Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches.

BACKGROUND: Posttranscriptional gene silencing (PTGS) is one of the most important mechanisms for plants during viral infection. However, viruses have also developed viral suppressors to negatively control PTGS by inhibiting microRNA (miRNA) and short-interfering RNA (siRNA) regulation in plants. The first identified viral suppressor, P1/HC-Pro, is a fusion protein that was translated from potyviral RNA. Upon infecting plants, the P1 protein itself is released from HC-Pro by the self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression. We performed proteomics to identify P1-interacting proteins. We also performed transcriptomics that were generated from Col-0 and various P1/HC-Pro-related transgenic plants to identify novel genes. The results showed several novel genes were identified through the comparative network analysis that might be involved in P1/HC-Pro-mediated PTGS suppression. RESULTS: First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might work through P1 binding to VERNALIZATION INDEPENDENCE 3/SUPERKILLER 8 (VIP3/SKI8), a subunit of the exosome, to interfere with the 5'-fragment of the PTGS-cleaved RNA degradation product. Second, the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant). Third, the comparative network highlighted potentially critical genes in PTGS, including miRNA targets, calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response. CONCLUSION: Through these genetic and omics approaches, we revealed an overall perspective to identify many critical genes involved in PTGS. These new findings significantly impact in our understanding of P1/HC-Pro-mediated PTGS suppression.

The complete mitochondrial genome of wild mung bean (Vigna radiata var. sublobata TC1966).

The entire mitogenome of wild mung bean (Vigna radiata var. sublobata TC1966) was identified as a circular molecule of 402,981 bp length. The wild mung bean mitogenome encoded 3 rRNAs, 16 tRNAs, and 33 proteins. A phylogenetic tree was reconstructed using the 18 protein-coding genes of 14 legumes and one close species, Ricinus communis. Our phylogenetic analysis suggests that the wild mung bean clustered with the Vigna radiata var. radiata, as well as, the species of Vigna and Glycine appeared as a monophyletic group. This complete mitogenome sequence provides a genomic resource for further studies in mung bean breeding and domestication.

Transcriptomic and Proteomic Research To Explore Bruchid-Resistant Genes in Mungbean Isogenic Lines.

Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene.

Identification of single nucleotide polymorphism markers associated with resistance to bruchids (Callosobruchus spp.) in wild mungbean (Vigna radiata var. sublobata) and cultivated V. radiata through genotyping by sequencing and quantitative trait locus analysis.

BACKGROUND: Bruchid beetles are an important storage pest of grain legumes. Callosobruchus sp. infect mungbean (Vigna radiata) at low levels in the field, multiply during grain storage and can destroy seed stocks in a few months. Resistance against bruchid beetles has been found in wild mungbean V. radiata var. sublobata TC1966 and in cultivated mungbean line V2802. RESULTS: Bruchid resistance data were obtained from recombinant inbred line populations TC1966 (V. radiata var. sublobata) × NM92 (F12) and V2802 (V. radiata) × NM94 (F7). More than 6,000 single nucleotide polymorphic markers were generated through genotyping by sequencing (GBS) for each of these populations and were used to map bruchid resistance genes. One highly significant quantitative trait locus (QTL) associated with bruchid resistance was mapped to chromosome 5 on genetic maps of both populations, suggesting that TC1966 and V2802 contain the same resistance locus. Co-segregation of all markers associated with resistance indicated the presence of only one major resistance QTL on chromosome 5, while QTL analysis based on physical map positions of the markers suggested the presence of multiple QTLs on different chromosomes. The diagnostic capacity of the identified molecular markers located in the QTL to correctly predict resistance was up to 100 %. CONCLUSIONS: Molecular markers tightly linked to bruchid resistance loci of two different mungbean resistance sources were developed and validated. These markers are highly useful for developing resistant lines.

Genomic and transcriptomic comparison of nucleotide variations for insights into bruchid resistance of mungbean (Vigna radiata [L.] R. Wilczek).

BACKGROUND: Mungbean (Vigna radiata [L.] R. Wilczek) is an important legume crop with high nutritional value in South and Southeast Asia. The crop plant is susceptible to a storage pest caused by bruchids (Callosobruchus spp.). Some wild and cultivated mungbean accessions show resistance to bruchids. Genomic and transcriptomic comparison of bruchid-resistant and -susceptible mungbean could reveal bruchid-resistant genes (Br) for this pest and give insights into the bruchid resistance of mungbean. RESULTS: Flow cytometry showed that the genome size varied by 61 Mb (mega base pairs) among the tested mungbean accessions. Next generation sequencing followed by de novo assembly of the genome of the bruchid-resistant recombinant inbred line 59 (RIL59) revealed more than 42,000 genes. Transcriptomic comparison of bruchid-resistant and -susceptible parental lines and their offspring identified 91 differentially expressed genes (DEGs) classified into 17 major and 74 minor bruchid-resistance-associated genes. We found 408 nucleotide variations (NVs) between bruchid-resistant and -susceptible lines in regions spanning 2 kb (kilo base pairs) of the promoters of 68 DEGs. Furthermore, 282 NVs were identified on exons of 148 sequence-changed-protein genes (SCPs). DEGs and SCPs comprised genes involved in resistant-related, transposable elements (TEs) and conserved metabolic pathways. A large number of these genes were mapped to a region on chromosome 5. Molecular markers designed for variants of putative bruchid-resistance-associated genes were highly diagnostic for the bruchid-resistant genotype. CONCLUSIONS: In addition to identifying bruchid-resistance-associated genes, we found that conserved metabolism and TEs may be modifier factors for bruchid resistance of mungbean. The genome sequence of a bruchid-resistant inbred line, candidate genes and sequence variations in promoter regions and exons putatively conditioning resistance as well as markers detecting these variants could be used for development of bruchid-resistant mungbean varieties.

The complete mitochondrial genome of mungbean Vigna radiata var. radiata NM92 and a phylogenetic analysis of crops in angiosperms.

The entire mitogenome of the Vigna radiata var. radiata NM92 was identified as a circular molecule of 401 262 bp length (DDBJ accession number: AP014716). The contents of A, T, C, and G in the NM92 mitogenome were found to be 27.48%, 27.41%, 22.63%, and 22.48%, respectively. The NM92 mitogenome encoded 3 rRNAs, 16 tRNAs and 33 proteins. Eight protein-coding genes (nad1, nad2, nad4, nad5, nad7, rps3, and rps10) centain introns. Among them, three (nad1, nad2, and nad5) are trans-spliced genes. A phylogenetic tree was reconstructed using the 21 protein-coding genes of 16 crops. A species of gymnosperms, Cycas, was selected as the outgroup. This complete mitogenome sequence provides useful information to understand the cultivation of Vigna radiata and other crops.

Mapping quantitative trait loci for fruit traits and powdery mildew resistance in melon (Cucumis melo).

BACKGROUND: Fruit characters affect consumer preferences and the market value of melons is determined by fruit quality. Most fruit quality-related traits are controlled by multiple genes, and are influenced by environmental factors. Furthermore, powdery mildew is another limiting factor in melon production. To develop new melon cultivars with disease resistance and high quality fruits using the molecular marker-assisted breeding strategy, identification of quantitative trait loci for fruit quality and disease resistance is required. RESULTS: The F2 populations from the cross of TARI-08874 (Cucumis melo ssp. melo) and 'Bai-li-gua' (C. melo ssp. agrestis) were used to map the quantitative trait loci (QTLs) for fruit-related traits and powdery mildew resistance in two trials. All traits were significantly different (P < 0.05) between parents. The generated linkage map consisted of twelve major linkage groups (LGs), spanning 626.1 cM in total, with an average distance of 8.3 cM between flanking markers. Nineteen QTLs were detected for seven melon traits, among which ten QTLs were localized to the same positions as the corresponding QTLs described in other studies. Four of these QTLs were detected in both trials. The results of identified QTLs in this study suggested that fruit size in the tested populations were mainly determined by fruit diameter and flesh thickness. All of the major QTLs for fruit diameter and flesh thickness were identified on LG5 and LG11. Four QTLs identified responsible for netting width of fruit rind were co-localized with the QTLs for netting density, suggesting similar genetic mechanisms affecting these two traits. Additionally, only one major QTL for powdery mildew resistance was detected on LG2, and it was closely linked to a simple sequence repeat (SSR) marker CMBR120 which was identified in a previous study. CONCLUSION: Because the netting feature is a crucial factor for external appearance of fruits in Asia market, we focus on mining the genetic information of fruit netting. This is the first report of QTL mapping to netting width. Furthermore, new QTLs were identified for netting density (qND4, qND6, and qND7) and netting width (qNW2, qNW4, qNW6, and qNW7) successfully. In addition, novel QTLs for fruit diameter (qFD5), flesh thickness (qFT11) were also detected.

Development of a Mild Viral Expression System for Gain-Of-Function Study of Phytoplasma Effector In Planta.

PHYL1 and SAP54 are orthologs of pathogenic effectors of Aster yellow witches'-broom (AYWB) phytoplasma and Peanut witches'-broom (PnWB) phytoplasma, respectively. These effectors cause virescence and phyllody symptoms (hereafter leafy flower) in phytoplasma-infected plants. T0 lines of transgenic Arabidopsis expressing the PHYL1 or SAP54 genes (PHYL1 or SAP54 plants) show a leafy flower phenotype and result in seedless, suggesting that PHYL1 and SAP54 interfere with reproduction stage that restrict gain-of-function studies in the next generation of transgenic plants. Turnip mosaic virus (TuMV) mild strain (TuGK) has an Arg182Lys mutation in the helper-component proteinase (HC-ProR182K) that blocks suppression of the miRNA pathway and prevents symptom development in TuGK-infected plants. We exploited TuGK as a viral vector for gain-of-function studies of PHYL1 and SAP54 in Arabidopsis plants. TuGK-PHYL1- and TuGK-SAP54-infected Arabidopsis plants produced identical leafy flower phenotypes and similar gene expression profiles as PHYL1 and SAP54 plants. In addition, the leafy flower formation rate was enhanced in TuGK-PHYL1- or TuGK-SAP54-infected Arabidopsis plants that compared with the T0 lines of PHYL1 plants. These results provide more evidence and novel directions for further studying the mechanism of PHYL1/SAP54-mediated leafy flower development. In addition, the TuGK vector is a good alternative in transgenic plant approaches for rapid gene expression in gain-of-function studies.

The effect of red light and far-red light conditions on secondary metabolism in agarwood.

BACKGROUND: Agarwood, a heartwood derived from Aquilaria trees, is a valuable commodity that has seen prevalent use among many cultures. In particular, it is widely used in herbal medicine and many compounds in agarwood are known to exhibit medicinal properties. Although there exists much research into medicinal herbs and extraction of high value compounds, few have focused on increasing the quantity of target compounds through stimulation of its related pathways in this species. RESULTS: In this study, we observed that cucurbitacin yield can be increased through the use of different light conditions to stimulate related pathways and conducted three types of high-throughput sequencing experiments in order to study the effect of light conditions on secondary metabolism in agarwood. We constructed genome-wide profiles of RNA expression, small RNA, and DNA methylation under red light and far-red light conditions. With these profiles, we identified a set of small RNA which potentially regulates gene expression via the RNA-directed DNA methylation pathway. CONCLUSIONS: We demonstrate that light conditions can be used to stimulate pathways related to secondary metabolism, increasing the yield of cucurbitacins. The genome-wide expression and methylation profiles from our study provide insight into the effect of light on gene expression for secondary metabolism in agarwood and provide compelling new candidates towards the study of functional secondary metabolic components.

Proteomic analysis of broccoli (Brassica oleracea) under high temperature and waterlogging stresses.

BACKGROUND: The production of broccoli (Brassica oleracea) is largely reduced by waterlogging and high temperature stresses. Heat-tolerant and heat-susceptible broccoli cultivars TSS-AVRDC-2 and B-75, respectively, were used for physiological and proteomic analyses. The objective of this study was to identify TSS-AVRDC-2 and B-75 proteins differentially regulated at different time periods in response to waterlogging at 40 °C for three days. RESULTS: TSS-AVRDC-2 exhibited significantly higher chlorophyll content, lower stomatal conductance, and better H2O2 scavenging under stress in comparison to B-75. Two-dimensional liquid phase fractionation analyses revealed that Rubisco proteins in both varieties were regulated under stressing treatments, and that TSS-AVRDC-2 had higher levels of both Rubisco large and small subunit transcripts than B-75 when subjected to high temperature and/or waterlogging. CONCLUSIONS: This report utilizes physiological and proteomic approaches to discover changes in the protein expression profiles of broccoli in response to heat and waterlogging stresses. Higher levels of Rubisco proteins in TSS-AVRDC-2 could lead to increased carbon fixation efficiency to provide sufficient energy to enable stress tolerance under waterlogging at 40 °C.

Identification of cucurbitacins and assembly of a draft genome for Aquilaria agallocha.

BACKGROUND: Agarwood is derived from Aquilaria trees, the trade of which has come under strict control with a listing in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. However, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood. RESULTS: In this study, we present a draft genome and a putative pathway for cucurbitacin E and I, compounds with known medicinal value, from in vitro Aquilaria agallocha agarwood. DNA and RNA data are utilized to annotate many genes and protein functions in the draft genome. The expression changes for cucurbitacin E and I are shown to be consistent with known responses of A. agallocha to biotic stress and a set of homologous genes in Arabidopsis thaliana related to cucurbitacin bio-synthesis is presented and validated through qRT-PCR. CONCLUSIONS: This study is the first attempt to identify cucurbitacin E and I from in vitro agarwood and the first draft genome for any species of Aquilaria. The results of this study will aid in future investigations of secondary metabolite pathways in Aquilaria and other non-model medicinal plants.

Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytes.

BACKGROUND: Herbaceous plants containing antioxidants can protect against DNA damage. The purpose of this study was to evaluate the antioxidant substances, antioxidant activity, and protection of DNA from oxidative damage in human lymphocytes induced by hydrogen peroxide (H2O2). Our methods used acidic methanol and water extractions from six herbaceous plants, including Bidens alba (BA), Lycium chinense (LC), Mentha arvensis (MA), Plantago asiatica (PA), Houttuynia cordata (HC), and Centella asiatica (CA). METHODS: Antioxidant compounds such as flavonol and polyphenol were analyzed. Antioxidant activity was determined by the inhibition percentage of conjugated diene formation in a linoleic acid emulsion system and by trolox-equivalent antioxidant capacity (TEAC) assay. Their antioxidative capacities for protecting human lymphocyte DNA from H2O2-induced strand breaks was evaluated by comet assay. RESULTS: The studied plants were found to be rich in flavonols, especially myricetin in BA, morin in MA, quercetin in HC, and kaemperol in CA. In addition, polyphenol abounded in BA and CA. The best conjugated diene formation inhibition percentage was found in the acidic methanolic extract of PA. Regarding TEAC, the best antioxidant activity was generated from the acidic methanolic extract of HC. Water and acidic methanolic extracts of MA and HC both had better inhibition percentages of tail DNA% and tail moment as compared to the rest of the tested extracts, and significantly suppressed oxidative damage to lymphocyte DNA. CONCLUSION: Quercetin and morin are important for preventing peroxidation and oxidative damage to DNA, and the leaves of MA and HC extracts may have excellent potential as functional ingredients representing potential sources of natural antioxidants.

Proteomics and transcriptomics of broccoli subjected to exogenously supplied and transgenic senescence-induced cytokinin for amelioration of postharvest yellowing.

Previously, we investigated transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and mimics the action of exogenous supplied CK in delaying postharvest senescence of broccoli. Here, we used proteomics and transcriptomics to compare the mechanisms of ipt-transgenic and N(6)-benzylaminopurine (BA) CK treatment of broccoli during postharvest storage. The 2 treatments conferred common and distinct mechanisms. BA treatment decreased the quantity of proteins involved in energy and carbohydrate metabolism and amino acid metabolism, and ipt-transgenic treatment increased that of stress-related proteins and molecular chaperones and slightly affected levels of carbohydrate metabolism proteins. Both treatments regulated genes involved in CK signaling, sugar transport, energy and carbohydrate metabolism, amino acid metabolism and lipid metabolism, although ipt-transgenic treatment to a lesser extent. BA treatment induced genes encoding molecular chaperones, whereas ipt-transgenic treatment induced stress-related genes for cellular protection during storage. Both BA and ipt-transgenic treatments acted antagonistically on ethylene functions. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. BIOLOGICAL SIGNIFICANCE: Transgenic broccoli harboring senescence-associated-gene (SAG) promoter-triggered isopentenyltransferase (ipt), which encodes the key enzyme for cytokinin (CK) synthesis and N(6)-benzylaminopurine (BA) CK treated broccoli both showed retardation of postharvest senescence during storage. The mechanisms underlying the two treatments were compared. The combination of proteomic and transcriptomic evidences revealed that the 2 treatments conferred common and distinct mechanisms in delaying senescence of broccoli florets. We propose a long-term acclimation of metabolism and protection systems with ipt-transgenic treatment of broccoli and short-term modulation of metabolism and establishment of a protection system with both BA and ipt-transgenic treatments in delaying senescence of broccoli florets. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Paclobutrazol pre-treatment enhanced flooding tolerance of sweet potato.

The objective of this experiment was to study changes of antioxidants and antioxidative enzymes in the flooding-stressed sweet potato leaf, as affected by paclobutrazol (PBZ) treatment at 24 h prior to flooding. Sweet potato 'Taoyuan 2' were treated with 0 and 0.5 mg/plant of PBZ, afterwards subjected to non-flooding and flooding-stress conditions for 0, 1, 3, and 5 d, followed by a 2 d drainage period. The study was conducted as a factorial experiment in completely randomized blocks with three replications maintained within a screen house. Plants with various antioxidative systems responded differently to flooding stress according to the duration of the flooding period and subsequent drainage period. The increased levels of antioxidants and antioxidative enzymes observed on different days of flooding afforded the sweet potato leaf with improved flooding tolerance. Glutathione reductase activity in the leaf was significantly enhanced over 5 d continuous flooding followed by a drainage period, in comparison with non-flooding conditions. Under non-flooding conditions, antioxidative system of leaf was regulated and elevated by PBZ pre-treatment. PBZ treatment may enable sweet potato 'Taoyuan 2' to maintain the balance between the formation and the detoxification of activated oxygen species. Our results also show that under flooding-stress conditions, the level of 'Taoyuan 2' antioxidative system is linked to PBZ treatment. Pre-treating with PBZ may increase levels of various components of antioxidative systems after exposure to different durations of flooding and drainage, thus inducing flooding tolerance. PBZ exhibited the important function of enhancing the restoration of leaf oxidative damage under flooding stress after the pre-application of 0.5 mg/plant. These findings may have greater significance for farming in frequently flooded areas.

RAPD markers for the identification of yield traits in tomatoes under heat stress via bulked segregant analysis.

Tomato production is limited to a large extent by climates with high temperatures. Yield-related traits in tomatoes are generally thought to be quantitative inherited traits that are significantly affected by variation in environmental factors. Breeding for heat tolerance is restricted due to the complexity of the traits. The objective of this study was to identify random amplified polymorphic DNA (RAPD) markers linked to heat-tolerance traits in tomatoes under heat stress. Forty-three F(7) recombinant inbred lines (RILs) derived from a wild cross between CL 5915 (heat-tolerant) and L4422 (heat-sensitive) were obtained and scored for flower number, fruit number, fruit set, fruit weight and yield under screen house conditions during the hot (summer) season of 2003. The distributions of average fruit weight, fruit number, fruit set and yield in the F(7) population were strongly skewed towards heat susceptibility, characteristic of L4422. Significant positive correlation was observed between fruit weight and yield, and between fruit number, fruit set and yield. However, the increase in yield and fruit set by selecting for large flower number may be fairly minor due to non-significant correlation between these traits. Selecting for flower number may not be a useful tool for improving yield under heat stress. A total of 200 RAPD primers were screened, among which 14 were identified as associated with heat-tolerance using bulk segregant analysis (BSA) based on the F(7) population grown in a heat-stressed environment. Some RAPD markers were unique to one specific trait, and others were linked to two traits. All markers for heat tolerance related traits had positive gene effects as a result of the contribution of the CL5915 gene that bolstered these traits. One hundred F(2) plants derived from the same parents (CL5915xL4422) were grown in the same location during the summer of 2004 to test for the stability and reliability of the 14 markers identified. Selection for the desired heat-tolerance genotypes corresponded well with targeting heat tolerance traits using the selected heat tolerance RAPD markers identified. Marker-assisted selection (MAS) for heat tolerance may be efficiently conducted by selecting individuals that contain high fruit number, high fruit weight, and high yield markers (P06, X01, D06 and D11), which would thus facilitate conventional breeding using CL5915 as a donor parent.