Genome-Wide Analysis of the Apple CBL Family Reveals That Mdcbl10.1 Functions Positively in Modulating Apple Salt Tolerance.

Abiotic stresses are increasingly harmful to crop yield and quality. Calcium and its signaling pathway play an important role in modulating plant stress tolerance. As specific Ca2+ sensors, calcineurin B-like (CBL) proteins play vital roles in plant stress response and calcium signaling. The CBL family has been identified in many plant species; however, the characterization of the CBL family and the functional study of apple MdCBL proteins in salt response have yet to be conducted in apple. In this study, 11 MdCBL genes were identified from the apple genome. The coding sequences of these MdCBL genes were cloned, and the gene structure and conserved motifs were analyzed in detail. The phylogenetic analysis indicated that these MdCBL proteins could be divided into four groups. The functional identification in Na+-sensitive yeast mutant showed that the overexpression of seven MdCBL genes could confer enhanced salt stress resistance in transgenic yeast. The function of MdCBL10.1 in regulating salt tolerance was also verified in cisgenic apple calli and apple plants. These results provided valuable insights for future research examining the function and mechanism of CBL proteins in regulating apple salt tolerance.

Salt Tolerance Improvement in Rice through Efficient SNP Marker-Assisted Selection Coupled with Speed-Breeding.

Salinity critically limits rice metabolism, growth, and productivity worldwide. Improvement of the salt resistance of locally grown high-yielding cultivars is a slow process. The objective of this study was to develop a new salt-tolerant rice germplasm using speed-breeding. Here, we precisely introgressed the hst1 gene, transferring salinity tolerance from "Kaijin" into high-yielding "Yukinko-mai" (WT) rice through single nucleotide polymorphism (SNP) marker-assisted selection. Using a biotron speed-breeding technique, we developed a BC3F3 population, named "YNU31-2-4", in six generations and 17 months. High-resolution genotyping by whole-genome sequencing revealed that the BC3F2 genome had 93.5% similarity to the WT and fixed only 2.7% of donor parent alleles. Functional annotation of BC3F2 variants along with field assessment data indicated that "YNU31-2-4" plants carrying the hst1 gene had similar agronomic traits to the WT under normal growth condition. "YNU31-2-4" seedlings subjected to salt stress (125 mM NaCl) had a significantly higher survival rate and increased shoot and root biomasses than the WT. At the tissue level, quantitative and electron probe microanalyzer studies indicated that "YNU31-2-4" seedlings avoided Na+ accumulation in shoots under salt stress. The "YNU31-2-4" plants showed an improved phenotype with significantly higher net CO2 assimilation and lower yield decline than WT under salt stress at the reproductive stage. "YNU31-2-4" is a potential candidate for a new rice cultivar that is highly tolerant to salt stress at the seedling and reproductive stages, and which might maintain yields under a changing global climate.

Increasing cyclic electron flow is related to Na+ sequestration into vacuoles for salt tolerance in soybean.

In land plants, the NAD(P)H dehydrogenase (NDH) complex reduces plastoquinones and drives cyclic electron flow (CEF) around PSI. It also produces extra ATP for photosynthesis and improves plant fitness under conditions of abiotic environmental stress. To elucidate the role of CEF in salt tolerance of the photosynthetic apparatus, Na(+) concentration, chlorophyll fluorescence, and expression of NDH B and H subunits, as well as of genes related to cellular and vacuolar Na(+) transport, were monitored. The salt-tolerant Glycine max (soybean) variety S111-9 exhibited much higher CEF activity and ATP accumulation in light than did the salt-sensitive variety Melrose, but similar leaf Na(+) concentrations under salt stress. In S111-9 plants, ndhB and ndhH were highly up-regulated under salt stress and their corresponding proteins were maintained at high levels or increased significantly. Under salt stress, S111-9 plants accumulated Na(+) in the vacuole, but Melrose plants accumulated Na(+) in the chloroplast. Compared with Melrose, S111-9 plants also showed higher expression of some genes associated with Na(+) transport into the vacuole and/or cell, such as genes encoding components of the CBL10 (calcineurin B-like protein 10)-CIPK24 (CBL-interacting protein kinase 24)-NHX (Na(+)/H(+) antiporter) and CBL4 (calcineurin B-like protein 4)-CIPK24-SOS1 (salt overly sensitive 1) complexes. Based on the findings, it is proposed that enhanced NDH-dependent CEF supplies extra ATP used to sequester Na(+) in the vacuole. This reveals an important mechanism for salt tolerance in soybean and provides new insights into plant resistance to salt stress.

RAP2.6 enhanced salt stress tolerance by reducing Na+ accumulation and stabilizing the electron transport in Arabidopsis thaliana.

The transcription factors of the AP2/ERF family are involved in plant growth and development and responses to biotic and abiotic stresses. Here, we found RAP2.6, a transcription factor which belongs to the ERF subfamily, was responsive to salt stress in Arabidopsis. Under salt stress conditions, rap2.6 mutant seedlings were the sensitivity deficiency to salt stress which was reflected in higher germination rate and longer root length compared to the wild type. Also, the expressions of salt-related gene including SOS1, SOS2, SOS3, NHX1, NHX3, NHX5 and HKT1 in rap2.6 mutant seedlings were lower than the wild type under salt stress. rap2.6 mutant adult lacked salt stress tolerance based on the results of the phenotype, survival rates and ion leakage. Compared to wild type, rap2.6 mutant adult accumulated more Na+ in leaves and roots while the salt-related gene expressions were lower. In addition, the photosynthetic electron transport and PSII energy distribution in rap2.6 mutant plant leaves had been more seriously affected under salt stress conditions compared to the wild type. In summary, this study identified essential roles of RAP2.6 in regulating salt stress tolerance in Arabidopsis.

Functional identification of the calcineurin B-like protein PavCBL4 in modulating salt tolerance in sweet cherry.

Abiotic stresses, such as high salinity, pose a significant threat to plant growth and development, reducing crop yield and quality. Calcineurin B-like (CBL) proteins serve as crucial calcium sensors in plant responses to diverse environmental stresses. However, the CBL family in sweet cherry has not been identified at the genome-wide level, and the regulatory role of CBL proteins in cherry plants' salt response is unclear. Here, we identified 10 CBL family genes (PavCBLs) from the Prunus avium genome and cloned seven of them. We comprehensively analyzed PavCBL genes for collinearity, phylogenetic relationships, gene structure, and conserved motifs. Expression analysis revealed significant induction of transcription under abiotic stress, with PavCBL4 displaying the most substantial expression change. Additionally, we identified PavCBL4 as a PavSOS2 (Salt Overly Sensitive 2)-interacting protein through Y2H and Split-LUC assays. Subcellular localization analysis indicated that PavCBL4 is present in both the cytoplasm and nucleus. Functional assessment of PavCBL4 in the PavCBL4-overexpressing transgenic 'Gisela 6' plants showed its positive role in enhancing salt tolerance in cherry plants. Measurements of Na+ content and antioxidant enzyme activity under salt stress indicated that PavCBL4 functions positively by inhibiting Na+ accumulation and promoting ROS scavenging in response to salt stress. These findings lay the groundwork for a deeper understanding of the molecular mechanisms underlying PavCBL-mediated salt tolerance in sweet cherry.

Cation/Ca2+ Exchanger 1 (MdCCX1), a Plasma Membrane-Localized Na+ Transporter, Enhances Plant Salt Tolerance by Inhibiting Excessive Accumulation of Na+ and Reactive Oxygen Species.

High salinity causes severe damage to plant growth and significantly reduces crop yields. The CCX family proteins can facilitate the transport of multiple ions to prevent toxicity. CCX proteins play an important role in regulating plant salt tolerance, but no detailed studies on CCX proteins in apples have been reported. Here, the CCX family gene MdCCX1 was cloned from apple (Malus domestica). It is constitutively expressed in various apple tissues and is significantly induced by salt stress. As a plasma membrane-localized protein, MdCCX1-overexpression could complement the Na+-sensitive phenotype of yeast mutants and reduce the Na+ content in yeast cells under NaCl treatment, suggesting that MdCCX1 could be a plasma membrane-localized Na+ transporter. To identify the function of MdCCX1 in salt response, we transformed this gene into Arabidopsis, apple calli, and apple plants. Overexpression of MdCCX1 significantly improved the salt tolerance of these transgenic materials. The significantly reduced Na+ content under NaCl treatment indicated that MdCCX1 overexpression could enhance plant salt tolerance by inhibiting the excessive accumulation of Na+. Besides, MdCCX1 overexpression could also enhance plant salt tolerance by promoting ROS scavenging. These findings provide new insight and rich resources for future studies of CCX proteins in plant species.

Cold stress increases salt tolerance of the extremophytes Eutrema salsugineum (Thellungiella salsuginea) and Eutrema (Thellungiella) botschantzevii.

A comparative study was performed to analyze the effect of cold acclimation on improving the resistance of Arabidopsis thaliana, Eutrema salsugineum and Eutrema botschantzevii plants to salt stress. Shoot FW, sodium and potassium accumulation, metabolite content, expression of proton pump genes VAB1, VAB2,VAB3, VP2, HA3 and genes encoding ion transporters SOS1, HKT1, NHX1, NHX2, NHX5 located in the plasma membrane or tonoplast were determined just after the cold treatment and the onset of the salt stress. In the same cold-acclimated E. botschantzevii plants, the Na+ concentration after salt treatment was around 80% lower than in non-acclimated plants, whereas the K+ concentration was higher. As a result of cold acclimation, the expression of, VAB3, NHX2, NHX5 genes and of SOS1, VP2, HA3 genes was strongly enhanced in E. botschantzevii and in E. salsugineum plants correspondently. None of the 10 genes analyzed showed any expression change in A. thaliana plants after cold acclimation. Altogether, the results indicate that cold-induced adaptation to subsequent salt stress exists in the extremophytes E. botschantzevii and to a lesser extend in E. salsugineum and is absent in Arabidopsis. This phenomenon may be attributed to the increased expression of ion transporter genes during cold acclimation in the Eutrema species.

The coordinated regulation of Na+ and K+ in Hordeum brevisubulatum responding to time of salt stress.

Hordeum brevisubulatum, called as wild barley, is a useful monocotyledonous halophyte for soil improvement in northern China. Although previously studied, its main salt tolerance mechanism remained controversial. The current work showed that shoot Na+ concentration was increased rapidly with stress time and significantly higher than in wheat during 0-168h of 100mM NaCl treatment. Similar results were also found under 25 and 50mM NaCl treatments. Even K+ was increased from 0.01 to 50mM in the cultural solution, no significant effect was found on tissue Na+ concentrations. Interestingly, shoot growth was improved, and stronger root activity was maintained in H. brevisubulatum compared with wheat after 7days treatment of 100mM NaCl. To investigate the long-term stress impact on tissue Na+, 100mM NaCl was prolonged to 60 days. The maximum values of Na+ concentrations were observed at 7th in shoot and 14th day in roots, respectively, and then decreased gradually. Micro-electrode ion flux estimation was used and it was found that increasing Na+ efflux while maintaining K+ influx were the major strategies to reduce the Na+ concentration during long-term salt stress. Moreover, leaf Na+ secretions showed little contribution to the tissue Na+ decrease. Thereby, the physiological mechanism for H. brevisubulatum to survive from long-term salt stress was proposed that rapid Na+ accumulation occurred in the shoot to respond the initial salt shock, then Na+ efflux was triggered and K+ influx was activated to maintain a stable K+/Na+ ratio in tissues.

PDH45 transgenic rice maintain cell viability through lower accumulation of Na(+), ROS and calcium homeostasis in roots under salinity stress.

Salinity severely affects the growth/productivity of rice, which is utilized as major staple food crop worldwide. PDH45 (pea DNA helicase 45), a member of the DEAD-box helicase family, actively provides salinity stress tolerance, but the mechanism behind this is not well known. Therefore, in order to understand the mechanism of stress tolerance, sodium ion (Na(+)), reactive oxygen species (ROS), cytosolic calcium [Ca(2+)]cyt and cell viability were analyzed in roots of PDH45 transgenic-IR64 rice lines along with wild-type (WT) IR64 rice under salinity stress (100mM and 200 mM NaCl). In addition, the roots of salinity-tolerant (FL478) and susceptible (Pusa-44) rice varieties were also analyzed under salinity stress for comparative analysis. The results reveal that, under salinity stress (100mM and 200 mM NaCl), roots of PDH45 transgenic lines accumulate lower levels of Na(+), ROS and maintain [Ca(2+)]cyt and exhibit higher cell viability as compared with roots of WT (IR64) plants. Similar results were also obtained in the salinity-tolerant FL478 rice. However, the roots of WT and salinity-susceptible Pusa-44 rice accumulated higher levels of Na(+), ROS and [Ca(2+)]cyt imbalance and lower cell viability during salinity stress, which is in contrast to the overexpressing PDH45 transgenic lines and salinity-tolerant FL478 rice. Further, to understand the mechanism of PDH45 at molecular level, comparative expression profiling of 12 cation transporters/genes was also conducted in roots of WT (IR64) and overexpressing PDH45 transgenic lines (L1 and L2) under salt stress (24h of 200 mM NaCl). The expression analysis results show altered and differential gene expression of cation transporters/genes in salt-stressed roots of WT (IR64) and overexpressing transgenic lines (L1 and L2). These observations collectively suggest that, under salinity stress conditions, PDH45 is involved in the regulation of Na(+) level, ROS production, [Ca(2+)]cyt homeostasis, cell viability and cation transporters in roots of PDH45 transgenic-IR64 rice and consequently provide salinity tolerance. Elucidating the detailed regulatory mechanism of PDH45 will provide a better understanding of salinity stress tolerance and further open new ways to manipulate genome to achieve higher agricultural production under stress.

Beneficial soil bacterium Bacillus subtilis (GB03) augments salt tolerance of white clover.

Soil salinity is an increasingly serious problem worldwide that reduces agricultural output potential. Selected beneficial soil bacteria can promote plant growth and augment tolerance to biotic and abiotic stresses. Bacillus subtilis strain GB03 has been shown to confer growth promotion and abiotic stress tolerance in the model plant Arabidopsis thaliana. Here we examined the effect of this beneficial soil bacterium on salt tolerance in the legume forage crop, white clover. Plants of white clover (Trifolium repens L. cultivar Huia) were grown from seeds with or without soil inoculation of the beneficial soil bacterium Bacillus subtilis GB03 supplemented with 0, 50, 100, or 150 mM NaCl water into soil. Growth parameters, chlorophyll content, malondialdehyde (MDA) content and osmotic potential were monitored during the growth cycle. Endogenous Na(+) and K(+) contents were determined at the time of harvest. White clover plants grown in GB03-inoculated soil were significantly larger than non-inoculated controls with respect to shoot height, root length, plant biomass, leaf area and chlorophyll content; leaf MDA content under saline condition and leaf osmotic potential under severe salinity condition (150 mM NaCl) were significantly decreased. Furthermore, GB03 significantly decreased shoot and root Na(+) accumulation and thereby improved K(+)/Na(+) ratio when GB03-inoculated plants were grown under elevated salt conditions. The results indicate that soil inoculation with GB03 promotes white clover growth under both non-saline and saline conditions by directly or indirectly regulating plant chlorophyll content, leaf osmotic potential, cell membrane integrity and ion accumulation.