Role of ET1 in chloroplasts

Since the chloroplast import experiments show that ET1 is plastid localised and very probably in the stroma, its functional sphere would probably involve physiological activities being carried out in the stroma. Plastids are semi-autonomous organelles, which are believed to have originated from oxygenic photosynthetic prokaryotes (Howe, 1996). Therefore, the stroma represents an equivalent of the prokaryotic cytoplasm and carries out a number of functions independent of the cell. These functions also include transcription and translation. Based on its structural homology to known eukaryotic as well as prokaryotic zinc binding proteins, involved in

(B) Ribbon diagram of the zinc ribbon domain of human CK2β with its three-stranded β-sheet structure (Chantalat et al., 1999). The zinc ion (Zn²⁺) is shown as a blue ball, whereas the β strands in red. The remaining peptide chain is shown in brown. The N-terminal (N) and C-terminal (C) ends are indicated.

transcription and translation, it could be postulated that ET1 might also be taking part in these processes being carried out in the plastids.

The Northern analyses with et1 probe have shown that apart from the leaves, tassels and endosperm, where a strong et1 expression is observed, et1 shows a basal expression level in all the remaining tissues examined. It is not unusual that a gene shows basal expression level in the absence of the requirement of its gene product.

However, another explanation for this expression pattern could also be that ET1 influences the housekeeping functions of all the plastids, but its requirement during increased physiological activity in the plastids is also increased, as in the amyloplasts of the developing kernels, and the chloroplasts in the leaves. This increased expression is probably stimulated by upstream elements of the et1 gene, which respond to external (light) or internal (endosperm development) stimulatory factors.

Increased et1 expression in response to light has been shown through Northern analysis (da Costa e Silva et al., 2001), where etiolated seedlings, grown in dark, or seedlings grown in light showed absence or strong expression of the et1 transcripts respectively. All these possibilities also indicate that ET1 might be controlling plastid development either at the transcriptional level or at the post-transcriptional level.

Although a large majority of plastid genes are believed to have been transferred to the nucleus during evolution, it still maintains a large number of vital genes, most of which encode components of the photosynthetic electron transport machinery, and transcriptional and translational apparatus (Stern et al., 1997). Transcription of the remaining plastid genes is driven by at least three different plastid RNA polymerases.

One transcription apparatus comprises a multi-subunit homologue of E. coli RNA polymerase, called PEP (Plastid Encoded RNA Polymerase), which is the only plastid encoded polymerase (Howe, 1996; Isono et al., 1997; Tan and Troxler, 1999; Hu and Bogorad, 1990). Another transcriptional apparatus comprises a NEP (Nuclear Encoded RNA Polymerase), which appears to be a single subunit homologue of the T7/T3 phage RNA polymerase (Allison et al., 1996; Maliga, 1998; Chang et al., 1999). Various studies indicate the presence of a third nucleus encoded RNA polymerase (Bligny et al., 2000; Hedtke et al., 2000). In Arabidopsis thaliana, whose complete genome has now been sequenced, showed the presence of three different phage type RNA polymerase genes, of which two had already been characterised as encoding plastid and mitochondrial NEPs respectively. The third RNA polymerase

was found to show dual targeting to plastids as well as mitochondria (Hedtke et al., 2000).

All these polymerases seem to work independent of each other, under the control of different regulatory mechanisms, and show differential activity in different tissues as well as developmental stages. Moreover, analyses of plastid promoters also revealed that these systems use different promoters for transcription. Many plastid genes also seem to contain promoters for more than one plastid RNA polymerase (Pfannschmidt and Link, 1994; Stern et al., 1997; Hajdukiewicz et al., 1997; Serino and Maliga, 1998; Maliga, 1998; Silhavy and Maliga, 1998; Bligny et al., 2000). A NEP is supposed to be the first polymerase to become active during early plastid development, which transcribes the genes for ribosomal rRNAs and the PEP transcription machinery. It is then soon followed by the PEP, whose activity upon illumination increases greatly (Stern et al., 1997). Since Northern analyses indicate that ET1 might be active right from a very early leaf developmental stage, it could be involved in transcription by acting as a transcription factor for either a NEP or PEP, or as their subunit. Moreover, the leaves of et1 mutants remain pale during early seedling development, showing the lack of chloroplast development, like the absence of organised thylakoids, as observed in et1 mutant seedlings (EM micrographs, Fig.

1.3). Since it also influences amyloplast development, it is very likely not a part of the photosynthetic apparatus. However, the genes for photosystems I and II are transcribed by the PEP, and PEP encoding genes, in turn, are transcribed by an NEP. Therefore, ET1 could be interacting with either the PEP or any of the NEPs.

One explanation for the recovery of the et1 seedlings from et1 phenotype after about 15 DAG could be that ET1 functions as a transcription factor that is replaced by another transcription factor in its absence. On the other hand, a large number of plastid genes are known to possess more than one type of promoter elements and are transcribed by more than one type of transcriptional apparatus (Krupinska and Falk, 1994; Stern et al., 1997; Maliga, 1998; Silhavy and Maliga, 1998; Bligny et al., 2000). Therefore, it could be possible that in the absence of ET1, another transcription apparatus is activated, which replaces the ET1-associated transcription apparatus and transcribes those genes, thus bringing the plastid system back to normal. However, it is also possible that ET1, as a transcription factor, is crucial to the plastid system during the early seedling development. In its absence, the

seedling just manages to tide-over this early period of growth with the help of the storage reserves of the endosperm. Thus, providing it with the needed relief until the next transcriptional apparatus can become active to carry out its function.

However, based on its homology to the zinc ribbon domain, the ET1 protein could also be involved at the post-transcriptional or translational level, instead of during transcription. The relative transcription rates of most chloroplast genes have been found to be almost constant, which indicates that post-transcriptional and translational mechanisms play an important role in the differential expression of chloroplast genes (Stern et al., 1997). Analysis of nuclear mutations have revealed that a large number of nuclear-encoded factors are involved in plastid development, which principally influence and regulate plastid expression pattern at the post-transcriptional level (Rochaix, 1996). ET1 could be involved at various steps, which demand an interaction of the proteins with the transcribed RNA molecules, like in RNA stability, RNA processing, RNA splicing and translation. In eukaryotic systems, a number of zinc ribbon or zinc finger proteins are known to be involved in post-transcriptional processing and modification. For example, SRD1 is a yeast pre-rRNA processing protein and the translation initiation factor eIF-2β, containing a C2-C2 zinc finger domain, and have bacterial homologues (Hess et al., 1994).

One possible function of ET1 could be an involvement of the zinc ribbon domain in protein-protein interaction, like in CK2β. If ET1 does happen to possess a CK2β type structure, and undergo dimerisation to constitute a subunit of a multimeric regulatory protein, then it could constitute a part of a CK2 like protein kinase or some other similar regulatory protein, whose catalytic domain, as also in CK2, is present in another subunit. Similar to CK2β subunit, ET1 also possesses α helices at both its ends, which cluster together in case of CK2β. However, in ET1 the α-helical region is much smaller. The indications of the presence of CK2-like and other protein kinases in chloroplasts is available from a number of studies. The PEP polymerase from mustard (Sinapis alba) has been found to be associated with a Ser/Thr protein kinase, called plastid transcription kinase (PTK) (Baginsky et al., 1999). Reversible phosphorylation of the photosystem II protein components has been observed associated with a CK2 like Ser/Thr protein kinase, which also indicates involvement in signal transduction (Testi et al., 1996).