Yuriko Kano-Murakami

Evolution and Expression of C4 Photosynthesis Genes

Based on the differences in the mechanism of CO, assimilation, land plants can be divided into three major photosynthetic types, namely C,, C,, and Crassulacean acid metabolism (CAM) plants. Each photosynthetic type possesses a unique set of anatomical, physiological, and biochemical features that allow them to adapt to a specific ecological niche. C, plants perform well in temperate climates, whereas C, plants thrive in high-light, arid, and warm environments. CAM plants, characterized by CO, uptake in the night, adapt to extreme arid conditions. Taxonomical and phylogenetic studies suggest that CAM and C, plants were derived from C, plants and the transitions occurred many times in diverse taxa during the course of evolution (Moore, 1982). A drastic decline in atmospheric CO, leve1 during the late Cretaceous period (65-85 million years ago), a time of major expansion of the angiosperms, has been proposed to account for the increase of C, plants (Ehleringer et al., 1991). The major function of the C, pathway is thought to overcome the limitation of low CO,, which results in significant increases in photorespiration and thus reduces competitiveness. Although it is widely assumed that CAM evolved in response to selection for increased water-use efficiency, the occurrence of CAM in aquatic plants (Keeley and Morton, 1982), in which photosynthesis is often limited by low CO, rather than water, strongly argues that the driving force for its evolution may be low CO, as well. Both C, and CAM evolved a very similar photosynthetic biochemistry for concentrating CO, in the leaf, except that the C0,-concentrating steps are spatially separated in C, plants but temporally separated in CAM plants. C, photosynthesis and CAM occur in only one cell type, the mesophyll cells, whereas C, photosynthesis requires the coordination of two photosynthetic cell types, the mesophyll and bundle-sheath cells. Therefore, the genetic modifications required for achieving the C0,-concentrating mechanism are considered relatively small for CAM, as compared with those required for C, photosynthesis. Comparative phylogenetic studies also suggest that CAM evolved earlier than C, photosynthesis (Moore, 1982).

A Metal-Dependent DNA-Binding Protein Interacts with a Constitutive Element of a Light-Responsive Promoter

We have used DNase I footprinting to characterize nuclear factors that bind to the light-responsive promoter of pea rbcS-3A, one member of the gene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. A sequence-specific binding activity, designated 3AF1, binds to an AT-rich sequence present at the -45 region of the rbcS-3A promoter. A tetramer of the 3AF1 binding site, designated as Box VI, can form multiple complexes with tobacco leaf and root nuclear extracts. Mutations of 3 base pairs in Box VI severely reduce DNA-protein complex formation in vitro. The wild-type Box VI tetramer, but not the mutant tetramer, is active in transgenic tobacco plants when placed upstream of the cauliflower mosaic virus 35S promoter truncated at -90. These results correlate binding of 3AF1 to the in vivo function of Box VI. The Box VI tetramer/35S chimeric construct confers expression in diverse cell types and organs and its activity is not dependent on light. By using the Box VI tetramer as a probe to screen a cDNA expression library, we have obtained a putative cDNA clone for the 3AF1 DNA-binding activity. Lysogen extracts of Escherichia coli expressing the cDNA clone give sequence-specific complexes with Box VI. The deduced amino acid sequence of the protein encoded by the cDNA contains two stretches of about 100 residues that are 80% homologous. Moreover, in each of the two repeats, there is an arrangement of histidines and cysteines, which may be related to the two known types of zinc-finger motifs found in many DNA-binding proteins. Consistent with the expectation that metal coordination plays an important role in DNA binding by this protein, we found that 1,10-phenanthroline can abolish the formation of DNA-protein complexes. Interestingly, we found that the same treatment did not abolish the DNA binding activity of 3AF1 in crude nuclear extracts of tobacco. These data indicate that the nuclear 3AF1 activity is likely due to multiple DNA-binding proteins all interacting with Box VI in vitro. RNA gel blot analysis shows that multiple transcripts homologous to this cDNA clone are expressed in different tobacco organs.

A rice homeotic gene, OSH1, causes unusual phenotypes in transgenic tobacco

A rice gene, OSH1, which shares homology with animal homeobox genes, has been isolated. We have introduced the OSH1 cDNA into tobacco in order to examine its function. Expression of the OSH1 cDNA in tobacco induced morphological abnormalities in the leaves, petals and stems of the transformants suggesting that OSH1 functions as a morphological regulator. OSH1 cDNA expression was analyzed under the control of three different promoters. This work revealed that not only the level of OSH1 expression but also the site and timing of the expression affect the morphology of the plant.

Nucleotide sequence of the PR-1 gene of Nicotiana tabacum

A gene encoding one of the pathogenesis‐related proteins, PR1a, and two related pseudogenes were isolated from Nicotiana tabacum. The cloned PR1a gene (pPR‐γ) and one of the pseudogenes (pPR‐α) were sequenced and found to have similar structures. The sequence of pPR‐γ was quite similar to that of the cDNA clone of PR1a. The plasmid pPR‐γ did not contain an intron and had a typical promoter sequence in the 5′‐flanking region.

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.

SEQUENCE-SPECIFIC INTERACTIONS OF A MAIZE FACTOR WITH A GC-RICH REPEAT IN THE PHOSPHOENOLPYRUVATE CARBOXYLASE GENE

SummaryA plant nuclear protein PEP-I, which binds specifically to the promoter region of the phosphoenolpyruvate carboxylase (PEPC) gene, was identified. Methylation interference analysis and DNA binding assays using synthetic oligonucleotides revealed that PEP-I binds to GC-rich elements. These elements are directly repeated sequences in the promoter region of the PEPC gene and we have suggested that they may be cis-regulatory element of this gene. The consensus sequence of the element is CCCTCTCCACATCC and the CTCC is essential for binding of PEP-I. PEP-I is present in the nuclear extracts of green leaves, where the PEPC gene is expressed. However, no binding was detected in tissues where the PEPC gene is not expressed in vivo, such as roots or etiolated leaves. Thus, PEP-1 is the first factor identified in plants which has different binding activity in light-grown compared with dark-grown tissue. PEP-I binding is also tissue-specific, suggesting that PEP-1 may function to coordinate PEPC gene expression with respect to light and tissue specificity. This report describes the identification and characterization of the sequences required for PEP-1 binding.

Expression of rice OSH1 gene is localized in developing vascular strands and its ectopic expression in transgenic rice causes altered morphology of leaf

Transgenic rice plants (Oryza sativa cv. Nipponbare) carrying 1 or 2 copies of a rice homeobox gene, OSH1, under the control of the CaMV 35S promoter were generated. The transgene caused altered morphology of leaf, such as ligule-replacement and abnormal division of sclerenchyma cells. The phenotype of these leaves resembles that of maize leaf morphological mutant, Knotted 1, which is caused by duplication of the KN1 gene (Veit et al., 1990). The in situ hybridization analysis has revealed that the expression of endogenous OSH1 is mainly localized in developing vascular strands of stem. We have discussed the biological roles of OSH1 in rice based on these results.