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.     

Vascular expression of the grp1.8 promoter is controlled by three specific regulatory elements and one unspecific activating sequence

The bean grp1.8 full-length promoter is specifically active in vascular tissue during normal development of tobacco. Deletion of a negative regulatory element resulted in ectopic activity of the promoter in cortical cells of hypocotyls, roots and stems. A 169 bp fragment (–205 to –36) of the grp1.8 promoter conferred vascular-specific expression to CaMV 35S minimal promoters whereas a 141 bp fragment (–205 to –64) strongly activated these minimal promoters both in vascular and cortical cells. These experiments defined a new regulatory element (VSE) that is essential for vascular-specific expression and is located between –64 and –36. The 141 bp grp1.8 promoter sequence had enhancer-like properties as it was active in both orientations. A 24 bp sequence (bp –119 to –96, corresponding to the SE1 regulatory element) enhanced expression from several minimal promoters strongly but unspecifically, whereas a 26 bp sequence (–98 to –73, corresponding to the RSE regulatory element) induced vascular-specific expression. Thus, the grp1.8 promoter is regulated by a combinatorial mechanism that can integrate the action of different, non-additively acting regulatory elements into vascular-specific expression.     

Activation of a tobacco glycine-rich protein gene by a fungal glucan preparation

A Phytophthora megasperma f.sp. glycinea cell wall glucan preparation was previously shown to protect tobacco plants against viral infection. Eleven plant defense-related genes were assayed for elevated mRNA accumulation levels in response to glucan treatment of tobacco plants. The expression of only one of these genes, a glycine-rich protein (GRP) gene, was induced by glucan application. Elevated GRP gene mRNA levels could be detected within 15 min of glucan treatment and reached maximum levels at 4 h post-treatment followed by a slow decline to 8 h. The maximum induction of the GRP gene was approximately ninefold above H₂O-treated control plants. Northern blot analysis showed that a single mRNA species of 1.4 kb was responding to the glucan treatment. GRP genes occur in tobacco as members of a multigene family, but only one specific GRP gene was induced by the glucan treatment. A genomic copy of this responding GRP gene was cloned and sequenced. This tobacco GRP gene is homologous to the petunia ptGRP1 gene and the French bean GRP1.8 gene, but is not closely related to the French bean GRP1.0 gene. GRP gene expression has previously been associated with disease resistance in plants, but it remains to be determined whether β-glucan activation of the tobacco GRP gene results in the observed resistance to virus.     

Hydrophobic interactions of the structural protein GRP1.8 in the cell wall of protoxylem elements

The glycine-rich structural protein GRP1.8 of French bean (Phaseolus vulgaris) is specifically localized in the modified primary cell walls of protoxylem elements. Continuous deposition of GRP1.8 into the cell walls during elongation growth of the plant suggests that GRP1.8 is part of a repair mechanism to strengthen the protoxylem. In this work, a reporter-protein system was developed to study the interaction of GRP1.8 with the extracellular matrix. Fusion proteins of a highly soluble chitinase with different domains of GRP1.8 were expressed in the vascular tissue of tobacco (Nicotiana tabacum), and the chemical nature of the interaction of these fusion proteins in the cell wall compartment was analyzed. In contrast with chitinase that required only low-salt conditions for complete extraction, the different chitinase/GRP1.8 fusion proteins were completely extracted only by a nonionic or ionic detergent, indicating hydrophobic interactions of GRP1.8. The same interactions were found with the endogenous GRP1.8 in bean hypocotyls. In addition, in vitro experiments indicate that oxidative cross-linking of tyrosines might account for the insolubilization of GRP1.8 observed in later stages of protoxylem development. Our data suggest that GRP1.8 forms a hydrophobic protein-layer in the cell wall of protoxylem vessels.     

Isolation and Characterization of OsGSTL1 Promoter from Rice

OsGSTL1 gene was isolated from the rice genomic library. Semi-quantitative RT-PCR analysis demonstrated that the expression of the OsGSTL1 in rice was not induced by chlorsulfuron, ethylene, abscisic acid, salicylic acid, and methyl jasmonate. In order to investigate the cis-elements of OsGSTL1 promoter, the promoter regions with different lengths were fused to the β-glucuronidase (GUS) reporter gene. All constructs were transformed into onion epidermal cells or A. thaliana plants to detect the expression patterns. In onion epidermal cells, the 160 bp fragment and longer ones were functional for directing GUS expression. In transgenic A. thaliana, the 2?155 bp upstream region of OsGSTL1 gene directed the GUS expression only in cotyledon after germination, but not in the root of young seedlings. In the later seedling, the 2?155 bp upstream region of OsGSTL1 gene directed GUS expression in roots, stems, and leaves. However, the GUS gene directed by a 1?224 bp upstream fragment is expressed in all the checked tissues. These results suggest that the spatiotemporal expression response elements of OsGSTL1 existed in the 5′-upstream region between −2?155 and −1?224 bp.     

The promoter of the geneItr1 from barley confers a different tissue specificity in transgenic tobacco

Tissue-specific expression of the gene coding for trypsin inhibitor BTI-CMe in barley (Itr1) occurs during the first half of endosperm development. In transgenic tobacco, theItr1 promoter drives expression of the β-glucuronidase reporter gene not only in developing endosperm but also in embryo, cotyledons and the meristematic intercotyledonary zone of germinating seedlings. A promoter fragment extending 343 bp upstream of the translation initiation ATG codon was sufficient for full transgene expression, whereas, the proximal 83 bp segment of the promoter was inactive. Possible reasons for the differences in expression patterns are discussed.     

Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

Heat shock responsiveness analysis of <Emphasis Type="Italic">Athsp70b</Emphasis> upstream region

A 1431-bp upstream fragment of Athsp70b was cloned via PCR amplification and expressed in onion epidermis by particle bombardment. Furthermore, the progressive deletions of the Athsp70b upstream fragment linked to the β-glucuronidase (GUS) coding region were performed. Then, a stable GUS expression was analyzed in tobacco BY2 cells and Arabidopsis. Our present results showed that about a 500-bp region upstream ATG of Athsp70b is suitable to confer heat inducibility to the GUS reporter gene in plants and around 116 bp contain nonperfect heat-sensitive element. This promoter responds to heat, salicylic acid, and benzyladenine. GUS staining was mainly observed in the vascular tissues and root tips, implying that Athsp70b is related to water transportation.