Journal of Plant Biochemistry and Biotechnology最新文献

In transgenic plants, the transgene copy numbers can highly affect the level of expression and genetic stability of the transgene. Hence, the first step in their characterization is the estimation of transgene copy numbers integrated in the plant genome. Quantitative real-time PCR (qRT-PCR) was used to determine the copy numbers of human interferon-γ (hIFN-γ) and hygromycin phosphortransferase II (hptII) transgenes in the genome of the T₀ generation of 18 transgenic tobacco lines using the axi1 gene as an endogenous control. With optimized PCR conditions, we attained highly exact estimates of one, two, three, and/or four transgene copies in the T₀ transformants. Moreover, estimation of copy numbers of the hIFN-γ transgene and the hptII selective marker gene indicated that rearrangements of the T-DNA has regularly happened in transgenic tobacco. Transgene copy number was also estimated using Southern blot analysis of gDNA derived from transformants. The transcript level and expression amount of recombinant hIFN-γ protein were evaluated in various events using RT-PCR and enzyme-linked immunosorbent assay (ELISA) techniques. A disagreement between the transcript level and the amount of recombinant protein with an inverse correlation between transgene copy number and expression level observed in some events, probably showing translational gene silencing and co-suppression or silencing, respectively. These results were also compared with segregation ratios of hygromycin-resistant phenotype in T₁ plants of each line and found to be, in general, consistent.

Increased hair in Arabidopsis roots during phosphorus deficiency is an important adaptation mechanism. The CAPRICE (CPC) gene, along with its six homologs, plays a pivotal role in root hair formation. Our previous study indicated upregulated expression of ENHANCER OF TRY AND CPC1 (ETC1) and ENHANCER OF TRY AND CPC3 (ETC3) homologous genes in response to phosphorus deficiency. ETC1 translocates from the root sub-epidermis to epidermal cells, thereby increasing the number of root hairs. In this study, we specifically focused on ETC3 to explore its function in this process. Our results showed that etc1, etc3, and etc1 etc3 mutants did not induce as much root hairs in response to phosphorus deficiency as the wild type. The upregulation of ETC1 and ETC3 in response to phosphorus deficiency was restricted to the roots, with no such response observed in leaves. Furthermore, examination of ETC3:GFP protein localization revealed that ETC3 expression was induced in the internal root tissues under phosphorus deficiency, without migrating to epidermal cells as observed for ETC1. This finding suggests that ETC3 may indirectly contribute to root hair formation through a different mechanism. Therefore, elucidating the complex process that governs root hair increase in phosphorus-deficient plants can lead to future advances in plant breeding and nutrient stress adaptation.

Abstract

Glucosyltransferases (GTs) are enzymes that use UDP-glucose to glucosylate wide variety of substrates, including the aglycones of anthocyanins. Anthocyanins are glycosylated polyphenolic plant pigments possessing potential health benefits to humans. The berries of Vaccinium species plants are rich in anthocyanins. Although the flavonoid content of bilberries is well characterized, the enzymes responsible for carrying out anthocyanin modifications are not thoroughly studied. In this study, a predicted sequence of an anthocyanin glucosyltransferase was identified from the genomic data of Vaccinium corymbosum. The codon-optimized gene sequence of the protein was integrated into the genome of P. pastoris. Constitutive expression in yeast extract-peptone-dextrose based media gave satisfactory amount of recombinant protein. The enzyme activity assays revealed that the V. corymbosum GT transferred glucosyl moieties to up to three positions of diverse flavonoids, such as naringenin, kaempferol, eriodictyol and cyanidin 3-O-glucoside, being therefore a rather unique enzyme among GTs described so far. The enzyme preferred cyanidin 3-O-glucoside, peonidin 3-O-glucoside and eriodictyol as substrates. This enzyme could find application in biotechnological production of glucosylated flavonoids.

Plants in the Allium genus have a significant economic impact due to their production of important food and medicinal compounds. Despite their agricultural and pharmaceutical value, breeding programs for Allium species are lagging behind those of other major crop plants. MicroRNAs (miRNAs) are a group of small non-coding RNAs that regulate post-transcriptional gene expression. In order to identify distinct miRNAs and their target proteins involved in Allicin biosynthesis pathways under heat-stress conditions, available Allium species Expressed Sequence Tags (ESTs) and miRNA databases were extensively searched and bioinformatically analyzed. Three distinct miRNA candidates targeting seven genes in garlic were identified. The real-time RT-PCR analysis results revealed that miR5021 and miR565 regulate the Alliinase and Heat shock protein 70 (HSP70) genes under cold stress (4°C), while miR5184 regulates the same genes at elevated temperatures (37 and 45°C). Based on the High-Performance Liquid Chromatography (HPLC) results, the expression of the Alliinase gene was found to be higher in both stem and leaf tissues compared to the expression of miR5021 under cold stress conditions. This indicates the potential involvement of miR5021 in the biosynthetic pathway of Allicin production. Furthermore, the results of the elevated heat-stress treatment showed that the expression of the HSP70 gene under thermal stress conditions at 37°C in both stem and leaf tissue was significantly increased compared to the expression of miR5658 and miR5184 at 45°C. However the expression of the HSP70 gene in the stem tissue was significantly decreased compared to the expression of miR5658 and miR5184 genes. This experimental study aims to identify miRNAs associated with the allicin production pathway in two tissues of garlic: leaves and cloves. Among the main findings of this research, we can mention the identification of 3 miRNAs that are likely to play a role in the allicin synthesis pathway. The results of this study suggest that environmental factors may affect the biosynthesis of allicin in plants of the Allium family.

Vegetables play a crucial role in global health and nutrition. Transcriptomics, a branch of functional genomics, has emerged as a powerful tool for studying gene expression patterns in various organisms, including vegetables. It is a dynamic genomics field that studies the entire set of RNA molecules in a cell, known as the transcriptome. This review offers a thorough overview of transcriptomics applications in vegetables, covering insights gained, challenges faced and future directions. It explores experimental techniques, bioinformatics tools and key findings from transcriptomic studies in various vegetable species. We summarize influential studies in vegetable crop transcriptomics and discuss how transcriptomics enhances crop productivity, disease resistance and nutritional quality in vegetables. Recent advances in RNA-seq technology have led to significant progress in characterizing the transcriptomes of diverse cell types. Transcriptomic analyses of vegetables have unveiled the dynamic patterns of gene expression, exposing temporal and spatial regulatory mechanisms. Transcriptomics identifies differentially expressed genes, aiding in finding candidate genes for desirable traits, enabling targeted breeding and genetic engineering. This review highlights its significance in understanding complex regulatory networks in vegetable development and response to environmental cues. This overview serves as a valuable resource for researchers and practitioners interested in transcriptomic studies.

Abstract

The pea (Pisum sativum L.) is one of widely cultivated crops over the world, but the mechanism of pod coloration is still unclear. Understanding the underlying mechanism regulating pea pod coloration can broaden pea breeding. In this study, the comparative analysis of transcriptome and metabolome was performed to examine the changes in metabolites accumulation and gene expression at two pod developmental stages in two pea accessions with green pods (GP) and yellow pods (YP). At the two omics levels, a total of 4504 differential metabolites (DEMs) were detected in four samples, and 2709 and 3725 differential expression genes (DEGs) were detected at two pod developmental stages between GP and YP, respectively. The verification results of RT-qPCR showed that the relative expression of ten genes randomly detected showed statistically high correlations between RT-qPCR and RNA-seq. The functional analysis of DEMs and DEGs found that the flavonoid metabolism pathway was the most direct metabolic pathway for the variation of pod coloration between two pea accessions. The 34 DEMs and 21 DEGs related to flavonoid metabolism pathway exhibited different expression pattern between GP and YP samples. Additionally, the combined analysis of transcriptome and metabolome in flavonoid biosynthesis pathway revealed that a direct correlation between the decreased expression level of genes and reduction accumulation of metabolites in the delphinidin biosynthesis pathway. The obtained results of this study provided a foundation to reveal the candidate genes controlling the pod color variation, and provide new germplasm resources for genetic improvement in pea.

Fusarium wilt, Ascochyta blight and Botrytis grey mould caused by fungal pathogens are major biotic stresses that lead to 100% yield loss in chickpea (Cicer arietinum L.). Several independent studies reported the genomic regions, using heterogeneous populations, that are of limited use in breeding programs for the development of enhanced disease resistance in climate change scenarios. In order to identify the consensus genomic regions, we developed a consensus integrated map and performed Meta-QTL analysis using 52 QTLs reported in 17 independent studies. The consensus integrated map spanned 1292.04 cM with 669 markers on all eight linkage groups. In total, we report 10 Meta-QTLs distributed on four linkage groups (CaLG02, CaLG03, CaLG04 and CaLG06) and 172 genes belonging to a wide range of gene-families that are involved in multiple disease resistance. Among these, the key genes such as Glutaredoxin, Reticuline oxidase-like protein, and RING-Finger proteins, which have been previously reported for disease resistance. The confidence interval of reported Meta-QTLs decreased 4.89 folds from the original QTL studies whose confidence interval ranged from 0.84 to 28.94 cM. Among these genes, Ca_13066 and Ca_05186 present in Meta-QTL4_6 and Meta-QTL6_8, that play major role in biotic stress resistance pathways possessed 3 and 8 haplotypes respectively. Our study provides a better understanding of the genetic mechanisms underlying these three important biotic stresses. Further, the Meta-QTLs, candidate genes and haplotypes reported in this study can be used for developing climate resilient and disease resistant chickpea cultivars.

Luteolin is an important secondary metabolite of maize, which can be glycosylated through uridine diphosphate glycosyltransferase (UGT). Extensive research on UGT has been centered in Arabidopsis thaliana, but little in maize. We cloned two maize glycosyltransferase genes, ZmUGT84A3 and ZmUGT84A4. In vitro experiments revealed that ZmUGT84A3 and ZmUGT84A4 glycosylated luteolin to both luteolin-7-O-glucoside and luteolin-4′,7-di-O-glucoside. Notably, both ZmUGT84A3 and ZmUGT84A4 catalyzed glycosylation of many flavonoids including apigenin, naringenin, eriodyctiol, kaempferol, and quercetin. Increased temperature enhanced activities of both ZmUGT84A3 and ZmUGT84A4, and elevated a production of luteolin-4′,7-di-O-glucoside. ZmUGT84A3 activity was optimal under acidic conditions but ZmUGT84A4 preferred alkaline environments. ZmUGT84A4 exhibited higher catalytic activities (1.3 to 2.6-fold) at various substrate concentrations than did ZmUGT84A3; the latter enzyme evidenced a unique activity pattern. Enzyme-catalyzed reaction processes of ZmUGT84A3 and ZmUGT84A4 in vitro for luteolin were depicted. This study unveiled two novel multifunctional glycosyltransferases identified in maize, which exhibited extensive substrate specificity, possessed multiple catalytic sites, and offered valuable insights for studying their enzymatic characteristics.

Abstract

Rice production is drastically affected by biotic stresses, namely, bacterial leaf blight (BB), blast (BL) and brown planthopper (BPH). As marker-assisted selection for uniplex gene screening in breeding population is often tedious, time-consuming and expensive, multiplex PCR is practically a reasonable choice for the testing of genes in the pyramided lines as it offers significant time- and cost-saving advantages. Therefore, a cost-effective multiplex PCR-based screening for simultaneous detection of three BB (Xa21, xa13, and Xa4), two BL (Pi1and Pi54) and two BPH resistance genes (Bph20 and Bph21) has been developed. This multiplex system can detect different combinations of BB, BL and BPH resistance. In addition to this most cost-effective method, a five-gene (Xa21, xa13, Pi54, Bph20 and Bph21) multiplex assay in a single PCR tube for three biotic stresses, BB, BL and BPH, has been developed. These standardized multiplex assays have been incorporated into our breeding programs for accelerated results. These assays have displayed no deviation and are in perfect correlation with the individual marker screenings. As one of the most efficient and cost-saving technologies, this technology will have a great impact in the seed industry.

Iron and zinc deficiency is a major problem among large populations in rice-consuming countries. Development micronutrient dense rice varieties with high yield is a key target area in breeding programmes and QTL mapping studies using backcross inbred lines to transfer beneficial genes from wild relatives is one of the potential strategy. In this study, 136 BC₄F₁₀ backcross inbred lines (BILs) from BPT5204 x Oryza rufipogon WR119 were field evaluated for 3 years for nine yield related traits. Grain Fe and Zn were estimated using ED-XRF. In all, 11 major QTLs with phenotypic variance from 10 to 16.8% were identified for Fe, Zn, and 5 yield related traits. O. rufipogon alleles were trait-enhancing in 18% of all QTLs and an allele at qFe2.1 increased iron concentration. Major effect QTLs qFe1.1 for grain Fe and qZn5.1, qZn8.1, and qZn10.1 for grain Zn explained 11 to 16% PVE, qZn8.1 and qZn10.1 were co-located with QTLs for grain yield related traits. Seven chromosomal regions showed QTLs for more than two traits. QTLs were associated with several high priority candidate genes for grain Fe, Zn and yield. One elite BIL [IET 24775 RP4920-Bio51B] was tested in AICRIP bio fortification trials for 4 years [2014–2017], and three BILs [IET 28715 RP4920-Bio61-1B], [IET28706 RP4920-Bio83B] and [IET28695 RP4920-Bio88B] are evaluated for 2 years of trials. The significant BILs and QTLs are useful in rice bio fortification and for gene discovery.