Independent flavonoid and anthocyanin biosynthesis in the flesh of a red-fleshed table grape revealed by metabolome and transcriptome co-analysis.

BACKGROUND: Red flesh is a desired fruit trait, but the regulation of red flesh formation in grape is not well understood. 'Mio Red' is a seedless table grape variety with light-red flesh and blue-purple skin. The skin color develops at veraison whereas the flesh color develops at a later stage of berry development. The flesh and skin flavonoid metabolomes and transcriptomes were analyzed. RESULTS: A total of 161 flavonoids were identified, including 16 anthocyanins. A total of 66 flavonoids were found at significantly different levels in the flesh and skin (fold change ≥ 2 or ≤ 0.5, variable importance in projection (VIP) ≥ 1). The main anthocyanins in the flesh were pelargonidin and peonidin, and in the skin were peonidin, delphinidin, and petunidin. Transcriptome comparison revealed 57 differentially expressed structural genes of the flavonoid-metabolism pathway (log2fold change ≥ 1, FDR < 0.05, FPKM ≥ 1). Two differentially expressed anthocyanin synthase (ANS) genes were annotated, ANS2 (Vitvi02g00435) with high expression in flesh and ANS1 (Vitvi11g00565) in skin, respectively. One dihydro flavonol 4-reductase (DFR, Vitvi18g00988) gene was differentially expressed although high in both skin and flesh. Screened and correlation analysis of 12 ERF, 9 MYB and 3 bHLH genes. The Y1H and dual luciferase assays showed that MYBA1 highly activates the ANS2 promoter in flesh and that ERFCBF6 was an inhibitory, EFR23 and bHLH93 may activate the DFR gene. These genes may be involved in the regulation of berry flesh color. CONCLUSIONS: Our study revealed that anthocyanin biosynthesis in grape flesh is independent of that in the skin. Differentially expressed ANS, MYB and ERF transcription factors provide new clues for the future breeding of table grapes that will provide the health benefits as red wine.

RNA sequencing reveals high resolution expression change of major plant hormone pathway genes after young seedless grape berries treated with gibberellin.

Seedless varieties are of particular importance to the table-grape and raisin industries. Gibberellin (GA) application is widely used in the early stages of seedless berry development to increase berry size and economic value. However, the underlying mechanism of GA induction of berry enlargement is not well understood. Here, RNA-sequencing analysis of 'Centennial Seedless' (Vitis vinifera L.) berries treated with GA3 12 days after flowering is reported. Pair-wise comparison of GA3-treated and control samples detected 165, 444, 463 genes with an over two-fold change in expression 1, 3, and 7 days after GA3 treatment, respectively. The number of differentially expressed genes increased with time after GA3 treatment, and the differential expression was dominated by downregulation. Significantly modulated expression included genes encoding synthesis and catabolism to manage plant hormone homeostasis, hormone transporters, receptors and key components in signaling pathways; exogenous GA3 induced multipoint cross talk with auxin, cytokinin, brassinosteroid, ABA and ethylene. The temporal gene-expression patterns of cell-wall-modification enzymes, cytoskeleton and membrane components and transporters revealed a pivotal role for cell-wall-relaxation genes in GA3-induced berry enlargement. Our results provide the first sequential transcriptomic atlas of exogenous GA3-induced berry enlargement and reveal the complexity of GA3's effect on berry sizing.

Proteomic analysis of berry-sizing effect of GA3 on seedless Vitis vinifera L.

Gibberellin (GA) is widely used in the table grape and raisin industries to enlarge the berries of seedless varieties. However, the mechanism underlying its berry-sizing effect is poorly understood. In this study, clusters of Centennial Seedless (Vitis vinifera L.) were treated with 30 ppm GA3 on day 12 after flowering, and berries were sampled at development stages I, II and III for proteomic analysis. Among the 1479 proteins detected on 2-DE maps, 19, 70 and 69 spots in stages I, II and III, respectively, showed an at least twofold difference in volume between treatments and controls. Of these, 125 proteins were successfully identified and assigned to eight functional groups, chief among them are metabolism and energy, stress response, expression regulation and cytoskeleton proteins. Stress-response proteins were predominantly down-regulated in GA3-treated berries in stages I and II, and significantly up-regulated in stage III. Up-regulation of cytoskeleton, cell-wall modification and other important proteins was found in the two latter stages of berry development. Our proteomic results and subsequent validation revealed, for the first time, the role of redox homeostasis in GA3-induced berry enlargement and markedly remodeled cellular protein expression in treated berries.

Proteomic changes in grape embryogenic callus in response to Agrobacterium tumefaciens-mediated transformation.

Agrobacterium tumefaciens-mediated transformation is highly required for studies of grapevine gene function and of huge potential for tailored variety improvements. However, grape is recalcitrant to transformation, and the underlying mechanism is largely unknown. To better understand the overall response of grapevine to A. tumefaciens-mediated transformation, the proteomic profile of cv. Prime embryogenic callus (EC) after co-cultivation with A. tumefaciens was investigated by two-dimensional electrophoresis and MALDI-TOF-MS analysis. Over 1100 protein spots were detected in both inoculated and control EC, 69 of which showed significantly differential expression; 38 of these were successfully identified. The proteins significantly up-regulated 3 d after inoculation were PR10, resistance protein Pto, secretory peroxidase, cinnamoyl-CoA reductase and different expression regulators; down-regulated proteins were ascorbate peroxidase, tocopherol cyclase, Hsp 70 and proteins involved in the ubiquitin-associated protein-degradation pathway. A. tumefaciens transformation-induced oxidative burst and modified protein-degradation pathways were further validated with biochemical measurements. Our results reveal that agrobacterial transformation markedly inhibits the cellular ROS-removal system, mitochondrial energy metabolism and the protein-degradation machinery for misfolded proteins, while the apoptosis signaling pathway and hypersensitive response are strengthened, which might partially explain the low efficiency and severe EC necrosis in grape transformation.

Improved tolerance toward fungal diseases in transgenic Cavendish banana (Musa spp. AAA group) cv. Grand Nain.

The most devastating disease currently threatening to destroy the banana industry worldwide is undoubtedly Sigatoka Leaf spot disease caused by Mycosphaerella fijiensis. In this study, we developed a transformation system for banana and expressed the endochitinase gene ThEn-42 from Trichoderma harzianum together with the grape stilbene synthase (StSy) gene in transgenic banana plants under the control of the 35S promoter and the inducible PR-10 promoter, respectively. The superoxide dismutase gene Cu,Zn-SOD from tomato, under control of the ubiquitin promoter, was added to this cassette to improve scavenging of free radicals generated during fungal attack. A 4-year field trial demonstrated several transgenic banana lines with improved tolerance to Sigatoka. As the genes conferring Sigatoka tolerance may have a wide range of anti-fungal activities we also inoculated the regenerated banana plants with Botrytis cinerea. The best transgenic lines exhibiting Sigatoka tolerance were also found to have tolerance to B. cinerea in laboratory assays.

Glutathione peroxidase regulation of reactive oxygen species level is crucial for in vitro plant differentiation.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is overexpressed in plants under abiotic and biotic stress conditions that mediate oxidative stress. To study its biological role and its ability to confer stress resistance in plants, we tried to obtain transgenic plants overexpressing citrus (Citrus sinensis) PHGPx (cit-PHGPx). All attempts to obtain regenerated plants expressing this enzyme constitutively failed. However, when the enzyme's catalytic activity was abolished by active site-directed mutagenesis, transgenic plants constitutively expressing inactive cit-PHGPx were successfully regenerated. Constitutive expression of enzymatically active cit-PHGPx could only be obtained when transformation was based on non-regenerative processes. These results indicate that overexpression of the antioxidant enzyme PHGPx interferes with shoot organogenesis and suggests the involvement of reactive oxygen species (ROS) in this process. Using transgenic tobacco (Nicotiana tabacum) leaves obtained from plants transformed with a beta-estradiol-inducible promoter, time-dependent induction of cit-PHGPx expression was employed. A pronounced inhibitory effect of cit-PHGPx on shoot formation was found to be limited to the early stage of the regeneration process. Monitoring the ROS level during regeneration revealed that upon cit-PHGPx induction, the lowest level of ROS correlated with the maximal level of shoot inhibition. Our results clearly demonstrate the essential role of ROS in the early stages of in vitro shoot organogenesis and the possible involvement of PHGPx in maintaining ROS homeostasis at this point.

The ubiquitin extension protein S27a is differentially expressed in developing flower organs of Thompson seedless versus Thompson seeded grape isogenic clones.

In Vitis vinifera L. cv. Thompson Seedless, fertilization occurs but seeds abort, a type of stenospermocarpy. To clone transcripts with differential expression during flower development, suppressive subtractive hybridization was carried out using two isogenic clones 'Thompson seedless' and 'Thompson seeded', at three stages of inflorescence development (from bud break to ~20 days prior to anthesis). Differential screening and sequencing of a forward and reverse subtractive cDNA library yielded several singleton ESTs. One differentially expressed clone in 'Thompson' seeded versus seedless isogenic clones was the ubiquitin extension protein S27a. In situ hybridization demonstrated its significantly higher expression in the carpel and ovaries of 'Thompson' seedless versus seeded isogenic clones during flower development. Overexpression of this gene resulted in abnormal plant regeneration and inhibited shoot development compared to controls; its silencing in embryogenic callus induced cell necrosis and callus death, evidencing tight regulation of this gene in developing organs of grape. S27a overexpression in carpels and integuments of the seedless flower may interfere with normal development of these organs, leading to embryo abortion and seedlessness.

Suppression and overexpression of ubiquitin extension protein S27a affects cell proliferation and in vitro regeneration in Nicotiana benthamiana.

Ubiquitin is a highly conserved 76-amino-acid protein found in all eukaryotic cells. Ubiquitin's expression is encoded and expressed as multimeric head-to-tail repeats (polyubiquitins) that are post-translationally cleaved into monomers, or fused with ribosomal proteins S27a and L40. S27a is highly expressed in meristematic tissues, pollen and ovules and its ubiquitin moiety is thought to act as a chaperone in ribosome biogenesis prior to cleavage. This study suggests that the ribosomal protein S27a plays a critical role in the allocation of meristematic cells that differentiate into lateral structures such as leaves and flowers. S27a was also found to regulate floral meristem development, possibly through the control of cell proliferation as well as cell identity. Overexpression of S27a was correlated with increased proliferation of undifferentiated cells and arrest of morphologically "normal" shoot and leaf development. The ubiquitin moiety did not affect the localization of S27a, but it did affect its protein level: expression of S27a without the ubiquitin moiety caused a severe reduction in S27a protein level.

Spirodela (duckweed) as an alternative production system for pharmaceuticals: a case study, aprotinin.

Aprotinin is a small serine protease inhibitor used in human health. Spirodela were transformed, via Agrobacterium, with a synthetic gene encoding the mature aprotinin sequence and a signal peptide for secretion which was driven by the CaMV 35S promoter. A total of 25 transgenic Spirodela lines were generated and aprotinin production was confirmed by northern and western blot analyses. Expression levels of up to 3.7% of water soluble proteins were detected in the plant and 0.65 mg/l in the growth medium. In addition, immunoaffinity purification of the protein followed by amino acid sequencing confirmed the correct splicing of the aprotinin produced in Spirodela and secreted into the growth medium.

High expression of transgene protein in Spirodela.

The monocot family Lemnaceae (duckweed) is composed of small, edible, aquatic plants. Spirodela oligorrhiza SP is a duckweed with a biomass doubling time of about 2 days under controlled, axenic conditions. Stably transformed Spirodela plants were obtained following co-cultivation of regenerative calli with Agrobacterium tumefaciens. GFP activity was successfully monitored in different subcellular compartments of the plant and correlated with different targeting sequences. Transgenic lines were followed for a period of at least 18 months and more than 180 vegetative doublings (generations). The lines are stable in morphology, growth rate, transgene expression, and activity as measured by DNA-DNA and immunoblot hybridizations, fluorescence activity measurements, and antibiotic resistance. The level of transgene expression is a function of leader sequences rather than transgene copy number. A stable, transgenic, GFP expression level >25% of total soluble protein is demonstrated for the S. oligorrhiza system, making it among the higher expressing systems for nuclear transformation in a higher plant.

Silencing of chaperonin 21, that was differentially expressed in inflorescence of seedless and seeded grapes, promoted seed abortion in tobacco and tomato fruits.

Vitis vinifera L. cv. 'Thompson Seedless' presents a type of stenospermocarpy in grape where fertilization occurs but seeds abort and fail to develop. To unravel the molecular basis for stenospermocarpy in grapes, subtractive hybridization was carried out in order to isolate differentially regulated genes that participate in the seedlessness machinery. Two 'Thompson' lines, a seeded and a seedless, were screened during different flower developmental stages. One of the genes, that was differentially expressed between the seeded and seedless lines, was the chloroplast chaperonin 21 (ch-Cpn21). ch-Cpn21 is a 21-kDa co-chaperonin polypeptide formed by two GroES-like domains fused together in tandem. Silencing of ch-Cpn21 in Nicotiana benthamiana plants resulted in leaf stunting, chlorosis, as well as ovary necrogenesis leading to seed abortion. Moreover, organ-specific silencing of ch-Cpn21 only in Lycopersicum esculentum fruits resulted in the development of seedless tomatoes. These results suggest that ch-Cpn21 may play a role in seed abortion in stenospermocarpic grapes.

Cryopreservation of embryogenic cell suspensions by encapsulation-vitrification.

Encapsulation-vitrification, which is a combination of encapsulation-dehydration and vitrification procedures, is a newly developed technique for cryopreservation of plant germoplasm. Here, we describe the protocol of this methodology, using grapevine (Vitis) as a model plant. Cell suspensions at the exponential growth stage were encapsulated with 2.5% sodium alginate solution in 0.1 M calcium chloride solution for 20 min to form beads of about 4 mm in diameter containing 25% cells. The beads were stepwise precultured in increasing sucrose concentrations of 0.25, 0.5, and 0.75 M for 3 yr, with 1 d for each step. Precultured beads were treated with a loading solution for 60 min at room temperature and then dehydrated with PVS2 at 0 degrees C for 270 min, followed by direct immersion in liquid nitrogen for 1 h. The beads were rapidly rewarmed at 40 degrees C in a water bath for 3 min and then diluted with 1 M sucorse solution at room temperature for 30 min. Rewarmed, washed beads were post-cultured on a recovery medium for 3 d at 25 degrees C in the dark for survival. Surviving cells were transferred to a regrowth medium to induce cell proliferation. Embryogenic cell suspensions were re-established by suspending the cells in a cell suspension maintenance medium maintained on a gyratory shaker at 25 degrees C in the dark. For plant regeneration, surviving cells were transferred from the recovery medium to an embryo maturation medium and maintained at 25 degrees C under light conditions. Embryos at the torpedo stage were cultured on rooting medium until whole plantlet was developed.

Expression of streptavidin in tomato resulted in abnormal plant development that could be restored by biotin application.

Biotin is an essential cofactor for a variety of carboxylase and decarboxylase reactions and is involved in diverse metabolic pathways of all organisms. In the present study we tested the hypothesis that controlling biotin availability by the expression of Streptomyces avidinii streptavidin, would impede plant development. Transient expression of streptavidin fused to plant signal peptide, bacterial signal peptide or both, in tomato (Lycopersicon esculentum cv. VF36) plants resulted in various levels of tissue impairment, exhibited as lesion development on 1-week-old tomato seedlings. The least toxic construct was introduced to tomato (stable transformation) under the constitutive CaMV 35S promoter, and lesions appeared on stems, flower morphologies were modified and numbers and sizes of fruits were altered. Furthermore, tissue-specific expression of the streptavidin, by means of the beta-phaseolin or TobRB7 promoters, resulted in localised effects, i.e., impaired seed formation or seedless fruits, respectively, with no alteration in the morphology of the other plant organs. External application of biotin on streptavidin-expressing tomato plants prevented the degeneration symptoms and facilitated normal plant development. It can be concluded that expression of streptavidin in the plant cell can lead to local and temporal deficiencies in biotin availability, impairing developmental processes while biotin application restores plant growth cycle.

Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region.

Dihydroflavonol reductase (DFR) is a key enzyme involved in anthocyanin biosynthesis and proanthocyanidin synthesis in grape. DFR catalyses the reduction of dihydroflavonols to leucoanthocyanidins in the anthocyanin pathway. The DFR products, the leucoanthocyanidins, are substrates for the next step in the anthocyanin pathway and are also the substrates for the proanthocyanidin pathway. In the present study the promoter of the grape dfr gene was cloned. Analysis of the dfr promoter sequence revealed the existence of several putative DNA binding motifs. The dfr promoter was fused to the uidA gene and the control of this fusion and the endogenous dfr gene expression, was studied in transformed plants and in red cell suspension originated from fruits. The dfr promoter-uidA gene fusion was expressed in leaves, roots and stems. Deletions of the dfr promoter influenced the specificity of the expression of the GUS gene fusion in plantlet roots and the level of expression in plants and in the red cell suspension originated from fruits. The deletion analysis of the dfr promoter suggests that a specific sequence located between -725 to -233 might be involved in expression of the dfr gene in fruits. Light, calcium and sucrose induced the dfr gene expression. In the transformed suspension cultures, expression of both the endogenous dfr gene and the dfr promoter-uidA gene fusions was induced by white light. The induction by both light and calcium suggests the possible involvement of a UV receptors signal transduction pathway in the induction of the dfr gene. The induction of the dfr gene and the dfr promoter-uidA gene fusions by light and sucrose indicates a close interaction between sucrose and light signalling pathways.