cinnamoyl-CoA Reductase Research Articles

Transcriptomic analysis identifies differentially expressed genes in purple tender shoots and green mature leaves of Zijuan tea

Purple shoots of tea germplasm Zijuan (Camellia sinensis var. asssamica) have high anthocyanin content and gradually turn green with continued growth and development. To uncover the mechanism of this colour change, we performed transcriptome analysis to identify differentially expressed genes (DEGs). A total of 18 328 DEGs were identified in purple tender shoots of Zijuan (ZJP) compared with mature green leaves of Zijuan (ZJG). These included 85 DEGs associated with phenylpropanoid biosynthesis, 30 DEGs associated with flavonoid biosynthesis, and 3 DEGs involved in flavone and flavonol biosynthesis. We detected higher expression in ZJP than in ZJG of genes encoding the following key enzymes regulating anthocyanin biosynthesis and their corresponding metabolites: chalcone synthase (CHS), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), adenosine triphosphate (ATP)-binding cassette (ABC) transporters, bifunctional 3-dehydroquinate dehydratase (DHQ), chorismate mutase (CM), cinnamoyl-CoA reductase 1 (CCR), and cinnamyl alcohol dehydrogenase (CAD). In addition, upregulated expression of carbohydrate metabolism, glycolysis pathway, sucrose metabolism, and pyruvate metabolism suggested that glycolysis and upregulation of the sucrose synthase (SUS) gene may provide more intermediates as substrates for promoting anthocyanin accumulation in ZJP. Moreover, DEGs involved in anthocyanin biosynthesis, including phenylalanine ammonialyase (PAL), cinnamate acid 4-hydroxylase (C4H), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), ANR, ANS, CHS, and flavonoid 3',5'-hydroxylase (F3'5'H) were significantly (P < 0.01) correlated with the contents of anthocyanin components such as cyanidin (Cy), delphinidin (Dp), peonidin (Pn), petunidin (Pt), and malvidin (Mv). The above results suggested that these upregulated DEGs may contribute to anthocyanin accumulation in ZJP and may play important roles in the colour changes in Zijuan tea. This research provided a foundation for clarifying the mechanisms underlying colour changes in Zijuan tea.

Key genes for phenylpropanoid metabolite biosynthesis during half-highbush blueberry (Vaccinium angustifolium×Vaccinium corymbosum) fruit development

BACKGROUND: Blueberry fruits contain large amounts of phenolic compounds derived from the phenylpropanoid pathway. Their biosynthesis is complex, involving many enzymes. OBJECTIVE: We sought to investigate the content of phenylpropanoid-derived compounds and identify key genes involved in the phenylpropanoid metabolite pathway during half-highbush blueberry fruit development. METHODS: Phenylpropanoid metabolite contents were determined by high-performance liquid chromatography (HPLC) and spectrophotometry. Gene expression was examined through reverse-transcription PCR. RESULTS: Phloretin, chlorogenic acid, total flavonol, quercetin, catechin, and proanthocyanidin contents were highest in small-sized green fruits; myricetin and epicatechin contents were highest in pink fruits; and lignin and anthocyanin were highest in blue fruits. Genes from the 4-coumarate CoA ligase (4CL) family regulate the biosynthesis of phenylpropanoid metabolites. Phenylalanine ammonia-lyase (PAL) and cinnamoyl-CoA reductase (CCR) are key genes in the lignin biosynthetic pathway. Flavonol synthase (FLS) is a key gene affecting total flavonols and the quercetin biosynthetic pathway. PAL and chalcone isomerase (CHI) are key genes in the epicatechin and anthocyanin biosynthetic pathways, respectively. CONCLUSION: Phenylpropanoid metabolites are regulated by multiple genes from the same or different families. Enzymes in different metabolic pathways compete for precursors to form a complex regulatory network for phenylalanine metabolism.

Analyses of Pepper Cinnamoyl-CoA Reductase Gene Family and Cloning of CcCCR1/2 and Their Function Identification in the Formation of Pungency

Cinnamoyl-CoA reductases (CCR) have a possible role in pungency formation of pepper because they can convert feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA into lignin, which are also competitive precursors of capsaicin biosynthesis in phenylpropanoid metabolism. In this study, genome-wide CCR gene family, exon–intron structures, sequence homology, phylogenetic characterization, and promoters were analyzed in pepper. Two CCR genes were cloned from Capsicum chinense, their enzymic kinetic parameters and regulatory function were identified by heterologous expression, ectopic expression, and VIGS. In total, 38 genes were found as predicted CCRs or CCR-like proteins and were composed of 2–10 exons. The promoters of pepper CCRs contained growth, stress, hormone, and light-response elements. The affinity and catalytic efficiency of CcCCR1/2 to feruolyl-CoA was the highest. The analysis of metabolic substances showed that capsaicin content was negatively correlated with lignin and positively correlated with flavonoids. The highest expression of CcCCR1 was found in stems, the higher expression of CcCCR2 was found in stem and early fruit than other organs. CCR1, 2 had certain effects on capsaicin content by regulating related enzyme activity, CCR2 played a more important role in regulating pungency formation. Our results clarify the competitive mechanism between lignin and capsaicin biosynthesis and provide an explanation for spice regulation.

Comparative transcriptome and tissue-specific expression analysis of genes reveal tissue-cultured plants as an alternative source for phenylethanoids and phenylpropanoids in Rhodiola imbricata (Edgew.)

Rhodiola imbricata (Crassulaceae) is a traditional trans-Himalayan endangered medicinal herb with immense therapeutic applications. Over the years, over-exploitation, un-managed harvesting, and lack of captive cultivation procedures persuaded threat to its wild habitat. Plant tissue culture and RNA-Seq-based molecular bioprospection of key regulatory genes aid the understanding of molecular dynamics involved in specialized metabolites (phenylethanoids and phenylpropanoids) biosynthesis and its sustainable production. Hence, comparative transcriptomic analysis was performed using leaf and root tissues from the wild and tissue-cultured plants, revealing tissue-specific production of salidroside and rosavin. The transcriptome profiling resulted in 345 million high-quality reads yielding 92,380 unique transcripts with an N50 of 1260 bp. Tissue-specific gene expression analysis revealed that both phenylethanoids and phenylpropanoids biosynthesis are predominantly associated with the shikimate pathway. In addition to RNA-Seq data, the downstream biosynthesis pathways genes viz., phospho-2-dehydro-3-deoxyheptonate aldolase (DAHPS), 3-dehydroquinate synthase (DHQS), shikimate kinase (SK), chorismate mutase (CM), arogenate dehydrogenase (TYRAAT), aromatic-L-amino-acid decarboxylase (TDC), phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4-CL), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) showed higher expression pattern in wild plant tissues compared to tissue-cultured plants. The transcript fold expression determined by RT-qPCR results followed similar patterns as those observed in RNA-seq and targeted metabolite profiling data. Salidroside and rosavin content in wild plants exhibited 2.40 fold and 1.77 fold increase accumulation compared to the tissue-cultured plant. The present investigation explained the tissue and condition-specific significant differences between the expression of proposed biosynthetic pathway genes and salidroside and rosavin content. Additionally, NAC, bHLH, and ARF were the most abundant transcription factor families found in the transcriptomic analysis of R. imbricata. The generated transcriptome dataset provides a valuable gene(s)/transcription factors hub that can be used for the sustainable production of salidroside and rosavin in R. imbricata under tissue culture conditions.

Proteomics analysis reveals new insights into surface pitting of sweet cherry cultivars displaying contrasting susceptibility

ABSTRACT Surface pitting in sweet cherry (Prunus avium L.) is characterised by depressions development on the skin surface. Pitting damage happens during harvest and postharvest handling and develops during cold storage. This study compared two cultivars with contrasting susceptibility to pitting (Kordia: tolerant; Sweetheart: susceptible) using a LC-MS/MS proteomic approach to identify key metabolic and signalling pathways related to this disorder during ripening and postharvest storage. The variability observed in the principal component analysis was driven by the cultivars suggesting that surface pitting susceptibility is triggered by the inherent differences between cultivars. Proteins involved in anthocyanin biosynthesis were more abundant in Kordia. Moreover, CCR (cinnamoyl-CoA reductase) and FLS (flavonol synthase) proteins showed higher abundance in Sweetheart. An overexpression in enzymes related to the synthesis of abscisic acid (ABA), jasmonic acid (JA) and ethylene were found in Kordia. A higher abundance of sucrose synthase (SUS), UDP-glycosyltransferases (UDP-GT) and polygalacturonase-inhibiting proteins (PGIP) were observed in Kordia, while a higher content of invertase (INV) was observed in Sweetheart. The overexpression of the studied pathways suggests that tolerance to surface pitting could be correlated to one or more of these factors, and susceptibility might be given by the inherent differences in the metabolic processes of each cultivar.

Molecular cloning and characterization of Cinnamoyl-CoA reductase promoter gene from Asarum sieboldii Miq.

Asarum sieboldii Miq., a perennial herb of the family Aristolochiaceae, is widely used in China to treat cold, fever, aphthous stomatitis, toothache, gingivitis, and rheumatoid arthritis. Methyleugenol is the most representative pharmacological constituent of this medicinal herb. Cinnamoyl-CoA reductase (CCR), which has been well known for occupying a critical position in the lignin biosynthesis pathway, is also shared with the biosynthesis of methyleugenol. To better understand the regulatory mechanisms of methyleugenol biosynthesis, a 1530-bp long promoter region of the AsCCR1 gene was isolated. PLACE and PlantCARE analysis affirmed the existence of the core promoter elements such as TATA and CAAT boxes, abiotic stress-responsive cis-regulation elements like abscisic acid-responsive element, G-box, and MBS in the isolated sequence. The histochemical assay suggested that it was a constitutive promoter, highly expressed in the root tissue. Moreover, the region of -200 bp to ATG (start codon) was enough to drive the expression of It GUS gene. Treatments with low temperature and high concentration of gibberellin or abscisic acid demonstrated the abiotic stress-induced expression of the AsCCR1 promoter. Overall, this study revealedthe isolation and characterization of the AsCCR1 promoter. Moreover, it also provided a candidate gene for molecular breeding in A. sieboldii to enhance its pharmacological potential.

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.

Chitosan Treatment Promotes Wound Healing of Apple by Eliciting Phenylpropanoid Pathway and Enzymatic Browning of Wounds.

Chitosan is an elicitor that induces resistance in fruits against postharvest diseases, but there is little knowledge about the wound healing ability of chitosan on apple fruits. Our study aimed at revealing the effect of chitosan on the phenylpropanoid pathway by determining some enzyme activities, products metabolites, polyphenol oxidase activity, color (L*, b*, a*), weight loss, and disease index during healing. Apple (cv. Fuji) fruits wounded artificially were treated with 2.5% chitosan and healed at 21–25°C, relative humidity = 81–85% for 7 days, and non-wounded fruits (coated and non-coated) were used as control. The result shows that chitosan treatment significantly decreased weight loss of wounded fruits and disease index of Penicillium expansum inoculated fruits. The activities of phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumaryl coenzyme A ligase (4CL), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) were elicited throughout the healing period by chitosan, which increased the biosynthesis of cinnamic acid, caffeic acid, ferulic acid, sinapic acid, p-coumaric acid, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. Also, total phenol, flavonoid, and lignin contents were significantly increased at the fruits wounds. In addition, chitosan’s ability to enhance polyphenol oxidase activity stimulated enzymatic browning of wounds. Although wounding increased phenylpropanoid enzymes activities before healing, chitosan caused higher enzyme activities for a significant healing effect compared with the control. These findings imply that chitosan accelerates apple wound healing by activating the phenylpropanoid pathway and stimulating enzymatic browning of wounds.

Arbuscular mycorrhizal fungi enhanced cadmium uptake in Photinia frase through altering root transcriptomes and root-associated microbial communities

As a non-essential metal, cadmium (Cd) pollution poses severe threats to plant growth, environment, and human health. Phytoextraction using nursery stocks prior to their transplantation is a potential useful approach for bioremediation of Cd contaminated soil. A greenhouse pot experiment was performed to investigate the growth, Cd accumulation, profiles of transcriptome as well as root-associated microbiomes of Photinia frase in Cd-added soil, upon inoculation of two types of arbuscular mycorrhizal fungi (AMF) Sieverdingia tortuosa and Funneliformis mosseae. Compared with the control, inoculation of F. mosseae increased Cd concentrations in root, stem and leaf by 57.2%, 44.1% and 71.1%, respectively, contributing to a total Cd content of 182 μg/plant. KEGG pathway analysis revealed that hundreds of genes involved in 'Mitogen-activated protein kinase (MAPK) signaling pathway', 'plant hormone signal transduction', 'biosynthesis of secondary metabolites' and 'glycolysis/gluconeogenesis' were enriched upon inoculation of F. mosseae. The relative abundance of Acidobacteria was increased upon inoculation of S. tortuosa, while Chloroflexi and Patescibacteria were increased upon inoculation of F. mosseae, and the abundance of Glomerales increased from 23.0% to above 70%. Correlation analysis indicated that ethylene-responsive transcription factor, alpha-aminoadipic semialdehyde synthase, isoamylase and agmatine deiminase related genes were negatively associated with the relative abundance of Glomerales operational taxonomic units (OTUs) upon inoculation of F. mosseae. In addition, plant cysteine oxidase, heat shock protein, cinnamoyl-CoA reductase and abscisic acid receptor related genes were positively associated with the relative abundance of Patescibacteria OTUs upon inoculation of F. mosseae. These finding suggested that AMF can enhance P. frase Cd uptake by modulating plant gene expression and altering the structure of the soil microbial community. This study provides a theoretical basis for better understanding the relationship between root-associated microbiomes and root transcriptomes of P. frase, from which a cost-effective and environment-friendly strategy for phytoextraction of Cd in Cd-polluted soil might be developed.

Transcriptome and metabolite profiling to identify genes associated with rhizome lignification and the function of ZoCSE in ginger (Zingiber officinale).

Ginger (Zingiber officinale Roscoe) is an important spice crop in China, and fresh ginger rhizomes are consumed as vegetable in Sichuan and Chongqing. However, tissue lignification accelerates with rhizome maturation, resulting in the loss of edible quality. To understand the molecular mechanisms of texture modification during rhizome development, we investigated lignin accumulation patterns and identified the key genes associated with lignin biosynthesis using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA-sequencing (RNA-Seq). Results showed that the contents of total lignin and its precursors exhibited notable declines with tissue maturation. However, the lignin composition was remarkably modified and syringyl lignin was deposited in mature rhizomes, leading to ginger lignification. Transcriptome analysis displayed 32 lignin biosynthetic genes were dramatically downregulated with rhizome development, including caffeoylshikimate esterase (CSE ), 4-coumarate-CoA ligase , laccase , cinnamoyl-CoA reductase , cinnamyl-alcohol dehydrogenase , peroxidase and caffeic acid 3-O-methyltransferase , indicating that lignin reduction might be attributed to deficiency in intermediates or the downregulation of key biosynthetic enzymes. Furthermore, overexpressing ZoCSE in Nicotiana benthamiana L. enhanced the total lignin content, suggesting its fundamental role in lignin biosynthesis. RNA-Seq also identified candidate lignin production regulators, including hormone-related genes and NAC/MYB transcription factors (ZoNAC1 , ZoNAC4 , ZoMYB14 and ZoMYB17 ). This result provides a molecular basis for lignin accumulation in ginger.

The SlHB8 Acts as a Negative Regulator in Stem Development and Lignin Biosynthesis.

The stem is an important organ in supporting plant body, transporting nutrients and communicating signals for plant growing. However, studies on the regulation of stem development in tomato are rather limited. In our study, we demonstrated that SlHB8 negatively regulated tomato stem development. SlHB8 belongs to homeo domain-leucine zipper Class III gene family transcription factors and expressed in all the organs examined including root, stem, leaves, flower, and fruit. Among these tissues, SlHB8 showed stable high expression level during tomato stem development. Overexpression of SlHB8 gene decreased stem diameter with inhibited xylem width and xylem cell layers, while loss of function of SlHB8 gene increased the stem diameter and xylem width. The contents of lignin were decreased both in leaves and stems of SlHB8 overexpression plants. RNA-seq analysis on the stems of wild type and SlHB8 transgenic plants showed that the 116 DEGs (differential expressed genes) with reversible expression profiles in SlHB8-ox and SlHB8-cr plants were significantly enriched in the phenylpropanoid biosynthesis pathway and plant-pathogen pathway which were related to lignin biosynthesis and disease resistance. Meanwhile, the key genes involved in the lignin biosynthesis pathway such as SlCCR (cinnamoyl-CoA reductase), SlCYP73A14/C4H (cinnamate 4-hydroxylase), SlC3H (coumarate 3-hydroxylase) and SlCAD (cinnamoyl alcohol dehydrogenase) were down-regulated in both stem and leaves of SlHB8 overexpression plants, indicating a negative regulatory role of SlHB8 in the lignin biosynthesis and stem development.

Two types of cinnamoyl-CoA reductase function divergently in accumulation of lignins, flavonoids and glucosinolates and enhance lodging resistance in Brassica napus

Brassica crops, which are of worldwide importance, provide various oil, vegetable and ornamental products, as well as feedstocks for animal husbandry and biofuel industry. Cinnamoyl-CoA reductase (CCR) is the entry point to the lignin pathway and a crucial locus in manipulation of associated traits, but CCR-associated metabolism and traits in Brassica crops have remained largely unstudied except in Arabidopsis thaliana. We report the identification of 16 CCR genes from Brassica napus and its parental species B. rapa and B. oleracea. The BnCCR1 and BnCCR2 subfamilies displayed divergent organ-specificity and participation in the yellow-seed trait. Their functions were dissected via overexpression of representative paralogs in B. napus. BnCCR1 was expressed preferentially in G- and H-lignin biosynthesis and vascular development, while BnCCR2 was expressed in S-lignin biosynthesis and interfascicular fiber development. BnCCR1 showed stronger effects on lignification-related development, lodging resistance, phenylpropanoid flux control, and seed coat pigmentation, whereas BnCCR2 showed a stronger effect on sinapate biosynthesis. BnCCR1 upregulation delayed bolting and flowering time, while BnCCR2 upregulation weakened the leaf vascular system in consequence of suppressed G-lignin accumulation. BnCCR1 and BnCCR2 were closely but almost oppositely linked with glucosinolate metabolism via inter-pathway crosstalk. We conclude that BnCCR1 and BnCCR2 subfamilies offer great but differing potential for manipulating traits associated with phenylpropanoids and glucosinolates. This study reveals the CCR1–CCR2 divergence in Brassicaceae and offers a resource for rapeseed breeding for lodging resistance, yellow-seed traits, and glucosinolate traits.

CRISPR- mediated Mutation in Cinnamoyl- CoA Reductase 4 in Allohexaploid Oilseed Crop Camelina sativa, Revealed its Pivotal Role in Resistance Against Sclerotinia sclerotiorum.

Background: Sclerotinia sclerotiorum (Ss) is a broad host range necrotrophic ascomycete fungus affecting over 400 plant species. Ss causes stem rot disease on Camelina sativa (Cs) an allohexaploid crucifer species that is promoted as a low input crop and industrial oil attributes suitable as biofuel and lubricant. Histochemical and molecular studies has linked resistance to Ss in C. sativa with the cell wall lignification (Eynck et al., 2012) and reported constitutive expression of Cinnamoyl-CoA Reductase 4 (CsCCR4) gene, in the Cs resistant line CN114263. Modern breeding efforts, such as gene editing, are needed to improve commercial lines and to limit the risk of crop loss which would be substantial to producers.Objectives: To investigate the importance of monolignol biosynthesis and the role of CsCCR4 in Camelina resistance to Ss we generated CsCCR4 knockout mutants of CN114263 Camelina line using CRISPR/Cas9-mediated gene editing.Materials and Methods: Thirty T1 plants were produced via floral dip transformation followed by glyphosate spraying that was used in the first step of screening procedures and were confirmed by PCR method. Transgene’s T-DNA copy number variation, T-DNA CNV, in T1 and T2 progenitors were determined using digital droplet PCR (ddPCR) and the occurrence of mutation in the three copies of CsCCR4 homeologues in T1 and T2 generations were scrutinized by drop-off assay technique. To make sure that if the created mutants in T2 plants are real, TOPO TA sequencing flanking the Cas9/gRNA specific hot point of cleavage for three of them was conducted.Results: In the T1 generation, 25 plants were confirmed which had between one to nine T-DNA copies in the corresponding Camelina genome. In T2 generation the population were screened for potential mutation in CsCCR4 gene. Various types of mutations, including insertions and deletions, were demonstrated in three copies of CsCCR4. In fact, CRISPR system could have cut one, two or three copies of the gene in events numbered T2-plant 10, T2-plant 15 and T2-plant 19, respectively. The T3-plant 19 which showed mutation in all versions of CsCCR4 in previous generation had susceptibility to S. sclerotiorum invasion and was kept as real csccr4 mutant material for further investigations of Camelina-Sclerotinia interaction. Mutation in CsCCR4 had occurred through error-prone none- homologous end joining (NHEJ) nucleus DNA repair pathway. Ss challenge on the early flowering T3 generation. The T3 plants with mutation causing premature stop codon at position 217 of CsCCR4 were compromised in their resistance to Ss compared to the wildtype resistant control parent CN114263.Conclusion: Using ddPCR it easily was possible to identify both the T-DNA CNV and occurrence of mutation in CsCCR4 homeologues in T1 and T2 progenitors. We illustrated that CRISPR/Cas9-mediated mutation is a decent technique that can be utilized to expedite the mutant line development which could assist to figure out the activity of a CsCCR4 gene in defense responses to the pathogens in C. sativa as prospective oilseed crop for biodiesel production.

Overexpression of Cinnamoyl-CoA Reductase 2 in Brassica napus Increases Resistance to Sclerotinia sclerotiorum by Affecting Lignin Biosynthesis

Sclerotinia sclerotiorum causes severe yield and economic losses for many crop and vegetable species, especially Brassica napus. To date, no immune B. napus germplasm has been identified, giving rise to a major challenge in the breeding of Sclerotinia resistance. In the present study, we found that, compared with a Sclerotinia-susceptible line (J902), a Sclerotinia-resistant line (J964) exhibited better xylem development and a higher lignin content in the stems, which may limit the invasion and spread of S. sclerotiorum during the early infection period. In addition, genes involved in lignin biosynthesis were induced under S. sclerotiorum infection in both lines, indicating that lignin was deposited proactively in infected tissues. We then overexpressed BnaC.CCR2.b, which encodes the first rate-limiting enzyme (cinnamoyl-CoA reductase) that catalyzes the reaction of lignin-specific pathways, and found that overexpression of BnaC.CCR2.b increased the lignin content in the stems of B. napus by 2.28–2.76% under normal growth conditions. We further evaluated the Sclerotinia resistance of BnaC.CCR2.b overexpression lines at the flower-termination stage and found that the disease lesions on the stems of plants in the T2 and T3 generations decreased by 12.2–33.7% and 32.5–37.3% compared to non-transgenic control plants, respectively, at 7days post-inoculation (dpi). The above results indicate that overexpression of BnaC.CCR2.b leads to an increase in lignin content in the stems, which subsequently leads to increased resistance to S. sclerotiorum. Our findings demonstrate that increasing the lignin content in the stems of B. napus is an important strategy for controlling Sclerotinia.

Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar.

Lignin is one of the main factors determining recalcitrance to processing of lignocellulosic biomass towards bio-based materials and fuels. Consequently, wood of plants engineered for low lignin content is typically more amenable to processing. However, lignin-modified plants often exhibit collapsed vessels and associated growth defects. Vessel-specific reintroduction of lignin biosynthesis in dwarfed low-lignin cinnamoyl-CoA reductase1 (ccr1) Arabidopsis mutants using the ProSNBE:AtCCR1 construct overcame the yield penalty while maintaining high saccharification yields, and showed that monolignols can be transported between the different xylem cells acting as 'good neighbors' in Arabidopsis. Here, we translated this research into the bio-energy crop poplar. By expressing ProSNBE:AtCCR1 into CRISPR/Cas9-generated ccr2 poplars, we aimed for vessel-specific lignin biosynthesis to: (i) achieve growth restoration while maintaining high saccharification yields; and (ii) study the existence of 'good neighbors' in poplar wood. Analyzing the resulting ccr2 ProSNBE:AtCCR1 poplars showed that vessels and rays act as good neighbors for lignification in poplar. If sufficient monolignols are produced by these cells, monolignols migrate over multiple cell layers, resulting in a restoration of the lignin amount to wild-type levels. If the supply of monolignols is limited, the monolignols are incorporated into the cell walls of the vessels and rays producing them and their adjoining cells resulting in fiber hypolignification. One such fiber-hypolignified line had 18% less lignin and, despite its small yield penalty, had an increase of up to 71% in sugar release on a plant base upon saccharification.

Molecular mechanisms of mesocotyl elongation induced by brassinosteroid in maize under deep-seeding stress by RNA-sequencing, microstructure observation, and physiological metabolism

Deep-seeding is an important way to improve maize drought resistance, mesocotyl elongation can significantly enhance its seedling germination. To improve our understanding of transcription-mediated maize mesocotyl elongation under deep-seeding stress. RNA-sequencing was used to identify differentially expressed genes (DEGs) in both deep-seeding tolerant W64A and intolerant K12 mesocotyls following culture for 10 days after 2.0 mg·L−1 24-epibrassinolide (EBR) induced stress at the depths of 3 and 20 cm. Phenotypically, the mesocotyl length of both maize significantly increased under 20 cm stress and in the presence of EBR. Microstructure observations revealed that the mesocotyls underwent programmed cell death under deep-seeding stress, which was alleviated by EBR. This was found to be regulated by multiple DEGs encoding cysteine protease/senescence-specific cysteine protease, aspartic protease family protein, phospholipase D, etc. and transcription factors (TFs; MYB, NAC). Additionally, some DEGs associated with cell wall components, i.e., cellulose synthase/cellulose synthase like protein (CESA/CSL), fasciclin-like arabinogalactan (APG), leucine-rich repeat protein (LRR) and lignin biosynthesis enzymes including phenylalanine ammonia-lyase, S-adenosyl-L-methionine-dependent methyltransferases, 4-coumarate-CoA ligase, cinnamoyl CoA reductase, cinnamyl alcohol dehydrogenase, catalase, peroxiredoxin/peroxidase were found to control cell wall sclerosis. Moreover, in auxin, ethylene, brassinosteriod, cytokinin, zeatin, abscisic acid, gibberellin, jasmonic acid, and salicylic acid signaling transduction pathways, the corresponding DEGs were activated/inhibited by TFs (ARF, BZR1/2, B-ARR, A-ARR, MYC2, ABF, TGA) and synthesis of phytohormones-related metabolites. These findings provide information on the molecular mechanisms controlling maize deep-seeding tolerance and will aid in the breeding of deep-seeding maize varieties.

Amplification, sequencing and characterization of pectin methyl esterase inhibitor 51 gene in Tectona grandis L.f.

Tectona grandis L.f. (Teak), a very important source of incomparable timber, withstands a wide range of tropical deciduous conditions. We achieved partial amplification of pectin methylesterase inhibitor 51 (PMEI) gene in teak by E. pilularis cinnamoyl Co-A reductase (CCR) gene specific primer. The amplified teak gene was of 750bp, 79% identity and 97% query cover with PMEI of Sesamum indicum. The phylogenetic tree clustered the amplified gene with PMEI of database plant species, Erythranthe guttata and Sesamum indicum (87% bootstrap value). On conversion to amino acid sequence, the obtained protein comprised 237 amino acids. However, PMEI region spanned from 24 to 171 amino acids, 15.94kDa molecular weight, 8.97 pI value and C697H1117N199O211S9 molecular formula with four conserved cysteine residues as disulfide bridges. 25.9 % protein residues were hydrophilic, 42.7% hydrophobic and 31.2% neutral. Teak 3D PMEI protein structure corresponded well with Arabidopsis thaliana and Actinidia deliciosa PMEIs. The gene maintains integrity of pectin component of middle lamella of primary cell wall and confers tolerance against various kinds of stresses. Teak conferred with overexpression of PMEI may secure a wide adaptability as well as luxuriant timber productivity and quality in adverse/ fluctuating/ scarce climatic and environmental conditions of tropical forests.

Stacking AsFMT overexpression with BdPMT loss of function enhances monolignol ferulate production in Brachypodiumdistachyon.

SummaryTo what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML‐FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML‐FA production are as follows: (1) The presence of an active FERULOYL‐CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16‐fold increase in lignin‐bound ML‐FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon. On its own, knocking out the native p‐COUMAROYL‐CoA MONOLIGNOL TRANSFERASE (BdPMT) pathway that competes for monolignols and the p‐coumaroyl‐CoA intermediate did not change ML‐FA incorporation, nor did partial loss of CINNAMOYL‐CoA REDUCTASE1 (CCR1) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt‐1 mutant resulted in a 32‐fold increase in ML‐FA incorporation into lignin over the wild‐type level.

The Regulation of Adaptation to Cold and Drought Stresses in Poa crymophila Keng Revealed by Integrative Transcriptomics and Metabolomics Analysis.

Poa crymophila Keng is highly adaptable to long-term low temperature and drought conditions, making it a desirable foraging grass of the Qinghai-Tibet Plateau. Here, the widely targeted metabolomics and comparative transcriptome analyses were utilized for the discovery of metabolites and genes in P. crymophila in response to cold and drought stresses. P. crymophila were exposed to −5°C for 24 h and recovered to 22°C for 48 h, as well as drought for 10 days followed by re-watering for 1 day. In total, 779 metabolic features were assigned to metabolites and 167,845 unigenes were generated. Seventeen compounds showed significant up-regulation (variable importance in project >1) under both stresses in the metabolic profiling, mainly annotated as carbohydrates, flavones, and phenylpropanoids. The genes which were positively correlated with these metabolites were assigned to pathways (sucrose-starch, raffinose, phenylpropanoid, and flavone metabolism) using the Mapman software package. Alpha-amylase, beta-fructofuranosidase, and sugar transport genes degraded the glucose and starch to small molecule sugars for the purpose of osmotic adjustment and to provide more energy for the growth of P. crymophila in an adverse environment. The induction of cinnamoyl-CoA reductase (CCR) and the MYB gene as well as the sharp increase in schizandrin, a kind of lignan, showed that this likely has the closest connection with the tolerance to both stresses. Four significantly induced flavone compounds are probably involved in reducing oxidative damage. Our results indicated that activation of the phenlypropanoid pathway plays the primary role in P. crymophila adapting to harsh environments. This study showed the mechanism of P. crymophila responding to both cold and drought stresses and showed the discovery of a new biological regulator against stresses.

Screening for a Novel Gene, OsPSLSq6, Using QTL Analysis for Lodging Resistance in Rice

Lodging is the most common factor that affects crop productivity, reducing yield, grain quality, and harvesting efficiency of rice and other cereal crops. The Cheongcheong (Indica)/Nagdong (Japonica) doubled haploid (CNDH) genetic map was used to develop a lodging-resistant variety. The major agricultural traits of rice related to lodging resistance, such as the pushing strength of the lower stem before the heading date (PSLSB) at reproductive growth period and pushing strength of the lower stem after the heading date (PSLSA) at full ripe period were investigated. A quantitative trait locus (QTL) analysis of PSLSA and PSLSB detected on RM439-RM20318 on chromosome 6 has overlap in three consecutive years. RM439-RM20318 on chromosome 6 contained 15 lodging resistance candidate genes. Among the candidate genes, Os06g0623200, named OsPSLSq6, which is similar to Cinnamoyl-CoA reductase, involved lignin biosynthesis in defense responses. Lignin is the main structural component of vascular plants’ secondary cell wall, which is not only related to plant growth and development but also to mechanical strength. OsPSLSq6 opens new possibilities to control lignin synthesis to improve lodging resistance. OsPSLSq6 can be used as a target gene for further studies to provide important information for the marker-assisted improvement of target traits and cloning genes underlying the QTL of interest.