Shotgun proteomics of peach fruit reveals major metabolic pathways associated to ripening.

BACKGROUND: Fruit ripening in Prunus persica melting varieties involves several physiological changes that have a direct impact on the fruit organoleptic quality and storage potential. By studying the proteomic differences between the mesocarp of mature and ripe fruit, it would be possible to highlight critical molecular processes involved in the fruit ripening. RESULTS: To accomplish this goal, the proteome from mature and ripe fruit was assessed from the variety O'Henry through shotgun proteomics using 1D-gel (PAGE-SDS) as fractionation method followed by LC/MS-MS analysis. Data from the 131,435 spectra could be matched to 2740 proteins, using the peach genome reference v1. After data pre-treatment, 1663 proteins could be used for comparison with datasets assessed using transcriptomic approaches and for quantitative protein accumulation analysis. Close to 26% of the genes that code for the proteins assessed displayed higher expression at ripe fruit compared to other fruit developmental stages, based on published transcriptomic data. Differential accumulation analysis between mature and ripe fruit revealed that 15% of the proteins identified were modulated by the ripening process, with glycogen and isocitrate metabolism, and protein localization overrepresented in mature fruit, as well as cell wall modification in ripe fruit. Potential biomarkers for the ripening process, due to their differential accumulation and gene expression pattern, included a pectin methylesterase inhibitor, a gibbellerin 2-beta-dioxygenase, an omega-6 fatty acid desaturase, a homeobox-leucine zipper protein and an ACC oxidase. Transcription factors enriched in NAC and Myb protein domains would target preferentially the genes encoding proteins more abundant in mature and ripe fruit, respectively. CONCLUSIONS: Shotgun proteomics is an unbiased approach to get deeper into the proteome allowing to detect differences in protein abundance between samples. This technique provided a resolution so that individual gene products could be identified. Many proteins likely involved in cell wall and sugar metabolism, aroma and color, change their abundance during the transition from mature to ripe fruit.

Whole genome comparison between table and wine grapes reveals a comprehensive catalog of structural variants.

BACKGROUND: Grapevine (Vitis vinifera L.) is the most important Mediterranean fruit crop, used to produce both wine and spirits as well as table grape and raisins. Wine and table grape cultivars represent two divergent germplasm pools with different origins and domestication history, as well as differential characteristics for berry size, cluster architecture and berry chemical profile, among others. 'Sultanina' plays a pivotal role in modern table grape breeding providing the main source of seedlessness. This cultivar is also one of the most planted for fresh consumption and raisins production. Given its importance, we sequenced it and implemented a novel strategy for the de novo assembly of its highly heterozygous genome. RESULTS: Our approach produced a draft genome of 466 Mb, recovering 82% of the genes present in the grapevine reference genome; in addition, we identified 240 novel genes. A large number of structural variants and SNPs were identified. Among them, 45 (21 SNPs and 24 INDELs) were experimentally confirmed in 'Sultanina' and six SNPs in other 23 table grape varieties. Transposable elements corresponded to ca. 80% of the repetitive sequences involved in structural variants and more than 2,000 genes were affected in their structure by these variants. Some of these genes are likely involved in embryo development, suggesting that they may contribute to seedlessness, a key trait for table grapes. CONCLUSIONS: This work produced the first structural variants and SNPs catalog for grapevine, constituting a novel and very powerful tool for genomic studies in this key fruit crop, particularly useful to support marker assisted breeding in table grapes.

Small RNA Differential Expression Analysis Reveals miRNAs Involved in Dormancy Progression in Sweet Cherry Floral Buds.

In sweet cherry (Prunus avium), as in other temperate woody perennials, bud dormancy allows for survival in adverse environmental conditions during winter. During this process, environmental signals such as short days and/or low temperatures trigger internal signals that enable buds to become tolerant to the cold. The process involves tracking chilling units up to chilling the requirement fulfillment to resume growth, a transition involving transcriptional regulation, metabolic signaling, and epigenetic-related regulatory events. Massive sequencing of small RNAs was performed to identify miRNAs involved in sweet cherry dormancy by comparing their expression in field (regular seasonal) and controlled non-stop (continuous) chilling conditions. miRNAs highlighted by sequencing were validated using specific stem-loop PCR quantification, confirming expression patterns for known miRNAs such as miR156e, miR166c, miR172d, miR391, miR482c, and miR535b, as well as for newly proposed miRNAs. In silico prediction of the target genes was used to construct miRNA/target gene nodes. In particular, the involvement of the sweet cherry version for the miR156/SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN genes whose expression was opposite in the two conditions suggests their involvement on dormancy regulation in sweet cherry. miRNA levels indicate that the regulation of stress-related genes and hormone synthesis modulates the expression of calcium metabolism and cell development-associated genes. Understanding the regulatory networks involved in sweet cherry dormancy, particularly in the context of miRNA involvement, represents the first step in the development of new agricultural strategies that may help overcome the increasing challenges presented by global climate change.

Dormant but Active: Chilling Accumulation Modulates the Epigenome and Transcriptome of Prunus avium During Bud Dormancy.

Temperate deciduous fruit tree species like sweet cherry (Prunus avium) require long periods of low temperatures to trigger dormancy release and flowering. In addition to sequence-based genetic diversity, epigenetic variation may contribute to different chilling requirements among varieties. For the low chill variety 'Royal Dawn' and high chill variety 'Kordia', we studied the methylome of floral buds during chilling accumulation using MethylC-seq to identify differentially methylated regions (DMRs) during chilling hours (CH) accumulation, followed by transcriptome analysis to correlate changes in gene expression with DNA methylation. We found that during chilling accumulation, DNA methylation increased from 173 CH in 'Royal Dawn' and 443 CH in 'Kordia' and was mostly associated with the CHH context. In addition, transcriptional changes were observed from 443 CH in 'Kordia' with 1,210 differentially expressed genes, increasing to 4,292 genes at 1,295 CH. While 'Royal Dawn' showed approximately 5,000 genes differentially expressed at 348 CH and 516 CH, showing a reprogramming that was specific for each genotype. From conserved upregulated genes that overlapped with hypomethylated regions and downregulated genes that overlapped with hypermethylated regions in both varieties, we identified genes related to cold-sensing, cold-signaling, oxidation-reduction process, metabolism of phenylpropanoids and lipids, and a MADS-box SVP-like gene. As a complementary analysis, we used conserved and non-conserved DEGs that presented a negative correlation between DNA methylations and mRNA levels across all chilling conditions, obtaining Gene Ontology (GO) categories related to abiotic stress, metabolism, and oxidative stress. Altogether, this data indicates that changes in DNA methylation precedes transcript changes and may occur as an early response to low temperatures to increase the cold tolerance in the endodormancy period, contributing with the first methylome information about the effect of environmental cues over two different genotypes of sweet cherry.

Metabolite Fruit Profile Is Altered in Response to Source-Sink Imbalance and Can Be Used as an Early Predictor of Fruit Quality in Nectarine.

Peaches and nectarines [Prunus persica (L.) Batsch] are among the most exported fresh fruit from Chile to the Northern Hemisphere. Fruit acceptance by final consumers is defined by quality parameters such as the size, weight, taste, aroma, color, and juiciness of the fruit. In peaches and nectarines, the balance between soluble sugars present in the mesocarp and the predominant organic acids determines the taste. Biomass production and metabolite accumulation by fruits occur during the different developmental stages and depend on photosynthesis and carbon export by source leaves. Carbon supply to fruit can be potentiated through the field practice of thinning (removal of flowers and young fruit), leading to a change in the source-sink balance favoring fruit development. Thinning leads to fruit with increased size, but it is not known how this practice could influence fruit quality in terms of individual metabolite composition. In this work, we analyzed soluble metabolite profiles of nectarine fruit cv "Magique" at different developmental stages and from trees subjected to different thinning treatments. Mesocarp metabolites were analyzed throughout fruit development until harvest during two consecutive harvest seasons. Major polar compounds such as soluble sugars, amino acids, organic acids, and some secondary metabolites were measured by quantitative 1H-NMR profiling in the first season and GC-MS profiling in the second season. In addition, harvest and ripening quality parameters such as fruit weight, firmness, and acidity were determined. Our results indicated that thinning (i.e., source-sink imbalance) mainly affects fruit metabolic composition at early developmental stages. Metabolomic data revealed that sugar, organic acid, and phenylpropanoid pathway intermediates at early stages of development can be used to segregate fruits impacted by the change in source-sink balance. In conclusion, we suggest that the metabolite profile at early stages of development could be a metabolic predictor of final fruit quality in nectarines.

DNA methylation and small interference RNAs participate in the regulation of MADS-box genes involved in dormancy in sweet cherry (Prunus avium L.).

Epigenetic modifications can yield information about connections between genotype, phenotype variation and environmental conditions. Bud dormancy release in temperate perennial fruit trees depends on internal and environmental signals such as cold accumulation and photoperiod. Previous investigations have noted the participation of epigenetic mechanisms in the control of this physiological process. We examined whether epigenetic modifications were modulated in MADS-box genes, potential candidates for the regulation of bud dormancy and flowering in sweet cherry (Prunus avium L.). We identified and cloned two MADS-box genes homologous to the already-characterized dormancy regulators DORMANCY-ASSOCIATED MADS-box (DAM3 and DAM5) from Prunus persica (L.) Batsch. Bisulfite sequencing of the identified genes (PavMADS1 and PavMADS2), Methylated DNA Immunoprecipitation and small RNA deep sequencing were performed to analyze the presence of DNA methylations that could be guided by non-coding RNAs in the floral buds exposed to differential chilling hours. The results obtained reveal an increase in the level of DNA methylation and abundance of matching small interference RNAs (siRNAs) in the promoter of PavMADS1 when the chilling requirement is complete. For the first intron and 5' UTR of PavMADS1, de novo DNA methylation could be associated with the increase in the abundance of 24-nt siRNA matching the promoter area. Also, in the second large intron of PavMADS1, maintenance DNA methylation in all cytosine contexts is associated with the presence of homologous siRNAs in that zone. For PavMADS2, only maintenance methylation was present in the CG context, and no matching siRNAs were detected. Silencing of PavMADS1 and PavMADS2 coincided with an increase in Flowering Locus T expression during dormancy. In conclusion, DNA methylations and siRNAs appear to be involved in the silencing of PavMADS1 during cold accumulation and dormancy release in sweet cherry.

Ecophysiological responses to excess iron in lowland and upland rice cultivars.

Iron (Fe) is an essential nutrient for plants but under high concentrations, such as that found naturally in clay and waterlogged soils, its toxic effect can limit production. This study aimed to investigate the stress tolerance responses exhibited by different rice cultivars. Both lowland and upland cultivars were grown under excess Fe and hypoxic conditions. Lowland cultivars showed higher Fe accumulation in roots compared with upland cultivars suggesting the use of different strategies to tolerate excess Fe. The upland Canastra cultivar displayed a mechanism to limit iron translocation from roots to the shoots, minimizing leaf oxidative stress induced by excess Fe. Conversely, the cultivar Curinga invested in the increase of R1/A, as an alternative drain of electrons. However, the higher iron accumulation in the leaves, was not necessarily related to high toxicity. Nutrient uptake and/or utilization mechanisms in rice plants are in accordance with their needs, which may be defined in relation to crop environments. Alterations in the biochemical parameters of photosynthesis suggest that photosynthesis in rice under excess Fe is primarily limited by biochemical processes rather than by diffusional limitations, particularly in the upland cultivars. The electron transport rate, carboxylation efficiency and electron excess dissipation by photorespiration demonstrate to be good indicators of iron tolerance. Altogether, these chemical and molecular patterns suggests that rice plants grown under excess Fe exhibit gene expression reprogramming in response to the Fe excess per se and in response to changes in photosynthesis and nutrient levels to maintain growth under stress.

The sweet cherry (Prunus avium) FLOWERING LOCUS T gene is expressed during floral bud determination and can promote flowering in a winter-annual Arabidopsis accession.

KEY MESSAGE: FT gene is expressed in leaves and buds and is involved in floral meristem determination and bud development in sweet cherry. In woody fruit perennial trees, floral determination, dormancy and bloom, depends on perception of different environmental and endogenous cues which converge to a systemic signaling gene known as FLOWERING LOCUS T (FT). In long-day flowering plants, FT is expressed in the leaves on long days. The protein travels through the phloem to the shoot apical meristem, where it induces flower determination. In perennial plants, meristem determination and flowering are separated by a dormancy period. Meristem determination takes place in summer, but flowering occurs only after a dormancy period and cold accumulation during winter. The roles of FT are not completely clear in meristem determination, dormancy release, and flowering in perennial plants. We cloned FT from sweet cherry (Prunus avium) and analyzed its expression pattern in leaves and floral buds during spring and summer. Phylogenetic analysis shows high identity of the FT cloned sequence with orthologous genes from other Rosaceae species. Our results show that FT is expressed in both leaves and floral buds and increases when the daylight reached 12 h. The peak in FT expression was coincident with floral meristem identity genes expression and morphological changes typical of floral meristem determination. The Edi-0 Arabidopsis ecotype, which requires vernalization to flower, was transformed with a construct for overexpression of PavFT. These transgenic plants showed an early-flowering phenotype without cold treatment. Our results suggest that FT is involved in floral meristem determination and bud development in sweet cherry. Moreover, we show that FT is expressed in both leaves and floral buds in this species, in contrast to annual plants.

Proteomic analysis of a segregant population reveals candidate proteins linked to mealiness in peach.

Peaches are stored at low temperatures to delay ripening and increase postharvest life. However some varieties are susceptible to chilling injury,which leads to fruit mealiness, browning and flesh bleeding. In order to identify potentialmarkers associated with chilling injury,we performed proteomic analyses on a segregating population with contrasting susceptibility to chilling-induced mealiness. Chilling-induced mealiness was assessed by measuring juiciness in fruits that have been stored in cold and then allowed to ripen. Fruitmesocarp and leaf proteome from contrasting segregants were analyzed using 2-DE gels. Comparison of protein abundance between segregants revealed 133 spots from fruit mesocarp and 36 from leaf. Thirty four fruit mesocarp proteins were identified from these spots. Most of these proteins were related to ethylene synthesis, ABA response and stress response. Leaf protein analyses identified 22 proteins, most of which related to energy metabolism. Some of the genes that code for these proteins have been previously correlated with chilling injury through transcript analyses and co-segregation with mealiness QTLs. The results from this study, further deciphers the molecular mechanisms associated with chilling response in peach fruit, and identifies candidate proteins linked to mealiness in peach which may be used as putative markers for this trait.

Iron excess affects rice photosynthesis through stomatal and non-stomatal limitations.

Iron toxicity is the most important stressor of rice in many lowland environments worldwide. Rice cultivars differ widely in their ability to tolerate excess iron. A physiological evaluation of iron toxicity in rice was performed using non-invasive photosynthesis, photorespiration and chlorophyll a fluorescence imaging measurements and chlorophyll content determination by SPAD. Four rice cultivars (BR IRGA 409; BR IRGA 412; BRA 041171 and BRA 041152) from the Brazilian breeding programs were used. Fe(2+) was supplied in the nutrient solution as Fe-EDTA (0.019, 4, 7 and 9 mM). Increases in shoot iron content due to Fe(2+) treatments led to changes in most of the non-invasive physiological variables assessed. The reduction in rice photosynthesis can be attributed to stomatal limitations at moderate Fe(2+) doses (4mM) and both stomatal and non-stomatal limitations at higher doses. Photorespiration was an important sink for electrons in rice cultivars under iron excess. A decreased chlorophyll content and limited photochemical ability to cope with light excess were characteristic of the more sensitive and iron accumulator cultivars (BRA 041171 and BRA 041152). Chlorophyll fluorescence imaging revealed a spatial heterogeneity of photosynthesis under excessive iron concentrations. The results showed the usefulness of non-invasive physiological measurements to assess differences among cultivars. The contributions toward understanding the rice photosynthetic response to toxic levels of iron in the nutrient solution are also discussed.

Energy conservation and dissipation in mitochondria isolated from developing tomato fruit of ethylene-defective mutants failing normal ripening: the effect of ethephon, a chemical precursor of ethylene.

Alternative oxidase (AOX) and uncoupling protein (UCP) are present simultaneously in tomato fruit mitochondria. In a previous work, it has been shown that protein expression and activity of these two energy-dissipating systems exhibit large variations during tomato fruit development and ripening on the vine. It has been suggested that AOX and UCP could be responsible for the respiration increase at the end of ripening and that the cytochrome pathway could be implicated in the climacteric respiratory burst before the onset of ripening. In this study, the use of tomato mutants that fail normal ripening because of deficiencies in ethylene perception or production as well as the treatment of one selected mutant with a chemical precursor of ethylene have revealed that the bioenergetics of tomato fruit development and ripening is under the control of this plant hormone. Indeed, the evolution pattern of bioenergetic features changes with the type of mutation and with the introduction of ethylene into an ethylene-synthesis-deficient tomato fruit mutant during its induced ripening.

The energy-conserving and energy-dissipating processes in mitochondria isolated from wild type and nonripening tomato fruits during development on the plant.

Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, non-climacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-microM linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.

Caracterizaçäo funcional da proteína desacopladora mitocondrial de plantas (UCP) e sua interaçäo com oxidase alternativa (AOX) / Physiological characterization of the plant uncoupling mitochondrial protein (UCP) and its interaction with the alternative oxidase (AOX)

Mitocôndrias de plantas possuem dois sistemas dissipadores de energia que diminuem a eficiência da fosforilaçao oxidativa: a oxidase alternativa (AOX) insensível ao cianeto e a proteína desacopladora (UCP). No presente trabalho estabelecemos as condiçoes que permitiram determinar as contribuiçoes da via dos citocromos, da AOX e da UCP na respiraçao fosforilativa (estado 3) medindo-se a razao ADP/O (estequiometria entre o número de moléculas de ADP fosforiladas por átomo de oxigênio consumido). Demonstramos que a atividade da UCP é capaz de dissipar eficientemente a energia de óxido-reduçao destinada a fosforilaçao do ADP de uma maneira puramente protonofórica e dependente da presença de ácidos graxos livres. Observamos que quando uma diminuiçao de até 60 por cento na velocidade de respiraçao é causada por inibiçao do transporte do substrato succinato para o interior da mitocôndria, a contribuiçao da UCP permanece constante, em contraste com a AOX que cai drasticamente a zero. Estes resultados mostram que estes dois sistemas dissipadores de energia presentes em mitocôndrias de plantas, possuem uma cinética distinta e provavelmente papéis fisiológicos diferentes in vivo. Observamos também que estas proteínas têm suas atividades co-reguladas por ácidos graxos livres. Enquanto ativam a UCP, os ácidos graxos livres, na mesma faixa de concentraçao, também inibem a atividade da AOX de uma maneira dose dependente. Estes resultados indicam que estes dois sistemas nunca trabalham simultaneamente na sua capacidade máxima. As atividades e a expressao da AOX, UCP e da via dos citocromos também foram acompanhadas durante o amadurecimento de tomates na própria planta e pós-colheita. Durante o amadurecimento pós-colheita as atividades da AOX, UCP e da via dos citocromos gradativamente. Quando o tomate amadurece na própria planta, observa-se um pico na respiraçao mitocondrial total que coincide com o burst climatérico da respiraçao no fruto inteiro. Demonstramos que o aumento na velocidade de respiraçao em mitocôndrias isoladas de tomates no período do burst climatério, é regulada por um aumento na atividade da via dos citocromos. Além disso, como a quantidade de ácidos graxos livres presentes no pericarpo apresenta um pico neste mesmo período, a UCP deve contribuir na respiraçao climatérica de tomates in vivo. Por outro lado, o aumento da expressao e da atividade da AOX observados no final do processo de amadurecimento...(au)