Physiological and Molecular Function of HAP3b in Flowering Time Regulation and Cold Stress Response

Heme-activated proteins (HAPs) are transcription factors that have multiple roles in plant growth and development, such as embryogenesis, flowering time control, and drought tolerance. In the present study I found that HAP3b was also involved in controlling response to cold stress. Transcript profiling and gene expression analyses indicated that HAP3b repressed the CBF3 regulon under normal growth conditions. As a result, plants with HAP3b-overexpressed showed decreased survival rates while plants homozygous for the null allele hap3b showed an improved freezing tolerance compared to wild-type plants. To understand the mechanism of HAP3b in Arabidopsis, i.e. whether it also acts through forming a heterotrimer, the yeast two-hybrid system and the protein coimmunoprecipitation method were used to identify the proteins that could interact with HAP3b. From yeast two-hybrid analyses, it was found that HAP3b could interact with one (At3g14020) of ten HAP2s and all ten HAP5s tested. Further analyses showed that the newly identified HAP2 protein could only interact with two HAP5 proteins, those encoded by At5g63470 and At1g56170. To address whether HAPs also play important roles in major crop plants, HAP3 genes in barley (Hordeum vulgare L.) were identified and characterized. From database sequence analyses, cloning, and sequencing, it was found that barley plants have at least six full-length members in the HAP3 family. Phylogenetic analyses showed that each barley HAP3 was different, forming its own cluster with the HAP3s from other plant species. Each barley HAP3 also showed its own expression pattern in different tissues, at different developmental stages and under various environmental stresses. In particular, TC176294 showed the highest sequence similarity to HAP3b in Arabidopsis and its high expression was associated with flowering. In addition, TC176294 was upregulated by various abiotic stresses and by abscisic acid (ABA). Thus, TC176294 might be a barley ortholog of HAP3b. TC191694 showed the highest sequence similarity to HAP3c and might be a barley ortholog of HAP3c. TC191694 overexpression plants were early flowering compared to HAP3b-overexpression and wild-type plants while overexpression of TC176294 plants were not.