Genome-Wide Identification, Primary Functional Characterization of the NHX Gene Family in Canavalia rosea, and Their Possible Roles for Adaptation to Tropical Coral Reefs

Canavalia rosea, distributed in the coastal areas of tropical and subtropical regions, is an extremophile halophyte with good adaptability to high salinity/alkaline and drought tolerance. Plant sodium/hydrogen (Na+/H+) exchanger (NHX) genes encode membrane transporters involved in sodium ion (Na+), potassium ion (K+), and lithium ion (Li+) transport and pH homeostasis, thereby playing key roles in salinity tolerance. However, the NHX family has not been reported in this leguminous halophyte. In the present study, a genome-wide comprehensive analysis was conducted and finally eight CrNHXs were identified in C. rosea genome. Based on the bioinformatics analysis about the chromosomal location, protein domain, motif organization, and phylogenetic relationships of CrNHXs and their coding proteins, as well as the comparison with plant NHXs from other species, the CrNHXs were grouped into three major subfamilies (Vac-, Endo-, and PM-NHX). Promoter analyses of cis-regulatory elements indicated that the expression of different CrNHXs was affected by a series of stress challenges. Six CrNHXs showed high expression levels in five tested tissues of C. rosea in different levels, while CrNHX1 and CrNHX3 were expressed at extremely low levels, indicating that CrNHXs might be involved in regulating the development of C. rosea plant. The expression analysis based on RNA-seq showed that the transcripts of most CrNHXs were obviously decreased in mature leaves of C. rosea plant growing on tropical coral reefs, which suggested their involvement in this species’ adaptation to reefs and specialized islands habitats. Furthermore, in the single-factor stress treatments mimicking the extreme environments of tropical coral reefs, the RNA-seq data also implied CrNHXs holding possible gene-specific regulatory roles in the environmental adaptation. The qRT-PCR based expression profiling exhibited that CrNHXs responded to different stresses to varying degrees, which further confirmed the specificity of CrNHXs’ in responding to abiotic stresses. Moreover, the yeast functional complementation test proved that some CrNHXs could partially restore the salt tolerance of the salt-sensitive yeast mutant AXT3. This study provides comprehensive bio-information and primary functional identification of NHXs in C. rosea, which could help improve the salt/alkaline tolerance of genetically modified plants for further studies. This research also contributes to our understanding of the possible molecular mechanism whereby NHXs maintain the ion balance in the natural ecological adaptability of C. rosea to tropical coral islands and reefs.

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli . By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we mapped the topology of the protein. Using these data, we experimentally validated a structural model of YqjA generated using evolutionary covariance, which consists of an α-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggest that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

Isolation, Characterization and Acid/Alkaline Tolerance of Lactic Acid Bacteria Present in Fermented Rye, Wheat, Oat and Barley

Objective: This study determined the lactic acid bacteria (LAB) present in fermented rye, wheat, oat and barley grains, and evaluated their survival in simulated gastric juice and pancreatic juice. Methods: Samples of rye, wheat, oat and barley grains were fermented for 72 hours at room temperature. Lactic acid bacteria (LAB) strains were isolated using MRS agar and were enumerated. Isolated LAB strains were cultured with MRS broth and the fermentation patterns of the isolated strains were characterized using API 50 CH kit (Biomerieux, France). Each isolated LAB strain was exposed to simulated gastric juice at pH of 2.0 for 80 minutes at 370C, followed by exposure to simulated pancreatic juice at pH of 8.0 for 120 minutes at 370C. Aliquots were taken at 0 minute and 80 minutes at pH of 2.0 and 0 minutes and 120 minutes at pH of 8.0 for enumeration of LAB strains. Results: The total LAB cell count ranged from 6.6 * 108 ± 11 cfu/ml in the rye sample to 9.5*109 ± 7 cfu/ml in the oat sample. 13 LAB strains were isolated from the four selected cereal grains and were characterized as six strains of Lactobacillus plantarum1, five strains of L. brevis 1 and one strain each of L. collinoides and Leuconostoc citreum. All the isolated LAB strains from the four selected cereals survived in the simulated gastric juice at pH of 2.0 (before and after incubation at 0min and 80a min) and after addition of simulated pancreatic juice at pH of 8.0 (before and after incubation at 80b min and 200 min respectively). The mean viable counts of all the strains ranged from 2.0 *108 in R3 at 80b min to 1.54 * 1010 in B4 at 80b minutes. Conclusion: LAB associated with fermentation of rye, wheat, oat and barley grains are likely to survive transport through the harsh acidic and alkaline conditions of the GIT.

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance.

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake, and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we have performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli. By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we have mapped the topology of the protein. Using these data, we have experimentally validated a structural model of YqjA generated using evolutionary co-variance, which consists of an a-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggests that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

Abscisic Acid Priming Creates Alkaline Tolerance in Alfalfa Seedlings (Medicago sativa L.)

Soil alkalization triggers ion toxicity and osmotic and alkaline (high pH) stresses in plants, damaging their growth and productivity. Therefore, we investigated whether priming with abscisic acid (ABA) increases the tolerance of alfalfa seedlings to alkaline stress, and then examined the underlying molecular mechanisms. Alfalfa seedlings were pretreated with ABA (10 μM) for 16 h and then subjected to alkaline stress using a 15 mM Na2CO3 solution (pH 10.87). Compared with the control, ABA pretreatment significantly alleviated leaf damage and improved the fresh weight, water content, and survival rate of alfalfa seedlings under alkaline conditions. Abscisic acid pretreatment reduced accumulation of reactive oxygen species (ROS), increased activities of the antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD), maintained higher ratios of K+/Na+, Ca2+/Na+, and Mg2+/Na+, and increased accumulation of proline. In addition, ABA upregulated the expression of genes involved in proline biosynthesis (P5CS) and the sequestration of Na+ in vacuoles (NHX1 and AVP) under alkaline conditions. Abscisic acid priming increased tolerance to alkaline stress by maintaining homeostasis of ROS and metal ions and upregulating osmoprotection and the expression of stress tolerance-related genes.

Saline–alkaline stress in growing maize seedlings is alleviated by Trichoderma asperellum through regulation of the soil environment

A significant proportion of the land area of Heilongjiang Province, China, is composed of saline–alkaline soil, which severely inhibits maize growth. Although Trichoderma treatment is widely regarded as a promising strategy for improving the soil environment and promoting plant growth, the mechanism through which Trichoderma asperellum enhances maize resistance to saline–alkaline stress is not clear. In this study, we explored the effect of T. asperellum application at different concentrations to soil saline–alkaline environment on the seedlings of two maize cultivars, assessing the biochemical parameters related to oxidation resistance. Increasing spore densities of T. asperellum suspension effectively regulated the soil ion balance in the rhizosphere of maize seedlings, reduced the soil pH by 2.15–5.76% and sodium adsorption ratios by 22.70–54.13%, increased soil nutrient content and enzyme activity, and improved the soil environment for seedling growth. Additionally, T. asperellum treatment increased the maize seedling content of osmo-regulating substances and rate of glutathione:oxidised glutathione (43.86–88.25%) and ascorbate:oxidised ascorbate (25.26–222.32%) by affecting the antioxidant enzyme activity in the roots, increasing reactive oxygen species scavenging, and maintaining the osmotic balance and metabolic homeostasis under saline–alkaline stress. T. asperellum also improved the saline–alkaline tolerance of maize seedlings by improving the root growth characteristics. Moreover, results showed that Trichoderma applied at high concentration had the greatest effect. In conclusion, improvement in the saline–alkaline tolerance of maize seedlings by T. asperellum under saline–alkaline soil conditions may be achieved through diverse effects that vary among maize cultivars.

Physiological and transcriptomic analyses reveal novel insights into the cultivar-specific response to alkaline stress in alfalfa (Medicago sativa L.)

Key message Candidate pathways for alkaline tolerance in alfalfa seedlings were identified; these included those for homeostasis of ions and redox status, biosynthesis of phenylpropanoids, flavonoids, and amino acids, and MAPK signaling.Abstract Soil alkalization severely limits plant growth and development; however, the mechanisms of alkaline response remain largely unknown. In this study, we performed physiological and transcriptomic analyses using two alfalfa cultivars (Medicago sativa L.) with different sensitivities to alkaline conditions. The chlorophyll content and shoot fresh weight drastically declined in the alkaline-sensitive cultivar Algonquin (AG) following alkaline treatment (0-25 mM Na2CO3 solution), while the alkaline-tolerant cultivar Gongnong NO.1 (GN) maintained relatively stable growth and chlorophyll content. Physiological analysis revealed that compared with AG, GN had higher contents of Ca2+ and Mg2+; the ratios of Ca2+ and Mg2+ to Na+, proline and soluble sugar, and enzyme activities of peroxidase (POD) and catalase (CAT) decreased under the alkaline conditions. Further, transcriptomic analysis identified three categories of alkaline-responsive differentially expressed genes (DEGs) between the two cultivars: 48 genes commonly induced in both the cultivars (CAR), 574 genes from the tolerant cultivar (TAR), and 493 genes from the sensitive cultivar (SAR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that CAR genes were mostly involved in phenylpropanoid biosynthesis, lipid metabolism, and DNA replication and repair; TAR genes were significantly enriched in metabolic pathways, biosynthesis of secondary metabolites, MAPK signaling pathway, and flavonoid and amino acid biosynthesis; the SAR genes were specifically enriched in vitamin B6 metabolism. Taken together, the results identified candidate pathways associated with genetic variation in response to alkaline stress, providing novel insights into the mechanisms underlying alkaline tolerance in alfalfa.

Transcription Factor ChbZIP1 from Alkaliphilic Microalgae Chlorella sp. BLD Enhancing Alkaline Tolerance in Transgenic Arabidopsis thaliana

Saline-alkali soil has become an important environmental problem for crop productivity. One of the most effective approaches is to cultivate new stress-tolerant plants through genetic engineering. Through RNA-seq analysis and RT-PCR validation, a novel bZIP transcription factor ChbZIP1, which is significantly upregulated at alkali conditions, was obtained from alkaliphilic microalgae Chlorella sp. BLD. Overexpression of ChbZIP1 in Saccharomyces cerevisiae and Arabidopsis increased their alkali resistance, indicating ChbZIP1 may play important roles in alkali stress response. Through subcellular localization and transcriptional activation activity analyses, we found that ChbZIP1 is a nuclear-localized bZIP TF with transactivation activity to bind with the motif of G-box 2 (TGACGT). Functional analysis found that genes such as GPX1, DOX1, CAT2, and EMB, which contained G-box 2 and were associated with oxidative stress, were significantly upregulated in Arabidopsis with ChbZIP1 overexpression. The antioxidant ability was also enhanced in transgenic Arabidopsis. These results indicate that ChbZIP1 might mediate plant adaptation to alkali stress through the active oxygen detoxification pathway. Thus, ChbZIP1 may contribute to genetically improving plants’ tolerance to alkali stress.

MdTyDc Overexpression Improves Alkalinity Tolerance in Malus domestica

Tyrosine is decarboxylated to tyramine by TYDC (Tyrosine decarboxylase) and then hydroxylated to dopamine, which is involved in plant response to abiotic stress. However, little is known about the function of MdTyDc in response to alkaline stress in plants. In our study, it was found that the expression of MdTyDc was induced by alkaline stress. Therefore, the apple plants overexpressing MdTyDc was treated with alkali stress, and we found that MdTyDc played an important role in apple plants’ resistance to alkali stress. Our results showed that the restriction on the growth, the decrease of membrane permeability and the accumulation of Na+ were alleviated to various degrees in MdTyDc transgenic plants under alkali stress. In addition, overexpression of MdTyDc enhanced the root activity and photosynthetic capacity, and improved the enzyme activity related to N metabolism, thus promoting N absorption. It is noteworthy that the dopamine content of these three transgenic lines is significantly higher than that of WT. In summary, these findings indicated that MdTyDc may enhance alkaline tolerance of apples by mediating dopamine content, mainly by maintaining high photosynthetic capacity, normal ion homeostasis and strong nitrogen absorption capacity.

Cytoplasmic glucose-6-phosphate dehydrogenase plays an important role in the silicon-enhanced alkaline tolerance in highland barley

Glucose-6-phosphate dehydrogenase (G6PDH), as a key enzyme in the pentose phosphate pathway, extensively responds to the biotic and abiotic stresses. In this study we focussed on the G6PDH role in the alleviation of alkaline stress induced by silicon (Si) in highland barley. Application of Si reduced the water loss and malondialdehyde (MDA) and reactive oxygen species (ROS) contents, improved the fresh weight, photosynthesis, K+ content, and the superoxide dismutase (SOD) and catalase (CAT) activities, thus alleviating the damage caused by alkaline stress. The G6PDH activity, especially the cytoplasmic G6PDH, significantly increased under alkaline stress, and was further stimulated by the addition of exogenous Si. Meanwhile, the levels of NADPH and reduced glutathione (GSH) showed similar profiles to G6PDH activity under NaHCO3 and NaHCO3 + Si treatments. The inhibition of G6PDH activity by glucosamine abolished the relieving effect of Si on alkaline stress, which was manifested in the increase of ROS and the decrease of GSH content. Together, our results suggest that Si-enhanced tolerance of alkaline stress may be related to the regulation of GSH levels by the cytoplasmic G6PDH in highland barley.