Transcriptional analysis of differentially expressed genes in response to stem inclination in young seedlings of pine

The gravitropic response in trees is a widely studied phenomenon, however understanding of the molecular mechanism involved remains unclear. The purpose of this work was to identify differentially expressed genes in response to inclination using a comparative approach for two conifer species. Young seedlings were subjected to inclination and samples were collected at four different times points. First, suppression subtractive hybridisation (SSH) was used to identify differentially regulated genes in radiata pine (Pinus radiata D. Don). cDNA libraries were constructed from the upper and lower part of inclined stems in a time course experiment, ranging from 2.5 h to 1 month. From a total of 3092 sequences obtained, 2203 elements were assembled, displaying homology to a public database. A total of 942 unigene elements were identified using bioinformatic tools after redundancy analysis. Of these, 614 corresponded to known function genes and 328 to unknown function genes, including hypothetical proteins. Comparative analysis between radiata pine and maritime pine (Pinus pinaster Ait.) was performed to validate the differential expression of relevant candidate genes using qPCR. Selected genes were involved in several functional categories: hormone regulation, phenylpropanoid pathway and signal transduction. This comparative approach for the two conifer species helped determine the molecular gene pattern generated by inclination, providing a set of Pinus gene signatures that may be involved in the gravitropic stress response. These genes may also represent relevant candidate genes involved in the gravitropic response and potentially in wood formation.     

Characterization of a pine multigene family containing elicitor- responsive stilbene synthase genes

Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial -glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.     

Selection and testing of reference genes for accurate RT-qPCR in adult needles and seedlings of maritime pine

Quantitative real-time PCR (RT-qPCR) techniques have revolutionized gene expression analyses. To obtain accurate results, raw RT-qPCR results need to be normalized by using endogenous reference genes whose expression is assumed invariable in all studied samples. However, there are no universal reference genes, and candidate genes need to be evaluated for each experimental condition. In this work, we tested a set of possible reference genes for use in different organs and tissues of Pinus pinaster (needles from adult trees and different organs and developmental stages of seedlings). The putative reference genes were selected using microarray analyses and from those commonly used in previous works. To achieve reproducible and reliable results, Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed. To highlight the importance of these rules, 10 alternative primer pairs to be evaluated in pine samples were designed by following or not following the MIQE guidelines. Twenty-four candidate reference genes were tested in pine needles and 14 were also tested in pine seedlings. In both cases, valid reference genes were found, but differences in the stability and expression levels were also observed. Furthermore, a few of the best genes had unknown functions. The five most stable genes in the pine seedlings as well as four new candidate reference genes were evaluated in isolated tissues using laser capture microdissection. The results showed that the appropriate reference genes in different maritime pine organs were not invariable when sourced from the different tissues forming the organs.     

Expressed sequences from the basidiomycetous tree pathogen Heterobasidion annosum during early infection of scots pine

The pattern of gene expression of the basidiomycete Heterobasidion annosum, causal agent of the root rot of conifers, was analysed during its interaction with pine roots. A complementary DNA (cDNA) library was constructed from total RNA extracted from H. annosum mycelia challenged with Scots pine seedling roots for 6 and 72h. Single pass sequencing of 1148 randomly selected cDNA clones resulted in 923 expressed sequence tags (ESTs). Contig analysis and sequence comparisons identified 318 unigene sequences, of which 62 were repeatedly sampled. A putative cellular function was assigned to 223 contigs (70%) that showed a moderate to high homology to protein sequences from public databases. Variations in expression levels during the infection process were monitored on a set of 96 unigenes by reverse northern using dot hybridisation. Seven unigenes (7%) were shown to be either up (4) or down (3) regulated during interaction of the fungus with pine roots. Fungal genes differentially expressed during contact with roots include genes encoding mitochondrial proteins, a cytochrome P450 and a vacuolar ATP synthase.     

Differential expression of heat shock protein genes in preconditioning for photosynthetic acclimation in water-stressed loblolly pine

Heat shock proteins (HSPs) are induced not only under heat stress conditions but also under other environmental stresses such as water stress. In plants, HSPs families are larger than those of other eukaryotes. In order to elucidate a possible connection between HSP expression and photosynthetic acclimation or conditioning, we conducted a water stress experiment in loblolly pine (Pinus taeda L.) seedlings involving progressive treatment consisting of one cycle of mild stress (?1 MPa) followed by two cycles of severe stress (?1.7 MPa). Net photosynthesis was measured at each stress level. Photosynthetic acclimation occurred in the progressive treatment after the first cycle, but not in the severe treatment, suggesting that a cycle of mild stress conditioned the trees to adapt to a more severe stress. Real time results indicated specific patterns in needles in the expression of HSP70, HSP90 and sHSP genes for each treatment, both at maximum stress and at recovery. We identified a pine homolog to GRP94 (ER resident HSP90) that was induced after rehydration coincident with acclimation. Further analysis of the promoter region of the pine GRP94 showed putative cis-elements associated with water stress and rehydration, corresponding to the expression pattern observed in our experiment.     

Gene expression profiling in the stem of young maritime pine trees: detection of ammonium stress-responsive genes in the apex

The shoots of young conifer trees represent an interesting model to study the development and growth of conifers from meristematic cells in the shoot apex to differentiated tissues at the shoot base. In this work, microarray analysis was used to monitor contrasting patterns of gene expression between the apex and the base of maritime pine shoots. A group of differentially expressed genes were selected and validated by examining their relative expression levels in different sections along the stem, from the top to the bottom. After validation of the microarray data, additional gene expression analyses were also performed in the shoots of young maritime pine trees exposed to different levels of ammonium nutrition. Our results show that the apex of maritime pine trees is extremely sensitive to conditions of ammonium excess or deficiency, as revealed by the observed changes in the expression of stress-responsive genes. This new knowledge may be used to precocious detection of early symptoms of nitrogen nutritional stresses, thereby increasing survival and growth rates of young trees in managed forests.     

Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms

Specific plant cellulose synthases (CesA), encoded by a multigene family, are necessary for secondary wall synthesis in vascular tissues and are critical to wood production. We obtained full-length clones for the three CesAs that are highly expressed in developing xylem and examined their phylogenetic relationships and expression patterns in loblolly pine tissues. Full-length CesA clones were isolated from cDNA of developing loblolly pine (Pinus taeda) xylem and phylogenetic inferences made from plant CesA protein sequences. Expression of the three genes was examined by Northern blot analysis and semiquantitative RT-PCR. Each of three PtCesA genes is orthologous to one of the three angiosperm secondary cell wall CesAs. The PtCesAs are coexpressed in tissues of loblolly pine with tissues undergoing secondary cell wall biosynthesis showing the highest levels of expression. Phylogenetic and expression analyses suggest that functional roles for these loblolly pine CesAs are analogous to those of orthologs in angiosperm taxa. Based upon evidence from this and other studies, we suggest division of seed plant CesA genes into six major paralogous groups, each containing orthologs from various taxa. Available evidence suggests that paralogous CesA genes and their distinct functional roles evolved before the divergence of gymnosperm and angiosperm lineages.     

Expressed sequence tag analysis of the response of apple (Malus x domestica'Royal Gala') to low temperature and water deficit

Leaf, bark, xylem and root tissues were used to make nine cDNA libraries from non-stressed (control) 'Royal Gala' apple trees, and from 'Royal Gala' trees exposed to either low temperature (5 degrees C for 24 h) or water deficit (45% of saturated pot mass for 2 weeks). Over 22 600 clones from the nine libraries were subjected to 5' single-pass sequencing, clustered and annotated using blastx. The number of clusters in the libraries ranged from 170 to 1430. Regarding annotation of the sequences, blastx analysis indicated that within the libraries 65-72% of the clones had a high similarity to known function genes, 6-15% had no functional assignment and 15-26% were completely novel. The expressed sequence tags were combined into three classes (control, low-temperature and water deficit) and the annotated genes in each class were placed into 1 of 10 different functional categories. The percentage of genes falling into each category was then calculated. This analysis indicated a distinct downregulation of genes involved in general metabolism and photosynthesis, while a significant increase in defense/stress-related genes, protein metabolism and energy was observed. In particular, there was a three-fold increase in the number of stress genes observed in the water deficit libraries indicating a major shift in gene expression in response to a chronic stress. The number of stress genes in response to low temperature, although elevated, was much less than the water deficit libraries perhaps reflecting the shorter (24 h) exposure to stress. Genes with greater than five clones in any specific library were identified and, based on the number of clones obtained, the fold increase or decrease in expression in the libraries was calculated and verified by semiquantitative polymerase chain reaction. Genes, of particular note, that code for the following proteins were overexpressed in the low-temperature libraries: dehydrin and metallothionein-like proteins, ubiquitin proteins, a dormancy-associated protein, a plasma membrane intrinsic protein and an RNA-binding protein. Genes that were upregulated in the water deficit libraries fell mainly into the functional categories of stress (heat shock proteins, dehydrins) and photosynthesis. With few exceptions, the overall differences in downregulated genes were nominal compared with differences in upregulated genes. The results of this apple study are similar to other global studies of plant response to stress but offer a more detailed analysis of specific tissue response (bark vs xylem vs leaf vs root) and a comparison between an acute stress (24-h exposure to low temperature) and a chronic stress (2 weeks of water deficit).