Cloning and Characterization of Two Putative P-Type ATPases from the Marine Microalga Dunaliella maritima Similar to Plant H+-ATPases and Their Gene Expression Analysis under Conditions of Hyperosmotic Salt Shock

The green microalga genus Dunaliella is mostly comprised of species that exhibit a wide range of salinity tolerance, including inhabitants of hyperhaline reservoirs. Na+ content in Dunaliella cells inhabiting saline environments is maintained at a fairly low level, comparable to that in the cells of freshwater organisms. However, despite a long history of studying the physiological and molecular mechanisms that ensure the ability of halotolerant Dunaliella species to survive at high concentrations of NaCl, the question of how Dunaliella cells remove excess Na+ ions entering from the environment is still debatable. For thermodynamic reasons it should be a primary active mechanism; for example, via a Na+-transporting ATPase, but the molecular identification of Na+-transporting mechanism in Dunaliella has not yet been carried out. Formerly, in the euryhaline alga D. maritima, we functionally identified Na+-transporting P-type ATPase in experiments with plasma membrane (PM) vesicles which were isolated from this alga. Here we describe the cloning of two putative P-type ATPases from D. maritima, DmHA1 and DmHA2. Phylogenetic analysis showed that both ATPases belong to the clade of proton P-type ATPases, but the similarity between DmHA1 and DmHA2 is not high. The expression of DmHA1 and DmHA2 in D. maritima cells under hyperosmotic salt shock was studied by qRT-PCR. Expression of DmHA1 gene decreases and remains at a relatively low level during the response of D. maritima cells to hyperosmotic salt shock. In contrast, expression of DmHA2 increases under hyperosmotic salt shock. This indicates that DmHA2 is important for overcoming hyperosmotic salt stress by the algal cells and as an ATPase it is likely directly involved in transport of Na+ ions. We assume that it is the DmHA2 ATPase that represents the Na+-transporting ATPase.

Integrative analysis of the salt stress response in cyanobacteria

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.

Transcriptome Profiling of the Salt Stress Response in the Leaves and Roots of Halophytic Eutrema salsugineum

Eutrema salsugineum can grow in natural harsh environments; however, the underlying mechanisms for salt tolerance of Eutrema need to be further understood. Herein, the transcriptome profiling of Eutrema leaves and roots exposed to 300 mM NaCl is investigated, and the result emphasized the role of genes involved in lignin biosynthesis, autophagy, peroxisome, and sugar metabolism upon salt stress. Furthermore, the expression of the lignin biosynthesis and autophagy-related genes, as well as 16 random selected genes, was validated by qRT-PCR. Notably, the transcript abundance of a large number of lignin biosynthesis genes such as CCoAOMT, C4H, CCR, CAD, POD, and C3′H in leaves was markedly elevated by salt shock. And the examined lignin content in leaves and roots demonstrated salt stress led to lignin accumulation, which indicated the enhanced lignin level could be an important mechanism for Eutrema responding to salt stress. Additionally, the differentially expressed genes (DEGs) assigned in the autophagy pathway including Vac8, Atg8, and Atg4, as well as DEGs enriched in the peroxisome pathway such as EsPEX7, EsCAT, and EsSOD2, were markedly induced in leaves and/or roots. In sugar metabolism pathways, the transcript levels of most DEGs associated with the synthesis of sucrose, trehalose, raffinose, and xylose were significantly enhanced. Furthermore, the expression of various stress-related transcription factor genes including WRKY, AP2/ERF-ERF, NAC, bZIP, MYB, C2H2, and HSF was strikingly improved. Collectively, the increased expression of biosynthesis genes of lignin and soluble sugars, as well as the genes in the autophagy and peroxisome pathways, suggested that Eutrema encountering salt shock possibly possess a higher capacity to adjust osmotically and facilitate water transport and scavenge reactive oxidative species and oxidative proteins to cope with the salt environment. Thus, this study provides a new insight for exploring the salt tolerance mechanism of halophytic Eutrema and discovering new gene targets for the genetic improvement of crops.

Integrative analysis of the salt stress response in cyanobacteria

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome and proteome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.

Salt stress and salt shock differently affect DNA methylation in salt-responsive genes in sugar beet and its wild, halophytic ancestor

Here we determined the impact of salt shock and salt stress on the level of DNA methylation in selected CpG islands localized in promoters or first exons of sixteen salt-responsive genes in beets. Two subspecies differing in salt tolerance were subjected for analysis, a moderately salt-tolerant sugar beet Beta vulgaris ssp. vulgaris cv. Huzar and a halophytic beet, Beta vulgaris ssp. maritima. The CpG island methylation status was determined. All target sequences were hyper- or hypomethylated under salt shock and/or salt stress in one or both beet subspecies. It was revealed that the genomic regions analyzed were highly methylated in both, the salt treated plants and untreated controls. Methylation of the target sequences changed in a salt-dependent manner, being affected by either one or both treatments. Under both shock and stress, the hypomethylation was a predominant response in sugar beet. In Beta vulgaris ssp. maritima, the hypermethylation occurred with higher frequency than hypomethylation, especially under salt stress and in the promoter-located CpG sites. Conversely, the hypomethylation of the promoter-located CpG sites predominated in sugar beet plants subjected to salt stress. This findings suggest that DNA methylation may be involved in salt-tolerance and transcriptomic response to salinity in beets.

Defining the Cell Wall, Cell Cycle and Chromatin Landmarks in the Responses of Brachypodium distachyon to Salinity

Excess salinity is a major stress that limits crop yields. Here, we used the model grass Brachypodium distachyon (Brachypodium) reference line Bd21 in order to define the key molecular events in the responses to salt during germination. Salt was applied either throughout the germination period (“salt stress”) or only after root emergence (“salt shock”). Germination was affected at ≥100 mM and root elongation at ≥75 mM NaCl. The expression of arabinogalactan proteins (AGPs), FLA1, FLA10, FLA11, AGP20 and AGP26, which regulate cell wall expansion (especially FLA11), were mostly induced by the “salt stress” but to a lesser extent by “salt shock”. Cytological assessment using two AGP epitopes, JIM8 and JIM13 indicated that “salt stress” increases the fluorescence signals in rhizodermal and exodermal cell wall. Cell division was suppressed at >75 mM NaCl. The cell cycle genes (CDKB1, CDKB2, CYCA3, CYCB1, WEE1) were induced by “salt stress” in a concentration-dependent manner but not CDKA, CYCA and CYCLIN-D4-1-RELATED. Under “salt shock”, the cell cycle genes were optimally expressed at 100 mM NaCl. These changes were consistent with the cell cycle arrest, possibly at the G1 phase. The salt-induced genomic damage was linked with the oxidative events via an increased glutathione accumulation. Histone acetylation and methylation and DNA methylation were visualized by immunofluorescence. Histone H4 acetylation at lysine 5 increased strongly whereas DNA methylation decreased with the application of salt. Taken together, we suggest that salt-induced oxidative stress causes genomic damage but that it also has epigenetic effects, which might modulate the cell cycle and AGP expression gene. Based on these landmarks, we aim to encourage functional genomics studies on the responses of Brachypodium to salt.

Spatial Variability of Microbial Communities and Salt Distributions Across a Latitudinal Aridity Gradient in the Atacama Desert

Over the past 150 million years, the Chilean Atacama Desert has been transformed into one of the most inhospitable landscapes by geophysical changes, which makes it an ideal Mars analog that has been explored for decades. However, a heavy rainfall that occurred in the Atacama in 2017 provides a unique opportunity to study the response of resident extremophiles to rapid environmental change associated with excessive water and salt shock. Here we combine mineral/salt composition measurements, amendment cell culture experiments, and next-generation sequencing analyses to study the variations in salts and microbial communities along a latitudinal aridity gradient of the Atacama Desert. In addition, we examine the reshuffling of Atacama microbiomes after the rainfall event. Analysis of microbial community composition revealed that soils within the southern arid desert were consistently dominated by Actinobacteria, Chloroflexi, Proteobacteria, Firmicutes, Bacteroidetes, Gemmatimonadetes, Planctomycetes, and Acidobacteria, and Verrucomicrobia. Intriguingly, the hyperarid microbial consortia exhibited a similar pattern to the more southern desert. Salts at the shallow subsurface were dissolved and leached down to a deeper layer, challenging indigenous microorganisms with the increasing osmotic stress. Microbial viability was found to change with aridity and rainfall events. This study sheds light on the structure of xerotolerant, halotolerant, and radioresistant microbiomes from the hyperarid northern desert to the less arid southern transition region, as well as their response to changes in water availability.

Loss of cell wall integrity genes CpxA and MrcB causes flocculation in Escherichia coli

Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc related gene by conventional mutant screening. To overcome this, we performed a two-step E. coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB, encoding a peptidoglycan-synthesizing enzyme and cpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, growth of ΔmrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was upregulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.