Non-climacteric Species Research Articles

Estimating Transcriptome Diversity and Specialization in Capsicum annuum L.

Chili pepper fruits of the genus Capsicum represent excellent experimental models to study the growth, development, and ripening processes in a non-climacteric species at the physiological, biochemical, and molecular levels. Fruit growth, development, and ripening involve a complex, harmonious, and finely controlled regulation of gene expression. The purpose of this study was to estimate the changes in transcriptome diversity and specialization, as well as gene specificities during fruit development in this crop, and to illustrate the advantages of estimating these parameters. To achieve these aims, we programmed and made publicly available an R package. In this study, we applied these methods to a set of 179 RNA-Seq libraries from a factorial experiment that includes 12 different genotypes at various stages of fruit development. We found that the diversity of the transcriptome decreases linearly from the flower to the mature fruit, while its specialization follows a complex and non-linear behavior during this process. Additionally, by defining sets of genes with different degrees of specialization and applying Gene Ontology enrichment analysis, we identified processes, functions, and components that play a central role in particular fruit development stages. In conclusion, the estimation of diversity, specialization, and specificity summarizes the global properties of the transcriptomes, providing insights that are difficult to achieve by other means.

Non-climacteric fruit development and ripening regulation. The Phytohormones Show.

Fruit ripening involves numerous physiological, structural and metabolic changes that result in the formation of edible fruits. This process is controlled at different molecular levels, with essential roles for phytohormones, transcription factors and epigenetic modifications. Fleshy fruits are classified as either climacteric or non-climacteric species. Climacteric fruits are characterized by a burst in respiration and ethylene production at the onset of ripening, while regulation of non-climacteric fruit ripening has been commonly attributed to abscisic acid (ABA). However, there is controversy as to whether mechanisms regulating fruit ripening are shared between non-climacteric species, and to what extent other hormones contribute alongside ABA. In this review, we summarize classic and recent studies on the accumulation profile and role of ABA and other important hormones in the regulation of non-climacteric fruit development and ripening, as well as their cross-talk, paying special attention to the two main non-climacteric plant models, strawberry and grape. We highlight both the common and different roles of these regulators in these two crops, and discuss the importance of the transcriptional and environmental regulation of fruit ripening, as well as the need to optimize genetic transformation methodologies to facilitate gene functional analyses.

Insights into transcription factors controlling strawberry fruit development and ripening.

Fruit ripening is a highly regulated and complex process involving a series of physiological and biochemical changes aiming to maximize fruit organoleptic traits to attract herbivores, maximizing therefore seed dispersal. Furthermore, this process is of key importance for fruit quality and therefore consumer acceptance. In fleshy fruits, ripening involves an alteration in color, in the content of sugars, organic acids and secondary metabolites, such as volatile compounds, which influence flavor and aroma, and the remodeling of cell walls, resulting in the softening of the fruit. The mechanisms underlying these processes rely on the action of phytohormones, transcription factors and epigenetic modifications. Strawberry fruit is considered a model of non-climacteric species, as its ripening is mainly controlled by abscisic acid. Besides the role of phytohormones in the regulation of strawberry fruit ripening, a number of transcription factors have been identified as important regulators of these processes to date. In this review, we present a comprehensive overview of the current knowledge on the role of transcription factors in the regulation of strawberry fruit ripening, as well as in compiling candidate regulators that might play an important role but that have not been functionally studied to date.

ABA influences color initiation timing in P. avium L. fruits by sequentially modulating the transcript levels of ABA and anthocyanin-related genes

In sweet cherry, as in most non-climacteric species, abscisic acid (ABA) plays a major role in the control of fruit ripening and color development. Although the ABA treatment of sweet cherry fruits has been reported to upregulate anthocyanin pathway-related genes or ABA pathway-related genes, the temporality of molecular and physiological events occurring during color development and the ABA control of these events during the color initiation are lacking in this species. In this work, we analyzed variations in the Index of Absorbance Difference (IAD), a maturity index, and total anthocyanins along with changes in transcript abundance of ABA and anthocyanin pathway-related genes, from light green to red fruit stages. PavNCED1 and ABA signaling pathway-related genes upregulated when fruits transitioned from light green to pink stage, whereas anthocyanin pathway-related transcripts increased from pink to the red stage, together with increases in the anthocyanin content and IAD, suggesting sequentiality in molecular and physiological events during color development. Additionally, ABA applied at color initiation in planta advanced IAD, increased anthocyanin content, and yielded darker fruits at harvest. These changes were accompanied by changes in the transcript accumulation of ABA and anthocyanin pathway-related genes. This in planta treatment of sweet cherry fruits with ABA confirms that ABA is a central player in the control of color initiation in sweet cherries, associated with the transcript accumulation of genes involved in ABA homeostasis and signaling, which is followed by the up-regulation of anthocyanin pathway-related genes and color development.

Biomass composition explains fruit relative growth rate and discriminates climacteric from non-climacteric species.

Fleshy fruits are very varied, whether in terms of their composition, physiology, or rate and duration of growth. To understand the mechanisms that link metabolism to phenotypes, which would help the targeting of breeding strategies, we compared eight fleshy fruit species during development and ripening. Three herbaceous (eggplant, pepper, and cucumber), three tree (apple, peach, and clementine) and two vine (kiwifruit and grape) species were selected for their diversity. Fruit fresh weight and biomass composition, including the major soluble and insoluble components, were determined throughout fruit development and ripening. Best-fitting models of fruit weight were used to estimate relative growth rate (RGR), which was significantly correlated with several biomass components, especially protein content (R=84), stearate (R=0.72), palmitate (R=0.72), and lignocerate (R=0.68). The strong link between biomass composition and RGR was further evidenced by generalized linear models that predicted RGR with R-values exceeding 0.9. Comparison of the fruit also showed that climacteric fruit (apple, peach, kiwifruit) contained more non-cellulosic cell-wall glucose and fucose, and more starch, than non-climacteric fruit. The rate of starch net accumulation was also higher in climacteric fruit. These results suggest that the way biomass is constructed has a major influence on performance, especially growth rate.

Model-assisted comparison of sugar accumulation patterns in ten fleshy fruits highlights differences between herbaceous and woody species.

Sugar concentration is a key determinant of fruit quality. Soluble sugars and starch concentrations in fruits vary greatly from one species to another. The aim of this study was to investigate similarities and differences in sugar accumulation strategies across ten contrasting fruit species using a modelling approach. We developed a coarse-grained model of primary metabolism based on the description of the main metabolic and hydraulic processes (synthesis of compounds other than sugar and starch, synthesis and hydrolysis of starch, and water dilution) involved in the accumulation of soluble sugars during fruit development. Statistical analyses based on metabolic rates separated the species into six groups according to the rate of synthesis of compounds other than sugar and starch. Herbaceous species (cucumber, tomato, eggplant, pepper and strawberry) were characterized by a higher synthesis rate than woody species (apple, nectarine, clementine, grape and kiwifruit). Inspection of the dynamics of the processes involved in sugar accumulation revealed that net sugar importation, metabolism and dilution processes were remarkably synchronous in most herbaceous plants, whereas in kiwifruit, apple and nectarine, processes related to starch metabolism were temporally separated from other processes. Strawberry, clementine and grape showed a distinct dynamic compared with all other species. Overall, these results provide fresh insights into species-specific regulatory strategies and into the role of starch metabolism in the accumulation of soluble sugars in fleshy fruits. In particular, inter-specific differences in development period shape the co-ordination of metabolic processes and affect priorities for carbon allocation across species. The six metabolic groups identified by our analysis do not show a clear separation into climacteric and non-climacteric species, possibly suggesting that the metabolic processes related to sugar concentration are not greatly affected by ethylene-associated events.

Integrative Transcriptome and Proteome Analysis Identifies Major Metabolic Pathways Involved in Pepper Fruit Development

Pepper ( Capsicum annuum L.) fruit development is a complex and genetically programmed process. In this study, we conducted integrative analysis of transcriptome and proteome profiles during pepper fruit development. A total of 23 349 transcripts and 5455 protein groups were identified in four fruit developmental stages of two pepper varieties. The numbers of transcripts and proteins identified were decreased gradually across fruit development, and the most significant changes in transcript and protein levels happened from the mature green (MG) to breaker (Br) stages. Poor correlation between differentially expressed transcript and differentially expressed protein profiles was observed during pepper fruit development. We then analyzed expression profiles of transcripts and proteins involved in cell wall metabolism, and capsanthin, tocopherol, and ascorbate biosynthetic pathways during fruit development, and identified key regulatory proteins in these pathways. We presented a dynamic picture of pepper fruit development via comprehensive analysis of transcriptome and proteome profiles at different fruit developmental stages and in different varieties, revealing the temporal specificity of key protein expression. Our report provides insight into the transcription and translation dynamics of pepper fruit development and a reference for other nonclimacteric species.

The pineapple AcMADS1 promoter confers high level expression in tomato and Arabidopsis flowering and fruiting tissues, but AcMADS1 does not complement the tomato LeMADS-RIN (rin) mutant.

A previous EST study identified a MADS box transcription factor coding sequence, AcMADS1, that is strongly induced during non-climacteric pineapple fruit ripening. Phylogenetic analyses place the AcMADS1 protein in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of ethylene in climacteric tomato. LeMADS-RIN has been proposed to be a global ripening regulator shared among climacteric and non-climacteric species, although few functional homologs of LeMADS-RIN have been identified in non-climacteric species. AcMADS1 shares 67 % protein sequence similarity and a similar expression pattern in ripening fruits as LeMADS-RIN. However, in this study AcMADS1 was not able to complement the tomato rin mutant phenotype, indicating AcMADS1 may not be a functionally conserved homolog of LeMADS-RIN or has sufficiently diverged to be unable to act in the context of the tomato network of interacting proteins. The AcMADS1 promoter directed strong expression of the GUS reporter gene to fruits and developing floral organs in tomato and Arabidopsis thaliana, suggesting AcMADS1 may play a role in flower development as well as fruitlet ripening. The AcMADS1 promoter provides a useful molecular tool for directing transgene expression, particularly where up-regulation in developing flowers and fruits is desirable.

The N-glycan processing enzymes alpha-mannosidase and beta-D-N-acetylhexosaminidase are involved in ripening-associated softening in the non-climacteric fruits of capsicum.

Excessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening: however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and β-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and β-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and β-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and β-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and β-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and β-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and β-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses.

Influence of harvest time and after-ripening on the seed quality of eggplant

Eggplant cv. Emi and Tsakoniki were cultivated for seed in an unheated greenhouse and fruits were harvested at 25–65 days after anthesis (DAA) in order to determine the optimum harvest time. In addition, the effect of after-ripening on seed quality (i.e. seed size and germination) was examined by storing harvested fruit at 25 °C for 20 days prior to seed extraction. From the results, it was concluded that the optimum time of harvest for seed production is 55 DAA. Seeds extracted from fruits that were harvested at 25–35 DAA did not germinate, but when fruits harvested at the same age were stored for 20 days at 25 °C prior to seed extraction (i.e. seeds were after-ripened) germination was induced. Seeds extracted from fruits harvested at 45 DAA showed a high percent germination, which decreased after storage at 25 °C for 3 months. This decrease, however, was reduced by after-ripening prior to extraction. It is concluded that although eggplant is a non-climacteric species and fruit do not ripen after harvest, nevertheless seeds within the fruit continue to fill and mature after harvest; hence storage of prematurely harvested fruit prior to seed extraction permits the seeds of these fruits to after-ripen in situ and thereby increases seed size and germination. The implication of this result for eggplant seed production is discussed.