B-type granule containing protrusions and interconnections between amyloplasts in developing wheat endosperm revealed by transmission electron microscopy and GFP expression.

Starch granules in mature wheat endosperm show a bimodal size distribution. The formation of small starch granules in wheat endosperm cells was studied by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) after expression and targeting of fluorescent protein into amyloplasts. Both techniques demonstrated the presence of protrusions emanating from A-type granules-containing amyloplasts and the presence of B-type starch granules in these evaginations. Moreover, CLSM recordings demonstrated the interconnection of the amyloplasts by these protrusions, suggesting a possible role of these protrusions in interplastid communication.

RAP-1 is an Arabidopsis MYC-like R protein homologue, that binds to G-box sequence motifs.

An Arabidopsis cDNA clone encoding a DNA-binding protein, RAP-1, was isolated by southwestern screening of an Escherichia coli cDNA expression library. The protein contains a bHLH DNA-binding domain and is homologous to R proteins, regulating anthocyanin biosynthesis. RAP-1 binds to the sequence CACNTG. It is encoded by a single gene, which is expressed to high levels in root and stem and to low levels in leaf and flower. No expression could be detected in siliques. Rap-1 does not correspond to one of the known loci involved in anthocyanin biosynthesis, since it is located at a different map position. In contrast to the maize R protein Lc, RAP-1 did not induce anthocyanin biosynthesis in pea cotyledons. Thus, RAP-1 is a novel member of the bHLH class of DNA-binding proteins.

Characterization of a zinc-dependent transcriptional activator from Arabidopsis.

The C2-H2 zinc-finger is a widely occurring DNA binding motif, usually present as tandem repeats. The majority of C2-H2 zinc-finger proteins that have been studied are derived from animals. Here, we characterize a member of a distinct class of plant C2-H2 zinc-finger proteins in detail. A cDNA clone encoding a DNA binding protein from Arabidopsis was isolated by SouthWestern screening. The protein, termed ZAP1 (Zinc-dependent Activator Protein-1), is encoded by a single copy gene, which is expressed to similar levels in root and flower, to a somewhat lower level in stem and to low levels in leaf and siliques. The optimal binding site was determined by random binding site selection, and the consensus sequence found is CGTTGACCGAG. The homology between ZAP1 and other DNA binding proteins is restricted to a repeated region of a stretch of 24 highly conserved amino acids followed by a zinc-finger motif (C-X4-C-X22-23-H-X1-H). The C-terminal zinc-finger region is essential for DNA binding, whereas deletion of the N-terminal one resulted in 2.5-fold reduced binding affinity. Binding of ZAP1 to DNA was abolished by metal-chelating agents. The activation domain as determined in yeast is adjacent to and possibly overlapping with the DNA binding domain. Particle bombardment experiments with plant cells showed that ZAP1 increases expression of a gusA reporter gene that is under control of ZAP1 binding sites. We conclude that ZAP1 is a plant transcriptional activator with a C2-H2 zinc-finger DNA binding domain.

The 22 bp W1 element in the pea lectin promoter is necessary and, as a multimer, sufficient for high gene expression in tobacco seeds.

The pea lectin (Psl) gene encodes an abundant seed protein. Its seed-specific expression pattern is conserved in transgenic tobacco plants. Progressive 5' promoter deletions resulted in a gradual decrease of transcriptional activity in tobacco seed. A fragment of 115 bp still conferred seed-specific expression albeit at a low level. This fragment contains a 22 bp element (W1), which has been demonstrated to be important for seed-specific expression when coupled as a trimer to a heterologous TATA box (de Pater et al., Plant Cell 5:877-886, 1993). Here we show that deletion of W1 in the natural promoter context resulted in a strongly decreased level of gene expression. A 4 bp mutation of W1 reduced the expression of truncated derivatives of the Psl promoter. A single copy of W1 coupled to the TATA box of the CaMV 35S promoter directed low gene expression in seeds and leaves. Multimerization enhanced the expression in seeds up to 100-fold, to levels found with the Psl promoter, whereas the expression level in leaves remained low. These results demonstrate that the W1 element is an essential control element in the Psl promoter. When taken out of its natural context and multimerized, it is sufficient for high expression in seeds.

Binding specificity and tissue-specific expression pattern of the Arabidopsis bZIP transcription factor TGA2.

The binding specificity and tissue-specific expression pattern of TGA2 (AHBP-1b), an Arabidopsis bZIP transcription factor have been determined. Filter-binding and gel-shift assays showed that TGA2 has high affinity for C-boxes (ATGACGTCAT). In this respect TGA2 is similar to other members of the Arabidopsis TGA family (such as TGA1, TGA3 and OBF4) and to tobacco TGA1a. Genomic Southern blot analysis confirmed that TGA2 is a member of the gene family. Northern blot analysis showed that the gene is expressed at similar levels in root, stem, leaf and flower t at somewhat lower levels in siliques. TGA3 was also found to be expressed at the same level throughout the plant, whereas genes encoding TGA1 and OBF4 have relatively high RNA expression levels in root. The differential expression of these genes suggests that they have distinct functions.

bZIP proteins bind to a palindromic sequence without an ACGT core located in a seed-specific element of the pea lectin promoter.

Previously, it has been shown that a trimer of a 22 bp fragment of the promoter of the seed-specific pea lectin gene confers high expression in seed. Here it is reported that this fragment contains a binding site for the cloned basic domain/leucine zipper (bZIP) proteins TGA1a and Opaque-2 (O2). Gel shift assays, DNasel footprinting and methylation interference assays using purified TGA1a were performed to determine whether additional binding sites are present in the psl promoter. Within the 469 bp upstream region only one other TGA1a binding site was found, which is much weaker than the one present in the 22 bp element. Both O2 and TGA1a bound to the odd base palindromic C-box sequence, ATGAGTCAT, present within the 22 bp fragment. The 22 bp fragment also contains the sequence CACGTA, which contains the ACGT core usually found in binding sites for bZIP proteins. However, this sequence did not significantly contribute to bZIP protein binding. The binding affinity of TGA1a for the odd base palindromic sequence was low relative to a high-affinity C-box (ATGACGTCAT). By contrast, O2 strongly bound to the odd base C-box; the affinity was comparable with that for high-affinity G-(GACACGTGTC) and C-boxes. It is concluded that the presence of an ACGT core sequence is not a prerequisite for high-affinity binding of O2.

A 22-bp fragment of the pea lectin promoter containing essential TGAC-like motifs confers seed-specific gene expression.

To elucidate the molecular mechanisms responsible for seed-specific gene expression in plants, the promoter of the pea lectin (psl) gene, encoding an abundant seed protein, was used as a model. Leaf and seed nuclear proteins bound to a region in the psl promoter containing three overlapping TGAC-like motifs, which have been shown to be a binding site for basic/leucine zipper proteins, including TGA1a. A trimer of a 22-bp region of the psl promoter, containing the TGAC-like motifs, coupled to a heterologous minimal promoter conferred low reporter gene expression in root, stem, and leaf and high expression in seed of transgenic tobacco. Expression increased during the midmaturation stage of seed development and was observed in the endosperm as well as in the embryo, where it strongly decreased within a few days after germination. This expression pattern is qualitatively identical to the expression pattern conferred by a 2000-bp fragment of the psl promoter. Nucleotides within the TGAC-like motifs important for in vitro binding are also essential for in vivo transcription activation in vegetative tissue as well as in seed. The electrophoretic mobility of a DNA-protein complex containing seed nuclear protein was different from that formed with leaf nuclear protein. Furthermore, the TGA1a steady state mRNA level in immature seed was relatively low. These results suggest that a seed-specific factor different from TGA1a, but with similar binding specificity, is responsible for gene activation in seed. We conclude that the 22-bp region contains all the information, including an essential TGAGTCATCA sequence, necessary for seed-specific expression and very likely plays an essential role in the seed-specific expression pattern of the psl gene.

Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L. cv. Désirée) is strongly influenced by plant culture conditions.

The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for beta-glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3' flanking DNA. The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L. cv. Désirée). In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca. 11,000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca. 7000 units). GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca. 3000 units). Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca. 100 units), and lowest of all in leaves of soil-grown plants (ca. 10-15 units). However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures. Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene. In particular, it was silenced in leaves of soil-grown plants. The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S. tuberosum cv. Désirée cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives. A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5' expression control sequences.

Structure and expression of a light-inducible shoot-specific rice gene.

By differential screening of a cDNA library of two-week-old rice seedlings cDNA clones were obtained, corresponding to shoot-specific mRNAs. By sequence analysis two of these clones were found to be rbcS cDNA clones. The mRNA corresponding to a third cDNA clone (COS5) displayed an expression pattern similar to the expression pattern of rbcS genes. The mRNA (800 bases) was light-inducible and encoded by a single-copy gene. The genomic clone (GOS5) was isolated and the intron/exon structure was determined by comparing the nucleotide sequences of the mRNA and the genomic clone. The gene contains two introns. Transcription start sites were determined by S1-nuclease mapping and primer extension. The start site obtained by both methods is located 87 bp upstream of the translation start site and 23 bp downstream of TATA box-like sequence. In the 5' non-coding region motifs can be found that are homologous to sequences in promoters that are light- or UV-inducible or confer leaf-specific expression. The open reading frame present in GOS5 codes for a protein (15 kDa) that contains a putative chloroplast transit peptide and does not show any significant homology to protein sequences in the NBRF protein database.

The relationship between region E1a and E1b of human adenoviruses in cell transformation.

Baby rat kidney (BRK) cells were transfected either with intact region E1 DNA of adenovirus type 5 (Ad5) or with mixtures of DNA fragments containing the separated E1a and E1b regions. The results showed that mixtures of regions E1a and E1b transform with a similar efficiency as intact region E1. DNA fragments containing region E1b alone have no detectable transforming activity in primary BRK cells nor in established rat cell lines. When region E1a and Ad5 was combined with region E1b and Ad12 complete transformation was also obtained. Characterization of the cell lines transformed by separated E1a and E1b regions have led to the following conclusions: (1) Expression of region E1b is not dependent on specific linkage to region E1a as it occurs in the intact E1 region. (2) Region E1b is normally expressed into the corresponding major adenovirus T antigens (65,000 and 19,000 Mr with region E1b of Ad5; 60,000 and 19,000 Mr with E1b or AD12). (3) Region E1b of Ad12 can be activated by region E1a of Ad5 indicating that the Ela regions of both serotypes are functionally similar in transformation. (4) Cell lines containing region E1b of Ad5 are weakly oncogenic in nude mice whereas cells containing E1b of Ad12 are highly oncogenic in nude mice, indicating that the degree of oncogenicity is determined by region E1b.