José G. Vallarino

Ethylene is involved in strawberry fruit ripening in an organ-specific manner

The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.

Alteration of the Interconversion of Pyruvate and Malate in the Plastid or Cytosol of Ripening Tomato Fruit Invokes Diverse Consequences on Sugar But Similar Effects on Cellular Organic Acid, Metabolism, and Transitory Starch Accumulation

Summary: Normal tomato ripening is influenced by alterations on both cytosolic phosphoenolpyruvate carboxykinase and plastidic NADP-malic enzyme. This study provides compelling evidence of their roles in starch biosynthesis, respiration rates, and tricarboxylic acid cycle flux. The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects on fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate, which suggests that an excess of oxaloacetate is converted to aspartate and reintroduced in the TCA cycle via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-ME antisense lines were characterized by no changes in respiration rates and TCA cycle flux, which together with increases of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicate that pyruvate is supplied through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening.

Transcriptomic Analysis in Strawberry Fruits Reveals Active Auxin Biosynthesis and Signaling in the Ripe Receptacle

The role of auxin in ripening strawberry (Fragaria ×ananassa) fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and ripening receptacle.

Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry ( Fragaria × ananassa )

RNA-seq has been used to perform global expression analysis of the achene and the receptacle at four stages of fruit ripening, and of the roots and leaves of strawberry (Fragaria × ananassa). About 967 million reads and 191 Gb of sequence were produced, using Illumina sequencing. Mapping the reads in the related genome of the wild diploid Fragaria vesca revealed differences between the achene and receptacle development program, and reinforced the role played by ethylene in the ripening receptacle. For the strawberry transcriptome assembly, a de novo strategy was followed, generating separate assemblies for each of the ten tissues and stages sampled. The Trinity program was used for these assemblies, resulting in over 1.4 M isoforms. Filtering by a threshold of 0.3 FPKM, and doing Blastx (E-value < 1 e-30) against the UniProt database of plants reduced the number to 472,476 isoforms. Their assembly with the MIRA program (90% homology) resulted in 26,087 contigs. From these, 91.34 percent showed high homology to Fragaria vesca genes and 87.30 percent Fragaria iinumae (BlastN E-value < 1 e-100). Mapping back the reads on the MIRA contigs identified polymorphisms at nucleotide level, using FREEBAYES, as well as estimate their relative abundance in each sample.

Signaling role of oligogalacturonides derived during cell wall degradation.

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGAs) are essential components to trigger signaling pathways that induce rapid defense responses. Many pathogens directly penetrate the cell wall to access water and nutrients of the plant protoplast, and a rigid cell wall can fend off pathogen attack by forming an impenetrable physical barrier. Thus, cell wall integrity sensing is one mechanism by which plants may detect pathogen attack. Moreover, when the plant-pathogen interaction occurred, OGAs released during cell wall modification can trigger plant defense (e.g., production of reactive oxygen species, production of anti-microbial metabolites and synthesis of pathogenesis-related proteins). This review documents and discusses studies suggesting that OGAs play a dual signaling role during pathogen attack by inducing defense responses and plant architecture adjustment.

Simultaneous Determination of Plant Hormones by GC-TOF-MS.

Phytohormones are key signaling molecules that coordinate plant growth and development through a range of complex interactions. Since the vast majority of plant responses to given stimuli result, amongst other factors, from a crosstalk between hormones, simultaneous analysis of multiple hormones is vital to improve our understanding of these interactions. This chapter describes a sensitive, reliable, and inexpensive method for quantification of multiple phytohormones by gas chromatography-mass spectrometry (GC-MS).

Postharvest changes in LIN5-down-regulated plants suggest a role for sugar deficiency in cuticle metabolism during ripening

The cell wall invertase gene (LIN5) was reported to be a key enzyme influencing sugar uptake of tomato (Solanum lycopersicum) fruit. It was additionally revealed to be a key regulator of total soluble solids content in fruit as well as for reproductive development, being mainly involved in flower development, early fruit and seed development but also in ripening. Here, we demonstrate that silencing of the LIN5 gene promotes changes affecting fruit cuticle development which has a direct effect on postharvest properties. Transformants were characterized by reduced transpirational water loss in mature fruits accompanied by several other changes in the cuticle. Quantitative chemical composition, coupled with microscopy of isolated cuticle fruits revealed that the cuticle of the transformants were characterized by an increase of the thickness as well as significant increase in the content of cuticle components (cutin, phenolic compounds, and waxes). Furthermore, detailed analysis of the waxes revealed that the transformants displayed changes in waxes composition, showing higher levels of n-alkanes and triterpenoids which can shift the proportion of crystalline and amorphous waxes and change the water flux through the cuticle. Expression of the genes involved in cuticle biosynthesis indicated that LIN5 influences the biosynthesis of components of the cuticle, indicating that this process is coupled to sugar uploading via a mechanism which links carbon supply with the capacity for fruit expansion.

Genetic diversity of strawberry germplasm using metabolomic biomarkers

High-throughput metabolomics technologies can provide the quantification of metabolites levels across various biological processes in different tissues, organs and species, allowing the identification of genes underpinning these complex traits. Information about changes of metabolites during strawberry development and ripening processes is key to aiding the development of new approaches to improve fruit attributes. We used network-based methods and multivariate statistical approaches to characterize and investigate variation in the primary and secondary metabolism of seven domesticated and seven wild strawberry fruit accessions at three different fruit development and ripening stages. Our results demonstrated that Fragaria sub-species can be identified solely based on the gathered metabolic profiles. We also showed that domesticated accessions displayed highly similar metabolic changes due to shared domestication history. Differences between domesticated and wild accessions were detected at the level of metabolite associations which served to rank metabolites whose regulation was mostly altered in the process of domestication. The discovery of comprehensive metabolic variation among strawberry accessions offers opportunities to probe into the genetic basis of variation, providing insights into the pathways to relate metabolic variation with important traits.

Fruit Ripening and QTL for Fruit Quality in the Octoploid Strawberry

Fruit development and ripening is a unique developmental process to flowering plants that ensures the propagation of seeds and plant survival. In addition, fruits are an essential part of human diet. In particular, strawberry is a rich source of nutraceuticals such as vitamin C, folate and phenolic compounds. Strawberry production and breeding is becoming an extremely competing area of economic development worldwide. Cost of production in many countries is increasing due to a number of challenges such as rising labour costs, pest control or water availability. One way to increase competiveness is increasing fruit quality of new strawberry cultivars. Amazing advances have been made in our knowledge of the different metabolic pathways that take place in the final stages of fruit development and that lead to a flavourful and ripe strawberry fruit. Similarly, different genes involved in gene regulation during ripening have been discovered and characterized. In parallel, the discovery of loci responsible for natural variation among strawberry germplasm is producing a growing amount of DNA markers that after validation could be used in accelerating the selection of new cultivars with improved fruit quality. This chapter summarizes main advances in the study of fruit ripening in the octoploid strawberry and QTL controlling fruit quality traits.