The translocation of a chloride channel from the Golgi to the plasma membrane helps plants adapt to salt stress.

A key mechanism employed by plants to adapt to salinity stress involves maintaining ion homeostasis via the actions of ion transporters. While the function of cation transporters in maintaining ion homeostasis in plants has been extensively studied, little is known about the roles of their anion counterparts in this process. Here, we describe a mechanism of salt adaptation in plants. We characterized the chloride channel (CLC) gene AtCLCf, whose expression is regulated by WRKY transcription factor under salt stress in Arabidopsis thaliana. Loss-of-function atclcf seedlings show increased sensitivity to salt, whereas AtCLCf overexpression confers enhanced resistance to salt stress. Salt stress induces the translocation of GFP-AtCLCf fusion protein to the plasma membrane (PM). Blocking AtCLCf translocation using the exocytosis inhibitor brefeldin-A or mutating the small GTPase gene AtRABA1b/BEX5 (RAS GENES FROM RAT BRAINA1b homolog) increases salt sensitivity in plants. Electrophysiology and liposome-based assays confirm the Cl-/H+ antiport function of AtCLCf. Therefore, we have uncovered a mechanism of plant adaptation to salt stress involving the NaCl-induced translocation of AtCLCf to the PM, thus facilitating Cl- removal at the roots, and increasing the plant's salinity tolerance.

Arabidopsis class II TPS controls root development and confers salt stress tolerance through enhanced hydrophobic barrier deposition.

KEY MESSAGE: The mechanism of conferring salt tolerance by AtTPS9 involves enhanced deposition of suberin lamellae in the Arabidopsis root endodermis, resulting in reduction of Na+ transported to the leaves. Members of the class I trehalose-6-phosphate synthase (TPS) enzymes are known to play an important role in plant growth and development in Arabidopsis. However, class II TPSs and their functions in salinity stress tolerance are not well studied. We characterized the function of a class II TPS gene, AtTPS9, to understand its role in salt stress response and root development in Arabidopsis. The attps9 mutant exhibited significant reduction of soluble sugar levels in the leaves and formation of suberin lamellae (SL) in the endodermis of roots compared to the wild type (WT). The reduction in SL deposition (hydrophobic barriers) leads to increased apoplastic xylem loading, resulting in enhanced Na+ content in the plants, which explains salt sensitivity of the mutant plants. Conversely, AtTPS9 overexpression lines exhibited increased SL deposition in the root endodermis along with increased salt tolerance, showing that regulation of SL deposition is one of the mechanisms of action of AtTPS9 in conferring salt tolerance to Arabidopsis plants. Our data showed that besides salt tolerance, AtTPS9 also regulates seed germination and root development. qRT-PCR analyses showed significant downregulation of selected SNF1-RELATED PROTEIN KINASE2 genes (SnRK2s) and ABA-responsive genes in the mutant, suggesting that AtTPS9 may regulate the ABA-signaling intermediates as part of the mechanism conferring salinity tolerance.

All-organic transparent plant e-skin for noninvasive phenotyping.

Real-time in situ monitoring of plant physiology is essential for establishing a phenotyping platform for precision agriculture. A key enabler for this monitoring is a device that can be noninvasively attached to plants and transduce their physiological status into digital data. Here, we report an all-organic transparent plant e-skin by micropatterning poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) on polydimethylsiloxane (PDMS) substrate. This plant e-skin is optically and mechanically invisible to plants with no observable adverse effects to plant health. We demonstrate the capabilities of our plant e-skins as strain and temperature sensors, with the application to Brassica rapa leaves for collecting corresponding parameters under normal and abiotic stress conditions. Strains imposed on the leaf surface during growth as well as diurnal fluctuation of surface temperature were captured. We further present a digital-twin interface to visualize real-time plant surface environment, providing an intuitive and vivid platform for plant phenotyping.

High-affinity potassium transporter from a mangrove tree Avicennia officinalis increases salinity tolerance of Arabidopsis thaliana.

Salinity reduces the growth and productivity of crop plants worldwide. Mangroves have evolved efficient ion homeostasis mechanisms to survive under their natural saline growth habitat. Information obtained from them may be utilized for increasing the salt tolerance of crop plants. We identified and characterized a high-affinity potassium transporter gene (AoHKT1) from Avicennia officinalis. The expression of AoHKT1 was induced by NaCl mainly in the leaves. Functional study by heterologous expression of AoHKT1 in Arabidopsis T-DNA insertional mutants athkt1-1 and athkt1-4 revealed that it could enhance the salt tolerance of the mutant plants. This was accompanied by an increase in K+ accumulation in the leaves. AoHKT1 was localized to the plasma membrane in Arabidopsis, and when expressed in yeast, it could complement the functions of both Na+ and K+ transporters. An attempt was made to identify the upstream regulator of AtHKT1, a close homolog of AoHKT1. Using chromatin immunoprecipitation, luciferase assay and yeast one-hybrid assays, WRKY9 was identified as the main transcription factor in the process. Furthermore, this was corroborated by the observation that AtHKT1 levels were significantly reduced in the atwrky9 seedlings. These findings revealed a part of the molecular regulatory mechanism of HKT1 induction in response to salt treatment in Arabidopsis. Our study suggests that AoHKT1 is a potential candidate for generating crop plants with increased salt tolerance.

Identification of two QTLs, BPH41 and BPH42, and their respective gene candidates for brown planthopper resistance in rice.

The brown planthopper (BPH) is the leading cause of insect damage to rice plants and BPH infestations have caused profound losses in rice production since the 1970's. There is an urgent need to discover new BPH resistance genes to ensure the successful production of rice. Here, a new BPH resistance source provided by SeedWorks International Pvt. Ltd., SWD10, was used for this purpose. QTL mapping using 232 F2 progenies and 216 polymorphic markers revealed two dominant BPH resistance QTLs, BPH41 and BPH42, located on chromosome 4. BPH resistance mechanism test revealed that antibiosis and antixenosis mechanisms both play a role in BPH resistance conferred by these two QTLs. The QTLs were delimited between markers SWRm_01617 and SWRm_01522 for BPH41, and SWRm_01695 and SWRm_00328 for BPH42. Additionally, using RNA-seq data of lines containing the resistant QTLs, we shortlisted four and three gene candidates for BPH41 and BPH42, respectively. Differential gene expression analysis of lines containing the QTLs suggested that SWD10 BPH resistance is contributed by the plant's innate immunity and the candidate genes may be part of the rice innate immunity pathway. Currently, the newly identified QTLs are being utilized for breeding BPH resistant rice varieties and hybrids.

Regulation of primary seed dormancy by MAJOR LATEX PROTEIN-LIKE PROTEIN329 in Arabidopsis is dependent on DNA-BINDING ONE ZINC FINGER6.

Seeds exhibit primary dormancy to prevent germination under unfavourable conditions. Previous studies have shown that the gibberellin signalling intermediate RGA-LIKE2 (RGL2) forms a transcription factor complex with DNA-BINDING ONE ZINC FINGER6 (DOF6) in regulating seed dormancy in Arabidopsis. Using an RNA-sequencing approach, we identified MAJOR LATEX PROTEIN-LIKE PROTEIN329 (MLP329) as a downstream target of DOF6. MLP329 was found to be a positive regulator of primary seed dormancy, because freshly harvested unstratified mlp329 mutant seeds showed early germination, while unstratified transgenic seeds overexpressing MLP329 showed poor germination. MLP329 expression level was reduced in wild-type seeds upon dry storage and cold stratification. MLP329 expression level was enhanced by DOF6; however, DOF6-dependent MLP329 expression was suppressed in the presence of RGL2. MLP329 expression was enhanced in seeds treated with ABA and auxin IAA. Moreover, the mlp329 mutant seeds exhibited enhanced expression of the GA biosynthetic gene GA1 and suppression of the ABA biosynthetic gene ZEP compared to the overexpression lines. The observed suppression of DOF6-dependent MLP329 expression by RGL2 reveals a possible negative feedback mechanism to modulate seed dormancy. MLP329 also probably enhances the endogenous ABA/GA ratio to positively regulate primary seed dormancy.

Systems-based rice improvement approaches for sustainable food and nutritional security.

KEY MESSAGE: An integrated research approach to ensure sustainable rice yield increase of a crop grown by 25% of the world's farmers in 10% of cropland is essential for global food security. Rice, being a global staple crop, feeds about 56% of the world population and sustains 40% of the world's poor. At ~ $200 billion, it also accounts for 13% of the annual crop value. With hunger and malnutrition rampant among the poor, rice research for development is unique in global food and nutrition security. A systems-based, sustainable increase in rice quantity and quality is imperative for environmental and biodiversity benefits. Upstream 'discovery' through biotechnology, midstream 'development' through breeding and agronomy, downstream 'dissemination and deployment' must be 'demand-driven' for 'distinct socio-economic transformational impacts'. Local agro-ecology and livelihood nexus must drive the research agenda for targeted benefits. This necessitates sustained long-term investments by government, non-government and private sectors to secure the future food, nutrition, environment, prosperity and equity status.

Ethylene-Mediated Modulation of Bud Phenology, Cold Hardiness, and Hormone Biosynthesis in Peach (Prunus persica).

Spring frosts exacerbated by global climate change have become a constant threat to temperate fruit production. Delaying the bloom date by plant growth regulators (PGRs) has been proposed as a practical frost avoidance strategy. Ethephon is an ethylene-releasing PGR found to delay bloom in several fruit species, yet its use is often coupled with harmful effects, limiting its applicability in commercial tree fruit production. Little information is available regarding the mechanisms by which ethephon influences blooming and bud dormancy. This study investigated the effects of fall-applied ethephon on bud phenology, cold hardiness, and hormonal balance throughout the bud dormancy cycle in peach. Our findings concluded that ethephon could alter several significant aspects of peach bud physiology, including accelerated leaf fall, extended chilling accumulation period, increased heat requirements, improved cold hardiness, and delayed bloom date. Ethephon effects on these traits were primarily dependent on its concentration and application timing, with a high concentration (500 ppm) and an early application timing (10% leaf fall) being the most effective. Endogenous ethylene levels were induced significantly in the buds when ethephon was applied at 10% versus 90% leaf fall, indicating that leaves are essential for ethephon uptake. The hormonal analysis of buds at regular intervals of chilling hours (CH) and growing degree hours (GDH) also indicated that ethephon might exert its effects through an abscisic acid (ABA)-independent way in dormant buds. Instead, our data signifies the role of jasmonic acid (JA) in mediating budburst and bloom in peach, which also appears to be influenced by ethephon treatment. Overall, this research presents a new perspective in interpreting horticultural traits in the light of biochemical and molecular data and sheds light on the potential role of JA in bud dormancy, which deserves further attention in future studies that aim at mitigating spring frosts.

WRKY9 transcription factor regulates cytochrome P450 genes CYP94B3 and CYP86B1, leading to increased root suberin and salt tolerance in Arabidopsis.

Salinity affects crop productivity worldwide and mangroves growing under high salinity exhibit adaptations such as enhanced root apoplastic barrier to survive under such conditions. We have identified two cytochrome P450 family genes, AoCYP94B3 and AoCYP86B1 from the mangrove tree Avicennia officinalis and characterized them using atcyp94b3 and atcyp86b1, which are mutants of their putative Arabidopsis orthologs and the corresponding complemented lines with A. officinalis genes. CYP94B3 and CYP86B1 transcripts were induced upon salt treatment in the roots of both A. officinalis and Arabidopsis. Both AoCYP94B3 and AoCYP86B1 were localized to the endoplasmic reticulum. Heterologous expression of 35S::AoCYP94B3 and 35S::AoCYP86B1 in their respective Arabidopsis mutants (atcyp94b3 and atcyp86b1) increased the salt tolerance of the transgenic seedlings by reducing the amount of Na+ accumulation in the shoots. Moreover, the reduced root suberin phenotype of atcyp94b3 was rescued in the 35S::AoCYP94B3;atcyp94b3 transgenic Arabidopsis seedlings. Gas-chromatography and mass spectrometry analyses showed that the amount of suberin monomers (C-16 ω-hydroxy acids, C-16 α, ω-dicarboxylic acids and C-20 eicosanol) were increased in the roots of 35S::AoCYP94B3;atcyp94b3 Arabidopsis seedlings. Using chromatin immunoprecipitation and electrophoretic mobility shift assays, we identified AtWRKY9 as the upstream regulator of AtCYP94B3 and AtCYP86B1 in Arabidopsis. In addition, atwrky9 showed suppressed expression of AtCYP94B3 and AtCYP86B1 transcripts, and reduced suberin in the roots. These results show that AtWRKY9 controls suberin deposition by regulating AtCYP94B3 and AtCYP86B1, leading to salt tolerance. Our data can be used for generating salt-tolerant crop plants in the future.

Functional characterization and expression profiling of glyoxalase III genes in date palm grown under abiotic stresses.

Methylglyoxal (MG), a by-product of various metabolic processes, including glycolysis, is a highly reactive cytotoxic metabolite. The level of MG in the cell is maintained at a non-toxic level via MG detoxification pathways such as the universal glyoxalase system, including glyoxalase I/II/III enzymes. Glyoxalase III (DJ-1) can breakdown MG to d-lactate in a single step without reducing glutathione (GSH). Elucidating the function of the DJ-1 gene family may provide further knowledge about its role in plants under abiotic stresses. Here, we characterize four glyoxalase III genes (PdDJ-1B1, PdDJ-1B2, PdDJ-1C, and PdDJ-1D) encoding the conserved DJ-1 domain in the genome of the date palm, a crop with high drought and salinity tolerance. The expression level of the PdDJ-1 genes increased in date palm leaves upon salinity treatment. In addition, overexpression of PdDJ-1 genes in Escherichia coli and the complementation in yeast hsp31Δ knockout mutant cells enhanced their growth rate and reduced the accumulation of reactive oxygen species (ROS) under MG and oxidative stress conditions as shown by the flow cytometry assay. Subcellular localization using confocal microscopy revealed the accumulation of PdDJ-1B1, PdDJ-1C, and PdDJ-1D in the chloroplast, whereas PdDJ-1B2 was localized to the cytosol. Remarkably, constitutive expression of the PdDJ-1C gene in Arabidopsis thaliana Columbia (Col-0) resulted in the generation of non-viable albino plants implying that PdDJ-1C plays a critical function in chloroplast development. These findings suggest that PdDJ-1 protein has an important function in MG-detoxification and maintaining the redox balance in date palm plants under abiotic stress conditions.

Regulation of AtKUP2 Expression by bHLH and WRKY Transcription Factors Helps to Confer Increased Salt Tolerance to Arabidopsis thaliana Plants.

Potassium transporters play an essential role in maintaining cellular ion homeostasis, turgor pressure, and pH, which are critical for adaptation under salt stress. We identified a salt responsive Avicennia officinalis KUP/HAK/KT transporter family gene, AoKUP2, which has high sequence similarity to its Arabidopsis ortholog AtKUP2. These genes were functionally characterized in mutant yeast cells and Arabidopsis plants. Both AoKUP2 and AtKUP2 were induced by salt stress, and AtKUP2 was primarily induced in roots. Subcellular localization revealed that AoKUP2 and AtKUP2 are localized to the plasma membrane and mitochondria. Expression of AtKUP2 and AoKUP2 in Saccharomyces cerevisiae mutant strain (BY4741 trk1Δ::loxP trk2Δ::loxP) helped to rescue the growth defect of the mutant under different NaCl and K+ concentrations. Furthermore, constitutive expression of AoKUP2 and AtKUP2 conferred enhanced salt tolerance in Arabidopsis indicated by higher germination rate, better survival, and increased root and shoot length compared to the untreated controls. Analysis of Na+ and K+ contents in the shoots and roots showed that ectopic expression lines accumulated less Na+ and more K+ than the WT. Two stress-responsive transcription factors, bHLH122 and WRKY33, were identified as direct regulators of AtKUP2 expression. Our results suggest that AtKUP2 plays a key role in enhancing salt stress tolerance by maintaining cellular ion homeostasis.

Regulation of a Cytochrome P450 Gene CYP94B1 by WRKY33 Transcription Factor Controls Apoplastic Barrier Formation in Roots to Confer Salt Tolerance.

Salinity is an environmental stress that causes decline in crop yield. Avicennia officinalis and other mangroves have adaptations such as ultrafiltration at the roots aided by apoplastic cell wall barriers to thrive in saline conditions. We studied a cytochrome P450 gene from A. officinalis, AoCYP94B1, and its putative ortholog in Arabidopsis (Arabidopsis thaliana), AtCYP94B1, which are involved in apoplastic barrier formation. Both genes were induced by 30 min of salt treatment in the roots. Heterologous expression of AoCYP94B1 in the atcyp94b1 Arabidopsis mutant and wild-type rice (Oryza sativa) conferred increased NaCl tolerance to seedlings by enhancing root suberin deposition. Histochemical staining and gas chromatography-tandem mass spectrometry quantification of suberin precursors confirmed the role of CYP94B1 in suberin biosynthesis. Using chromatin immunoprecipitation and yeast one-hybrid and luciferase assays, we identified AtWRKY33 as the upstream regulator of AtCYP94B1 in Arabidopsis. In addition, atwrky33 mutants exhibited reduced suberin and salt-sensitive phenotypes, which were rescued by expressing 35S::AtCYP94B1 in the atwrky33 background. This further confirmed that AtWRKY33-mediated regulation of AtCYP94B1 is part of the salt tolerance mechanism. Our findings may help efforts aimed at generating salt-tolerant crops.

Molecular Characterization of a Date Palm Vascular Highway 1-Interacting Kinase (PdVIK) Under Abiotic Stresses.

The date palm (Khalas) is an extremophile plant that can adapt to various abiotic stresses including drought and salinity. Salinity tolerance is a complex trait controlled by numerous genes. Identification and functional characterization of salt-responsive genes from the date palm is fundamental to understand salinity tolerance at the molecular level in this plant species. In this study, a salt-inducible vascular highway 1-interacting kinase (PdVIK) that is a MAP kinase kinase kinase (MAPKKK) gene from the date palm, was functionally characterized using in vitro and in vivo strategies. PdVIK, one of the 597 kinases encoded by the date palm genome possesses an ankyrin repeat domain and a kinase domain. The recombinant PdVIK protein exhibited phosphotyrosine activity against myelin basic protein (MBP) substrate. Overexpression of PdVIK in yeast significantly improved its tolerance to salinity, LiCl, and oxidative stresses. Transgenic Arabidopsis seedlings overexpressing PdVIK displayed improved tolerance to salinity, osmotic, and oxidative stresses as assessed by root growth assay. The transgenic lines grown in the soil also displayed modulated salt response, compared to wild-type controls as evaluated by the overall plant growth and proline levels. Likewise, the transgenic lines exhibited drought tolerance by maintaining better relative water content (RWC) compared to non-transgenic control plants. Collectively, these results implicate the involvement of PdVIK in modulating the abiotic stress response of the date palm.

A novel tonoplast Na+/H+ antiporter gene from date palm (PdNHX6) confers enhanced salt tolerance response in Arabidopsis.

KEY MESSAGE: A sodium hydrogen exchanger (NHX) gene from the date palm enhances tolerance to salinity in Arabidopsis plants. Plant sodium hydrogen exchangers/antiporters (NHXs) are pivotal regulators of intracellular Na+/K+ and pH homeostasis, which is essential for salt stress adaptation. In this study, a novel orthologue of Na+/H+ antiporter was isolated from date palm (PdNHX6) and functionally characterized in mutant yeast cells and Arabidopsis plants to assess the behavior of the transgenic organisms in response to salinity. Genetically transformed yeast cells with PdNHX6 were sensitive to salt stress when compared to the empty vector (EV) yeast cells. Besides, the acidity value of the vacuoles of the transformant yeast cells has significantly (p ≤ 0.05) increased, as indicated by the calibrated fluorescence intensity measurements and the fluorescence imagining analyses. This observation supports the notion that PdNHX6 might regulate proton pumping into the vacuole, a crucial salt tolerance mechanism in the plants. Consistently, the transient overexpression and subcellular localization revealed the accumulation of PdNHX6 in the tonoplast surrounding the central vacuole of Nicotiana benthamiana leaf epidermal cells. Stable overexpression of PdNHX6 in Arabidopsis plants enhanced tolerance to salt stress and retained significantly higher chlorophyll, water contents, and increased seed germination under salinity when compared to the wild-type plants. Despite the significant increase of Na+, transgenic Arabidopsis lines maintained a balanced Na+/K+ ratio under salt stress conditions. Together, the results obtained from this study imply that PdNHX6 is involved in the salt tolerance mechanism in plants by controlling K+ and pH homeostasis of the vacuoles.

An LRR-only protein regulates abscisic acid-mediated abiotic stress responses during Arabidopsis seed germination.

KEY MESSAGE: LRRop-1, induced by DOF6 transcription factor, negatively regulates abiotic stress responses during Arabidopsis seed germination. The lrrop-1 mutant has reduced ABA signaling, which is part of the underlying stress-remediation mechanism. The large family of leucine-rich repeat (LRR) proteins plays a role in plant immune responses. Most LRR proteins have multiple functional domains, but a subfamily is known to possess only the LRR domain. The roles of these LRR-only proteins in Arabidopsis remain largely uncharacterized. In the present study, we have identified 44 LRR-only proteins in Arabidopsis and phylogenetically classified them into nine sub-groups. We characterized the function of LRRop-1, belonging to sub-group V. LRRop-1 encodes a predominantly ER-localized LRR domain-containing protein that is highly expressed in seeds and rosette leaves. Promoter motif analysis revealed an enrichment in binding sites for several GA-responsive and stress-responsive transcription factors. The lrrop-1 mutant seeds showed enhanced seed germination on medium containing abscisic acid (ABA), paclobutrazol and NaCl compared to the wild type (WT), demonstrating higher abiotic stress tolerance. Also, the lrrop-1 mutant seeds have lower levels of endogenous ABA, but higher levels of gibberellic acid (GA) and jasmonic acid-Ile (JA-Ile) compared to the WT. Furthermore, lrrop-1 mutant seeds imbibed with ABA exhibited reduced expression of ABA-responsive genes compared to similarly treated WT seeds, suggesting suppressed ABA signaling events in the mutant. Furthermore, chromatin immunoprecipitation (ChIP) data showed that DNA BINDING1 ZINC FINGER6 (DOF6), a negative regulator of seed germination, could directly bind to the LRRop-1 promoter and up-regulate its expression. Thus, our results show that LRRop-1 regulates ABA-mediated abiotic stress responses during Arabidopsis seed germination.

Systems Metabolic Alteration in a Semi-Dwarf Rice Mutant Induced by OsCYP96B4 Gene Mutation.

Dwarfism and semi-dwarfism are among the most valuable agronomic traits in crop breeding, which were adopted by the "Green Revolution". Previously, we reported a novel semi-dwarf rice mutant (oscyp96b4) derived from the insertion of a single copy of Dissociator (Ds) transposon into the gene OsCYP96B4. However, the systems metabolic effect of the mutation is not well understood, which is important for understanding the gene function and developing new semi-dwarf mutants. Here, the metabolic phenotypes in the semi-dwarf mutant (M) and ectopic expression (ECE) rice line were compared to the wild-type (WT) rice, by using nuclear magnetic resonance (NMR) metabolomics and quantitative real-time polymerase chain reaction (qRT-PCR). Compared with WT, ECE of the OsCYP96B4 gene resulted in significant increase of γ-aminobutyrate (GABA), glutamine, and alanine, but significant decrease of glutamate, aromatic and branched-chain amino acids, and some other amino acids. The ECE caused significant increase of monosaccharides (glucose, fructose), but significant decrease of disaccharide (sucrose); induced significant changes of metabolites involved in choline metabolism (phosphocholine, ethanolamine) and nucleotide metabolism (adenosine, adenosine monophosphate, uridine). These metabolic profile alterations were accompanied with changes in the gene expression levels of some related enzymes, involved in GABA shunt, glutamate and glutamine metabolism, choline metabolism, sucrose metabolism, glycolysis/gluconeogenesis pathway, tricarboxylic acid (TCA) cycle, nucleotide metabolism, and shikimate-mediated secondary metabolism. The semi-dwarf mutant showed corresponding but less pronounced changes, especially in the gene expression levels. It indicates that OsCYP96B4 gene mutation in rice causes significant alteration in amino acid metabolism, carbohydrate metabolism, nucleotide metabolism, and shikimate-mediated secondary metabolism. The present study will provide essential information for the OsCYP96B4 gene function analysis and may serve as valuable reference data for the development of new semi-dwarf mutants.

Expression of AoNHX1 increases salt tolerance of rice and Arabidopsis, and bHLH transcription factors regulate AtNHX1 and AtNHX6 in Arabidopsis.

KEY MESSAGE: Expression of AoNHX1 from the mangrove Avicennia increases salt tolerance of rice and Arabidopsis, and specific bHLH transcription factors regulate AtNHX1 and AtNHX6 in Arabidopsis to mediate the salinity response. Improving crop plants to better tolerate soil salinity is a challenging task. Mangrove trees such as Avicennia officinalis have special adaptations to thrive in high salt conditions, which include subcellular compartmentalization of ions facilitated by specialized ion transporters. We identified and characterized two genes encoding Na+/H+ exchangers AoNHX1 and AoNHX6 from Avicennia. AoNHX1 was present in the tonoplast, while, AoNHX6 was localized to the ER and Golgi. Both NHXs were induced by NaCl treatment, with AoNHX1 showing high expression levels in the leaves and AoNHX6 in the seedling roots. Yeast deletion mutants (ena1-5Δ nha1Δ nhx1Δ and ena1-5Δ nha1Δ vnx1Δ) complemented with AoNHX1 and AoNHX6 showed increased tolerance to both NaCl and KCl. Expression of AoNHX1 and AoNHX6 in the corresponding Arabidopsis mutants conferred enhanced NaCl tolerance. The underlying molecular regulatory mechanism was investigated using AtNHX1 and AtNHX6 in Arabidopsis. We identified two basic helix-loop-helix (bHLH) transcription factors AtMYC2 and AtbHLH122 as the ABA-mediated upstream regulators of AtNHX1 and AtNHX6 by chromatin immunoprecipitation. Furthermore, expression of AtNHX1 and AtNHX6 transcripts was reduced in the atmyc2 and atbhlh122 mutants. Lastly, transgenic rice seedlings harboring pUBI::AoNHX1 showed enhanced salt tolerance, suggesting that this gene can be exploited for developing salt-tolerant crops.

OsTPS8 controls yield-related traits and confers salt stress tolerance in rice by enhancing suberin deposition.

Class I TREHALOSE-PHOSPHATE-SYNTHASE (TPS) genes affect salinity tolerance and plant development. However, the function of class IITPS genes and their underlying mechanisms of action are unknown. We report the identification and functional analysis of a rice class IITPS gene (OsTPS8). The ostps8 mutant was characterised by GC-MS analysis, an abscisic acid (ABA) sensitivity test and by generating transgenic lines. To identify the underlying mechanism, gene expression analyses, genetic complementation and examination of suberin deposition in the roots were conducted. The ostps8 mutant showed salt sensitivity, ABA sensitivity and altered agronomic traits compared to the wild-type (WT), which could be rescued upon complementation. The dsRNAi line phenocopied the mutant, while the overexpression lines exhibited enhanced salt tolerance. The ostps8 mutant showed significantly reduced soluble sugars, Casparian bands and suberin deposition in the roots compared to the WT and overexpression lines. The mutant also showed downregulation of SAPKs (rice SnRK2s) and ABA-responsive genes. Furthermore, ostps8pUBI::SAPK9 rescued the salt-sensitive phenotype of ostps8. Our results suggest that OsTPS8 may regulate suberin deposition in rice through ABA signalling. Additionally, SAPK9-mediated regulation of altered ABA-responsive genes helps to confer salinity tolerance. Overexpression of OsTPS8 was adequate to confer enhanced salinity tolerance without any yield penalty, suggesting its usefulness in rice genetic improvement.