(Lahiri and Allison 2000)
Q ues lion m;irks signify a proposed ye I unproved function.
Q ues lion m;irks signify a proposed ye I unproved function.
in developing leaves was reported for both Arabidopsis and rice, suggesting an early function of Sig2 in seedling development (Kanamaru et al. 1999; Kasai et al. 2004). This was supported by recent findings by Demarsy et al. (2006) showing that the mRNAs of AthSig2 and AthSig5 are already present in dry Arabidopsis seeds. Interestingly, unlike AthSig1 and AthSig2, AthSig3 protein accumulates in seeds and during early germination (Homann and Link 2003; Privat et al. 2003) as was shown for SolSig2 in spinach (Demarsy et al. 2006). A similar expression pattern was observed for the mustard SalSig3 factor, which accumulates rather in the dark than in light grown seedlings (Homann and Link 2003). Hence, Sig3 may play a distinctive role in regulation of gene expression in etio- and/or proplastids, and might be regulated by posttranslational processes (Homann and Link 2003; Privat et al. 2003). Similarly, ZmaSig6 was detected in root, leaf base, and etiolated leaf tissue in maize (Lahiri and Allison 2000). Therefore, it might be possible that Sig3 and Sig6 represent light-independent, early c-factors regulating plas-tid gene expression during seedling growth and development. In opposite, AthSig5 transcripts are expressed later in plant development, controlled via the plastidial redox state (Fey et al. 2005). Furthermore, AthSig5 is rapidly induced by blue, but not red light, which coincides with the blue-light-activated expression of psbD (Tsunoyama et al. 2002, 2004). AthSig5 expression is also activated by various stress cues (Nagashima et al. 2004b). Expression of some plastid genes in higher plants seems to be regulated by circadian rhythms (Nakahira et al. 1998). Cir-cadian timing of plastid gene expression is expected to be mediated by nuclear factors. c-factors are good candidates to represent such factors. Indeed, TaeSig1, NtaSig1, AthSig1, AthSig2, and PpaSig5 transcripts were shown to exhibit cir-cadian or diurnal expression patterns (Kanamaru et al. 1999; Morikawa et al. 1999; Oikawa et al. 2000; Ichikawa et al. 2004).
Increasingly, functions of c-factor genes in plants are investigated by analyses of knockout mutants, overexpression, or anti-sense lines. If plastidial gene expression would be controlled by a principal c-factor similar to the situation in most bacteria, one would assume that inactivation of this gene would result in a drastic, most likely albino phenotype by causing defects in PEP-dependent transcription of photosynthesis related genes. However, examination of various Arabidopsis mutants of AthSig2, AthSig3, AthSig4, AthSig5, and AthSig6 did not reveal such a severe phenotype (Shirano et al. 2000; Kanamaru et al. 2001; Hanaoka et al. 2003; Privat et al. 2003; Nagashima et al. 2004b; Tsunoyama et al. 2004; Favory et al. 2005; Ishizaki et al. 2005; Loschelder et al. 2006; Zghidi et al. 2006). Yet, a major break-through in revealing the specificity of c-factors in transcription came by characterization of these plants.
AthSig2 knockout mutants. AthSig2 mutants displayed a pale green phenotype accompanied by reduced accumulation of some plastid-encoded photosynthesis genes (Shirano et al. 2000; Kanamaru et al. 2001; Privat et al. 2003; Nagashima et al. 2004a). Furthermore, several PEP-transcribed tRNAs including trnD-GUC, trnE-UUC, trnM-CAU, and trnV-UAC were prominently reduced in AthSig2 knockout mutants (Kanamaru et al. 2001; Hanaoka et al. 2003) and anti-sense plants (Privat et al. 2003). Vice versa, overexpression of AthSig2 enhanced transcription of trnE-trnD (Tsunoyama et al. 2004). It has been suggested that reduc-
tion of the photosynthesis-related components is caused by defects in chlorophyll biosynthesis and plastid translation due to the decrease of trnE, an initiator of ALA and consequently chlorophyll synthesis. Hence, AthSig2 may have a primary role in driving transcription of certain plastid tRNAs. It cannot be excluded, however, that Sig2 is able to recognize other PEP promoters as suggested for psbA, psbD, and rbcL (Kanamaru et al. 2001; Hanaoka et al. 2003; Tsunoyama et al.
AthSig3 knockout mutants. In opposite, characterization of AthSig3 knockout mutants revealed a distinct reduction of transcript levels of the plastid psbN gene (Zghidi et al. 2006). Further analyses of transcript initiation sites in these mutants not only showed a loss of transcription initiation from AthPpsbN-32 but also from AthPatpH-413, one of the two PEP promoters upstream of atpH in Arabidopsis. Therefore, it seems likely that AthSig3 directly controls psbN and partially atpH gene expression. The function of PsbN is still unknown and its suggested presence in photosystem II has been challenged (Kashino et al. 2002).
AthSig4 knockout mutants. Similarly, characterization of an AthSig4 knockout mutant revealed a specific reduction in transcription of the plastid ndhF gene resulting in a strong downregulation of the plastid NDH activity (Favory et al.
2005). Therefore, ndhF expression and thus NDH activity seems to be regulated at transcriptional level, controlled by specific c-factor AthSig4. Interestingly, NDH is involved in plant stress response (Casano et al. 2001) and leaf senescence (Zapata et al. 2005). Whether AthSig4 expression is modulated by such environmental or developmental parameters remains to be investigated.
AthSig5 knockout mutants. Apart from AthSig3 and AthSig4, AthSig5 might be an additional c-factor tied to a specific function in regulation of plastid gene expression. As shown by analyses of transcription in light-treated plants (Tsunoyama et al. 2002; Nagashima et al. 2004b), AthSig5 knockout plants, and overexpression studies (Nagashima et al. 2004b; Tsunoyama et al. 2004), AthSig5 is regulated by blue light and specifically activates transcription from the psbD blue-light responsive promoter (BLRP). Interestingly, analysis of a further Ath-Sig5 knockout mutant showed embryo lethality (Yao et al. 2003). AthSig5 has recently been identified as one of 250 genes required for normal embryo development in Arabidopsis (Tzafrir et al. 2004) and its mRNA is present in seeds (Demarsy et al. 2006) indicating a substantial role of AthSig5 in seed development. However, it is not yet understood why the different AthSig5 mutants exhibit these diverse phenotypes.
AthSig6 knockout mutants. Cotyledons of AthSig6 knockout mutants displayed a transient pale green phenotype during early plant development combined with a delay in light-dependent chloroplast development (Ishizaki et al. 2005; Lo-schelder et al. 2006). During this developmental stage the transcript pattern was found to be similar to that of Arpo mutants, since transcript levels of most PEP-dependent genes for photosynthesis components, rRNAs, and some tRNAs were decreased. Since the maize homologue ZmSig6 is expressed exclusively in tissue containing immature plastids (Lahiri and Allison 2000), it was proposed that (Ath)Sig6 might be a general c-factor serving PEP in an early, initial developmental stage. Nonetheless, given that after eight days the mutant phenotype is restored to wild type it is plausible that other CT-factor(s) are able to take over AthSig6 function later in seedling development and plant growth (Shiina et al. 2005). However, characterization of a second Arabidopsis knockout line with a Sig6 mutant allele throughout leaf development (sig6-2) suggested a second (persistent or long-term) role of AthSig6 (Loschelder et al. 2006). While transcript accumulation of genes such as psbA and rbcL was only affected early in development, RNA levels of atpB and ndhC originating from their corresponding PEP promoters declined during plant development. Interestingly, emerging transcripts which originated further upstream of atpB suggested a SOS promoter switch (Schweer et al. 2006).
AthSigl overexpressing mutants. Knockout or anti-sense mutants of AthSig1 have yet to be characterized. Thus far, data on the role of AthSigl in plastidial gene expression have been derived from mutant plants overexpressing the AthSig1 gene (Tsunoyama et al. 200l). Investigation of transcription activity by run-on analyses revealed enhanced initiation from psaA, psbB, psbE, and rbcL promoters indicating a more general role of this a-factor in transcription of genes encoding components of the photosynthesis complexes.
Taken together, only five genes in Arabidopsis seem to be controlled by a distinct a-factor with specific function: psaJ by AthSig2, psbN by AthSig3, ndhF by AthSig4, and psbD (BLRP) by AthSig5 (Table 2). However, some other genes appear to be controlled by several a-factors thereby possessing overlapping functions. Most prominent are genes such as psbA, controlled by AthSig2, AthSig5, and AthSig6; rbcL controlled by AthSigl and AthSig6; trnV-UAC and trnE-UUC, controlled by AthSig2 and AthSig6. Consequently, overlapping functions of a-factors are generally believed to be the reason for the weak phenotype of a-factor knockout mutants.
Regulation of a-factors. PEP activity depends on the developmental stage of the plastids: it is down regulated in etioplasts and is more active in chloroplasts (Rapp et al. 1992; DuBell and Mullet 1995). Furthermore, rates of PEP transcription are higher in the light than in the dark (Shiina et al. 1998). Changes in PEP transcription activity have been suggested to be partly resulting from changes in the phosphorylation state of a-factors. Phosphorylation of a-factors and the PEP enzyme itself have been shown to be an important regulatory event in chloroplast transcription (Tiller and Link 1993a; Baginsky et al. 1997; Christopher et al. 1997). In mustard, a CK2-type kinase has been identified to be part of the chloroplast PEP-A complex (Ogrzewalla et al. 2002). This plastid transcription kinase activity (PTK), termed cpCK2, is able to phosphorylate purified sigma-like factors (SLFs) as well as subunits of the PEP-A complex in vitro. Based on the observation that cpCK2 itself is differentially regulated by phosphorylation and redox state, cpCK2 was proposed to be part of a signaling pathway controlling PEP activity (Baginsky et al. 1999). Phosphorylation and SH-group redox state were shown to work antagonistically. A non-phosphorylated cpCK2 appears to be more active, but is inhibited by treatment with reduced glutathione (GSH). Vice versa, a phosphorylated non-active enzyme could be re-activated by adding GSH. In opposite to cpCK2 isolated from plants grown under high light conditions, cpCK2 iso lated from plants grown under moderate light conditions effectively phosphory-lated the associated PEP-A, therefore corroborating these findings (Baena-Gonzalez et al. 2001). Thus, light dependent reduction of GSH would inactivate cpCK2, while dephosphorylation of PEP under high light conditions would enhance PEP-dependent transcription. It remains unknown whether cpCK2 is also regulated via extraplastidic signal chains mediated by phyto- and/or chrypto-chromes. Since cpCK2 orthologs have been identified in various plant species (Loschelder et al. 2004) it might well be that this kinase has an evolutionary conserved role in plastid redox-sensitive signal transduction.
In bacteria, a-factor activity is controlled by anti-a factors (Ishihama 2000). Plastid a-factor AthSig1 associated proteins with plastid localization were identified in Arabidopsis (SibI and T3K9.5; Morikawa et al. 2002). They are not related to any proteins of known function and are light-dependent, developmental, and tissue-specifically expressed, and thus may be involved in regulation of AthSig1 activity.
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