Callose and cellulose synthase gene expression analysis from the tight cluster to the full bloom stage and during early fruit development in Malus × domestica.

Apple (Malus × domestica) is an economically important temperate fruit-bearing crop which belongs to the family of Rosaceae and its pomaceous fruit is one of the most commonly cultivated. Several studies have demonstrated that the cell wall plays a pivotal role during flower and fruit development. It takes active part in pollen tube growth and contributes to determine the fruit firmness trait through the action of cell wall-related enzymes (i.e. polygalacturonase and pectinmethylesterase). We have investigated the expression of callose and cellulose synthase genes during flowering from tight cluster to anthesis and during early fruit development in domesticated apple. We also link the changes observed in gene expression to the profile of soluble non-structural carbohydrates at different developmental stages of flowers/fruitlets and to the qualitative results linked to wall polysaccharides' composition obtained through near-infrared spectroscopy. This work represents an important addition to the study of tree physiology with respect to the analysis of the expression of callose and cellulose synthase genes during flower and early fruit development in domesticated apple.

Heat shock transcriptional factors in Malus domestica: identification, classification and expression analysis.

BACKGROUND: Heat shock transcriptional factors (Hsfs) play a crucial role in plant responses to biotic and abiotic stress conditions and in plant growth and development. Apple (Malus domestica Borkh) is an economically important fruit tree whose genome has been fully sequenced. So far, no detailed characterization of the Hsf gene family is available for this crop plant. RESULTS: A genome-wide analysis was carried out in Malus domestica to identify heat shock transcriptional factor (Hsf) genes, named MdHsfs. Twenty five MdHsfs were identified and classified in three main groups (class A, B and C) according to the structural characteristics and to the phylogenetic comparison with Arabidopsis thaliana and Populus trichocarpa. Chromosomal duplications were analyzed and segmental duplications were shown to have occurred more frequently in the expansion of Hsf genes in the apple genome. Furthermore, MdHsfs transcripts were detected in several apple organs, and expression changes were observed by quantitative real-time PCR (qRT-PCR) analysis in developing flowers and fruits as well as in leaves, harvested from trees grown in the field and exposed to the naturally increased temperatures. CONCLUSIONS: The apple genome comprises 25 full length Hsf genes. The data obtained from this investigation contribute to a better understanding of the complexity of the Hsf gene family in apple, and provide the basis for further studies to dissect Hsf function during development as well as in response to environmental stimuli.

Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A.

Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus×domestica, was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53-CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.×domestica are discussed.

Developmental and heat stress-regulated expression of HsfA2 and small heat shock proteins in tomato anthers.

The high sensitivity of male reproductive cells to high temperatures may be due to an inadequate heat stress response. The results of a comprehensive expression analysis of HsfA2 and Hsp17-CII, two important members of the heat stress system, in the developing anthers of a heat-tolerant tomato genotype are reported here. A transcriptional analysis at different developmental anther/pollen stages was performed using semi-quantitative and real-time PCR. The messengers were localized using in situ RNA hybridization, and protein accumulation was monitored using immunoblot analysis. Based on the analysis of the gene and protein expression profiles, HsfA2 and Hsp17-CII are finely regulated during anther development and are further induced under both short and prolonged heat stress conditions. These data suggest that HsfA2 may be directly involved in the activation of protection mechanisms in the tomato anther during heat stress and, thereby, may contribute to tomato fruit set under adverse temperatures.

Purification of triacylglycerols for biodiesel production from Nannochloropsis microalgae by membrane technology.

Triacylglycerols recovery from wet microalgae is a key aspect of biodiesel production, because of the energetic balance gained from avoiding biomass drying. In order to isolate TAG from Nannochloropsis cells, the possibility to concentrate biomass and to recover TAG in a single step by membrane process was studied. Different polymeric membranes were selected and screened on the basis of adsorption test and permeation flux. Results showed that membrane of regenerated cellulose (RC) with nominal molecular weight cutoff of 100 kDa and 30 kDa gave the best performance. Indeed, permeate flux was stable during ultrafiltration experiment in concentration mode and no severe fouling/cake deposition was observed. Both membranes allowed to recover permeates with high content of triacylglicerols. However, a more purity of the triacylglicerols from the other co-products was only obtained with the 30 kDa RC membrane because the retention of the unwanted proteins was in the range of 89%.

Ensuring Reproduction at High Temperatures: The Heat Stress Response during Anther and Pollen Development.

Sexual reproduction in flowering plants is very sensitive to environmental stresses, particularly to thermal insults which frequently occur when plants grow in field conditions in the warm season. Although abnormalities in both male and female reproductive organs due to high temperatures have been described in several crops, the failure to set fruits has mainly been attributed to the high sensitivity of developing anthers and pollen grains, particularly at certain developmental stages. A global view of the molecular mechanisms involved in the response to high temperatures in the male reproductive organs will be presented in this review. In addition, transcriptome and proteomic data, currently available, will be discussed in the light of physiological and metabolic changes occurring during anther and pollen development. A deep understanding of the molecular mechanisms involved in the stress response to high temperatures in flowers and, particularly, in the male reproductive organs will be a major step towards development of effective breeding strategies for high and stable production in crop plants.

Gene expression and biochemical changes of carbohydrate metabolism in in vitro micro-propagated apple plantlets infected by 'Candidatus Phytoplasma mali'.

'Candidatus Phytoplasma mali' (Ca. P. mali) is the disease agent causing apple proliferation (AP), which has detrimental effects on production in many apple growing areas of Central and Southern Europe. The present study investigated transcriptional and biochemical changes related to the sugar metabolism as well as expression of pathogenesis-related (PR) protein genes in in vitro micro-propagated AP-infected and healthy control plantlets with the aim of shedding light on host plant response to 'Ca. P. mali' infection. Expression changes between infected and control plantlets were detected by quantitative real-time PCR analysis. The most significant transcriptional changes were observed for genes coding for pathogenesis-related proteins and for heat shock protein 70, as well as for some genes related to the sugar metabolism, such as a sorbitol transporter (SOT5), hexokinase, sucrose-phosphate synthase or granule bound starch synthase. Furthermore, biochemical analyses revealed that infected plantlets were characterized by a significant accumulation of starch and sucrose, while hexoses, such as glucose and fructose, and sorbitol were present at lower concentrations. In summary, the present analysis provides an overview of a gene set that is involved in response to phytoplasma infection and, therefore, it may help for a better understanding of the molecular mechanisms involved in phytoplasma-host plant interaction in apple.

A gene expression analysis of cell wall biosynthetic genes in Malus x domestica infected by 'Candidatus Phytoplasma mali'.

Apple proliferation (AP) represents a serious threat to several fruit-growing areas and is responsible for great economic losses. Several studies have highlighted the key role played by the cell wall in response to pathogen attack. The existence of a cell wall integrity signaling pathway which senses perturbations in the cell wall architecture upon abiotic/biotic stresses and activates specific defence responses has been widely demonstrated in plants. More recently a role played by cell wall-related genes has also been reported in plants infected by phytoplasmas. With the aim of shedding light on the cell wall response to AP disease in the economically relevant fruit-tree Malus × domestica Borkh., we investigated the expression of the cellulose (CesA) and callose synthase (CalS) genes in different organs (i.e., leaves, roots and branch phloem) of healthy and infected symptomatic outdoor-grown trees, sampled over the course of two time points (i.e., spring and autumn 2011), as well as in in vitro micropropagated control and infected plantlets. A strong up-regulation in the expression of cell wall biosynthetic genes was recorded in roots from infected trees. Secondary cell wall CesAs showed up-regulation in the phloem tissue from branches of infected plants, while either a down-regulation of some genes or no major changes were observed in the leaves. Micropropagated plantlets also showed an increase in cell wall-related genes and constitute a useful system for a general assessment of gene expression analysis upon phytoplasma infection. Finally, we also report the presence of several 'knot'-like structures along the roots of infected apple trees and discuss the occurrence of this interesting phenotype in relation to the gene expression results and the modalities of phytoplasma diffusion.