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.

Nucleoside 5′-Phosphoramidates Control the Phenylpropanoid Pathway in Vitis vinifera Suspension-Cultured Cells

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN’s) and adenosine 5′-phosphoramidate (NH2-pA) belonging to nucleoside 5′-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN’s induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5′-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5′-phosphoramidates could be considered as new signaling molecules.

Plant Growth-Promoting Endophytic Bacillus Aryabhattai Triggers Novel Genes to Induce Plant Growth

Background: In nature, plants interact with a wide range of microorganisms. Most of these microorganisms have the ability to promote plant growth through the induction of important molecular pathways. The current work evaluated whether the endophytic bacterium Bacillus aryabhattai encourages plant growth and how transcriptional changes might be implicated in this effect.Results: The endophytic bacterium showed a significant effect on plant growth. Our results revealed that B. aryabhattai promotes the growth of Arabidopsis and tobacco plants. Notably, transcriptional changes in Arabidopsis plants treated with the bacterium were identified. Genes such as cinnamyl alcohol dehydrogenase, apyrase, thioredoxin H8, benzaldehyde dehydrogenase, indoleacetaldoxime dehydratase, berberine bridge enzyme-like and gibberellin-regulated protein were highly expressed. Additionally, endophytic bacterial genes such as arginine decarboxylase, D-hydantoinase, ATP synthase gamma chain and 2-hydroxyhexa-2,4-dienoate hydratase were activated during the interaction with Arabidopsis.Conclusions: The results show that new plant growth-related genes are induced during the interaction endophytic bacterium B. aryabhattai, and these changes may promote plant growth in sustainable agriculture.

Whole-genome sequencing of Acer catalpifolium reveals evolutionary history of endangered species

Acer catalpifolium is an endangered species restricted to remote localities of West China. Understanding the genomic content and evolution of A. catalpifolium is essential to conservation efforts of this rare and ecologically valuable plant. Here, we report a high-quality genome of A. catalpifolium consisting of ∼654 Mbps and ∼35,132 protein-coding genes. We detected 969 positively-selected genes in two Acer genomes compared with four other eudicots, 65 of which were transcription factors. We hypothesize that these positively-selected mutations in transcription factors might affect their function and thus contribute to A. catalpifolium’s decline-type population. We also identified 179 significantly expanded gene families compared to 12 other eudicots, some of which are involved in stress responses, such as the FRS-FRF family. We inferred that A. catalpifolium has experienced gene family expansions to cope with environmental stress in its evolutionary history. Finally, 109 candidate genes encoding key enzymes in the lignin biosynthesis pathway were identified in A. catalpifolium; of particular note were the large range and high copy number of cinnamyl alcohol dehydrogenase genes. The chromosome-level genome of A. catalpifolium presented here may serve as a fundamental genomic resource for better understanding endangered Acer species, informing future conservation efforts.

Homo- and Hetero-Dimers of CAD Enzymes Regulate Lignification and Abiotic Stress Response in Moso Bamboo

Lignin biosynthesis enzymes form complexes for metabolic channelling during lignification and these enzymes also play an essential role in biotic and abiotic stress response. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme that catalyses the reduction of aldehydes to alcohols, which is the final step in the lignin biosynthesis pathway. In the present study, we identified 49 CAD enzymes in five Bambusoideae species and analysed their phylogenetic relationships and conserved domains. Expression analysis of Moso bamboo PheCAD genes in several developmental tissues and stages revealed that among the PheCAD genes, PheCAD2 has the highest expression level and is expressed in many tissues and PheCAD1, PheCAD6, PheCAD8 and PheCAD12 were also expressed in most of the tissues studied. Co-expression analysis identified that the PheCAD2 positively correlates with most lignin biosynthesis enzymes, indicating that PheCAD2 might be the key enzyme involved in lignin biosynthesis. Further, more than 35% of the co-expressed genes with PheCADs were involved in biotic or abiotic stress responses. Abiotic stress transcriptomic data (SA, ABA, drought, and salt) analysis identified that PheCAD2, PheCAD3 and PheCAD5 genes were highly upregulated, confirming their involvement in abiotic stress response. Through yeast two-hybrid analysis, we found that PheCAD1, PheCAD2 and PheCAD8 form homo-dimers. Interestingly, BiFC and pull-down experiments identified that these enzymes form both homo- and hetero- dimers. These data suggest that PheCAD genes are involved in abiotic stress response and PheCAD2 might be a key lignin biosynthesis pathway enzyme. Moreover, this is the first report to show that three PheCAD enzymes form complexes and that the formation of PheCAD homo- and hetero- dimers might be tissue specific.

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

BACKGROUND: Strawberry fruits are perishable with a short post-harvest life. Cinnamyl alcohol dehydrogenase (CAD) is the key enzyme for lignin biosynthesis strengthening plant cell wall. A systematic characterization of strawberry CAD family is absent and their involvement in fruit firmness is largely elusive. OBJECTIVE: Current work aims for a genome-wide identificationof CAD family and its expression correlation with fruit firmness in strawberry varieties. METHODS: A genome-scale identification and molecular characterization for CADs were performed in the commercial strawberry (Fragaria×ananassa) and woodland strawberry (F. vesca). qPCR analysis of CAD homoeologs in three cultivars varying with fruit firmness revealed candidate CAD members positively correlated with lignin content and fruit firmness. RESULTS: A total of 14 and 24 CAD loci were identified in the genomes of F.vesca var. Hawaii4 and F. ×ananassa cv. Camarosa, respectively.Phylogenetic analysis supported a division of this family into three classes. Class I FvCAD each has four homoeologs in commercial strawberry, while those of Class II and Class III have only one or two homoeologs. Except for FvCAD2 and -6, there exits at least one pair of CADs sharing ∼97% or above amino acid identity between F. vesca and F. ×ananassa.The flesh firmness and lignin content varied greatly among strawberry germplasm. Distinct dynamic changes in fruit lignin content were observed before the large green stage, but fruit firmness displayed a similar decrease profile during fruit development in three varieties. Of the eight genes detected in F.×ananassa, FvCAD3 and -12 did not display a F. vesca-biased expression pattern during fruit development.FvCAD4 of Class I was expressed at levels positively correlated with variation in fruit lignin content at white stage.Transcript abundance of five Class IIgenes including FvCAD3, -8, -10, -11, and -12 was positively correlated with lignin content and fruit firmness, with FvCAD10 and -11 (FaCAD in previous publication) reaching an extremely significant correlation with the genetic variation in fruit firmness across three varieties. CONCLUSION: Strawberry Class II CADs were significantly correlated with the genetic variation in fruit firmness,whichmight expand the potential choices for improving strawberry shelf life.

Improved chemical pulping and saccharification of a natural mulberry mutant deficient in cinnamyl alcohol dehydrogenase

Lignin content and its molecular structure influence various wood characteristics. In this study, the anatomical and physicochemical properties of wood derived from a naturally occurring mulberry mutant deficient in cinnamyl alcohol dehydrogenase (CAD), a key enzyme in lignin biosynthesis, were analyzed using conventional staining assays on stem sections, length and width measurements of xylem fiber cells, wood pulping and saccharification assays, and sugar compositional analysis of extractive-free wood powder. The present data indicate that the mutation in the CAD gene leads to improved wood delignification efficiency, increased pulp yield under alkaline pulping conditions, and enhanced saccharification efficiency following alkaline pretreatment. This study opens up new avenues for the multipurpose use of the mulberry CAD-deficient mutant as a raw material for biorefinery processes, in addition to its traditional use as a favored feed for silkworms.

Assessment of potential winter hardiness in winter bread wheat cultivars and genotypes by analyzing autofluorescence in seedling tissues

Background.Winter crops are the most productive component of agricultural biocenoses. In Russia, winter wheat suffers the greatest losses in winter, so a search for traits marking high or low winter hardiness in autumn-sown genotypes, including improved cultivars, is needed to assess their potential for overwintering. One of such markers of high winter hardiness is an increased lignin content in plant tissues. The terminal enzyme in the phenylpropanoid pathway of metabolism, wherein lignin components are formed, is cinnamyl-alcohol dehydrogenase (CAD, EC 1.1.1.195). In plants, the CAD enzyme is one of the links in the aromatic metabolism, which generates, in addition to lignin, a number of aromatic compounds, such as lignans, aromatic glycosides, etc. Many of these compounds, like lignin, contain chromophore groups and are capable of autofluorescence.Correlations of the genotypes that incorporate CAD1-F with overwintering are studied in this work.Materials and methods.The winter bread wheat cultivars ‘Zitnica’ (Yugoslavia) and ‘Novosibirskaya 9’ (ICG SB RAS, Russia), contrasting in winter hardiness and CAD isozyme spectra, their hybrids, and 28 improved winter cultivars developed in Krasnodar were selected for the study. Fluorescence analysis of 28 winter wheat cultivars was also performed. Correlation coefficients between fluorescence and frost tolerance were calculated using the results of the analysis of 7 most contrasting cultivars.Conclusions. The tested winter bread wheat genotypes demonstrated the interplay between CAD1-F and successful overwintering: a correlation was found in the genotypes carrying the 00 CAD1-F allele with higher percentage of overwintered plants. This dependence was not observed in every season. The analysis of seedling sections for fluorescence can also be used for preliminary assessment of winter tolerance in winter bread wheat under laboratory conditions.

The Cysteine-Rich Peptide Snakin-2 Negatively Regulates Tubers Sprouting through Modulating Lignin Biosynthesis and H2O2 Accumulation in Potato

Potato tuber dormancy is critical for the post-harvest quality. Snakin/Gibberellic Acid Stimulated in Arabidopsis (GASA) family genes are involved in the plants’ defense against pathogens and in growth and development, but the effect of Snakin-2 (SN2) on tuber dormancy and sprouting is largely unknown. In this study, a transgenic approach was applied to manipulate the expression level of SN2 in tubers, and it demonstrated that StSN2 significantly controlled tuber sprouting, and silencing StSN2 resulted in a release of dormancy and overexpressing tubers showed a longer dormant period than that of the control. Further analyses revealed that the decrease expression level accelerated skin cracking and water loss. Metabolite analyses revealed that StSN2 significantly down-regulated the accumulation of lignin precursors in the periderm, and the change of lignin content was documented, a finding which was consistent with the precursors’ level. Subsequently, proteomics found that cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase (COMT) and peroxidase (Prx), the key proteins for lignin synthesis, were significantly up-regulated in silencing lines, and gene expression and enzyme activity analyses also supported this effect. Interestingly, we found that StSN2 physically interacts with three peroxidases catalyzing the oxidation and polymerization of lignin. In addition, SN2 altered the hydrogen peroxide (H2O2) content and the activities of superoxide dismutase (SOD) and catalase (CAT). These results suggest that StSN2 negatively regulates lignin biosynthesis and H2O2 accumulation, and ultimately inhibits the sprouting of potato tubers.

Drought-induced ABA, H2O2 and JA positively regulate CmCAD genes and lignin synthesis in melon stems

Background Cinnamyl alcohol dehydrogenase (CAD) is an important enzyme functions at the last step in lignin monomer synthesis pathway. Our previous work found that drought induced the expressions of CmCAD genes and promoted lignin biosynthesis in melon stems. Results Here we studied the effects of abscisic acid (ABA), hydrogen peroxide (H2O2) and jasmonic acid (JA) to CmCADs under drought stress. Results discovered that drought-induced ABA, H2O2 and MeJA were prevented efficiently from increasing in melon stems pretreated with fluridone (Flu, ABA inhibitor), imidazole (Imi, H2O2 scavenger) and ibuprofen (Ibu, JA inhibitor). ABA and H2O2 are involved in the positive regulations to CmCAD1, 2, 3, and 5, and JA is involved in the positive regulations to CmCAD2, 3, and 5. According to the expression profiles of lignin biosynthesis genes, ABA, H2O2 and MeJA all showed positive regulations to CmPAL2-like, CmPOD1-like, CmPOD2-like and CmLAC4-like. In addition, positive regulations were also observed with ABA to CmPAL1-like, CmC4H and CmCOMT, with H2O2 to CmPAL1-like, CmC4H, CmCCR and CmLAC17-like, and with JA to CmCCR, CmCOMT, CmLAC11-like and CmLAC17-like. As expected, the signal molecules positively regulated CAD activity and lignin biosynthesis under drought stress. Promoter::GUS assays not only further confirmed the regulations of the signal molecules to CmCAD1~3, but also revealed the important role of CmCAD3 in lignin synthesis due to the strongest staining of CmCAD3 promoter::GUS. Conclusions CmCADs but CmCAD4 are positively regulated by ABA, H2O2 and JA under drought stress and participate in lignin synthesis.