Structural variations in wheat HKT1;5 underpin differences in Na+ transport capacity.

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na+) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na+-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K m) for the Na+ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D471/a gap and D474/G473 that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na+ exclusion in wheat that leads to an improved salinity tolerance in the field.

Foxtail millet SiHAK1 excites extreme high-affinity K+ uptake to maintain K+ homeostasis under low K+ or salt stress.

KEY MESSAGE: This is the first evidence that SiHAK1 acts as a K+ transporter and is modulated by internal and external K+, which expands our understanding of the significant physiological roles of large HAK/KUP/KT transporters in crops. Crop genomes have shown the richness of K+ transporters in HAK/KUP/KT (High Affinity K+/K+ Uptake Proteins/K+ Transporter) family, and much progress have been achieved toward understanding the diverse roles of K+ uptake and translocation, and abiotic stresses resistance in this family. The HAK/KUP/KT family has increasingly been recognized to be at a pivotal status in the mediation of K+ translocation and long-term transport; however, our understanding of the molecular mechanisms remains limited. Foxtail millet is an ideal plant for studying long-distance potassium (K) transport because of its small diploid genome and better adaptability to arid lands. Here, we identified 29 putative HAK/KUP/KT proteins from the Setaria italica genome database. These genes were distributed in seven chromosomes of foxtail millet and divided into five clusters. SiHAK1 exhibited widespread expression in various tissues and significant up-regulation in the shoots under low K condition. SiHAK1 was localized in the cell membrane and low K elicited SiHAK1-meidated high-affinity K+ uptake activity in Cy162 yeast cells and Arabidopsis athak5 mutants. The transport activity of SiHAK1 was coordinately modulated by external K+ supply and internal K+ content in the cell under low K and high salt environment. Our findings reveal the K uptake mechanisms of SiHAK1 and indicated that it may be involved in the mediation of K homeostasis in S. italica under K+-deficiency and salt stress.

T-DNA Tagging-Based Gain-of-Function of OsHKT1;4 Reinforces Na Exclusion from Leaves and Stems but Triggers Na Toxicity in Roots of Rice Under Salt Stress.

The high affinity K⁺ transporter 1;4 (HKT1;4) in rice (Oryza sativa), which shows Na⁺ selective transport with little K⁺ transport activity, has been suggested to be involved in reducing Na in leaves and stems under salt stress. However, detailed physiological roles of OsHKT1;4 remain unknown. Here, we have characterized a transfer DNA (T-DNA) insertion mutant line of rice, which overexpresses OsHKT1;4, owing to enhancer elements in the T-DNA, to gain an insight into the impact of OsHKT1;4 on salt tolerance of rice. The homozygous mutant (the O/E line) accumulated significantly lower concentrations of Na in young leaves, stems, and seeds than the sibling WT line under salt stress. Interestingly, however, the mutation rendered the O/E plants more salt sensitive than WT plants. Together with the evaluation of biomass of rice lines, rhizosphere acidification assays using a pH indicator bromocresol purple and 22NaCl tracer experiments have led to an assumption that roots of O/E plants suffered heavier damages from Na which excessively accumulated in the root due to increased activity of Na⁺ uptake and Na⁺ exclusion in the vasculature. Implications toward the application of the HKT1-mediated Na⁺ exclusion system to the breeding of salt tolerant crop cultivars will be discussed.

Increased tolerance to low K+, and to cationic stress of Arabidopsis plants by expressing the F130S mutant version of the K+ transporter AtHAK5.

Potassium (K+) selectivity of high-affinity K+ uptake systems is crucial for plant growth under low K+ and in the presence of inhibitors of K+ uptake that are toxic to plants such as Na+ or Cs+. Here, we express a mutated version of the Arabidopsis AtHAK5 high-affinity K+ transporter consisting on a change of phenylalanine 130 to serine (F130S) in athak5 akt1 double mutant plants. F130S-expressing plants show better growth, increased K+ uptake from low external concentrations and higher K+ contents when grown at low K+ (10 µM) and when grown at low K+ in the presence of Na+ (15 mM) or Cs+ (1 µM). In addition, these plants accumulate less Na+ and Cs+, resulting in lower Na+/K+ and Cs+/K+ ratios, which are important determinants of plant tolerance to salt stress and to Cs+-polluted soils. Structure analysis of AtHAK5 suggest that the F130 residue approaches the intracellular gate of the K+ tunnel of AtHAK5, affecting somehow its ionic selectivity. Modification of transport systems has a large potential to face challenges of future agriculture such as sustainable production under abiotic stress conditions imposed by climate change.

Identification of the High-Affinity Potassium Transporter Gene Family (HKT) in Brassica U-Triangle Species and Its Potential Roles in Abiotic Stress in Brassica napus L.

Members of the high-affinity potassium transporter (HKT) protein family regulate the uptake and homeostasis of sodium and potassium ions, but little research describes their roles in response to abiotic stresses in rapeseed (Brassica napus L.). In this study, we identified and characterized a total of 36 HKT genes from the species comprising the triangle of U model (U-triangle species): B. rapa, B. nigra, B. oleracea, B. juncea, B. napus, and B. carinata. We analyzed the phylogenetic relationships, gene structures, motif compositions, and chromosomal distributions of the HKT family members of rapeseed. Based on their phylogenetic relationships and assemblage of functional domains, we classified the HKT members into four subgroups, HKT1;1 to HKT1;4. Analysis of the nonsynonymous substitutions (Ka), synonymous substitutions (Ks), and the Ka/Ks ratios of HKT gene pairs suggested that these genes have experienced strong purifying selective pressure after duplication, with their evolutionary relationships supporting the U-triangle theory. Furthermore, the expression profiles of BnaHKT genes varies among potassium, phytohormone and heavy-metal treatment. Their repression provides resistance to heavy-metal stress, possibly by limiting uptake. Our results systematically reveal the characteristics of HKT family proteins and their encoding genes in six Brassica species and lay a foundation for further exploration of the role of HKT family genes in heavy-metal tolerance.

Genome-wide identification of a novel Na+ transporter from Bienertia sinuspersici and overexpression of BsHKT1;2 improved salt tolerance in Brassica rapa.

Salt stress is an ever-increasing stressor that affects both plants and humans. Therefore, developing strategies to limit the undesirable effects of salt stress is essential. Sodium ion exclusion is well known for its efficient salt-tolerance mechanism. The High-affinity K+ Transporter (HKT) excludes excess Na+ from the transpiration stream. This study identified and characterized the HKT protein family in Bienertia sinuspersici, a single-cell C4 plant. The HKT and Salt Overly Sensitive 1 (SOS1) expression levels were examined in B. sinuspersici and Arabidopsis thaliana leaves under four different salt stress conditions: 0, 100, 200, and 300 mM NaCl. Furthermore, BsHKT1;2 was cloned, thereby producing stable transgenic Brassica rapa. Our results showed that, compared to A. thaliana as a glycophyte, the HKT family is expanded in B. sinuspersici as a halophyte with three paralogs. The phylogenetic analysis revealed three paralogs belonging to the HKT subfamily I. Out of three copies, the expression of BsHKT1;2 was higher in Bienertia under control and salt stress conditions than in A. thaliana. Stable transgenic plants overexpressing 35S::BsHKT1;2 showed higher salt tolerance than non-transgenic plants. Higher biomass and longer roots were observed in the transgenic plants under salt stress than in non-transgenic plants. This study demonstrates the evolutionary and functional differences in HKT proteins between glycophytes and halophytes and associates the role of BsHKT1;2 in imparting salt tolerance and productivity.

[Genome-wide identification of BvHAK gene family in sugar beet (Beta vulgaris) and their expression analysis under salt treatments].

High-affinity K+ transporter (HAK) is one of the most important K+ transporter families in plants and plays an important role in plant K+ uptake and transport. To explore the biological functions and gene expression patterns of the HAK gene family members in sugar beet (Beta vulgaris), physicochemical properties, the gene structure, chromosomal location, phylogenetic evolution, conserved motifs, three-dimensional structure, interaction network, cis-acting elements of promoter of BvHAKs were predicted by bioinformatic analysis, and their expression levels in different tissues of sugar beet under salt stress were analyzed by qRT-PCR. A total of 10 BvHAK genes were identified in the sugar beet genome. They contained 8-10 exons and 7-9 introns. The average number of amino acids was 778.30, the average molecular weight was 88.31 kDa, and the isoelectric point was 5.38-9.41. The BvHAK proteins contained 11-14 transmembrane regions. BvHAK4, -5, -7 and -13 were localized on plasma membrane, while others were localized on tonoplast. Phylogenetic analysis showed that HAK in higher plants can be divided into five clusters, namely cluster Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅴ, among which the members of cluster Ⅱ can be divided into three subclusters, including Ⅱa, Ⅱb, and Ⅱc. The BvHAK gene family members were distributed in cluster Ⅰ-Ⅳ with 1, 6, 1, and 2 members, respectively. The promoter of BvHAK gene family mainly contained stress responsive elements, hormone responsive elements, and growth and development responsive elements. The expression pattern of the BvHAK genes were further analyzed in different tissues of sugar beet upon salt treatment, and found that 50 and 100 mmol/L NaCl significantly induced the expression of the BvHAK genes in both shoots and roots. High salt (150 mmol/L) treatment clearly down-regulated their expression levels in shoots, but not in roots. These results suggested that the BvHAK gene family plays important roles in the response of sugar beet to salt stress.

Rice Cultivars Under Salt Stress Show Differential Expression of Genes Related to the Regulation of Na+/K+ Balance.

Soil salinity is a major problem in agriculture because high accumulation of Na+ ions in plants causes toxicity that can result in yield reduction. Na+/K+ homeostasis is known to be important for salt tolerance in plants. Na+/K+ homeostasis in rice (Oryza sativa L.) involves nine high-affinity K+ transporter (HKT) encoding Na+-K+ symporter, five OsNHX Na+/H+ antiporters, and OsSOS1 Na+/K+ antiporter genes. In the present study, we investigated various molecular and physiological processes to evaluate germination rate, growth pattern, ion content, and expression of OsHKT, OsNHX, and OsSOS1genes related to Na+/K+ homeostasis in different rice genotypes under salt stress. We found a significant increase in the germination percentage, plant vigor, Na+/K+ ratio, and gene expression of the OsHKT family in both the roots and shoots of the Nagdong cultivar and salt-tolerant cultivar Pokkali. In the roots of Cheongcheong and IR28 cultivars, Na+ ion concentrations were found to be higher than K+ ion concentrations. Similarly, high expression levels of OsHKT1, OsHKT3, and OsHKT6 were observed in Cheongcheong, whereas expression levels of OsHKT9 was high in IR28. The expression patterns of OsNHX and OsSOS1 and regulation of other micronutrients differed in the roots and shoots regions of rice and were generally increased by salt stress. The OsNHX family was also expressed at high levels in the roots of Nagdong and in the roots and shoots of Pokkali; in contrast, comparatively low expression levels were observed in the roots and shoots of Cheongcheong and IR28 (with the exception of high OsNHX1 expression in the roots of IR28). Furthermore, the OsSOS1 gene was highly expressed in the roots of Nagdong and shoots of Cheongcheong. We also observed that salt stress decreases chlorophyll content in IR28 and Pokkali but not in Cheongcheong and Nagdong. This study suggests that under salt stress, cultivar Nagdong has more salt-tolerance than cultivar Cheongcheong.

Restructuring the Cellular Responses: Connecting Microbial Intervention With Ecological Fitness and Adaptiveness to the Maize (Zea mays L.) Grown in Saline-Sodic Soil.

Salt stress hampers plant growth and development. It is now becoming one of the most important threats to agricultural productivity. Rhizosphere microorganisms play key roles in modulating cellular responses and enable plant tolerant to salt stress, but the detailed mechanisms of how this occurs need in-depth investigation. The present study elucidated that the microbe-mediated restructuring of the cellular responses leads to ecological fitness and adaptiveness to the maize (Zea mays L.) grown in saline-sodic soil. In the present study, effects of seed biopriming with B. safensis MF-01, B. altitudinis MF-15, and B. velezensis MF-08 singly and in consortium on different growth parameters were recorded. Soil biochemical and enzymatic analyses were performed. The activity and gene expression of High-Affinity K+ Transporter (ZmHKT-1), Sodium/Hydrogen exchanger 1 (zmNHX1), and antioxidant enzymes (ZmAPX1.2, ZmBADH-1, ZmCAT, ZmMPK5, ZmMPK7, and ZmCPK11) were studied. The expression of genes related to lateral root development (ZmHO-1, ZmGSL-1, and ZmGSL-3) and root architecture were also carried out. Seeds bioprimed with consortium of all three strains have been shown to confer increased seed germination (23.34-26.31%) and vigor indices (vigor index I: 38.71-53.68% and vigor index II: 74.11-82.43%) as compared to untreated control plant grown in saline-sodic soil at 30 days of sowing. Results indicated that plants treated with consortium of three strains induced early production of adventitious roots (tips: 4889.29, forks: 7951.57, and crossings: 2296.45) in maize compared to plants primed with single strains and untreated control (tips: 2019.25, forks: 3021.45, and crossings: 388.36), which was further confirmed by assessing the transcript level of ZmHO-1 (7.20 folds), ZmGSL-1 (4.50 folds), and ZmGSL-3 (12.00 folds) genes using the qPCR approach. The uptake and translocation of Na+, K+, and Ca2+ significantly varied in the plants treated with bioagents alone or in consortium. qRT-PCR analysis also revealed that the ZmHKT-1 and zmNHX1 expression levels varied significantly in the maize root upon inoculation and showed a 6- to 11-fold increase in the plants bioprimed with all the three strains in combination. Further, the activity and gene expression levels of antioxidant enzymes were significantly higher in the leaves of maize subjected seed biopriming with bioagents individually or in combination (3.50- to 12.00-fold). Our research indicated that ZmHKT-1 and zmNHX1 expression could effectively enhance salt tolerance by maintaining an optimal Na+/K+ balance and increasing the antioxidant activity that keeps reactive oxygen species at a low accumulation level. Interestingly, up-regulation of ZmHKT-1, NHX1, ZmHO-1, ZmGSL-1, and ZmGSL-3 and genes encoding antioxidants regulates the cellular responses that could effectively enhance the adaptiveness and ultimately leads to better plant growth and grain production in the maize crop grown in saline-sodic soil.

A calmodulin-like CmCML13 from Cucumis melo improved transgenic Arabidopsis salt tolerance through reduced shoot's Na+, and also improved drought resistance.

The calmodulin-like proteins (CMLs) are a large family involved in plant biological processes. A calmodulin-like gene CmCML13 (GenBank accession number: MT340534) from melon (Cucumis melo L.) was isolated and functionally analyzed. CmCML13 was predicted to possess 3 EF-hands in which only the first EF-hand could bind with Ca2+. Subcellular localization assay revealed that CmCML13 was localized in nucleus, cell membrane, vacuolar membrane and cytoplasmic strand. The transcript level of CmCML13 was temporally and spatially regulated under salt stress. Constitutive expression of CmCML13 in the Arabidopsis thaliana enhanced salt tolerance at seeds germination. CmCML13 improved the transgenic Arabidopsis plants salt tolerance by significantly reducing Na+ content of shoots, which was unrelated to HKT1-involving pathway. Moreover, overexpressing of CmCML13 in Arabidopsis showed stronger drought tolerance. This study demonstrates that the CmCML13 is an important multifunctional protein associated with salt and drought stress, which may play a key role in stress signaling pathway.