Bud formation is an adaptive trait that temperate forest trees have acquired to facilitate seasonal synchronization. We have characterized transcriptome-level changes that occur during bud formation of white spruce [Picea glauca (Moench) Voss], a primarily determinate species in which preformed stem units contained within the apical bud constitute most of next season's growth. Microarray analysis identified 4460 differentially expressed sequences in shoot tips during short day-induced bud formation. Cluster analysis revealed distinct temporal patterns of expression, and functional classification of genes in these clusters implied molecular processes that coincide with anatomical changes occurring in the developing bud. Comparing expression profiles in developing buds under long day and short day conditions identified possible photoperiod-responsive genes that may not be essential for bud development. Several genes putatively associated with hormone signalling were identified, and hormone quantification revealed distinct profiles for abscisic acid (ABA), cytokinins, auxin and their metabolites that can be related to morphological changes to the bud. Comparison of gene expression profiles during bud formation in different tissues revealed 108 genes that are differentially expressed only in developing buds and show greater transcript abundance in developing buds than other tissues. These findings provide a temporal roadmap of bud formation in white spruce.

Two CBF (CRT/DRE-binding factor) homologues isolated from Eucalyptus gunnii were designated EguCBF1a and EguCBF1b and belong to a gene family which includes at least five members. Both promoter and coding sequences were found to exhibit the main characteristics of a CBF transcription activator gene and, as expected, the corresponding protein targeted the nucleus. Gene expression was quantitatively analysed using real-time reverse transcription-polymerase chain reaction (RT-PCR) after a short exposure to different environmental conditions or along a two-step cold acclimation programme with either short or long daylengths. A very strong and fast response to cold was observed, with dark conditions and cold intensity (down to 0 degrees C) having a positive effect on the magnitude of induction. The two genes under study exhibited several similar features such as light response. However, interestingly, their regulation by cold proved differential and complementary as EguCBF1a was more transiently induced by a direct and intense exposure while EguCBF1b responded to milder treatments and exhibited a longer (i.e. which started earlier and finished later) time course. During acclimation, the short daylength positively affected the freezing tolerance in the same way as it positively affected the CBF transcript accumulation, suggesting a potential involvement of these genes in the adaptive response. Although very quick after the first signal, the up-regulation of the two EguCBF1 genes unexpectedly lasted throughout the chilling culture, and new inductions were seen during the thermoperiod transitions. Using a quantitative and highly sensitive measurement of gene expression combined with the application of a cold treatment consistent with natural environmental conditions, this study provides new information on the regulation of CBF-like genes by cold in planta.

In the autumn, stems of woody perennials such as forest trees undergo a transition from active growth to dormancy. We used microarray transcriptomic profiling in combination with a proteomics analysis to elucidate processes that occur during this growth-to-dormancy transition in a conifer, white spruce (Picea glauca [Moench] Voss). Several differentially expressed genes were likely associated with the developmental transition that occurs during growth cessation in the cambial zone and the concomitant completion of cell maturation in vascular tissues. Genes encoding for cell wall and membrane biosynthetic enzymes showed transcript abundance patterns consistent with completion of cell maturation, and also of cell wall and membrane modifications potentially enabling cells to withstand the harsh conditions of winter. Several differentially expressed genes were identified that encoded putative regulators of cambial activity, cell development and of the photoperiodic pathway. Reconfiguration of carbon allocation figured centrally in the tree's overwintering preparations. For example, genes associated with carbon-based defences such as terpenoids were down-regulated, while many genes associated with protein-based defences and other stress mitigation mechanisms were up-regulated. Several of these correspond to proteins that were accumulated during the growth-to-dormancy transition, emphasizing the importance of stress protection in the tree's adaptive response to overwintering.

SummaryTwo C-repeat binding factor genes (EguCBF1a ⁄ b), isolated from E. gunnii and differentially cold-regulated, were constitutively overexpressed in a cold-sensitive Eucalyptus hybrid. In addition to the expected improvement on freezing tolerance, some resulting transgenic lines (EguCBF1a-OE and EguCBF1b-OE) exhibited a decrease in stomata density and an over-accumulation of anthocyanins also observed to a lesser extent in a cold-acclimated control plant. Given that the induction of five putative CBF target genes was observed in CBF-overexpressing lines as well as in the coldacclimated control line, these phenotypes might be related to cold acclimation. In comparison with the control plant, the most altered transgenic line (EguCBF1a-OE A1 line), exhibited reduced growth and better water retention capacity. This modified phenotype includes reduced leaf area and thickness associated with a decrease in cell size, as well as a higher oil gland density and a wax deposition on the cuticle. Surprisingly, the EguCBF1b-OE B9 line, with a level of transgene expression equivalent to the A1 line, showed a less marked phenotype, suggesting a difference in transactivation efficiency between EguCBF1A and B factors. The features of these transgenic lines provide the first signs of adaptive mechanisms controlled by CBF transcription factors in an evergreen broad-leaved tree. These data also open new prospects towards genetic improvement on Eucalyptus for freezing tolerance.

Several dwarf plum genotypes (Prunus salicina L.), due to deficiency of unknown gibberellin (GA) signalling, were identified. A cDNA encoding GA 2-oxidase (PslGA2ox), the major gibberellin catabolic enzyme in plants, was cloned and used to screen the GA-deficient hybrids. This resulted in the identification of a dwarf plum hybrid, designated as DGO24, that exhibits a markedly elevated PslGA2ox signal. Grafting ‘Early Golden’ (EG), a commercial plum cultivar, on DGO24 (EG/D) enhanced PslGA2ox accumulation in the scion part and generated trees of compact stature. Assessment of active GAs in such trees revealed that DGO24 and EG/D accumulated relatively much lower quantities of main bioactive GAs (GA1 and GA4) than control trees (EG/M). Moreover, the physiological function of PslGA2ox was studied by determining the molecular and developmental consequences due to ectopic expression in Arabidopsis. Among several lines, two groups of homozygous transgenics that exhibited contrasting phenotypes were identified. Group-1 displayed a dwarf growth pattern typical of mutants with a GA deficiency including smaller leaves, shorter stems, and delay in the development of reproductive events. In contrast, Group-2 exhibited a ‘GA overdose’ phenotype as all the plants showed elongated growth, a typical response to GA application, even under limited GA conditions, potentially due to co-suppression of closely related Arabidopsis homologous. The studies reveal the possibility of utilizing PslGA2ox as a marker for developing size-controlling rootstocks in Prunus.