Structure and expression of the nuclear gene coding for the chloroplast ribosomal protein L21: developmental regulation of a housekeeping gene by alternative promoters.

Abstract

We have cloned and sequenced the nuclear gene of the chloroplast ribosomal protein L21 (rpI2l) of Spinacia oleracea. The gene consists of five exons and four introns. All introns are located in the sequence which corresponds to the Escherichia coli-like central core of the protein. L21 mRNA is present in photosynthetic (leaves) and nonphotosynthetic (roots and seeds) plant organs, although large quantitative differences exist. Primer extension and Si nuclease mapping experiments revealed the existence of two types of transcripts in leaves. The two corresponding start sites were defined as P1 and P2. In roots and seeds, we found only the shorter of the two transcripts (initiated at P2). The nucleotide sequence surrounding P2 resembles promoters for housekeeping and vertebrate r-protein genes. Analysis of several promoter constructions by transient expression confirmed that both transcripts originate from transcription initiation. Results are interpreted to mean that the expression of the rpI21 gene is regulated by alternative promoters. One of the promoters (P2) is constitutive, and the other one (P1) is specifically induced in leaves, i.e., its activation should be related to the transformation of amyloplasts or proplastids to chloroplasts. The gene thus represents the first example of a housekeeping gene which is regulated by the organ-specific usage of alternative promoters. Primer extension analysis and S1 nuclease mapping of another nucleus-encoded chloroplast ribosomal protein gene (rpsl) give evidence that the same type of regulation by two-promoter usage might be a more general phenomenon of plant chloroplast-related ribosomal protein genes. Preliminary results indicate the presence of conserved sequences within the rpl2l and rpsl promoter regions which compete for the same DNA binding activities.Plastids are essential components of plant cells. Depending on their function within a defined plant tissue or organ, plastids differentiate into several types of organelles. They are present as chloroplasts in photosynthetically active cells of leaves, as amyloplasts and proplastids in root cells and seeds, and as chromoplasts in fruit and flower cells.The transformation of plastids into these specialized types of organelles is necessarily based on changes in gene expression within the organelles as well as changes in the expression of nuclear-encoded plastid proteins. These changes concern the induction and/or repression of the expression of plastid-specific genes, but they also have to include modulated expression of genes which are necessary for the genetic functions of all types of organelles. In this respect, we are especially interested in how different types of plastids maintain their translational capacities, e.g., in the regulation of ribosomal gene expression during plastid differentiation.The present knowledge on ribosomal gene expression originates mainly from studies of procaryotic and animal systems. It has been shown previously that the rate of ribosome formation is adapted to the individual cell...

S2F, a leaf-specific trans-acting factor, binds to a novel cis-acting element and differentially activates the RPL21 gene.

“…This result indicates the presence of a positive cis-regulatory element(s) in this region, hereafter referred to as RPL27-distal positive domain (RfL27-DPD). Further deletion of the promoter region span-ning from -204 to -89 (pCHA5 and pCHAG), which contains the previously characterized negative S1 F sites (S1 F1 and S1 F2 sites in Figure 1 ; Lagrange et al, 1993), increased transcriptional activity (Figure 28). These sites might play a role in downregulating RPL27 promoter activity.…”

“…We have previously observed that RfL27 gene expression is upregulated in photosynthetic tissues by use of an alternative promoter (Lagrange et al, 1993). To elucidate the mechanisms involved in the developmental regulation of the RPL27 gene, we initiated a functional analysis of the 5' flanking sequence by transient expression of promoter deletions (Figures 1 and 2A) into tobacco mesophyll cell protoplasts.…”

“…To identify tissue-specific factors regulating plastid development, we analyzed the promoter organization of the nuclear RPL21 gene encoding the plastid ribosomal protein L21. We focused our attention on the RPL21 gene because we have shown previously that its expression is under the control of the plant developmental program, that is, the RPL21 gene is expressed independently of light, and its level of expression correlates with the amount of plastid ribosomes present in different plastid types (Franzetti et al, 1992;Lagrange et al, 1993). Previous results have shown that this gene is expressed differentially by the use of two transcriptional initiators, one at P2, providing a low level of constitutive expression, and a second at P1, providing a high level of leaf-specific expression (Lagrange et al, 1993).…”

“…In mesophyll cells, the RfL27 gene uses a second promoter, P1, that is activated early during chloroplast differentiation (Harrak et al, 1995). The accumulation of P1 transcripts in dark-or lightgrown cotyledons and leaves is -30-to 50-fold that of P2 transcripts in root cells (Lagrange et al, 1993). Thus, the expression of the RPL27 gene is organ dependent and does not require the presence of photosynthetically active chloroplasts.…”

“…This gene is present in one copy per haploid genome, and its expression is light independent (Lagrange et al, 1993). The RPL27 gene lacks a canonical TATA box, and its expression is driven by a constitutive promoter, P2, that initiates low-leve1 transcription in a polypyrimidine initiator stretch.…”