Evidence of alternative splicing for a Sorghum bicolor gene encoding putative GAMYB transcription factor

MARÍA V. RODRIGUEZ; GUILLERMINA M. MENDIONDO; GUSTAVO E. GUDESBLAT; ROBERTO L. BENECH-ARNOLD

California, Davis. USA

Simposio; Plant Sciences Symposium; 2007

University of California

Transcription factor GAMYB has been widely studied in cereal species such as wheat and barley. It is known that GAMYB regulates positively the expression of GA response genes involved in seed germination like ALPHA-AMYLASE among many others, by directly binding to the GARE element in their promoters. Also, expression of TaGAMYB has been shown to be down-regulated through protein kinase PKABA1 in wheat, which links ABA and GA signalling in a negative interaction. We analysed the transcript levels of a GAMYB orthologue in grain sorghum seeds, and found that its expression was higher in more dormant seeds, which exhibited lower levels of active GA; also, expression pattern of GAMYB was very similar to that of SbPKABA orthologue. These results are in clear contradiction with what is known about this component of GA signalling in cereal seeds.             The presence of two different EST types aligned with the GSS sequence covering the 2nd and third exons of putative SbGAMYB suggested that alternative splicing might be occurring. We designed a pair of primers that would distinguish both transcripts, and again obtained contradictory results with a positive role of SbGAMYB in GA-promoted germination. Germination experiments with GA and paclobutrazol were carried out and SbGAMYB levels determined with each of two pairs of primers; GA and paclobutrazol application both induced significant changes in germination and also in transcript levels, but no proper correlation between them could be established. The reason seems to be that there are more than two transcript types, and though primers were designed to distinguish between two of them, they probably amplify several other transcript types. After PCR with combined extreme primers of both pairs (Fw, located in exon 2 and Rev, in non-coding 3´ region), five bands were detected in the gel, sizes ranging from ca.150 to 620 nt. Two of these bands have the expected size as deduced from the position of PCR primers on the available EST´s sequences: 620 and 320 nt. The shortest sequence lacks the last 302 nt of exon 3 encoding the transactivation domain BOX3 which is present in the longer transcript. Other authors have shown that an artificially truncated version of GAMYB is able to inhibit normal GAMYB function in transient assays; this suggests that SbGAMYB alternative transcripts may have negative effects on GA signalling, and that the relative contribution of these transcripts may be regulated during germination. To further explore this hypothesis we are sequencing the different transcripts. Future experiments to study their function include expression analysis of individual transcripts under different hormone treatments and dormancy conditions.   Gómez-Cadenas A, Zentella R, Walker-Simmons MK, Ho T-HD (2001) Plant Cell 13: 667–679