The Carthamus tinctorius L. genome sequence provides insights into synthesis of unsaturated fatty acids.

Domesticated safflower (Carthamus tinctorius L.) is a widely cultivated edible oil crop. However, despite its economic importance, the genetic basis underlying key traits such as oil content, resistance to biotic and abiotic stresses, and flowering time remains poorly understood. Here, we present the genome assembly for C. tinctorius variety Jihong01, which was obtained by integrating Oxford Nanopore Technologies (ONT) and BGI-SEQ500 sequencing results. The assembled genome was 1,061.1 Mb, and consisted of 32,379 protein-coding genes, 97.71% of which were functionally annotated. Safflower had a recent whole genome duplication (WGD) event in evolution history and diverged from sunflower approximately 37.3 million years ago. Through comparative genomic analysis at five seed development stages, we unveiled the pivotal roles of fatty acid desaturase 2 (FAD2) and fatty acid desaturase 6 (FAD6) in linoleic acid (LA) biosynthesis. Similarly, the differential gene expression analysis further reinforced the significance of these genes in regulating LA accumulation. Moreover, our investigation of seed fatty acid composition at different seed developmental stages unveiled the crucial roles of FAD2 and FAD6 in LA biosynthesis. These findings offer important insights into enhancing breeding programs for the improvement of quality traits and provide reference resource for further research on the natural properties of safflower.

Genome-Wide Identification and Comprehensive Analysis of the FtsH Gene Family in Soybean (Glycine max).

The filamentation temperature-sensitive H (FtsH) gene family is critical in regulating plant chloroplast development and photosynthesis. It plays a vital role in plant growth, development, and stress response. Although FtsH genes have been identified in a wide range of plants, there is no detailed study of the FtsH gene family in soybean (Glycine max). Here, we identified 34 GmFtsH genes, which could be categorized into eight groups, and GmFtsH genes in the same group had similar structures and conserved protein motifs. We also performed intraspecific and interspecific collinearity analysis and found that the GmFtsH family has large-scale gene duplication and is more closely related to Arabidopsis thaliana. Cis-acting elements analysis in the promoter region of the GmFtsH genes revealed that most genes contain developmental and stress response elements. Expression patterns based on transcriptome data and real-time reverse transcription quantitative PCR (qRT-PCR) showed that most of the GmFtsH genes were expressed at the highest levels in leaves. Then, GO enrichment analysis indicated that GmFtsH genes might function as a protein hydrolase. In addition, the GmFtsH13 protein was confirmed to be localized in chloroplasts by a transient expression experiment in tobacco. Taken together, the results of this study lay the foundation for the functional determination of GmFtsH genes and help researchers further understand the regulatory network in soybean leaf development.

CePP2C19 confers tolerance to drought by regulating the ABA sensitivity in Cyperus esculentus.

BACKGROUND: Tiger nut (Cyperus esculentus) is widely known as an additional source of food, oil and feed worldwide. The agricultural production of tiger nut has been greatly hindered by drought stress, reducing both yield and quality. Protein phosphatase 2 C (PP2Cs) plays an important role in plant responses to drought stress however, the molecular mechanism of PP2Cs in tiger nuts still unclear. RESULTS: In this study, we identified a putative group A PP2C-encoding gene (CePP2C19) from tiger nut using transcriptome analysis, which is highly induced by drought stress. The transient expression assay suggested that CePP2C19 was localized to nucleus. Furthermore, the interaction between CePP2C19 and CePYR1, a coreceptor for ABA signaling, was first detected using a yeast two-hybrid assay and then verified using a bimolecular fluorescence complementation (BiFC) analysis. In addition, the transgenic Arabidopsis lines overexpressing CePP2C19 exhibited extreme tolerance to ABA and mannitol stresses during seed germination and root growth. At the mature stage, overexpression of CePP2C19 resulted in a higher tolerance to drought stress in transgenic Arabidopsis, as confirmed by a visible phenotype and several physiological parameters. Noticeably, the silencing of CePP2C19 by virus-induced gene silencing (VIGS) showed obvious reduction in drought tolerance in tiger nut plants. CONCLUSIONS: The CePP2C19 emerges as a pivotal gene involved in the ABA signaling pathway, which likely reduce ABA sensitivity and thus enhances drought tolerance in Cyperus esculentus.

Comparative Proteomic Analysis of Two Wild Soybean (Glycine soja) Genotypes Reveals Positive Regulation of Saline-Alkaline Stress Tolerance by Tonoplast Transporters.

Soil saline-alkalization is a significant constraint for soybean production. Owing to higher genetic diversity of wild soybean, we compared the proteomic landscape of saline-alkaline stress-tolerant (SWBY032) and stress-sensitive (SWLJ092) wild soybean (Glycine soja) strains under saline and saline-alkaline stress. Out of 346 differentially expressed proteins (DEPs) specifically involved in saline-alkaline stress, 159 and 133 DEPs were identified in only SWLJ092 and SWBY032, respectively. Functional annotations revealed that more ribosome proteins were downregulated in SWLJ092, whereas more membrane transporters were upregulated in SWBY032. Moreover, protein-protein interaction analysis of 133 DEPs revealed that 14 protein-synthesis- and 2 TCA-cycle-related DEPs might alter saline-alkaline tolerance by affecting protein synthesis and amino acid metabolism. Furthermore, we confirmed G. soja tonoplast intrinsic protein (GsTIP2-1 and GsTIP2-2), inositol transporter (GsINT1), sucrose transport protein (GsSUC4), and autoinhibited Ca2+-ATPase (GsACA11) as tonoplast transporters can synergistically improve saline-alkaline tolerance in soybean, possibly by relieving the inhibition of protein synthesis and amino acid metabolism. Overall, our findings provided a foundation for molecular breeding of a saline-alkaline stress-tolerant soybean.

A Soybean Sucrose Non-Fermenting Protein Kinase 1 Gene, GmSNF1, Positively Regulates Plant Response to Salt and Salt-Alkali Stress in Transgenic Plants.

Soybean is one of the most widely grown oilseed crops worldwide. Several unfavorable factors, including salt and salt-alkali stress caused by soil salinization, affect soybean yield and quality. Therefore, exploring the molecular basis of salt tolerance in plants and developing genetic resources for genetic breeding is important. Sucrose non-fermentable protein kinase 1 (SnRK1) belongs to a class of Ser/Thr protein kinases that are evolutionarily highly conserved direct homologs of yeast SNF1 and animal AMPKs and are involved in various abiotic stresses in plants. The GmPKS4 gene was experimentally shown to be involved with salinity tolerance. First, using the yeast two-hybrid technique and bimolecular fluorescence complementation (BiFC) technique, the GmSNF1 protein was shown to interact with the GmPKS4 protein. Second, the GmSNF1 gene responded positively to salt and salt-alkali stress according to qRT-PCR analysis, and the GmSNF1 protein was localized in the nucleus and cytoplasm using subcellular localization assay. The GmSNF1 gene was then heterologously expressed in yeast, and the GmSNF1 gene was tentatively identified as having salt and salt-alkali tolerance function. Finally, the salt-alkali tolerance function of the GmSNF1 gene was demonstrated by transgenic Arabidopsis thaliana, soybean hairy root complex plants overexpressing GmSNF1 and GmSNF1 gene-silenced soybean using VIGS. These results indicated that GmSNF1 might be useful in genetic engineering to improve plant salt and salt-alkali tolerance.

GmUFO1 Regulates Floral Organ Number and Shape in Soybean.

The UNUSUAL FLORAL ORGANS (UFO) gene is an essential regulatory factor of class B genes and plays a vital role in the process of inflorescence primordial and flower primordial development. The role of UFO genes in soybean was investigated to better understand the development of floral organs through gene cloning, expression analysis, and gene knockout. There are two copies of UFO genes in soybean and in situ hybridization, which have demonstrated similar expression patterns of the GmUFO1 and GmUFO2 genes in the flower primordium. The phenotypic observation of GmUFO1 knockout mutant lines (Gmufo1) showed an obvious alteration in the floral organ number and shape and mosaic organ formation. By contrast, GmUFO2 knockout mutant lines (Gmufo2) showed no obvious difference in the floral organs. However, the GmUFO1 and GmUFO2 double knockout lines (Gmufo1ufo2) showed more mosaic organs than the Gmufo1 lines, in addition to the alteration in the organ number and shape. Gene expression analysis also showed differences in the expression of major ABC function genes in the knockout lines. Based on the phenotypic and expression analysis, our results suggest the major role of GmUFO1 in the regulation of flower organ formation in soybeans and that GmUFO2 does not have any direct effect but might have an interaction role with GmUFO1 in the regulation of flower development. In conclusion, the present study identified UFO genes in soybean and improved our understanding of floral development, which could be useful for flower designs in hybrid soybean breeding.

Disruption of CHORISMATE SYNTHASE1 leads to yellow-green variegation in soybean leaves.

Yellow-green variegation leaf phenotype adds more value to ornamental plants, but it is regarded as an undesirable trait in crop plants, affecting their yields. Until recently, the underlying mechanism regulating the yellow-green variegation phenotype has remained largely unexplored in soybean. In the present study, we indentified four Glycine max leaf yellow/green variegation mutants, Gmvar1, Gmvar2, Gmvar3, and Gmvar4, from artificial mutagenesis populations. Map-based cloning, together with the allelic identification test and CRISPR-based gene knockout, proved that mutated GmCS1 controls yellow-green variegation phenotype of the Gmvar mutants. GmCS1 encodes a chorismate synthase in soybean. The content of Phe, Tyr, and Trp were dramatically decreased in Gmcs1 mutants. Exogenous supply of three aromatic amino acid mixtures, or only Phe to Gmvar mutants, leads to recovery of the mutant phenotype. The various biological processes and signalling pathways related to metabolism and biosynthesis were altered in Gmvar mutants. Collectively, our findings provide new insights about the molecular regulatory network of yellow-green variegation leaf phenotype in soybean.

Genome-Wide Identification of MADS-Box Family Genes in Safflower (Carthamus tinctorius L.) and Functional Analysis of CtMADS24 during Flowering.

Safflower is an important economic crop with a plethora of industrial and medicinal applications around the world. The bioactive components of safflower petals are known to have pharmacological activity that promotes blood circulation and reduces blood stasis. However, fine-tuning the genetic mechanism of flower development in safflower is still required. In this study, we report the genome-wide identification of MADS-box transcription factors in safflower and the functional characterization of a putative CtMADS24 during vegetative and reproductive growth. In total, 77 members of MADS-box-encoding genes were identified from the safflower genome. The phylogenetic analysis divided CtMADS genes into two types and 15 subfamilies. Similarly, bioinformatic analysis, such as of conserved protein motifs, gene structures, and cis-regulatory elements, also revealed structural conservation of MADS-box genes in safflower. Furthermore, the differential expression pattern of CtMADS genes by RNA-seq data indicated that type II genes might play important regulatory roles in floral development. Similarly, the qRT-PCR analysis also revealed the transcript abundance of 12 CtMADS genes exhibiting tissue-specific expression in different flower organs. The nucleus-localized CtMADS24 of the AP1 subfamily was validated by transient transformation in tobacco using GFP translational fusion. Moreover, CtMADS24-overexpressed transgenic Arabidopsis exhibited early flowering and an abnormal phenotype, suggesting that CtMADS24 mediated the expression of genes involved in floral organ development. Taken together, these findings provide valuable information on the regulatory role of CtMADS24 during flower development in safflower and for the selection of important genes for future molecular breeding programs.

Genome-Wide Identification and Expression Analysis of Fatty Acid Desaturase (FAD) Genes in Camelina sativa (L.) Crantz.

Camelina sativa (L.) Crantz is an indispensable oilseed crop, and its seeds contain many unsaturated fatty acids. FAD (fatty acid desaturase) regulates the synthesis of unsaturated fatty acids. In this research, we performed CsFAD gene family analysis and identified 24 CsFAD genes in Camelina, which were unevenly distributed on 14 of the 19 total chromosomes. Phylogenetic analysis showed that CsFAD includes four subfamilies, supported by the conserved structures and motifs of CsFAD genes. In addition, we investigated the expression patterns of the FAD family in the different tissues of Camelina. We found that CsFAD family genes were all expressed in the stem, and CsFAD2-2 was highly expressed in the early stage of seed development. Moreover, during low temperature (4 °C) stress, we identified that the expression level of CsFAD2-2 significantly changed. By observing the transient expression of CsFAD2-2 in Arabidopsis protoplasts, we found that CsFAD2-2 was located on the nucleus. Through the detection and analysis of fatty acids, we prove that CsFAD2-2 is involved in the synthesis of linolenic acid (C18:3). In conclusion, we identified CsFAD2-2 through the phylogenetic analysis of the CsFAD gene family and further determined the fatty acid content to find that CsFAD2-2 is involved in fatty acid synthesis in Camelina.

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.

Soybean F-Box-Like Protein GmFBL144 Interacts With Small Heat Shock Protein and Negatively Regulates Plant Drought Stress Tolerance.

The F-box gene family is one of the largest gene families in plants. These genes regulate plant growth and development, as well as biotic and abiotic stress responses, and they have been extensively researched. Drought stress is one of the major factors limiting the yield and quality of soybean. In this study, bioinformatics analysis of the soybean F-box gene family was performed, and the role of soybean F-box-like gene GmFBL144 in drought stress adaptation was characterized. We identified 507 F-box genes in the soybean genome database, which were classified into 11 subfamilies. The expression profiles showed that GmFBL144 was highly expressed in plant roots. Overexpression of GmFBL144 increased the sensitivity of transgenic Arabidopsis to drought stress. Under drought stress, the hydrogen peroxide (H2O2) and malonaldehyde (MDA) contents of transgenic Arabidopsis were higher than those of the wild type (WT) and empty vector control, and the chlorophyll content was lower than that of the control. Y2H and bimolecular fluorescence complementation (BiFC) assays showed that GmFBL144 can interact with GmsHSP. Furthermore, our results showed that GmFBL144 can form SCF FBL144 (E3 ubiquitin ligase) with GmSkp1 and GmCullin1. Altogether, these results indicate that the soybean F-box-like protein GmFBL144 may negatively regulate plant drought stress tolerance by interacting with sHSP. These findings provide a basis for molecular genetics and breeding of soybean.

The antimalarial activity of 1,2,4-trioxolane/trioxane hybrids and dimers: A review.

Malaria, a mosquito-borne parasitic infection caused by protozoan parasites belonging to the genus Plasmodium, is a dangerous disease that contributes to millions of hospital visits and hundreds and thousands of deaths across the world, especially in Sub-Saharan Africa. Antimalarial agents are vital for treating malaria and controlling transmission, and 1,2,4-trioxolane/trioxane-containing agents, especially artemisinin and its derivatives, own antimalarial efficacy and low toxicity with unique mechanisms of action. Moreover, artemisinin-based combination therapies were recommended by the World Health Organization as the first-line treatment for uncomplicated malaria infection and have remained as the mainstay of the treatment of malaria, demonstrating that 1,2,4-trioxolane/trioxane derivatives are useful prototypes for the control and eradication of malaria. However, malaria parasites have already developed resistance to almost all of the currently available antimalarial agents, creating an urgent need for the search of novel pharmaceutical interventions for malaria. The purpose of this review article is to provide an emphasis on the current scenario (January 2012 to January 2022) of 1,2,4-trioxolane/trioxane hybrids and dimers with potential antimalarial activity and the structure-activity relationships are also discussed to facilitate further rational design of more effective candidates.

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.

Research Progress of UiO-66-Based Electrochemical Biosensors.

UiO-66, as a member of the MOFs families, is widely employed in sensing, drug release, separation, and adsorption due to its large specific surface area, uniform pore size, easy functionalization, and excellent stability. Especially in electrochemical biosensors, UiO-66 has demonstrated excellent adsorption capacity and response signal, which significantly improves the sensitivity and specificity of detection. However, the existing application research remains in its infancy, lacking systematic methods, and recycling utilization and exclusive sensing of UiO-66 still require further improvement. Therefore, one of the present research objectives is to explore the breakthrough point of existing technologies and optimize the performance of UiO-66-based electrochemical biosensors (UiO-66-EBs). In this work, we summarized current experimental methods and detection mechanisms of UiO-66-EBs in environmental detection, food safety, and disease diagnosis, analyzed the existing problems, and proposed some suggestions to provide new ideas for future research.

Tracing Key Molecular Regulators of Lipid Biosynthesis in Tuber Development of Cyperus esculentus Using Transcriptomics and Lipidomics Profiling.

Cyperus esculentus is widely representing one of the important oil crops around the world, which provides valuable resources of edible tubers called tiger nut. The chemical composition and high ability to produce fats emphasize the role of tiger nut in promoting oil crop productivity. However, the underlying molecular mechanism of the production and accumulation of lipids in tiger nut development still remains unclear. Here, we conducted comprehensive transcriptomics and lipidomics analyses at different developmental stages of tuber in Cyperus esculentus. Lipidomic analyses confirmed that the accumulation of lipids including glycolipids, phospholipids, and glycerides were significantly enriched during tuber development from early to mature stage. The proportion of phosphatidylcholines (PC) declined during all stages and phosphatidyl ethanolamine (PE) was significantly declined in early and middle stages. These findings implied that PC is actively involved in triacylglycerol (TAG) biosynthesis during the tubers development, whereas PE may participate in TAG metabolism during early and middle stages. Comparative transcriptomics analyses indicated several genomic and metabolic pathways associated with lipid metabolism during tuber development in tiger nut. The Pearson correlation analysis showed that TAG synthesis in different developmental stages was attributed to 37 candidate transcripts including CePAH1. The up-regulation of diacylglycerol (DAG) and oil content in yeast, resulted from the inducible expression of exogenous CePAH1 confirmed the central role of this candidate gene in lipid metabolism. Our results demonstrated the foundation of an integrative metabolic model for understanding the molecular mechanism of tuber development in tiger nut, in which lipid biosynthesis plays a central role.

Genome-wide identification of soybean Shaker K+ channel gene family and functional characterization of GmAKT1 in transgenic Arabidopsis thaliana under salt and drought stress.

Potassium is a major cationic nutrient involved in numerous physiological processes in plants. The uptake of K+ is mediated by K+ channels and transporters, and the Shaker K+ channel gene family plays an essential role in K+ uptake and stress resistance in plants. However, little is known regarding this family in soybean. In this study, 14 members of the Shaker K+ channel gene family were identified in soybean and were classified into five groups. Protein domain analysis revealed that Shaker K+ channel gene members have an ion transport domain (ion trans), a cyclic nucleotide-binding domain, ankyrin repeat domains, and a dimerization domain in the potassium ion channel. Quantitative real-time polymerase chain reaction analysis indicated that the expression of eight genes (notably GmAKT1) in soybean leaves and roots was significantly increased in response to salt and drought stress. Furthermore, the overexpression of GmAKT1 in Arabidopsis enhanced root length, K+ concentration, and fresh/dry weight ratio compared with wild-type plants subjected to salt and drought stress; this suggests that GmAKT1 improves the tolerance of soybean to abiotic stress. Our results provide important insight into the characterization of Shaker K+ channel gene family members in soybean and highlight the function of GmAKT1 in soybean plants under salt and drought stress.

Camelina lipid droplets as skin delivery system promotes wound repair by enhancing the absorption of hFGF2.

Human basic fibroblast growth factor (hFGF2) is widely recognized for accelerating skin wound healing in both animal models and randomized clinical trials. However, the low skin permeation and bioavailability of hFGF2 remain the most limiting factors in the pharmacological application. For the first time, Camelina Lipid Droplets (CLD) delivery system was displayed important virtue, by promoting the skin absorption of hFGF2, which is a key factor that accelerates the skin wound repair, and provide a new alternative for skin therapy. In this study, we used the CLD as a safer material to prepare the nanoparticles, which were characterized by size and morphology. Our data revealed that particle sizes of Camelina Lipid Droplets linked to hFGF2 (CLD-hFGF2) were around 133.5 nm; it also displayed that the complex of CLD-hFGF2 penetrates the skin barrier in deeper than an individual hFGF2. This suggests that once the hFGF2 is fixed onto the surface of CLD, it can cross the stratum corneum and play a therapeutic role into the dermis. Furthermore, we demonstrated that CLD-hFGF2 enhances fibroblast migration, and significantly improves skin regeneration for accelerating wound healing without any significant toxicity. This paper highlights the importance of CLD as an emerging delivery system; it is also providing a new and applicable therapeutic research direction through enhancing the skin permeation of hFGF2 to accelerate wound healing.

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.