Exploration of alternative splicing events in ten different grapevine cultivars.

BACKGROUND: The complex dynamics of gene regulation in plants are still far from being fully understood. Among many factors involved, alternative splicing (AS) in particular is one of the least well documented. For many years, AS has been considered of less relevant in plants, especially when compared to animals, however, since the introduction of next generation sequencing techniques the number of plant genes believed to be alternatively spliced has increased exponentially. RESULTS: Here, we performed a comprehensive high-throughput transcript sequencing of ten different grapevine cultivars, which resulted in the first high coverage atlas of the grape berry transcriptome. We also developed findAS, a software tool for the analysis of alternatively spliced junctions. We demonstrate that at least 44% of multi-exonic genes undergo AS and a large number of low abundance splice variants is present within the 131.622 splice junctions we have annotated from Pinot noir. CONCLUSIONS: Our analysis shows that ~70% of AS events have relatively low expression levels, furthermore alternative splice sites seem to be enriched near the constitutive ones in some extent showing the noise of the splicing mechanisms. However, AS seems to be extensively conserved among the 10 cultivars.

Antioxidant Defenses in Plants with Attention to Prunus and Citrus spp.

This short review briefly introduces the formation of reactive oxygen species (ROS) as by-products of oxidation/reduction (redox) reactions, and the ways in which the antioxidant defense machinery is involved directly or indirectly in ROS scavenging. Major antioxidants, both enzymatic and non enzymatic, that protect higher plant cells from oxidative stress damage are described. Biochemical and molecular features of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) are discussed because they play crucial roles in scavenging ROS in the different cell compartments and in response to stress conditions. Among the non enzymatic defenses, particular attention is paid to ascorbic acid, glutathione, flavonoids, carotenoids, and tocopherols. The operation of ROS scavenging systems during the seasonal cycle and specific developmental events, such as fruit ripening and senescence, are discussed in relation to the intense ROS formation during these processes that impact fruit quality. Particular attention is paid to Prunus and Citrus species because of the nutritional and antioxidant properties contained in these commonly consumed fruits.

Expression of a metallothionein A1 gene of Pisum sativum in white poplar enhances tolerance and accumulation of zinc and copper.

Metallothioneins (MT) play an important role in heavy metal detoxification and homeostasis of intracellular metal ions in plant. In this study, two transgenic lines expressing MT type 2 gene (PsMT(A1)) from Pisum sativum, a regenerated non transformed line NT and clone AL22, selected as heavy metal tolerant, were characterized in presence of the heavy metals for the ability to accumulate zinc and copper and to activate antioxidative enzyme defences: superoxide dismutase, catalase, ascorbate peroxidase. The levels of expression of MT type 2 gene assessed by RT-qPCR confirmed the gene over-expression in transgenic lines and evidenced in NT and AL22 the up-regulation of gene transcription by zinc and copper. Transgenic poplar lines during heavy metal stress displayed increased ability to translocate and accumulate zinc and copper compared with NT and AL22. The antioxidant enzyme defence was differently activated in response to metals in the transgenic lines without a significant increase of ROS. These results suggested that PsMT(A1) could play a role in ROS scavenging leading to enhanced metal tolerance and increased zinc and copper sequestration in root and leaf.

Type 3 metallothioneins respond to water deficit in leaf and in the cambial zone of white poplar (Populus alba).

The involvement of metallothioneins (MTs) in response to plant water stress and recovery was assessed by analyzing gene expression in leaves and in the cambial zone of white poplar. One-year-old plants were submitted to two different watering regimes: irrigation was withheld for 9d and then resumed until day 17, or soil moisture was maintained to field capacity by irrigation during the experiment. Changes in leaves and stem water relations, gas exchange and CO(2) assimilation were recorded. The expression profiles of MT genes were analyzed in developing leaves and the cambial zone at maximum stress levels and after recovery and compared with the watered controls. Whole-plant water relations were significantly affected by water deprivation, though a complete recovery of plant water status was reached after resumption of watering. Withholding irrigation resulted in a significant decrease of leaf turgor potential and relative water content without a significant increase of the osmotic potential at full turgor. Similarly, stem water content decreased, leading to a marked increase of stem shrinkage, confirming that mild water stress affected primarily tissue water status. Following water depletion, the transcript analysis of MT genes revealed increased expression of type 3a and 3b MT genes in cambial tissues, and particularly in leaves. After water resumption, transcription decreased, suggesting that the changes in gene expression were related to water deficit. The results indicate that in leaves and, for the first time, in the cambial zone, type 3 MTs respond in a specific manner to changes in water status. These results are consistent with the regulatory cis-elements present in the 5' flanking region of type 3 MT genes.

New evidence of similarity between human and plant steroid metabolism: 5alpha-reductase activity in Solanum malacoxylon.

The physiological role of steroid hormones in humans is well known, and the metabolic pathway and mechanisms of action are almost completely elucidated. The role of plant steroid hormones, brassinosteroids, is less known, but an increasing amount of data on brassinosteroid biosynthesis is showing unexpected similarities between human and plant steroid metabolic pathways. Here we focus our attention on the enzyme 5alpha-reductase (5alphaR) for which a plant ortholog of the mammalian system, DET2, was recently described in Arabidopsis thaliana. We demonstrate that campestenone, the natural substrate of DET2, is reduced to 5alpha-campestanone by both human 5alphaR isozymes but with different affinities. Solanum malacoxylon, which is a calcinogenic plant very active in the biosynthesis of vitamin D-like molecules and sterols, was used to study 5alphaR activity. Leaves and calli were chosen as examples of differentiated and undifferentiated tissues, respectively. Two separate 5alphaR activities were found in calli and leaves of Solanum using campestenone as substrate. The use of progesterone allowed the detection of both activities in calli. Support for the existence of two 5alphaR isozymes in S. malacoxylon was provided by the differential actions of inhibitors of the human 5alphaR in calli and leaves. The evidence for the presence of two isozymes in different plant tissues extends the analogies between plant and mammalian steroid metabolic pathways.