Detection and validation of cis-regulatory motifs in osmotic stress-inducible synthetic gene switches via computational and experimental approaches.

Synthetic cis -regulatory modules can improve our understanding of gene regulatory networks. We applied an ensemble approach for de novo cis motif discovery among the promoters of 181 drought inducible differentially expressed soybean (Glycine max L.) genes. A total of 43 cis motifs were identified in promoter regions of all gene sets using the binding site estimation suite of tools (BEST). Comparative analysis of these motifs revealed similarities with known cis -elements found in PLACE database and led to the discovery of cis -regulatory motifs that were not yet implicated in drought response. Compiled with the proposed synthetic promoter design rationale, three synthetic assemblies were constructed by concatenating multiple copies of drought-inducible cis motifs in a specific order with inter-motif spacing using random bases and placed upstream of 35s minimal core promoter. Each synthetic module substituted 35S promoter in pBI121 and pCAMBIA3301 to drive glucuronidase expression in soybean hairy roots and Arabidopsis thaliana L. Chimeric soybean seedlings and 3-week-old transgenic Arabidopsis plants were treated with simulated with different levels of osmotic stress. Histochemical staining of transgenic soybean hairy roots and Arabidopsis displayed drought-inducible GUS activity of synthetic promoters. Fluorometric assay and expression analysis revealed that SP2 is the better manual combination of cis -elements for stress-inducible expression. qRT-PCR results further demonstrated that designed synthetic promoters are not tissue-specific and thus active in different parts upon treatment with osmotic stress in Arabidopsis plants. This study provides tools for transcriptional upgradation of valuable crops against drought stress and adds to the current knowledge of synthetic biology.

Overview of the roles of calcium sensors in plants’ response to osmotic stress signalling.

Calcium signals serve an important function as secondary messengers between cells in various biological processes due to their robust homeostatic mechanism, maintaining an intracellular free Ca2+ concentration. Plant growth, development, and biotic and abiotic stress are all regulated by Ca2+ signals. Ca2+ binding proteins decode and convey the messages encoded by Ca2+ ions. In the presence of high quantities of Mg2+ and monovalent cations, such sensors bind to Ca2+ ions and modify their conformation in a Ca2+ -dependent manner. Calcium-dependent protein kinases (CPKs), calmodulins (CaMs), and calcineurin B-like proteins are all calcium sensors (CBLs). To transmit Ca2+ signals, CPKs, CBLs, and CaMs interact with target proteins and regulate the expression of their genes. These target proteins may be protein kinases, metabolic enzymes, or cytoskeletal-associated proteins. Beyond its role in plant nutrition as a macroelement and its involvement in the plant cell wall structure, calcium modulates many aspects of development, growth and adaptation to environmental constraints such as drought, salinity and osmotic stresses. This review summarises current knowledge on calcium sensors in plant responses to osmotic stress signalling.

Calmodulin binding transcription activators: An interplay between calcium signalling and plant stress tolerance.

In plants, next to the secondary messengers lies an array of signal relaying molecules among which Calmodulins convey the unequivocal alarms of calcium influxes to Calmodulin-Binding Transcription Activators (CAMTA). Upon reception, CAMTA transcription factors decode the calcium signatures by transcribing the genes corresponding to the specific stimulus, thus have direct/indirect engagement in the complex signalling crosstalk. CAMTA transcription factors make an important contribution to the genome of all eukaryotes, including plants, from Brassica napus (18) to Carica papaya (2), the number of CAMTA genes varies across the plant species, however they exhibit a similar evolutionarily conserved domain organization including a DNA-Binding Domain (CG-1), a Transcription Factor Immunoglobulin Binding Domain (TIG), a Calmodulin-Binding Domain (CaMBD/IQ) and several Ankyrin repeats. The regulatory region of CAMTA genes possess multiple stress-responsive cis motifs including ABRE, SARE, G-box, W-box, AuXRE, DRE and others. CAMTA TFs in Arabidopsis have been studied extensively, however in other plants (with a few exceptions), the evidence merely bases upon expression analyses. CAMTAs are reported to orchestrate biotic as well as abiotic stresses including those occurring due to water and temperature fluctuations as well as heavy metals, light and salinity. Through CG-1 domain, CAMTA TFs bind the CG-box in the promoter of their target genes and modulate their expression under adverse conditions. Here we present a glimpse of how calcium signatures are coded and decoded and translated into necessary responses. In addition, we have emphasized on exploitation of the multiple-stress responsive nature of CAMTAs in engineering plants with desired traits.

A soybean calcineurin B-like protein-interacting protein kinase, GmPKS4, regulates plant responses to salt and alkali stresses.

Calcineurin B-like protein-interacting protein kinases (CIPKs) are key elements of plant abiotic stress signaling pathways. CIPKs are SOS2 (Salt Overly Sensitive 2)-like proteins (protein kinase S [PKS] proteins) which all contain a putative FISL motif. It seems that the FISL motif is found only in the SOS2 subfamily of protein kinases. In this study, the full-length cDNA of a soybean CIPK gene (GmPKS4) was isolated and was revealed to have an important role in abiotic stress responses. A qRT-PCR analysis indicated that GmPKS4 expression is upregulated under saline conditions or when exposed to alkali, salt-alkali, drought, or abscisic acid (ABA). A subcellular localization assay revealed the presence of GmPKS4 in the nucleus and cytoplasm. Further studies on the GmPKS4 promoter suggested it affects soybean resistance to various stresses. Transgenic Arabidopsis thaliana and soybean hairy roots overexpressing GmPKS4 had increased proline content as well as high antioxidant enzyme activities but decreased malondialdehyde levels following salt and salt-alkali stress treatments. Additionally, GmPKS4 overexpression activated reactive oxygen species scavenging systems, thereby minimizing damages due to oxidative and osmotic stresses. Moreover, upregulated stress-related gene expression levels were detected in lines overexpressing GmPKS4 under stress conditions. In conclusion, GmPKS4 improves soybean tolerance to salt and salt-alkali stresses. The overexpression of GmPKS4 enhances the scavenging of reactive oxygen species, osmolyte synthesis, and the transcriptional regulation of stress-related genes.

Integration of sRNA, degradome, transcriptome analysis and functional investigation reveals gma-miR398c negatively regulates drought tolerance via GmCSDs and GmCCS in transgenic Arabidopsis and soybean.

BACKGROUND: Drought conditions adversely affect soybean growth, resulting in severe yield losses worldwide. Increasing experimental evidence indicates miRNAs are important post-transcriptional regulators of gene expression. However, the drought-responsive molecular mechanism underlying miRNA-mRNA interactions remains largely uncharacterized in soybean. Meanwhile, the miRNA-regulated drought response pathways based on multi-omics approaches remain elusive. RESULTS: We combined sRNA, transcriptome and degradome sequencing to elucidate the complex regulatory mechanism mediating soybean drought resistance. One-thousand transcripts from 384 target genes of 365 miRNAs, which were enriched in the peroxisome, were validated by degradome-seq. An integrated analysis showed 42 miRNA-target pairs exhibited inversely related expression profiles. Among these pairs, a strong induction of gma-miR398c as a major gene negatively regulates multiple peroxisome-related genes (GmCSD1a/b, GmCSD2a/b/c and GmCCS). Meanwhile, we detected that alternative splicing of GmCSD1a/b might affect soybean drought tolerance by bypassing gma-miR398c regulation. Overexpressing gma-miR398c in Arabidopsis thaliana L. resulted in decreased percentage germination, increased leaf water loss, and reduced survival under water deficiency, which displayed sensitivity to drought during seed germination and seedling growth. Furthermore, overexpressing gma-miR398c in soybean decreased GmCSD1a/b, GmCSD2a/b/c and GmCCS expression, which weakened the ability to scavenge O2.-, resulting in increased relative electrolyte leakage and stomatal opening compared with knockout miR398c and wild-type soybean under drought conditions. CONCLUSION: The study indicates that gma-miR398c negatively regulates soybean drought tolerance, and provides novel insights useful for breeding programs to improve drought resistance by CRISPR technology.

A psychrotolerant Ni-resistant Bacillus cereus D2 induces carbonate precipitation of nickel at low temperature.

Psychrotolerant bacteria play a particularly important role in the remediation of heavy metal in contamination sites at low temperatures. In the current study, a psychrotolerant Ni-resistant bacterial strain, identified as Bacillus cereus D2, was isolated from a nickel mining area in China. The isolated strain could produce a large amount of urease enzyme (194.6 U/mL), grow well in harsh environmental conditions at a temperature of 10 °C, and at a Ni (II) concentration up to 400 mg/L. Also, under the low temperature (10 °C), this strain has been revealed to induce carbonate precipitation (Ni2CO3(OH)2·H2O) through biomineralization for removing the high efficiency of Ni ions (73.47%) from the culture solution. Furthermore, strain D2 could immobilize the DTPA-Ni in contaminated soil under the case of the laboratory condition at 10 °C. These data support that the psychrotolerant bacterial strain D2 may play an important role in remediation technology by eliminating Ni ions from the contaminated soil at low temperatures.

Comprehensive Genomic Analysis and Expression Profiling of Diacylglycerol Kinase (DGK) Gene Family in Soybean (Glycine max) under Abiotic Stresses.

Diacylglycerol kinase (DGK) is an enzyme that plays a pivotal role in abiotic and biotic stress responses in plants by transforming the diacylglycerol into phosphatidic acid. However, there is no report on the characterization of soybean DGK genes in spite of the availability of the soybean genome sequence. In this study, we performed genome-wide analysis and expression profiling of the DGK gene family in the soybean genome. We identified 12 DGK genes (namely GmDGK1-12) which all contained conserved catalytic domains with protein lengths and molecular weights ranging from 436 to 727 amino acids (aa) and 48.62 to 80.93 kDa, respectively. Phylogenetic analyses grouped GmDGK genes into three clusters-cluster I, cluster II, and cluster III-which had three, four, and five genes, respectively. The qRT-PCR analysis revealed significant GmDGK gene expression levels in both leaves and roots coping with polyethylene glycol (PEG), salt, alkali, and salt/alkali treatments. This work provides the first characterization of the DGK gene family in soybean and suggests their importance in soybean response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of this gene family.