Victoriano Valpuesta

Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase

L-Ascorbic acid (vitamin C) in fruits and vegetables is an essential component of human nutrition. Surprisingly, only limited information is available about the pathway(s) leading to its biosynthesis in plants. Here, we report the isolation and characterization of GalUR, a gene from strawberry that encodes an NADPH-dependent D-galacturonate reductase. We provide evidence that the biosynthesis of L-ascorbic acid in strawberry fruit occurs through D-galacturonic acid, a principal component of cell wall pectins. Expression of GalUR correlated with changing ascorbic acid content in strawberry fruit during ripening and with variations in ascorbic acid content in fruit of different species of the genus Fragaria. Reduced pectin solubilization in cell walls of transgenic strawberry fruit with decreased expression of an endogenous pectate lyase gene resulted in lower ascorbic acid content. Overexpression of GalUR in Arabidopsis thaliana enhanced vitamin C content two- to threefold, demonstrating the feasibility of engineering increased vitamin C levels in plants using this gene.

Manipulation of Strawberry Fruit Softening by Antisense Expression of a Pectate Lyase Gene

Strawberry (Fragaria ×ananassa, Duch., cv Chandler) is a soft fruit with a short postharvest life, mainly due to a rapid lost of firm texture. To control the strawberry fruit softening, we obtained transgenic plants that incorporate an antisense sequence of a strawberry pectate lyase gene under the control of the 35S promoter. Forty-one independent transgenic lines (Apel lines) were obtained, propagated in the greenhouse for agronomical analysis, and compared with control plants, non-transformed plants, and transgenic lines transformed with the pGUSINT plasmid. Total yield was significantly reduced in 33 of the 41 Apel lines. At the stage of full ripen, no differences in color, size, shape, and weight were observed between Apel and control fruit. However, in most of the Apel lines, ripened fruits were significantly firmer than controls. Six Apel lines were selected for further analysis. In all these lines, the pectate lyase gene expression in ripened fruit was 30% lower than in control, being totally suppressed in three of them. Cell wall material isolated from ripened Apel fruit showed a lower degree of in vitro swelling and a lower amount of ionically bound pectins than control fruit. An analysis of firmness at three different stages of fruit development (green, white, and red) showed that the highest reduction of softening in Apel fruit occurred during the transition from the white to the red stage. The postharvest softening of Apel fruit was also diminished. Our results indicate that pectate lyase gene is an excellent candidate for biotechnological improvement of fruit softening in strawberry.

Vitamin deficiencies in humans: can plant science help?

The term vitamin describes a small group of organic compounds that are absolutely required in the human diet. Although for the most part, dependency criteria are met in developed countries through balanced diets, this is not the case for the five billion people in developing countries who depend predominantly on a single staple crop for survival. Thus, providing a more balanced vitamin intake from high-quality food remains one of the grandest challenges for global human nutrition in the coming decade(s). Here, we describe the known importance of vitamins in human health and current knowledge on their metabolism in plants. Deficits in developing countries are a combined consequence of a paucity of specific vitamins in major food staple crops, losses during crop processing, and/or overreliance on a single species as a primary food source. We discuss the role that plant science can play in addressing this problem and review successful engineering of vitamin pathways. We conclude that while considerable advances have been made in understanding vitamin metabolic pathways in plants, more cross-disciplinary approaches must be adopted to provide adequate levels of all vitamins in the major staple crops to eradicate vitamin deficiencies from the global population.

Developing salt tolerant plants in a new century: a molecular biology approach

Soil salinity is a major abiotic stress in plant agriculture strongly, influencing plant productivity world-wide. Classical breeding for salt tolerance in crop plants has been attempted to improve field performance without success. Therefore, an alternative strategy is to generate salt tolerant plants through genetic engineering. Several species and experimental approaches have been used in order to identify those genes that are important for salt tolerance. Due to high level of salt tolerance, halophytes are good candidates to identify salt tolerance genes. However, other species such as yeast and glycophytes have also been employed. Three approaches are commonly used to identify genes important for salt tolerance. The first approach is to identify genes involved in processes known to be critical for salt tolerance (osmolyte synthesis, ion homeostasis, etc.). The second approach is to identify genes whose expression is regulated by salt stress. This is relatively simply and applicable to any plant species. Genetic amenability of some species allows the third approach, which consists in the identification of salt tolerance determinants based on functionality. At the moment, there is a large number of reports in the literature claiming that plants with increased salt tolerance have been obtained. The main problem is that different plant species, stage of development, organs, promoters and salt conditions used it is difficult to compare the degree of salt tolerance conferred by different genes. In this review, we discuss progress made towards understanding the molecular elements involved in salt stress responses that have been used in transgenic approaches to improve salt tolerance.

Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca2+-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca2+-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.

Up-regulation of a cysteine protease accompanies the ethylene-insensitive senescence of daylily (Hemerocallis) flowers

The flowers of daylily (Hemerocallis × hybrida cv. Cradle Song) open at midnight, start to senesce 12 h later, and are completely senescent by the following midnight. Differential screening of a cDNA library constructed from tepals of flowers showing incipient senescence revealed 25 clones that were strongly up-regulated in senescent tepals. Re-screening and interactive Southern analysis of these clones revealed 3 families of up-regulated clones. Transcripts of one clone, SEN10, were not detectable at midnight, but increased dramatically as senescence proceeded. The derived amino acid sequence of the full-length cDNA (SEN102) has strong homology with cysteine proteases that have been reported from other plant tissues. The sequence contains a secretory signal peptide and a probable prosequence upstream of the mature protein. Amino acids critical to the active site and structure of cysteine proteases are conserved, and the C-terminus of the polypeptide has a unique putative endoplasmic reticulum retention signal -RDEL.

Analysis of the expression of two thiolprotease genes from daylily (Hemerocallis spp.) during flower senescence.

A cDNA clone encoding a daylily (Hemerocallis spp.) thiolprotease (SEN11), whose expression is strongly up-regulated in flower tepal senescence, has been isolated. The amino acid sequence, deduced from the nucleotide sequence, showed highest similarity to plant thiolproteases of Vigna mungo, Phaseolus vulgaris and Hemerocallis (SEN102), and contains a putative ER retention signal that has been described in Vigna mungo. SEN102 and SEN11 transcripts were not detectable in flower buds at the opening stage, but two peaks of transcripts were seen after 9 h and 19 h, in both petals and sepals, when wilting symptoms were apparent. The pattern of protease activity migrating on a 26.3 kDa protein was similar to the SEN102 and SEN11 transcript profiles. These two genes were also expressed in stamens and leaves, but their transcripts were undetectable in carpels and rhizomes. The expression of SEN102 was lower in the senescent leaf than in the green leaf. The pattern of expression of these genes suggests their involvement in the protein hydrolysis occurring in tepals at the late senescence stage, whereas in leaves they could be involved in the constitutive protein turnover machinery. Exogenous gibberellic acid application to cut flowers increased transcripts of both genes.

Developing a core collection of olive (Olea europaea L.) based on molecular markers (DArTs, SSRs, SNPs) and agronomic traits

Molecular markers (SSR, SNP and DArT) and agronomical traits have been used in the world’s largest olive (Olea europaea L.) germplasm collection (IFAPA, Centre Alameda del Obispo, Cordoba, Spain) to study the patterns of genetic diversity and underlying genetic structure among 361 olive accessions. In addition the marker data were used to construct a set of core collections by means of two different algorithms (MSTRAT and PowerCore) based on M (maximization) strategy. Our results confirm that the germplasm collection is a useful source of genetically diverse material. We also found that geographical origin is an important factor structuring genetic diversity in olive. Subsets of 18, 27, 36, 45 and 68 olive accessions, representing respectively 5%, 7.5%, 10%, 12.5% and 19% of the whole germplasm collection, were selected based on the information obtained by all the data set as well as each marker type considered individually. According to our results, the core collections that represent between 19% and 10% of the total collection size could be considered as optimal to retain the bulk of the genetic diversity found in this collection. Due to its high efficiency at capturing all the alleles/traits states found in the whole collection, the core size of 68 accessions could be of special interest for genetic conservation applications in olive. The high average genetic distance and diversity and the almost equal representation of accessions from different geographical regions indicate that the core size of 36 accessions, could be the working collection for olive breeders.

Improved germination under osmotic stress of tobacco plants overexpressing a cell wall peroxidase

The cell wall is a fundamental component in the response of plants to environmental changes. To directly assess the role of the cell wall we have increased the expression and activity of a cell wall associated peroxidase (TPX2), an enzyme involved in modifying cell wall architecture. Overexpression of TPX2 had no effect on wild‐type development, but greatly increased the germination rate under high salt or osmotic stress. Differential scanning calorimetry showed that transgenic seeds were able to retain more water available for germination than wild‐type seeds. Thermoporometry calculations indicated that this could be due to a lower mean pore size in the walls of transgenic seeds. Therefore, the higher capacity of transgenic seeds in retaining water could result in higher germination rates in conditions where the availability of water is restricted.

Regulation of L-ascorbic acid content in strawberry fruits

Plants have several L-ascorbic acid (AsA) biosynthetic pathways, but the contribution of each one to the synthesis of AsA varyies between different species, organs, and developmental stages. Strawberry (Fragaria×ananassa) fruits are rich in AsA. The pathway that uses D-galacturonate as the initial substrate is functional in ripe fruits, but the contribution of other pathways to AsA biosynthesis has not been studied. The transcription of genes encoding biosynthetic enzymes such as D-galacturonate reductase (FaGalUR) and myo-inositol oxygenase (FaMIOX), and the AsA recycling enzyme monodehydroascorbate reductase (FaMDHAR) were positively correlated with the increase in AsA during fruit ripening. Fruit storage for 72 h in a cold room reduced the AsA content by 30%. Under an ozone atmosphere, this reduction was 15%. Ozone treatment increased the expression of the FaGalUR, FaMIOX, and L-galactose-1-phosphate phosphatase (FaGIPP) genes, and transcription of the L-galactono-1,4-lactone dehydrogenase (FaGLDH) and FAMDHAR genes was higher in the ozone-stored than in the air-stored fruits. Analysis of AsA content in a segregating population from two strawberry cultivars showed high variability, which did not correlate with the transcription of any of the genes studied. Study of GalUR protein in diverse cultivars of strawberry and different Fragaria species showed that a correlation between GalUR and AsA content was apparent in most cases, but it was not general. Three alleles were identified in strawberry, but any sequence effect on the AsA variability was eliminated by analysis of the allele-specific expression. Taken together, these results indicate that FaGalUR shares the control of AsA levels with other enzymes and regulatory elements in strawberry fruit.