Light and Potassium Improve the Quality of Dendrobium officinale through Optimizing Transcriptomic and Metabolomic Alteration.

BACKGROUND: Dendrobium officinale is a perennial epiphytic herb in Orchidaceae. Cultivated products are the main alternative for clinical application due to the shortage of wild resources. However, the phenotype and quality of D. officinale have changed post-artificial cultivation, and environmental cues such as light, temperature, water, and nutrition supply are the major influencing factors. This study aims to unveil the mechanisms beneath the cultivation-induced variation by analyzing the changes of the metabolome and transcriptome of D. officinale seedlings treated with red- blue LED light and potassium fertilizer. RESULTS: After light- and K-treatment, the D. officinale pseudobulbs turned purple and the anthocyanin content increased significantly. Through wide-target metabolome analysis, compared with pseudobulbs in the control group (P), the proportion of flavonoids in differentially-accumulated metabolites (DAMs) was 22.4% and 33.5% post light- and K-treatment, respectively. The gene modules coupled to flavonoids were obtained through the coexpression analysis of the light- and K-treated D. officinale transcriptome by WGCNA. The KEGG enrichment results of the key modules showed that the DEGs of the D. officinale pseudobulb were enriched in phenylpropane biosynthesis, flavonoid biosynthesis, and jasmonic acid (JA) synthesis post-light- and K-treatment. In addition, anthocyanin accumulation was the main contribution to the purple color of pseudobulbs, and the plant hormone JA induced the accumulation of anthocyanins in D. officinale. CONCLUSIONS: These results suggested that light and potassium affected the accumulation of active compounds in D. officinale, and the gene-flavone network analysis emphasizes the key functional genes and regulatory factors for quality improvement in the cultivation of this medicinal plant.

Integrative analyses of targeted metabolome and transcriptome of Isatidis Radix autotetraploids highlighted key polyploidization-responsive regulators.

BACKGROUND: Isatidis Radix, the root of Isatis indigotica Fort. (Chinese woad) can produce a variety of efficacious compound with medicinal properties. The tetraploid I. indigotica plants exhibit superior phenotypic traits, such as greater yield, higher bioactive compounds accumulation and enhanced stress tolerance. In this study, a comparative transcriptomic and metabolomic study on Isatidis Radix autotetraploid and its progenitor was performed. RESULTS: Through the targeted metabolic profiling, 283 metabolites were identified in Isatidis Radix, and 70 polyploidization-altered metabolites were obtained. Moreover, the production of lignans was significantly increased post polyploidization, which implied that polyploidization-modulated changes in lignan biosynthesis. Regarding the transcriptomic shift, 2065 differentially expressed genes (DEGs) were identified as being polyploidy-responsive genes, and the polyploidization-altered DEGs were enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. The further integrative analysis of polyploidy-responsive metabolome and transcriptome showed that 1584 DEGs were highly correlated with the 70 polyploidization-altered metabolites, and the transcriptional factors TFs-lignans network highlighted 10 polyploidy-altered TFs and 17 fluctuated phenylpropanoid pathway compounds. CONCLUSIONS: These results collectively indicated that polyploidization contributed to the high content of active compounds in autotetraploid roots, and the gene-lignan pathway network analysis highlighted polyploidy-responsive key functional genes and regulators.

Bioinformatic Exploration of the Targets of Xylem Sap miRNAs in Maize under Cadmium Stress.

Cadmium (Cd) has the potential to be chronically toxic to humans through contaminated crop products. MicroRNAs (miRNAs) can move systemically in plants. To investigate the roles of long-distance moving xylem miRNAs in regulating maize response to Cd stress, three xylem sap small RNA (sRNA) libraries were constructed for high-throughput sequencing to identify potential mobile miRNAs in Cd-stressed maize seedlings and their putative targets in maize transcriptomes. In total, about 199 miRNAs (20⁻22 nucleotides) were identified in xylem sap from maize seedlings, including 97 newly discovered miRNAs and 102 known miRNAs. Among them, 10 miRNAs showed differential expression in xylem sap after 1 h of Cd treatment. Two miRNAs target prediction tools, psRNAtarget (reporting the inhibition pattern of cleavage) and DPMIND (discovering Plant MiRNA-Target Interaction with degradome evidence), were used in combination to identify, via bioinformatics, the targets of 199 significantly expressed miRNAs in maize xylem sap. The integrative results of these two bioinformatic tools suggested that 27 xylem sap miRNAs inhibit 34 genes through cleavage with degradome evidence. Moreover, nearly 300 other genes were also the potential miRNAs cleavable targets without available degradome data support, and the majority of them were enriched in abiotic stress response, cell signaling, transcription regulation, as well as metal handling. These approaches and results not only enhanced our understanding of the Cd-responsive long-distance transported miRNAs from the view of xylem sap, but also provided novel insights for predicting the molecular genetic mechanisms mediated by miRNAs.

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.

Comparative analysis of Cd-responsive maize and rice transcriptomes highlights Cd co-modulated orthologs.

BACKGROUND: Metal tolerance is often an integrative result of metal uptake and distribution, which are fine-tuned by a network of signaling cascades and metal transporters. Thus, with the goal of advancing the molecular understanding of such metal homeostatic mechanisms, comparative RNAseq-based transcriptome analysis was conducted to dissect differentially expressed genes (DEGs) in maize roots exposed to cadmium (Cd) stress. RESULTS: To unveil conserved Cd-responsive genes in cereal plants, the obtained 5166 maize DEGs were compared with 2567 Cd-regulated orthologs in rice roots, and this comparison generated 880 universal Cd-responsive orthologs groups composed of 1074 maize DEGs and 981 rice counterparts. More importantly, most of the orthologous DEGs showed coordinated expression pattern between Cd-treated maize and rice, and these include one large orthologs group of pleiotropic drug resistance (PDR)-type ABC transporters, two clusters of amino acid transporters, and 3 blocks of multidrug and toxic compound extrusion (MATE) efflux family transporters, and 3 clusters of heavy metal-associated domain (HMAD) isoprenylated plant proteins (HIPPs), as well as all 4 groups of zinc/iron regulated transporter protein (ZIPs). Additionally, several blocks of tandem maize paralogs, such as germin-like proteins (GLPs), phenylalanine ammonia-lyases (PALs) and several enzymes involved in JA biosynthesis, displayed consistent co-expression pattern under Cd stress. Out of the 1074 maize DEGs, approximately 30 maize Cd-responsive genes such as ZmHIPP27, stress-responsive NAC transcription factor (ZmSNAC1) and 9-cis-epoxycarotenoid dioxygenase (NCED, vp14) were also common stress-responsive genes reported to be uniformly regulated by multiple abiotic stresses. Moreover, the aforementioned three promising Cd-upregulated genes with rice counterparts were identified to be novel Cd-responsive genes in maize. Meanwhile, one maize glutamate decarboxylase (ZmGAD1) with Cd co-modulated rice ortholog was selected for further analysis of Cd tolerance via heterologous expression, and the results suggest that ZmGAD1 can confer Cd tolerance in yeast and tobacco leaves. CONCLUSIONS: These novel findings revealed the conserved function of Cd-responsive orthologs and paralogs, which would be valuable for elucidating the genetic basis of the plant response to Cd stress and unraveling Cd tolerance genes.

The role of ZmWRKY4 in regulating maize antioxidant defense under cadmium stress.

WRKY transcription factors act as positive regulators in abiotic stress responses by activation of the cellular antioxidant systems. However, there are few reports on the response of WRKY genes to cadmium (Cd) stress. In this study, the role of maize ZmWRKY4 in regulating antioxidant enzymes in Cd stress was investigated. The results indicated that Cd induced up-regulation of the expression and the activities of ZmWRKY4 and superoxide dismutase (SOD) and ascorbate peroxidase (APX). Transient expression and RNA interference (RNAi) silencing of ZmWRKY4 in maize mesophyll protoplasts further revealed that ZmWRKY4 was required for the abscisic acid (ABA)-induced increase in expression and activity of SOD and APX. Overexpression of ZmWRKY4 in protoplasts upregulated the expression and the activities of antioxidant enzymes, whereas ABA induced increases in the expression and the activities of antioxidant enzymes were blocked by the RNAi silencing of ZmWRKY4. Bioinformatic analysis indicated that ZmSOD4 and ZmcAPX both harbored two W-boxes, binding motif for WRKY transcription factors, in their promoter region. Intriguingly, ZmWRKY4 belongs to group I WRKYs with two WRKY domains. Moreover, the synchronized expression patterns indicate that ZmWRKY4 might play a critical role in either regulating the ZmSOD4 and ZmcAPX expression or cooperating with them in response to stress and phytohormone.

Co-expression network analysis of the transcriptomes of rice roots exposed to various cadmium stresses reveals universal cadmium-responsive genes.

BACKGROUND: The migration of cadmium (Cd) from contaminated soil to rice is a cause for concern. However, the molecular mechanism underlying the response of rice roots to various Cd stresses remains to be clarified from the viewpoint of the co-expression network at a system-wide scale. RESULTS: We employed a comparative RNAseq-based approach to identify early Cd-responsive differentially expressed genes (DEGs) in rice 'Nipponbare' seedling roots after 1 h of high-Cd treatment. A multiplicity of the identified 1772 DEGs were implicated in hormone signaling and transcriptional regulation, particularly NACs and WRKYs were all upregulated under Cd stress. All of the 6 Cd-upregulated ABC transporters were pleiotropic drug resistance proteins (PDRs), whereas all of the 6 ZRT/IRT-like proteins (ZIPs) were consistently downregulated by Cd treatment. To further confirm our results of this early transcriptomic response to Cd exposure, we then conducted weighted gene co-expression network analysis (WGCNA) to re-analyze our RNAseq data in combination with other 11 previously published RNAseq datasets for rice roots exposed to diverse concentrations of Cd for extended treatment periods. This integrative approach identified 271 transcripts as universal Cd-regulated DEGs that are key components of the Cd treatment coupled co-expression module. A global view of the 164 transcripts with annotated functions in pathway networks revealed several Cd-upregulated key functional genes, including transporter ABCG36/OsPDR9, 12-oxo-phytodienoic acid reductases (OPRs) for JA synthesis, and ZIM domain proteins JAZs in JA signaling, as well as OsWRKY10, NAC, and ZFP transcription factors. More importantly, 104 of these, including ABCG36/OsPDR9, OsNAC3, as well as several orthologs in group metalloendoproteinase, plastocyanin-like domain containing proteins and pectin methylesterase inhibitor, may respond specifically to various Cd pressures, after subtracting the 60 general stress-responsive genes reported to be commonly upregulated following multiple stresses. CONCLUSION: An integrative approach was implemented to identify DEGs and co-expression network modules in response to various Cd pressures, and 104 of the 164 annotatable universal Cd-responsive DEGs may specifically respond to various Cd pressures. These results provide insight into the universal molecular mechanisms beneath the Cd response in rice roots, and suggest many promising targets for improving the rice acclimation process against Cd toxicity.

Comparative transcriptome profiling of the fertile and sterile flower buds of a dominant genic male sterile line in sesame (Sesamum indicum L.).

BACKGROUND: Sesame (Sesamum indicum L.) is a globally important oilseed crop with highly-valued oil. Strong hybrid vigor is frequently observed within this crop, which can be exploited by the means of genic male sterility (GMS). We have previously developed a dominant GMS (DGMS) line W1098A that has great potential for the breeding of F1 hybrids. Although it has been genetically and anatomically characterized, the underlying molecular mechanism for male sterility remains unclear and therefore limits the full utilization of such GMS line. In this study, RNA-seq based transcriptome profiling was carried out in two near-isogenic DGMS lines (W1098A and its fertile counterpart, W1098B) to identify differentially expressed genes (DEGs) related to male sterility. RESULTS: A total of 1,502 significant DEGs were detected, among which 751 were up-regulated and 751 were down-regulated in sterile flower buds. A number of DEGs were implicated in both ethylene and JA synthesis & signaling pathway; the expression of which were either up- or down-regulated in the sterile buds, respectively. Moreover, the majority of NAC and WRKY transcription factors implicated from the DEGs were up-regulated in sterile buds. By querying the Plant Male Reproduction Database, 49 sesame homologous genes were obtained; several of these encode transcription factors (bHLH089, MYB99, and AMS) that showed reduced expression in sterile buds, thus implying the possible role in specifying or determining tapetal fate and development. The predicted effect of allelic variants on the function of their corresponding DEGs highlighted several Insertions/Deletions (InDels), which might be responsible for the phenotype of sterility/fertility in DGMS lines. CONCLUSION: The present comparative transcriptome study suggested that both hormone signaling pathway and transcription factors control the male sterility of DGMS in sesame. The results also revealed that several InDels located in DEGs prone to cause loss of function, which might contribute to male sterility. These findings provide valuable genomic resources for a deeper insight into the molecular mechanism underlying DGMS.

ZmABA2, an interacting protein of ZmMPK5, is involved in abscisic acid biosynthesis and functions.

In maize (Zea mays), the mitogen-activated protein kinase ZmMPK5 has been shown to be involved in abscisic acid (ABA)-induced antioxidant defence and to enhance the tolerance of plants to drought, salt stress and oxidative stress. However, the underlying molecular mechanisms are poorly understood. Here, using ZmMPK5 as bait in yeast two-hybrid screening, a protein interacting with ZmMPK5 named ZmABA2, which belongs to a member of the short-chain dehydrogenase/reductase family, was identified. Pull-down assay and bimolecular fluorescence complementation analysis and co-immunoprecipitation test confirmed that ZmMPK5 interacts with ZmABA2 in vitro and in vivo. Phosphorylation of Ser173 in ZmABA2 by ZmMPK5 was shown to increase the activity of ZmABA2 and the protein stability. Various abiotic stimuli induced the expression of ZmABA2 in leaves of maize plants. Pharmacological, biochemical and molecular biology and genetic analyses showed that both ZmMPK5 and ZmABA2 coordinately regulate the content of ABA. Overexpression of ZmABA2 in tobacco plants was found to elevate the content of ABA, regulate seed germination and root growth under drought and salt stress and enhance the tolerance of tobacco plants to drought and salt stress. These results suggest that ZmABA2 is a direct target of ZmMPK5 and is involved in ABA biosynthesis and functions.

ABA Affects Brassinosteroid-Induced Antioxidant Defense via ZmMAP65-1a in Maize Plants.

Brassinosteroids (BRs) and ABA co-ordinately regulate water deficit tolerance in maize leaves. ZmMAP65-1a, a maize microtubule-associated protein (MAP) which plays an essential role in BR-induced antioxidant defense, has been characterized previously. However, the interactions among BR, ABA and ZmMAP65-1a in water deficit tolerance remain unexplored. In this study, we demonstrated that ABA was required for BR-induced antioxidant defense via ZmMAP65-1a by using biochemical blocking and ABA biosynthetic mutants. The expression of ZmMAP65-1a in maize leaves and mesophyll protoplasts could be increased under polyethylene glycol- (PEG) stimulated water deficit and ABA treatments. Furthermore, the importance of ABA in the early pathway of BR-induced water deficit tolerance was demonstrated by limiting ABA availability. Blocking ABA biosynthesis biochemically or by a null mutation inhibited the downstream gene expression of ZmMAP65-1a and the activity of ZmMAPK5 in the pathway. It also affected the activities of BR-induced antioxidant defense-related enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD) and NADPH oxidase. In addition, combining results from transiently overexpressed or silenced ZmMAP65-1a in mesophyll protoplasts, we discovered that ZmMAP65-1a mediated the ABA-induced gene expression and activities of APX and SOD. Surprisingly, silencing of ZmMAP65-1a in mesophyll protoplasts did not alter the gene expression of ZmCCaMK and vice versa in response to ABA. Taken together, our data indicate that water deficit-induced ABA is a key mediator in BR-induced antioxidant defense via ZmMAP65-1a in maize.