Mechanisms controlling C4 gene expression

Genes coding for C4 enzymes probably arose by gene duplication followed by the addition of new gene regulatory elements to confer high expression and cell specificity. Given that the nonphotosynthetic isoforms of most C4 enzymes are found at low levels in several plant tissues, this dramatic change in gene regulation is the major difference between C4 genes and their relatives. In principle, this evolutionary change could have been accomplished by the addition of a common set of DNA elements controlling BSC or MC specificity coupled with DNA elements for high expression. The expression programs of genes coding for most of the C4 enzymes have been studied in detail (reviewed in Dengler and Taylor 2000, Sheen 1999). These studies indicate that there is no common mechanism; rather, a diverse set of mechanisms controls the expression of different genes.

There are, however, some common themes in C4 gene regulation. Several lines of evidence have shown that C4 genes are up-regulated in chlorenchymous tissue before the differentiation of BSC and MC, in some cases showing expression in the precursors of both cell types (Langdale et al 1988, Ramsperger et al 1996, Sheen and Bogorad 1987). These observations suggest that part of the enhanced expression of C, genes could be due to the acquisition of DNA elements similar to those found on C, photosynthetic genes. The C, elements could provide the appropriate gene control during leaf development that results in high expression in cells containing chloroplasts.

Dengler and Taylor (2000) presented a hierarchical model to explain C, gene regulation. The first level can be considered the C, default program. The potential for high gene expression is established in those cells that will become photosynthetically active, including BSC. This level involves positional signaling that distinguishes ground from dermal and provascular tissue, light signals, and signaling between the nucleus and chloroplast. Nuclear genes coding for chloroplast proteins are activated, but not expressed at high levels; chloroplast genes are activated; and chloroplast replication is induced. The fact that some C4 genes are expressed in a leaf-specific fashion in transgenic C, plants provides evidence for this first level of regulation.

The second level of regulation involves BSC and MC differentiation. The same intercellular signaling that promotes cell differentiation also promotes high cell-specific expression of C4 genes (C4 signal, Fig. 3). Regulation for some C4 genes involves both increased expression in one cell type and repression of gene expression in the other.

An additional level of regulation is exerted by the products of photosynthesis (Koch 1996, Jang and Sheen 1997). Nitrogen also affects the expression of C4 genes and the nitrogen-sensing mechanism has been shown to act through cytokinin (Sakakibara et al 1998).

Several recent reviews provide comprehensive discussions of our current understanding of C4 gene structure and mechanisms of regulation, including analyses of promoter activities in transgenic C, plants (Dengler and Taylor 2000. Ku et al 1996. Sheen 1999). Selected examples will be discussed here.

PEP (phosphoeno/pyruvate) carboxylase is encoded by a gene family in maize and Flaveria species (Hermans and Westhoff 1990, Ku et al 1996). Ppc genes coding for the C4 isoform have been analyzed in transgenic plants and shown to be transcriptionally regulated by sequences located at the 5' end of the gene (Schaffner and Sheen 1992, Stockhaus et al 1997). The 5' promoter region controls both high-level expression and MC specificity. Yanagisawa and Sheen (1998) have identified a maize transcription factor, Dofl, that activates expression of the C4 Ppc gene but has no effect on Pdk or Cab (chlorophyll a/b apoprotein of photosystem II) promoters.

Matsuoka et al (1994) demonstrated that the maize C4 Ppc gene promoter directs high reporter gene expression in rice. Of particular interest was their observation that this expression was confined to leaf MC. Stockhaus et al (1994) isolated 5'promoter regions from two PpcAl genes, one from the C4 species Flaveria trinenia. which encodes the C4 isoform, and one from the orthologous PpcAl gene from the C, species F. pringlei, which encodes a nonphotosynthetic isoform of PEP carboxylase. The F. trinen ia promoter directed high leaf-specific expression in transgenic tobacco. This expression was found primarily in palisade mesophyll. Low expression in leaves, stems, and roots was found with the F. pringlei promoter. Both research groups concluded that the major change in the evolution of C4 Ppc genes was the acquisition of ¿■«-acting DNA sequences in the 5' promoter region. Furthermore, these sequences are recognized by irans-acting factors in unrelated C, plants to give a C4-like pattern of expression.

The C4 isoform of pyruvate, orthophosphate dikinase is encoded by a single dual-function gene in maize and Flaveria (Glackin and Grula 1990, Rosche and Westhoff 1995, Sheen 1991). These Pdk genes are transcribed from two separate promoters, one producing an abundant mRNA specific to MC that encodes the chloroplast-localized C4 isoform. The other promoter is located in the large first intron and in roots and stems directs the low accumulation of a shorter mRNA encoding a cytoplasmic isoform of unknown function. The 5' promoter region of the F. trinervia Pdk gene directs high MC-specific expression in transformed F. bidentis (C4 species) plants (Rosche et al 1998). Low expression is also seen in stems and roots, suggesting that the chloroplast isoform also has nonphotosynthetic functions. The same region from the maize Pdk gene directs high expression in MC protoplasts (Sheen 1991). The Flaveria and maize C4 Pdk genes did not evolve through a gene duplication; rather, the existing gene gained new, ris-acting regulatory elements in the 5' promoter region that function along with the original promoter elements.

Matsuoka and colleagues (1993) demonstrated that the maize C4 Pdk promoter directed high MC-specific expression in rice. They have also achieved high expression in rice by transferring the entire maize C4 Ppc and Pdk genes (Matsuoka et al, this volume).

These experimental results provide encouragement for the possibility of expressing C4 genes in a C4-like pattern in rice. However, a very different story has emerged for the gene encoding the C4 isoform of NADP-ME. Marshall et al (1996) determined that Flaveria species, regardless of photosynthetic type, have two genes coding for chloroplast NADP-ME. The Mel gene codes for the C4 isoform and appears to have arisen by a gene duplication that occurred before speciation within the genus. Experiments in transgenic F. bidentis plants showed the regulation of this gene to be complex and different from that of other C, genes (Marshall et al 1997, S. Ali and W.C. Taylor, manuscript submitted). Cell specificity is controlled by sequences in the 5' region, either upstream of the transcription initiation site (promoter) or in the 5' untranslated (5' UTR) site of the transcribed part of the gene (Fig. 4). These sequences probably interact with a BSC-specific trans factor. Quantitative levels of gene expression are controlled by an interaction of sequences at the N terminus, including the first intron, and at the 3' UTR. This interaction increases transgene expression in C4 Flaveria several hundred-fold. This mechanism controls the accumulation of mRNA, but it is unknown whether it acts through transcriptional orpost-transcriptional processes. Surprisingly, none of the transgene constructs showing high BSC-specific expression in Flaveria showed any detectable expression in tobacco. We concluded that the interaction between 5' and 3' sequences requires a trans-acting factor present only in C4 Flaveria. Evolution of the C4 Mel gene therefore involved acquisition of ds-acting DNA elements and at least one rra«.v-acting factor. Our data do not provide any clues as to whether the hypothesized trans factor controlling quantitative expression is BSC-

BSC factor i

(BSC) factor 1

Cell specificity


1 _

ATG . i * mm ■


■ I

5' upstream

First intron

3' UTR

Fig. 4. DNA sequence elements and trans-acting factors that regulate expression of the Flaveria C4 Mel gene. Exons, present in Mel mRNA, are indicated by black boxes. ATG = initiation codon, UTR = untranslated region.

Fig. 4. DNA sequence elements and trans-acting factors that regulate expression of the Flaveria C4 Mel gene. Exons, present in Mel mRNA, are indicated by black boxes. ATG = initiation codon, UTR = untranslated region.

specific, or whether it is related to the proposed trans factor that controls BSC-specific expression (Fig. 4). Although these results with the Flaveria Mel gene do not prove that the orthologous maize gene will be regulated by similar mechanisms, the similarities between maize and Flaveria Ppc and Pdk genes suggest that might be the case. If so, expression of a C4 Me gene in rice may be a challenge.

Expression of genes coding for both subunits of Rubisco is altered in C4 plants such that expression is confined to BSC. In maize and in the C4 dicot Amaranthus hypochondriacus, regulation of RbcS genes (coding for the small subunit) involves gene repression in MC and up-regulation in BSC (Langdale et al 1988. Ramsperger et al 1996. Sheen and Bogorad 1987). Positive and negative regulatory DNA elements have been defined (Schaffner and Sheen 1991) and sequences at the 3' end of the RbcS-m3 gene have been shown to be responsible for repressing gene activity in MC (Viret et al 1994). Sheen (1990) found evidence of a post-transcriptional step in repression of MC activity of RbcS.

In a detailed study of the patterns of accumulation of mRNAs and proteins during early stages of leaf development in A. hypochondriacus. Ramsperger et al (1996) found evidence for translational control of cell-specific accumulation of Rubisco. Translational control is also involved in light regulation of RbcS (Berry et al 1997. Wang et al 1993). To explore this level of regulation further, reporter gene constructs have been made to determine which parts of RbcS are responsible for translational control. Identification of sequences in the RbcS mRNA will then facilitate the study of the mechanism. The 5' and 3' UTRs from an A. hypochondriacus RbcS gene have been added to a construct with the cauliflower mosaic virus 35S promoter driving the gusA reporter gene and the nos 3' terminator (A.C. Corey, J.O. Berry, S. Ali, W.C. Taylor, unpublished). This construct was compared with the original 35S-gus in transgenic C4 Flaveria. The addition of the RbcS 5' and 3' UTRs increased gene expression about fortyfold. It remains to be determined if the 5' and 3' UTRs will increase gene expression in C, plants, or if they are involved in C4-specific regulation.

When the promoter activity of a maize RbcS gene was tested in rice, it was found to direct high expression in MC (Matsuoka et al 1994). The authors concluded that rrans-acting factors responsible for repressing MC expression are absent in rice because it is a C, plant.

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