An expansin gene expressed in ripening strawberry fruit

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.     

Changes in gene expression during strawberry fruit ripening and their regulation by auxin

Changes in messenger RNA during the development of the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, were analysed by extracting total RNA and separating the in-vitro translated products by two-dimensional polyacrylamide gel electrophoresis. Alterations in numerous messenger RNAs accompanied fruit development between the immature green stage and the overripe stage, with prominent changes detected at or before the onset of ripening. A number of messenger RNAs undetectable in immature green fruit increased as the fruit matured and ripened. Others showed a marked decrease in advance of the ripening phase. A further group of messenger RNAs was prominent in immature and ripe fruit but absent just prior to the turning stage. Removing the achenes from a segment of the fruit accelerated anthocyanin accumulation in the de-achened portion and produced a pattern of translated polypeptides similar to normal ripe fruit. Application of the synthetic auxin 1-naphthaleneacetic acid to the de-achened receptacle produced a translation pattern similar to that in mature green fruit. These findings indicate that ripening in strawberry is associated with the expression of specific genes.     

Extensive transcriptomic studies on the roles played by abscisic acid and auxins in the development and ripening of strawberry fruits

Strawberry is an ideal model for studying the molecular biology of the development and ripening of non-climacteric fruits. Hormonal regulation of gene expression along all these processes in strawberries is still to be fully elucidated. Although auxins and ABA have been pointed out as the major regulatory hormones, few high-throughput analyses have been carried out to date. The role for ethylene and gibberellins as regulatory hormones during the development and ripening of the strawberry fruit remain still elusive. By using a custom-made and high-quality oligo microarray platform done with over 32,000 probes including all of the genes actually described in the strawberry genome, we have analysed the expression of genes during the development and ripening in the receptacles of these fruits. We classify these genes into two major groups depending upon their temporal and developmental expression. First group are genes induced during the initial development stages. The second group encompasses genes induced during the final maturation and ripening processes. Each of these two groups has been also divided into four sub-groups according their pattern of hormonal regulation. By analyzing gene expression, we clearly show that auxins and ABA are the main and key hormones that combined or independently are responsible of the development and ripening process. Auxins are responsible for the receptacle fruit development and, at the same time¸ prevent ripening by repressing crucial genes. ABA regulates the expression of the vast majority of genes involved in the ripening. The main genes expressed under the control of these hormones are presented and their physiological rule discussed. We also conclude that ethylene and gibberellins do not seem to play a prominent role during these processes.     

Hormonal changes during non-climacteric ripening in strawberry

In contrast to climacteric fruits, where ethylene is known to be pivotal, the regulation of ripening in non-climacteric fruits is not well understood. In the non-climacteric strawberry (Fragaria anannassa), auxin and abscisic acid (ABA) are thought to be important, but the roles of other hormones suggested to be involved in fruit development and ripening are not clear. Here changes in the levels of indole-3-acetic acid (IAA), ABA, GA(1), and castasterone from anthesis to fully ripened fruit are reported. The levels of IAA and GA(1) rise early in fruit development before dropping to low levels prior to colour accumulation. Castasterone levels are highest at anthesis and drop to very low levels well before ripening commences, suggesting that brassinosteroids do not play an important role in ripening in strawberry. ABA levels are low at anthesis and gradually rise through development and ripening. The synthetic auxin, 1-naphthaleneacetic acid (NAA), can delay ripening, but the application of GA(3), the gibberellin biosythesis inhibitor paclobutrazol, and ABA had no significant effect. IAA and ABA levels are higher in the developing achenes than in the receptacle tissue and may be important for receptacle enlargement and ripening, and seed maturation, respectively. Contrary to a recent report, the biologically active GA(4) was not detected. The pattern of changes in the levels of the hormones are different from those reported in another well studied non-climateric fruit, grape, suggesting that a single consistent pattern of hormone changes does not occur in this group of fruit during ripening.     

Cloning and molecular characterization of a strawberry fruit ripening-related cDNA corresponding a mRNA for a low-molecular-weight heat-shock protein

We have isolated and characterized a cDNA from a strawberry fruit subtractive library that shows homology to class-I low-molecular-weight (LMW) heat-shock protein genes from other higher plants. The strawberry cDNA (clone njjs4) was a 779 bp full-length cDNA with a single open reading frame of 468 bp that is expected to encode a protein of ca. 17.4 kDa with a pI of 6.57. Southern analysis with genomic DNA showed several high-molecular-weight hybridization bands, indicating that the corresponding njjs4 gene is not present as a single copy in the genome. This strawberry gene was not expressed in roots, leaves, flowers and stolons but in fruits at specific stages of elongation and ripening. However, a differential pattern of mRNA expression was detected in the fruit tissues achenes and receptacle. The njjs4 gene expression increased in achenes accompanying the process of seed maturation whereas in the receptacle, a high mRNA expression was detected in the W2 stage, during which most of the metabolic changes leading to the fruit ripening are occurring. Our results clearly show a specific relationship of this njjs4 strawberry gene with the processes of seed maturation and fruit ripening, and strongly support that at least some of the class-I LMW heat-shock protein-like genes have a heat-stress-independent role in plant development, including fruit ripening.     

Downregulation of RdDM during strawberry fruit ripening

Background

Recently, DNA methylation was proposed to regulate fleshy fruit ripening. Fleshy fruits can be distinguished by their ripening process as climacteric fruits, such as tomatoes, or non-climacteric fruits, such as strawberries. Tomatoes undergo a global decrease in DNA methylation during ripening, due to increased expression of a DNA demethylase gene. The dynamics and biological relevance of DNA methylation during the ripening of non-climacteric fruits are unknown.

Results

Here, we generate single-base resolution maps of the DNA methylome in immature and ripe strawberry. We observe an overall loss of DNA methylation during strawberry fruit ripening. Thus, ripening-induced DNA hypomethylation occurs not only in climacteric fruit, but also in non-climacteric fruit. Application of a DNA methylation inhibitor causes an early ripening phenotype, suggesting that DNA hypomethylation is important for strawberry fruit ripening. The mechanisms underlying DNA hypomethylation during the ripening of tomato and strawberry are distinct. Unlike in tomatoes, DNA demethylase genes are not upregulated during the ripening of strawberries. Instead, genes involved in RNA-directed DNA methylation are downregulated during strawberry ripening. Further, ripening-induced DNA hypomethylation is associated with decreased siRNA levels, consistent with reduced RdDM activity. Therefore, we propose that a downregulation of RdDM contributes to DNA hypomethylation during strawberry ripening.

Conclusions

Our findings provide new insight into the DNA methylation dynamics during the ripening of non-climacteric fruit and suggest a novel function of RdDM in regulating an important process in plant development.

     

Identification of a strawberry gene encoding a non-specific lipid transfer protein that responds to ABA,wounding and cold stress

cDNA and genomic clones encoding a strawberry (Fragariaxananassa cv. Chandler) non-specific lipid transfer protein (Fxaltp gene) were isolated and characterized. The spatio-temporal expression pattern and structural features of this gene were studied for the first time in strawberry, a non-climacteric fruit of agricultural importance. The architecture and the encoded amino acid sequence of this non-climacteric fruit ltp gene were similar to those of other plant LTPs previously reported, and presents the eight cysteine residues and other features characteristic of plant LTPs. In addition, the deduced protein posseses an N-terminal signal peptide and lacks the K/HDEL retention signal, indicating that the strawberry LTP protein would enter the secretory pathway. In situ studies have shown that the Fxaltp gene is expressed in the epidermal cell layer of the strawberry fruit receptacle and achenes, flowers, and within the cell layer surrounding the endosperm. These results suggest that this Fxaltp gene promoter could be used as an endogenous promoter for biotechnological purposes in strawberry. Computer analysis using the PLACE database predicted the presence of several putative cis-regulatory sequences in response to abscisic acid and cold or wounding stresses within the Fxaltp 5'-flanking region. Accordingly, the strawberry gene responds to ABA and SA, but not to salt and heat stresses. It is also reported that ltp gene expression in strawberry is stimulated by wounding and repressed by cold stresses.     

Abscisic acid perception and signaling transduction in strawberry: A model for non-climacteric fruit ripening

On basis of fruit differential respiration and ethylene effects, climacteric and non-climacteric fruits have been classically defined. Over the past decades, the molecular mechanisms of climacteric fruit ripening were abundantly described and found to focus on ethylene perception and signaling transduction. In contrast, until our most recent breakthroughs, much progress has been made toward understanding the signaling perception and transduction mechanisms for abscisic acid (ABA) in strawberry, a model for non-climacteric fruit ripening. Our reports not only have provided several lines of strong evidences for ABA-regulated ripening of strawberry fruit, but also have demonstrated that homology proteins of Arabidopsis ABA receptors, including PYR/PYL/RCAR and ABAR/CHLH, act as positive regulators of ripening in response to ABA. These receptors also trigger a set of ABA downstream signaling components, and determine significant changes in the expression levels of both sugar and pigment metabolism-related genes that are closely associated with ripening. Soluble sugars, especially sucrose, may act as a signal molecular to trigger ABA accumulation through an enzymatic action of 9-cis-epoxycarotenoid dioxygenase 1 (FaNCED1). This mini-review offers an overview of these processes and also outlines the possible, molecular mechanisms for ABA in the regulation of strawberry fruit ripening through the ABA receptors.     

The fruit ripening-related gene FaAAT2 encodes an acyl transferase involved in strawberry aroma biogenesis

Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour.     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid