Characterization of the Glehnia littoralis Non-specific Phospholipase C Gene GlNPC3 and Its Involvement in the Salt Stress Response

Glehnia littoralis is a medicinal halophyte that inhabits sandy beaches and has high ecological and commercial value. However, the molecular mechanism of salt adaptation in G. littoralis remains largely unknown. Here, we cloned and identified a non-specific phospholipase C gene (GlNPC3) from G. littoralis, which conferred lipid-mediated signaling during the salt stress response. The expression of GlNPC3 was induced continuously by salt treatment. Overexpression of GlNPC3 in Arabidopsis thaliana increased salt tolerance compared to wild-type (WT) plants. GlNPC3-overexpressing plants had longer roots and higher fresh and dry masses under the salt treatment. The GlNPC3 expression pattern revealed that the gene was expressed in most G. littoralis tissues, particularly in roots. The subcellular localization of GlNPC3 was mainly at the plasma membrane, and partially at the tonoplast. GlNPC3 hydrolyzed common membrane phospholipids, such as phosphotidylserine (PS), phosphoethanolamine (PE), and phosphocholine (PC). In vitro enzymatic assay showed salt-induced total non-specific phospholipase C (NPC) activation in A. thaliana GlNPC3-overexpressing plants. Plant lipid profiling showed a significant change in the membrane-lipid composition of A. thaliana GlNPC3-overexpressing plants compared to WT after the salt treatment. Furthermore, downregulation of GlNPC3 expression by virus-induced gene silencing in G. littoralis reduced the expression levels of some stress-related genes, such as SnRK2, P5SC5, TPC1, and SOS1. Together, these results indicated that GlNPC3 and GlNPC3-mediated membrane lipid change played a positive role in the response of G. littoralis to a saline environment.

Changes in natural transformation after salt adaptation

The exchange of genes between potentially unrelated bacteria is termed horizontal gene transfer (HGT) and is a driving force in bacterial evolution. Natural transformation is one mechanism of HGT where extracellular DNA (eDNA) from the environment is recombined into a host genome. The widespread conservation of transformation in bacterial lineages implies there is a fitness benefit. However, the nature of these benefits and the evolutionary origins of transformation are still unknown. Here, I examine how ∼330 generations or 100 days of serial passage in either constant or increasing salinities impacts the growth rate and transformation efficiency of Pseudomonas stutzeri. While the growth rate generally improved in response to serial transfer, the transformation efficiency of the evolved lineages varied extensively, with only 39-64% of populations undergoing transformation at the end of adaptive evolution. In comparison, 100% of the ancestral populations were able to undergo natural transformation. I also found that evolving P. stutzeri with different cell lysates (or populations of dead cells) minimally affected the growth rate and transformation efficiency, especially in comparison to the pervasiveness with which transformation capacity was lost across the evolved populations. Taken together, I show that the efficiency of eDNA uptake changes over relatively rapid timescales, suggesting that transformation is an adaptive and selectable trait that could be lost in environments where it is not beneficial.

Non-CG DNA methylation-deficiency mutations enhance mutagenesis rates during salt adaptation in cultured Arabidopsis cells

Much has been learned about how plants acclimate to stressful environments, but the molecular basis of stress adaptation and the potential involvement of epigenetic regulation remain poorly understood. Here, we examined if salt stress induces mutagenesis in suspension cultured plant cells and if DNA methylation affects the mutagenesis using whole genome resequencing analysis. We generated suspension cell cultures from two Arabidopsis DNA methylation-deficient mutants and wild-type plants, and subjected the cultured cells to stepwise increases in salt stress intensity over 40 culture cycles. We show that ddc (drm1 drm2 cmt3) mutant cells can adapt to grow in 175 mM NaCl-containing growth medium and exhibit higher adaptability compared to wild type Col-0 and nrpe1 cells, which can adapt to grow in only 125 mM NaCl-containing growth medium. Salt treated nrpe1 and ddc cells but not wild type cells accumulate more mutations compared with their respective untreated cells. There is no enrichment of stress responsive genes in the list of mutated genes in salt treated cells compared to the list of mutated genes in untreated cells. Our results suggest that DNA methylation prevents the induction of mutagenesis by salt stress in plant cells during stress adaptation.

Comparative Transcriptomics and Metabolomics Reveal an Intricate Priming Mechanism Involved in PGPR-Mediated Salt Tolerance in Tomato

Plant-associated beneficial strains inhabiting plants grown under harsh ecosystems can help them cope with abiotic stress factors by positively influencing plant physiology, development, and environmental adaptation. Previously, we isolated a potential plant growth promoting strain (AXSa06) identified as Pseudomonas oryzihabitans, possessing 1-aminocyclopropane-1-carboxylate deaminase activity, producing indole-3-acetic acid and siderophores, as well as solubilizing inorganic phosphorus. In this study, we aimed to further evaluate the effects of AXSa06 seed inoculation on the growth of tomato seedlings under excess salt (200 mM NaCl) by deciphering their transcriptomic and metabolomic profiles. Differences in transcript levels and metabolites following AXSa06 inoculation seem likely to have contributed to the observed difference in salt adaptation of inoculated plants. In particular, inoculations exerted a positive effect on plant growth and photosynthetic parameters, imposing plants to a primed state, at which they were able to respond more robustly to salt stress probably by efficiently activating antioxidant metabolism, by dampening stress signals, by detoxifying Na+, as well as by effectively assimilating carbon and nitrogen. The primed state of AXSa06-inoculated plants is supported by the increased leaf lipid peroxidation, ascorbate content, as well as the enhanced activities of antioxidant enzymes, prior to stress treatment. The identified signatory molecules of AXSa06-mediated salt tolerance included the amino acids aspartate, threonine, serine, and glutamate, as well as key genes related to ethylene or abscisic acid homeostasis and perception, and ion antiporters. Our findings represent a promising sustainable solution to improve agricultural production under the forthcoming climate change conditions.

Biotechnological Potential of Bacterial Endoglucanase from Marine and Hypersaline Environments in Saccharification of Lignocellulosic Biomass Pre-Treated with Alkali or Ionic Liquid

In second-generation biofuel production, the recalcitrant plant biomass requires pretreatment prior to enzymatic hydrolysis. Pretreatment with alkali or ionic liquids (IL) such as 1-butyl 3-methyl immidazolium chloride ([Bmim][Cl]), are efficient but the residual salt in former, or IL in later process inhibits downstream enzymatic saccharification and thus require extensive washing. Recent studies have established IL tolerance by moderate halophilic bacteria being contributed by their general salt adaptation strategies. Objective of the present study is to examine whether the same holds true for their extracellular enzymes, and eventually select a few for future exploitation. In this direction, ten distinct endoglucanase positive (≥3 mm halo zone in congo-red cellulolytic assay) colonies were picked each from decomposed wood material of the hypersaline Sambhar Lake (SLW), in Rajasthan; and Bichitrapur (BPW) mangrove in Orissa. SLW and BPW samples had total salinities of 21.54% and 2.18%; and their isolates had optimum NaCl requirement of 10-15% and 1-5% respectively. The extracellular endoglucanase of SLW isolates were active in 5-25% NaCl but those from BPW remain active in only up to 5% NaCl. Interestingly, SLW endoglucanases also performed better in 10% and some even in 30% (v/v)[Bmim][Cl]. Endoglucanase secreted by two SLW isolates, identified by their 16S rRNA gene sequence as Salipaludibacillus sp. were effectively used for in situ enzymatic hydrolysis of both alkali and [Bmim][Cl] pretreated rice straw. However, endoglucanase from BPW isolate Salinicola sp. could hydrolyze seawater washed alkali-pretreated biomass thereby expanding their industrial applicability in coastal areas.

Transcriptome analysis reveals the main metabolic pathway of c-GMP induced by salt stress in tomato seedlings

Tomato is a model crop, as well as important food worldwide. In the arid areas, aggravation of soil salinity has become the primary problem that threatens the high yield in tomato production. As a second messenger substance, cyclic guanosine monophosphate (c-GMP) plays an indispensable role in plant response to salt stress through regulating cell processes to promote plant growth and development. However, this mechanism has not been fully explored in tomato seedlings. In this experiment, the tomato seeds were cultured in distilled water (CK), 20 μM c-GMP (T1), 50 mM NaCl (T2), 20 μM c-GMP + 50 mM NaCl (T3). The results show that 20 μM c-GMP effectively alleviated the inhibition of 50 mM NaCl on tomato growth and development, inducing the expression of 1580 DEGs. 95 DEGs were up-regulated and 442 DEGs were down-regulated (CK vs T1), whereas in the T2 vs T3 comparison 271 DEGs were up-regulated and 772 DEGs were down-regulated. Based on KEGG analysis, the majority of DEGs were involved in metabolism; exogenous c-GMP induced significant enrichment of pathways associated with carbohydrates, phenylpropanoids and fatty acid metabolism. Most PMEs, acCoA, PAL, PODs, FADs, and AD were up-regulated, and GAPDHs, PL, PG, BXL4, and β-G were down-regulated, which reduced susceptibility of tomato seedlings to salt and promoted their salt adaptation. The application of c-GMP promoted soluble sugar, flavonoids and lignin content, reduced accumulation of MDA, and enhanced the activity of POD. Thus, our results provide insights into the molecular mechanisms associated with salt tolerance of tomato seedlings.

Nanotube mediated cell-to-cell communication and cannibalism in Halobacillus sp. GSS1 isolated from Sundarbans, India: A cryptic story of survival under nutrient-limiting condition

Microorganisms play a self-protective role by evolving their genetic and metabolic machinery to thrive in extreme environmental habitats. Halophiles are such salt-loving extremophilic microorganisms able to adapt, survive, and tend to grow at high salt concentrations. In this study, we have isolated Halobacillus sp. GSS1 from Sundarbans mangrove, India having a strong salt-tolerant ability (up to 4M) in Zobell Marine 2216 medium. The salt adaptation mechanism of Halobacillus sp. was investigated by Confocal microscopy using [Na+] specific dye, ‘Sodium Green’ indicating the ‘salt-in’ strategy for their osmoadaptation. Electron microscopic studies revealed that a contact-dependent cell-to-cell communication was profound among the Halobacillus sp. under nutrient limiting condition. This communication is mediated by ‘nanotube’, which is highly recommended for the exchange of molecular information between the two individual bacteria. The existence of the ‘ymdB’ gene strongly supports our claim for nanotube formation by Halobacillus sp. GSS1. Surprisingly, Halobacillus sp. not only utilizing the nanotubes for communication, rather they desperately use nanotubes as a survival weapon under nutrient limiting conditions by triggering cannibalism. This is the first-ever report on the existence of nanotube mediated cell-to-cell communication and cannibalism in any halophilic bacteria, isolated from Sundarbans mangrove forest, India.HighlightsThe existence of nanotube mediated cell-to-cell communication was discovered in Halobacillus sp. GSS1, isolated from Sundarbans mangrove, India.The communication of Halobacillus sp. GSS1 was established through single or multiple nanotubes with the neighboring cells.Intercellular nanotube communication was possible only after the participation of two individual bacteria.Halobacillus sp. GSS1 also uses these nanotubes as a survival weapon by triggering the cannibalism to kill their genetically identical siblings.The presence of the ymdB gene in Halobacillus sp. GSS1 strongly confers the evidence of nanotube formation.Graphical Abstract

Natural variation in growth and physiology under salt stress in rice: QTL mapping in a Horkuch × IR29 mapping population at seedling and reproductive stages

Salinity has a significant negative impact on production of rice. To cope with the increased soil salinity due to climate change, we need to develop salt tolerant rice varieties that can maintain their high yield. Rice landraces indigenous to coastal Bangladesh can be a great resource to study the genetic basis of salt adaptation. In this study, we implemented a QTL analysis framework on a reciprocal mapping population between a salt tolerant landrace Horkuch and a high yieldingrice variety IR29. Our aim was to detect genetic loci that contributes to the salt adaptive responses of the two different developmental stages of rice which are very sensitive to salinity stress. We identified 14 QTL for 9 traits and found that most are unique to the specific developmental stage. In addition, we detected a significant effect of the cytoplasmic genome on the QTL model for some traits such as leaf total potassium and filled grain weight. This underscores the importance of considering cytoplasm-nuclear interaction for breeding programs. Along with this, we identified QTL co-localization for multiple traits that highlights the possible constraint of multiple QTL selection for breeding programs due to different contributions of a donor allele for different traits.HighlightsWe identified genetic loci for the salt tolerance response of two different developmental stages of the rice plant and detected significant contribution of cytoplasm-nuclear genome interaction for a few traits.