The Etched1 gene of Zea mays (L.) encodes a zinc ribbon protein that belongs to the transcriptionally active chromosome (TAC) of plastids and is similar to the transcription factor TFIIS

Abstract

SummaryEtched1 (et1) is a pleiotropic, recessive mutation of maize that causes ®ssured and cracked mature kernels and virescent seedlings. Microscopic examinations of the et1 phenotype revealed an aberrant plastid development in mutant kernels and mutant leaves. Here, we report on the cloning of the et1 gene by transposon tagging, the localization of the gene product in chloroplasts, and its putative function in the plastid transcriptional apparatus. Several alleles of Mutator (Mu)-induced et1 mutants, the et1-reference (et1-R) mutant, and Et1 wild-type were cloned and analyzed at the molecular level. Northern analyses with wildtype plants revealed that Et1 transcripts are present in kernels, leaves, and other types of tissue, and no Et1 expression could be detected in the et1 mutants analyzed. The ET1 protein is imported by chloroplasts and has been immunologically detected in transcriptionally active chromosome (TAC) fractions derived from chloroplasts. Accordingly, the relative transcriptional activity of TAC fractions was signi®cantly reduced in chloroplasts of et1-R plants. ET1 is the ®rst zinc ribbon (ZR) protein shown to be targeted to plastids. With regard to its localization and its striking structural similarity to the eukaryotic transcription elongation factor TFIIS, it is feasible that ET1 functions in plastid transcription elongation by reactivation of arrested RNA polymerases.Keywords: etched1, plastid nucleoids, transcriptional active chromosome, transposon tagging, plastid transcription, TFIIS. IntroductionThe analysis of genes involved in the development of the maize endosperm is of particular interest in order to understand the structural and regulatory features of seed growth. In maize, many mutants and a number of genes affecting tissue and organ development have been isolated. Numerous mutants have been described, which exhibit abnormal endosperm development (for review: Coe et al., 1988). Among these, a few are pleiotropic and thus of particular interest because they affect the development of different tissue types. Etched1 (et1) is one such mutation, which affects the development of kernels as well as of seedlings.Etched1 is a recessive mutation that was ®rst identi®ed and described by Stadler (1940). The mutant reference allele (et1-reference (et1-R)) was isolated from the progeny of a population of maize plants pollinated with X-ray irradiated pollen. Kernels homozygous for the et1-R allele are ®ssured because of depressions and crevices on the endosperm surface (Figure 1a±c). Prior biochemical and structural analyses of the et1-R kernels revealed that starch synthesis is reduced and starchless endosperm cells are present around the cracks and scars in the kernels (Figure 1; Sangeetha and Reddy, 1991). The et1 kernel phenotype becomes visible approximately 15 days after pollination (DAP). The degree of etching differs among the kernels on an ear and can vary from a weak to a very severe phenotype. This phenotypic variation is apparently not correlated with any speci®c genetic background ...

pTAC2, -6, and -12 Are Components of the Transcriptionally Active Plastid Chromosome That Are Required for Plastid Gene Expression

“…We could not identify the TAC component ET1, which was recently detected immunologically in maize (Zea mays) (da Costa e Silva et al, 2004). However, it cannot be excluded that this protein is present in the TAC fraction.…”

“…Forty to sixty polypeptides appear to be present in the TAC from chloroplasts, along with two core subunits of PEP and ETCHED1 (ET1) with high homology to the nuclear transcription elongation factor TFSII (Suck et al, 1996;da Costa e Silva et al, 2004). Together with the findings that the ET1-R mutants exhibit quantitative, but no qualitative, differences in TAC activity, it was suggested that ET1 may be involved in plastid mRNA elongation (da Costa e Silva et al, 2004).…”

“…TAC preparations from various organisms were analyzed on SDS gels, and in this way, at least 40 polypeptides associated with TAC have been detected. However, only the a-and b-subunits of the PEP (Suck et al, 1996;, a homolog of the nuclear transcription elongation factor TFIIS (da Costa e Silva et al, 2004), and few plastid DNA-bound proteins, including PEND (Sato et al, 1993), sulfite reductase (Cannon et al, 1999;Chi-Ham et al, 2002;Sekine et al, 2002), CND41 (Murakami et al, 2000), and MFP1 (Jeong et al, 2003), have been identified until now. We used different gel filtration and affinity chromatography purification steps for the isolation of TACs from mustard and Arabidopsis.…”

“…Together with the findings that the ET1-R mutants exhibit quantitative, but no qualitative, differences in TAC activity, it was suggested that ET1 may be involved in plastid mRNA elongation (da Costa e Silva et al, 2004). Furthermore, analyses of TAC preparations from ribosomedeficient mutants suggest that the fraction also contains a nuclearencoded RNA polymerase activity (Suck et al, 1996).…”

Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase

“…The Arabidopsis genome encodes only one putative bacterial-type elongation factor, pTAC13 (At3g09210) containing a KOW domain, which is characteristic for members of the NusG protein family (40). Furthermore, maize ET1, which resembles the eukaryotic elongation factor TFIIS, was identified as a component of the chloroplast transcriptionally active fraction, and plants deficient in ET1 showed an aberrant chloroplast development phenotype and decreased PEP-dependent transcription (21), suggesting that bacterial-type pTAC13 and eukaryotic-type ET1 may be involved in PEP elongation. In this study, we demonstrate that pTAC3 is associated with PEP through transcription initiation and elongation steps, suggesting a possible role for pTAC3 in the regulation of PEP transcription elongation in chloroplasts.…”