ARAG1 is a rice DREB gene.
- ARAG1 encodes a DREB-like protein containing the characterized AP2 DNA binding domain. It includes an open reading frame of 678 bp capable of encoding a 225-amino-acid protein. The N- and C-terminal regions of ARAG1 contain a serine/threonine-rich motif of 21 amino acids and a highly acidic region of 74 amino acids, respectively.
- ARAG1 was involved in the ABA signalling and stress responsive pathways. ARAG1 was involved in the process associated with tolerance in the seedlings.
- Germination and a-amylase activity are separate processes although they are closely linked in seeds. The presence of ARAG1 transcripts in embryos and endosperms of germinating seeds suggests that the gene is probably regulated differently in these tissues.
ARAG1 knockdown rice was hypersensitive to ABA inhibition during seed germination and seedling growt:
- Three over-expression lines:
- four antisense lines:
- ARAG1 transcripts decreased in AS lines and increased in OE lines, implying that the constructs worked effectively. Western blot with antibody specific to ARAG1 revealed that the protein level was reduced significantly in AS lines; however, compared with ARAG1 in WT, its increase in OE lines was very limited (<20 %).
- All concentrations of ABA caused WT seeds to germinate more quickly than the transgenics, and their mean germination rate decreased proportionally with increased ABA concentration. At 4 d after imbibition, the germination rate of all the seeds reached 100% in the absence of ABA application. However, when treated with ABA, it decreased obviously in WT and OE lines. No seed germinated in AS lines.
- It is worth noticing that the inhibition was more serious in the AS seedlings compared with their WT and OE counterparts. When treated with 4 or 8 mM ABA, very few adventitious roots were observed in any of the seedlings.
- Semi-quantitative RT-PCR showed that ARAG1 expressed in roots, inflorescences, endosperms of germinating seeds and developing and germinating embryos but not in coleoptiles, leaves and mature embryos.
- compared with the control, expression of ARAG1 was up-regulated by drought and 100 mM ABA treatment after 30 min. However, it seemed insensitive to low temperature.
- Overall, drought stress and ABA treatment increased transcript levels of the gene rapidly. ARAG1 knockdown line was hypersensitive to ABA application during seed germination and seedling growth. However, the line over-expressing ARAG1 behaved similarly to wild type in these circumstances. Knockdown of ARAG1 weakened tolerance of the transgenic seedlings to drought stress, while over-expression of it increased the tolerance slightly. In addition, activity of a-amylases was enhanced in germinating seeds of the knockdown and over-expression lines.
Besides the AP2 DNA binding domain, the protein has a putative nuclear localization signal (NLS) KKKRPRK.
- The similarities were intensively restricted to their AP2/EREBP DNA-binding domain regions in which about 73–92% amino acids were identical. In addition, ARAG1 contained the 14th valine, 19th glutamic and the quartet amino acids SEIR between the 14th and 19th amino acids. These amino acids, which are commonly present in other known DREB proteins, are identified as essential for the recognition and binding of the protein to the target DNA fragment.
- Phylogenetic analysis based on multi-sequence alignments of the domains revealed that ARAG1 formed a clade with DBF2 of maize, and it was more close to DREB1 than to DREB2 (Fig. 1), implying that ARAG1 was a novel DREB-like protein of rice that might be involved in drought response.
- Other members of AP2 TFs, which have been shown to be involved in drought response, are AP37, AP59 and ARAG1 (ABA-responsive AP2-like). The transcript levels of all these genes increased after 2 h of drought treatment. Transgenic rice overexpressing AP37 displayed improved tolerance and higher grain yield under drought conditions.
- By contrast, although overexpression of AP59 in rice resulted in drought tolerance, the AP59 transgenic plants produced lower yield under both normal and drought conditions in comparison with WT due to disrupted spikelet development caused by AP59 overexpression.
Labs working on this gene
- Research Center for Molecular & Developmental Biology, Key Laboratory of Photosynthesis & Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China
- College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, China
- Zhao L, Hu Y, Chong K, et al. ARAG1, an ABA-responsive DREB gene, plays a role in seed germination and drought tolerance of rice[J]. Annals of botany, 2010: mcp303.
- Sakuma Y, Liu Q, Dubouzet J G, et al. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression[J]. Biochemical and biophysical research communications, 2002, 290(3): 998-1009.
- Oh S J, Kim Y S, Kwon C W, et al. Overexpression of the transcription factor AP37 in rice improves grain yield under drought conditions[J]. Plant Physiology, 2009, 150(3): 1368-1379.