Enhancing grain size in durum wheat using RNAi to knockdown GW2 genes.

KEY MESSAGE: Knocking down GW2 enhances grain size by regulating genes encoding the synthesis of cytokinin, gibberellin, starch and cell wall. Raising crop yield is a priority task in the light of the continuing growth of the world's population and the inexorable loss of arable land to urbanization. Here, the RNAi approach was taken to reduce the abundance of Grain Weight 2 (GW2) transcript in the durum wheat cultivar Svevo. The effect of the knockdown was to increase the grains' starch content by 10-40%, their width by 4-13% and their surface area by 3-5%. Transcriptomic profiling, based on a quantitative real-time PCR platform, revealed that the transcript abundance of genes encoding both cytokinin dehydrogenase 1 and the large subunit of ADP-glucose pyrophosphorylase was markedly increased in the transgenic lines, whereas that of the genes encoding cytokinin dehydrogenase 2 and gibberellin 3-oxidase was reduced. A proteomic analysis of the non-storage fraction extracted from mature grains detected that eleven proteins were differentially represented in the transgenic compared to wild-type grain: some of these were involved, or at least potentially involved, in cell wall development, suggesting a role of GW2 in the regulation of cell division in the wheat grain.

Protein Hydrolysate Stimulates Growth in Tomato Coupled With N-Dependent Gene Expression Involved in N Assimilation.

Plant-derived protein hydrolysates (PHs) have received increased attention in the last decade because of their potential to improve yield, nutritional quality as well as tolerance to abiotic stressors. The current study investigated the effects and the molecular mechanisms of a legume-derived PH under optimal and sub-optimal nitrogen (N) concentrations (112 and 7 mg L-1, respectively) in tomato (Solanum lycopersicum L.). Growth and mineral composition of tomato plants treated with PHs by foliar spray or substrate drench were compared to untreated plants. In addition, the expression was determined of genes encoding ammonium and nitrate transporters and seven enzymes involved in N metabolism: nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase 1 (GS1), glutamine synthetase 2 (GS2), ferredoxin-dependent glutamate synthase (GLT), NADH-dependent glutamate synthase (GLS), and glutamate dehydrogenase (GDH). The root and total plant dry weight, SPAD index and leaf nitrogen content were higher by 21, 17, 7, and 6%, respectively, in plants treated by a substrate drench in comparison to untreated tomato plants, whereas foliar application of PH gave intermediate values. PH-treated plants grown with lower N availability showed reduced expression of NR and NiR as well as of nitrate and ammonium transporter transcripts in both leaf and root tissues in comparison with untreated plants; this was especially pronounced after application of PH by substrate drench. Conversely, the transcript level of an amino acid transporter gene was up-regulated in comparison with untreated plants. At high N regime, the transcript levels of the ammonium and amino acid transporters and also NR, NiR, and GLT were significantly up-regulated in root after PH foliar and substrate drench applications compared with untreated plants. An up-regulation was also observed for GS1, GS2, and GDH transcripts in leaf after substrate drench. These results highlighted the potential benefits of using legume PH in vegetable production systems to increase growth and N-nutritional status of plants.

Molecular characterisation of two novel starch granule proteins 1 in wild and cultivated diploid A genome wheat species.

Starch synthase IIa, also known as starch granule protein 1 (SGP-1), plays a key role in amylopectin biosynthesis. The absence of SGP-1 in cereal grains is correlated to dramatic changes in the grains' starch content, structure, and composition. An extensive investigation of starch granule proteins in this study revealed a polymorphism in the electrophoretic mobility of SGP-1 between two species of wheat, Triticum urartu and T. monococcum; this protein was, however, conserved among all other Triticum species that share the A genome inherited from their progenitor T. urartu. Two different electrophoretic profiles were identified: SGP-A1 proteins of T. urartu accessions had a SDS-PAGE mobility similar to those of tetraploid and hexaploid wheat species; conversely, SGP-A1 proteins of T. monococcum ssp. monococcum and ssp. boeoticum accessions showed a different electrophoretic mobility. The entire coding region of the two genes was isolated and sequenced in an attempt to explain the polymorphism identified. Several single nucleotide polymorphisms (SNPs) responsible for amino acid changes were identified, but no indel polymorphism was observed to explain the difference in electrophoretic mobility. Amylose content did not differ significantly among T. urartu, T. monococcum ssp. boeoticum and T. monococcum ssp. monococcum, except in one accession of the ssp. boeoticum. Conversely, several interspecific differences were observed in viscosity properties (investigated as viscosity profiles using a rapid visco analyzer-RVA profiles) of these cereal grains. T. monococcum ssp. boeoticum accessions had the lowest RVA profiles, T. urartu accessions had an intermediate RVA profile, whereas T. monococcum ssp. monococcum showed the highest RVA profile. These differences could be associated with the numerous amino acid and structural changes evident among the SGP-1 proteins.

Phenotypic, genetic and molecular characterization of 7B-1, a conditional male-sterile mutant in tomato.

KEY MESSAGE: We characterized the photoperiod-sensitive 7B - 1 male-sterile mutant in tomato, showing its allelism with stamenless - 2 . Mapping experiments indicated SlGLO2 , a B-class MADS-box family member, as a strong candidate to underlie the 7B - 1 mutation. The interest in male sterility (MS) dates back to a long time due to its perspective use in hybrid seed production. Here, we characterize 7B-1, a photoperiod-sensitive male-sterile (ms) mutant in tomato (Solanum lycopersicum L.), in which stamens are restored to fertility by permissive growth conditions in short days (SD). This system represents a useful strategy to facilitate the maintenance of the ms line. Examination of 7B-1 and other structural mutants, vms, sl, sl-2 and tap3, showed carpellization of stamens in the third floral whorl. 7B-1 exhibits strong expressivity in long days (LD), producing 100% aberrant anthers and virtually no seed production under open pollination, whereas it recovered fertility in SD. By genetic analysis, we demonstrate that 7B-1 is not allelic to sl nor to vms; instead it shows allelism to sl-2. Because the homeotic phenotype of the mutation resembles lesions to members of the B-class MADS-box transcription factor family, that specify petal and stamen identity, we pursued a candidate gene approach towards these targets. Using an interspecific backcross mapping population and markers linked to B-class MADS-box genes, significant linkage was found between 7B-1 and the SlGLO2 gene on Chr6. This result was supported by the 7B-1 phenotype that is similar to that of SlGLO2 knock outs and by the strong downregulation of the gene in the mutant. Although the lesion underlying the mutant phenotype is still elusive, our results pave the way for the final demonstration that SlGLO2 underlies 7B-1 and further the use of 7B-1 mutant in tomato hybrid seed production schemes.

Transcriptional regulation of male-sterility in 7B-1 male-sterile tomato mutant.

The 7B-1 tomato (Solanum lycopersicum L. cv Rutgers) is a male-sterile mutant with enhanced tolerance to abiotic stress, which makes it a potential candidate for hybrid seed breeding and stress engineering. To underline the molecular mechanism regulating the male-sterility in 7B-1, transcriptomic profiles of the 7B-1 male-sterile and wild type (WT) anthers were studied using mRNA sequencing (RNA-Seq). In total, 768 differentially expressed genes (DEGs) were identified, including 132 up-regulated and 636 down-regulated transcripts. Gene ontology (GO) enrichment analysis of DEGs suggested a general impact of the 7B-1 mutation on metabolic processes, such as proteolysis and carbohydrate catabolic process. Sixteen candidates with key roles in regulation of anther development were subjected to further analysis using qRT-PCR and in situ hybridization. Cytological studies showed several defects associated with anther development in the 7B-1 mutant, including unsynchronized anther maturation, dysfunctional meiosis, arrested microspores, defect in callose degradation and abnormal tapetum development. TUNEL assay showed a defect in programmed cell death (PCD) of tapetal cells in 7B-1 anthers. The present study provides insights into the transcriptome of the 7B-1 mutant. We identified several genes with altered expression level in 7B-1 (including beta-1,3 glucanase, GA2oxs, cystatin, cysteine protease, pectinesterase, TA29, and actin) that could potentially regulate anther developmental processes, such as meiosis, tapetum development, and cell-wall formation/degradation.

Metabolite Profiling of Italian Tomato Landraces with Different Fruit Types.

Increased interest toward traditional tomato varieties is fueled by the need to rescue desirable organoleptic traits and to improve the quality of fresh and processed tomatoes in the market. In addition, the phenotypic and genetic variation preserved in tomato landraces represents a means to understand the genetic basis of traits related to health and organoleptic aspects and improve them in modern varieties. To establish a framework for this approach, we studied the content of several metabolites in a panel of Italian tomato landraces categorized into three broad fruit type classes (flattened/ribbed, pear/oxheart, round/elongate). Three modern hybrids, corresponding to the three fruit shape typologies, were included as reference. Red ripe fruits were morphologically characterized and biochemically analyzed for their content in glycoalkaloids, phenols, amino acids, and Amadori products. The round/elongate types showed a higher content in glycoalkaloids, whereas flattened types had higher levels of phenolic compounds. Flattened tomatoes were also rich in total amino acids and in particular in glutamic acid. Multivariate analysis of amino acid content clearly separated the three classes of fruit types. Making allowance of the very low number of genotypes, phenotype-marker relationships were analyzed after retrieving single nucleotide polymorphisms (SNPs) among the landraces available in the literature. Sixty-six markers were significantly associated with the studied traits. The positions of several of these SNPs showed correspondence with already described genomic regions and QTLs supporting the reliability of the association. Overall the data indicated that significant changes in quality-related metabolites occur depending on the genetic background in traditional tomato germplasm, frequently according to specific fruit shape categories. Such a variability is suitable to harness association mapping for metabolic quality traits using this germplasm as an experimental population, paving the way for investigating their genetic/molecular basis, and facilitating breeding for quality-related compounds in tomato fruits.

A new "functional" pasta containing tartary buckwheat sprouts as an ingredient improves the oxidative status and normalizes some blood pressure parameters in spontaneously hypertensive rats.

Epidemiological studies have reported that some foods, particularly those rich in (poly)phenols, may reduce cardiovascular risk and metabolic disorders such as hypertension. Buckwheat sprouts have been suggested as a new raw material for the production of functional foods due to their high content of healthy compounds such as rutin and quercetin. The aim of this paper is to evaluate the biological hypotensive and antioxidant responses of pasta containing tartary buckwheat sprouts (TBSP) on spontaneously hypertensive rats (SHR). In this study, dry tartary buckwheat sprouts were milled to obtain a powder that was used in the production of pasta containing 30% dry buckwheat sprouts and 70% durum wheat semolina. Afterwards, we analyzed the in vitro TBSP features compared with the control (durum wheat flour pasta, DWFP), and the in vivo effects of TBSP on SHR and their normotensive counterpart, Wistar Kyoto rats (WKY rats). The total phenolic content and antioxidant activity were higher in TBSP compared to DWFP. The results showed that SHR fed TBSP exhibited higher plasma levels of the endogenous vasodilators bradykinin (BK) and nitric oxide (NO), a lower level of the vasoconstrictor endothelin-1 (ET-1), and an improved antioxidant capacity. These data suggest that TBSP may help reduce hypertension and oxidative stress in vivo.

Constitutive co-suppression of the GA 20-oxidase1 gene in tomato leads to severe defects in vegetative and reproductive development.

To dissect the role of gibberellins in tomato development, we have constitutively down-regulated the gene GA 20-oxidase1 (GA20ox1). Plants co-suppressed for GA20ox1 (referred to as CO-6 plants) showed vegetative defects typical of GA deficiency such as darker and mis-shaped leaves and dwarfism. CO-6 plants flowered as the controls, although their flowers had subtle defects in the pedicel and in organ insertion. Analysis of male development revealed defects before, during and after meiosis, and a final pollen viability of 22%. The development of female organs and gametes appeared normal. Pollination experiments indicated that the pollen produced by CO-6 plants was able to fertilize control ovaries, but the analysis of the progeny showed that the construct was not transmitted. Ovaries of CO-6 plants showed high fruit set and normal fruit development when pollinated with control pollen. However these fruits were completely seedless due to a stenospermocarpic behaviour that was evidenced by callose layering in the endothelium between 7 and 15 days after pollination. We conclude that GA20ox1 in tomato exerts specific developmental roles that are not redundantly shared with other members of this gene family. For reproductive male development, silencing of this gene is detrimental for pollen production and either gametophytically lethal or severely hampering seed germination. In the pistil, the co-suppression construct does not affect the progamic phase, nor fruit set and growth, but it interferes with seed development after fertilization leading to seed abortion.