Stress Response Element Research Articles

A Comprehensive Identification and Expression Analysis of the WUSCHEL Homeobox-Containing Protein Family Reveals Their Special Role in Development and Abiotic Stress Response in Zea mays L.

Maize is an important food and cash crop worldwide. The WUSCHEL (WUS)-related homeobox (WOX) transcription factor (TF) family plays a significant role in the development process and the response to abiotic stress of plants. However, few studies have been reported on the function of WOX genes in maize. This work, utilizing the latest maize B73 reference genome, results in the identification of 22 putative ZmWOX gene family members. Except for chromosome 5, the 22 ZmWOX genes were homogeneously distributed on the other nine chromosomes and showed three tandem duplication and 10 segmental duplication events. Based on phylogenetic characteristics, ZmWOXs are divided into three clades (e.g., WUS, intermediate, and ancient groups), and the majority of ZmWOXs in same group display similar gene and protein structures. Cross-species collinearity results indicated that some WOX genes might be evolutionarily conservative. The promoter region of ZmWOX family members is enriched in light, plant growth/hormone, and abiotic stress-responsive elements. Tissue-specific expression evaluation showed that ZmWOX genes might play a significant role in the occurrence of maize reproductive organs. Transcriptome data and RT-qPCR analysis further showed that six ZmWOX genes (e.g., ZmWOX1, 4, 6, 13, 16, and 18) were positively or negatively modulated by temperature, salt, and waterlogging stresses. Moreover, two ZmWOXs, ZmWOX1 and ZmWOX18, both were upregulated by abiotic stress. ZmWOX18 was localized in the nucleus and had transactivation activities, while ZmWOX1 was localized in both the cytoplasm and nucleus, without transactivation activity. Overall, this work offers new perspectives on the evolutionary relationships of ZmWOX genes and might provide a resource for further detecting the biological functions of ZmWOXs.

Genome-Wide Identification, Characterization, and Expression Analysis of the Amino Acid Permease Gene Family in Soybean

Amino acid permeases (AAPs) play important roles in transporting amino acids in plant species, leading to increased low-nitrogen tolerance, grain yield, or protein content. However, very few AAPs have been characterized in soybean (Glycine max). In this study, we scanned the soybean reference genome and identified a total of 36 AAP genes (named GmAAP). The GmAAPs were phylogenetically divided into three evolutionary clades, with the genes in the same clades sharing similar gene structures and domain organization. We also showed that seventeen GmAAP genes on ten chromosomes were in collinearity, likely due to whole-genome duplication. Further analysis revealed a variety of cis-acting regulatory elements (such as hormone response elements (ABRE, ERE, GARE, P-box, and TGA-element), stress response elements (LTR, MBS, MYB-related components, TC-rich repeats, TCA-element, and WUN-motif), the tissue expression element (GCN4-motif), and the circadian regulatory element (circadian) present in the 2 kb region of the GmAAP promoter region, demonstrating functional diversity and expression specificity. RNA-Seq data and quantitative real-time PCR identified five GmAAPs showing differential expression under nitrogen limitation, including GmAAP3, GmAAP5, and GmAAP8 showing downregulation while GmAAP14, GmAAP29 showed upregulation, suggesting their involvement in low-nitrogen stress response. These results provide comprehensive information on soybean AAP genes in nitrogen stress, and provide putative candidates with possible roles in enhancing amino acid delivery to seeds for yield improvement.

Molecular cloning, expression and Insilco analysis of drought stress inducible MYB transcription factor encoding gene from C4 plant Eleusine coracana

Drought is one of the key abiotic stresses that critically influences the crops by restraining their growth and yield potential. Being sessile, plant tackle the detrimental effects of drought stress by modulating the cellular state by changing the gene expression. The transcriptional syndicate essentially drives such alteration of gene expression. Transcription factors (TF) are the key regulatory protein that controls the expression of their target gene by binding to the cis-regulatory elements present in the promoter region. Myb-TF, ubiquitously present in all eukaryotes belong to one of the largest TF family, and play a wide array of biological functions in plants, including anthocyanin biosynthesis, vasculature system, cell signalling, seed maturation and abiotc stress responses. The present performed isolation and molecular cloning of full length Myb TF from Eleusine corocana. The isolated full-length coding sequence has 1053 bp and 350 aa was submitted to NCBI (Accession number MT312253). The transcript level of EcMYB increases under different abiotic stress treatments including dehydration, salinity, and high-temperature stress. The promoter region of EcMyb1 was found to be enriched in stress-responsive cis-regulatory elements such as DRE, HSE, ABRE etc. In phylogenetic analysis, EcMyb1 appeared to have high homology with its monocot orthologs particularly Sateria italica, Hordeum vulgare, Saccharum barberi and Oryza sativa. The three-dimension protein structure was generated based on homology modeling and structural aspects were discussed. Further, Insilco analysis was conducted to explore the physiological properties, subcellular localization, potential posttranslational modification sites (phosphorylation and glycosylation sites), and molecular and biological function of the full-length protein. Overall, the expression profiling and Insilco analysis of EcMyb1 strongly indicated its potential role in abiotic stress response in Eleusine corocana.

Genome-wide identification and expression analysis of the JMJ-C gene family in melon (Cucumis melo L.) reveals their potential role in fruit development

BackgroundProteins with the jumonji (JMJ)-C domain belong to the histone demethylase family and contribute to reverse histone methylation. Although JMJ-C family genes have an essential role in regulating plant growth and development, the characterization of the JMJ-C family genes in melon has not been uncovered.ResultsIn this study, a total of 17 JMJ-C proteins were identified in melon (Cucumis melo L.). CmJMJs were categorized into five subfamilies based on the specific conserved domain: KDM4/JHDM3, KDM5/JARID1, JMJD6, KDM3/JHDM2, and JMJ-C domain-only. The chromosome localization analyses showed that 17 CmJMJs were distributed on nine chromosomes. Cis-acting element analyses of the 17 CmJMJ genes showed numerous hormone, light, and stress response elements distributed in the promoter region. Covariance analysis revealed one pair of replicated fragments (CmJMJ3a and CmJMJ3b) in 17 CmJMJ genes. We investigated the expression profile of 17 CmJMJ genes in different lateral organs and four developmental stages of fruit by RNA-seq transcriptome analysis and RT-qPCR. The results revealed that most CmJMJ genes were prominently expressed in female flowers, ovaries, and developing fruits, suggesting their active role in melon fruit development. Subcellular localization showed that the fruit-related CmJMJ5a protein is specifically localized in the cell nucleus.ConclusionsThis study provides a comprehensive understanding of the gene structure, classification, and evolution of JMJ-C in melon and supports the clarification of the JMJ-C functions in further research.

Genome-wide identification and expression analysis of the KCS gene family in soybean (Glycine max) reveal their potential roles in response to abiotic stress.

Very long chain fatty acids (VLCFAs) are fatty acids with chain lengths of 20 or more carbon atoms, which are the building blocks of various lipids that regulate developmental processes and plant stress responses. 3-ketoacyl-CoA synthase encoded by the KCS gene is the key rate-limiting enzyme in VLCFA biosynthesis, but the KCS gene family in soybean (Glycine max) has not been adequately studied thus far. In this study, 31 KCS genes (namely GmKCS1 - GmKCS31) were identified in the soybean genome, which are unevenly distributed on 14 chromosomes. These GmKCS genes could be phylogenetically classified into seven groups. A total of 27 paralogous GmKCS gene pairs were identified with their Ka/Ks ratios indicating that they had undergone purifying selection during soybean genome expansion. Cis-acting element analysis revealed that GmKCS promoters contained multiple hormone- and stress-responsive elements, indicating that GmKCS gene expression levels may be regulated by various developmental and environmental stimuli. Expression profiles derived from RNA-seq data and qRT-PCR experiments indicated that GmKCS genes were diversely expressed in different organs/tissues, and many GmKCS genes were found to be differentially expressed in the leaves under cold, heat, salt, and drought stresses, suggesting their critical role in soybean resistance to abiotic stress. These results provide fundamental information about the soybean KCS genes and will aid in their further functional elucidation and exploitation.

Decoding VaCOLD1 Function in Grapevines: A Membrane Protein Enhancing Cold Stress Tolerance.

In globally cultivated grapevines, low-temperature stress poses a persistent challenge. Although COLD1 is recognized as a cold receptor in rice, its function in grapevine cold signaling is unclear. Here, we identified VaCOLD1, a transmembrane protein from the cold-tolerant Vitis amurensis Rupr, which is primarily located on plasma and endoplasmic reticulum membranes. Broadly expressed across multiple tissues, VaCOLD1 responds to various environmental stresses, particularly to cold. Its promoter contains distinct hormone- and stress-responsive elements, with GUS assays confirming widespread expression in Arabidopsis thaliana. Validation of interaction between VaCOLD1 and VaGPA1, together with their combined expression in yeast and grape calli, notably improved cold endurance. Overexpression of VaCOLD1 enhances cold tolerance in Arabidopsis by strengthening the CBF-COR signaling pathway. This is achieved through shielding against osmotic disturbances and modifying the expression of ABA-mediated genes. These findings emphasize the critical role of the VaCOLD1-VaGPA1 complex in mediating the response to cold stress via the CBF-COR pathway.

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.

Identification of Flavanone 3-Hydroxylase Gene Family in Strawberry and Expression Analysis of Fruit at Different Coloring Stages

The color of strawberry fruit is an important appearance quality index that affects the marketability of fruit, and the content and type of anthocyanin are two of the main reasons for the formation of fruit color. At present, the research on anthocyanin synthesis mainly focuses on the phenylpropane metabolic pathway, and the F3H gene family is an important member of this metabolic pathway. Therefore, in order to clarify the role of flavanone 3-hydroxylase (F3H) in regulating anthocyanin accumulation in strawberry, we identified F3H gene family members in strawberry and analyzed their bioinformatics and expression at different fruit color stages. The results showed that the strawberry F3H family contains 126 members, which are distributed on seven chromosomes and can be divided into six subgroups. The promoter region of strawberry F3H gene family contains light response elements, abiotic stress response elements and hormone response elements. Intraspecic collinearity analysis showed that there were six pairs of collinearity of the F3H gene. Interspecific collinearity analysis showed that there were more collinearity relationships between strawberry and apple, grape and Arabidopsis, but less collinearity between strawberry and rice. Via tissue-specific expression analysis, we found that the expression levels of FvF3H48, FvF3H120 and FvF3H74 were higher in the stages of germination, growth, flowering and fruit setting. The expression levels of FvF3H42 and FvF3H16 were higher in seeds. The expression levels of FvF3H16 and FvF3H11 were higher in the ovary wall of stage 1, stage 2, stage 3 and stage 5. FvF3H15 and FvF3H48 were highly expressed in the pericardium, anther, receptacle and anther. Real-time fluorescence quantitative PCR showed the expression changes in F3H in the fruit coloring process. The results indicate that the expression levels of most members were higher during the S3 stage, such as FvF3H7, FvF3H16, FvF3H32, FvF3H82, FvF3H89, FvF3H92 and FvF3H112. FvF3H63 and FvF3H104 exhibited particularly high expression levels during the S1 stage, with some genes also showing elevated expression during the S4 stage, including FvF3H13, FvF3H27, FvF3H66 and FvF3H103. FvF3H58, FvF3H69, FvF3H79 and FvF3H80 showed higher expression levels during the S2 stage. These findings lay the groundwork for elucidating the biological functions of the strawberry F3H gene family and the selection of related genes.

Genome-wide identification, expression analysis, and functional study of the bZIP transcription factor family and its response to hormone treatments in pea (Pisum sativum L.)

BackgroundBasic leucine zipper (bZIP) protein is a plant-specific transcription factor involved in various biological processes, including light signaling, seed maturation, flower development, cell elongation, seed accumulation protein, and abiotic and biological stress responses. However, little is known about the pea bZIP family.ResultsIn this study, we identified 87 bZIP genes in pea, named PsbZIP1 ~ PsbZIP87, via homology analysis using Arabidopsis. The genes were divided into 12 subfamilies and distributed unevenly in 7 pea chromosomes. PsbZIPs in the same subfamily contained similar intron/exon organization and motif composition. 1 tandem repeat event and 12 segmental duplication events regulated the expansion of the PsbZIP gene family. To better understand the evolution of the PsbZIP gene family, we conducted collinearity analysis using Arabidopsis thaliana, Oryza sativa Japonica, Fagopyrum tataricum, Solanum lycopersicum, Vitis vinifera, and Brachypodium distachyon as the related species of pea. In addition, interactions between PsbZIP proteins and promoters containing hormone- and stress-responsive cis-acting elements suggest that the regulation of PsbZIP expression was complex. We also evaluated the expression patterns of bZIP genes in different tissues and at different fruit development stages, all while subjecting them to five hormonal treatments.ConclusionThese results provide a deeper understanding of PsbZIP gene family evolution and resources for the molecular breeding of pea. The findings suggested that PsbZIP genes, specifically PSbZIP49, play key roles in the development of peas and their response to various hormones.

Molecular characterization of the cytokinin responsive type-B response regulators in perennial ryegrass highlights LpARR10 conferring salt tolerance via trans-activating SOS1 and SOS3

The type-B response regulator (ARR) family members represent the elements of two-component regulatory systems and play crucial roles in the cytokinin signaling, plant growth, and stress responses. The objective of this study was to characterize type-B ARR genes from perennial ryegrass, and to investigate their roles in regulating to salt tolerance. Twelve type-B ARRs, namely LpARR1-12, were identified in perennial ryegrass. They were clustered into three subgroups and were unevenly distributed on chromosomes. The stress responsive elements (SREs) and hormone responsive elements (HREs) were identified in LpARRs promoters and the transcription of LpARRs genes were responsive to various abiotic stress (heat, salt, and osmotic) and hormonal (cytokinin, ABA, and ethylene) treatments. LpARR10 was strong induced by salt stress, therefore it was selected for function analysis. Overexpressing LpARR10 in Arabidopsis led to a significant improvement in salt tolerance. The LpARR10-overexpressing (LpARR10-OE) lines exhibited lower levels of Na+ content compared to WT plants, and the expression levels of SOS1 and SOS3 were significantly higher in the LpARR10-OE lines. Results of yeast auto-transactivation and plant cell transcriptional activity assays shows LpARR10 is a nucleus localized transcriptional activator and directly bind and transactivated the promoter of SOS1 and SOS3. Together, LpARR10 enhances salt tolerance may via trans-activating SOS1/SOS3 in Arabidopsis. Knowledge gained in this study will facilitate further functional analysis of LpARRs for molecular breeding of ryegrass and other related grass species.

A Genome-Wide Analysis and Expression Profile of Heat Shock Transcription Factor (Hsf) Gene Family in Rhododendron simsii.

Heat shock transcription factors are key players in a number of transcriptional regulatory pathways that function during plant growth and development. However, their mode of action in Rhododendron simsii is still unclear. In this study, 22 RsHsf genes were identified from genomic data of R. simsii. The 22 genes were randomly distributed on 12 chromosomes, and were divided into three major groups according to their phylogenetic relationships. The structures and conserved motifs were predicted for the 22 genes. Analysis of cis-acting elements revealed stress-responsive and phytohormone-responsive elements in the gene promoter regions, but the types and number varied among the different groups of genes. Transcriptional profile analyses revealed that RsHsfs were expressed in a tissue-specific manner, with particularly high transcript levels in the roots. The transcriptional profiles under abiotic stress were detected by qRT-PCR, and the results further validated the critical function of RsHsfs. This study provides basic information about RsHsf family in R. simsii, and paves the way for further research to clarify their precise roles and to breed new stress-tolerant varieties.

Bioinformatics and expression analysis of proline metabolism-related gene families in alfalfa under saline-alkali stress

Regulation of the proline metabolic pathway is essential for the accumulation of proline under abiotic stress and for the amelioration of plant stress resistance. Δ1-pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), ornithine transaminase (δ-OAT), proline dehydrogenase (PDH), pyrroline-5-carboxylate dehydrogenase (P5CDH), and proline transporter (ProT) are the key enzymes in the proline metabolic pathway. However, the gene families responsible for proline metabolism have not yet been identified or reported in alfalfa. In this study, a total of 12 MsP5CSs, 4 MsP5CRs, 3 MsOATs, 6 MsPDHs, 2 MsP5CDHs, and 5 MsProTs were identified in the genome of alfalfa, and the members of the same subfamily had similar gene structures and conserved motifs. Analysis of cis-regulatory elements revealed the presence of light-responsive, hormone-regulated, and stress-responsive elements in the promoter regions of alfalfa proline metabolism-related genes. Following treatment with saline-alkali, the expression of MsP5CSs, MsP5CRs, MsOATs, and MsProTs was significantly upregulated, whereas the expression of MsPDH1.1, MsPDH1.3, and MsP5CDH was significantly downregulated. The proline content and enzyme activity of P5CS gradually increased, whereas the enzyme activity of PDH gradually decreased as the duration of stress increased. Root growth rates decreased upon MsP5CS1a suppression (MsP5CS1a-RNAi) in the hairy roots of alfalfa compared to the empty vector line under saline-alkali stress. These results show that proline metabolism-related genes play an important role in the saline-alkali stress tolerance of alfalfa and provide a theoretical basis for further research on the functions of proline metabolism-related genes in alfalfa in response to saline-alkali stress.

Genome-Wide Characterization of Tomato FAD Gene Family and Expression Analysis under Abiotic Stresses.

The fatty acid desaturase (FAD) gene family plays a crucial regulatory role in the resistance process of plant biomembranes. To understand the role of FADs in tomato growth and development, this study identified and analyzed the tomato FAD gene family based on bioinformatics analysis methods. In this study, 26 SlFADs were unevenly distributed on 10 chromosomes. Phylogenetic analysis showed that the SlFAD gene family was divided into six branches, and the exon-intron composition and conserved motifs of SlFADs clustered in the same branch were quite conservative. Several hormone and stress response elements in the SlFAD promoter suggest that the expression of SlFAD members is subject to complex regulation; the construction of a tomato FAD protein interaction network found that SlFAD proteins have apparent synergistic effects with SPA and GPAT proteins. qRT-PCR verification results show that SlFAD participates in the expression of tomato root, stem, and leaf tissues; SlFAD8 is mainly highly expressed in leaves; SlFAD9 plays a vital role in response to salt stress; and SlFAB5 regulates all stages of fruit development under the action of exogenous hormones. In summary, this study provides a basis for a systematic understanding of the SlFAD gene family. It provides a theoretical basis for in-depth research on the functional characteristics of tomato SlFAD genes.

Decoding the guardians of cotton resilience: A comprehensive exploration of the βCA genes and its role in Verticillium dahliae resistance.

Plant Carbonic anhydrases (Cas) have been shown to be stress-responsive enzymes that may play a role in adapting to adverse conditions. Cotton is a significant economic crop in China, with upland cotton (Gossypium hirsutum) being the most widely cultivated species. We conducted genome-wide identification of the βCA gene in six cotton species and preliminary analysis of the βCA gene in upland cotton. In total, 73 βCA genes from six cotton species were identified, with phylogenetic analysis dividing them into five subgroups. GHβCA proteins were predominantly localized in the chloroplast and cytoplasm. The genes exhibited conserved motifs, with motifs 1, 2, and 3 being prominent. GHβCA genes were unevenly distributed across chromosomes and were associated with stress-responsive cis-regulatory elements, including those responding to light, MeJA, salicylic acid, abscisic acid, cell cycle regulation, and defence/stress. Expression analysis indicated that GHβCA6, GHβCA7, GHβCA10, GHβCA15, and GHβCA16 were highly expressed under various abiotic stress conditions, whereas GHβCA3, GHβCA9, GHβCA10, and GHβCA18 had higher expression patterns under Verticillium dahliae infection at different time intervals. In Gossypium thurberi, GthβCA1, GthβCA2, and GthβCA4 showed elevated expression across stress conditions and tissues. Silencing GHβCA10 through VIGS increased Verticillium wilt severity and reduced lignin deposition compared to non-silenced plants. GHβCA10 is crucial for cotton's defense against Verticillium dahliae. Further research is needed to understand the underlying mechanisms and develop strategies to enhance resistance against Verticillium wilt.

Genome-wide analysis of the PtHMGR gene family and functional validation of PtHMGR5 improving drought tolerance in Populus trichocarpa

Terpenoids are one of the main types of secondary metabolites in plants and are crucial for plant growth, development and defense. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a rate-limiting enzyme for terpene biosynthesis in the MVA pathways. In this study, six PtHMGR genes (PtHMGR1-PtHMGR6) were identified in Populus trichocarpa and distributed on six chromosomes. Collinear analysis of PtHMGR genes suggested that six replication gene pairs were acquired by segmental duplication events in the Populus genome. In addition, the promoters of PtHMGRs contained abundant light-response elements, hormone-response elements and abiotic stress-response elements. Expression profile analysis demonstrated that the PtHMGR genes were induced by various abiotic stresses and hormone treatments. Furthermore, transgenic expression of PtHMGR5 in Populus significantly reduced membrane damage and increased drought (mannitol) tolerance. In conclusion, our findings provide valuable information regarding PtHMGR genes, which is of great significance for the cultivation of improved plants in response to drought (mannitol) stress.

Genome-wide investigation and analysis of C2H2 Zinc Finger Protein gene family in apple: Expression profiles during Penicillium expansum infection process

The gene family known as C2H2 zinc finger genes is widely recognized for its significant role in facilitating plant growth, development and reproduction. Many C2H2 zinc finger proteins (C2H2-ZFPs) have been identified and intensively studied in other plants, but little is known about their function in apples. In this study, the C2H2-ZFPs genes of apples were identified and analyzed by whole genome sequence analysis. In our study, 70 members of the C2H2-ZFPs gene family were identified in apples. Phylogenetic analyses divided the apple C2H2-ZFPs gene family into 6 subgroups, with the same subgroups having similar gene structures and motif patterns and speculating on their possible functions. After analysis based on numerous cis-elements on gene promoters, the apple C2H2-ZFPs family contained many light-responsive elements, phytohormone-responsive elements, plant growth and development-related elements, and stress-responsive elements, which indicated the diversity of their functions. The RNA-seq results of apples under P. expansum treatment revealed that fifteen C2H2-ZFPs genes were differentially expressed. The RT-qPCR further validated the same trend as transcriptome data. C2H2-ZFPs genes were significantly expressed in the process of P. expansum infestation. Our analysis evidenced that the apple C2H2-ZFPs gene family plays a vital role in the process of P. expansum infestation in apples. The identified genes can serve as potential candidates for further investigation. The present study provides new ideas for further research on the function of C2H2-ZFPs genes and the innovation of disease resistance technology in apples.

A novel natural variation in the promoter of GmCHX1 regulates conditional gene expression to improve salt tolerance in soybean.

Identification and characterization of soybean germplasm and gene(s)/allele(s) for salt tolerance is an effective way to develop improved varieties for saline soils. Previous studies identified GmCHX1 (Glyma03g32900) as a major salt tolerance gene in soybean, and two main functional variations were found in the promoter region (148/150 bp insertion) and the third exon with a retrotransposon insertion (3.78 kb). In the current study, we identified four salt-tolerant soybean lines, including PI 483460B (Glycine soja), carrying the previously identified salt-sensitive variations at GmCHX1, suggesting new gene(s) or new functional allele(s) of GmCHX1 in these soybean lines. Subsequently, we conducted quantitative trait locus (QTL) mapping in a recombinant-inbred line population (Williams 82 (salt-sensitive) × PI 483460B) to identify the new salt tolerance loci/alleles. A new locus, qSalt_Gm18, was mapped on chromosome 18 associated with leaf scorch score. Another major QTL, qSalt_Gm03, was identified to be associated with chlorophyll content ratio and leaf scorch score in the same chromosomal region of GmCHX1 on chromosome 3. Novel variations in a STRE (stress response element) cis-element in the promoter region of GmCHX1 were found to regulate the salt-inducible expression of the gene in these four newly identified salt-tolerant lines including PI 483460B. This new allele of GmCHX1 with salt-inducible expression pattern provides an energy cost efficient (conditional gene expression) strategy to protect soybean yield in saline soils without yield penalty under non-stress conditions. Our results suggest that there might be no other major salt tolerance locus similar to GmCHX1 in soybean germplasm, and further improvement of salt tolerance in soybean may rely on gene-editing techniques instead of looking for natural variations.

Genome-Wide Identification and Expression Analysis of BnPP2C Gene Family in Response to Multiple Stresses in Ramie (Boehmeria nivea L.).

The protein phosphatase 2C (PP2C), a key regulator of the ABA signaling pathway, plays important roles in plant growth and development, hormone signaling, and abiotic stress response. Although the PP2C gene family has been identified in many species, systematic analysis was still relatively lacking in ramie (Boehmeria nivea L.). In the present study, we identified 63 BnPP2C genes from the ramie genome, using bioinformatics analysis, and classified them into 12 subfamilies, and this classification was consistently supported by their gene structures and conserved motifs. In addition, we observed that the functional differentiation of the BnPP2C family of genes was restricted and that fragment replication played a major role in the amplification of the BnPP2C gene family. The promoter cis-regulatory elements of BnPP2C genes were mainly involved in light response regulation, phytohormone synthesis, transport and signaling, environmental stress response and plant growth and development regulation. We identified BnPP2C genes with tissue specificity, using ramie transcriptome data from different tissues, in rhizome leaves and bast fibers. The qRT-PCR results showed that the BnPP2C1, BnPP2C26 and BnPP2C27 genes had a strong response to drought, high salt and ABA, and there were a large number of stress-responsive elements in the promoter region of BnPP2C1 and BnPP2C26. The results suggested that BnPP2C1 and BnPP2C26 could be used as the candidate genes for drought and salt tolerance in ramie. These results provide a reference for further studies on the function of the PP2C gene and advance the development of the mechanism of ramie stress response, with a view to providing candidate genes for the molecular breeding of ramie for drought and salt tolerance.

Promoter cloning and activities analysis of JmLFY, a key gene for flowering in Juglans mandshurica.

Juglans mandshurica (Manchurian walnut) is a precious timber and woody grain and oil species in Northeast China. The heterodichogamous characteristic phenomenon resulted in the non-synchronous flowering and development of male and female flowers, which limited the mating and the yield and quality of fruits. LFY is a core gene in the flowering regulatory networks, which has been cloned in J. mandshurica, and the function has also been verified preliminarily. In this study, the JmLFY promoter sequence with different lengths of 5'-deletion (pLFY1-pLFY6) were cloned and conducted bioinformatics analysis, the promoter activities were analyzed by detecting their driving activity to GUS gene in the tobacco plants that transformed with different promoter sequence stably or transiently. After that, the interaction between JmSOC1 and JmLFY gene promoter was also analyzed via yeast single-hybrid. The results showed that the promoter sequence contains core cis-acting elements essential for eukaryotic promoters, hormone response elements, defense- and stress-responsive elements, flowering-related elements, etc. Transgenic tobacco plants with pLFY1 were obtained by Agrobacterium infection using the pCAMBIA1301 expression vector, and the GUS gene driven by the JmLFY promoter was detected to express in the leaf, stem, flower, and root of the transformed tobacco plant, which indicated that the obtained JmLFY promoter had driving activity. GUS histochemical staining and enzyme activity detection showed that promoter fragments with different lengths had promoter activity and could respond to the induction of long photoperiod, low temperature, salicylic acid (SA), IAA, GA3, and methyl jasmonate (MeJA). The core regulatory region of JmLFY gene promoter in J. mandshurica was between -657 bp and -1,904 bp. Point-to-point validation of yeast single-hybrid confirmed the interaction between JmSOC1 and JmLFY gene promoter, which indicated that JmLFY gene is the downstream target of JmSOC1. These results reveal relevant factors affecting JmLFY gene expression and clarify the molecular mechanism of JmLFY gene regulation in the flower developmental partially, which will provide a theoretical basis for regulating the flowering time by regulating JmLFY gene expression in J. mandshurica.

Comprehensive Analysis of GH3 Gene Family in Potato and Functional Characterization of StGH3.3 under Drought Stress.

As an important hormone response gene, Gretchen Hagen 3 (GH3) maintains hormonal homeostasis by conjugating excess auxin with amino acids during plant stress-related signaling pathways. GH3 genes have been characterized in many plant species, but they are rarely reported in potato. Here, 19 StGH3 genes were isolated and characterized. Phylogenetic analysis indicated that StGH3s were divided into two categories (group I and group III). Analyses of gene structure and motif composition showed that the members of a specific StGH3 subfamily are relatively conserved. Collinearity analysis of StGH3 genes in potato and other plants laid a foundation for further exploring the evolutionary characteristics of the StGH3 genes. Promoter analysis showed that most StGH3 promoters contained hormone and abiotic stress response elements. Multiple transcriptome studies indicated that some StGH3 genes were responsive to ABA, water deficits, and salt treatments. Moreover, qRT-PCR analysis indicated that StGH3 genes could be induced by phytohormones (ABA, SA, and MeJA) and abiotic stresses (water deficit, high salt, and low temperature), although with different patterns. Furthermore, transgenic tobacco with transient overexpression of the StGH3.3 gene showed positive regulation in response to water deficits by increasing proline accumulation and reducing the leaf water loss rate. These results suggested that StGH3 genes may be involved in the response to abiotic stress through hormonal signal pathways. Overall, this study provides useful insights into the evolution and function of StGH3s and lays a foundation for further study on the molecular mechanisms of StGH3s in the regulation of potato drought resistance.