Non-climacteric Fruit Ripening Research Articles

The draft genome of MD-2 pineapple using hybrid error correction of long reads.

The introduction of the elite pineapple variety, MD-2, has caused a significant market shift in the pineapple industry. Better productivity, overall increased in fruit quality and taste, resilience to chilled storage and resistance to internal browning are among the key advantages of the MD-2 as compared with its previous predecessor, the Smooth Cayenne. Here, we present the genome sequence of the MD-2 pineapple (Ananas comosus (L.) Merr.) by using the hybrid sequencing technology from two highly reputable platforms, i.e. the PacBio long sequencing reads and the accurate Illumina short reads. Our draft genome achieved 99.6% genome coverage with 27,017 predicted protein-coding genes while 45.21% of the genome was identified as repetitive elements. Furthermore, differential expression of ripening RNASeq library of pineapple fruits revealed ethylene-related transcripts, believed to be involved in regulating the process of non-climacteric pineapple fruit ripening. The MD-2 pineapple draft genome serves as an example of how a complex heterozygous genome is amenable to whole genome sequencing by using a hybrid technology that is both economical and accurate. The genome will make genomic applications more feasible as a medium to understand complex biological processes specific to pineapple.

Structural and Functional Analysis of the GRAS Gene Family in Grapevine Indicates a Role of GRAS Proteins in the Control of Development and Stress Responses.

GRAS transcription factors are involved in many processes of plant growth and development (e.g., axillary shoot meristem formation, root radial patterning, nodule morphogenesis, arbuscular development) as well as in plant disease resistance and abiotic stress responses. However, little information is available concerning this gene family in grapevine (Vitis vinifera L.), an economically important woody crop. We performed a model curation of GRAS genes identified in the latest genome annotation leading to the identification of 52 genes. Gene models were improved and three new genes were identified that could be grapevine- or woody-plant specific. Phylogenetic analysis showed that GRAS genes could be classified into 13 groups that mapped on the 19 V. vinifera chromosomes. Five new subfamilies, previously not characterized in other species, were identified. Multiple sequence alignment showed typical GRAS domain in the proteins and new motifs were also described. As observed in other species, both segmental and tandem duplications contributed significantly to the expansion and evolution of the GRAS gene family in grapevine. Expression patterns across a variety of tissues and upon abiotic and biotic conditions revealed possible divergent functions of GRAS genes in grapevine development and stress responses. By comparing the information available for tomato and grapevine GRAS genes, we identified candidate genes that might constitute conserved transcriptional regulators of both climacteric and non-climacteric fruit ripening. Altogether this study provides valuable information and robust candidate genes for future functional analysis aiming at improving the quality of fleshy fruits.

Complex Interplay of Hormonal Signals during Grape Berry Ripening.

Grape and wine production and quality is extremely dependent on the fruit ripening process. Sensory and nutritional characteristics are important aspects for consumers and their development during fruit ripening involves complex hormonal control. In this review, we explored data already published on grape ripening and compared it with the hormonal regulation of ripening of other climacteric and non-climacteric fruits. The roles of abscisic acid, ethylene, and brassinosteroids as promoters of ripening are discussed, as well as the role of auxins, cytokinins, gibberellins, jasmonates, and polyamines as inhibitors of ripening. In particular, the recently described role of polyamine catabolism in grape ripening is discussed, together with its putative interaction with other hormones. Furthermore, other recent examples of cross-talk among the different hormones are presented, revealing a complex interplay of signals during grape development and ripening.

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.

Preharvest application of abscisic acid promotes anthocyanins accumulation in pericarp of litchi fruit without adversely affecting postharvest quality

Pericarp colour of litchi fruit is an important quality attribute that determines its market value and consumer acceptance. Plant growth regulators (PGR) such as abscisic acid (ABA) and ethephon are known to play important roles in peel colour development during maturation and ripening of non-climacteric fruits (e.g. grape and litchi). Our aim was to investigate the effects of preharvest application of ABA, ethephon and their combination on pericarp colour and fruit quality of litchi (cv. Calcuttia) and also to assess the potential effects on postharvest performance of fruit. Exogenous application of ABA (150 or 300mgL−1) at the colour-break stage significantly increased the concentration of total anthocyanins and cyanidin-3-O-rutinoside, the major anthocyanin contributing ∼71–96% of the total anthocyanins, in litchi pericarp compared to ethephon (500μLL−1). Among different anthocyanins quantified, the relative contribution of cyanidin-3,5-diglucoside to the total anthocyanins was significantly higher in all PGR-treated fruit compared to the control, but the concentration of cyanidin-3-O-glucoside was specifically enhanced by ABA. No significant effect on the concentrations of epicatechin, and quercetin-3-O-rutinoside was observed in response to PGR treatments. Ethephon (500μLL−1) treatment did not significantly increase the anthocyanin levels in pericarp, but it caused more degradation of chlorophyll pigments than control. Aril quality with regard to firmness, soluble solids and acidity was not significantly affected by PGR treatments, except that ethephon-treated fruit showed significant softening and lower acidity. Postharvest changes in fruit quality attributes including pericarp browning during cold storage at 5°C for 14 d were mainly related to the storage duration effect, rather than PGR treatment. In conclusion, ABA treatment (150 or 300mgL−1) at the colour-break stage enhanced anthocyanins accumulation in litchi pericarp without adversely affecting postharvest quality and storage stability for 14 d.

Hormonal regulation during plant fruit development

The modern concept of the hormonal regulation of fruit set, growth, maturation, and ripening is considered. Pollination and fertilization induce ovule activation by surmounting the blocking action of ethylene and ABA to be manifested in auxin accumulation. Active fruit growth by pericarp cell division and elongation is due to the syntheses ofauxin in the developing seed and ofgibberellins in the pericarp. In climacteric fleshy fruits, the maturation is controlled by ethylene via so-called System 1 combining the possibilities of autoinhibition and autocatalysis by ethylene of its own biosynthesis. Transition of tomato fruits from maturation to ripening is characterized by highly active synthesis of ethylene and its receptors due to the functioning of regulatory System 2 resulting in the up-regulation of much greater number of ethylene-inducible genes. In peach fruits, the hormonal regulation of ripening includes also an active auxin involvement in the ethylene biosynthesis, which is combined with the ethylene-induced expression of genes encoding both auxin biosynthesis and the response to auxin. Ethylene induces the expression of genes responsible for the fruit softening, its taste, color, and flavor. Nonclimacteric fleshy fruits produce very small amounts ofethylene; its evolution increases only by the very end of ripening and can be described by a reduced System 1. The ripening of nonclimacteric fruits only weakly depends on ethylene but is stimulated by abscisic acid.

Perturbation of polyamine catabolism affects grape ripening of Vitis vinifera cv. Trincadeira

Grapes are economically the most important fruit worldwide. However, the complexity of biological events that lead to ripening of nonclimacteric fruits is not fully understood, particularly the role of polyamines' catabolism.The transcriptional and metabolic profilings complemented with biochemical data were studied during ripening of Trincadeira grapes submitted to guazatine treatment, a potent inhibitor of polyamine oxidase activity. The mRNA expression profiles of one time point (EL 38) corresponding to harvest stage was compared between mock and guazatine treatments using Affymetrix GrapeGen® genome array. A total of 2113 probesets (1880 unigenes) were differentially expressed between these samples. Quantitative RT-PCR validated microarrays results being carried out for EL 35 (véraison berries), EL 36 (ripe berries) and EL 38 (harvest stage berries). Metabolic profiling using HPLC and 1H NMR spectroscopy showed increase of putrescine, proline, threonine and 1-O-ethyl- β -glucoside in guazatine treated samples.Genes involved in amino acid, carbohydrate and water transport were down-regulated in guazatine treated samples suggesting that the strong dehydrated phenotype obtained in guazatine treated samples may be due to impaired transport mechanisms. Genes involved in terpenes' metabolism were differentially expressed between guazatine and mock treated samples. Altogether, results support an important role of polyamine catabolism in grape ripening namely in cell expansion and aroma development.

Application of a Rapid and Sensitive Method for Hormonal and Vitamin E Profiling Reveals Crucial Regulatory Mechanisms in Flower Senescence and Fruit Ripening

Knowledge of ripeness and regulation of postharvest processes is an important tool to prevent loss of commercial value in both fruit and cut flower markets. The joint analysis of hormones and vitamin E levels can reveal complex interactions between hormones and oxidative stress as key regulators of postharvest processes. Profiling of both groups of metabolic compounds was performed during the ripening of non-climacteric fruits (red raspberry, Rubus idaeus L.) and senescence of ethylene-insensitive flowers (Dutch Iris, Iris x hollandica L.). After an initial extraction of the sample, without further purification steps, the hormonal profile was analyzed by UPLC-MS/MS and vitamin E levels were measured by HPLC. This methodological approach was very fast and had enough sensitivity for the analysis of small samples. Raspberry fruit maturation was characterized by a decline of cytokinin levels [zeatin, zeatin riboside, 2-isopentenyl adenine, and isopentenyl adenosine (Z, ZR, 2-iP, and IPA, respectively)] and gibberellins (GA1 in particular). Exogenous application of ABA prevented δ-tocopherol loss during fruit ripening. Iris floral senescence was also under strict hormonal control, also mediated by cytokinins and gibberellins. Z, ZR, 2-iP, GA9, and GA24 levels decreased in inner tepals, whereas the level of IPA decreased in style-merged-to-stigma tissues, thus suggesting tissue-specific roles for different hormones. α-Tocopherol levels decreased during senescence of inner tepals, hence suggesting enhanced oxidative stress. In conclusion, the rapid and sensitive hormonal and vitamin E profiling presented here can help in understanding the key physiological processes underlying fruit ripening and floral senescence.

Changes in the abscisic acid levels and related gene expression during fruit development and ripening in bilberry (Vaccinium myrtillus L.)

Abscisic acid (ABA) is a natural plant hormone playing an important role in many physiological processes including fruit ripening and is also recently found to be potential for biomedical applications. This study was aimed to measure ABA levels and its biosynthesis in bilberry (Vaccinium myrtillus L.), which is one of the best sources of anthocyanins. Five ABA biosynthetic genes were isolated from bilberry and their expression profiles were studied in bilberry tissues, particularly during berry development. The level of ABA highly increased at the onset of bilberry fruit ripening, at the stage when expression of anthocyanin biosynthetic genes, chalcone synthase (VmCHS) and anthocyanidin synthase (VmANS), also increased. In fully ripe berries and leaves, ABA levels were lower but none was detected in bilberry stem or rhizome. The expression of 9-cis-epoxycarotenoid dioxygenase (VmNCED1) and putative neoxanthin synthase (VmNSY) was high in berry tissues and their expression increased markedly at the onset of berry ripening along with the accumulation of ABA. In contrast, the expression of zeaxanthin epoxidase (VmZEP), short-chain dehydrogenase/reductase (VmSDR/ABA2) and aldehyde oxidase (VmAO) were most highly associated with leaf tissues with no obvious relation to ABA content during berry development. The obtained results indicate that the ABA biosynthesis may play an important role in the regulation of ripening of non-climacteric bilberry fruits through transcriptional regulation of key ABA biosynthetic genes.

Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit

To investigate the role of jasmonates (JAs) in the ripening of Fragaria chiloensis fruit, two concentrations of methyl jasmonate (MeJA, 10 and 100μM) were evaluated at 2, 5 and 9dusing an invitro ripening system. Fruit quality parameters; the contents of anthocyanin, lignin and cell wall polymers; and the transcriptional profiles of several ripening-related genes were analyzed. MeJA accelerated fruit ripening by means of a transitory increase in the soluble solid content/titratable acidity ratio, anthocyanin accumulation and an increase in softening at day 5. The expression of several phenylpropanoid-related genes, primarily those associated with anthocyanin biosynthesis, was increased under MeJA treatment, which correlated with an increased accumulation of anthocyanin. MeJA also altered the expression profiles of some cell wall-modifying genes, namely, EG1 and XTH1, and these changes correlated with a transient reduction in the firmness of MeJA-treated fruits. MeJA-responsive elements were observed in the promoter region of the EG1 gene. MeJA also increased the expression of LOX, AOS and OPR3, genes involved in the biosynthesis of JAs, and these changes correlated with the transient activation of fruit ripening observed. Conversely, the expression of ethylene and lignin biosynthesis genes (ACS, ACO, CAD and POD27) increased in MeJA-treated fruits at day 9. The present findings suggest that JAs promote the ripening of non-climacteric fruits through their involvement in anthocyanin accumulation, cell wall modification and the biosynthesis of ethylene and JAs.

Study of polyamines during grape ripening indicate an important role of polyamine catabolism

Grapevine (Vitis species) is the most economically important fruit crop worldwide. Ripening of non-climacteric fruits such as grapes has been the subject of intense research. Despite this interest, little is known on the role played by polyamines in the onset of ripening of non-climacteric fruits. These growth regulators have been involved in plant development and stress responses. Molecular and biochemical studies were developed in three important Portuguese cultivars (Trincadeira, Touriga Nacional and Aragonês) during the year 2008 and in Trincadeira during 2007 in order to gather insights on the role of polyamines in grape ripening. Microarray and real-time qPCR studies revealed up-regulation of a gene coding for arginine decarboxylase (ADC) during grape ripening in all the varieties. This increase was not accompanied by an increase in free and conjugated polyamines that presented a strong decrease. Putrescine and Spermidine levels were higher at earlier stages of development, while Spermine level remained constant. Berries of Trincadeira cultivar presented the highest content in total free and conjugated polyamines at earlier stages of fruit development in particular in the year 2007. The decrease in polyamines content during grape ripening was accompanied by up-regulation of genes coding for diamine oxidase (CuAO) and polyamine oxidase (PAO), together with a significant increase in their enzymatic activity and in the hydrogen peroxide content. These results provide, for the first time, strong evidence of a role of polyamine catabolism in grape ripening possibly through interaction with other growth regulators.

Sucrose functions as a signal involved in the regulation of strawberry fruit development and ripening

Fleshy fruits are classically divided into climacteric and nonclimacteric types. It has long been thought that the ripening of climacteric and nonclimacteric fruits is regulated by ethylene and abscisic acid (ABA), respectively. Here, we report that sucrose functions as a signal in the ripening of strawberry (Fragaria × ananassa), a nonclimacteric fruit. Pharmacological experiments, as well as gain- and loss-of-function studies, were performed to demonstrate the critical role of sucrose in the regulation of fruit ripening. Fruit growth and development were closely correlated with a change in sucrose content. Exogenous sucrose and its nonmetabolizable analog, turanose, induced ABA accumulation in fruit and accelerated dramatically fruit ripening. A set of sucrose transporters, FaSUT1-7, was identified and characterized, among which FaSUT1 was found to be a major component responsible for sucrose accumulation during fruit development. RNA interference-induced silencing of FaSUT1 led to a decrease in both sucrose and ABA content, and arrested fruit ripening. By contrast, overexpression of FaSUT1 led to an increase in both sucrose and ABA content, and accelerated fruit ripening. In conclusion, this study demonstrates that sucrose is an important signal in the regulation of strawberry fruit ripening.

Proteomic analysis during capsicum ripening reveals differential expression of ACC oxidase isoform 4 and other candidates.

Capsicum (Capsicum annuum L.) is categorised as a non-climacteric fruit that exhibits limited ethylene production during ripening and the molecular mechanisms associated with this process are poorly understood. A proteomic approach was used to identify the differentially expressed proteins during various ripening stages (Green (G), Breaker Red 1 (BR1) and Light Red (LR)) and the genes associated with their synthesis. From 2D gel electrophoresis (2DGE), seven protein spots were identified as selectively present either in G or BR1 and are involved in carbon metabolism, colour and fruit development, protein synthesis and chaperones or biosynthesis of amino acids and polyamines. One candidate of interest, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) is known to be involved in ethylene biosynthesis and was only present in BR1 and is related to the tomato ACO isoform 4 (LeACO4) and hence named CaACO4. CaACO4 RNA expression as well as total ACO protein expression in multiple stages of ripening (G, Breaker (B), BR1, Breaker Red 2 (BR2), LR and Deep Red (DR)) corresponded to the 2DGE protein spot abundance in breaker stages. Our findings highlight the involvement of the ethylene pathway in non-climacteric fruit ripening.

Transcript and metabolite analysis in Trincadeira cultivar reveals novel information regarding the dynamics of grape ripening.

BackgroundGrapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses.ResultsRipening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening.ConclusionsAltogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit.

The citrus fruit proteome: insights into citrus fruit metabolism

Fruit development and ripening are key processes in the production of the phytonutrients that are essential for a balanced diet and for disease prevention. The pathways involved in these processes are unique to plants and vary between species. Climacteric fruit ripening, especially in tomato, has been extensively studied; yet, ripening of non-climacteric fruit is poorly understood. Although the different species share common pathways; developmental programs, physiological, anatomical, biochemical composition and structural differences must contribute to the operation of unique pathways, genes and proteins. Citrus has a non-climacteric fruit ripening behavior and has a unique anatomical fruit structure. For the last few years a citrus genome-wide ESTs project has been initiated and consists of 222,911 clones corresponding to 19,854 contigs and 37,138 singletons. Taking advantage of the citrus database we analyzed the citrus proteome. Using LC-MS/MS we analyzed soluble and enriched membrane fractions of mature citrus fruit to identify the proteome of fruit juice cells. We have identified ca. 1,400 proteins from these fractions by searching NCBI-nr (green plants) and citrus ESTs databases, classified these proteins according to their putative function and assigned function according to known biosynthetic pathways.

Ripened with Steroids

Fruits are divided into two classes: climacteric and nonclimacteric. By definition, nonclimacteric fruit ripens without the peak in ethylene and increase in respiration associated with ripening of climacteric fruits. Symons et al. provide evidence that brassinosteroids stimulate ripening of grape berries, specifically field-grown Cabernet Sauvignon. The abundance of castasterone (CS), a bioactive brassinosteroid, and its precursor increased during the period of berry ripening. Two enzymes involved in brassinosteroid biosynthesis (VvDWF1 and VvBR6OX1) and the brassinosteroid receptor (VvBRI1) were cloned from grape. Transcript analysis showed that VvDWF1 and VvBRI1 increased during berry ripening, whereas the abundance of VvBR6OX1 transcripts decreased. The decrease in VvBR6OX1 was consistent with negative feedback regulation by CS. This type of feedback by brassinosteroids has been reported in other species of plants. Application of exogenous brassinosteroid enhanced the rate of berry ripening, whereas application of an inhibitor brassinosteroid biosynthesis delayed berry ripening. Thus, brassinosteroids appear to serve a role in stimulating ripening of nonclimacteric fruits. Understanding nonclimacteric fruit ripening has important economic and agricultural implications, because several nonclimacteric fruits are grown for food or beverage production, including strawberry, grape, and citrus. G. M. Symons, C. Davies, Y. Shavrukov, I. B. Dry, J. B. Reid, M. R. Thomas, Grapes on steroids: Brassinosteroids are involved in grape berry ripening. Plant Physiol. 140 , 150-158 (2006). [Abstract] [Full Text]

Grapes on steroids. Brassinosteroids are involved in grape berry ripening.

Fruit ripening is a unique plant developmental process with direct implications for our food supply, nutrition, and health. In contrast to climacteric fruit, where ethylene is pivotal, the hormonal control of ripening in nonclimacteric fruit, such as grape (Vitis vinifera), is poorly understood. Brassinosteroids (BRs) are steroidal hormones, essential for normal plant growth and development but not previously implicated in the ripening of nonclimacteric fruit. Here we show that increases in endogenous BR levels, but not indole-3-acetic acid (IAA) or GA levels, are associated with ripening in grapes. Putative grape homologs of genes encoding BR biosynthesis enzymes (BRASSINOSTEROID-6-OXIDASE and DWARF1) and the BR receptor (BRASSINOSTEROID INSENSITIVE 1) were isolated, and the function of the grape BRASSINOSTEROID-6-OXIDASE gene was confirmed by transgenic complementation of the tomato (Lycopersicon esculentum) extreme dwarf (dx/dx) mutant. Expression analysis of these genes during berry development revealed transcript accumulation patterns that were consistent with a dramatic increase in endogenous BR levels observed at the onset of fruit ripening. Furthermore, we show that application of BRs to grape berries significantly promoted ripening, while brassinazole, an inhibitor of BR biosynthesis, significantly delayed fruit ripening. These results provide evidence that changes in endogenous BR levels influence this key developmental process. This may provide a significant insight into the mechanism controlling ripening in grapes, which has direct implications for the logistics of grape production and down-stream processing.

The Effect of 1-MCP on the Expression of Several Ripening-related Genes in Strawberries

To elucidate the role of ethylene in nonclimacteric fruit development and ripening, quantitative (cDNA–amplified fragment length polymorphism) cDNA–AFLP was used to visualize differential gene expression in four stages of ripening of strawberries (Fragaria×ananassa Duch. `Elsanta') treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. The proportion of clones affected by 1-MCP treatment was much higher in green than in white, pink, and red receptacle tissue. Three major cell-wall-related genes were affected by 1-MCP and, thus, are putatively ethylene dependent: a ripening-repressed beta-galactosidase (Faßgal3), up-regulated by 1-MCP; a putative endo-1,3-1,4-beta-D-glucanase (EGase), up-regulated in green and down-regulated in red fruit by 1-MCP; and a pectate lyase B (plB), expressed only in the red stage and significantly down-regulated by 1-MCP. Furthermore, we have identified genes encoding an alcohol dehydrogenase, a protein kinase-related protein, and a putative glutathione S-transferase, all ripening-induced and down-regulated by 1-MCP, suggesting that their regulation is at least partly ethylene dependent.

Expression of ripening-related genes in prickly pear (Opuntia sp.) fruits.

To throw light on the expression of ripening-related genes in prickly pear (Opuntia sp.) fruits and on the possible role of the gaseous hormone ethylene in nonclimacteric fruit ripening, cDNA fragments that showed high homologies with 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase cDNAs from other plants were cloned and partially characterized. Thus, the corresponding genes were accordingly named opaccs-1 and opacco-1, after Opuntia ACC synthase-1 and Opuntia ACC oxidase-1, respectively. Southern analysis suggests the presence of at least one copy of both genes, as well as other related homologous sequences in the Opuntia genome. Northern analysis of the opaccs-1 gene shows an enhanced expression in ripening fruit tissues, whereas opacco-1 expression is highly induced in ripe tissues with respect to the green fruits and mature cladodes. These results are in agreement with an active metabolic role of ethylene during nonclimacteric prickly pear fruit ripening. This is the first report on the analysis at the molecular level of ripening-related genes of the Opuntia genus.

Cloning and characterisation of ripening-induced ethylene biosynthetic genes from non-climacteric pineapple (Ananas comosus) fruits

To gain a better understanding of non-climacteric fruit ripening, pineapple was used as a model system to clone and characterise two ripening-inducible cDNAs coding for two enzymes of the ethylene biosynthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) synthase (acacc-1) and 1-aminocyclo-propane- 1-carboxylate oxidase (acaco-1) respectively. Due to the extreme acidity and high polyphenolic content of pineapple fruits, a method was optimised for the extraction of high quality RNA from fruit tissue. acacc-1 is a 1080 bp ACC synthase cDNA fragment encoding 360 amino acids including 10 of the 12 amino acid residues conserved in all aminotransferases. Comparison of the deduced amino acid sequence with previously reported ACC synthases shows between 52 and 67% similarity at the protein level. Southern analysis suggests the presence of only one copy of acacc-1 in the pineapple genome. Although some acacc-1 expression is detected in green fruits, there is a 16-fold increase in the level of acacc-1 in ripe fruit tissue. acaco-1 is a partial length cDNA clone of 611 bp which codes for 203 amino acids representing approximately 66% of the ACC oxidase open reading frame. Southern analysis suggests the presence of one or two copies of the gene in the pineapple genome. Northern analysis shows the expression of acaco-1 to be highly induced in wounded leaf tissue and to a lesser extent in ripening fruit tissue. The accumulation of ACC-synthase and ACC oxidase mRNAs during pineapple fruit ripening raises new questions about the putative role of ethylene during non-climacteric fruit ripening.