caffeoyl-CoA O-methyltransferase Research Articles

Comparative Transcriptome Analysis of MeJA Responsive Enzymes Involved in Phillyrin Biosynthesis of Forsythia suspensa.

Forsythia suspensa (Thunb.) has been widely used in traditional medicines in Asia. According to the 2020 edition of Chinese Pharmacopoeia, phillyrin is the main active ingredient in F. suspensa, which is effective in clearing heat, reducing swelling, and dispersing nodules. F. suspensa leaf is a non-toxic substance and it can be used to make a health tea. Here, we combine elicitors and transcriptomics to investigate the inducible biosynthesis of the phillyrin from the F. suspensa. After the fruits and leaves of F. suspensa were treated with different concentrations of methyl jasmonate (MeJA), the content of phillyrin in the fruits reached a peak at 200 µM MeJA for 12 h, but which was decreased in leaves. To analyze the differences in key enzyme genes involved in the phillyrin biosynthesis, we sequenced the transcriptome of F. suspensa leaves and fruits treated with 200 µM MeJA for 12 h. We hypothesized that nine genes related to coniferin synthesis including: F. suspensa UDP-glycosyltransferase (FsUGT); F. suspensa 4-coumarate coenzyme CoA ligase (Fs4CL); and F. suspensa Caffeoyl-CoA O-methyltransferase (FsCCoAOMT) etc. The qRT-PCR analysis of genes related to phillyrin biosynthesis was consistent with RNA-seq analysis. We also investigated the dynamic changes of genes in F. suspensa leaves and fruits at different time points after 200 µM MeJA treatment, which laid the foundation for further study of the molecular mechanisms regulating the biosynthesis of phillyrin.

Transcriptome and metabolome reveal the effects of three canopy types on the flavonoids and phenolic acids in ‘Merlot’ (Vitis vinifera L.) berry pericarp

The flavonoids and phenolic acids in grape berries greatly influence the quality of wine. Various methods are used to shape and prune grapevines, but their effects on the flavonoids and phenolic acids remain unclear. The flavonoids and phenolic acids in the berry pericarps from grapevines pruned using three types of leaf canopy, namely, V-shaped, T-shaped, and vertical shoot-positioned (VSP) canopies, were compared in this study. Results showed that the V-shaped canopy was more favorable for the accumulation of flavonoids and phenolic acids. Transcriptome and metabolome analyses revealed that the differentially expressed genes (DEGs) and differentially regulated metabolites (DRMs) were significantly enriched in the flavonoid and phenylpropanoid biosynthesis pathways. A total of 96 flavonoids and 32 phenolic acids were detected among the DRMs. Their contents were higher in the V-shaped canopy than in the T-shaped and VSP canopies. Conjoint analysis of transcriptome and metabolome showed that nine DEGs (e.g., cytochrome P450 98A9 and 98A2) were significantly correlated to nine phenolic acids (e.g., gentisic acid and neochlorogenic acid) and three genes (i.e., chalcone isomerase, UDP-glycosyltransferase 88A1, and caffeoyl-CoA O-methyltransferase) significantly correlated to 15 flavonoids (e.g., baimaside and tricin-7-O-rutinoside). These genes may be involved in the regulation of various flavonoids and phenolic acids in grape berries, but their functions need validation. This study provides novel insights into the effects of leaf canopy on flavonoids and phenolic acids in the skin of grape berries and reveals the potential regulatory networks involved in this phenomenon.

Essential oils nano-emulsion confers resistance against Penicillium digitatum in 'Newhall' navel orange by promoting phenylpropanoid metabolism

Inducing natural resistance against pathogen infection in postharvest citrus by exogenous elicitor is a promising alternative to reduce postharvest losses. Here, the potential induced resistance mechanism of blended cinnamaldehyde, carvacrol and eugenol nano-emulsion against P. digitatum in ‘Newhall’ navel orange (Citrus sinensis (L.) Osbeck) was evaluated through combined transcriptomic and metabolomics analysis. Application of nano-emulsion maintained fruit firmness after P. digitatum inoculation, nano-emulsion reduced H2O2 and MDA accumulation of citrus fruit, increased PAL, C4H, 4CL and CAD activity, and higher contents of flavonoids, lignin and total phenolic compounds. Furthermore, higher activities of SOD, CAT, POD and APX in nano-emulsion treated fruit possibly benefited reducing ROS accumulation and lipid peroxidation. The activities of the defense enzymes CHI, GLU were higher in the nano-emulsion treated fruits. RNA-seq identified 653 differentially expressed genes (DEGs) between the control and the nano-emulsion-treated fruit at 48 hpi, including 444 up-regulated and 209 downregulated genes. Genes encoding shikimate O-hydroxycinnamoyltransferase (HCT), caffeoyl-CoA O-methyltransferase (CCoAOMT), caffeic acid 3-O-methyltransferase / acetylserotonin O-methyltransferase (COMT), cinnamyl-alcohol dehydrogenase (CAD) and peroxidase (POD) were differentially expressed by nano-emulsion treatment. In addition, 175 differential accumulated metabolites between the control and the nano-emulsion-treated fruit at 48 hpi also were identified, the different accumulated metabolites from phenylpropanoid biosynthesis pathway related with disease resistance including L-phenylalanine, caffeic acid, caffeoyl quinic acid, coniferyl alcohol, coniferaldehyde. The integrated transcriptomic and metabolomic profiling indicated that the differentially expressed genes (DEGs) and the differentially accumulated metabolites (DAMs) were mainly involved in phenylpropanoid biosynthesis pathway. Besides, the nano-emulsion significantly induced accumulation of primary metabolites including amino acids, soluble sugars, organic acids, lipids, sugar and alcohols. All these results indicated that blended essential oil nano-emulsion as an antifungal delivery system to induce resistance against P. digitatum infection of ‘Newhall’ navel orange by promoting phenylpropanoid metabolism.

RNA-Seq and Iso-Seq Reveal the Important Role of COMT and CCoAOMT Genes in Accumulation of Scopoletin in Noni (Morinda citrifolia).

Scopoletin, the main component of clinical drugs and the functional component of health products, is highly abundant in noni fruit (Morinda citrifolia). Multiple enzyme genes regulate scopoletin accumulation. In the present study, differentially expressed genes of noni were analyzed by RNA sequencing (RNA-Seq) and the full-length genes by isoform-sequencing (Iso-Seq) to find the critical genes in the scopoletin accumulation mechanism pathway. A total of 32,682 full-length nonchimeric reads (FLNC) were obtained, out of which 16,620 non-redundant transcripts were validated. Based on KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation and differential expression analysis, two differentially expressed genes, caffeic acid 3-O-methyltransferase (COMT) and caffeoyl-CoA O-methyltransferase (CCoAOMT), were found in the scopoletin accumulation pathway of noni. Real-time quantitative polymerase chain reaction (q-PCR), phylogenetic tree analysis, gene expression analysis, and the change in scopoletin content confirmed that these two proteins are important in this pathway. Based on these results, the current study supposed that COMT and CCoAOMT play a significant role in the accumulation of scopoletin in noni fruit, and COMT (gene number: gene 7446, gene 8422, and gene 6794) and CCoAOMT (gene number: gene 12,084) were more significant. These results provide the importance of COMT and CCoAOMT and a basis for further understanding the accumulation mechanism of scopoletin in noni.

Characterization of Abscisic Acid and Ethylene in Regulating the White Blush in Fresh-Cut Carrots.

The surface of fresh-cut carrots is apt to white blush, however the physiological and molecular mechanism for this process is not yet fully understood. In this study, exogenous abscisic acid (ABA) and ethylene separately promoted and inhibited the white-blush formation after three days after treatment, respectively. Metabolome analysis found that white-blush components mainly consist of p-hydroxyphenyl lignin and guaiacyl lignin. Transcriptome analysis found an increase in the whiteness values was consistent with the higher expression of genes encoding O-methyltransferase, trans-anol O-methyltransferase, bergaptol O-methyltransferase, caffeic acid 3-O-methyltransferase, phenylalanine ammonia-lyase, and ferulate-5-hydroxylase, together with the lower expression of genes encoding cinnamic acid 4-hydroxylase caffeoyl-CoA O-methyltransferase and 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase. In conclusion, ABA plays an important role in lignin biosynthesis essential to the formation of white blush in fresh-cut carrots. This is the first report that uncovers the physiological and molecular causes of white blush in fresh-cut carrots, providing a basis for white-blush control in fresh-cut carrots.

Metabolomic and transcriptomic changes in mungbean (Vigna radiata (L.) R. Wilczek) sprouts under salinity stress.

Mungbean (Vigna radiata) sprouts are consumed globally as a healthy food with high nutritional values, having antioxidant and anticancer capacity. Under mild salinity stress, plants accumulate more secondary metabolites to alleviate oxidative stress. In this study, metabolomic and transcriptomic changes in mungbean sprouts were identified using a reference cultivar, sunhwa, to understand the regulatory mechanisms of secondary metabolites in response to salinity stress. Under salinity conditions, the contents of phenylpropanoid-derived metabolites, including catechin, chlorogenic acid, isovitexin, p-coumaric acid, syringic acid, ferulic acid, and vitexin, significantly increased. Through RNA sequencing, 728 differentially expressed genes (DEGs) were identified and 20 DEGs were detected in phenylpropanoid and flavonoid biosynthetic pathways. Among them, 11 DEGs encoding key enzymes involved in the biosynthesis of the secondary metabolites that increased after NaCl treatment were significantly upregulated, including dihydroflavonol 4-reductase (log2FC 1.46), caffeoyl-CoA O-methyltransferase (1.38), chalcone synthase (1.15), and chalcone isomerase (1.19). Transcription factor families, such as MYB, WRKY, and bHLH, were also identified as upregulated DEGs, which play a crucial role in stress responses in plants. Furthermore, this study showed that mild salinity stress can increase the contents of phenylpropanoids and flavonoids in mungbean sprouts through transcriptional regulation of the key enzymes involved in the biosynthetic pathways. Overall, these findings will provide valuable information for molecular breeders and scientists interested in improving the nutritional quality of sprout vegetables.

Grafting with an invasive Xanthium strumarium improves tolerance and phytoremediation of native congener X. sibiricum to cadmium/copper/nickel tailings

Invasive plants could play an important role in the restoration of tailings, but their invasiveness limits their practical application. In this study, the phytoremediation potentials and invasive risks of an exotic invasive plant (Xanthium strumarium, LT), a native plant (X. sibiricum, CR), and combinations of inoculations (EG, with CR as the scion and LT as the rootstock; SG, with CR as both the scion and rootstock) were evaluated on Cd/Cu/Ni tailings. LT rootstock has a stronger nutrient and metal transport capacity, compared with CR. EG not only had higher biomass and Cd/Cu/Ni accumulation, but also abundant rhizosphere microbial communities. Hydroponic and common garden experiments showed that the growth and metal enrichment characteristics of EG are not inherited by plant offspring, which reduces the risk of the biological diffusion in the process of using exotic species. Transcriptome analysis shows that a large number of differentially-expressed genes in EG leaves and roots are involved in phenylpropanoid biosynthesis, secondary metabolite generation, and signal transduction. The genes induced in EG leaves, including cyclic nucleotide-gated ion channel, calcium-binding protein, and WRKY transcription factor, were found to be differentially expressed compared to CR. The genes induced in EG roots, included phenylalanine ammonia-lyase, cinnamoyl-CoA reductase, caffeoyl-CoA O-methyltransferase, and beta-glucosidase. We speculate that lignin and glucosinolates play an important role in the metal accumulation and transportation of EG. The results demonstrate that grafting with LT not only improved CR tolerance and accumulation of Cd, Cu, and Ni, but also created a beneficial microbial environment for plants in tailings. More importantly, grafting with LT did not enhance the invasiveness of CR. Our results provide an example of the safe use of invasive plants in the restoration of Cd/Cu/Ni tailings.

Metabolome and transcriptome analysis of eleutheroside B biosynthesis pathway in Eleutherococcus senticosus

Eleutheroside B (syringin) is a medicinal active ingredient extracted from Eleutherococcus senticosus (Ruper. et Maxim.) Maxim with high clinical application value. However, its synthesis pathway remains unknown. Here, we analyzed the eleutheroside B biosynthesis pathway in E. senticosus. Consequently, metabolomic and transcriptomic analyses identified 461 differentially expressed genes (DEGs) and 425 metabolites. Further, we identified 7 DEGs and 67 metabolites involved in the eleutheroside B biosynthetic pathway in the eleutheroside B high and low plants. The correlation between the gene and metabolites was explored using the pearson correlation coefficient (PCC) analysis. Caffeoyl-CoA O-methyltransferase, caffeic acid-O-methyltransferase, β-amyrin synthase (β-AS) genes, NAC5, and HB5 transcription factors were identified as candidate genes and transcription factors related to the eleutheroside B synthesis. Eleutheroside B content was the highest at the young stage of the leaves both in the high and low eleutheroside B plants. Quantitative real-time polymerase chain reaction revealed that phenylalanine ammonia-lyase1, cinnamate 4-hydroxylase, β-AS, and leucoanthocyanidin reductase gene had higher expression levels at the young stage of the leaves in the low eleutheroside B plants but lower expression levels in the high eleutheroside B plants. In the present study, we complemented the eleutheroside B biosynthetic pathway by analyzing the expression levels of relevant genes and metabolite accumulation patterns.

Essential Roles of Cupredoxin Family Proteins in Soybean Cyst Nematode Resistance.

Soybean cyst nematode (SCN, Heterodera glycines), one of the most devastating soybean pathogens, causes a significant yield loss in soybean production. One of the most effective ways to manage SCN is to grow resistant cultivars. Therefore, comparative study using resistant and susceptible soybean cultivars provides a powerful tool to identify new genes involved in soybean SCN resistance. In the present study, a transcriptome analysis was carried out using both the resistant (PI88788) and susceptible (Williams 82) soybean cultivars to characterize the responses to nematode infection. Various defense-related genes and different pathways involved in nematode resistance were recognized as being highly expressed in resistant cultivar. Promoter-GUS analysis was conducted to monitor the spatial expression pattern of the genes highly induced by nematode infection. Two nematode-inducible promoters for Glyma.05g147000 (encoding caffeoyl-CoA O-methyltransferase) and Glyma.06g036700 (encoding cupredoxin superfamily protein) were characterized, and the promoters could efficiently drive the expression of known nematode resistance genes (α-SNAPRhg1HC or GmSHMT) to affect soybean SCN resistance. Interestingly, expression of the cupredoxin family genes was upregulated not only by SCN, but also by jasmonic acid treatment. DNA sequence analysis identified that a conserved motif (GGTGCATG) with high similarity to SCNbox1 and GC-rich element is enriched in their promoter regions, suggesting its potential to serve as a nematode-responsive regulatory element. Overexpression of Glyma.06g036700 significantly enhanced soybean resistance to cyst nematode. Overall, our findings not only highlight the essential role of cupredoxin family genes in SCN resistance, but also offer potential functional tools to develop nematode resistance in crops.

Transcriptome Analysis Reveals Candidate Lignin-Related Genes and Transcription Factors during Fruit Development in Pomelo (Citrus maxima).

Juice sac granulation (a physiological disorder) leads to large postharvest losses of pomelo (Citrus maxima). Previous studies have shown that juice sac granulation is closely related to lignin accumulation, while the molecular mechanisms underlying this disorder remain elusive in pomelo. Our results showed that the lignin content in NC (near the core) and FC (far away from the core) juice sacs overall increased from 157 DPA (days post anthesis) to 212 DPA and reached a maximum at 212 DPA. Additionally, the lignin content of NC juice sacs was higher than that of FC juice sacs. In this study, we used transcriptome-based weighted gene co-expression network analysis (WGCNA) to address how lignin formation in NC and FC juice sacs is generated during the development of pomelo. After data assembly and bioinformatic analysis, we found a most correlated module (black module) to the lignin content, then we used the 11 DEGs in this module as hub genes for lignin biosynthesis. Among these DEGs, PAL (phenylalanine ammonia lyase), HCT (hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase), 4CL2 (4-coumarate: CoA ligase), C4H (cinnamate 4-hydroxylase), C3′H (p-coumarate 3-hydroxylase), and CCoAOMT1 (caffeoyl CoA 3-Omethyltransferase) were the most distinct DEGs in granulated juice sacs. Co-expression analysis revealed that the expression patterns of several transcription factors such as MYB, NAC, OFP6, and bHLH130 are highly correlated with lignin formation. In addition, the expression patterns of the DEGs related to lignin biosynthesis and transcription factors were validated by qRT-PCR, and the results were highly concordant with the RNA-seq results. These results would be beneficial for further studies on the molecular mechanism of lignin accumulation in pomelo juice sacs and would help with citrus breeding.

The Genetic Architecture of Nitrogen Use Efficiency in Switchgrass (Panicum virgatum L.).

Switchgrass (Panicum virgatum L.) has immense potential as a bioenergy crop with the aim of producing biofuel as an end goal. Nitrogen (N)-related sustainability traits, such as nitrogen use efficiency (NUE) and nitrogen remobilization efficiency (NRE), are important factors affecting switchgrass quality and productivity. Hence, it is imperative to develop nitrogen use-efficient switchgrass accessions by exploring the genetic basis of NUE in switchgrass. For that, we used 331 diverse field-grown switchgrass accessions planted under low and moderate N fertility treatments. We performed a genome wide association study (GWAS) in a holistic manner where we not only considered NUE as a single trait but also used its related phenotypic traits, such as total dry biomass at low N and moderate N, and nitrogen use index, such as NRE. We have evaluated the phenotypic characterization of the NUE and the related traits, highlighted their relationship using correlation analysis, and identified the top ten nitrogen use-efficient switchgrass accessions. Our GWAS analysis identified 19 unique single nucleotide polymorphisms (SNPs) and 32 candidate genes. Two promising GWAS candidate genes, caffeoyl-CoA O-methyltransferase (CCoAOMT) and alfin-like 6 (AL6), were further supported by linkage disequilibrium (LD) analysis. Finally, we discussed the potential role of nitrogen in modulating the expression of these two genes. Our findings have opened avenues for the development of improved nitrogen use-efficient switchgrass lines.

Transcriptome characterization of Larrea tridentata and identification of genes associated with phenylpropanoid metabolic pathways.

Larrea tridentata (Sesse and Moc. ex DC.) Coville (family: Zygophyllaceae) is an aromatic evergreen shrub with resin-covered leaves, known to use in traditional medicine for diverse ailments. It also has immense pharmacological significance due to presence of powerful phenylpropanoids antioxidant, nordihydroguaiaretic acid (NDGA). The RNA sequence/transcriptome analyses connect the genomic information into the discovery of gene function. Hence, the acquaint analysis of L. tridentata is in lieu to characterize the transcriptome, and to identify the candidate genes involved in the phenylpropanoid biosynthetic pathway. To gain molecular insight, the bioinformatics analysis of transcriptome was performed. The total bases covered 48,630 contigs of length greater than 200 bp and above came out to 21,590,549 with an average GC content of 45% and an abundance of mononucleotide, SSR, including C3H, FAR1, and MADS transcription gene families. The best enzyme commission (EC) classification obtained from the assembled sequences represented major abundant enzyme classes e.g., RING-type E3 ubiquitin transferase and non-specific serine/threonine protein kinase. The KEGG pathway analysis mapped into 377 KEGG different metabolic pathways. The enrichment of phenylpropanoid biosynthesis pathways (22 genes i.e., phenylalanine ammonia-lyase, trans-cinnamate 4-monooxygenase, 4-coumarate-CoA ligase, cinnamoyl-CoA reductase, beta-glucosidase, shikimate O-hydroxycinnamoyl transferase, 5-O-(4-coumaroyl)-D-quinate 3’-monooxygenase, cinnamyl-alcohol dehydrogenase, peroxidase, coniferyl-alcohol glucosyltransferase, caffeoyl shikimate esterase, caffeoyl-CoA O-methyltransferase, caffeate O-methyltransferase, coniferyl-aldehyde dehydrogenase, feruloyl-CoA 6-hydroxylase, and ferulate-5-hydroxylase), and expression profile indicated antioxidant, anti-arthritic, and anticancer properties of L. tridentata. The present results could provide an important resource for squeezing biotechnological applications of L. tridentata.

Simultaneous elevation of acid-insoluble lignin and syringyl lignin is the preliminary Cd detoxification strategy in a Cd pollution-safe cultivar (Cd-PSC) of Brassica parachinensis L.

Cadmium (Cd) immobilization in the cell wall is responsible for Cd detoxification in Cd-accumulating cultivars. Lignin is the dominant constituent of the secondary cell wall (SCW), but its role in Cd compartmentalization is poorly understood. The mechanism of Cd compartmentalization by lignin was thus investigated herein. Lignin content and composition, as well as related genes and proteins, were analyzed in the Cd pollution-safe cultivar Brassica parachinensis L. ‘SJ19’. The lignin microstructural regions and Cd affinities were characterized by 2D-heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) and density functional theory (DFT). Lignin combined with 14.2% of the total Cd in SJ19, with a higher Cd distribution in acid-insoluble lignin (AIL). Additionally, the proportion of AIL increased under Cd treatment. The increase in the proportion of the lignin aromatic region syringyl (S) and decrease in the guaiacyl (G) proportion were in accordance with the significant up-regulation of peroxidase P7 and down-regulation of caffeoyl-CoA O-methyltransferase At1g67980 under Cd stress. Moreover, the Cd affinity associating with the lignin aromatic region ranked as S > G> H (p-hydroxyphenyl). Therefore, rather than lignin biosynthesis, the simultaneous elevation of AIL and S lignin was recognized as the preliminary strategy for lignin compartmentalization of Cd in SCW for the first time herein. This is beneficial for Cd detoxification in the Cd pollution-safe cultivar of B. parachinensis.

Molecular cloning and characterization of two distinct caffeoyl CoA O-methyltransferases (CCoAOMTs) from the liverwort Marchantia paleacea

Caffeoyl CoA O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to a hydroxyl moiety of caffeoyl-CoA as part of the lignin biosynthetic pathway. CCoAOMT-like proteins also catalyze to a variety of flavonoids, coumarins, and phenylpropanoids. Several CCoAOMTs that prefer flavonoids as substrates have been characterized from liverworts. Here, we cloned two CCoAOMT genes, MpalOMT2 and MpalOMT3, from the liverwort Marchantia paleacea. MpalOMT3 has a second ATG codon downstream and the truncated version that lacks 11 amino acids was named MpalOMT3-Tr. Phylogenetic analysis placed MpalOMT3 at the root of the clade with true CCoAOMTs from vascular plants and placed MpalOMT2 between the CCoAOMT and CCoAOMT-like proteins. Recombinant OMTs methylated caffeoyl CoA, phenylpropanoids, and flavonoids containing two or three vicinal hydroxyl groups. MpalOMT3 showed higher catalytic activity for phenylpropanoids than MpalOMT2, but MpalOMT2 showed more promiscuous towards eriodictyol and myricetin. The lignin content in Arabidopsis thaliana stems increased with constitutive heterologous expression of MpalOMT3-Tr, but not MpalOMT2. Subcellular localization experiments indicated that the N-terminus of MpalOMT3 probably served as a chloroplast transit peptide and inhibited its enzymatic activity. Combining the phylogenetic analysis and functional characterization, we conclude that the liverwort M. paleacea harbors true CCoAOMT and CCoAOMT-like genes.

Long-term subculture affects rooting competence via changes in the hormones and protein profiles in Cedrela fissilis Vell. (Meliaceae) shoots

Long-term subculture plays an essential role in the large-scale multiplication and production of somatic plantlets. We investigated the effects of long-term subculture on in vitro shoot development and ex vitro rooting associated with changes in the hormones and protein profiles in Cedrela fissilis. The number of subcultures of shoots induced a decrease in the ex vitro rooting response. The reduction in adventitious root (AR) formation was associated with decreases in the contents of indole-3-acetic acid (IAA), abscisic acid (ABA), 12-oxo-phytodienoic acid (OPDA), putrescine (Put), and spermine and increases in jasmonic acid (JA), jasmonoyl-isoleucine, trans-cinnamic acid, and salicylic acid contents in shoots at the fourth subculture compared to the first. The ornithine decarboxylase enzyme preferentially functions in the Put biosynthesis pathway, and it was related to the highest AR formation in shoots at the first subculture. Down-accumulation of the auxin-binding protein ABP19a in shoots from the fourth subculture compared to the first subculture was related to a decrease in both the IAA content and AR formation. In addition, down-accumulation of glucose-6-phosphate isomerase; glutamine synthetase leaf isozyme, chloroplastic; 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase; l-ascorbate peroxidase, cytosolic; monodehydroascorbate reductase; and 2-Cys peroxiredoxin BAS1-like, chloroplastic and up-accumulation of caffeoyl-CoA O-methyltransferase 1 and isoforms of peroxidase 4 proteins in shoots from the fourth relative to the first subculture were associated with a reduction in AR formation. These results showed that the understanding of hormonal and molecular mechanisms related to the potential of AR formation in shoots under successive subcultures is relevant to improving large-scale plantlet production in C. fissilis.

Comparative physiology and transcriptome analysis reveals the regulatory mechanism of genome duplication enhancing cold resistance in Fragaria nilgerrensis

Polyploidization plays an important role in plant adaption to adverse environments, but the mechanism by which polyploidization confers adaptation to cold stress remains elusive. Here, autotetraploid strawberry (2n = 4x = 28) was successfully synthesized from wild strawberry (Fragaria nilgerrensis; 2n = 2x = 14) using a primary meristem processing technique. The autotetraploid plants exhibited a higher resistance to cold stress compared to their parental diploids, revealed by a series of physiological-biochemical examinations. Transcriptome analysis showed that expression pattern changes of genes in several pathways were the key contributors to the high cold resistance of autotetraploid strawberry. These main players included Ca2+ signal transduction, transcription factors (TFs), reactive oxygen species (ROS) scavenging enzyme, phenylpropanoid biosynthesis, abscisic acid (ABA) signaling and carbohydrate metabolism. The coordinate action of these factors enhanced the antioxidant defense system and protected the integrity of membranes and cellular organelles. Notably, gene co-expression network analysis (WGCNA) demonstrated that four hub genes (regulating cinnamyl-alcohol dehydrogenase (CAD), peroxidase (POD), caffeoyl-CoA O-methyltransferase (CCoAMT) and calmodulin-like protein (CML)) function much importantly in the network regulating this high cold resistance. Therefore, a model is proposed to elucidate the regulatory mechanisms of high cold resistance in the autotetraploid strawberry. The present findings provide new insights into understanding the complex regulatory network responsible for enhancing cold resistance in ployploid plants.

Arabidopsis CCoAOMT1 Plays a Role in Drought Stress Response via ROS- and ABA-Dependent Manners.

Plants possess adaptive reprogramed modules to prolonged environmental stresses, including adjustment of metabolism and gene expression for physiological and morphological adaptation. CCoAOMT1 encodes a caffeoyl CoA O-methyltransferase and is known to play an important role in adaptation of Arabidopsis plants to prolonged saline stress. In this study, we showed that the CCoAOMT1 gene plays a role in drought stress response. Transcript of CCoAOMT1 was induced by salt, dehydration (drought), and methyl viologen (MV), and loss of function mutants of CCoAOMT1, ccoaomt1-1, and ccoaomt1-2 exhibit hypersensitive phenotypes to drought and MV stresses. The ccoaomt1 mutants accumulated higher level of H2O2 in the leaves and expressed lower levels of drought-responsive genes including RD29B, RD20, RD29A, and ERD1, as well as ABA3 3 and NCED3 encoding ABA biosynthesis enzymes during drought stress compared to wild-type plants. A seed germination assay of ccoaomt1 mutants in the presence of ABA also revealed that CCoAOMT1 functions in ABA response. Our data suggests that CCoAOMT1 plays a positive role in response to drought stress response by regulating H2O2 accumulation and ABA signaling.

Transcriptome profiling reveals histone deacetylase 1 gene overexpression improves flavonoid, isoflavonoid, and phenylpropanoid metabolism in Arachis hypogaea hairy roots.

BackgroundThe peanut (Arachis hypogaea) is a crop plant of high economic importance, but the epigenetic regulation of its root growth and development has not received sufficient attention. Research on Arabidopsis thaliana has shown that histone deacetylases (HDACs) are involved in cell growth, cell differentiation, and stress response. Few studies have focused on the role of HDACs in the root development of other plants, particularly crop plants. In earlier studies, we found large accumulations of A. hypogaea histone deacetylase 1 (AhHDA1) mRNA in peanut roots. However, we did not explore the role of AhHDA1 in peanut root development.MethodsIn this paper, we investigated the role of the peanut AhHDA1 gene and focused on the effect of altered AhHDA1 expression in hairy roots at both the phenotypic and transcriptional levels. We analyzed the transformation of A. hypogaea hairy roots using Agrobacterium rhizogenes and RNA sequencing to identify differentially expressed genes that were assigned to specific metabolic pathways. Transgenic hairy roots were used as experimental material to analyze the downstream genes expression and histone acetylation levels. To thoroughly understand AhHDA1 function, we also simultaneously screened the AhHDA1-interacting proteins using a yeast two-hybrid system.ResultsAhHDA1-overexpressing hairy roots were growth-retarded after 20 d in vitro cultivation, and they had a greater accumulation of superoxide anions and hydrogen peroxide than the control and RNAi groups. AhHDA1 overexpression in hairy roots accelerated flux through various secondary synthetic metabolic pathways, as well as inhibited the primary metabolism process. AhHDA1 overexpression also caused a significant upregulation of genes encoding the critical enzyme chalcone synthase (Araip.B8TJ0, CHS) in the flavonoid biosynthesis pathway, hydroxyisoflavanone synthase (Araip.0P3RJ) in the isoflavonoid biosynthesis pathway, and caffeoyl-CoA O-methyltransferase (Aradu.M62BY, CCoAOMT) in the phenylpropanoid biosynthesis pathway. In contrast, ferredoxin 1 (Araip.327XS), the polypeptide of the oxygen-evolving complex of photosystem II (Araip.N6ZTJ), and ribulose bisphosphate carboxylase (Aradu.5IY98) in the photosynthetic pathway were significantly downregulated by AhHDA1 overexpression. The expression levels of these genes had a positive correlation with histone acetylation levels.ConclusionOur results revealed that the relationship between altered gene metabolism activities and AhHDA1 overexpression was mainly reflected in flavonoid, isoflavonoid, and phenylpropanoid metabolism. AhHDA1 overexpression retarded the growth of transgenic hairy roots and may be associated with cell metabolism status. Future studies should focus on the function of AhHDA1-interacting proteins and their effect on root development.

Expression of CCoAOMT from Zingiber officinale Roscoe under NaCl Stress and Its Regulatory Role in 6-Gingerol Biosynthesis

Ginger (Zingiber officinale Roscoe) is of high economic value as a medicinal and food resource. 6‑Gingerol is the predominant bioactive component in the rhizome of ginger. A crucial enzyme involved in 6-gingerol biosynthesis is caffeoyl coenzyme A O-methyltransferase (CCoAOMT). In the present study, the cDNA sequence of ZoCCoAOMT from ginger was cloned. The expression level of ZoCCoAOMT and the contents of 6-gingerol in the rhizome were analyzed in ginger plants treated with different concentrations of NaCl. The full-length ZoCCoAOMT cDNA consisted of 738 bp and showed 96.34% sequence homology with Curcuma longa CCoAOMT. Transcription of ZoCCoAOMT was highly consistent with the trend in accumulation of 6-gingerol. The ZoCCoAOMT transcript level and 6-gingerol content in the rhizome were highest under treatment with 25 g/L NaCl but decreased under higher concentrations of NaCl, which suggested a positive regulatory relationship between ZoCCoAOMT and 6-gingerol synthesis. Virus-induced gene silencing (VIGS) analysis further verified the positive regulatory role of ZoCCoAOMT in 6-gingerol synthesis. The results provide fundamental information for clarification of the role of ZoCCoAOMT in the 6-gingerol biosynthesis pathway of Z. officinale, and also gene resources for 6-gingerol biosynthesis.

Engineering Alfalfa to Produce 2-O-Caffeoyl-L-Malate (Phaselic Acid) for Preventing Post-harvest Protein Loss via Oxidation by Polyphenol Oxidase.

Many plants accumulate high levels of hydroxycinnamoyl esters and amides in their tissues, presumably to protect against biotic and abiotic stress. Red clover (Trifolium pretense) leaves accumulate high levels [5–15 mmol/kg fresh weight (FW)] of caffeic acid derivatives, including phaselic acid (2-O-caffeoyl-L-malate). Oxidation of caffeoyl-malate by an endogenous polyphenol oxidase (PPO) has been shown to help preserve forage protein after harvest and during storage as silage, which should improve N use efficiency in dairy and other ruminant production systems. The widely grown forage alfalfa lacks both PPO and PPO substrates and experiences substantial loss of protein following harvest. We previously identified a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT, previously called HCT2) responsible for phaselic accumulation in red clover. With the goal of producing PPO-oxidizable compounds in alfalfa to help preserve forage protein, we expressed red clover HMT in alfalfa. Leaves of these alfalfa accumulated mainly p-coumaroyl- and feruloyl-malate (up to 1.26 and 0.25 mmol/kg FW, respectively). Leaves of HMT-expressing alfalfa supertransformed with an RNA interference (RNAi) construct to silence endogenous caffeoyl-CoA acid O-methyltransferase (CCOMT) accumulated high levels of caffeoyl-malate, as well as the p-coumaroyl and feruloyl esters (up to 2.16, 2.08, and 3.13 mmol/kg FW, respectively). Even higher levels of caffeoyl- and p-coumaroyl-malate were seen in stems (up to 8.37 and 3.15 mmol/kg FW, respectively). This level of caffeoyl-malate accumulation was sufficient to inhibit proteolysis in a PPO-dependent manner in in vitro experiments, indicating that the PPO system of post-harvest protein protection can be successfully adapted to alfalfa.