Exogenous γ-aminobutyric acid treatment improves the cold tolerance of zucchini fruit during postharvest storage.

This work examines the effect of a treatment with 1 mM of γ-aminobutyric acid (GABA) on zucchini fruit during postharvest cold storage. Specifically, the effect of GABA on postharvest quality was measured, as well as its implication in the GABA shunt and other related metabolic pathways. The treatments were performed in Sinatra, a variety of zucchini highly sensitive to low-temperature storage. The application of GABA improved the quality of zucchini fruit stored at 4 °C, with a reduction of chilling-injury index, weight loss, and cell death, as well as a lower rate of electrolyte leakage. GABA content was significantly higher in the treated fruit than in the control fruit at all times analyzed. At the end of the storage period, GABA-treated fruit had higher contents of both proline and putrescine. The catabolism of this polyamine was not affected by exogenous GABA. Also, over the long term, the treatment induced the GABA shunt by increasing the activities of the enzymes GABA transaminase (GABA-T) and glutamate decarboxylase (GAD). GABA-treated fruit contained higher levels of fumarate and malate than did non-treated fruit, as well as higher ATP and NADH contents. These results imply that the GABA shunt is involved in providing metabolites to produce energy, reduce power, and help the fruit to cope with cold stress over the long term.

Thiol-based redox regulation in sexual plant reproduction: new insights and perspectives.

The success of sexual reproduction in plants involves (i) the proper formation of the plant gametophytes (pollen and embryo sac) containing the gametes, (ii) the accomplishment of specific interactions between pollen grains and the stigma, which subsequently lead to (iii) the fusion of the gametes and eventually to (iv) the seed setting. Owing to the lack of mobility, plants have developed specific regulatory mechanisms to control all developmental events underlying the sexual plant reproduction according to environmental challenges. Over the last decade, redox regulation and signaling have come into sight as crucial mechanisms able to manage critical stages during sexual plant reproduction. This regulation involves a complex redox network which includes reactive oxygen species (ROS), reactive nitrogen species (RNS), glutathione and other classic buffer molecules or antioxidant proteins, and some thiol/disulphide-containing proteins belonging to the thioredoxin superfamily, like glutaredoxins (GRXs) or thioredoxins (TRXs). These proteins participate as critical elements not only in the switch between the mitotic to the meiotic cycle but also at further developmental stages of microsporogenesis. They are also implicated in the regulation of pollen rejection as the result of self-incompatibility. In addition, they display precise space-temporal patterns of expression and are present in specific localizations like the stigmatic papillae or the mature pollen, although their functions and subcellular localizations are not clear yet. In this review we summarize insights and perspectives about the presence of thiol/disulphide-containing proteins in plant reproduction, taking into account the general context of the cell redox network.

Co-ordination of developmental processes by small RNAs during leaf development.

Leaf development entails the transition from a small group of undifferentiated cells to a structure of defined size and shape, highly organized into different cell types with specialized functions. During this developmental sequence, patterning, growth, and differentiation have to be tightly coordinated by intricate regulatory networks in which small RNAs [microRNAs (miRNAs) and trans-acting small interfering RNAs (ta-siRNAs)] have emerged during the last years as essential players. In this review, after having given an overview of miRNA and ta-siRNA biogenesis and mode of action, their contribution to the life of a leaf from initiation to senescence is described. MiRNA and ta-siRNA are not merely regulators of gene expression patterns, but, by acting both locally and at the whole organ scale, they have an essential role in the coordination of complex developmental processes and are fully integrated in genetic networks and signalling pathways.

Arabidopsis monomeric G-proteins, markers of early and late events in cell differentiation.

In Schizosaccharomyces pombe, septum formation is intricately controlled by proteins which constitute the SIN (Septum Initiation Network) signalling cascade. The SIN ensures the coordination between mitotic exit and cytokinesis. Yeast spg1p is a core component of the SIN pathway and we have previously characterized the two orthologs of this G-protein in Arabidopsis thaliana (named AtSGP1 and 2). In this work, the cell and tissue expression of AtSGP genes during plant development has been analysed using AtSGP promoter::GUS fusions in stably transformed A. thaliana lines. AtSGP1 promoter activity was restricted to the quiescent centre, collumella cells, stomata guard cells and the stele while AtSGP2 promoter activity was detected in atrichoblasts, trichomes and pollen. The observed promoter activities are in accordance with publicly available pollen, stomata guard cell and root transcriptome data. Two-hybrid experiments previously evidenced an interaction between AtMAP3Kepsilon1 and AtSGP1. The AtMAP3Kepsilon1 promoter activity was detected in root apices, trichomes and ovule integuments. A genetic approach involving both markers of these specialized cells and mutant backgrounds was used to reinforce our hypothesis. It appears that, although highly conserved between plants and fungi, the spg1p G-protein has evolved in plants to perform a function different from the SIN pathway. Interestingly, cells expressing AtSGPs possessed limited or null mitotic activity. Our data suggests that AtSGP are crucial signalling components involved either in early cell fate specification, or in the final steps of cell differentiation. This is an interesting starting point for a wider study devoted to functional experiments designed to test these hypotheses.

HvPG1 and ECA1: two genes activated transcriptionally in the transition of barley microspores from the gametophytic to the embryogenic pathway.

Microspores genetically programmed to produce male gametes can be switched to the embryogenic pathway to give rise to haploid embryos. Microspore embryogenesis is usually induced in barley by stress pre-treatment applied to vacuolated microspores. We studied the expression of two genes during the early stages of microspore embryogenesis to gain further insight into the microspore transition from the gametophytic to the embryogenic pathway. RT-PCR together with in situ hybridization on sections (ISH) and whole-mount in situ hybridization (WISH) were used to analyse the expression of the early-culture abundant gene (ECA1), which is expressed in barley during microspore embryogenesis, and a polygalacturonase gene (HvPG1), a late pollen gene expressed during gametogenesis only after microspore division. Both ECA1 and HvPG1 genes were transcriptionally active after stress pre-treatment in the same populations of microspore-derived structures, representing the sporophytically induced ones. ECA1 transcripts were also detected after 3 days' culture. Our results point to the possibility of using ECA1 gene expression as a marker for the induction of microspore embryogenesis and the earliest stages of this process. Finally, we demonstrate that WISH is a suitable technique for studying gene expression in embryogenic microspore populations and, because different structures can be examined individually, is an appropriate complement to transcriptomic profile analyses in the study of early microspore embryogenesis.

A conserved molecular framework for compound leaf development.

Diversity in leaf shape is produced by alterations of the margin: for example, deep dissection leads to leaflet formation and less-pronounced incision results in serrations or lobes. By combining gene silencing and mutant analyses in four distantly related eudicot species, we show that reducing the function of NAM/CUC boundary genes (NO APICAL MERISTEM and CUP-SHAPED COTYLEDON) leads to a suppression of all marginal outgrowths and to fewer and fused leaflets. We propose that NAM/CUC genes promote formation of a boundary domain that delimits leaflets. This domain has a dual role promoting leaflet separation locally and leaflet formation at distance. In this manner, boundaries of compound leaves resemble boundaries functioning during animal development.

PsTRXh1 and PsTRXh2 are both pea h-type thioredoxins with antagonistic behavior in redox imbalances.

Thioredoxins (TRXs) are small ubiquitous oxidoreductases involved in disulfide bond reduction of a large panel of target proteins. The most complex cluster in the family of plant TRXs is formed by h-type TRXs. In Arabidopsis (Arabidopsis thaliana), nine members of this subgroup were described, which are less well known than their plastidial counterparts. The functional study of type-h TRXs is difficult because of the high number of isoforms and their similar biochemical characteristics, thus raising the question whether they have specific or redundant functions. Type-h TRXs are involved in seed germination and self incompatibility in pollen-pistil interaction. Their function as antioxidants has recently been proposed, but further work is needed to clarify this function in plants. In this study, we describe two new h-type TRXs from pea (Pisum sativum; stated PsTRXh1 and PsTRXh2). By functional complementation of a yeast (Saccharomyces cerevisiae) trx1Delta trx2Delta double mutant, we demonstrate that PsTRXh1 is involved in the redox-imbalance control, possibly through its interaction with peroxiredoxins. In contrast, PsTRXh2 provokes a phenotype of hypersensitivity to hydrogen peroxide in the yeast mutant. Furthermore, we show differential gene expression and protein accumulation of the two isoforms, PsTRXh1 protein being abundantly detected in vascular tissue and flowers, whereas PsTRXh2 gene expression was hardly detectable. By comparison with previous data of additional PsTRXh isoforms, our results indicate specific functions for the pea h-type TRXs so far described.

Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development.

Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time hordein expression during microspore embryogenesis. Surprisingly, these hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.

Programmed cell death during the transition from multicellular structures to globular embryos in barley androgenesis.

Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos.