Chunguo Wang

Ectopic Expression of DREB Transcription Factor, AtDREB1A, Confers Tolerance to Drought in Transgenic Salvia miltiorrhiza.

Drought decreases crop productivity more than any other type of environmental stress. Transcription factors (TFs) play crucial roles in regulating plant abiotic stress responses. The Arabidopsis thaliana gene DREB1A/CBF3, encoding a stress-inducible TF, was introduced into Salvia miltiorrhiza Ectopic expression of AtDREB1A resulted in increased drought tolerance, and transgenic lines had higher relative water content and Chl content, and exhibited an increased photosynthetic rate when subjected to drought stress. AtDREB1A transgenic plants generally displayed lower malondialdehyde (MDA), but higher superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under drought stress. In particular, plants with ectopic AtDREB1A expression under the control of the stress-induced RD29A promoter exhibited more tolerance to drought compared with p35S::AtDREB1A transgenic plants, without growth inhibition or phenotypic aberrations. Differential gene expression profiling of wild-type and pRD29A::AtDREB1A transgenic plants following drought stress revealed that the expression levels of various genes associated with the stress response, photosynthesis, signaling, carbohydrate metabolism and protein protection were substantially higher in transgenic plants. In addition, the amount of salvianolic acids and tanshinones was significantly elevated in AtDREB1A transgenic S. miltiorrhiza roots, and most of the genes in the related biosynthetic pathways were up-regulated. Together, these results demonstrated that inducing the expression of a TF can effectively regulate multiple genes in the stress response pathways and significantly improve the resistance of plants to abiotic stresses. Our results also suggest that genetic manipulation of a TF can improve production of valuable secondary metabolites by regulating genes in associated pathways.

Modulating AtDREB1C Expression Improves Drought Tolerance in Salvia miltiorrhiza

Dehydration responsive element binding proteins are transcription factors of the plant-specific AP2 family, many of which contribute to abiotic stress responses in several plant species. We investigated the possibility of increasing drought tolerance in the traditional Chinese medicinal herb, Salvia miltiorrhiza, through modulating the transcriptional regulation of AtDREB1C in transgenic plants under the control of a constitutive (35S) or drought-inducible (RD29A) promoter. AtDREB1C transgenic S. miltiorrhiza plants showed increased survival under severe drought conditions compared to the non-transgenic wild-type (WT) control. However, transgenic plants with constitutive overexpression of AtDREB1C showed considerable dwarfing relative to WT. Physiological tests suggested that the higher chlorophyll content, photosynthetic capacity, and superoxide dismutase, peroxidase, and catalase activity in the transgenic plants enhanced plant drought stress resistance compared to WT. Transcriptome analysis of S. miltiorrhiza following drought stress identified a number of differentially expressed genes (DEGs) between the AtDREB1C transgenic lines and WT. These DEGs are involved in photosynthesis, plant hormone signal transduction, phenylpropanoid biosynthesis, ribosome, starch and sucrose metabolism, and other metabolic pathways. The modified pathways involved in plant hormone signaling are thought to be one of the main causes of the increased drought tolerance of AtDREB1C transgenic S. miltiorrhiza plants.

Characteristics of cytosine methylation status and methyltransferase genes in the early development stage of cauliflower (Brassica oleracea L. var. botrytis)

DNA methylation is one of the most important epigenetic modifications involved in the development and differentiation in plants. Hypocotyl and cotyledon are the two major tissues of cauliflower (Brassica oleracea L. var. botrytis) seedlings. Both tissues show significantly different tissue specificity and regenerative abilities in vitro. However, the characteristics of DNA methylation modification and its roles in regulating the organ development in cauliflower remain largely unknown. In the present study, the DNA methylation status between the hypocotyl and cotyledon of cauliflower seedlings were analyzed. The results indicated that although the hypocotyl and cotyledon of cauliflower seedlings share the same genome, the genomic DNA methylation levels and patterns at CCGG sites were different. Compared with the cotyledon, the hypocotyl showed higher DNA methylation level, and more loci showing methylation pattern adjustments were also discovered. Twelve loci with changes of DNA methylation patterns were further explored. The quantitative expression analysis indicated that eight out of twelve sequenced fragments showed differential expression between the hypocotyl and cotyledon, of which the expression of six sequences was identified to be negative correlation with their DNA methylation status. In addition, three main DNA methyltransferase genes MET1, CMT3 and DRM were first explored in cauliflower. The results indicated that the expression of these three genes was closely associated with the different DNA methylation status in the hypocotyl and cotyledon. These findings provided more information to further explore the roles of DNA methylation modification in tissue differentiation and development of cauliflower.

Cloning and transcript analyses of the chimeric gene associated with cytoplasmic male sterility in cauliflower (Brassica oleracea var. botrytis)

To gain a preliminary understanding of cytoplasmic male sterility (CMS) in cauliflower NKC-A, seven primer pairs were designed to amplify atpa, atp6, atp9, coxI, coxII, orfB and orf224, which have been reported in other CMS systems. The amplification pattern of orfB is different in the CMS cauliflower and its maintainer line NKC-B. Results suggest that orfB only expresses in the CMS line, in both flowers and leaves. Sequence analysis indicated that orfB is highly homologous with orf138, a chimeric gene believed to closely associate with Ogura cytoplasmic male sterility in radish. Full-length sequence was obtained to elucidate the molecular basis of orfB. The sequence includes three open reading frames, trnfM, orf138 and orf158. Study of transcriptional patterns and RNA editing of this sequence revealed that orf138 and orf158 co-transcribe, similar to orf138 in Ogura CMS radish. Based on this, we concluded that the molecular basis of cytoplasmic male sterility in cauliflower NKC-A may be similar to that of the Ogura CMS in radish. However, the nuclear backgrounds and RNA editing of corresponding sequences in radish and cauliflower are different. The results of this study also provide important information for breeders selecting new CMS lines in cauliflower. A specific molecular marker was developed according to the CMS specific fragment in NKC-A.

Genome-Wide Identification of AP2/ERF Transcription Factors in Cauliflower and Expression Profiling of the ERF Family under Salt and Drought Stresses

The AP2/ERF transcription factors (TFs) comprise one of the largest gene superfamilies in plants. These TFs perform vital roles in plant growth, development, and responses to biotic and abiotic stresses. In this study, 171 AP2/ERF TFs were identified in cauliflower (Brassica oleracea L. var. botrytis), one of the most important horticultural crops in Brassica. Among these TFs, 15, 9, and 1 TFs were classified into the AP2, RAV, and Soloist family, respectively. The other 146 TFs belong to ERF family, which were further divided into the ERF and DREB subfamilies. The ERF subfamily contained 91 TFs, while the DREB subfamily contained 55 TFs. Phylogenetic analysis results indicated that the AP2/ERF TFs can be classified into 13 groups, in which 25 conserved motifs were confirmed. Some motifs were group- or subgroup- specific, implying that they are significant to the functions of the AP2/ERF TFs of these clades. In addition, 35 AP2/ERF TFs from the 13 groups were selected randomly and then used for expression pattern analysis under salt and drought stresses. The majority of these AP2/ERF TFs exhibited positive responses to these stress conditions. In specific, Bra-botrytis-ERF054a, Bra-botrytis-ERF056, and Bra-botrytis-CRF2a demonstrated rapid responses. By contrast, six AP2/ERF TFs were showed to delay responses to both stresses. The AP2/ERF TFs exhibiting specific expression patterns under salt or drought stresses were also confirmed. Further functional analysis indicated that ectopic overexpression of Bra-botrytis-ERF056 could increase tolerance to both salt and drought treatments. These findings provide new insights into the AP2/ERF TFs present in cauliflower, and offer candidate AP2/ERF TFs for further studies on their roles in salt and drought stress tolerance.

Small RNA Sequencing Reveals Differential miRNA Expression in the Early Development of Broccoli (Brassica oleracea var. italica) Pollen

Pollen development is an important and complex biological process in the sexual reproduction of flowering plants. Although the cytological characteristics of pollen development are well defined, the regulation of its early stages remains largely unknown. In the present study, miRNAs were explored in the early development of broccoli (Brassica oleracea var. italica) pollen. A total of 333 known miRNAs that originated from 235 miRNA families were detected. Fifty-five novel miRNA candidates were identified. Sixty of the 333 known miRNAs and 49 of the 55 predicted novel miRNAs exhibited significantly differential expression profiling in the three distinct developmental stages of broccoli pollen. Among these differentially expressed miRNAs, miRNAs that would be involved in the developmental phase transition from uninucleate microspores to binucleate pollen grains or from binucleate to trinucleate pollen grains were identified. miRNAs that showed significantly enriched expression in a specific early stage of broccoli pollen development were also observed. In addition, 552 targets for 127 known miRNAs and 69 targets for 40 predicted novel miRNAs were bioinformatically identified. Functional annotation and GO (Gene Ontology) analysis indicated that the putative miRNA targets showed significant enrichment in GO terms that were related to plant organ formation and morphogenesis. Some of enriched GO terms were detected for the targets directly involved in plant male reproduction development. These findings provided new insights into the functions of miRNA-mediated regulatory networks in broccoli pollen development.

Genome-wide identification and characterization of miRNAs in the hypocotyl and cotyledon of cauliflower (Brassica oleracea L. var. botrytis) seedlings

MicroRNAs (miRNAs) are a class of small endogenous, non-coding RNAs that have key regulatory functions in plant growth, development, and other biological processes. Hypocotyl and cotyledon are the two major tissues of cauliflower (Brassica oleracea L. var. botrytis) seedlings. Tissue culture experiments have indicated that the regenerative abilities of these two tissues are significantly different. However, the characterization of miRNAs and their roles in regulating organ development in cauliflower remain unexplored. In the present study, two small RNA libraries were sequenced by Solexa sequencing technology. 99 known miRNAs belonging to 28 miRNA families were identified, in which 6 miRNA families were detected only in Brassicaceae. A total of 162 new miRNA sequences with single nucleotide substitutions corresponding to the known miRNAs, and 32 potentially novel miRNAs were also first discovered. Comparative analysis indicated that 42 of 99 known miRNAs and 17 of 32 novel miRNAs exhibited significantly differential expression between hypocotyl and cotyledon, and the differential expression of several miRNAs was further validated by stem-loop RT-PCR. In addition, 235 targets for 89 known miRNAs and 198 targets for 24 novel miRNAs were predicted, and their functions were further discussed. The expression patterns of several representative targets were also confirmed by qRT-PCR analysis. The results identified that the transcriptional expression patterns of miRNAs were negatively correlated with their targets. These findings gave new insights into the characteristics of miRNAs in cauliflower, and provided important clues to elucidate the roles of miRNAs in the tissue differentiation and development of cauliflower.

Transcriptome Analyses from Mutant Salvia miltiorrhiza Reveals Important Roles for SmGASA4 during Plant Development

Salvia miltiorrhiza (S. miltiorrhiza) is an important Chinese herb that is derived from the perennial plant of Lamiaceae, which has been used to treat neurasthenic insomnia and cardiovascular disease. We produced a mutant S. miltiorrhiza (MT), from breeding experiments, that possessed a large taproot, reduced lateral roots, and defective flowering. We performed transcriptome profiling of wild type (WT) and MT S. miltiorrhiza using second-generation Illumina sequencing to identify differentially expressed genes (DEGs) that could account for these phenotypical differences. Of the DEGs identified, we investigated the role of SmGASA4, the expression of which was down-regulated in MT plants. SmGASA4 was introduced into Arobidopsis and S. militiorrhiza under the control of a CaMV35S promoter to verify its influence on abiotic stress and S. miltiorrhiza secondary metabolism biosynthesis. SmGASA4 was found to promote flower and root development in Arobidopsis. SmGASA4 was also found to be positively regulated by Gibberellin (GA) and significantly enhanced plant resistance to salt, drought, and paclobutrazol (PBZ) stress. SmGASA4 also led to the up-regulation of the genes involved in salvianolic acid biosynthesis, but inhibited the expression of the genes involved in tanshinone biosynthesis. Taken together, our results reveal SmGASA4 as a promising candidate gene to promote S. miltiorrhiza development.

Curd development associated gene (CDAG1) in cauliflower (Brassica oleracea L. var. botrytis) could result in enlarged organ size and increased biomass

The curd is a specialized organ and the most important product organ of cauliflower (Brassica oleracea L. var. botrytis). However, the mechanism underlying the regulation of curd formation and development remains largely unknown. In the present study, a novel homologous gene containing the Organ Size Related (OSR) domain, namely, CDAG1 (Curd Development Associated Gene 1) was identified in cauliflower. Quantitative analysis indicated that CDAG1 showed significantly higher transcript levels in young tissues. Functional analysis demonstrated that the ectopic overexpression of CDAG1 in Arabidopsis and cauliflower could significantly promote organ growth and result in larger organ size and increased biomass. Organ enlargement was predominantly due to increased cell number. In addition, 228 genes involved in the CDAG1-mediated regulatory network were discovered by transcriptome analysis. Among these genes, CDAG1 was confirmed to inhibit the transcriptional expression of the endogenous OSR genes, ARGOS and ARL, while a series of ethylene-responsive transcription factors (ERFs) were found to increased expression in 35S:CDAG1 transgenic Arabidopsis plants. This implies that CDAG1 may function in the ethylene-mediated signal pathway. These findings provide new insight into the function of OSR genes, and suggest potential applications of CDAG1 in breeding high-yielding crops.

Ectopic Overexpression of bol-miR171b Increases Chlorophyll Content and Results in Sterility in Broccoli (Brassica oleracea L var. italica)

MiR171 plays pleiotropic roles in the growth and development of several plant species. However, the mechanism underlying the miR171-mediated regulation of organ development in broccoli remains unknown. In this study, bol-miR171b was characterized and found to be differentially expressed in various broccoli organs. The ectopic overexpression of bol-miR171b in Arabidopsis affected the leaf and silique development of transgenic lines. In particular, the chlorophyll content of leaves from overexpressed bol-miR171b transgenic Arabidopsis was higher than that of the vector controls. The fertility and seed yield of Arabidopsis with overexpressed bol-miR171b were markedly lower than those of the vector controls. Similarly, overexpressed bol-miR171b transgenic broccoli exhibited dark green leaves with high chlorophyll content, and nearly all of the flowers were sterile. These results demonstrated that overexpression of bol-miR171b could increase the chlorophyll content of transgenic plants. Degradome sequencing was conducted to identify the targets of bol-miR171b. Two members of the GRAS gene family, BolSCL6 and BolSCL27, were cleaved by bol-miR171b-3p in broccoli. In addition to the genes targeted by bol-miR171b-3p, adenylylsulfate reductase 3 ( APSR3), which played important roles in plant sulfate assimilation and reduction, was speculated to be cleaved by bol-miR171b-5p, suggesting that the star sequence of bol-miR171b may also have functions in broccoli. Comparative transcriptome analysis further revealed that the genes involved in chloroplast development and sulfate homeostasis should participate in the bol-miR171b -mediated regulatory network. Taken together, these findings provided new insights into the function and regulation of bol-miR171b in broccoli and indicated the potential of bol-miR171b as a small RNA molecule that increased leaf chlorophyll in plants by genetic engineering.