Global analysis of gene expression during development and ripening of citrus fruit flesh. A proposed mechanism for citric acid utilization (original) (raw)
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Scientific Reports
Citrus fruit ripening is a complex process involving biochemical, physiological and molecular events that differ between the flesh and the peel of the fruit. We characterized sweet orange peel maturation by means of a comparative transcriptomic analysis between Navelate orange (Citrus sinensis L. osbeck) and its mutant fruit pinalate, which presents a severe blockage at early steps of the carotenoid biosynthetic pathway and consequently reduced ABA levels. Peel ripening involved the decrease of the photosynthetic activity and the transmembrane transport processes, as well as the buildup of starch and cuticular waxes and the cell wall modification. In addition, a number of biotic and abiotic stress responses, including the defense response, and the response to blue light, water deprivation and abscisic acid stimulus were modulated in a ripening-stage specific manner. The regulation of energyrelated processes and secondary metabolism pathways was attenuated in pinalate, while the molecular mechanisms underlying stress responses displayed dependency on ABA levels. These results indicate that ABA is a key signal inducing stress responses along orange peel ripening, which might determine the fruit postharvest performance. Fruits from the genus Citrus are of main relevance worldwide because they are grown in more than 130 countries, covering more than 9.5 million ha harvested area and with an estimated production over 125 million Tm per year (FAOSTAT, 2016). Biochemical and physiological events involved in citrus fruit development and ripening have been extensively studied for decades. Citrus fruit growth and development follows three stages: first, a cell division phase; then, a cell expansion stage of major increase in size and weight; and the third phase during which fruit growth bottoms down and most of the characteristic events of fruit maturation or ripening takes place 1-3. Among these changes, the main affected processes include the increase in sugars and other soluble solids, and the reduction of organic acids 2. Peel pigmentation develops as a result of chlorophyll degradation and activation of carotenoid biosynthesis, while the increase of specific volatile compounds, and an exhaustive modification of the cell wall metabolism occurs 4,5. However, defining the criteria to uniformly determine citrus fruit maturation may not be simple because physiological changes and metabolic processes occurring in peel and pulp during maturation are autonomous and independent, mainly due to the lack of vascular connections between both tissues 6. In spite of flavedo (outer colored cell layers of the fruit peel) is the first tissue of citrus fruit interacting with the environment and the first protection barrier of the fruit against biotic and abiotic stresses, most of the studies have been focused on the molecular processes underlying flesh maturation, and a limited number of reports investigated the molecular changes occurring along peel ripening, being most of them centered on changes in pigmentation 7-12. Citrus are defined as non-climacteric fruit, since respiration rate declines progressively during fruit development and full ripen fruit produce a very low and constant amount of ethylene. In contrast to the well-known role of ethylene controlling most of the maturation processes in climacteric fruits 13 , reports pointing to abscisic acid
Frontiers in plant science, 2016
Organic acids, such as citrate and malate, are important contributors for the sensory traits of fleshy fruits. Although their biosynthesis has been illustrated, regulatory mechanisms of acid accumulation remain to be dissected. To provide transcriptional architecture and identify candidate genes for citrate accumulation in fruits, we have selected for transcriptome analysis four varieties of sweet orange (Citrus sinensis L. Osbeck) with varying fruit acidity, Succari (acidless), Bingtang (low acid), and Newhall and Xinhui (normal acid). Fruits of these varieties at 45 days post anthesis (DPA), which corresponds to Stage I (cell division), had similar acidity, but they displayed differential acid accumulation at 142 DPA (Stage II, cell expansion). Transcriptomes of fruits at 45 and 142 DPA were profiled using RNA sequencing and analyzed with three different algorithms (Pearson correlation, gene coexpression network and surrogate variable analysis). Our network analysis shows that the...
Transcriptome and metabolome analysis of Citrus fruit to elucidate puffing disorder
Plant Science, 2014
A systems-level analysis reveals details of molecular mechanisms underlying puffing disorder in Citrus fruit. Flavedo, albedo and juice sac tissues of normal fruits and fruits displaying symptoms of puffing disorder were studied using metabolomics at three developmental stages. Microarrays were used to compare normal and puffed fruits for each of the three tissues. A protein-protein interaction network inferred from previous work on Arabidopsis identified hub proteins whose transcripts show significant changes in expression. Glycolysis, the backbone of primary metabolism, appeared to be severely affected by the disorder, based on both transcriptomic and metabolomic results. Significantly less citric acid was observed consistently in puffed fruits. Gene set enrichment analysis suggested that glycolysis and carbohydrate metabolism were significantly altered in puffed samples in both albedo and flavedo. Expression of invertases and genes for sucrose export, amylose-starch and starch-maltose conversion was higher in puffed fruits. These changes may significantly alter source-sink communications. Genes associated with gibberellin and cytokinin signaling were downregulated in symptomatic albedo tissues, suggesting that these hormones play key roles in the disorder. Findings may be applied toward the development of early diagnostic methods based on host response genes and metabolites (i.e. citric acid), and toward therapeutics based on hormones.
'Fengjiewancheng' (FJWC) is a late-ripening bud sport of 'Fengjie72-1' navel orange (FJ72-1). To investigate transcript differences during fruit ripening between the two cultivars and to get clues as to the mechanisms monitoring the late-ripening traits, we compared their transcript profiles during fruit ripening using cDNA-AFLP. As a result, 144 transcript-derived fragments (TDFs) were found differently expressed in FJWC compared with FJ72-1 of which 129 TDFs were recovered and assembled into 51 uniTDFs. Blast annotation indicated that 39 uniTDFs were assigned with putative function in signal transduction, control of gene expression and carbohydrate biosynthesis. Some TDFs shared relatively high identities with known functional genes and showed differential expression patterns in fruit pulp or peel between FJWC and FJ72-1 during fruit ripening, implying a potential role in the formation of the late-ripening phenotype.
Citric acid plays an important role in fresh fruit flavor and its adaptability to post‐harvest storage conditions. In order to explore organic acid regulatory mechanisms in post‐harvest citrus fruit, systematic biological analyses were conducted on stored Hirado Buntan Pummelo (HBP; Citrus grandis) fruits. Highperformance capillary electrophoresis, subcellular organelle expression microarray, real‐time quantitative reverse transcription polymerase chain reaction, gas chromatography mass spectrometry (GC‐MS), and conventional physiological and biochemical analyses were undertaken. The results showed that the concentration of organic acids in HBP underwent a regular fluctuation. GC‐MS‐based metabolic profiling indicated that succinic acid, g‐aminobutyric acid (GABA), and glutamine contents increased, but 2‐ oxoglutaric acid content declined, which further confirmed that the GABA shunt may have some regulatory roles in organic acid catabolismprocesses. In addition, the concentration of organic acids was significantly correlated with senescence‐related physiological processes, such as hydrogen peroxide content as well as superoxide dismutase and peroxidase activities, which showed that organic acids could be regarded as important parameters for measuring citrus fruit post‐harvest senescence processes.
Journal of Experimental Botany, 2009
Bud mutations often arise in citrus. The selection of mutants is one of the most important breeding channels in citrus. However, the molecular basis of bud mutation has rarely been studied. To identify differentially expressed genes in a spontaneous sweet orange [C. sinensis (L.) Osbeck] bud mutation which causes lycopene accumulation, low citric acid, and high sucrose in fruit, suppression subtractive hybridization and microarray analysis were performed to decipher this bud mutation during fruit development. After sequencing of the differentially expressed clones, a total of 267 non-redundant transcripts were obtained and 182 (68.2%) of them shared homology (E-value <1310 210 ) with known gene products. Few genes were constitutively up-or down-regulated (fold change >2) in the bud mutation during fruit development. Self-organizing tree algorithm analysis results showed that 95.1% of the differentially expressed genes were extensively coordinated with the initiation of lycopene accumulation. Metabolic process, cellular process, establishment of localization, response to stimulus, and biological regulation-related transcripts were among the most regulated genes. These genes were involved in many biological processes such as organic acid metabolism, lipid metabolism, transport, and pyruvate metabolism, etc. Moreover, 13 genes which were differentially regulated at 170 d after flowering shared homology with previously described signal transduction or transcription factors. The information generated in this study provides new clues to aid in the understanding of bud mutation in citrus.
Proteome analysis of Citrus sinensis L. (Osbeck) flesh at ripening time
Journal of Proteomics, 2009
A combination of 2-DE and LC-MSMS approaches was used to identify the differentially expressed proteome of a pigmented sweet orange (Citrus sinensis, cv. Moro) in comparison with a common cultivar (Cadenera) at ripening time. The comparison of the protein patterns of Moro and Cadenera showed 64 differential expressed protein spots. Fifty-five differentially expressed proteins were identified. Proteins were classified according to their putative function and known biosynthetic pathways. Most of the proteins related to sugar metabolism were overexpressed in Moro, while those related to stress responses were overexpressed in Cadenera. The abundance of proteins belonging to Unknown/Unnamed and Hypothetical classes could be associated to the incomplete data available on the Citrus genome. The relative abundance of Secondary metabolism and Oxidative process proteins substantiated the key role of the anthocyanin pathway in Moro, which is characterized by a strong pigmentation at ripening time. The potential role of protein differential expression between Moro and Cadenera fruits was discussed, and proteomic results were compared with the known variations of transcripts of the same fruits. The latter analyses highlighted many discrepancies, confirming the necessity to associate both proteomic and transcriptomic approaches in order to achieve a more complete characterization of the biological system.
II International Symposium on Citrus Biotechnology, 2011
The survival of the citrus industry is critically dependent on genetically superior cultivars. Improvements in these traits through traditional techniques are unfortunately extremely difficult due to the unusual combination of biological characteristics of citrus. Genomic science holds promise of improvements in breeding and the main goal of our group is to develop genomics tools for the generation of new genotypes. We pursue the identification of candidate genes, alleles and genotypes improving citrus fruit quality and performance, correlating phenotypic analyses, metabolic profiling and gene expression. At completion, genes and alleles with major functions in nutritional quality and stress tolerance could be selected and genotypes with improved fruit composition searched among existing or generated collections. This goal is supported by two complementary strategies the identification of genes of agricultural interest by the use of mutants and microarrays and the elucidation of the citrus genome sequence. In this communication the efforts we are developing in this first approach are revised while the other strategy will be resumed elsewhere. GOALS OF CITRUS BREEDING Major goals of variety breeding in citrus are mostly related to fruit quality, productivity and harvesting period. In a broad sense, citrus fruit quality includes many physical attributes like fruit size, shape, colour, texture, seedlessness, peelability and durability. The chemical characteristics of the fruit, such as sugar and acid content, flavour and aroma compounds (organoleptic properties) are also important. Citrus contain the largest number of carotenoids in addition to an extensive array of secondary compounds with pivotal nutritional properties that greatly contribute to the supply of anticancer agents and other nutraceutical substances essential to prevent cardiovascular and degenerative diseases, thrombosis, cancer, atherosclerosis and obesity. These traits are acquired along fruit growth, from pollination to ripening although they can be strongly modified during post-harvest storage. Extension of the ripening period is also a key target for the market while abscission behaviour plays a major role in fruit production. Citrus Industry basically all over the world is claiming for much more knowledge on the characteristics related to fruit quality. In addition, development of more modern know-how and protocols to generate both phenotypic variability to generate new citrus lines and original procedures for variety authentication are also mayor requirements of the sector. BREEDING PROBLEMS IN CITRUS The reasons for the low impact of traditional breeding of this major fruit crop are related to the peculiarities of citrus reproductive biology (see Talon and Gmitter, 2008). Citrus show apomixy, heterozygosis, facultative parthenocarpy, sterility, and gametophytic self-and cross-incompatibility. In addition to this unusual reproductive biology, Citrus also possess a rare combination of intriguing biological characteristics including non-climacteric fruit ripening, juvenility, dormancy, surprising root/shoot
Journal of Agricultural and Food Chemistry, 2009
Peel color is one of the main features affecting citrus quality. Clementine is a widespread citrus species with several mutants showing a delay in pigmentation and harvesting. This work characterizes the fruit development and ripening of two clementine clones, 'Comune', a widespread variety, and one of its natural mutations, 'Tardivo', which differ by a delayed color-break and extended harvest period. Morphological, chemical, and molecular analyses were carried out on fruits of both genotypes during the whole maturation process. Analysis showed that mutation did not affect ripening characteristics such as juice acidity and TSS. However, biochemical and molecular analysis revealed marked differences in the flavedo regarding carotenogenesis and chlorophyllase gene expression. Carotenoid showed quantitative differences at biochemical and molecular levels. Results demonstrated that the mutation in 'Tardivo' influenced the transcriptional activation of PSY, a key step in carotenoid biosynthesis. The differential PSY expression led to a significant quantitative difference in phytoene accumulation between the two genotypes. Also, 'Tardivo' showed delayed accumulation of carotenes, lutein, and β,β-xanthophylls. The differential expression of genes involved in ethylene biosynthesis and perception suggested differing responses to ethylene signaling between the two genotypes. Moreover, exogenous application of ethylene revealed a different sensitivity of the two varieties to this hormone. The analysis added new information to better understand the complex process of ripening in citrus.
Frontiers in Plant Science
Peel degreening is the most conspicuous aspect of fruit ripening in many citrus fruits because of its importance for marketability. In this study, peel degreening in response to propylene (an ethylene analog) and at varying storage temperatures was characterized in Satsuma mandarin (Citrus unshiu Marc.) fruit. Propylene treatment triggered rapid peel degreening (within 4–6 days), indicated by an increase in the citrus color index (CCI) and chlorophyll loss. Peel degreening was also observed in fruit at 10°C and 15°C after 28–42 days, with gradual CCI increase and chlorophyll reduction. However, fruit at 5°C, 20°C, and 25°C remained green, and no substantial changes in peel CCI and chlorophyll content were recorded during the 42-day storage duration. The transcriptomes of peels of fruit treated with propylene for 4 days and those stored at varying temperatures for 28 days were then analyzed by RNA-Seq. We identified three categories of differentially expressed genes that were regulat...