Identification of grapevine aquaporins and expression analysis in developing berries

Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. In this work, nine full-length cDNAs encoding putative aquaporins were isolated from grape berry cDNA libraries. A phylogenetic analysis conducted with 28 aquaporin genes identified in the grapevine genome and previously characterized aquaporins from Arabidopsis indicates that three cDNAs encode putative tonoplast aquaporins (TIPs) whereas six cDNAs belong to the plasma membrane aquaporin subfamily (PIPs). Specific probes designed on the 3' untranslated regions of each cDNA were used for the preparation of cDNA macroarray filters and in situ hybridization experiments. Macroarray data indicate that expression levels of most TIP and PIP genes depend on grape berry developmental stages and point out to a global decrease of aquaporin gene expression during berry ripening. In young berries, high expression of aquaporin genes was preferentially observed in dividing and elongating cells and in cells involved in water and solutes transport. Taken together, the data provided in this paper indicate that aquaporins are implicated in various physiological aspects of grape berry development.     

Identification of salt stress-induced transcripts in potato leaves by cDNA-AFLP

Potato (Solanum tuberosum L.) is highly sensitive to salt stress, which is one of the most important factors limiting plant cultivation. The investigation of plant response to high salinity was envisaged in this report using cDNA-amplified fragment length polymorphism (AFLP). This technique was applied to salt- stressed and control potato plants (cv. Nicola). The expression profiles showed approx 5000 bands. Of these, 154 were upregulated and 120 were repressed by salt stress. In this study we have only considered cDNA fragments that seem to be originated from salt-induced mRNA. Eighteen fragments were then reamplified, cloned, and sequenced. Sequence comparison of these cDNA, identified in response to salt stress in potato, revealed that some of them present homologies with proteins in other species that are involved in cell wall structure and turnover such as proline-rich proteins and beta-galactosidase. A number of identified clones encoded putative stress response proteins such as NADP-dependant glyceraldehyde dehydrogenase and wound-induced protein. In addition, some of them encode proteins related to hypersensitive response against pathogens such as putative late blight and nematode as well as putative pathogenesis-related proteins. These cDNA seem to be differentially expressed in the presence of salt stress as shown by Northern blot or reverse Northern hybridization experiments.     

Isolation and characterization of mRNAs differentially expressed during ripening of wild strawberry (Fragaria vesca L.) fruits

Wild strawberry (Fragaria vesca L.) is an attactive model system for studying ripening in non-climacteric fruit, because of its small diploid genome, its short reproductive cycle, and its capacity for transformation. We have isolated eight ripening-induced cDNAs from this species after differential screening of a cDNA library. The predicted polypeptides of seven of the clones exhibit similarity to database protein sequences, including acyl carrier protein, caffeoyl- CoA 3-O-methyltransferase, sesquiterpene cyclase, major latex protein, cystathionine -synthase, dehydrin and an auxin- induced gene. A ninth cDNA clone that was constitutively expressed is predicted to encode a metallothionein-like protein. None of these proteins appear to be directly related to events generally associated with ripening such as cell wall metabolism or the accumulation of sugars and pigments, rather, their putative functions are indicative of the wide range of processes upregulated during fruit ripening.     

Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.     

Isolation of a set of ripening-related genes from strawberry: their identification and possible relationship to fruit quality traits

The ripening of strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, is a complex developmental process that involves many changes in gene expression. To understand how these changes relate to the biochemistry and composition of the fruit the specific genes involved have been examined. A high-quality cDNA library prepared from ripe strawberry fruit was differentially screened for ripening-related clones using cDNA from ripe and white fruits. From 112 up-regulated clones obtained in the primary screen, 66 differentially expressed clones were isolated from the secondary screen. The partial sequences of these cDNAs were compared with database sequences and 26 families of non-redundant clones were identified. Northern analysis confirmed that all of these cDNAs were ripening-enhanced. The expression of many of their corresponding genes was negatively regulated in auxin-treated fruit. These sequences, several of which are novel to fruits, encode proteins involved in key metabolic events including anthocyanin biosynthesis, cell wall degradation, sucrose and lipid metabolism, protein synthesis and degradation, and respiration. These findings are discussed in relation to the role of these genes in determining fruit quality characteristics. Received: 19 January 1998 / Accepted: 5 February 1998     

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.     

cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin

Microarray analysis of Vitis vinifera cv. Shiraz developing berries has revealed the expression patterns of several categories of genes. Microarray slides were constructed from 4,608 PCR-amplified cDNA clones derived from a ripening grape berry cDNA library. The mRNA expression levels of the genes represented by these cDNAs were measured in flowers, week 2 post-flowering whole berries, week 5, week 8, week 10 (véraison, green berries), week 12 and week 13 berry skin. In addition, a comparison of RNA expression in pigmented and unpigmented berry skin at véraison (week 10) was undertaken. Image and statistical analysis revealed four sets of genes with distinctive and similar expression profiles over the course of berry development. The first set was composed of genes which had maximum RNA expression in flowers, followed by a steady decrease in expression. The most prominent group within this set were genes which have a role in photosynthesis. The second set of cDNAs was dominated by genes involved in flavonoid biosynthesis and had a peak of expression week 2 post-flowering. The data indicate co-ordinate regulation of flavonoid biosynthetic genes which code for the enzymes 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, flavonone hydroxylase, anthocyanidin reductase and cytochrome b5. The third set of cDNAs exhibited maximum expression week 5 post-flowering, midway between flowering and véraison, a period of rapid berry growth. This set of cDNAs is dominated by genes which code for structural cell wall proteins. The fourth set of genes was dramatically up-regulated at véraison and remained up-regulated until 13 weeks post-flowering. This set of genes was composed of a diverse range of genes, a reflection of the complexity of ripening, most with no known function.     

A shift of Phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry

It remains unclear whether the phloem unloading pathway alters to adapt to developmental transition in fleshy fruits that accumulate high level of soluble sugars. Using a combination of electron microscopy, transport of the phloem-mobile symplasmic tracer carboxyfluorescein, movement of the companion cell-expressed and the green fluorescent protein-tagged viral movement protein, and assays of the sucrose cleavage enzymes, the pathway of phloem unloading was studied in the berries of a hybrid grape (Vitis vinifera x Vitis labrusca). Structural investigations showed that the sieve element-companion cell complex is apparently symplasmically connected through plasmodesmata with surrounding parenchyma cells throughout fruit development, though a small portion of plasmodesmata are apparently blocked in the ripening stage. Both carboxyfluorescein and the green fluorescent protein-tagged viral movement protein were released from the functional phloem strands during the early and middle stages of fruit development, whereas the two symplasmic tracers were confined to the phloem strands during the late stage. This reveals a shift of phloem unloading from symplasmic to apoplasmic pathway during fruit development. The turning point of the phloem unloading pathways was further shown to be at or just before onset of ripening, an important developmental checkpoint of grape berry. In addition, the levels of both the expression and activities of cell wall acid invertase increased around the onset of ripening and reached a high level in the late stage, providing further evidence for an operation of the apoplasmic unloading pathway after onset of ripening. These data demonstrate clearly the occurrence of an adaptive shift of phloem unloading pathway to developmental transition from growing phase to ripening in grape berry.     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

Proteome analysis of grape skins during ripening

The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and beta-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

Coordinate- and elicitor-dependent expression of stilbene synthase and phenylalanine ammonia-lyase genes in Vitis cv. Optima

The mechanisms controlling the induction of stilbene synthase and phenylalanine ammonia-lyase (PAL), two putative key regulatory enzymes of the biosynthetic pathway to stilbene phytoalexins, have been investigated. The induction was studied in cell suspension cultures of grape (Vitis cv. Optima) by treatment with fungal cell wall. Several independent cDNA clones for PAL and stilbene synthase were isolated from a cDNA library of fungal cell wall-induced grape cells and identified by sequence analysis. The stilbene synthase cDNA sequence of pSV21 predicted a protein of 392 amino acids and Mr 42,791, similar in size to that observed experimentally for immunodetected stilbene synthase. The cDNA sequences of pSV21 and pSV25 differed in 76 bp in the coding region. The sequences of grape stilbene synthase cDNAs exhibited significant homology to the sequence reported for the peanut stilbene synthase cDNA. Both PAL and stilbene synthase mRNA, measured by RNA blot hybridizations, were induced within 1 h of addition of fungal cell wall preparations to the cell cultures, rose to a maximum by the sixth hour, then declined slowly over the next 20 h. The activities of PAL and stilbene synthase were also induced in parallel, but reached their maximum at different times after fungal cell wall addition to the cell cultures. The induction patterns of stilbene synthase and PAL in grape and peanut are discussed.     

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.     

Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process.