GSBF1, a seedling-specific bZIP DNA-binding protein with preference for a ?split? G-box-related element in Brassica napus RbcS promoters

Abstract

Promoters of RbcS genes may contain a GS-box, which is a cis element with a core sequence related to the G-box, but split by a spacer of about 14 bp. Here we describe GSBF1, a DNA-binding protein that specifically interacts with a GS-box element located proximal to the G-box in the Brassica napus RbcS IV promoter. Sequence analysis of GSBF1 revealed a basic region/leucine zipper (bZIP) domain that displays structural features distinct from that of G-box binding factors (GBFs). Gel shift experiments showed that recombinant GSBF1 does not efficiently bind to the continuous G-box motif. RNA gel blot analysis indicated that GSBF1 transcripts are cotyledon-specific and accumulate to the highest levels during late seedling development in a ligh-dependent manner. During the same time period, RbcS IV transcript levels decreased simultaneously, suggesting that GSBF1 acts as a developmental, stage-specific, negative regulator of RbcS IV gene expression in rape seedlings.

New insights into plastid nucleoid structure and functionality

“…The PEND protein was initially discovered in developing pea chloroplasts . Homologs were later detected in other angiosperms, e.g., in Brassica napus (Waldmüller et al 1996) while functional homologs in algae and in nonflowering plants are still not known (Terasawa and Sato 2005a). The cbZIP domain of PEND was shown to bind selectively to AT-rich regions of plastidic DNA containing the canonical sequence TAAGAAGT (Sato and Ohta 2001).…”

“…The cbZIP domain of PEND was shown to bind selectively to AT-rich regions of plastidic DNA containing the canonical sequence TAAGAAGT (Sato and Ohta 2001). Interestingly, both the PEND protein from Brassica napus and its rapeseed homolog, GSBF1, were found to repress the expression of the nuclear-encoded rbcS gene (Waldmüller et al 1996;Wycliffe et al 2005). That PEND indeed fulfills a direct role in the nucleus was supported by the recent observation that a PEND:GFP fusion protein is targeted to the nucleus when the N-terminal presequence is deleted (Terasawa and Sato 2009).…”

Occurrence and characterization of PEND proteins in angiosperms

“…The Brassica napus homolog of PEND or BNGSBF1 was reported previously (Waldmüller et al 1996), but we did not notice that it was a true homolog (or ortholog) of pea PEND protein because of the difference in structure of the central region (Sato et al 1998). In addition, a sequence error at the termination codon in the original pea PEND sequence made the putative translated sequence longer in the original database.…”

“…Immunodetection of PEND homologs in chloroplasts of crucifers BNGSBF1, which is highly similar to Brassica PEND homolog 1, was previously shown to be expressed in B. napus leaves (Waldmüller et al 1996). No clear evidence has been presented that the Arabidopsis thaliana homolog is expressed, although the PEND homolog At3g52170 is described as expressed sequence in the TAIR database.…”

“…The PEND protein was initially identified biochemically in pea, but a homolog of PEND is also encoded by the Arabidopsis genome (At3g52170) (Sato and Ohta 2001). A DNA-binding protein called BNGSBF1 from Brassica napus (Waldmüller et al 1996), which was originally isolated as a nuclear transcription factor regulating the RbcS gene, is also a homolog of PEND. However, phylogenetic occurrence of PEND protein in plants remained obscure.…”