroponic conditions. Mineral analysis showed that the oscasp1 mutants accumulated more Ca, but less Mn, Zn, Fe, Cd, and As in the shoots compared with the wild type. The growth inhibition of the mutants was further aggravated by high Ca in growth medium. The polar localization of the Si transporter Low Si 1 at the distal side of the endodermis was not altered in the mutant, but the protein abundance was decreased, resulting in a substantial reduction in silicon uptake. These results indicated that OsCASP1 is required for CS formation at the endodermis and that the CS in rice plays an important role in root selective uptake of mineral elements, especially Ca and Si.

A Simple Method to Detect the Inhibition of Transcription Factor-DNA Binding Due to Protein-Protein Interactions In Vivo.

Binding of transcription factors (TFs) to cis-regulatory elements (DNA) could modulate the expression of downstream genes, while interactions between TFs and other proteins might inhibit them binding to DNA. Nowadays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) approaches are usually employed to detect the inhibitory effect. However, EMSA might not reflect the inhibitory effect in vivo. ChIP requires preparation of specific antibody or stable genetic transformation and complicated experimental steps, making it laborious and time-consuming. Here, based on the yeast one-hybrid (Y1H) system, we present a simple method to detect the inhibition of TF-DNA binding due to protein-protein interactions in vivo. When interactions between TFs and other proteins inhibit TFs binding to DNA, the reporter (Aureobasidin A resistance) gene is not activated, thereby inhibiting yeast growth on media containing the AbA antibiotic. Two examples were tested with the newly developed method to demonstrate its feasibility. In conclusion, this method provides an alternative strategy for detecting the inhibition of DNA-binding of TFs due to their interactions with other proteins in vivo.

The ABC transporter ABCG36 is required for cadmium tolerance in rice.

Cadmium (Cd) is a highly toxic heavy metal in nature, which causes severe damage to plant growth. The molecular mechanisms for Cd detoxification are poorly understood. Here, we report that a G-type ATP-binding cassette transporter, OsABCG36, is involved in Cd tolerance in rice. OsABCG36 was expressed in both roots and shoots at a low level, but expression in the roots rather than the shoots was greatly up-regulated by a short exposure to Cd. A spatial expression analysis showed that Cd-induced expression of OsABCG36 was found in both the root tip and the mature root region. Transient expression of OsABCG36 in rice protoplast cells showed that it was localized to the plasma membrane. Immunostaining showed that OsABCG36 was localized in all root cells except the epidermal cells. Knockout of OsABCG36 resulted in increased Cd accumulation in root cell sap and enhanced Cd sensitivity, but did not affect tolerance to other metals including Al, Zn, Cu, and Pb. The concentration of Cd in the shoots was similar between the knockout lines and wild-type rice. Heterologous expression of OsABCG36 in yeast showed an efflux activity for Cd, but not for Zn. Taken together, our results indicate that OsABCG36 is not involved in Cd accumulation in the shoots, but is required for Cd tolerance by exporting Cd or Cd conjugates from the root cells in rice.

Disruption of an amino acid transporter LHT1 leads to growth inhibition and low yields in rice.

BACKGROUND: Research on plant amino acid transporters was mainly performed in Arabidopsis, while our understanding of them is generally scant in rice. OsLHT1 (Lysine/Histidine transporter) has been previously reported as a histidine transporter in yeast, but its substrate profile and function in planta are unclear. The aims of this study are to analyze the substrate selectivity of OsLHT1 and influence of its disruption on rice growth and fecundity. RESULTS: Substrate selectivity of OsLHT1 was analyzed in Xenopus oocytes using the two-electrode voltage clamp technique. The results showed that OsLHT1 could transport a broad spectrum of amino acids, including basic, neutral and acidic amino acids, and exhibited a preference for neutral and acidic amino acids. Two oslht1 mutants were generated using CRISPR/Cas9 genome-editing technology, and the loss-of-function of OsLHT1 inhibited rice root and shoot growth, thereby markedly reducing grain yields. QRT-PCR analysis indicated that OsLHT1 was expressed in various rice organs, including root, stem, flag leaf, flag leaf sheath and young panicle. Transient expression in rice protoplast suggested OsLHT1 was localized to the plasma membrane, which is consistent with its function as an amino acid transporter. CONCLUSIONS: Our results indicated that OsLHT1 is an amino acid transporter with wide substrate specificity and with preference for neutral and acidic amino acids, and disruption of OsLHT1 function markedly inhibited rice growth and fecundity.

OsC2DP, a Novel C2 Domain-Containing Protein Is Required for Salt Tolerance in Rice.

Salt stress is one of the major factors limiting crop production globally, including rice (Oryza sativa). Although a number of genes involved in salt tolerance have been functionally identified, the mechanism underlying salt tolerance in rice is still poorly understood. Here, we reported a novel C2 domain-containing protein, OsC2DP required for salt tolerance in rice. OsC2DP was predominately expressed in the roots and its expression was repressed by salt stress. Transient expression of OsC2DP in rice protoplast cells showed that it was localized in the cytosol. Immunostaining further showed that OsC2DP was able to translocate from the cytosol to plasma membrane under salt conditions. Knockout of OsC2DP did not affect Na+ concentration in the roots, but increased shoot Na+ concentration, resulting in a significant sensitivity of rice to salt stress. Furthermore, the quantitative Real-time PCR and transcriptomic analysis showed that the expression level of some genes related to salt tolerance were indirectly regulated by OsC2DP, especially OsSOS1 and OsNHX4. These results indicate that OsC2DP has an important role in salt tolerance and these findings provide new insights into the regulation of OsC2DP gene for rice breeding with high salt tolerance.

Bioinformatic and Functional Analysis of a Key Determinant Underlying the Substrate Selectivity of the Al Transporter, Nrat1.

Nrat1 is a member of the natural resistance-associated macrophage protein (Nramp) family of metal ion transporters in all organisms. Different from other Nramp members capable of transporting divalent metals, Nrat1 specifically transports trivalent aluminum (Al) ion. However, molecular mechanism underlying the Al transport selectivity of Nrat1 remains unknown. Here, we performed structure-function analyses of Nrat1 and other Nramp members to gain insights into the determinants of ion selectivity. A phylogenetic analysis showed that plant Nramp transporters could be divided into five groups. OsNrat1 was found in one of the individual clades and clustered with SbNrat1 and ZmNrat1 on the evolutionary tree. Structural modeling revealed that Nrat1 transporters adopted a common LeuT fold shared by many Nramp-family transporters that likely employed an identical transport mechanism. Sequence alignment and evolutionary conservation analysis of amino acids identified a metal-permeation pathway of Nrat1 centered at the metal binding site. The metal binding site of Nrat1 was characterized by two conserved sequence motifs, i.e., the Asp-Pro-Ser-Asn motif (motif A) and the Ala-Ile-Ile-Thr motif (motif B). Replacement of the Ala-Met-Val-Met motif B of the OsNramp3 manganese (Mn) transporter to that of Nrat1 resulted in a partial gain of Al transport activity and a total loss of Mn in yeast. Conversely, substitution of the motif B of OsNrat1 with that of OsNramp3 altered the Al transport activity. These observations indicated the metal binding site, particularly the motif B, as a key determinant of Al selectivity of Nrat1.

Functional characterization of the SbNrat1 gene in sorghum.

The Natural Resistance Associated Macrophage Protein (Nramp) members play diverse roles in metal transport in plants. Recent studies have showed that OsNrat1 (OsNramp4) encodes an Al transporter, which is required for rice Al tolerance. In this study, we functionally characterized a Nramp member in sorghum, SbNrat1, which is homologous to OsNrat1 with 88% identity. SbNrat1 was expressed in both roots and shoots, and its expression was not induced by Al treatment. When expressed in yeast, SbNrat1 transports trivalent Al ion, but not Mn and Cd. Furthermore, introduction of SbNrat1 into the rice mutant osnrat1 can rescue its sensitivity to Al. However, no correlation between Al tolerance and the expression level of SbNrat1 was found in thirteen sorghum cultivars tested. These results indicate that SbNrat1 functions as an Al transporter that is possibly involved in basic Al tolerance in sorghum.

An ABC transporter, OsABCG26, is required for anther cuticle and pollen exine formation and pollen-pistil interactions in rice.

Wax, cutin and sporopollenin are essential components for the formation of the anther cuticle and the pollen exine, respectively. Their lipid precursors are synthesized by secretory tapetal cells and transported to the anther and microspore surface for deposition. However, the molecular mechanisms involved in the formation of the anther cuticle and pollen exine are poorly understood in rice. Here, we characterized a rice male sterile mutant osabcg26. Molecular cloning and sequence analysis revealed a point mutation in the gene encoding an ATP binding cassette transporter G26 (OsABCG26). OsABCG26 was specifically expressed in the anther and pistil. Cytological analysis revealed defects in tapetal cells, lipidic Ubisch bodies, pollen exine, and anther cuticle in the osabcg26 mutant. Expression of some key genes involved in lipid metabolism and transport, such as UDT1, WDA1, CYP704B2, OsABCG15, OsC4 and OsC6, was significantly altered in osabcg26 anther, possibly due to a disturbance in the homeostasis of anther lipid metabolism and transport. Additionally, wild-type pollen tubes showed a growth defect in osabcg26 pistils, leading to low seed setting in osabcg26 cross-pollinated with the wild-type pollen. These results indicated that OsABCG26 plays an important role in anther cuticle and pollen exine formation and pollen-pistil interactions in rice.

Complex interactions among residues within pore region determine the K+ dependence of a KAT1-type potassium channel AmKAT1.

KAT1-type channels mediate K(+) influx into guard cells that enables stomatal opening. In this study, a KAT1-type channel AmKAT1 was cloned from the xerophyte Ammopiptanthus mongolicus. In contrast to most KAT1-type channels, its activation is strongly dependent on external K(+) concentration, so it can be used as a model to explore the mechanism for the K(+) -dependent gating of KAT1-type channels. Domain swapping between AmKAT1 and KAT1 reveals that the S5-pore-S6 region controls the K(+) dependence of AmKAT1, and residue substitutions show that multiple residues within the S5-Pore linker and Pore are involved in its K(+) -dependent gating. Importantly, complex interactions occur among these residues, and it is these interactions that determine its K(+) dependence. Finally, we analyzed the potential mechanism for the K(+) dependence of AmKAT1, which could originate from the requirement of K(+) occupancy in the selectivity filter to maintain its conductive conformation. These results provide new insights into the molecular basis of the K(+) -dependent gating of KAT1-type channels.

RNA-Seq analysis of differentially expressed genes in rice under varied nitrogen supplies.

Ammonium is the main inorganic nitrogen source in paddy soil. Rice (Oryza sativa), an ammonium-preferring and -tolerant grain crop, is a valuable resource for researching ammonium-uptake mechanism and understanding the molecular networks that the plant copes with ammonium variation. To generate a broad survey of early responses affected by varied ammonium supplies in rice, RNA samples were prepared from the roots and shoots of rice plants subjected to nitrogen-free (0mM ammonium), 1mM ammonium and high ammonium (10mM ammonium) for a short period of 4h (1mM ammonium treatment as the control), respectively, and the transcripts were sequenced using the Illumina/HiSeq™ 2000 RNA sequencing (RNA-Seq) platform. By comparative analysis, 394 differentially expressed genes (DEGs) were identified in roots, among which, 143 and 251 DEGs were up- and down-regulated under nitrogen-free condition, respectively. In shoots, 468 (119 up-regulated/349 down-regulated) DEGs were found under such condition. However, with high ammonium treatment, only 63 genes (6 up-regulated/57 down-regulated) in roots and 115 genes in shoots (93 up-regulated/22 down-regulated) were differentially expressed. According to KEGG analysis, when exposed to nitrogen-free condition, DEGs participating in the carbohydrate and amino acid metabolisms were down-regulated (with 1 exception) in roots as well as in shoots, implying reduced carbohydrate and nitrogen metabolisms. Under high ammonium supply, all DEGs associated with carbohydrate and amino acid metabolisms were down-regulated in roots and to the contrary, up-regulated in shoots. Aldehyde dehydrogenase (ALDH, NAD(+)) [EC: 1.2.1.3] seemed to have played an important role in rice shoots under high ammonium condition, analysis results implicated a coordinative regulation of carbohydrate with amino acid metabolisms under nitrogen deficiency as well as the high ammonium conditions during a short period of several hours in rice. Moreover, transcripts with abundance variation might be precious gene resources in responding to different ammonium supplies in rice.