Identification of candidate genes important for frost tolerance in Festuca pratensis Huds. by transcriptional profiling.

Studies of differential gene expression between cold acclimated (CA) and non-cold acclimated (NA) plants yield insight into how plants prepare for cold stress at the transcriptional level. Furthermore genes involved in the cold acclimation process are good candidate loci for genetic variation in frost tolerance and winter survival. In this study we combine different approaches to try to decode the genetics of cold acclimation and frost tolerance in meadow fescue (Festuca pratensis Huds). An EST library of cold acclimation responsive genes was established by suppression subtractive hybridization (SSH), and a microarray experiment was used to identify gene expression differences between high and low frost tolerance genotypes in response to cold acclimation. Many genes known to be involved in CA in other species were confirmed to be involved in CA in F. pratensis, however, 18% of the ESTs did not show significant homology to any database proteins. Seven genes were found to be differentially expressed (>2-fold) between high and low frost tolerance genotypes. Two of these genes, FpQM and FpTPT, represent interesting candidate genes for frost tolerance in perennial forage grasses.

Gene expression changes during short day induced terminal bud formation in Norway spruce.

The molecular basis for terminal bud formation in autumn is not well understood in conifers. By combining suppression subtractive hybridization and monitoring of gene expression by qRT-PCR analysis, we aimed to identify genes involved in photoperiodic control of growth cessation and bud set in Norway spruce. Close to 1400 ESTs were generated and their functional distribution differed between short day (SD-12 h photoperiod) and long day (LD-24 h photoperiod) libraries. Many genes with putative roles in protection against stress appeared differentially regulated under SD and LD, and also differed in transcript levels between 6 and 20 SDs. Of these, PaTFL1(TERMINAL FLOWER LIKE 1) showed strongly increased transcript levels at 6 SDs. PaCCCH(CCCH-TYPE ZINC FINGER) and PaCBF2&3(C-REPEAT BINDING FACTOR 2&3) showed a later response at 20 SDs, with increased and decreased transcript levels, respectively. For rhythmically expressed genes such as CBFs, such differences might represent a phase shift in peak expression, but might also suggest a putative role in response to SD. Multivariate analyses revealed strong differences in gene expression between LD, 6 SD and 20 SD. The robustness of the gene expression patterns was verified in 6 families differing in bud-set timing under natural light with gradually decreasing photoperiod.

Identification of PaCOL1 and PaCOL2, two CONSTANS-like genes showing decreased transcript levels preceding short day induced growth cessation in Norway spruce.

In woody plants of the temperate zone short photoperiod (SD) leads to growth cessation. In angiosperms CONSTANS (CO) or CO-like genes play an important role in the photoperiodic control of flowering, tuberisation and shoot growth. To investigate the role of CO-like genes in photoperiodic control of shoot elongation in gymnosperms, PaCOL1 and PaCOL2 were isolated from Norway spruce. PaCOL1 encodes a 3.9kb gene with a predicted protein of 444 amino acids. PaCOL2 encodes a 1.2kb gene with a predicted protein of 385 amino acids. Both genes consist of two exons and have conserved domains found in other CO-like genes; two zinc finger domains, a CCT and a COOH domain. PaCOL1 and PaCOL2 fall into the group 1c clade of the CO-like genes, and are thus distinct from Arabidopsis CO that belongs to group 1a. Transcript levels of both PaCOL-genes appear to be light regulated, an increasing trend was observed upon transition from darkness to light, and a decreasing trend during darkness. The increasing trend at dawn was observed both in needles and shoot tips, whereas the decreasing trend in darkness was most prominent in shoot tips, and limited to the late part of the dark period in needles. The transcript levels of both genes decreased significantly in both tissues under SD prior to growth cessation and bud formation. This might suggest an involvement in photoperiodic control of shoot elongation or might be a consequence of regulation by light.