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Heavy metal such as arsenite (AsIII) is a threat worldwide. Thus, to mitigate AsIII toxicity on plants, we investigated the interactive effect of olive solid waste (OSW) and arbuscular mycorrhizal fungi (AMF) on wheat plants under AsIII stress. To this end, wheat seeds were grown in soils treated with OSW (4% w/w), AMF-inoculation, and/or AsIII treated soil (100 mg/kg soil). AMF colonization is reduced by AsIII but to a lesser extent under AsIII + OSW. AMF and OSW interactive effects also improved soil fertility and increased wheat plants' growth, particularly under AsIII stress. The interactions between OSW and AMF treatments reduced AsIII-induced H2O2 accumulation. Less H2O2 production consequently reduced AsIII-related oxidative damages i.e., lipid peroxidation (malondialdehyde, MDA) (58%), compared to As stress. This can be explained by the increase in wheat's antioxidant defense system. OSW and AMF increased total antioxidant content, phenol, flavonoids, and α-tocopherol by approximately 34%, 63%, 118%, 232%, and 93%, respectively, compared to As stress. The combined effect also significantly induced anthocyanins accumulation. The combination of OSW+AMF improved antioxidants enzymes activity, where superoxide dismutase (SOD, catalase (CAT), peroxidase (POX), glutathione reductase (GR), and glutathione peroxidase (GPX) were increased by 98%, 121%, 105%, 129%, and 110.29%, respectively, compared to AsIII stress. This can be explained by induced anthocyanin percussors phenylalanine, cinamic acid and naringenin, and biosynthesic enzymes (phenylalanine aminolayse (PAL) and chalcone synthase (CHS)). Overall, this study suggested the effectiveness of OSW and AMF as a promising approach to mitigate AsIII toxicity on wheat growth, physiology, and biochemistry.

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Drought is an important threat worldwide, therefore, it is vital to create workable solutions to mitigate the negative effects of drought stress. To this end, we investigated the interactive effect of compost (Comp), arbuscular mycorrhizal fungi (AMF) and carbon nanoparticles (CNPs) on maize plant crops under drought stress. The combined treatments were more effective at increasing soil fertility and promoting the growth of maize plants under both control and drought stress conditions by 20.1% and 39.4%, respectively. The interactions between treatments, especially the effects of Comp-AMF-CNPs mixture, reduce the activity of photorespiration induced H2O2 production that consequently reduces drought-related oxidative damages (lipid peroxidation and protein oxidation). Plants treated with Comp-AMF or Comp-AMF-CNPs showed an increase in their antioxidant defense system. Comp-AMF-CNPs increased enzyme activities by 50.3%, 30.1%, and 71% for ascorbate peroxidase (APX), dehydro-ASC reductase (DHAR), and monodehydro-ASC reductase (MDHAR), respectively. Comp-AMF-CNPs also induced the highest increase in anthocyanins (69.5%) compared to the control treatment. This increase was explained by increased anthocyanin percussor, by 37% and 13% under control and drought, respectively. While the increases in biosynthetic key enzymes, phenylalanine aminolayse (PAL) and chalcone synthase (CHS) were 77% and 5% under control and 69% and 89% under drought, respectively. This work advanced our understanding on how Comp-AMF-CNPs improve growth, physiology, and biochemistry of maize plants under drought stress conditions. Overall, this study suggests the effectiveness of Comp-AMF-CNPs as a promising approach to enhance the growth of maize plants in dry areas.

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MicroRNAs (miRNAs) are types of endogenous non-coding small RNAs found in eukaryotes that are 18-25 nucleotides long. miRNAs are considered to be key regulatory factors of the expression of target mRNA. The roles of miRNAs involved in the regulation of anthocyanin accumulation in pigmented potatoes have not been systematically reported. In this study, the differentially expressed miRNAs and their target genes involved in the accumulation of anthocyanin during different developmental stages in purple potato (Solanum tuberosum L.) were identified using small RNA (sRNA) and degradome sequencing. A total of 275 differentially expressed miRNAs were identified in the sRNA libraries. A total of 69,387,200 raw reads were obtained from three degradome libraries. The anthocyanin responsive miRNA-mRNA modules were analyzed, and 37 miRNAs and 23 target genes were obtained. Different miRNAs regulate the key enzymes of anthocyanin synthesis in purple potato. The structural genes included phenylalanine ammonia lyase, chalcone isomerase, flavanone 3-hydroxylase, and anthocyanidin 3-O-glucosyltransferase. The regulatory genes included WD40, MYB, and SPL9. stu-miR172e-5p_L-1R-1, stu-miR828a, stu-miR29b-4-5p, stu-miR8019-5p_L-4R-3, stu-miR396b-5p, stu-miR5303f_L-7R + 2, stu-miR7997a_L-3, stu-miR7997b_L-3, stu-miR7997c_L + 3R-5_2ss2TA3AG, stu-miR156f-5p_L + 1, stu-miR156a, stu-miR156a_R-1, stu-miR156e, stu-miR858, stu-miR5021, stu-miR828 and their target genes were validated by qRT-PCR. They play important roles in the coloration and accumulation of purple potatoes. These results provide new insights into the biosynthesis of anthocyanins in pigmented potatoes.

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In this study, we performed an association analysis of metabolomics and transcriptomics to reveal the anthocyanin biosynthesis mechanism in a new purple-leaf tea cultivar Zikui (Camellia sinensis cv. Zikui) (ZK). Three glycosylated anthocyanins were identified, including petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside, and their contents were the highest in ZK leaves at 15 days. This is the first report on petunidin 3-O-glucoside in purple-leaf tea. Integrated analysis of the transcriptome and metabolome identified eleven dependent transcription factors, among which CsMYB90 had strong correlations with petunidin 3-O-glucoside, cyanidin 3-O-galactoside, and cyanidin 3-O-glucoside (PCC > 0.8). Furthermore, we also identified key correlated structural genes, including two positively correlated F3'H (flavonoid-3'-hydroxylase) genes, two positively correlated ANS (anthocyanin synthase) genes, and three negatively correlated PPO (polyphenol oxidase) genes. Overexpression of CsMYB90 in tobacco resulted in dark-purple transgenic calluses. These results showed that the increased accumulation of three anthocyanins in ZK may promote purple-leaf coloration because of changes in the expression levels of genes, including CsMYB90, F3'Hs, ANSs, and PPOs. These findings reveal new insight into the molecular mechanism of anthocyanin biosynthesis in purple-leaf tea plants and provide a series of candidate genes for the breeding of anthocyanin-rich cultivars.

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The MYB family, one of the largest transcription factor (TF) families, plays an important role in plant growth, development, and stress response. Although genome-wide analysis of the MYB family has been performed in many species based on sequence similarity, predicting the potential functions of the MYB genes and classifying the regulators into specific metabolic pathways remains difficult. In this study, using a hidden Markov model search and co-expression regulatory network analysis, we demonstrated a process to screen and identify potential MYB TFs in the anthocyanin biosynthesis pathway of Gossypium hirsutum. As a result, we identified 617 and 784 MYB genes (812 in total) from the previously reported and recently released genomes, respectively. Using 126 structural genes involved in the anthocyanin biosynthesis pathway as targets for several co-expression network analyses, we sorted out 31 R2R3-MYB genes, which are potential regulators in the specific pathway. Phylogenetic and collinearity analyses indicated that 83.9% of the 31 MYB genes originated from whole genome duplication or polyploidization. In addition, we revealed relatively specific regulatory relationships between the MYB TFs and their target structural genes. Approximately, 71% of the MYBs could regulate only a single anthocyanin-related structural gene. Moreover, we found that the A- and D- subgenome homoeologs of MYB TFs in G. hirsutum rarely co-regulate the same target gene. The current study not only demonstrated an easy method to rapidly predict potential TFs in a specific metabolic pathway, but also enhanced our understanding of the evolution, gene characteristics, expression, and regulatory pattern of MYB TFs in G. hirsutum.

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Sweet potato [Ipomoea batatas (L.) Lam.] (2n = 6x = 90) is an economic important autopolyploid species and its varieties differ regarding storage root skin and flesh colors. Two sweet potato genetic lines, Sushu8 (with red skin) and its mutant Zhengshu20, which produced different colored storage roots, were used in this study. The total flavonoid, carotenoid, and anthocyanin contents of the two lines were analyzed and revealed that anthocyanin was primarily responsible for the skin color difference. In addition, the early storage root expanding stage was the key period for anthocyanin accumulation in Sushu8. A total of 24 samples, including the skins of the fibrous root and the storage root at the early and middle expanding stages as well as the flesh of the storage root at the middle expanding stage, were analyzed based on differentially expressed genes identified by transcriptome sequencing and a weighted gene co-expression network analysis. Two gene modules highly related with the regulation of sweet potato skin color through stress responses as well as starch synthesis and glucose metabolism were identified. Furthermore, the WRKY75 transcription factor gene, fructose-bisphosphate aldolase 2 gene, and other DEGs highly related to the regulation of anthocyanin metabolism were enriched in the brown and green modules.

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Given the potential health benefits of polyphenolic compounds in the diet, there is a growing interest in the generation of food crops enriched with health-protective flavonoids. We undertook a series of metabolite analyses of tomatoes ectopically expressing the Delila and Rosea1 transcription factor genes from snapdragon (Antirrhinum majus), paying particular attention to changes in phenylpropanoids compared to controls. These analyses revealed multiple changes, including depletion of rutin and naringenin chalcone, and enhanced levels of anthocyanins and phenylacylated flavonol derivatives. We isolated and characterized the chemical structures of the two most abundant anthocyanins, which were shown by NMR spectroscopy to be delphinidin-3-(4'''-O-trans-p-coumaroyl)-rutinoside-5-O-glucoside and petunidin-3-(4'''-O-trans-p-coumaroyl)-rutinoside-5-O-glucoside. By performing RNA sequencing on both purple fruit and wild-type fruit, we obtained important information concerning the relative expression of both structural and transcription factor genes. Integrative analysis of the transcript and metabolite datasets provided compelling evidence of the nature of all anthocyanin biosynthetic genes, including those encoding species-specific anthocyanin decoration enzymes. One gene, SlFdAT1 (Solyc12g088170), predicted to encode a flavonoid-3-O-rutinoside-4'''-phenylacyltransferase, was characterized by assays of recombinant protein and over-expression assays in tobacco. The combined data are discussed in the context of both our current understanding of phenylpropanoid metabolism in Solanaceous species, and evolution of flavonoid decorating enzymes and their transcriptional networks in various plant species.

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