Identification of a root-specific glycosyltransferase from Arabidopsis and characterization of its promoter

A set of Ds-element enhancer trap lines of Arabidopsis thaliana was generated and screened for expression patterns leading to the identification of a line that showed root-specific expression of the bacterial uidA reporter gene encoding beta-glucuronidase (GUS). The insertion of the Ds element was found to be immediately downstream to a glycosyltransferase gene At1g73160. Analysis of At1g73160 expression showed that it is highly root-specific. Isolation and characterization of the upstream region of the At1g73160 gene led to the definition of a 218 bp fragment that is sufficient to confer root-specific expression. Sequence analysis revealed that several regulatory elements were implicated in expression in root tissue. The promoter identified and characterized in this study has the potential to be applied in crop biotechnology for directing the root-specific expression of transgenes.     

Gene trap lines identify Arabidopsis genes expressed in stomatal guard cells

We employed a gene trap approach to identify genes expressed in stomatal guard cells of Arabidopsis thaliana . We examined patterns of reporter gene expression in approximately 20 000 gene trap lines, and recovered five lines with exclusive or preferential expression in stomata. The screen yielded two insertions in annotated genes, encoding the CYTOCHROME P450 86A2 (CYP86A2) mono-oxygenase, and the PLEIOTROPIC DRUG RESISTANCE 3 (AtPDR3) transporter. Expression of the trapped genes in guard cells was confirmed by RT-PCR experiments in purified stomata. Examination of homozygous mutant lines revealed that abscisic acid (ABA)-induced stomatal closure was impaired in the atpdr3 mutant. In three lines, insertions occurred outside transcribed units. Expression analysis of the genes surrounding the trapping inserts identified two genes selectively expressed in guard cells, corresponding to a PP2C PROTEIN PHOSPHATASE and an unknown expressed protein gene. Statistical analyses of the chromosomal regions tagged by the gene trap insertions revealed an over-represented [A/T]AAAG motif, previously described as an essential cis -active element for gene expression in stomata. The lines described in this work identify novel genes involved in the modulation of stomatal activity, provide useful markers for the study of developmental pathways in guard cells, and are a valuable source of guard cell-specific promoters.     

GAL4 GFP enhancer trap lines for analysis of stomatal guard cell development and gene expression

To facilitate the monitoring of guard cells during developmentand isolation, a population of 704 GAL4 GFP enhancer trap lineswas screened and four single insert lines with guard cell GFPexpression and one with developmentally-regulated guard cellGFP expression were identified. The location of the T-DNA inserts,the expression of the flanking genes, and the promoter activityof the genomic DNA upstream of the T-DNA were characterized.The results indicated that the GFP expression pattern in atleast one of the lines was due to elements in the intergenicDNA immediately upstream of the T-DNA, rather than due to theactivity of the promoters of genes flanking the insert, andprovide evidence for the involvement of Dof elements in regulatingguard cell gene expression. It is shown further that the GAL4GFP lines can be used to track the contribution of guard cellmaterial in vitro, and this method was used to assess the purityof guard cell samples obtained using two methods of guard cellisolation. Key words: Arabidopsis, development, enhancer trap, GFP, guard cells, stomata, T-DNA Received 21 July 2008; Revised 7 October 2008 Accepted 16 October 2008     

Generation of enhancer trap lines in Arabidopsis and characterization of expression patterns in the inflorescence

Eleven thousand, three hundred and seventy enhancer/promoter trap lines in Arabidopsis were generated via T-DNA transformation utilizing the binary vector pD991 that contains a minimal promoter fused to the uidA reporter gene. Overall 31% of the lines generated exhibit a staining pattern in the inflorescence. Flanking DNA has been cloned from 15 lines exhibiting inflorescence staining patterns by either thermal asymmetric interlaced PCR (TAIL-PCR), inverse PCR (IPCR), or partial library construction. Seeds from these lines are available from the ABRC and NASC Arabidopsis stock centers and DNA pools are available from the ABRC.     

Insertional tagging of regulatory sequences in tritordeum; a hexaploid cereal species

As an approach to isolate novel cereal promoters, promoterless uidA constructs and particle bombardment were used to transform tritordeum. Five of eight transgenic lines containing uidA sequences showed evidence of promoter tagging. Expression of uidA was detected in four lines as: constitutive expression, expression in short cells of the epidermis of the spikelets, expression in pollen grains and in cells of the epidermis of the spikelet, and expression in anther primordia and pollen grains. In the fifth line, the uidA was shown by RT-PCR to be transcribed, but no GUS activity was detected. The different patterns of uidA expression indicate that different regulatory sequences were tagged in each of these lines. Analysis of the progeny resulting from self-fertilisation of the primary tagged plants, indicate that the transgenes integrated at one or two loci and the patterns of expression were stably inherited. To our knowledge, this is the first report of promoter tagging in cereals by direct gene transfer.     

ABA-regulated promoter activity in stomatal guard cells

CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum . The CDeT6-19 gene promoter has been fused to the β- glucuronidase reporter gene ( GUS ) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum . This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed-specific, ABA-regulated gene, Em , or the promoter from the ABA-regulated CDeT27-45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA-regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA-induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca²⁺ and other second messengers in ABA-induced gene expression.     

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.     

Rice salT promoter is activated in Papaver somniferum and Nicotiana tabacum transgenic cells in the absence of exogenous ABA

With the aim of modifying secondary metabolism in Opium poppy (Papaver somniferum) and tobacco (Nicotiana tabacum) cells, gene transfer was performed using the sam1 gene from Arabidopsis thaliana under the control of the salT promoter. This promoter is induced by ABA in rice and in tobacco and we have shown that it is also induced in poppy cells (gus gene). Putatively transformed poppy and tobacco cell lines with the sam1 gene were obtained. In the absence of exogenous inducer we noticed the expression of the transgene resulting in a significant increase of SAM-S activity in all tested transformants of poppy and in half the transgenic tobacco cell lines tested. Addition of ABA to the culture medium failed to enhance the expression of the transgene in both species and resulted in a decrease of the sam1 gene expression in some cell lines. Since the salT promoter is induced by exogenous ABA in both species (gus reporter gene), we suggest a partial sam1 transgene inactivation in certain cell lines. These results show that the efficiency of a regulatory sequence may be different when fused with a reporter gene (gus) compared to fusion with a gene belonging to the housekeeping family (sam1).     

Vascular-specific expression of the bean GRP 1.8 gene is negatively regulated.

In French bean, the glycine-rich cell wall protein GRP 1.8 is specifically synthesized in the vascular tissue. To identify cis-acting sequences required for cell type-specific synthesis of GRP 1.8, expression patterns of fusion gene constructs were analyzed in transgenic tobacco. In these constructs, the uidA (beta-glucuronidase) gene was placed under control of 5' upstream deletions as well as internal deletions of the GRP 1.8 promoter. Four different cis-acting regulatory regions, SE1 and SE2 (stem elements), a negative regulatory element, and a root-specific element, were found to control the tissue-specific expression. Deletion of the negative regulatory element resulted in expression of the uidA gene in cell types other than vascular cells. The SE1 region was essential for expression in several cell types in the absence of further upstream regulatory sequences. Full-length promoters having insertions between the negative regulatory element and SE1 strongly expressed the gene in nonvascular cell types in stems and leaves. Thus, vascular-specific expression of the GRP 1.8 promoter is controlled by a complex set of positive and negative interactions between cis-acting regulatory regions. The disturbance of these interactions results in expression in additional cell types.     

Analysis of GDP-D-mannose pyrophosphorylase gene promoter from acerola (Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing the acerola gene

GDP-D-mannose pyrophosphorylase (GMP) is an important enzyme in the Smirnoff-Wheeler's pathway for the biosynthesis of ascorbic acid (AsA) in plants. We have reported recently that the expression of the acerola (Malpighia glabra) GMP gene, designated MgGMP, correlates with the AsA content of the plant. The acerola plant has very high levels of AsA relative to better studied model plants such as Arabidopsis. Here we found that the GMP mRNA levels in acerola are higher than those from Arabidopsis and tomato. Also, the transient expression of the uidA reporter gene in the protoplasts of Nicotiana tabacum cultures showed the MgGMP gene promoter to have higher activity than the cauliflower mosaic virus 35S and Arabidopsis GMP promoters. The AsA content of transgenic tobacco plants expressing the MgGMP gene including its promoter was about 2-fold higher than that of the wild type.