Cinnamyl Alcohol Dehydrogenase Gene Research Articles

Genome-wide identification of the CAD gene family and functional analysis of putative bona fide CAD genes in tobacco (Nicotiana tabacum L.).

Cinnamyl alcohol dehydrogenase (CAD) plays a crucial role in lignin biosynthesis, and the gene family encoding various CAD isozymes has been cloned and characterized in numerous plant species. However, limited information regarding the CAD gene family in tobacco is currently available. In this study, we identified 10 CAD genes in Nicotiana tabacum, four in N. tomentosiformis, and six in N. sylvestris. The nucleotide and amino acid sequences of these tobacco CADs demonstrate high levels of similarity, whereas the putative protein sequences conservatively possessed two Zn2+ binding motifs and an NADP(H) cofactor binding motif. Both NtCAD1 and NtCAD2 had conservative substrate binding sites, similar to those possessed by bona fide CADs, and evidence from phylogenetic analysis as well as expression profiling supported their role as bona fide CADs involved in lignin biosynthesis. NtCAD1 has two paralogous genes, NtCAD1-1 and NtCAD1-2. Enzyme activity analysis revealed that NtCAD1-1 and NtCAD1-2 had a high affinity to coniferyl aldehyde, p-coumaryl aldehyde, and sinapyl aldehyde, whereas NtCAD2 preferred coniferyl aldehyde and p-coumaryl aldehyde as substrates. The kinetic parameter assay revealed that NtCAD1-2 functions as the most efficient enzyme. Downregulation of both NtCAD1-1 and NtCAD1-2 resulted in reddish-brown stems without significant changes in lignin content. Furthermore, NtCAD1-1, NtCAD1-2, and NtCAD2 showed distinct expression patterns in response to biotic and abiotic stresses, as well as different phytohormones. Our findings suggest that NtCAD1-1 and NtCAD1-2 are involved in lignin biosynthesis, with NtCAD1-2 also participating in both biological and abiotic stresses, whereas NtCAD2 plays a distinct role mainly in responding to biological and abiotic stresses in tobacco.

Genome-wide identification of the CAD gene family and functional analysis of putative bona fide CAD genes in tobacco (Nicotiana tabacum L.)

Cinnamyl alcohol dehydrogenase (CAD) plays a crucial role in lignin biosynthesis, and the gene family encoding various CAD isozymes has been cloned and characterized in numerous plant species. However, limited information regarding the CAD gene family in tobacco is currently available. In this study, we identified 10 CAD genes in Nicotiana tabacum, four in N. tomentosiformis, and six in N. sylvestris. The nucleotide and amino acid sequences of these tobacco CADs demonstrate high levels of similarity, whereas the putative protein sequences conservatively possessed two Zn2+ binding motifs and an NADP(H) cofactor binding motif. Both NtCAD1 and NtCAD2 had conservative substrate binding sites, similar to those possessed by bona fide CADs, and evidence from phylogenetic analysis as well as expression profiling supported their role as bona fide CADs involved in lignin biosynthesis. NtCAD1 has two paralogous genes, NtCAD1–1 and NtCAD1–2. Enzyme activity analysis revealed that NtCAD1–1 and NtCAD1–2 had a high affinity to coniferyl aldehyde, p-coumaryl aldehyde, and sinapyl aldehyde, whereas NtCAD2 preferred coniferyl aldehyde and p-coumaryl aldehyde as substrates. The kinetic parameter assay revealed that NtCAD1–2 functions as the most efficient enzyme. Downregulation of both NtCAD1–1 and NtCAD1–2 resulted in reddish-brown stems without significant changes in lignin content. Furthermore, NtCAD1–1, NtCAD1–2, and NtCAD2 showed distinct expression patterns in response to biotic and abiotic stresses, as well as different phytohormones. Our findings suggest that NtCAD1–1 and NtCAD1–2 are involved in lignin biosynthesis, with NtCAD1–2 also participating in both biological and abiotic stresses, whereas NtCAD2 plays a distinct role mainly in responding to biological and abiotic stresses in tobacco.

The Integration of Transcriptome and Metabolome Analyses Provides Insights into the Determinants of the Wood Properties in Toona ciliata.

Toona ciliata, also known as Chinese mahogany, is a high-quality and fast-growing wood species with a high economic value. The wood properties of T. ciliata of different provenances vary significantly. In this study, we conducted comprehensive transcriptome and metabolome analyses of red and non-red T. ciliata wood cores of different provenances to compare their wood properties and explore the differential metabolites and genes that govern the variation in their wood properties. Through combined analyses, three differential genes and two metabolites were identified that are possibly related to lignin synthesis. The lignin content in wood cores from T. ciliata of different provenances shows significant variation following systematic measurement and comparisons. The gene Tci09G002190, one of the three differential genes, was identified as a member of the CAD (Cinnamyl alcohol dehydrogenase) gene family of T. ciliata, which is associated with lignin synthesis. Our data provide insights into the determinants of the wood properties in T. ciliata, providing a solid foundation for research into the subsequent mechanisms of the formation of T. ciliata wood.

The expression of lignin biosynthesis genes during plant development and effects of downregulated cinnamyl alcohol dehydrogenase (CAD) gene in bahiagrass (Paspalum notatum Flügge)

AbstractBahiagrass (Paspalum notatum Flügge) is a warm‐season grass where high lignin content limits its forage quality, negatively affecting animal performance. To create a new breeding method by genome editing, isolation and characterization of lignin biosynthesis genes and identification of their molecular relationships are essential. The caffeic acid O‐methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD) cDNA clones were isolated from bahiagrass, and their protein sequences showed high similarity to other C4 monocot species through phylogenetic analysis. Gene expression analysis of phenylalanine ammonia‐lyase (PAL), COMT and CAD, involved in crucial stages of lignin biosynthesis and lignin content, was conducted at different plant developmental stages. The highest gene expression levels of these genes were observed in the vegetative (V) and early reproductive stages, while the lignin content increased until the middle reproductive stage and remained constant thereafter. Additionally, to further understand the molecular relationships in lignin biosynthesis, the effect of CAD downregulation was analyzed in transgenic bahiagrass lines introduced with sorghum CAD antisense and RNAi vector obtained from a previous report. This led to a reduced lignin content and affected the expression of PAL and COMT working upstream of CAD. In the V stage, PAL expression was lower in transgenic lines compared to wild type (WT), while COMT expression showed no significant differences. However, PAL and COMT expression of transgenic lines in the middle reproductive stage (R2) was significantly higher than in the WT. These findings suggest that the downregulation of CAD gene expression affects PAL and COMT expression and induces a feedback system in the R2 stage. Lignin content influenced the phenotype of the transgenic plants with significantly reduced lignin, exhibiting a dwarf phenotype with shorter plant heights. The findings of this study can be applied to genome editing for the development of practical new breeding materials with improved digestibility in bahiagrass.

Comparative characterization and expression analysis revealed cinnamyl alcohol dehydrogenase genes differentially responding to Colletotrichum fructicola in woodland and cultivated strawberries

BACKGROUND: Strawberry (Fragaria×ananassa) production is hampered by anthracnose caused by Colletotrichum spp. Cinnamyl alcohol dehydrogenase (CAD) catalyzes lignin biosynthesis to fortify plant cell wall upon various stresses, but their involvement in strawberry resistance is elusive. OBJECTIVE: Current work aims to uncover the involvement of CAD genes in both woodland and cultivated strawberry responses to Colletotrichum spp. METHODS: Genome-wide identification of CAD genes was updated in cultivated strawberry. Comparative molecular characterization integrated with pathogenesis phenotyping after C. fructicola inoculation, were followed by RT-qPCR analysis and RNAseq data retrieval. RESULTS: Cultivated strawberry comprises 32 CAD genes of three classes. Thirteen syntenic relationships were observed between cultivated and woodland strawberry, with high homology in protein coding regions while promoters being greatly diversified. F. vesca ‘Hawaii4’ was least susceptible, followed by octoploid cultivar ‘ShenQi’, and ‘Benihoppe’ the most susceptible to C. fructicola. The class I CAD4 and CAD5, preferentially expressed in leaves and crowns, respectively, were up-regulated in susceptible varieties at 72 h post inoculation (hpi). Of class II, CAD8 was up-regulated in three genotypes at distinct timepoints, while CAD12 was quickly down-regulated in ‘ShenQi’ upon inoculation. CAD1 and CAD2 of class III were down-regulated in ‘ShenQi’ and ‘Hawaii4’ at distinct stages. The strong involvement of CAD genes in strawberry response to Colletotrichum spp. sets a basis for future functional study. CONCLUSION: F. vesca is less susceptible to Colletotrichum spp. than cultivated strawberries but the expression of CAD family in F. vesca and F.×ananassa were differentially provoked in timing and magnitude hinting no correlation with strawberry resistance to this pathogen.

Integrative analysis of metabolome, proteome, and transcriptome for identifying genes influencing total lignin content in Populus trichocarpa.

Lignin, a component of plant cell walls, possesses significant research potential as a renewable energy source to replace carbon-based products and as a notable pollutant in papermaking processes. The monolignol biosynthetic pathway has been elucidated and it is known that not all monolignol genes influence the total lignin content. However, it remains unclear which monolignol genes are more closely related to the total lignin content and which potential genes influence the total lignin content. In this study, we present a combination of t-test, differential gene expression analysis, correlation analysis, and weighted gene co-expression network analysis to identify genes that regulate the total lignin content by utilizing multi-omics data from transgenic knockdowns of the monolignol genes that includes data related to the transcriptome, proteome, and total lignin content. Firstly, it was discovered that enzymes from the PtrPAL, Ptr4CL, PtrC3H, and PtrC4H gene families are more strongly correlated with the total lignin content. Additionally, the co-downregulation of three genes, PtrC3H3, PtrC4H1, and PtrC4H2, had the greatest impact on the total lignin content. Secondly, GO and KEGG analysis of lignin-related modules revealed that the total lignin content is not only influenced by monolignol genes, but also closely related to genes involved in the "glutathione metabolic process", "cellular modified amino acid metabolic process" and "carbohydrate catabolic process" pathways. Finally, the cinnamyl alcohol dehydrogenase genes CAD1, CADL3, and CADL8 emerged as potential contributors to total lignin content. The genes HYR1 (UDP-glycosyltransferase superfamily protein) and UGT71B1 (UDP-glucosyltransferase), exhibiting a close relationship with coumarin, have the potential to influence total lignin content by regulating coumarin metabolism. Additionally, the monolignol genes PtrC3H3, PtrC4H1, and PtrC4H2, which belong to the cytochrome P450 genes, may have a significant impact on the total lignin content. Overall, this study establishes connections between gene expression levels and total lignin content, effectively identifying genes that have a significant impact on total lignin content and offering novel perspectives for future lignin research endeavours.

Cinnamyl Alcohol Dehydrogenase Gene Regulates Bursaphelenchus xylophilus Reproduction and Development

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a globally distributed destructive disease of pine forest. To study the PWD pathogenic mechanism, the cinnamyl alcohol dehydrogenase gene (BxCAD-1) from B. xylophilus was selected. The BxCAD-1 gene was amplified by PCR from the cDNA library of B. xylophilus and cloned into the expression vector pET-15b to construct the recombinant vector pET-15b-BxCAD-1. The recombinant cinnamyl alcohol dehydrogenase protein was purified by Ni-NTA affinity chromatography from Escherichia coli BL21 (DE3) harboring pET-15b-BxCAD-1 induced by IPTG. The effects of pH, temperature, metal ions and substrates on the activity of BxCAD-1 were determined, showing the highest catalytic activity at pH 8.0 and 40 °C with cinnamyl alcohol as substrate and Zn2+ as an activator. To elucidate the functions of BxCAD-1 in B. xylophilus, the expression of the gene was down-regulated by RNA interference. Results showed that the movement, feeding, reproduction, spawning rate, hatching rate, lifespan, infectivity and sensitivity to ethanol decreased compared with negative controls. RNA interference also affected the development of B. xylophilus from the larval stage to the adult stage. In situ hybridization showed that the gene was expressed in the digestive tract of male and female adults. This study revealed a promising target for PWD control.

Differential Responses of Antioxidant Enzymes and Lignin Metabolism in Susceptible and Resistant Sweetpotato Cultivars during Root-Knot Nematode Infection.

Root-knot nematodes (RKN) cause significant damage to sweetpotato plants and cause significant losses in yield and quality. Reactive oxygen species (ROS) play an important role in plant defenses, with levels of ROS-detoxifying antioxidant enzymes tightly regulated during pathogen infection. In this study, ROS metabolism was examined in three RKN-resistant and three RKN-susceptible sweetpotato cultivars. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were assessed, as was lignin-related metabolism. In RKN-infected roots, both resistant and susceptible cultivars increased SOD activity to produce higher levels of hydrogen peroxide (H2O2). However, H2O2 removal by CAT activity differed between cultivars, with susceptible cultivars having higher CAT activity and lower overall H2O2 levels. In addition, the expression of phenylpropanoid-related phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase genes, which encode enzymes involved in lignin metabolism, were higher in resistant cultivars, as were total phenolic and lignin contents. Enzyme activities and H2O2 levels were examined during the early (7 days) and late (28 days) phases of infection in representative susceptible and resistant cultivars, revealing contrasting changes in ROS levels and antioxidant responses in the different stages of infection. This study suggests that differences in antioxidant enzyme activities and ROS regulation in resistant and susceptible cultivars might explain reduced RKN infection in resistant cultivars, resulting in smaller RKN populations and overall higher resistance to infection and infestation by RKNs.

Lignification in Zinnia (Zinnia elegans Jacq.) Stem Sections of Different Age: Biochemical and Molecular Genetic Traits

Lignification of the stem in zinnia provides its mechanical properties due to xylem formation, which depends on the stage of plant development and is responsible for the transport of water and minerals. The study was aimed at the lignin deposition, anatomical traits, biochemical markers of lignification, as well as the genetic regulation of this process in zinnia stem cross sections of different age during their radial growth. The anatomical traits were assessed on cross sections. The content of lignin (Cysteine-assisted sulfuric method (CASA) and the thioglycolic acid (TGA) methods), the spectrum of phenolics (by thin layer chromatography (TLC)), the total activity and the variety of class III peroxidases were determined. The expression level of genes regulating phenylpropanoids and lignin biosynthesis were assessed. We suggest that time-specific and organ-specific lignification is determined by the metabolism of phenolic compounds and depends on the expression of genes of the phenylpropanoid pathway. It was shown that in the hypocotyl, during xylem ring formation, lignification was associated with increased expression of phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) genes responsible for the early stages of the phenylpropanoid pathway, and with the rise of class III peroxidases activity, including cationic isoforms. This caused increased content and diversity of phenolics in mature hypocotyl. In epicotyl, which is younger than the hypocotyl, the proportion of ferulic acid among phenolics increased, which could be considered as a marker of lignification in it. The high expression level of CAD and the activity of peroxidases, including anionic isoforms, led to accumulation of lignin. Thus, the hypocotyl and epicotyl, being characterized by different ages, differed in spectrum of phenolics, isoforms of class III peroxidases, expression of the PAL, cinnamate 4-hydroxylase (C4H), peroxidases III class (PRX), and laccase (LAC) genes, and lignin content.

Identification and Functional Analysis of CAD Gene Family in Pomegranate (Punica granatum).

[Objective] Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. The aim of this study was to identify CAD gene family members in pomegranate and its expression correlation with seed hardness. [Methods] Based on the reported CAD sequence of Arabidopsis, the CAD gene family of pomegranate was identified by homologous comparison, and then phylogenetic, molecular characterization, and expression profile analysis were performed. [Results] Pomegranate CAD gene family has 25 members, distributed on seven chromosomes of pomegranate. All pomegranate CAD proteins have similar physical and chemical properties. We divide the family into four groups based on evolutionary relationships. The member of group I, called bona fide CAD, was involved in lignin synthesis. Most of the members of group II were involved in stress resistance. The functions of groups III and IV need to be explored. We found four duplicated modes (whole genome duplication or segmental (WGD), tandem duplication (TD), dispersed duplication (DSD), proximal duplication (PD) in this family; TD (36%) had the largest number of them. We predicted that 20 cis-acting elements were involved in lignin synthesis, stress resistance, and response to various hormones. Gene expression profiles further demonstrated that the PgCAD gene family had multiple functions. [Conclusions] Pomegranate CAD gene family is involved in lignin synthesis of hard-seeded cultivar Hongyushizi and Baiyushizi, but its role in seed hardness of soft-seeded cultivar Tunisia needs to be further studied.

Genome-Wide Identification and Expression Analysis of the CAD Gene Family in Walnut (Juglans regia L.).

Lignin deficiency in the endocarp of walnuts causes kernel bare, leads to inconvenient processing and transportation of walnuts, and easily produces insect damage and mildew, thereby affecting the quality of walnuts. Cinnamyl alcohol dehydrogenase (CAD) is one of the key rate-limiting enzymes in lignin synthesis and plays an important role in the synthesis of lignin in the endocarp of walnut. However, knowledge about CAD gene family members and their evolutionary and functional characteristics in walnuts is limited. In this study, all 18 JrCADs were identified, and phylogenetic relationships, gene structure, protein motifs, collinearity analysis, and expression patterns of the JrCADs were also analyzed. All JrCADs could be divided into three groups based on the phylogenetic tree, gene structure, and motif analysis also support this grouping. Transcriptome data demonstrated that JrCADs have different expression patterns in walnut endocarps at different developmental stages. Combined with qRT-PCR data, we finally identified several candidate JrCADs involved in the process of endocarp sclerosis. This study showed that the JrCAD family members are highly conservative in evolutionary characteristics and they might participate in a variety of hormone responses. JrCAD17 and JrCAD18 are highly expressed in all periods of walnut endocarp harding, they are closely related to lignin accumulation.

Potential of By-product of Kappaphycus alvarezii Derived from Bioethanol Production as Biofertilizer in Growing of Ocimum basilicum in an Aquaponic System

Nutrient recycling from biowaste is one of the sustainable approaches to managing waste. The aquaponic system is one of the nutrient recycling methods that can reduce water consumption and reuse the nutrient available in its ecosystem. The nutrient to fertilize the plant in aquaponic depends on the activities of microbes to convert the waste into the nutrient. To enhance the growth of the plants, some aquaponics systems still rely on chemical fertilizers. Kappaphycus alvarezii is one of the red seaweeds abundantly found in East Malaysia. After numerous processes such as carrageenan extraction, the biowaste derived from K. alvarezii still contains a nutrient that can be recycled. The present study explores the potential of K. alvarezii solid waste as fertilizer to grow Ocimum basilicum in an aquaponics system. In this study, the macro- and micronutrients in K. alvarezii solid waste were determined, and the prevalence of microbes in the aquaponics system was monitored using inductively coupled plasma-optical emission spectrometer (ICP-OES) and 16S metagenomic sequencing method, respectively. Based on the findings, the growth of O. basilicum supplemented with K. alvarezii biofertilizer was significantly higher than the negative control. For genetic expression study in O. basilicum, cinnamyl alcohol dehydrogenase (CAD), phenylalanine ammonia-lyase (PAL), and cytochrome p450 reductase (CPR) genes were upregulated. The O. basilicum is free from mycotoxin and heavy metals. Since K. alvarezii solid waste is rich with macro- and micronutrients, which are essential for plant growth and can enhance the growth of O. basilicum, K. alvarezii solid waste produced from bioethanol production could be a potential fertilizer.

Phylogeny and functional characterization of the cinnamyl alcohol dehydrogenase gene family in Phryma leptostachya

Phryma leptostachya has attracted increasing attention because it is rich in furofuran lignans with a wide range of biological activities. Biosynthesis of furofuran lignans begins with the dimerization of coniferyl alcohol, one of the monolignol. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of monolignol biosynthesis, reducing cinnamyl aldehydes to cinnamyl alcohol. As it is in the terminal position of monolignol biosynthesis, its type and activity can cause significant changes in the total amount and composition of lignans. Herein, combined with bioinformatics analysis and in vitro enzyme assays, we clarified that CAD in P. leptostachya belonged to a multigene family, and identified nearly the entire CAD gene family. Our in-depth characterization about the functions and structures of two major CAD isoforms, PlCAD2 and PlCAD3, showed that PlCAD2 exhibited the highest catalytic activity, and coniferyl aldehyde was its preferred substrate, followed by PlCAD3, and sinapyl aldehyde was its preferred substrate. Considering the accumulation patterns of furofuran lignans and expression patterns of PlCADs, we speculated that PlCAD2 was the predominant CAD isoform responsible for furofuran lignans biosynthesis in P. leptostachya. Moreover, these CADs found here can also provide effective biological parts for lignans and lignins biosynthesis.

Expression analysis of sweetpotato cinnamyl alcohol dehydrogenase genes in response to infection with the root-knot nematode Meloidogyne incognita

Expression analysis of sweetpotato cinnamyl alcohol dehydrogenase genes in response to infection with the root-knot nematode Meloidogyne incognita

Erratum to: Genome-wide identification and expression analysis revealed cinnamyl alcohol dehydrogenase genes correlated with fruit-firmness in strawberry

Erratum to: Genome-wide identification and expression analysis revealed cinnamyl alcohol dehydrogenase genes correlated with fruit-firmness in strawberry

Mechanisms of resistance to powdery mildew in cucumber

Podosphaera xanthii causes powdery mildew of cucumber, and is associated with significant yield and quality losses. Development of resistant or tolerant varieties is the most effective and eco-friendly strategy for powdery mildew management. An important host resistance mechanism is based on the recognition of conserved resistance genes, resulting in durable resistance. To determine powdery mildew resistance mechanisms in cucumber, total RNAs were isolated from the powdery mildew resistant cultivar Meltem, the tolerant line VT18, and the susceptible local variety Camlica. Expression levels of nine genes in these plants were analysed by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The host reactions were assessed using microscope observations of stained specimens. Serine/threonine (STN7), transcription factor (WRKY22), serine/threonine-protein kinase (D6PKL1), and serine/threonine receptor kinase (NFP) genes were induced, as positive regulators in defence mechanisms against powdery mildew. Polygalacturonase Inhibitor (PGIP) did not express after P. xanthii inoculation of Camlica, resulting in susceptibility. After inoculation, callose synthase (CALLOSE) and cinnamyl alcohol dehydrogenase (CAD) gene expression levels were increased in resistant Meltem, but Hypersensitive Reaction (HR) and ROS formation were only linked in the tolerant VT18. Powdery mildew development was less in Meltem than in VT18, indicating that cell wall thickening and HR play separate roles in resistance to this disease.

Higher Phytohormone Contents and Weaker Phytohormone Signal Transduction Were Observed in Cold-Tolerant Cucumber.

Cucumbers (Cucumis sativus L.) originated from the South Asian subcontinent, and most of them are fragile to cold stress. In this study, we evaluated the cold tolerance of 115 cucumber accessions and screened out 10 accessions showing high resistance to cold stress. We measured and compared plant hormone contents between cold-tolerant cucumber CT90R and cold-sensitive cucumber CT57S in cold treatment. Most of the detected plant hormones showed significantly higher content in CT90R. To elucidate the role of plant hormones, we compared the leaf- and root-transcriptomes of CT90R with those of CT57S in cold stress treatment. In leaves, there were 1209 differentially expressed genes (DEGs) between CT90R and CT57S, while there were 703 in roots. These DEGs were not evenly distributed across the chromosomes and there were significant enrichments at particular positions, including qLTT6.2, a known QTL controlling cucumber cold tolerance. The GO and KEGG enrichment analysis showed that there was a significant difference in the pathway of plant hormone transductions between CT90R and CT57S in leaves. In short, genes involved in plant hormone transductions showed lower transcription levels in CT90R. In roots, the most significantly different pathway was phenylpropanoid biosynthesis. CT90R seemed to actively accumulate more monolignols by upregulating cinnamyl-alcohol dehydrogenase (CAD) genes. These results above suggest a new perspective on the regulation mechanism of cold tolerance in cucumbers.

Effect of Methyl Jasmonate in Gene Expression, and in Hormonal and Phenolic Profiles of Holm Oak Embryogenic Lines Before and After Infection With Phytophthora cinnamomi.

The dieback syndrome affecting Quercus ilex and other oak species impels the search for tolerant plant genotypes, as well as methods of plant immunization against such infections. Elicitation treatments can be an effective strategy to activate plant defense response and embryogenic lines represent a promising tool to generate new tolerant genotypes and also to study early markers involved in defense response. The aim of the presented work was to investigate changes in gene expression, and in hormonal and phenolic profiles induced in three holm oak embryogenic lines (ELs) elicited with methyl jasmonate (MeJA) before and after infection with the oomycete Phytophthora cinnamomi, which is the main biotic agent involved in this pathogenic process. The three ELs, derived from three genotypes, showed different basal profiles in all tested parameters, noting that the VA5 naïve genotype from a scape tree was characterized by a basal higher expression in NADPH-dependent cinnamyl alcohol dehydrogenase (CAD) and chalcone synthase (CHS) genes and also by higher caffeic acid content. Our work also identifies changes triggered by MeJA elicitation in holm oak embryogenic lines, such as increases in ABA and JA contents, as well as in levels of most of the determined phenolic compounds, especially in caffeic acid in Q8 and E00 ELs, but not in their biosynthesis genes. Irrespective of the EL, the response to oomycete infection in holm oak elicited plant material was characterized by a further increase in JA. Since JA and phenols have been described as a part of the Q. ilex defense response against P. cinnamomi, we propose that MeJA may act as an induced resistance (IR) stimulus and that in our embryogenic material induced both direct (detected prior to any challenge) and primed (detected after subsequent challenge) defense responses.

Cloning and in silico characterization of cinnamyl alcohol dehydrogenase gene involved in lignification of Tall fescue (Festuca arundinacea Schreb.).

Tall fescue, a promising temperate forage grass of Himalayan region, possesses extraordinary property of rapid growth with high biomass production, but its poor digestibility due to higher lignin content limits its utilization in livestock feeding. The lignification in Tall fescue is under the control of enzymatic cascade of different regulatory enzymes. Cinnamyl alcohol dehydrogenase (CAD) is a crucial regulatory enzyme that catalyzes the last step of monolignol biosynthesis and is a potential candidate for altering the content and types of lignin, and hence increasing the digestibility of fodder crops. Hence, the present investigation was conducted on isolation, cloning and characterization of CAD gene from Tall fescue. Isolation and amplification of CAD gene resulted in an amplicon of 1521bp. The CAD gene sequence was submitted to NCBI database with an accession number MW442831. Translation of the CAD gene sequence exhibited an ORF of 361 amino acids. The deduced CAD protein was predicted to be hydrophobic, acidic and thermally stable with molecular formula C1712H2734N460O520S23, molecular mass of 38.82kDa, theoretical pI of 5.60 and 3 strong transmembrane helices. The CAD protein was predicted to have a dimer forming behavior with putative NAD(P) binding site between amino acids 48 and 301, putative substrate-binding site between amino acids 48 and 301, catalytic zinc-binding site between amino acids 48 and 164 and structural zinc-binding site between amino acid residue 101 and 115. A conserved 189GLGGVG194 motif is the binding site for NADP(H). The conserved motif pattern of CAD's zinc catalytic center was found to be 69GHEVVGEV(X)EVG(X)2V83. The zinc-binding site was found to be conserved between amino acid 89 and 115 and was found to be 89G(X)2VG(X)G(X)2VGXC(X)2C(X)2C(X)5QYC115. The deciphered sequence and putative protein information might be useful in subsequent research in lignin bioengineering for enhanced digestibility, biomass conversion as well as impact of lignin on cell wall mechanics.

CsWRKY25 Improves Resistance of Citrus Fruit to Penicillium digitatum via Modulating Reactive Oxygen Species Production.

WRKY transcription factors (TFs) play crucial roles in the regulation of biotic stress. Citrus is the most productive fruit in the world. It is of great value to investigate the regulatory molecular mechanism of WRKYs in improving disease resistance. In this research, the transcription level of CsWRKY25 was upregulated in P. digitatum infected citrus peel, and CsWRKY25 activated the expression of three target genes (RbohB, RbohD, and PR10). Besides, the Agrobacterium-mediated transient overexpression of CsWRKY25 has also been shown to enhance resistance to P. digitatum in citrus, and caused the accumulation of hydrogen peroxide and lignin. The accumulation of ROS also activated the antioxidant system, the catalase (CAT), peroxidase (POD), and cinnamyl alcohol dehydrogenase (CAD) genes were significant upregulated, leading to activation of antioxidant enzymes. In addition, the up-regulated expression of MPK5 and MPK6 genes suggested that the regulatory role of CsWRKY25 might be related to the phosphorylation process. In conclusion, CsWRKY25 could enhance the resistance to P. digitatum via modulating ROS production and PR genes in citrus peel.