Transcriptome and metabolome analysis of the developmental changes in Cynanchum thesioides anther.

Cynanchum thesioides, a xerophytic species utilized both as a medicinal herb and a food source, plays a significant role in arid and desert ecosystem management. Its inflorescence is an umbellate cyme, each carrying nearly a thousand flowers; however, its fruiting rate remains remarkably low. The normal development of the anther is a necessary prerequisite for plants to produce seeds. However, our understanding of the anther development process in Cynanchum thesioides remains limited. To better understand the pollen development process in Cynanchum thesioides, the stages of pollen development were determined through paraffin sectioning, and observations were made on the distribution characteristics of polysaccharides and lipid droplets in the pollen development of Cynanchum thesioides using Periodic Acid-Schiff stain (PAS) and 0.5% Sudan Black B tissue staining. Concurrently, the gene expression patterns and metabolite profiles were delineated across various developmental stages of Cynanchum thesioides anthers (T1: microspore stage, T2: tetrad stage, T3: mononuclear stage, and T4: maturation stage). The findings revealed that Cynanchum thesioides pollen is in an aggregate form. Polysaccharides gradually accumulate during maturation and lipid droplets form a surrounding membrane, thereby preventing pollen dispersion. Furthermore, transcriptomic and metabolomic analyses across distinct developmental phases uncovered a plethora of differentially expressed genes and metabolites associated with the flavonoid biosynthesis pathway. Flavonoid levels exhibited dynamic changes concurrent with anther development, aligning with the gene regulatory patterns of the corresponding biosynthetic pathways. The study identified 63 differentially accumulated flavonoid compounds and 21 differentially expressed genes associated with flavonoid biosynthesis. Weighted gene co-expression network analysis revealed six MYB and ten bHLH transcription factors as key candidates involved in flavonoid biosynthesis, with CtbHLH (Cluster-6587.1050) and CtMYB (Cluster-6587.31743) specifically regulating structural genes within the pathway. These findings underscore the pivotal role of flavonoid biosynthesis in anther development of Cynanchum thesioides. In conclusion, this research offers a comprehensive insight into the anther development process in Cynanchum thesioides.

Transcriptome-wide identification of WRKY transcription factors and their expression profiles under different stress in Cynanchum thesioides.

Cynanchum thesioides (Freyn) K. Schum. is an important economic and medicinal plant widely distributed in northern China. WRKY transcription factors (TFs) play important roles in plant growth, development and regulating responses. However, there is no report on the WRKY genes in Cynanchum thesioides. A total of 19 WRKY transcriptome sequences with complete ORFs were identified as WRKY transcriptome sequences by searching for WRKYs in RNA sequencing data. Then, the WRKY genes were classified by phylogenetic and conserved motif analysis of the WRKY family in Cynanchum thesioides and Arabidopsis thaliana. qRT-PCR was used to determine the expression patterns of 19 CtWRKY genes in different tissues and seedlings of Cynanchum thesioides under plant hormone (ABA and ETH) and abiotic stresses (cold and salt). The results showed that 19 CtWRKY genes could be divided into groups I-III according to their structure and phylogenetic characteristics, and group II could be divided into five subgroups. The prediction of CtWRKY gene protein interactions indicates that CtWRKY is involved in many biological processes. In addition, the CtWRKY gene was differentially expressed in different tissues and positively responded to abiotic stress and phytohormone treatment, among which CtWRKY9, CtWRKY18, and CtWRKY19 were significantly induced under various stresses. This study is the first to identify the WRKY gene family in Cynanchum thesioides, and the systematic analysis lays a foundation for further identification of the function of WRKY genes in Cynanchum thesioides.

Responses of transcriptome and metabolome in the roots of Pugionium cornutum (L.) Gaertn to exogenously applied phthalic acid.

BACKGROUND: The yield and quality of Pugionium cornutum (L.) Gaertn., a healthy, green vegetable with low sugar and high protein contents and high medicinal value, is severely affected by autotoxicity, which is a leading factor in the formation of plant disease. To help characterize the autotoxicity mechanism of P. cornutum (L.) Gaertn., we performed transcriptomic and metabolic analysis of the roots of P. cornutum (L.) Gaertn. response to phthalic acid, an autotoxin from P. cornutum (L.) Gaertn. RESULTS: In this study, high-throughput sequencing of nine RNA-seq libraries generated from the roots.of P. cornutum (L.) Gaertn. under different phthalic acid treatments yielded 37,737 unigenes. In total, 1085 (703 upregulated and 382 downregulated) and 5998 (4385 upregulated and 1613 downregulated) DEGs were identified under 0.1 and 10 mmol·L- 1 phthalic acid treatment, respectively, compared with the control treatment. Glutathione metabolism was among the top five important enriched pathways. In total, 457 and 435 differentially accumulated metabolites were detected under 0.1 and 10 mmol·L- 1 phthalic acid treatment compared with the control, respectively, of which 223 and 253, respectively, increased in abundance. With the increase in phthalic acid concentration, the accumulation of ten metabolites increased significantly, while that of four metabolites decreased significantly, and phthalic acid, dambonitol, 4-hydroxy-butyric acid, homocitrulline, and ethyl ß-D-glucopyranoside were 100 times more abundant under the 10 mmol·L- 1 phthalic acid treatment than under the control. Seventeen differentially expressed genes significantly associated with phthalic acid content were identified. In addition, the L-histidinol content was highest under 0.1 mmol·L- 1 phthalic acid, and a total of eleven differentially expressed genes were significantly positively correlated with the L-histidinol content, all of which were annotated to heat shock proteins, aquaporins and cysteine proteases. CONCLUSIONS: Accumulation of autotoxins altered the metabolic balance in P. cornutum (L.) Gaertn. and influenced water absorption and carbon and nitrogen metabolism. These important results provide insights into the formation mechanisms of autotoxicity and for the subsequent development of new control measures to improve the production and quality of replanted plants.

TMT-based proteomic analysis of liquorice root in response to drought stress.

BACKGROUND: Drought stress is a serious threat to land use efficiency and crop yields worldwide. Understanding the mechanisms that plants use to withstand drought stress will help breeders to develop drought-tolerant medicinal crops. Liquorice (Glycyrrhiza uralensis Fisch.) is an important medicinal crop in the legume family and is currently grown mostly in northwest China, it is highly tolerant to drought. Given this, it is considered an ideal crop to study plant stress tolerance and can be used to identify drought-resistant proteins. Therefore, to understand the effects of drought stress on protein levels of liquorice, we undertook a comparative proteomic analysis of liquorice seedlings grown for 10 days in soil with different relative water content (SRWC of 80%, 65%, 50% and 35%, respectively). We used an integrated approach of Tandem Mass Tag labeling in conjunction with LC-MS/MS. RESULTS: A total of 7409 proteins were identified in this study, of which 7305 total proteins could be quantified. There were 837 differentially expressed proteins (DEPs) identified after different drought stresses. Compared with CK, 123 DEPs (80 up-regulated and 43 down-regulated) were found in LS; 353 DEPs (254 up-regulated and 99 down-regulated) in MS; and 564 DEPs (312 up-regulated and 252 down-regulated) in SS.The number of differentially expressed proteins increased with increasing water stress, and the number of up-regulated proteins was higher than that of down-regulated proteins in the different drought stress treatments compared with the CK. Used systematic bioinformatics analysis of these data to identify informative proteins we showed that osmolytes such as cottonseed sugars and proline accumulated under light drought stress and improved resistance. Under moderate and severe drought stress, oxidation of unsaturated fatty acids and accumulation of glucose and galactose increased in response to drought stress. Under moderate and severe drought stress synthesis of the terpene precursors, pentacene 2,3-epoxide and ß-coumarin, was inhibited and accumulation of triterpenoids (glycyrrhetinic acid) was also affected. CONCLUSIONS: These data provide a baseline reference for further study of the downstream liquorice proteome in response to drought stress. Our data show that liquorice roots exhibit specific response mechanisms to different drought stresses.

Physiological responses and transcriptome analysis of the Kochia prostrata (L.) Schrad. to seedling drought stress.

Kochia prostrata is a good forage plant, which has important economic and ecological value in arid and semi-arid regions of China. Drought is one of the main factors affecting its productivity. At present, there are few studies on the mechanism of drought resistance. In order to reveal the changes of physiological and biochemical indexes, stomatal structure and gene expression profiles of Kochia prostrata under drought treatment, the classical determination method and high-throughput Illumina Hiseq sequencing platform were applied to the control group (CK) and drought treatment group of Kochia prostrata. The results showed that under the condition of moderate to mild drought stress, the SOD activity reached the maximum value of 350.68 U/g min on the 5th day of stress, and under the condition of severe drought stress, the SOD activity reached the maximum on the 2nd day of stress. The accumulation of Proline remained at a high level on the 5th day of stress, and there was at least one epidermal cell interval between the two adult stomatal of the leaf epidermis, so that the evaporation shell of each stomatal did not overlap, it ensures the efficient gas exchange of the stomatal, indicating that the Kochia prostrata has strong drought resistance. A total of 1,177.46 M reads were obtained by sequencing, with a total of 352.25 Gbp data and Q30 of 85%. In the differential gene annotation to the biological process (BP), a total of 261 GO terms were enriched in the up-regulated genes, and a total of 231 GO terms were enriched in the down-regulated genes. The differentially expressed genes (DEGs) were obtained in 27 KEGG metabolic pathways, which laid a foundation for revealing the molecular mechanism of drought tolerance.

De novo transcriptome assembly and co-expression network analysis of Cynanchum thesioides: Identification of genes involved in resistance to drought stress.

Cynanchum thesioides are upright, xerophytic shrubs that are widely distributed in arid and semi-arid areas of China, North Korea, Mongolia and Siberia. To date, little is known about the molecular mechanisms of drought resistance in C. thesioides. To better understand drought resistance, we used transcriptome analysis and Illumina sequencing technology on C. thesioides, to identify drought-responsive genes. Using de novo assembly 55,268 unigenes were identified from 207.58 Gb of clean data. Amongst these, 36,265 were annotated with gene descriptions, conserved domains, gene ontology terms and metabolic pathways. The sequencing results showed that genes that were differentially expressed (DEGs) under drought stress were enriched in pathways such as carbon metabolism, starch and sucrose metabolism, amino acid biosynthesis, phenylpropanoid biosynthesis and plant hormone signal transduction. Moreover, many functional genes were up-regulated under severe drought stress to enhance tolerance. Weighted gene co-expression network analysis showed that there were key hub genes related to drought stress. Hundreds of candidate genes were identified under severe drought stress, including transcriptional factors such as MYB, G2-like, ERF, C2H2, NAC, NF-X1, GRF, HD-ZIP, HB-other, HSF, C3H, GRAS, WRKY, bHLH and Trihelix. These data are a valuable resource for further investigation into the molecular mechanism for drought stress in C. thesioides and will facilitate exploration of drought resistance genes.