Genome-Wide Mining of CULLIN E3 Ubiquitin Ligase Genes from Uncaria rhynchophylla.

CULLIN (CUL) protein is a subtype of E3 ubiquitin ligase that is involved in a variety of biological processes and responses to stress in plants. In Uncaria rhynchophylla, the CUL gene family has not been identified and its role in plant development, stress response and secondary metabolite synthesis has not been studied. In this study, 12 UrCUL gene members all contained the typical N-terminal domain and C-terminal domain identified from the U. rhynchophylla genome and were classified into four subfamilies based on the phylogenetic relationship with CULs in Arabidopsis thaliana. They were unevenly distributed on eight chromosomes but had a similar structural composition in the same subfamily, indicating that they were relatively conserved and potentially had similar gene functions. An interspecific and intraspecific collinearity analysis showed that fragment duplication played an important role in the evolution of the CUL gene family. The analysis of the cis-acting elements suggests that the UrCULs may play an important role in various biological processes, including the abscisic acid (ABA) response. To investigate this hypothesis, we treated the roots of U. rhynchophylla tissue-cultured seedlings with ABA. The expression pattern analysis showed that all the UrCUL genes were widely expressed in roots with various expression patterns. The co-expression association analysis of the UrCULs and key enzyme genes in the terpenoid indole alkaloid (TIA) synthesis pathway revealed the complex expression patterns of 12 UrCUL genes and some key TIA enzyme genes, especially UrCUL1, UrCUL1-likeA, UrCUL2-likeA and UrCUL2-likeB, which might be involved in the biosynthesis of TIAs. The results showed that the UrCULs were involved in the response to ABA hormones, providing important information for elucidating the function of UrCULs in U. rhynchophylla. The mining of UrCULs in the whole genome of U. rhynchophylla provided new information for understanding the CUL gene and its function in plant secondary metabolites, growth and development.

Transcriptome-Wide Integrated Analysis of the PgGT25-04 Gene in Controlling Ginsenoside Biosynthesis in Panax ginseng.

Panax ginseng is a valuable medicinal herb of the Araliaceae family with various pharmacological activities. The Trihelix transcription factors family is involved in growth and secondary metabolic processes in plants, but no studies have been reported on the involvement of Trihelix genes in secondary metabolic processes in ginseng. In this study, weighted co-expression network analysis, correlation analysis between PgGTs and ginsenosides and key enzyme genes, and interaction network analysis between PgGTs and key enzyme genes were used to screen out the PgGT25-04 gene, which was negatively correlated with ginsenoside synthesis. Using ABA treatment of ginseng hair roots, PgGT genes were found to respond to ABA signals. Analysis of the sequence characteristics and expression pattern of the PgGT25-04 gene in ginseng revealed that its expression is spatiotemporally specific. The interfering vector pBI121-PgGT25-04 containing the PgGT25-04 gene was constructed, and the ginseng adventitious roots were transformed using the Agrobacterium-mediated method to obtain the pBI121-PgGT25-04 positive hairy root monocot line. The saponin contents of positive ginseng hair roots were measured by HPLC, and the changes in PgGT25-04 and key enzyme genes in positive ginseng hair roots were detected via fluorescence quantitative RT-PCR. These results preliminarily identified the role of the PgGT25-04 gene in the secondary metabolism of ginseng in Jilin to provide a theoretical basis for the study of Trihelix transcription factors in Panax ginseng.

Genome-wide identification and expression analyses of late embryogenesis abundant (LEA) gene family in tobacco (Nicotiana tabacum L.) reveal their function in abiotic stress responses.

Late embryogenesis abundant (LEA) proteins play an important role in plant growth and response to abiotic stresses. However the late embryogenesis abundant (LEA) gene family in Nicotiana tabacum has not been systematically studied. In this study, 123 NtLEA genes were identified in Nicotiana tabacum, and divided into 8 groups, including LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, DHN (dehydratin) and SMP (Seed Maturation Protein). The LEA_2 group is the most abundant of the NtLEA family. The gene structure, conserved motifs, subcellular localization and physicochemical properties of the NtLEA genes were analyzed. RNA-seq and qPCR analyses showed that the NtLEA genes were significantly induced under two different abiotic stresses and showed different expression patterns. The expression patterns of 35 NtLEA genes responding to ABA and 3 NtLEA genes responding to NaCl abiotic stress, respectively, were characterized. The protein-protein interaction network revealed that most NtLEA proteins (>78%) had the potential function to enhance tobacco resistance to abiotic stress. The transcriptional regulatory network showed that 21 transcription factor families were involved in regulating the expression of the NtLEA genes. These results are beneficial for future studies of the function of the NtLEA genes.

Systematic screening and analysis of bZIP transcription factors in Glycyrrhiza uralensis and their response to ABA stress / 药学学报

Licorice, one of the most commonly used medicinal materials in China, grows mainly in arid and semi-arid regions and has important economic and ecological values. Basic leucine zipper (bZIP) transcription factors in plants play an important role in regulating biological or abiotic stress responses, growth, and secondary metabolite synthesis. bZIP transcription factors in the published whole genome database of Glycyrrhiza uralensis were identified using bZIP sequences found in Arabidopsis thaliana genome as reference, and ABA-dependent bZIP genes were identified by using Illumina high-throughput sequencing. The physical and chemical properties, structure of the encoded proteins, and the gene expression patterns with exogenous ABA stress were analyzed. A total of 69 bZIP transcription factor genes were identified in G. uralensis, named Gubzip1-69, and they were divided into 10 subfamilies (A-I and S) according to their similarity to bZIPs of A. thaliana. By calculating the relative expression levels of the 69 GubZIPs genes under different concentrations of exogenous ABA stress, genes that may be involved in the regulation of ABA signaling pathways were identified, namely GubZIP1, GubZIP5, GubZIP8, GubZIP30, GubZIP33 and GubZIP56. The results of expression pattern analysis of these GubZIPs genes under exogenous ABA stress showed that the expression pattern of GubZIPs genes changed significantly with 50 mg·L-1 ABA. The relative expression levels of these genes decreased 3 h after treatment, and gradually increased 6 h after treatment. Except for GubZIP8, the relative expression levels of these genes were significantly increased after 12 h. Further research on the function of bZIP transcription factors of G. uralensis and elucidating their regulatory mechanisms should be of interest and will provide a scientific basis for cultivating high-quality cultivars of G. uralensis through molecular breeding methods.

Identification of a 193 bp promoter region of TaNRX1-D gene from common wheat that contributes to osmotic or ABA stress inducibility in transgenic Arabidopsis.

BACKGROUND: Cloning and characterizing the drought-inducible promoters is essential for their use in crop resistance's genetic improvement. Previous studies have shown that the TaNRX1-D gene participates in regulating the response of wheat to drought stress. However, its promoter has not yet been identified. OBJECTIVE: In this study, we aimed to characterize the promoter of the TaNRX1-D gene. METHODS: The promoter of TaNRX1-D (named P0, 2081 bp) was isolated from common wheat with several cis-acting elements that regulate in response to abiotic stresses and some core cis-acting elements. Functional verification of the promoter, eight 5'-deletion fragments of TaNRX1-D promoter, was fused to the ß-glucuronidase (GUS) gene P0::GUS ~ P7::GUS and transformed into Arabidopsis, respectively. Agrobacterium-mediated GUS transient assay the P6a and P6b promoter regions in tobacco leaves under normal, osmotic or ABA stress. RESULTS: Activity analysis of the full-length promoter (P0) showed that the intensity of stronger ß-glucuronidase (GUS) staining in the roots and leaves was obtained during the growth of transgenic Arabidopsis. P0::GUS displayed the GUS activity was much higher in the roots and leaves than in other parts of the transgenic plant under normal conditions, which was similarly within wheat. Analysis of the 5'-deletion fragments revealed that P0::GUS ~ P6::GUS responded well upon exposure to osmotic (polyethylene glycol-6000, PEG6000) and abscisic acid (ABA) stress treatments and expressed significantly higher GUS activity than the CaMV35S promoter (35S::GUS), while P7::GUS did not. GUS transient assay in tobacco leaves showed that the GUS activities of P6a and P6b were lower than P6 in the PEG6000 and ABA stresses. CONCLUSION: The 193 bp (P6) segment was considered the core region of TaNRX1-D responding to PEG6000 or ABA treatment. GUS activity assay in transgenic Arabidopsis showed that this segment was sufficient for the PEG6000 or ABA stress response. The identified 193 bp promoter of TaNRX1-D in this study will help breed osmotic or ABA tolerant crops. The 36 bp segment between P6 and P6b (-193 to -157 bp) was considered the critical sequence for the TaNRX1-D gene responding to PEG6000 or ABA treatment.

Functional analysis of CgWRKY57 from Cymbidium goeringii in ABA response.

BACKGROUND: The orchid is one of the top ten Chinese flowers and has high ornamental value and elegant color. However, orchids are vulnerable to abiotic stresses during their growth and development, and the molecular mechanism of the abiotic stress response in orchids is unclear. WRKY proteins belong to a transcription factor family that plays important roles in biotic stress, abiotic stress, growth and development in plants, but little is known about the WRKY family in Cymbidium goeringii. METHODS: The specific fragment of the CgWRKY57 gene of C. goeringii was analyzed by bioinformatics. The expression of the CgWRKY57 gene of C. goeringii under 4 °C, 42 °C water and ABA stress as well as different tissues was detected by real-time fluorescence quantitative PCR. CgWRKY57 gene was overexpressed in wild type Arabidopsis thaliana by inflorescence infection method, and the function of transgenic lines under ABA stress was analyzed. RESULTS: CgWRKY57 was cloned from C. goeringii and found to encode 303 amino acids. The CgWRKY57 protein is an acidic, nonsecreted hydrophilic protein without a signal peptide or transmembrane domain. The CgWRKY57 protein is located to the nucleus and may function intracellularly according to its predicted subcellular localization. A domain analysis and homology comparison showed that the CgWRKY57 protein has a "WRKYGQK" domain and belongs to Group III of the WRKY family, and a phylogenetic analysis demonstrated that CgWRKY57 is closely related to OsWRKY47. CgWRKY57 was expressed in the roots, stems, leaves and floral organs of C. goeringii, and its expression level was highest in the roots according to real-time qPCR analysis. There were significant differences in CgWRKY57 expression under 4 °C, 42 °C ABA and water stress treatments, and its expression changed greatly under ABA stress. The expression of CgWRKY57 in transgenic plants was significantly higher than that in wild type plants under ABA stress, and the root length and germination rate were reduced in transgenic plants compared to wild type plants. CONCLUSIONS: These results indicate that CgWRKY57 overexpression is responsive to ABA stress, and they provide a foundation for future analyses of the biological functions of the WRKY family in C. goeringii.

Comprehensive Analysis of the Cadmium Tolerance of Abscisic Acid-, Stress- and Ripening-Induced Proteins (ASRs) in Maize.

In plants, abscisic acid-, stress-, and ripening-induced (ASR) proteins have been shown to impart tolerance to multiple abiotic stresses such as drought and salinity. However, their roles in metal stress tolerance are poorly understood. To screen plant Cd-tolerance genes, the yeast-based gene hunting method which aimed to screen Cd-tolerance colonies from maize leaf cDNA library hosted in yeast was carried out. Here, maize ZmASR1 was identified to be putative Cd-tolerant through this survival screening strategy. In silico analysis of the functional domain organization, phylogenetic classification and tissue-specific expression patterns revealed that maize ASR1 to ASR5 are typical ASRs with considerable expression in leaves. Further, four of them were cloned for testifying Cd tolerance using yeast complementation assay. The results indicated that they all confer Cd tolerance in Cd-sensitive yeast. Then they were transiently expressed in tobacco leaves for subcellular localization analysis and for Cd-challenged lesion assay, continuously. The results demonstrated that all 4 maize ASRs tested are localized to the cell nucleus and cytoplasm in tobacco leaves. Moreover, they were confirmed to be Cd-tolerance genes in planta through lesion analysis in Cd-infiltrated leaves transiently expressing them. Taken together, our results demonstrate that maize ASRs play important roles in Cd tolerance, and they could be used as promising candidate genes for further functional studies toward improving the Cd tolerance in plants.

Genome-wide analysis of maize CONSTANS-LIKE gene family and expression profiling under light/dark and abscisic acid treatment.

The CONSTANS-LIKE (COL) gene has an important role both in regulation flowering through photoperiodic control and response to abiotic stress. In the present study, we performed a genome-wide analysis of maize COL gene family and identified 19 non-redundant ZmCOL genes, which were unequally distributed on ten maize chromosomes. Analysis of compound phylogenetic tree (maize, sorghum, rice and Arabidopsis) showed high bootstrap, as well as conserved domain and semblable gene structures among members within the same clade, revealing that COL genes in same clade were from the common ancestral and prior to the divergence of monocots and dicots lineages. Calculation of Ka/Ks ratio demonstrated that most duplicated ZmCOLs experienced purifying selection, which suggested limited functional divergence after duplication events. Comparing interspecies gene collinearity between three major grasses species, extensive microsynteny was detected among maize, sorghum and rice COL-containing segments. Additionally, several light-responsive and one ABA-responsive cis-elements could be detected for ZmCOL genes in group A. Therefore, qRT-PCR was performed to explore the expression patterns of ZmCOL genes in group A under light/dark conditions and ABA treatment. Our results laid the foundation for functional characterization of ZmCOL proteins and uncovering the biological roles of COL genes in response to stress in future.

Overexpression of MpCYS4, A Phytocystatin Gene from Malus prunifolia (Willd.) Borkh., Enhances Stomatal Closure to Confer Drought Tolerance in Transgenic Arabidopsis and Apple.

Phytocystatins (PhyCys) comprise a group of inhibitors for cysteine proteinases in plants. They play a wide range of important roles in regulating endogenous processes and protecting plants against various environmental stresses, but the underlying mechanisms remain largely unknown. Here, we detailed the biological functions of MpCYS4, a member of cystatin genes isolated from Malus prunifolia. This gene was activated under water deficit, heat (40°C), exogenous abscisic acid (ABA), or methyl viologen (MV) (Tan et al., 2014a). At cellular level, MpCYS4 protein was found to be localized in the nucleus, cytoplasm, and plasma membrane of onion epidermal cells. Recombinant MpCYS4 cystatin expressed in Escherichia coli was purified and it exhibited cysteine protease inhibitor activity. Transgenic overexpression of MpCYS4 in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica) led to ABA hypersensitivity and series of ABA-associated phenotypes, such as enhanced ABA-induced stomatal closing, altered expression of many ABA/stress-responsive genes, and enhanced drought tolerance. Taken together, our results demonstrate that MpCYS4 is involved in ABA-mediated stress signal transduction and confers drought tolerance at least in part by enhancing stomatal closure and up-regulating the transcriptional levels of ABA- and drought-related genes. These findings provide new insights into the molecular mechanisms by which phytocystatins influence plant growth, development, and tolerance to stress.