Genome-Wide Identification and Characterization of the Salvia miltiorrhiza Histone Deacetylase (HDAC) Family in Response to Multiple Abiotic Stresses.

Salvia miltiorrhiza is a plant commonly used in traditional Chinese medicine. Its material bases for treating diseases are tanshinones and phenolic acids, including salvianolic acids. Histone deacetylase proteins (HDACs) are a class of specific functional enzymes that interact with acetylation groups on the N-terminal lysine of histone proteins further regulate gene transcription through structural changes at the chromatin level. HDACs involved in the growth and development of various plants, and induced by plant hormones to regulate the internal environment of plants to resist stress, at the same time affect the accumulation of some secondary metabolites. However, the role of SmHDACs on the accumulation of salvianolic acid in S. miltiorrhiza remains unclear. In this study, 16 SmHDACs genes were identified from the high-quality S. miltiorrhiza genome, their physicochemical properties were predicted. In phylogenetic trees co-constructed with HDACs proteins from other plants, SmHDACs was divided into three subfamilies, each with similar motif and conserved domain composition. The distribution of the three subfamilies is similar to that of dicotyledonous plants. Chromosome localization analysis showed that SmHDACs genes were randomly located. Cis-acting element analysis predicted that SmHDACs gene expression may be related to and induced by various phytohormones, such as MeJA and ABA. By combining the expression pattern and co-expression network induced by phytohormones, we speculate that SmHDACs may further influence the synthesis of salvianolic acid, and identified SmHDA5, a potential functional gene, then speculate its downstream target based on the co-expression network. In summary, we analyzed the SmHDACs gene family of S. miltiorrhiza and screened out the potential functional gene SmHDA5. From the perspective of epigenetics, we proposed the molecular mechanism of plant hormone promoting salvianolic acid synthesis, which filled the gap in the subdivision of histone deacetylase in S. miltiorrhiza research, provided a theoretical basis for the culture and transformation of S. miltiorrhiza germplasm resources.

Metabolic accumulation and related synthetic genes of O-acetyl groups in mannan polysaccharides of Dendrobium officinale.

Mannan polysaccharides (MPs), which contain substituted O-acetyl groups in their backbone, are abundant in the medicinal plant Dendrobium officinale. Acetyl groups can influence the physiological and biochemical properties of polysaccharides, which mainly accumulate in the stems of D. officinale at four developmental stages (S1-S4), showing an increasing trend and a link with water-soluble polysaccharides (WSPs) and mannose. The genes coding for enzymes that catalyze O-acetyl groups to MPs are unknown in D. officinale. The TRICHOME BIREFRINGENCE-LIKE (TBL) gene family contains TBL and DUF231 domains that can transfer O-acetyl groups to various polysaccharides. Based on an established D. officinale genome database, 37 DoTBL genes were identified. Analysis of cis-elements in the promoter region showed that DoTBL genes might respond to different hormones and abiotic stresses. Most of the genes with MeJA-responsive elements were upregulated or downregulated after treatment with MeJA. qRT-PCR results demonstrated that DoTBL genes had significantly higher expression levels in stems and leaves than in roots. Eight DoTBL genes showed relatively higher expression at S2-S4 stages, which showed a link with the content of WSPs and O-acetyl groups. DoTBL35 and its homologous gene DoTBL34 displayed the higher mRNA level in different organs and developmental stages, which might participate in the acetylation of MPs in D. officinale. The subcellular localization of DoTBL34 and DoTBL35 reveals that the endoplasmic reticulum may play an important role in the acetylation of MPs.

The methyl jasmonate-responsive transcription factor DobHLH4 promotes DoTPS10, which is involved in linalool biosynthesis in Dendrobium officinale during floral development.

Linalool is an aromatic monoterpene produced in the Chinese medicinal plant Dendrobium officinale, but little information is available on the regulation of linalool biosynthesis. Here, a novel basic helix-loop-helix (bHLH) transcription factor, DobHLH4 from D. officinale, was identified and functionally characterized. The expression profile of DobHLH4 was positively correlated with that of DoTPS10 (R2 = 0.985, p < 0.01), which encodes linalool synthase that is responsible for linalool production, during floral development. DobHLH4 was highly expressed in petals, and was significantly induced by methyl jasmonate. Analysis of subcellular localization showed that DobHLH4 was located in the nucleus. Yeast one-hybrid and dual-luciferase assays indicated that DobHLH4 bound directly to the DoTPS10 promoter harboring the G-box element, and up-regulated DoTPS10 expression. A yeast two-hybrid screen confirmed that DobHLH4 physically interacted with DoJAZ1, suggesting that DobHLH4 might function in the jasmonic acid-mediated accumulation of linalool. Furthermore, transient overexpression of DobHLH4 in D. officinale petals significantly increased linalool production by triggering linalool biosynthetic pathway genes, especially DoTPS10. We suggest a hypothetical model that depicts how jasmonic acid signaling may regulate DoTPS10 by interacting with DobHLH4 and DoJAZ1. In doing so, the formation of linalool is controlled. Our results indicate that DobHLH4 is a positive regulator of linalool biosynthesis and may be a promising target for in vitro-based metabolic engineering to produce linalool.

Transcriptome and Metabolome Reveal Salt-Stress Responses of Leaf Tissues from Dendrobium officinale.

Dendrobium officinale Kimura et Migo is a precious traditional Chinese medicine. Despite D. officinale displaying a good salt-tolerance level, the yield and growth of D. officinale were impaired drastically by the increasing soil secondary salinization. The molecular mechanisms of D. officinale plants' adaptation to salt stress are not well documented. Therefore, in the present study, D. officinale plants were treated with 250 mM NaCl. Transcriptome analysis showed that salt stress significantly altered various metabolic pathways, including phenylalanine metabolism, flavonoid biosynthesis, and α-linolenic acid metabolism, and significantly upregulated the mRNA expression levels of DoAOC, DoAOS, DoLOX2S, DoMFP, and DoOPR involved in the jasmonic acid (JA) biosynthesis pathway, as well as rutin synthesis genes involved in the flavonoid synthesis pathway. In addition, metabolomics analysis showed that salt stress induced the accumulation of some compounds in D. officinale leaves, especially flavonoids, sugars, and alkaloids, which may play an important role in salt-stress responses of leaf tissues from D. officinale. Moreover, salt stress could trigger JA biosynthesis, and JA may act as a signal molecule that promotes flavonoid biosynthesis in D. officinale leaves. To sum up, D. officinale plants adapted to salt stress by enhancing the biosynthesis of secondary metabolites.

Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale.

The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid, Dendrobium officinale, or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the D. officinale genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all DoAP2 genes contained cis-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of DoAP2-2, DoAP2-5, DoAP2-7, DoAP2-8 and DoAP2-12 genes in protocorm-like bodies (PLBs), while DoAP2-3, DoAP2-4, DoAP2-6, DoAP2-9, DoAP2-10 and DoAP2-11 expression was strong in plantlets. In addition, the expression of some DoAP2 genes was down-regulated during flower development. These results suggest that DoAP2 genes may play roles in plant regeneration and flower development in D. officinale. Four DoAP2 genes (DoAP2-1 from euAP2, DoAP2-2 from euANT, and DoAP2-6 and DoAP2-11 from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of Arabidopsis thaliana mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in Nicotiana benthamiana leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on AP2 genes in D. officinale shed light on the function of AP2 genes in this orchid and other plant species.

DoRWA3 from Dendrobium officinale Plays an Essential Role in Acetylation of Polysaccharides.

The acetylation or deacetylation of polysaccharides can influence their physical properties and biological activities. One main constituent of the edible medicinal orchid, Dendrobium officinale, is water-soluble polysaccharides (WSPs) with substituted O-acetyl groups. Both O-acetyl groups and WSPs show a similar trend in different organs, but the genes coding for enzymes that transfer acetyl groups to WSPs have not been identified. In this study, we report that REDUCED WALL ACETYLATION (RWA) proteins may act as acetyltransferases. Three DoRWA genes were identified, cloned, and sequenced. They were sensitive to abscisic acid (ABA), but there were no differences in germination rate and root length between wild type and 35S::DoRWA3 transgenic lines under ABA stress. Three DoRWA proteins were localized in the endoplasmic reticulum. DoRWA3 had relatively stronger transcript levels in organs where acetyl groups accumulated than DoRWA1 and DoRWA2, was co-expressed with polysaccharides synthetic genes, so it was considered as a candidate acetyltransferase gene. The level of acetylation of polysaccharides increased significantly in the seeds, leaves and stems of three 35S::DoRWA3 transgenic lines compared to wild type plants. These results indicate that DoRWA3 can transfer acetyl groups to polysaccharides and is a candidate protein to improve the biological activity of other edible and medicinal plants.

Transformation of catechins into theaflavins by upregulation of CsPPO3 in preharvest tea (Camellia sinensis) leaves exposed to shading treatment.

Catechins and theaflavins are important metabolites contributing to tea function and quality. Catechins are known to transform into theaflavins during the tea manufacturing process, but the same transformation in preharvest tea leaves is unknown. Herein, we determined that shade treatment (dark), an agronomic practise widely used in tea cultivation, reduced the contents of most catechins, but increased the theaflavin contents, in preharvest tea leaves (cv. Yinghong No.9). This was attributed to the activation of polyphenoloxidase (PPO) activity in darkness. Furthermore, CsPPO3 was highly expressed under darkness, and thus CsPPO3 had been cloned, sequenced, and characterization. The CsPPO3 recombinant protein exhibited PPO function. Furthermore, shade treatment also reduced the catechin contents and increased the theaflavin contents in Yabukita and Hoshinomidori, suggesting that this phenomenon might not be specific to certain tea cultivars. This information will aid in understanding of theaflavin formation and its response to environmental factors at the preharvest tea stage.

Understanding different regulatory mechanisms of proteinaceous and non-proteinaceous amino acid formation in tea (Camellia sinensis) provides new insights into the safe and effective alteration of tea flavor and function.

Amino acids are the main contributors to tea (Camellia sinensis) flavor and function. Tea leaves contain not only proteinaceous amino acids but also specialized non-proteinaceous amino acids such as L-theanine and γ-aminobutyric acid (GABA). Here, we review different regulatory mechanisms of proteinaceous and non-proteinaceous amino acid formation in tea. The key findings were: (1) High accumulations of proteinaceous amino acids mainly result from protein degradation, which occurs in each tea stage, including preharvest, postharvest, manufacturing, and deep processing; (2) L-Theanine is the most represented non-proteinaceous amino acid that contributes to tea taste and function. Its accumulation is influenced more by the variety than by exogenous factors; and (3) GABA is the second most represented non-proteinaceous amino acid that contributes to tea function. Its formation, and resulting accumulation, are responses to stress. The combination of anoxic stress and mechanical damage are essential for a high GABA accumulation. An understanding of the biosynthesis, metabolism, and regulatory mechanisms of the proteinaceous and non-proteinaceous amino acids during the whole process from raw materials to tea products is necessary to safely and effectively alter tea flavor and function.

Effect of Major Tea Insect Attack on Formation of Quality-Related Nonvolatile Specialized Metabolites in Tea ( Camellia sinensis) Leaves.

Insect attack is known to induce a high accumulation of volatile metabolites in tea ( Camellia sinensis). However, little information is available concerning the effect of insect attack on tea quality-related nonvolatile specialized metabolites. This study aimed to investigate the formation of characteristic nonvolatile specialized metabolites in tea leaves in response to attack by major tea insects, namely, tea green leafhoppers and tea geometrids, and determine the possible involvement of phytohormones in metabolite formation resulting from insect attack. Both tea green leafhopper and tea geometrid attacks increased the jasmonic acid and salicylic acid contents. The abscisic acid content was only increased under tea green leafhopper attack, perhaps due to special continuous piercing-sucking wounding. Tea green leafhopper attack induced the formation of theaflavins from catechins under the action of polyphenol oxidase, while tea geometrid attack increased the l-theanine content. Exogenous phytohormone treatments can affect the caffeine and catechin contents. These results will help to determine the influence of major tea pest insects on important tea quality-related metabolites and enhance understanding of the relationship of phytohormones and quality-related nonvolatile metabolite formation in tea exposed to tea pest insect attacks.

The GDP-mannose transporter gene (DoGMT) from Dendrobium officinale is critical for mannan biosynthesis in plant growth and development.

Dendrobium officinale is a precious traditional Chinese medicinal herb because it is abundant in mannose-containing polysaccharides (MCPs). GDP-mannose transporter (GMT), which translocates GDP-mannose into the Golgi lumen, is indispensable for the biosynthesis of MCPs. In this study, we found that the dominant polysaccharides in D. officinale were MCPs in a range of varieties and different physiological phases. After a positive correlation between the accumulation of mannose and the transcript levels of candidate GMT genes was found, three GMT genes (DoGMT1-3) were identified in D. officinale. DoGMT1, DoGMT2 and DoGMT3 exhibited the highest transcript level in stem that an organ for MCPs storage. All three DoGMT proteins were targeted to Golgi apparatus, and had a GDP binding domain (GXL/VNK) that was homologous to a specially characterized GMT protein GONST1 in Arabidopsis thaliana. Moreover, DoGMT1, DoGMT2 and DoGMT3 complemented a GDP-mannose transport-defective yeast mutant (vrg4-2), meanwhile they also demonstrated a higher GDP-mannose uptake activity. Therefore, we conclude that DoGMT1, DoGMT2 and DoGMT3 are able to transport GDP-mannose while the expression patterns of these genes correspond to the accumulation of MCPs in D. officinale. These findings support the importance of GMT genes from D. officinale in the biosynthesis of MCPs.

Differential Accumulation of Aroma Compounds in Normal Green and Albino-Induced Yellow Tea (Camellia sinensis) Leaves.

Tea (Camellia sinensis) cultivars with green leaves are the most widely used for making tea. Recently, tea mutants with white or yellow young shoots have attracted increasing interest as raw materials for making "high-quality" tea products. Albino teas are generallycharacterized as having metabolites of relatively high amino acid content and lower catechin content. However, little is known about aroma compounds in albino tea leaves. Herein, we compared original normal leaves (green) and light-sensitive albino leaves (yellow) of cv. Yinghong No. 9. GC-MS was employed to analyze endogenous tea aroma compounds and related precursors. Quantitative real time PCR was used to measure expression levels of genes involved in biosyntheses of tea aromas.The total contents of most endogenous free tea aromas, including aroma fatty acid derivatives, aroma terpenes, and aroma phenylpropanoids/benzenoids, and their glycosidically bound aroma compounds, were lower in yellow leaves than in green leaves. The content of the key precursor geranyl diphosphate (GDP) and expression levels of key synthetic genes involved in the formation of linalool, a major aroma compound in cv. Yinghong No. 9, were investigated. Linalool content was lower in albino-induced yellow leaves, which was due to the lower GDP content compared with normal green leaves.

Differential Accumulation of Anthocyanins in Dendrobium officinale Stems with Red and Green Peels.

Dendrobium officinale stems, including red and green stems, are widely used as a dietary supplement to develop nutraceutical beverages and food products. However, there is no detailed information on pigment composition of red and green stems. Here, we investigated the content and composition of pigments in red and green stems by Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry and assessed the differential accumulation of anthocyanins at the molecular level. The color of peels in red stems was caused by the presence of anthocyanins in epidermal cells unlike the peels of green stems. The glucoside derivatives delphinidin and cyanidin are responsible for the red color. Within the D. officinale anthocyanidin biosynthetic pathway, DoANS and DoUFGT, coding for anthocyanidin synthase and UDP-glucose flavonoid-3-O-glucosyltransferase, respectively, are critical regulatory genes related to the differential accumulation of anthocyanidin. These findings provide a more complete profile of pigments, especially anthocyanin, in D. officinale stems, and lay a foundation for producing functional foods.

Cytochemical Localization of Polysaccharides in Dendrobium officinale and the Involvement of DoCSLA6 in the Synthesis of Mannan Polysaccharides.

Dendrobium officinale is a precious traditional Chinese medicinal plant because of its abundant polysaccharides found in stems. We determined the composition of water-soluble polysaccharides and starch content in D. officinale stems. The extracted water-soluble polysaccharide content was as high as 35% (w/w). Analysis of the composition of monosaccharides showed that the water-soluble polysaccharides were dominated by mannose, to a lesser extent glucose, and a small amount of galactose, in a molar ratio of 223:48:1. Although starch was also found, its content was less than 10%. This result indicated that the major polysaccharides in D. officinale stems were non-starch polysaccharides, which might be mannan polysaccharides. The polysaccharides formed granules and were stored in plastids similar to starch grains, were localized in D. officinale stems by semi-thin and ultrathin sections. CELLULOSE SYNTHASE-LIKE A (CSLA) family members encode mannan synthases that catalyze the formation of mannan polysaccharides. To determine whether the CSLA gene from D. officinale was responsible for the synthesis of mannan polysaccharides, 35S:DoCSLA6 transgenic lines were generated and characterized. Our results suggest that the CSLA family genes from D. officinale play an important role in the biosynthesis of mannan polysaccharides.

Molecular cloning and functional analysis of the phosphomannomutase (PMM) gene from Dendrobium officinale and evidence for the involvement of an abiotic stress response during germination.

Phosphomannomutase (PMM, EC 5.4.2.8) catalyzes the interconversion of mannose-6-phosphate to mannose-1-phosphate, the precursor for the synthesis of GDP-mannose. In this study, the complementary DNA (cDNA) of the Phosphomannomutase (PMM) gene was initially cloned from Dendrobium officinale by RACE method. Transient transform result showed that the DoPMM protein was localized in the cytoplasm. The DoPMM gene was highly expressed in the stems of D. officinale both in vegetative and reproductive developmental stages. The putative promoter was cloned by TAIL-PCR and used for searched cis-elements. Stress-related cis-elements like ABRE, TCA-element, and MBS were found in the promoter regions. The DoPMM gene was up-regulated after treatment with abscisic acid, salicylic acid, cold, polyethylene glycol, and NaCl. The total ascorbic acid (AsA) and polysaccharide content in all of the 35S::DoPMM Arabidopsis thaliana transgenic lines #1, #2, and #5 showed a 40, 39, and 31% increase in AsA and a 77, 22, and 39% increase in polysaccharides, respectively more than wild-type (WT) levels. All three 35S::DoPMM transgenic lines exhibited a higher germination percentage than WT plants when seeded on half-strength MS medium supplemented with 150 mM NaCl or 300 mM mannitol. These results provide genetic evidence for the involvement of PMM genes in the biosynthesis of AsA and polysaccharides and the mediation of PMM genes in abiotic stress tolerance during seed germination in A. thaliana.

Transcriptome Analysis of Dendrobium officinale and its Application to the Identification of Genes Associated with Polysaccharide Synthesis.

Dendrobium officinale is one of the most important Chinese medicinal herbs. Polysaccharides are one of the main active ingredients of D. officinale. To identify the genes that maybe related to polysaccharides synthesis, two cDNA libraries were prepared from juvenile and adult D. officinale, and were named Dendrobium-1 and Dendrobium-2, respectively. Illumina sequencing for Dendrobium-1 generated 102 million high quality reads that were assembled into 93,881 unigenes with an average sequence length of 790 base pairs. The sequencing for Dendrobium-2 generated 86 million reads that were assembled into 114,098 unigenes with an average sequence length of 695 base pairs. Two transcriptome databases were integrated and assembled into a total of 145,791 unigenes. Among them, 17,281 unigenes were assigned to 126 KEGG pathways while 135 unigenes were involved in fructose and mannose metabolism. Gene Ontology analysis revealed that the majority of genes were associated with metabolic and cellular processes. Furthermore, 430 glycosyltransferase and 89 cellulose synthase genes were identified. Comparative analysis of both transcriptome databases revealed a total of 32,794 differential expression genes (DEGs), including 22,051 up-regulated and 10,743 down-regulated genes in Dendrobium-2 compared to Dendrobium-1. Furthermore, a total of 1142 and 7918 unigenes showed unique expression in Dendrobium-1 and Dendrobium-2, respectively. These DEGs were mainly correlated with metabolic pathways and the biosynthesis of secondary metabolites. In addition, 170 DEGs belonged to glycosyltransferase genes, 37 DEGs were related to cellulose synthase genes and 627 DEGs encoded transcription factors. This study substantially expands the transcriptome information for D. officinale and provides valuable clues for identifying candidate genes involved in polysaccharide biosynthesis and elucidating the mechanism of polysaccharide biosynthesis.

Identification of genes involved in biosynthesis of mannan polysaccharides in Dendrobium officinale by RNA-seq analysis.

Dendrobium officinale is a traditional Chinese medicinal plant. The stems of D. officinale contain mannan polysaccharides, which are promising bioactive polysaccharides for use as drugs. However, the genes involved in the biosynthesis of mannan polysaccharides in D. officinale have not yet been identified. In this study, four digital gene expression profiling analyses were performed on developing stems of greenhouse-grown D. officinale to identify such genes. Based on the accumulation of mannose and on gene expression levels, eight CELLULOSE SYNTHASE-LIKE A genes (CSLA), which are highly likely to be related to the biosynthesis of bioactive mannan polysaccharides, were identified from the differentially expressed genes database. In order to further analyze these DoCSLA genes, a full-length cDNA of each was obtained by RACE. The eight genes, belonging to the CSLA family of the CesA superfamily, contain conserved domains of the CesA superfamily. Most of the genes, which were highly expressed in the stems of D. officinale, were related to abiotic stress. Our results suggest that the CSLA family genes from D. officinale are involved in the biosynthesis of bioactive mannan polysaccharides.