GUS Reporter Gene Constructs Research Articles

Constitutive Contribution by the Rice OsHKT1;4 Na+ Transporter to Xylem Sap Desalinization and Low Na+ Accumulation in Young Leaves Under Low as High External Na+ Conditions.

HKT Na+ transporters correspond to major salt tolerance QTLs in different plant species and are targets of great interest for breeders. In rice, the HKT family is composed of seven or eight functional genes depending on cultivars. Three rice HKT genes, OsHKT1;1, OsHKT1;4 and OsHKT1;5, are known to contribute to salt tolerance by reducing Na+ accumulation in shoots upon salt stress. Here, we further investigate the mechanisms by which OsHKT1;4 contributes to this process and extend this analysis to the role of this transporter in plants in presence of low Na+ concentrations. By analyzing transgenic rice plants expressing a GUS reporter gene construct, we observed that OsHKT1;4 is mainly expressed in xylem parenchyma in both roots and leaves. Using mutant lines expressing artificial microRNA that selectively reduced OsHKT1;4 expression, the involvement of OsHKT1;4 in retrieving Na+ from the xylem sap in the roots upon salt stress was evidenced. Since OsHKT1;4 was found to be also well expressed in the roots in absence of salt stress, we extended the analysis of its role when plants were subjected to non-toxic Na+ conditions (0.5 and 5 mM). Our finding that the transporter, expressed in Xenopus oocytes, displayed a relatively high affinity for Na+, just above 1 mM, provided first support to the hypothesis that OsHKT1;4 could have a physiological role at low Na+ concentrations. We observed that progressive desalinization of the xylem sap along its ascent to the leaf blades still occurred in plants grown at submillimolar Na+ concentration, and that OsHKT1;4 was involved in reducing xylem sap Na+ concentration in roots in these conditions too. Its contribution to tissue desalinization from roots to young mature leaf blades appeared to be rather similar in the whole range of explored external Na+ concentrations, from submillimolar to salt stress conditions. Our data therefore indicate that HKT transporters can be involved in controlling Na+ translocation from roots to shoots in a much wider range of Na+ concentrations than previously thought. This asks questions about the roles of such a transporter-mediated maintaining of tissue Na+ content gradients in non-toxic conditions.

Characterization and Functional Analysis of Transcription Factor LoMYB80 Related to Anther Development in Lily (Lilium Oriental Hybrids)

Anther development is very precise and complicated procedure. In this study, the gene LoMYB80 was isolated from lily (Lilium Oriental Hybrids cv. cultivar ‘Siberia’) anther. Multiple alignment and phylogenetic tree analysis showed that LoMYB80 belongs to the R2R3 MYB family. Furthermore, the protein of LoMYB80 shares a 44 amino acid sequence immediately adjacent to the R3 domain with other MYB80s. Subcellular localization showed that LoMYB80 was predominantly localized in the nucleus. The transcriptional activation experiment revealed that LoMYB80 belongs to the transcription activation factors and its transactivation activity depends on the last 70 amino acids located at the end of the C-terminal region. Real-time quantitative PCR analysis showed that LoMYB80 gene expression was significantly higher in anther than in other organs; the highest level was in anthers of 10-cm flower buds and then was in anthers of 4-cm buds. The relative expression in microspores/pollen of 10-cm buds was much higher than the whole anther in the same stage and also much higher than the microspore/pollen of 8- and 9-cm buds. The gene was mainly expressed in young anthers at the development stages from 6 to 10 by analyzing transgenic Arabidopsis lines which were transformed with LoMYB80 promoter::GUS reporter gene constructs. Complementation experiments revealed that transgenic ms188 mutant lines could produce normal pollen grains and display partial fertility. These results indicated that LoMYB80 was a regulator of lily anther development and pollen formation.

IDA-like gene expression in soybean and tomato leaf abscission and requirement for a diffusible stelar abscission signal

The stimulatory and inhibitory role of ethylene and auxin, respectively, in leaf abscission (leaf drop) is well documented. More recently, IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) peptides and their putative interacting receptor-like-kinase partners, HAESA and HAESA-like2, were shown to be essential components in Arabidopsis floral organ abscission. Prior to research on IDA, it was reported that bean (Phaseolus vulgaris) leaf abscission required a diffusible signal that emanated from the vascular tissue. We were interested in determining whether the IDA signalling path might regulate abscission in plants other than Arabidopsis and whether IDA might act as a diffusible signal in abscission. Quantitative polymerase chain reaction was used to monitor gene expression and a GUS reporter gene construct used to determine the need for a diffusible signal in tomato. We identified 12 IDA-like and 11 HAESA-like genes in soybean (Glycine max) and monitored their gene expression in abscission in relation to the expression of several cell-wall-modifying proteins and aminocyclopropane-1-carboxylic acid synthases. Ethylene evoked the expression of several IDA-like genes in abscission zones (AZ), but also to a lesser degree in the adjacent petiole tissue. Surprisingly, IDA-like gene expression was very high in senescent soybean leaves. We identified five IDA-like genes in tomato (Solanum lycopersicum). Only one IDA-like gene was expressed in the tomato AZ and its expression was approximately equal in the AZ and petioles, but no IDA-like gene showed significant expression in leaves at up to 96 h of exposure to ethylene. IDA-like gene expression is up-regulated during soybean and tomato abscission but up-regulation was not limited to the AZ. Cell separation in the AZ cortex of tomato does not require a diffusible signal emanating from the stele. A role for IDA in soybean and tomato leaf abscission is discussed.

The E2F transcription factor family regulates CENH3 expression in Arabidopsis thaliana

To elucidate the epigenetic maintenance mechanism for functional plant centromeres, we studied transcriptional regulation of the centromere-specific histone H3 variant CENH3 in Arabidopsis thaliana. We focused on the structure and activity of the CENH3 promoter (CENH3pro) and its regulation by E2F transcription factors. Use of CENH3pro::GUS reporter gene constructs showed that CENH3pro is active in dividing tissues, and that full expression in root meristems depends on intragenic regulatory elements within the second intron. Chromatin immunoprecipitation identified CENH3 as an E2F target gene. Transient co-expression of a CENH3pro::GUS reporter gene construct with various E2F transcription factors in A.thaliana protoplasts showed that E2Fa and E2Fb (preferentially with dimerization protein DPb) activate CENH3pro. Stable over-expression of E2Fa and E2Fb increased the CENH3 transcript level in planta, whereas over-expression of E2Fc decreased the CENH3 transcript level. Surprisingly, mutation of the two E2F binding sites of CENH3pro, in particular the more upstream one (E2F2), caused an increase in CENH3pro activity, indicating E2F-dependent transcriptional repression. CENH3pro repression may be triggered by the interplay of typical and atypical E2Fs in a cell cycle-dependent manner, and/or by interaction of typical E2Fs with retinoblastoma-related (RBR) protein. We speculate that E2Fs are involved in differential transcriptional regulation of CENH3 versus H3, as H3 promoters lack E2F binding motifs. E2F binding motifs are also present in human and Drosophila CENH3pro regions, thus cell cycle-dependent transcriptional regulation of CENH3 may be highly conserved.

Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration

The present study describes the successful development of vacuum infiltration method in the oomycete-resistant wild Piper sp., Piper colubrinum, as a rapid transient method for expression of GUS (β-Glucuronidase) reporter gene and introduction of hairpin vector for endogenous gene silencing. The GUS reporter gene construct pCAMBIA 1305.2 was used as a positive control to test the efficiency of vacuum infiltration strategy. Agrobacteria (EHA 105) harbouring GUS binary vector were vacuum-infiltrated into young detached in vitro leaf explants, which showed detectable GUS gene activity within 4 days of infiltration. This paper also reports for the first time the application of transient gene silencing in P. colubrinum by the delivery of in vitro synthesized hairpin vector construct (pHELLSGATE) containing endogenous serine threonine protein kinase (STPK) gene homologue into in vitro shoots. Introduction of hairpin vectors for the STPK gene into in vitro plantlets by vacuum infiltration resulted in significant reduction in transcript accumulation of the endogenous gene. The results indicate that transient gene silencing could be used as a rapid, preliminary high-throughput tool for P. colubrinum functional genomics.

Acquiring competence for shoot development in Arabidopsis

Shoots can be regenerated from roots in Arabidopsis by treating root explants with cytokinin, however, shoot regeneration requires preincubation on callus induction medium (CIM) prior to induction on cytokinin-rich shoot induction medium (SIM). A cytokinin-inducible marker gene, RESPONSE REGULATOR 15 (ARR15), was identified through a "CIM dropout experiment" with similar requirements for CIM preincubation. The requirements for ARR15 contrasted to ARR5, another cytokinin-inducible ARR gene that does not require CIM preincubation. We show here that despite their differences, both ARR5 and ARR15 are direct targets of the transcriptional B-type response regulator, ARR2. This was demonstrated by identifying genes upregulated following β estradiol induced nuclear relocation of an ARR2-estradiol receptor fusion protein. The differences in CIM preincubation requirements for ARR5 and ARR15 expression indicate an additional layer of control for these A-type ARR genes during SIM incubation. For ARR15, the CIM requirement is a transcriptional effect, because the expression of ARR15 promoter:GUS reporter gene constructs is also affected by CIM preincubation. A testable model is that transcription of ARR15, but not ARR5, is blocked by a repressor and that the effects of the repressor are relieved by CIM preincubation.

Rapid bioassays to evaluate the plant growth promoting activity of Ascophyllum nodosum (L.) Le Jol. using a model plant, Arabidopsis thaliana (L.) Heynh

Ascophyllum nodosum extract products are used commercially in the form of liquid concentrate and soluble powder. These formulations are manufactured from seaweeds that are harvested from natural habitats with inherent environmental variability. The seaweeds by themselves are at different stages of their development life-cycle. Owing to these differences, there could be variability in chemical composition that could in turn affect product consistency and performance. Here, we have tested the applicability of using Arabidopsis thaliana as a model to study the activity of two different extracts from A. nodosum. Three different bioassays: Arabidopsis root-tip elongation bioassay, Arabidopsis liquid growth bioassay and greenhouse growth bioassay were evaluated as growth assays. Our results indicate that both extracts promoted root and shoot growth in comparison to controls. Further, using Arabidopsis plants with a DR5:GUS reporter gene construct, we provide evidence that components of the commercial A. nodosum extracts modulates the concentration and localisation of auxins which could account, at least in part, for the enhanced plant growth. The results suggest that A. thaliana could be used effectively as a rapid means to test the bioactivity of seaweed extracts and fractions.

The Douglas-fir BiP promoter is functional in Arabidopsis and responds to wounding.

A DNA sequence representing the promoter region of the Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) luminal binding protein PmBiP (PmBiPPro1) was isolated using inverse polymerase chain reaction (iPCR). Transient expression analysis of PmBiPPro1 fused to the beta-glucuronidase (GUS) reporter gene demonstrated that this promoter is functional in germinating Douglas-fir embryos. Transgenic Arabidopsis plants containing PmBiPPro1:GUS reporter gene constructs revealed strong staining associated with actively dividing/expanding cells and secretory tissues in developing seedlings. Wounding of cotyledons resulted in an increase in local staining associated with cells surrounding the wound site. Deletion analysis showed that elements necessary for basal-level expression reside within a -261 to +16 bp region, although upstream elements are necessary for higher-level expression in germinating Douglas-fir embryos, developing Arabidopsis seedlings and wounded cotyledons. Correlation of the observed expression pattern with the known function of BiP suggests that pathways controlling expression are highly conserved between angiosperms and gymnosperms.

The ROOT HAIRLESS 1 gene encodes a nuclear protein required for root hair initiation in Arabidopsis.

The epidermis of Arabidopsis wild-type primary roots, in which some cells grow hairs and others remain hairless in a position-dependent manner, has become an established model system to study cell differentiation. Here we present a molecular analysis of the RHL1 (ROOT HAIRLESS 1) gene that, if mutated, prevents the formation of hairs on primary roots and causes a seedling lethal phenotype. We have cloned the RHL1 gene by use of a T-DNA-tagged mutant and found that it encodes a protein that appears to be plant specific. The predicted RHL1 gene product is a small hydrophilic protein (38.9 kD) containing putative nuclear localization signals and shows no significant homology to any known amino acid sequence. We demonstrate that a 78-amino-acid sequence at its amino terminus is capable of directing an RHL1-GFP fusion protein to the nucleus. The RHL1 transcript is present throughout the wild-type plant and in suspension culture cells, but in very low amounts, suggesting a regulatory function for the RHL1 protein. Structural evidence suggests a role for the RHL1 gene product in the nucleolus. We have examined the genetic relationship between RHL1 and GL2, an inhibitor of root hair initiation in non-hair cells. Our molecular and genetic data with double mutants, together with the expression analysis of a GL2 promoter-GUS reporter gene construct, indicate that the RHL1 gene acts independently of GL2.

Hordein promoter methylation and transcriptional activity in wild-type and mutant barley endosperm.

B- and C-hordein gene transcription is severely reduced in the endosperm of the regulatory barley mutant lys3a, and this is correlated with persistent hypermethylation of the promoters. In contrast, D-hordein is expressed at normal levels in the mutant. To confirm the connection between methylation and transcriptional activity, a genomic D-hordein clone was isolated and sequenced. The nucleotide composition of the promoter region revealed a CpG island and methylation analysis, using bisulphite treatment of genomic DNA, confirmed that the D-hordein promoter is unmethylated in endosperm and leaf tissue. Immunocytochemical studies localized D-hordein to the reticular component of protein bodies in both the wild-type Bomi and lys3a. Transient expression of GUS reporter gene constructs in barley endosperm, following transfection by particle bombardment revealed the D-hordein promotors. Comparison of transient expression in Bomi and lys3a endosperm demonstrated that the activities of the unmethylated D-hordein and the Hor1-14 C-hordein promoters were equivalent, while the activities in the mutant of the Horl-17 C-hordein and the Hor2-4 B-hordein promoters were reduced two- and tenfold, respectively. Methylation of plasmids in vitro prior to expression severely inhibited B- and D-hordein promoter activities. Based on these observations two categories of promoters for endosperm-specific expression of storage proteins are recognized and a model involving methylation and modulation of chromatin structure in the regulation by the Lys3 gene is presented.

Tissue-specific expression of an oat 12S seed globulin gene in developing tobacco seeds: differential mRNA and protein accumulation.

We studied the expression of the oat globulin gene asglo5 in developing transgenic tobacco seeds. The asglo5 gene promoter directed transcription in the endosperm as well as in the provascular tissue, the presumptive root tip and the shoot apical meristem of the embryo as revealed by GUS reporter gene constructs and in situ hybridization. However, immunological tissue printing detected the oat protein exclusively in the tobacco endosperm, suggesting that extensive post-transcriptional regulatory processes influence the expression of the monocot transgene in the dicot host.

Gene Transfer Into Intact Sugarcane Cells Using Microprojectile Bombardment

A microprojectile accelerator has been constructed and used to bombard cultured sugarcane tissues with GUS reporter gene constructs. Design features useful to minimise target tissue damage and variation between shots are described. Transient expression of GUS occurred in pEmuGN-bombarded cells of nonregenerable suspension culture as well as in regenerable embryogenic callus of commercial sugarcane cultivar 463, and in suspension cultures capable of regeneration to plants. Parameters yielding transient GUS expression in up to 1055 cells per bombardment in homogeneous suspension cultures of sugarcane have been established with a mean of 206 expressing cells per bombardment over a series of 8 independent experiments. Approximately 4% of these transiently expressing cells continued to express GUS for extended periods, indicating probable stable transformation of intact cells of the commercial sugarcane cultivar. Microprojectile bombardment appears the most promising of the available gene transfer techniques for practical genetic transformation of sugarcane because most commercial cultivars readily form regenerable callus suitable for bombardment.