Auxin treatment of grapevine (Vitis vinifera L.) berries delays ripening onset by inhibiting cell expansion.

KEY MESSAGE: Auxin treatment of grape (Vitis vinifera L.) berries delays ripening by inducing changes in gene expression and cell wall metabolism and could combat some deleterious climate change effects. Auxins are inhibitors of grape berry ripening and their application may be useful to delay harvest to counter effects of climate change. However, little is known about how this delay occurs. The expression of 1892 genes was significantly changed compared to the control during a 48 h time-course where the auxin 1-naphthaleneacetic acid (NAA) was applied to pre-veraison grape berries. Principal component analysis showed that the control and auxin-treated samples were most different at 3 h post-treatment when approximately three times more genes were induced than repressed by NAA. There was considerable cross-talk between hormone pathways, particularly between those of auxin and ethylene. Decreased expression of genes encoding putative cell wall catabolic enzymes (including those involved with pectin) and increased expression of putative cellulose synthases indicated that auxins may preserve cell wall structure. This was confirmed by immunochemical labelling of berry sections using antibodies that detect homogalacturonan (LM19) and methyl-esterified homogalacturonan (LM20) and by labelling with the CMB3a cellulose-binding module. Comparison of the auxin-induced changes in gene expression with the pattern of these genes during berry ripening showed that the effect on transcription is a mix of changes that may specifically alter the progress of berry development in a targeted manner and others that could be considered as non-specific changes. Several lines of evidence suggest that cell wall changes and associated berry softening are the first steps in ripening and that delaying cell expansion can delay ripening providing a possible mechanism for the observed auxin effects.

Pectins, Hemicelluloses and Celluloses Show Specific Dynamics in the Internal and External Surfaces of Grape Berry Skin During Ripening.

Grapevine berry skin is a complex structure that contributes to the final size and shape of the fruit and affects its quality traits. The organization of cell wall polysaccharides in situ and their modification during ripening are largely uncharacterized. The polymer structure of Corvina berry skin, its evolution during ripening and related modifying genes were determined by combing mid-infrared micro-spectroscopy and multivariate statistical analysis with transcript profiling and immunohistochemistry. Spectra were acquired in situ using a surface-sensitive technique on internal and external sides of the skin without previous sample pre-treatment, allowing comparison of the related cell wall polymer dynamics. The external surface featured cuticle-related bands; the internal surface showed more adsorbed water. Application of surface-specific normalization revealed the major molecular changes related to hemicelluloses and pectins in the internal surface and to cellulose and pectins in the external surface and that they occur between mid-ripening and full ripening in both sides of the skin. Transcript profiling of cell wall-modifying genes indicated a general suppression of cell wall metabolism during ripening. Genes related to pectin metabolism-a ß-galactosidase, a pectin(methyl)esterase and a pectate lyase-and a xyloglucan endotransglucosylase/hydrolase, involved in hemicellulose modification, showed enhanced expression. In agreement with Fourier transform infrared spectroscopy, patterns due to pectin methyl esterification provided new insights into the relationship between pectin modifications and the associated transcript profile during skin ripening. This study proposes an original description of polymer dynamics in grape berries during ripening, highlighting differences between the internal and external sides of the skin.

A downstream process allowing the efficient isolation of a recombinant amphiphilic protein from tobacco leaves.

The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major autoantigen in autoimmune diabetes. The heterologous production of hGAD65 for diagnostic and therapeutic applications is hampered by low upstream productivity and the absence of a robust and efficient downstream process for product isolation. A tobacco-based platform has been developed for the production of an enzymatically-inactive form of the protein (hGAD65mut), but standard downstream processing strategies for plant-derived recombinant proteins cannot be used in this case because the product is amphiphilic. We therefore evaluated different extraction buffers and an aqueous micellar two-phase system (AMTPS) to optimize the isolation and purification of hGAD65mut from plants. We identified the extraction conditions offering the greatest selectivity for hGAD65mut over native tobacco proteins using a complex experimental design approach. Under our optimized conditions, the most efficient initial extraction and partial purification strategy achieved an overall hGAD65mut yield of 92.5% with a purification factor of 12.3 and a concentration factor of 23.8. The process also removed a significant quantity of phenols, which are major contaminants present in tobacco tissue. This is the first report describing the use of AMTPS for the partial purification of an amphiphilic recombinant protein from plant tissues and our findings could also provide a working model for the initial recovery and partial purification of hydrophobic recombinant proteins from transgenic tobacco plants.

The evolutionary history and diverse physiological roles of the grapevine calcium-dependent protein kinase gene family.

Calcium-dependent protein kinases (CDPKs) are molecular switches that bind Ca(2+), ATP, and protein substrates, acting as sensor relays and responders that convert Ca(2+) signals, created by developmental processes and environmental stresses, into phosphorylation events. The precise functions of the CDPKs in grapevine (Vitis vinifera) are largely unknown. We therefore investigated the phylogenetic relationships and expression profiles of the 17 CDPK genes identified in the 12x grapevine genome sequence, resolving them into four subfamilies based on phylogenetic tree topology and gene structures. The origins of the CDPKs during grapevine evolution were characterized, involving 13 expansion events. Transcriptomic analysis using 54 tissues and developmental stages revealed three types of CDPK gene expression profiles: constitutive (housekeeping CDPKs), partitioned functions, and prevalent in pollen/stamen. We identified two duplicated CDPK genes that had evolved from housekeeping to pollen-prevalent functions and whose origin correlated with that of seed plants, suggesting neofunctionalization with an important role in pollen development and also potential value in the breeding of seedless varieties. We also found that CDPKs were involved in three abiotic stress signaling pathways and could therefore be used to investigate the crosstalk between stress responses.

Cancer prevention in Europe: the Mediterranean diet as a protective choice.

In the coming years, European death rates because of cancer will further decline, but the overall number of cases will increase, mostly as a consequence of the ageing of the population. The target for cancer prevention in Europe will remain a healthy diet and control of obesity in addition to a decrease in smoking. A healthy diet model in European countries is the traditional Mediterranean diet, which is based on abundant and variable plant foods, high consumption of cereals, olive oil as the main (added) fat, low intake of (red) meat and moderate consumption of wine. The Mediterranean diet is associated with a reduced risk of cardiovascular disease and cancer. The biological mechanisms for cancer prevention associated with the Mediterranean diet have been related to the favourable effect of a balanced ratio of omega 6 and omega 3 essential fatty acids and high amounts of fibre, antioxidants and polyphenols found in fruit, vegetables, olive oil and wine. The Mediterranean diet also involves a 'Mediterranean way of drinking', that is, regular, moderate consumption of wine mainly with food. This pattern of drinking increases longevity, reduces the risk of cardiovascular disease and does not appreciably influence the overall risk of cancer. However, heavy alcohol drinking is associated with digestive, upper respiratory tract, liver and breast cancers; therefore, avoidance or restriction of alcohol consumption to two drinks/day in men and one drink/day in women is a global public health priority.

The grapevine expression atlas reveals a deep transcriptome shift driving the entire plant into a maturation program.

We developed a genome-wide transcriptomic atlas of grapevine (Vitis vinifera) based on 54 samples representing green and woody tissues and organs at different developmental stages as well as specialized tissues such as pollen and senescent leaves. Together, these samples expressed ∼91% of the predicted grapevine genes. Pollen and senescent leaves had unique transcriptomes reflecting their specialized functions and physiological status. However, microarray and RNA-seq analysis grouped all the other samples into two major classes based on maturity rather than organ identity, namely, the vegetative/green and mature/woody categories. This division represents a fundamental transcriptomic reprogramming during the maturation process and was highlighted by three statistical approaches identifying the transcriptional relationships among samples (correlation analysis), putative biomarkers (O2PLS-DA approach), and sets of strongly and consistently expressed genes that define groups (topics) of similar samples (biclustering analysis). Gene coexpression analysis indicated that the mature/woody developmental program results from the reiterative coactivation of pathways that are largely inactive in vegetative/green tissues, often involving the coregulation of clusters of neighboring genes and global regulation based on codon preference. This global transcriptomic reprogramming during maturation has not been observed in herbaceous annual species and may be a defining characteristic of perennial woody plants.

Sporophytic and gametophytic functions of the cell cycle-associated Mob1 gene in Arabidopsis thaliana L.

Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle.

Revealing impaired pathways in the an11 mutant by high-throughput characterization of Petunia axillaris and Petunia inflata transcriptomes.

Petunia is an excellent model system, especially for genetic, physiological and molecular studies. Thus far, however, genome-wide expression analysis has been applied rarely because of the lack of sequence information. We applied next-generation sequencing to generate, through de novo read assembly, a large catalogue of transcripts for Petunia axillaris and Petunia inflata. On the basis of both transcriptomes, comprehensive microarray chips for gene expression analysis were established and used for the analysis of global- and organ-specific gene expression in Petunia axillaris and Petunia inflata and to explore the molecular basis of the seed coat defects in a Petunia hybrida mutant, anthocyanin 11 (an11), lacking a WD40-repeat (WDR) transcription regulator. Among the transcripts differentially expressed in an11 seeds compared with wild type, many expected targets of AN11 were found but also several interesting new candidates that might play a role in morphogenesis of the seed coat. Our results validate the combination of next-generation sequencing with microarray analyses strategies to identify the transcriptome of two petunia species without previous knowledge of their genome, and to develop comprehensive chips as useful tools for the analysis of gene expression in P. axillaris, P. inflata and P. hybrida.

Two-dimensional differential in gel electrophoresis (2D-DIGE) analysis of grape berry proteome during postharvest withering.

The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement.

The ascorbic acid content of tomato fruits is associated with the expression of genes involved in pectin degradation.

BACKGROUND: High levels of ascorbic acid (AsA) in tomato fruits provide health benefits for humans and also play an important role in several aspects of plant life. Although AsA metabolism has been characterized in detail, the genetic mechanisms controlling AsA accumulation in tomatoes are poorly understood. The transcriptional control of AsA levels in fruits can be investigated by combining the advanced genetic and genomic resources currently available for tomato. A comparative transcriptomic analysis of fruit tissues was carried out on an introgression line containing a QTL promoting AsA accumulation in the fruit, using a parental cultivar with lower AsA levels as a reference. RESULTS: Introgression line IL 12-4 (S. pennellii in a S. lycopersicum background) was selected for transcriptomic analysis because it maintained differences in AsA levels compared to the parental genotypes M82 and S. pennellii over three consecutive trials. Comparative microarray analysis of IL 12-4 and M82 fruits over a 2-year period allowed 253 differentially-expressed genes to be identified, suggesting that AsA accumulation in IL 12-4 may be caused by a combination of increased metabolic flux and reduced utilization of AsA. In particular, the upregulation of a pectinesterase and two polygalacturonases suggests that AsA accumulation in IL12-4 fruit is mainly achieved by increasing flux through the L-galactonic acid pathway, which is driven by pectin degradation and may be triggered by ethylene. CONCLUSIONS: Based on functional annotation, gene ontology classification and hierarchical clustering, a subset of the 253 differentially-expressed transcripts was used to develop a model to explain the higher AsA content in IL 12-4 fruits in terms of metabolic flux, precursor availability, demand for antioxidants, abundance of reactive oxygen species and ethylene signaling.

Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation.

The effect of auxin on stamen and pistil development in tobacco flowers was investigated by means of the localized expression of rolB (root loci B), an Agrobacterium oncogene that increases auxin sensitivity in a cell-autonomous fashion. When rolB is driven by the promoter of the meiosis-specific Arabidopsis gene DMC1 (disrupted meiotic cDNA 1), expression occurs earlier in male than in female developing organs, resulting in a delay in anther dehiscence with respect to normal timing of pistil development. As a consequence of this developmental uncoupling, self-pollination is prevented in pDMC1:rolB plants. Histological analysis of pDMC1:GFP plants indicates that in tobacco, this promoter is active not only in meiocytes but also in somatic tissues of the anther. In contrast, simultaneous expression of rolB in anther and pistil somatic tissues, achieved by expressing a construct containing rolB under the control of the promoter of the petunia gene FBP7 (floral binding protein 7), results in a concomitant delay of both anther dehiscence and pistil development without affecting self-pollination of the plants. Analysis of plants harboring the pFBP7:GUS construct shows that in tobacco, this promoter is active not only in the ovules, as described for petunia, but also in pistil and anther somatic tissues involved in the dehiscence program. The delay in anther dehiscence and pistil development could be phenocopied by exogenous application of auxin. Jasmonic acid (JA) could not rescue the delay in anther dehiscence. These results suggest that auxin plays a key role in the timing of anther dehiscence, the dehiscence program is controlled by the somatic tissues of the anther, and auxin also regulates pistil development.

Dynamic 1-aminocyclopropane-1-carboxylate-synthase and -oxidase transcript accumulation patterns during pollen tube growth in tobacco styles.

In flowering plants, pollination of the stigma sets off a cascade of responses in the distal flower organs. Ethylene and its biosynthetic precursor 1-aminocyclopropane-1-carboxylate (ACC) play an important role in regulating these responses. Because exogenous application of ethylene or ACC does not invoke the full postpollination syndrome, the pollination signal probably consists of a more complex set of stimuli. We set out to study how and when the pollination signal moves through the style of tobacco (Nicotiana tabacum) by analyzing the expression patterns of pistil-expressed ACC-synthase and -oxidase genes. Results from this analysis showed that pollination induces high ACC-oxidase transcript levels in all cells of the transmitting tissue. ACC-synthase mRNA accumulated only in a subset of transmitting tract cells and to lower levels as compared with ACC-oxidase. More significantly, we found that although ACC-oxidase transcripts accumulate to uniform high levels, the ACC-synthase transcripts accumulate in a wave-like pattern in which the peak coincides with the front of the ingrowing pollen tube tips. This wave of ACC-synthase expression can also be induced by incongruous pollination and (partially) by wounding. This indicates that wounding-like features of pollen tube invasion might be part of the stimuli evoking the postpollination response and that these stimuli are interpreted differently by the regulatory mechanisms of the ACC-synthase and -oxidase genes.

Pollination modulates expression of the PPAL gene, a pistil-specific beta-expansin.

Using differential screening we isolated a pistil-specific cDNA clone corresponding to a 1.2 kb mRNA and encoding a 32.5 kDa protein. The amino acid sequence shared similarity with that of group-I grass pollen allergens, which are known to have expansin activity. This clone, which later showed to share homology also with beta-expansins, was named PPAL. The PPAL mRNA was specifically expressed in the secretory zone of the stigma and in the epidermal layer of the placenta. The accumulation level of the transcript increased during pollination, and the protein was secreted in the stigmatic exudate of the tobacco flower. We suggest here that PPAL is a new expansin, acting as a cell-wall-loosening agent during pollination.