Low Temperature Responsive Elements Research Articles

Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress

BackgroundDespite its known significance in plant abiotic stress responses, the role of the RAV gene family in the response of Capsicum annuum to chilling stress remains largely unexplored.ResultsIn this study, we identified and characterized six members of the CaRAV gene subfamily in pepper plants through genome-wide analysis. Subsequently, the CaRAV subfamily was classified into four branches based on homology with Arabidopsis thaliana, each exhibiting relatively conserved domains within the branch. We discovered that light response elements accounted for the majority of CaRAVs, whereas low-temperature response elements were specific to the NGA gene subfamily. After pepper plants were subjected to chilling stress, qRT‒PCR analysis revealed that CaRAV1, CaRAV2 and CaNGA1 were significantly induced in response to chilling stress, indicating that CaRAVs play a role in the response to chilling stress. Using virus-induced gene silencing (VIGS) vectors, we targeted key members of the CaRAV gene family. Under normal growth conditions, the MDA content and SOD enzyme activity of the silenced plants were slightly greater than those of the control plants, and the REC activity was significantly greater than that of the control plants. The levels of MDA and electrolyte leakage were greater in the silenced plants after they were exposed to chilling stress, and the POD and CAT enzyme activities were significantly lower than those in the control, which was particularly evident under repeated chilling stress. In addition, the relative expression of CaPOD and CaCAT was greater in V2 plants upon repeated chilling stress, especially CaCAT was significantly greater in V2 plants than in the other two silenced plants, with 3.29 and 1.10 increases within 12 and 24 h. These findings suggest that CaRAV1 and CaNGA1 positively regulate the response to chilling stress.ConclusionsSilencing of key members of the CaRAV gene family results in increased susceptibility to chilling damage and reduced antioxidant enzyme activity in plants, particularly under repeated chilling stress. This study provides valuable information for understanding the classification and putative functions of RAV transcription factors in pepper plants.

Identification of Stress Responsive NAC Genes in Casuarina equisetifolia L. and Its Expression Analysis under Abiotic Stresses

NAC (NAM, ATAF and CUC)-like transcription factors, a class of plant-specific transcription factors, play a pivotal role in plant growth, development, metabolism, and stress response. Notably, a specific subclass of NAC family, known as SNAC (stress-responsive NAC), is particularly involved in the plant’s response to abiotic stress. As a very useful tree, Casuarina equisetifolia L. also has excellent stress resistance properties. To explore gene resources of C. equisetifolia which are associated with stress resistance and the molecular mechanisms that it employed is very helpful to its molecular-assisted breeding. In this study, 10 CeSNAC transcription factors were identified by constructing the phylogenetic tree of 94 CeNACs from the genome of C. equisetifolia L. together with 79 SNAC in different plant species. Phylogenetic tree analysis revealed that these 10 CeSNAC genes are classified into the ATAF (Arabidopsis transcription activation factor), NAP (NAC-like, activated by AP3/P1), and AtNAC3 subfamilies of the NAC family, all featuring the typical NAM (no apical meristem) domain, with the exception of CeSNAC7. In addition, all NAC transcription factors, except CeSNAC9, were localized in the nucleus. Examination of the CeSNAC promoter unveiled the presence of stress response elements such as a STRE (stress responsive element), an MBS (MYB binding site), an ABRE (abscisic acid responsive element) and a LTR (low temperature responsive element). Under various stress treatments, the majority of CeSNAC expressions exhibited induction in response to low temperature, drought, and high salt treatments, as well as ABA (abscisic acid) treatment. However, CeSNAC6, CeSNAC7, and CeSNAC9 were found to be inhibited specifically by drought treatment. Additionally, only CeSNAC3 and CeNAC9 expression was hindered while the rest of the CeSNAC expression were induced by MeJA (methyl jasmonate) treatment. These findings shed light on the relationship between different CeSNAC genes and their responses to abiotic stress conditions, providing valuable insights for further research into CeSNAC functions and aiding the development of stress-resistant varieties in C. equisetifolia.

Genome-wide identification of late embryogenesis abundant protein family and their key regulatory network in Pinus tabuliformis cold acclimation.

Cold acclimation is a crucial biological process that enables conifers to overwinter safely. The late embryogenesis abundant (LEA) protein family plays a pivotal role in enhancing freezing tolerance during this process. Despite its importance, the identification, molecular functions and regulatory networks of the LEA protein family have not been extensively studied in conifers or gymnosperms. Pinus tabuliformis, a conifer with high ecological and economic values and with high-quality genome sequence, is an ideal candidate for such studies. Here, a total of 104 LEA genes were identified from P. tabuliformis, and we renamed them according to their subfamily group: PtLEA1-PtLEA92 (group LEA1-LEA6), PtSMP1-PtSMP6 (group seed maturation protein) and PtDHN1-PtDHN6 (group Dehydrin). While the sequence structure of P. tabuliformis LEA genes are conserved, their physicochemical properties exhibit unique characteristics within different subfamily groupings. Notably, the abundance of low-temperature responsive elements in PtLEA genes was observed. Using annual rhythm and temperature gradient transcriptome data, PtLEA22 was identified as a key gene that responds to low-temperature induction while conforming to the annual cycle of cold acclimation. Overexpression of PtLEA22 enhanced Arabidopsis freezing tolerance. Furthermore, several transcription factors potentially co-expressed with PtLEA22 were validated using yeast one-hybrid and dual-luciferase assays, revealing that PtDREB1 could directly bind PtLEA22 promoter to positively regulate its expression. These findings reveal the genome-wide characterization of P. tabuliformis LEA genes and their importance in the cold acclimation, while providing a theoretical basis for studying the molecular mechanisms of cold acclimation in conifers.

Comprehensive analysis and expression profiles of the AP2/ERF gene family during spring bud break in tea plant (Camellia sinensis)

BackgroundAP2/ERF transcription factors (AP2/ERFs) are important regulators of plant physiological and biochemical metabolism. Evidence suggests that AP2/ERFs may be involved in the regulation of bud break in woody perennials. Green tea is economically vital in China, and its production value is significantly affected by the time of spring bud break of tea plant. However, the relationship between AP2/ERFs in tea plant and spring bud break remains largely unknown.ResultsA total of 178 AP2/ERF genes (CsAP2/ERFs) were identified in the genome of tea plant. Based on the phylogenetic analysis, these genes could be classified into five subfamilies. The analysis of gene duplication events demonstrated that whole genome duplication (WGD) or segmental duplication was the primary way of CsAP2/ERFs amplification. According to the result of the Ka/Ks value calculation, purification selection dominated the evolution of CsAP2/ERFs. Furthermore, gene composition and structure analyses of CsAP2/ERFs indicated that different subfamilies contained a variety of gene structures and conserved motifs, potentially resulting in functional differences among five subfamilies. The promoters of CsAP2/ERFs also contained various signal-sensing elements, such as abscisic acid responsive elements, light responsive elements and low temperature responsive elements. The evidence presented here offers a theoretical foundation for the diverse functions of CsAP2/ERFs. Additionally, the expressions of CsAP2/ERFs during spring bud break of tea plant were analyzed by RNA-seq and grouped into clusters A-F according to their expression patterns. The gene expression changes in clusters A and B were more synchronized with the spring bud break of tea plant. Moreover, several potential correlation genes, such as D-type cyclin genes, were screened out through weighted correlation network analysis (WGCNA). Temperature and light treatment experiments individually identified nine candidate CsAP2/ERFs that may be related to the spring bud break of tea plant.ConclusionsThis study provides new evidence for role of the CsAP2/ERFs in the spring bud break of tea plant, establishes a theoretical foundation for analyzing the molecular mechanism of the spring bud break of tea plant, and contributes to the improvement of tea cultivars.

Characterization of a Stress-Enhanced Promoter from the Grass Halophyte, Spartina alterniflora L.

Stress-inducible promoters are vital for the desirable expression of genes, especially transcription factors, which could otherwise compromise growth and development when constitutively overexpressed in plants. Here, we report on the characterization of the promoter region of a stress-responsive gene SaAsr1 from monocot halophyte cordgrass (Spartina alterniflora). Several cis-acting elements, such as ABRE (ABA-responsive element), DRE-CRT (dehydration responsive-element/C-Repeat), LTRE (low temperature-responsive element), ERE (ethylene-responsive element), LRE (light-responsive element), etc. contributed at varying degrees to salt-, drought- and ABA-enhanced expression of gusA reporter gene in Arabidopsis thaliana under the full-length promoter, pAsr11875 and its deletion derivatives with an assortment of cis-regulatory motifs. The smallest promoter, pAsr1491, with three cis-acting elements (a CCAAT box-heat responsive, an LRE, and a copper responsive element) conferred drought-enhanced expression of gusA; pAsr1755 (with an ABRE and a DRE) presented the highest expression in ABA and drought; and pAsr1994 with seven ABREs and two DREs conferred optimal induction of gusA, especially under drought and ABA. Arabidopsis transgenics expressing a known abiotic stress-responsive gene, SaADF2 (actin depolymerization factor 2), under both pAsr11875 and p35S promoters outperformed the wild type (WT) with enhanced drought and salt tolerance contributed by higher relative water content and membrane stability with no significant difference between pAsr11875:SaADF2 or p35S:SaADF2 lines. However, pAsr11875:SaADF2 lines produced healthy plants with robust shoot systems under salt stress and control compared to slightly stunted growth of the p35S:SaADF2 plants. This reestablished the evidence that transgene expression under a stress-inducible promoter is a better strategy for the genetic manipulation of crops.

The molecular characteristics of soybean ARR-B transcription factors

The Type-B authentic response regulator (ARR-Bs) gene family is one of the important plant-specific transcription factor families involved in variety of physiological processes. However, study of ARR-Bs gene family in soybean is limited. Genome-wide analysis and expression profiling of the ARR-Bs gene family were performed in the soybean genome. 31 ARR-Bs genes (namely GmARR-B1-31) were identified, containing conserved catalytic domains with protein lengths and molecular weights ranging from 246 to 699 amino acids (aa) and 28.30 to 76.86 kDa, respectively. Phylogenetic analysis grouped ARR-Bs genes into three clusters—Cluster I, Cluster II, and Cluster III—which included 15, 12, and 4 genes, respectively, and were asymmetrically distributed on 17 chromosomes. Tissue specific expression analysis of GmARR-Bs family revealed a high transcription level in flowers, roots and seeds. The subcellular localization of GmARR5, GmARR14 were observed in the nucleus, and the promoter region of them included low-temperature responsive element (LTR), the C-repeat/dehydration responsive element (DRE), MBS, ABRE, MYB, MEJA and TCA-elements, which possibly participate in abiotic stress and hormones responses. qRT-PCR analysis showed that the expression of GmARR-B genes were affected by different abiotic stresses, especially cold stress and salt stress, and GmARR-B5 and GmARR-B14 were significantly induced by cold stress. This suggested that ARR-Bs genes were involved in multiple abiotic stress response pathways and acted as a positive factor under cold stress.

UNFERTILIZED EMBRYO SAC 12 phosphorylation plays a crucial role in conferring salt tolerance.

Arabidopsis (Arabidopsis thaliana) UNFERTILIZED EMBRYO SAC 12 (AtUNE12) belongs to the basic helix-loop-helix DNA-binding superfamily of proteins. However, its function is not well known. Here, we found that AtUNE12 plays an important role in mediating salt tolerance. AtUNE12 is a transcriptional activator located in the nucleus whose expression is induced by NaCl, mannitol, and abscisic acid. In addition to binding to the G-box "CACGTG", AtUNE12 also binds to the low temperature responsive element 15 (LTRE15) "CCGAC". Furthermore, the serine residue at position 108 of AtUNE12 is phosphorylated during the salt stress response, enabling AtUNE12 to trigger gene expression by binding to G-box and/or LTRE15 motifs. Phosphorylated AtUNE12 regulates the expression of the genes involved in ion transport leading to reduced Na+ accumulation and K+ loss. At the same time, phosphorylation of AtUNE12 also induces the expression of AtMYB61 to decrease stomatal aperture, leading to a reduced transpiration rate. Overall, AtUNE12 serves as a transcriptional activator that is induced and phosphorylated upon salt stress, and the induction and phosphorylation of AtUNE12 in turn activate the salt-overly-sensitive pathway and decrease the stomatal aperture, enabling improved salt tolerance.

Abiotic Stress-Induced Actin-Depolymerizing Factor 3 From Deschampsia antarctica Enhanced Cold Tolerance When Constitutively Expressed in Rice.

The Antarctic flowering plant Deschampsia antarctica is highly sensitive to climate change and has shown rapid population increases during regional warming of the Antarctic Peninsula. Several studies have examined the physiological and biochemical changes related to environmental stress tolerance that allow D. antarctica to colonize harsh Antarctic environments; however, the molecular mechanisms of its responses to environmental changes remain poorly understood. To elucidate the survival strategies of D. antarctica in Antarctic environments, we investigated the functions of actin depolymerizing factor (ADF) in this species. We identified eight ADF genes in the transcriptome that were clustered into five subgroups by phylogenetic analysis. DaADF3, which belongs to a monocot-specific clade together with cold-responsive ADF in wheat, showed significant transcriptional induction in response to dehydration and cold, as well as under Antarctic field conditions. Multiple drought and low-temperature responsive elements were identified as possible binding sites of C-repeat-binding factors in the promoter region of DaADF3, indicating a close relationship between DaADF3 transcription control and abiotic stress responses. To investigate the functions of DaADF3 related to abiotic stresses in vivo, we generated transgenic rice plants overexpressing DaADF3. These transgenic plants showed greater tolerance to low-temperature stress than the wild-type in terms of survival rate, leaf chlorophyll content, and electrolyte leakage, accompanied by changes in actin filament organization in the root tips. Together, our results imply that DaADF3 played an important role in the enhancement of cold tolerance in transgenic rice plants and in the adaptation of D. antarctica to its extreme environment.

Papain-Like Cysteine Protease Gene Family in Fig (Ficus carica L.): Genome-Wide Analysis and Expression Patterns.

The papain-like cysteine proteases (PLCPs) are the most abundant family of cysteine proteases in plants, with essential roles in biotic/abiotic stress responses, growth and senescence. Papain, bromelain and ficin are widely used in food, medicine and other industries. In this study, 31 PLCP genes (FcPCLPs) were identified in the fig (Ficus carica L.) genome by HMM search and manual screening, and assigned to one of nine subfamilies based on gene structure and conserved motifs. SAG12 and RD21 were the largest subfamilies with 10 and 7 members, respectively. The FcPCLPs ranged from 1,128 to 5,075 bp in length, containing 1–10 introns, and the coding sequence ranged from 624 to 1,518 bp, encoding 207–505 amino acids. Subcellular localization analysis indicated that 24, 2, and 5 PLCP proteins were targeted to the lysosome/vacuole, cytoplasm and extracellular matrix, respectively. Promoter (2,000 bp upstream) analysis of FcPLCPs revealed a high number of plant hormone and low temperature response elements. RNA-seq revealed differential expression of 17 FcPLCPs in the inflorescence and receptacle, and RD21 subfamily members were the major PLCPs expressed in the fruit; 16 and 5 FcPLCPs responded significantly to ethylene and light, respectively. Proteome analyses revealed 18 and 5 PLCPs in the fruit cell soluble proteome and fruit latex, respectively. Ficins were the major PLCP in fig fruit, with decreased abundance in inflorescences, but increased abundance in receptacles of commercial-ripe fruit. FcRD21B/C and FcALP1 were aligned as the genes encoding the main ficin isoforms. Our study provides valuable multi-omics information on the FcPLCP family and lays the foundation for further functional studies.

Galactinol synthase confers salt-stress tolerance by regulating the synthesis of galactinol and raffinose family oligosaccharides in poplar

Plants respond to abiotic stress through a series of transcriptional profile and metabolic process modifications through signaling events. Raffinose family oligosaccharides (RFOs) play a role in the regulatory mechanisms of abiotic stress tolerance and are catalyzed by the key enzyme, galactinol synthase (GolS), in plants. The amino acids sequences of GolS of Populus trichocarpa contained the common features of GolS proteins in other plant species, namely, a hydrophobic pentapeptide (APSAA). The PtrGolS3 promoter region contains several cis-elements for stress responses, including abscisic acid responsive element (ABRE), the dehydration and cold responsive elements (DRE/CRT), the low-temperature responsive element (LTRE), and the transcription factor MYB binding sites. Overexpression of AtGolS2 and PtrGolS3 enhanced abiotic stress tolerance along with the expression of stress-related genes in transgenic poplar. In addition, overexpression of AtGolS2 and PtrGolS3 resulted in higher contents of soluble sugars and other stress-associated metabolites (proline, salicylic acid, phenylalanine, and so forth) in two GolS overexpressing poplar lines compared with wild-type poplar under salt stress. Based on these results, a promising PtrGolS candidate gene for metabolic engineering of sugars to improve abiotic stress tolerance in poplar was identified and it may increase the understanding of RFO metabolism in woody plants under short-term salt treatment.

Genome-Wide Identification and Expression Pattern Analysis of the HAK/KUP/KT Gene Family of Cotton in Fiber Development and Under Stresses.

The potassium transporter family HAK/KUP/KT is a large group of proteins that are important in plant potassium transport and plays a crucial role in plant growth and development, especially in economic crops. Although HAK/KUP/KT genes have been identified in many species, research on these genes in cotton is still quite rare. In this study, in total, 21, 24, 45, and 44 HAK/KUP/KT genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. Phylogenetic analysis showed that these genes were divided into four clusters. The G. hirsutum gene promoters contained diverse cis-regulatory elements, such as drought-responsive elements, low temperature-responsive elements, and other elements. The RNA-seq data and qRT-PCR results showed that HAK/KUP/KT genes had different expression patterns in fiber development. The qRT-PCR results of drought and NaCl treatment indicated that HAK/KUP/KT genes might play important roles in abiotic stress responses. These results will provide molecular insights into potassium transporter research in cotton.

PkGPPS.SSU interacts with two PkGGPPS to form heteromeric GPPS in Picrorhiza kurrooa: Molecular insights into the picroside biosynthetic pathway

Geranyl geranyl pyrophosphate synthase (GGPPS) is known to form an integral subunit of the heteromeric GPPS (geranyl pyrophosphate synthase) complex and catalyzes the biosynthesis of monoterpene in plants. Picrorhiza kurrooa Royle ex Benth., a medicinally important high altitude plant is known for picroside biomolecules, the monoterpenoids. However, the significance of heteromeric GPPS in P. kurrooa still remains obscure. Here, transient silencing of PkGGPPS was observed to reduce picroside-I (P-I) content by more than 60% as well as picroside-II (P-II) by more than 75%. Thus, PkGGPPS was found to be involved in the biosynthesis of P-I and P-II besides other terpenoids. To unravel the mechanism, small subunit of GPPS (PkGPPS.SSU) was identified from P. kurrooa. Protein-protein interaction studies in yeast as well as bimolecular fluorescence complementation (BiFC) in planta have indicated that large subunit of GPPS PkGPPS.LSUs (PkGGPPS1 and PkGGPPS2) and PkGPPS.SSU form a heteromeric GPPS. Presence of similar conserved domains such as light responsive motifs, low temperature responsive elements (LTRE), dehydration responsive elements (DREs), W Box and MeJA responsive elements in the promoters of PkGPPS.LSU and PkGPPS.SSU documented their involvement in picroside biosynthesis. Further, the tissue specific transcript expression analysis vis-à-vis epigenetic regulation (DNA methylation) of promoters as well as coding regions of PkGPPS.LSU and PkGPPS.SSU has strongly suggested their role in picroside biosynthesis. Taken together, the newly identified PkGPPS.SSU formed the heteromeric GPPS by interacting with PkGPPS.LSUs to synthesize P-I and P-II in P. kurrooa.

Retrotransposon promoter of Ruby1 controls both light- and cold-induced accumulation of anthocyanins in blood orange.

Blood orange is generally recognized to accumulate anthocyanins in its fruit pulp in a cold-inducible manner. We observed that the fruit peel of blood orange can also accumulate anthocyanins under ample light conditions. Interestingly, purple pummelo can accumulate anthocyanins only in its fruit peel but not in its pulp. The mechanism underlying the tissue specificity of anthocyanin accumulation in citrus is unknown. Here, we show that the active promoter of Ruby1, a key activator of anthocyanin biosynthesis, is also light inducible in addition to its already known cold inducibility in blood orange. Electrophoretic mobility shift assays and transient expression assays showed that HY5 positively regulated the transcription of Ruby1 by binding to the G-box motif (CACGTC). The tissue specificity of anthocyanin accumulation in the peel of purple pummelo may be due to the lack of a low temperature responsive element and a MYC binding site, which were shown to be involved in cold inducibility of CsRuby1 in blood orange by insertion of a long terminal repeat type retrotransposon in the promoter. These results bring new insights into the regulatory mechanism of anthocyanin biosynthesis in response to environmental stimuli and provide cis-elements for genetic improvement of anthocyanin-stable fruits rich in antioxidant metabolites.

Genome-wide screening of hexokinase gene family and functional elucidation of HXK2 response to cold stress in Jatropha curcas.

Hexokinase, the key rate-limiting enzyme of plant respiration and glycolysis metabolism, has been found to play a vital role in plant sugar sensing and sugar signal transduction. Using Jatropha curcas genome database and bioinformatics method, J. curcas HXK gene family (JcHXK) was identified and its phylogenetic evolution, functional domain, signal peptide at the N-terminal, and expression analysis were conducted. The results showed that a total of 4 HXK genes (JcHXK1, JcHXK2, JcHXK3, and JcHKL1) with 9 exons were systematically identified from J. curcas. JcHXK1, JcHXK3, and JcHKL1 with putative transmembrane domain at the N-terminal belonged to the type of secretory pathway protein, and JcHXK2 contained putative chloroplast targeting peptide. Quantitative real-time PCR (qRT-PCR) analysis revealed that all the four JcHXKs were expressed in different tissues of the leaves, roots, and seeds; however, JcHXK1 and JcHKL1 expression were higher in the roots, whereas JcHXK2 and JcHXK3 showed over-expression in the leaves and seeds, respectively. Furthermore, all the four JcHXKs were up-regulated in the leaves after cold stress at 12°C; however, only JcHXK3 remarkably demonstrated cold-induced expression in the roots, which reached the highest expression level at 12h (2.28-fold). According to the cis-acting element analysis results, JcHXK2 contained the most low temperature responsive elements, which was closely related to the cold resistance in J. curcas. A pET-28a-JcHXK2 prokaryotic recombinant expression vector was successfully constructed and a 57.0kDa protein was obtained, JcHXK2 revealed catalytic activity towards glucose and fructose, with a higher affinity for glucose than fructose. The subcellular localization assays revealed that JcHXK2 was localized in the chloroplast. The results of this study might provide theoretical foundation for further studies on gene cloning and functional verification of HXK family in J. curcas.

Cloning and functional analysis of the promoter of a stress-inducible gene (Zmap) in maize.

The anionic peroxidases play an important role in a variety of plant physiological processes. We characterized and isolated the Zmap promoter (PZmap) at the 5′ flanking region in order to better understand the regulatory mechanisms of Zmap gene expression. A series of PZmap deletion derivatives, termed a1 –a6, at positions −1694, −1394, −1138, −784, −527 and −221 from the translation start site were blended to the β-glucuronidase reporter gene. Agrobacterium-mediated transformation method was used to study each deletion construct in tobaccos. Sequence analysis showed that several cis-acting elements (MYB binding site, Box-II, a TGACG-element, a CGTCA-element and a low temperature responsive element) were located within the promoter. Deletion analysis suggested the sequence between −1,694 and −1394bp may contain cis-elements associated with GUS up regulation. The MYB binding site (-757) might act as a negative drought-responsive element. There might be repressor elements located in the region (−1,694 to −1394bp) to repress Zmap expression under 4°C. The characterized promoter would be an ideal candidate for genetic engineering for improving the resistance of maize to different stressors.

Cloning and function analysis of promoter of DcCDPK8 from Dendrobium catenatum

DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.

Maize WRKY Transcription Factor ZmWRKY106 Confers Drought and Heat Tolerance in Transgenic Plants.

WRKY transcription factors constitute one of the largest transcription factor families in plants, and play crucial roles in plant growth and development, defense regulation and stress responses. However, knowledge about this family in maize is limited. In the present study, we identified a drought-induced WRKY gene, ZmWRKY106, based on the maize drought de novo transcriptome sequencing data. ZmWRKY106 was identified as part of the WRKYII group, and a phylogenetic tree analysis showed that ZmWRKY106 was closer to OsWRKY13. The subcellular localization of ZmWRKY106 was only observed in the nucleus. The promoter region of ZmWRKY106 included the C-repeat/dehydration responsive element (DRE), low-temperature responsive element (LTR), MBS, and TCA-elements, which possibly participate in drought, cold, and salicylic acid (SA) stress responses. The expression of ZmWRKY106 was induced significantly by drought, high temperature, and exogenous abscisic acid (ABA), but was weakly induced by salt. Overexpression of ZmWRKY106 improved the tolerance to drought and heat in transgenic Arabidopsis by regulating stress-related genes through the ABA-signaling pathway, and the reactive oxygen species (ROS) content in transgenic lines was reduced by enhancing the activities of superoxide dismutase (SOD), peroxide dismutase (POD), and catalase (CAT) under drought stress. This suggested that ZmWRKY106 was involved in multiple abiotic stress response pathways and acted as a positive factor under drought and heat stress.

Cotton Late Embryogenesis Abundant (LEA2) Genes Promote Root Growth and Confer Drought Stress Tolerance in Transgenic Arabidopsis thaliana.

Late embryogenesis abundant (LEA) proteins play key roles in plant drought tolerance. In this study, 157, 85 and 89 candidate LEA2 proteins were identified in G. hirsutum, G. arboreum and G. raimondii respectively. LEA2 genes were classified into 6 groups, designated as group 1 to 6. Phylogenetic tree analysis revealed orthologous gene pairs within the cotton genome. The cotton specific LEA2 motifs identified were E, R and D in addition to Y, K and S motifs. The genes were distributed on all chromosomes. LEA2s were found to be highly enriched in non-polar, aliphatic amino acid residues, with leucine being the highest, 9.1% in proportion. The miRNA, ghr-miR827a/b/c/d and ghr-miR164 targeted many genes are known to be drought stress responsive. Various stress-responsive regulatory elements, ABA-responsive element (ABRE), Drought-responsive Element (DRE/CRT), MYBS and low-temperature-responsive element (LTRE) were detected. Most genes were highly expressed in leaves and roots, being the primary organs greatly affected by water deficit. The expression levels were much higher in G. tomentosum as opposed to G. hirsutum. The tolerant genotype had higher capacity to induce more of LEA2 genes. Over expression of the transformed gene Cot_AD24498 showed that the LEA2 genes are involved in promoting root growth and in turn confers drought stress tolerance. We therefore infer that Cot_AD24498, CotAD_20020, CotAD_21924 and CotAD_59405 could be the candidate genes with profound functions under drought stress in upland cotton among the LEA2 genes. The transformed Arabidopsis plants showed higher tolerance levels to drought stress compared to the wild types. There was significant increase in antioxidants, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) accumulation, increased root length and significant reduction in oxidants, Hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations in the leaves of transformed lines under drought stress condition. This study provides comprehensive analysis of LEA2 proteins in cotton thus forms primary foundation for breeders to utilize these genes in developing drought tolerant genotypes.

A Genome-Wide Identification of the WRKY Family Genes and a Survey of Potential WRKY Target Genes in Dendrobium officinale

The WRKY family, one of the largest families of transcription factors, plays important roles in the regulation of various biological processes, including growth, development and stress responses in plants. In the present study, 63 DoWRKY genes were identified from the Dendrobium officinale genome. These were classified into groups I, II, III and a non-group, each with 14, 28, 10 and 11 members, respectively. ABA-responsive, sulfur-responsive and low temperature-responsive elements were identified in the 1-k upstream regulatory region of DoWRKY genes. Subsequently, the expression of the 63 DoWRKY genes under cold stress was assessed, and the expression profiles of a large number of these genes were regulated by low temperature in roots and stems. To further understand the regulatory mechanism of DoWRKY genes in biological processes, potential WRKY target genes were investigated. Among them, most stress-related genes contained multiple W-box elements in their promoters. In addition, the genes involved in polysaccharide synthesis and hydrolysis contained W-box elements in their 1-k upstream regulatory regions, suggesting that DoWRKY genes may play a role in polysaccharide metabolism. These results provide a basis for investigating the function of WRKY genes and help to understand the downstream regulation network in plants within the Orchidaceae.

Genome-wide identification of galactinol synthase (GolS) genes in Solanum lycopersicum and Brachypodium distachyon

GolS genes stand as potential candidate genes for molecular breeding and/or engineering programs in order for improving abiotic stress tolerance in plant species. In this study, a total of six galactinol synthase (GolS) genes/proteins were retrieved for Solanum lycopersicum and Brachypodium distachyon. GolS protein sequences were identified to include glyco_transf_8 (PF01501) domain structure, and to have a close molecular weight (36.40–39.59kDa) and amino acid length (318–347 aa) with a slightly acidic pI (5.35–6.40). The sub-cellular location was mainly predicted as cytoplasmic. S. lycopersicum genes located on chr 1 and 2, and included one segmental duplication while genes of B. distachyon were only on chr 1 with one tandem duplication. GolS sequences were found to have well conserved motif structures. Cis-acting analysis was performed for three abiotic stress responsive elements, including ABA responsive element (ABRE), dehydration and cold responsive elements (DRE/CRT) and low-temperature responsive element (LTRE). ABRE elements were found in all GolS genes, except for SlGolS4; DRE/CRT was not detected in any GolS genes and LTRE element found in SlGolS1 and BdGolS1 genes. AU analysis in UTR and ORF regions indicated that SlGolS and BdGolS mRNAs may have a short half-life. SlGolS3 and SlGolS4 genes may generate more stable transcripts since they included AATTAAA motif for polyadenylation signal POLASIG2. Seconder structures of SlGolS proteins were well conserved than that of BdGolS. Some structural divergences were detected in 3D structures and predicted binding sites exhibited various patterns in GolS proteins.