Isolation of GUS marker lines for genes expressed in Arabidopsis endosperm,embryo and maternal tissues

In order to identify marker lines expressing GUS in various endosperm compartments and at different developmental stages, a collection of Arabidopsis thaliana (L.) Heynh. promoter trap lines were screened. The screen identified 16 lines displaying GUS-reporter gene expression in the endosperm, embryo and other seed organs. The distinctive patterns of GUS expression in these lines provide molecular markers for most cell compartments in the endosperm of Arabidopsis seeds at all developmental stages, and represent a valuable research tool for characterizing present and future Arabidopsis seed mutants. GUS expression patterns of these 16 lines are presented here. One line showed chalazal endosperm-specific GUS activity at the heart stage of embryo development. In six lines embryo-specific GUS activity was detected. Six lines exhibited GUS activity predominantly in the endosperm and embryo while two lines showed strong GUS activity in all seed organs. In one line GUS activity was detected in integuments and syncytial endosperm, while the GUS activity at the cotyledonary stage of the embryo was seed coat-specific. In addition, two funiculus markers and two silique markers expressed in the abscission zone and the guard cells are also presented.     

The Arabidopsis AtEPR1 extensin-like gene is specifically expressed in endosperm during seed germination

Screening of 10 000 Arabidopsis transgenic lines carrying a gene-trap (GUS) construct has been undertaken to identify markers of seed germination. One of these lines showed GUS activity restricted to the endosperm, at the micropylar end of the germinating seed. The genomic DNA flanking the T-DNA insert was cloned by walking PCR and the insertion was shown to be located 70 bp upstream of a 2285 bp open reading frame (AtEPR1) sharing strong similarities with extensins. The AtEPR1 open reading frame consists of 40 proline-rich repeats and is expressed in both wild-type and mutant lines. The expression of the AtEPR1 gene appears to be under positive control of gibberellic acid, but is not downregulated by abscisic acid during seed germination. No expression was detected in organs other than endosperm during seed germination. The putative role of AtEPR1 is discussed in the light of its specific expression in relation to seed germination.     

Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes

Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.     

Gene trapping in Arabidopsis reveals genes involved in vascular development

The procambium is made up of stem cells that give rise to various vascular cells in plants. To understand the molecular nature of procambium cells, we tried to identify genes that characterize procambium cells using Arabidopsis gene trap lines. Among 26,000 gene trap lines, we found 67 lines in which beta-glucuronidase (GUS) staining occurred along vascular tissues in cotyledons and/or adult leaves. Although four gene trap lines showed procambium-preferential GUS expression, their expression patterns differed from each other during procambium development in root tips and young rosette leaves. Genomic regions flanking the gene trap insertion points in 25 of the 67 lines were determined, including three lines showing preferential GUS staining of the procambium. The three procambium-related genes encoded PINHEAD, katanin and an unknown DUF740 domain-containing protein. We discuss procambium development based on the functions and the differential GUS staining patterns of the procambium-related genes.     

GhKLCR1, a kinesin light chain-related gene,induces drought-stress sensitivity in Arabidopsis

Drought stress results in significant losses in agricultural production, and especially that of cotton. The molecular mechanisms that coordinate drought tolerance remain elusive in cotton. Here, we isolated a drought-response gene GhKLCR1, which is a close homolog of AtKLCR1, which encodes a kinesin light chain-related protein enriched with a tetratrico peptide-repeat region.A subcellular localization assay showed that GhKLCR1 is associated with the cell membrane. A tissue-specific expression profile analysis demonstrated that GhKLCR1 is a cotton root-specific gene. Further abiotic and hormonal stress treatments showed that GhKLCR1 was upregulated during abiotic stresses, especially after polyethylene glycol treatments. In addition, the glucuronidase(GUS) staining activity increased as the increment of mannitol concentration in transgenic Arabidopsis plants harboring the fusion construct PGhKLCR1::GUS. The root lengths of 35 S::GhKLCR1 lines were significantly reduced compared with that of wild type. Additionally, seed germination was strongly inhibited in 35 S::GhKLCR1 lines after 300-mmol L~(-1) mannitol treatments as compared with Columbia-0, indicating the sensitivity of GhKLCR1 to drought. These findings provide a better understanding of the structural, physiological and functional mechanisms of kinesin light chain-related proteins.     

T-DNA insertional mutagenesis for functional genomics in rice

We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.     

Downstream effectors of light- and phytochrome-dependent regulation of hypocotyl elongation in Arabidopsis thaliana

Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.     

GASA4, one of the 14-member Arabidopsis GASA family of small polypeptides, regulates flowering and seed development

Members of the plant-specific gibberellic acid-stimulated Arabidopsis (GASA) gene family play roles in hormone response, defense and development. We have identified six new Arabidopsis GASA genes, bringing the total number of family members to 14. Here we show that these genes all encode small polypeptides that share the common structural features of an N-terminal putative signal sequence, a highly divergent intermediate region and a conserved 60 amino acid C-terminal domain containing 12 conserved cysteine residues. Analysis of promoter::GUS (beta-glucuronidase) transgenic plants representing six different GASA loci reveals that the promoters are activated in a variety of stage- and tissue-specific patterns during development, indicating that the GASA genes are involved in diverse processes. Characterization of GASA4 shows that the promoter is active in the shoot apex region, developing flowers and developing embryos. Phenotypic analyses of GASA4 loss-of-function and gain-of-function lines indicate that GASA4 regulates floral meristem identity and also positively affects both seed size and total seed yield.     

Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter—gusA fusion constructs

Sedentary plant-parasitic nematodes are able to induce specialized feeding structures in the root system of their host plants by triggering a series of dramatic cellular responses. These changes presumably are accompanied by a reprogramming of gene expression. To monitor such changes, a variety of promoter— gus A fusion constructs were introduced into Arabidopsis and tobacco. Transgenic plants were analysed histochemically for GUS activity in the nematode feeding structures after infection with either Heterodera schachtii or Meloidogyne incognita . Promoters of the Cauliflower Mosaic Virus 35S gene, the bacterial nopaline synthase, rooting loci ( rol ) and T- cyt genes and the plant-derived phenylalanine ammonia-lyase I gene, which are highly active in non-infected roots, were all downregulated in the feeding structures as indicated by the strong decrease of GUS activity inside these structures. Less stringent down-regulation was observed with chimeric gus A fusion constructs harbouring truncated rol B and rol C promoter sequences. Similar observations were made with transgenic Arabidopsis lines that carried randomly integrated promoterless gus A constructs to identify regulatory sequences in the plant genome. Most of the lines that were selected for expression in the root vascular cylinder demonstrated local down-regulation in feeding structures after infection with H. schachtii . The reverse pattern of GUS activity, a blue feeding structure amidst unstained root cells, was also found in several lines. However, GUS activity that was entirely specific for the feeding structures was not observed. Our data show that the expression of a large number of genes is influenced during the development of the nematode feeding structures.     

pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage

ABSTRACT: BACKGROUND: The production of transgenic plants, either for the overproduction of the protein of interest, for promoter::reporter lines, or for the downregulation of genes is an important prerequisite in modern plant research but is also very time-consuming. RESULTS: We have produced additions to the pPZP family of vectors. Vector pPZP500 (derived from pPZP200) is devoid of NotI sites and vector pPZP600 (derived from pPZP500) contains a bacterial kanamycin resistance gene. Vector pMAA-Red contains a Pdf2.1::DsRed marker and a CaMV::GUS cassette within the T-DNA and is useful for the production of promoter::GUS lines and overexpression lines. The Pdf2.1 promoter is expressed in seeds and syncytia induced by the beet cyst nematode Heterodera schachti in Arabidopsis roots. Transgenic seeds show red fluorescence which can be used for selection and the fluorescence level is indicative of the expression level of the transgene. The advantage is that plants can be grown on soil and that expression of the marker can be directly screened at the seed stage which saves time and resources. Due to the expression of the Pdf2.1::DsRed marker in syncytia, the vector is especially useful for the expression of a gene of interest in syncytia. CONCLUSIONS: The vector pMAA-Red allows for fast and easy production of transgenic Arabidopsis plants with a strong expression level of the gene of interest.     

Global functional analyses of rice promoters by genomics approaches

Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. Promoters that are subject to specific regulations are useful for manipulating foreign gene expression in plant cells, tissues, or organs with desirable patterns and under controlled conditions, and have been important for both basic research and applications in agriculture biotechnology. Recent advances in genomics technologies have greatly facilitated identification and study of promoters in a genome scale with high efficiency. Previously we have generated a large T-DNA tagged rice mutant library (TRIM), in which the T-DNA was designed with a gene/promoter trap system, by placing a promoter-less GUS gene next to the right border of T-DNA. GUS activity screens of this library offer in situ and in planta identifications and analyses of promoter activities in their native configurations in the rice genome. In the present study, we systematically performed GUS activity screens of the rice mutant library for genes/promoters constitutively, differentially, or specifically active in vegetative and reproductive tissues. More than 8,200 lines have been screened, and 11% and 22% of them displayed GUS staining in vegetative tissues and in flowers, respectively. Among the vegetative tissue active promoters, the ratio of leaf active versus root active is about 1.6. Interestingly, all the flower active promoters are anther active, but with varied activities in different flower tissues. To identify tissue specific ABA/stress up-regulated promoters, we compared microarray data of ABA/stress induced genes with those of tissue-specific expression determined by promoter trap GUS staining. Following this approach, we showed that the peroxidase 1 gene promoter was ABA up-regulated by 4 fold within 1 day of exposure to ABA and its expression is lateral root specific. We suggest that this be an easy bioinformatics approach in identifying tissue/cell type specific promoters that are up-regulated by hormones or other factors. Su-May Yu and Swee-Suak Ko equally contributed to this work.     

The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia

The Arabidopsis thaliana MERISTEM LAYER 1 (ATML1) gene is expressed in the epidermis of developing embryos and shoot meristems. To identify regulatory sequences necessary for epidermis-specific expression, three fusions of overlapping ATML1 genomic sequences to the GUS reporter gene were introduced into Arabidopsis plants. All fusion genes conferred epidermis-specific expression of both GUS mRNA and protein activity but varied in both the timing and relative levels of expression, suggesting partial redundancy of ATML1 regulatory elements. This study defines L1-specific regulatory sequences that are sufficient to direct foreign gene expression in a layer-specific manner.