The arabidopsis ACT7 actin gene is expressed in rapidly developing tissues and responds to several external stimuli

ACT7 encodes one of the six distinct and ancient subclasses of actin protein in the complex Arabidopsis actin gene family. We determined the sequence and structure of the Arabidopsis thaliana ACT7 actin gene and investigated its tissue-specific expression and regulation. The ACT7 mRNA levels varied by 128-fold among several different tissues and organs. The highest levels of aCT7 mRNA were found in rapidly expanding vegetative organs, the lowest in pollen. A translational fusion with the 5' end of ACT 7 (1.9 kb) joined to the beta-glucuronidase reporter gene was strongly and preferentially expressed in all young, developing vegetative tissues of transgenic Arabidopsis plants. ACT7 was the only Arabidopsis actin gene strongly expressed in the hypocotyl and seed coat. Although no beta-glucuronidase expression was seen in developing ovules or immature seeds, strong expression was seen in dry seeds and immediately after imbibition in the entire seedling. ACT7 was the only Arabidopsis actin gene to respond strongly to auxin, other hormone treatments, light regime, and wounding, and may be the primary actin gene responding to external stimuli. The ACT7 promoter sequence contains a remarkable number of motifs with sequence similarity to putative phytohormone response elements.     

Identification of promoter elements involved in the cytosolic Ca(2+)-mediated photoregulation of maize cab-m1 expression

Changes in cytoplasmic Ca2+ levels are involved in the regulation of several plant genes. However, to our knowledge, no regions of genes or specific cis elements have been shown to be involved in the regulation of plant gene expression by cytosolic Ca2+ signaling. The maize (Zea mays) gene cab-m1, which encodes a light-harvesting chlorophyll a/b-binding apoprotein, is positively photoregulated in mesophyll cells (MC) but not in bundle-sheath cells (BSC). This gene is highly preferentially expressed in maize MC versus BSC. In situ transient expression assays have revealed that exposure of tissues to ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), which chelates Ca2+, blocks the photostimulation of cab-m1 full promoter (-1026 to + 14) activity in MC of leaf segments of dark-grown maize seedlings. EGTA has no effect on expression in BSC. These results suggest that light-induced elevation of the cytosolic Ca2+ concentration in MC is required for the enhancement of cab-m1 expression in MC. Deletion of the sequence from -1026 to -360 completely abolished Ca2+ responsiveness of cab-m1 expression in MC. On the other hand, a 54-bp fragment in the 5' flanking region (-953 to -899 relative to the translation start site) conferred Ca2+ responsiveness on a -359 core promoter: reporter gene, suggesting that Ca2+ signaling is mediated via specific sequences in this short fragment. Furthermore, possible involvement of Ca(2+)-calmodulin in the signal transduction chain for regulating cab-m1 expression was suggested by the results of inhibitor experiments.     

Higher order chromatin structures in maize and Arabidopsis

We are investigating the nature of plant genome domain organization by using DNase I- and topoisomerase II-mediated cleavage to produce domains reflecting higher order chromatin structures. Limited digestion of nuclei with DNase I results in the conversion of the >800 kb genomic DNA to an accumulation of fragments that represents a collection of individual domains of the genome created by preferential cleavage at super-hypersensitive regions. The median size of these fragments is approximately 45 kb in maize and approximately 25 kb in Arabidopsis. Hybridization analyses with specific gene probes revealed that individual genes occupy discrete domains within the distribution created by DNase I. The maize alcohol dehydrogenase Adh1 gene occupies a domain of 90 kb, and the maize general regulatory factor GRF1 gene occupies a domain of 100 kb in length. Arabidopsis Adh was found within two distinct domains of 8.3 and 6.1 kb, whereas an Arabidopsis GRF gene occupies a single domain of 27 kb. The domains created by topoisomerase II-mediated cleavage are identical in size to those created by DNase I. These results imply that the genome is not packaged by means of a random gathering of the genome into domains of indiscriminate length but rather that the genome is gathered into specific domains and that a gene consistently occupies a discrete physical section of the genome. Our proposed model is that these large organizational domains represent the fundamental structural loop domains created by attachment of chromatin to the nuclear matrix at loop basements. These loop domains may be distinct from the domains created by the matrix attachment regions that typically flank smaller, often functionally distinct sections of the genome.     

Expression of the Zinnia TED3 promoter in developing tracheary elements of transgenic Arabidopsis

To determine the regulatory mechanism of gene expression in the early stages of tracheary element (TE) differentiation, we isolated and characterized a genomic fragment of TED3 whose mRNA is expressed preferentially in differentiating TEs 12-24 h before morphological changes in the in vitro Zinnia system. Transgenic Arabidopsis plants with a chimeric gene of the 537 bp TED3 promoter and the beta-glucuronidase (GUS) reporter gene indicated the strong expression of the GUS gene by the TED3 promoter in TEs, in particular in immature TEs as well as stipules and trichomes. GUS expression driven by the promoter was also induced in callus, in which GUS activity was localized in immature TEs and slender cells around TEs that may be TE precursor cells. The TED3 promoter was not significantly activated by wounding. This pattern of expression differed clearly from that of other vascular tissue-related genes such as PAL, 4CL, and GRP1.8. The nature of TED3 promoter enables us to use it to monitor TE differentiation in tissue and to introduce foreign genes preferentially into immature TE.     

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable element Ds1 in maize plants has been established previously. In this system, the Ds1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of the Ac element, excision of Ds1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences required in cis for excision of Ds1. The Ds1 element contains the Ac transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5' subterminal region). We showed that mutation of these motifs abolished completely the excision capacity of Ds1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3') subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.