Genome-Wide Identification and Characterization of Soybean GmLOR Gene Family and Expression Analysis in Response to Abiotic Stresses

The LOR (LURP-one related) family genes encode proteins containing a conserved LOR domain. Several members of the LOR family genes are required for defense against Hyaloperonospora parasitica (Hpa) in Arabidopsis. However, there are few reports of LOR genes in response to abiotic stresses in plants. In this study, a genome-wide survey and expression levels in response to abiotic stresses of 36 LOR genes from Glycine max were conducted. The results indicated that the GmLOR gene family was divided into eight subgroups, distributed on 14 chromosomes. A majority of members contained three extremely conservative motifs. There were four pairs of tandem duplicated GmLORs and nineteen pairs of segmental duplicated genes identified, which led to the expansion of the number of GmLOR genes. The expansion patterns of the GmLOR family were mainly segmental duplication. A heatmap of soybean LOR family genes showed that 36 GmLOR genes exhibited various expression patterns in different tissues. The cis-acting elements in promoter regions of GmLORs include abiotic stress-responsive elements, such as dehydration-responsive elements and drought-inducible elements. Real-time quantitative PCR was used to detect the expression level of GmLOR genes, and most of them were expressed in the leaf or root except that GmLOR6 was induced by osmotic and salt stresses. Moreover, GmLOR4/10/14/19 were significantly upregulated after PEG and salt treatments, indicating important roles in the improvement of plant tolerance to abiotic stress. Overall, our study provides a foundation for future investigations of GmLOR gene functions in soybean.     

Genome-Wide Identification and Characterization of TALE Superfamily Genes in Soybean (Glycine max L.)

The three-amino-acid-loop-extension (TALE) superfamily genes broadly existed in plants, which played important roles in plant growth, development and abiotic stress responses. In this study, we identified 68 Glycine max TALE (GmTALE) superfamily members. Phylogenetic analysis divided the GmTALE superfamily into the BEL1-like (BLH/BELL homeodomain) and the KNOX (KNOTTED-like homeodomain) subfamilies. Moreover, the KNOX subfamily could be further categorized into three clades (KNOX Class I, KNOX Class II and KNOX Class III). The GmTALE genes showed similarities in the gene structures in the same subfamily or clade, whose coding proteins exhibited analogous motif and conserved domain compositions. Besides, synteny analyses and evolutionary constraint evaluations of the TALE members among soybean and different species provided more clues for GmTALE superfamily evolution. The cis-element analyses in gene promoter regions and relevant gene expression profiling revealed different regulating roles of GmTALE genes during soybean plant development, saline and dehydration stresses. Genome-wide characterization, evolution, and expression profile analyses of GmTALE genes can pave the way for future gene functional research and facilitate their roles for applications in genetic improvement on soybean in saline and dehydration stresses.     

Genome-Wide Identification,Characterization and Expression Analysis of Soybean CHYR Gene Family

The CHYR (CHY ZINC-FINGER AND RING FINGER PROTEIN) proteins have been functionally characterized in iron regulation and stress response in Arabidopsis, rice and Populus. However, their roles in soybean have not yet been systematically investigated. Here, in this study, 16 GmCHYR genes with conserved Zinc_ribbon, CHY zinc finger and Ring finger domains were obtained and divided into three groups. Moreover, additional 2–3 hemerythrin domains could be found in the N terminus of Group III. Phylogenetic and homology analysis of CHYRs in green plants indicated that three groups might originate from different ancestors. Expectedly, GmCHYR genes shared similar conserved domains/motifs distribution within the same group. Gene expression analysis uncovered their special expression patterns in different soybean tissues/organs and under various abiotic stresses. Group I and II members were mainly involved in salt and alkaline stresses. The expression of Group III members was induced/repressed by dehydration, salt and alkaline stresses, indicating their diverse roles in response to abiotic stress. In conclusion, our work will benefit for further revealing the biological roles of GmCHYRs.     

Oberflächenlipide und Lipidpolymere von Pflanzen

Surface Lipids and Lipid Polymers of Plants The protective skin of higher plants, or cuticle, consists of a lipid coating and the characteristic polymer lipids cutin and suberin. The lipid coating contains hydrocarbons, wax, triterpenes, sterols and sometimes aldehydes and ketones. Cutin, an estolid, unsoluble in water and organic solvents, is built up by hydroxylated fatty acids with 16 and 18 C-Atoms. Suberin is in its constitution very similar to cutin. Its characteristic compounds are hydroxylated dicarbonic acids. To the degradation of cutin and suberin by men and animals some references have been given.     

Selection and Validation of Reference Genes for RT-qPCR Analysis in Aegilops tauschii (Coss.) under Different Abiotic Stresses

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.     

Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis “Xuegan” plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.     

Wild Soybean Oxalyl-CoA Synthetase Degrades Oxalate and Affects the Tolerance to Cadmium and Aluminum Stresses

Acyl activating enzyme 3 (AAE3) was identified as being involved in the acetylation pathway of oxalate degradation, which regulates the responses to biotic and abiotic stresses in various higher plants. Here, we investigated the role of Glycine soja AAE3 (GsAAE3) in Cadmium (Cd) and Aluminum (Al) tolerances. The recombinant GsAAE3 protein showed high activity toward oxalate, with a K_m of 105.10 ± 12.30 μM and V_max of 12.64 ± 0.34 μmol min⁻¹ mg⁻¹ protein, suggesting that it functions as an oxalyl–CoA synthetase. The expression of a GsAAE3–green fluorescent protein (GFP) fusion protein in tobacco leaves did not reveal a specific subcellular localization pattern of GsAAE3. An analysis of the GsAAE3 expression pattern revealed an increase in GsAAE3 expression in response to Cd and Al stresses, and it is mainly expressed in root tips. Furthermore, oxalate accumulation induced by Cd and Al contributes to the inhibition of root growth in wild soybean. Importantly, GsAAE3 overexpression increases Cd and Al tolerances in A. thaliana and soybean hairy roots, which is associated with a decrease in oxalate accumulation. Taken together, our data provide evidence that the GsAAE3-encoded protein plays an important role in coping with Cd and Al stresses.     

Systematic Genome-Wide Study and Expression Analysis of SWEET Gene Family: Sugar Transporter Family Contributes to Biotic and Abiotic Stimuli in Watermelon

The SWEET (Sugars Will Eventually be Exported Transporter) proteins are a novel family of sugar transporters that play key roles in sugar efflux, signal transduction, plant growth and development, plant–pathogen interactions, and stress tolerance. In this study, 22 ClaSWEET genes were identified in Citrullus lanatus (Thunb.) through homology searches and classified into four groups by phylogenetic analysis. The genes with similar structures, conserved domains, and motifs were clustered into the same groups. Further analysis of the gene promoter regions uncovered various growth, development, and biotic and abiotic stress responsive cis-regulatory elements. Tissue-specific analysis showed most of the genes were highly expressed in male flowers and the roots of cultivated varieties and wild cultivars. In addition, qRT-PCR results further imply that ClaSWEET proteins might be involved in resistance to Fusarium oxysporum infection. Moreover, a significantly higher expression level of these genes under various abiotic stresses suggests its multifaceted role in mediating plant responses to drought, salt, and low-temperature stress. The genome-wide characterization and phylogenetic analysis of ClaSWEET genes, together with the expression patterns in different tissues and stimuli, lays a solid foundation for future research into their molecular function in watermelon developmental processes and responses to biotic and abiotic stresses.     

Stress-Responsive Expression,Subcellular Localization and Protein–Protein Interactions of the Rice Metacaspase Family

Metacaspases, a class of cysteine-dependent proteases like caspases in animals, are important regulators of programmed cell death (PCD) during development and stress responses in plants. The present study was focused on comprehensive analyses of expression patterns of the rice metacaspase (OsMC) genes in response to abiotic and biotic stresses and stress-related hormones. Results indicate that members of the OsMC family displayed differential expression patterns in response to abiotic (e.g., drought, salt, cold, and heat) and biotic (e.g., infection by Magnaporthe oryzae, Xanthomonas oryzae pv. oryzae and Rhizoctonia solani) stresses and stress-related hormones such as abscisic acid, salicylic acid, jasmonic acid, and 1-amino cyclopropane-1-carboxylic acid (a precursor of ethylene), although the responsiveness to these stresses or hormones varies to some extent. Subcellular localization analyses revealed that OsMC1 was solely localized and OsMC2 was mainly localized in the nucleus. Whereas OsMC3, OsMC4, and OsMC7 were evenly distributed in the cells, OsMC5, OsMC6, and OsMC8 were localized in cytoplasm. OsMC1 interacted with OsLSD1 and OsLSD3 while OsMC3 only interacted with OsLSD1 and that the zinc finger domain in OsMC1 is responsible for the interaction activity. The systematic expression and biochemical analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMCs in PCD that is related to abiotic and biotic stress responses.     

A Novel DUF569 Gene Is a Positive Regulator of the Drought Stress Response in Arabidopsis

In the last two decades, global environmental change has increased abiotic stress on plants and severely affected crops. For example, drought stress is a serious abiotic stress that rapidly and substantially alters the morphological, physiological, and molecular responses of plants. In Arabidopsis, several drought-responsive genes have been identified; however, the underlying molecular mechanism of drought tolerance in plants remains largely unclear. Here, we report that the “domain of unknown function” novel gene DUF569 (AT1G69890) positively regulates drought stress in Arabidopsis. The Arabidopsis loss-of-function mutant atduf569 showed significant sensitivity to drought stress, i.e., severe wilting at the rosette-leaf stage after water was withheld for 3 days. Importantly, the mutant plant did not recover after rewatering, unlike wild-type (WT) plants. In addition, atduf569 plants showed significantly lower abscisic acid accumulation under optimal and drought-stress conditions, as well as significantly higher electrolyte leakage when compared with WT Col-0 plants. Spectrophotometric analyses also indicated a significantly lower accumulation of polyphenols, flavonoids, carotenoids, and chlorophylls in atduf569 mutant plants. Overall, our results suggest that novel DUF569 is a positive regulator of the response to drought in Arabidopsis.     

Genome-Wide Identification and Characterization of the Brassinazole-resistant (BZR) Gene Family and Its Expression in the Various Developmental Stage and Stress Conditions in Wheat (Triticum aestivum L.)

Brassinosteroids (BRs) play crucial roles in various biological processes, including plant developmental processes and response to diverse biotic and abiotic stresses. However, no information is currently available about this gene family in wheat (Triticum aestivum L.). In the present investigation, we identified the BZR gene family in wheat to understand the evolution and their role in diverse developmental processes and under different stress conditions. In this study, we performed the genome-wide analysis of the BZR gene family in the bread wheat and identified 20 TaBZR genes through a homology search and further characterized them to understand their structure, function, and distribution across various tissues. Phylogenetic analyses lead to the classification of TaBZR genes into five different groups or subfamilies, providing evidence of evolutionary relationship with Arabidopsis thaliana, Zea mays, Glycine max, and Oryza sativa. A gene exon/intron structure analysis showed a distinct evolutionary path and predicted the possible gene duplication events. Further, the physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, and cis-acting regulatory elements were also examined using various computational approaches. In addition, an analysis of public RNA-seq data also shows that TaBZR genes may be involved in diverse developmental processes and stress tolerance mechanisms. Moreover, qRT-PCR results also showed similar expression with slight variation. Collectively, these results suggest that TaBZR genes might play an important role in plant developmental processes and various stress conditions. Therefore, this work provides valuable information for further elucidate the precise role of BZR family members in wheat.     

CaDHN3, a Pepper (Capsicum annuum L.) Dehydrin Gene Enhances the Tolerance against Salt and Drought Stresses by Reducing ROS Accumulation

Dehydrins (DHNs) play an important role in abiotic stress tolerance in a large number of plants, but very little is known about the function of DHNs in pepper plants. Here, we isolated a Y₁SK₂-type DHN gene “CaDHN3” from pepper. To authenticate the function of CaDHN3 in salt and drought stresses, it was overexpressed in Arabidopsis and silenced in pepper through virus-induced gene silencing (VIGS). Sub-cellular localization showed that CaDHN3 was located in the nucleus and cell membrane. It was found that CaDHN3-overexpressed (OE) in Arabidopsis plants showed salt and drought tolerance phenotypic characteristics, i.e., increased the initial rooting length and germination rate, enhanced chlorophyll content, lowered the relative electrolyte leakage (REL) and malondialdehyde (MDA) content than the wild-type (WT) plants. Moreover, a substantial increase in the activities of antioxidant enzymes; including the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and lower hydrogen peroxide (H₂O₂) contents and higher O₂^•− contents in the transgenic Arabidopsis plants. Silencing of CaDHN3 in pepper decreased the salt- and drought-stress tolerance, through a higher REL and MDA content, and there was more accumulation of reactive oxygen species (ROS) in the CaDHN3-silenced pepper plants than the control plants. Based on the yeast two-hybrid (Y2H) screening and Bimolecular Fluorescence Complementation (BiFC) results, we found that CaDHN3 interacts with CaHIRD11 protein in the plasma membrane. Correspondingly, the expressions of four osmotic-related genes were significantly up-regulated in the CaDHN3-overexpressed lines. In brief, our results manifested that CaDHN3 may play an important role in regulating the relative osmotic stress responses in plants through the ROS signaling pathway. The results of this study will provide a basis for further analyses of the function of DHN genes in pepper.