Research Article: Transcriptome Analysis of the Vernalization Response in Barley (Hordeum vulgare) Seedlings

Date Published: March 9, 2011

Publisher: Public Library of Science

Author(s): Aaron G. Greenup, Sharyar Sasani, Sandra N. Oliver, Sally A. Walford, Anthony A. Millar, Ben Trevaskis, Samuel Hazen.

Abstract: Temperate cereals, such as wheat (Triticum spp.) and barley (Hordeum vulgare), respond to prolonged cold by becoming more tolerant of freezing (cold acclimation) and by becoming competent to flower (vernalization). These responses occur concomitantly during winter, but vernalization continues to influence development during spring. Previous studies identified VERNALIZATION1 (VRN1) as a master regulator of the vernalization response in cereals. The extent to which other genes contribute to this process is unclear. In this study the Barley1 Affymetrix chip was used to assay gene expression in barley seedlings during short or prolonged cold treatment. Gene expression was also assayed in the leaves of plants after prolonged cold treatment, in order to identify genes that show lasting responses to prolonged cold, which might contribute to vernalization-induced flowering. Many genes showed altered expression in response to short or prolonged cold treatment, but these responses differed markedly. A limited number of genes showed lasting responses to prolonged cold treatment. These include genes known to be regulated by vernalization, such as VRN1 and ODDSOC2, and also contigs encoding a calcium binding protein, 23-KD jasmonate induced proteins, an RNase S-like protein, a PR17d secretory protein and a serine acetyltransferase. Some contigs that were up-regulated by short term cold also showed lasting changes in expression after prolonged cold treatment. These include COLD REGULATED 14B (COR14B) and the barley homologue of WHEAT COLD SPECIFIC 19 (WSC19), which were expressed at elevated levels after prolonged cold. Conversely, two C-REPEAT BINDING FACTOR (CBF) genes showed reduced expression after prolonged cold. Overall, these data show that a limited number of barley genes exhibit lasting changes in expression after prolonged cold treatment, highlighting the central role of VRN1 in the vernalization response in cereals.

Partial Text: In temperate regions, wheat (Triticum spp.) and barley (Hordeum vulgare) can be sown in autumn to grow vegetatively through winter before flowering in spring. Autumn sowing can enhance yield relative to later sowing times, but can also expose plants to freezing winter conditions [1]. Consequently, the capacity to survive winter frosts is an important trait for autumn-sown wheat and barley varieties grown in regions that experience cold winters [2]–[4].

The Affymetrix 22K Barley1 chip [39] was used to examine the effects of short or prolonged cold on the transcriptome of barley seedlings (see materials and methods, Figure 1A). Pair wise comparisons of the different treatments were used to identify contigs that have altered expression in the short term, prolonged or post-cold treatments (Tables S1, S2, S3, S4, S5, S6). Large numbers of contigs show altered expression (> two fold change relative to the non-treated control, p<0.01) in short (613 contigs, Table S1) or prolonged (786 contigs, Table S2) cold treatment (Figure 2). The contigs that showed altered expression after short term cold were predominantly up-regulated, whereas many contigs were down-regulated in the prolonged cold treatment (Figure 2). Fewer contigs showed altered expression in the post cold treatment relative to the non-treated control (150 contigs, Table S3). A limited number of contigs showed a lasting response to prolonged cold; i.e. significantly changed in both the prolonged cold and 1 day after prolonged cold samples, but not the short term cold sample (60 contigs) (Figure 3). Expression levels were verified for a subset of contigs with contrasting expression patterns, using quantitative RT-PCR (Figure S1). Previous studies investigated the effects of low-temperature on the transcriptome of wheat or barley by examining short to medium term cold responses (1 day–2 weeks) [42]–[45]. In this study, transcriptional responses to short or prolonged cold were assayed and compared (Figure 1). The data presented show that transcriptional responses to short or prolonged cold differ markedly. This is evident from the lists of contigs with significantly changed expression for each treatment relative to the control (Figure 2, Tables S1, S2, S3, S4, S5, S6) and is further highlighted by PCA (Figure 4). Comparing and contrasting the effects of different lengths of cold treatment identified contigs potentially involved in different low-temperature responses. Contigs that respond to short term cold treatment are likely to function during cold “shock”, to adjust homeostasis to rapid decreases in temperature, whereas contigs that show altered expression after prolonged cold are likely to be important for long term growth at low temperatures. Comparison of this dataset with previous microarray analyses of low-temperature responses in barley identified 55 contigs that showed a significant response to cold in all experiments (8 down and 47 up) (Table S10) (Plexdb accession no. BB65 and BB81; [42]). These contigs define a core set of low-temperature responsive genes from barley, including genes previously identified as cold responsive, such DHN5[43], that are likely to play critical roles in cold acclimation (HVSMEa0006I22r2_s_at and contig1717_s_at) (Table S10). Source: