Strength, Stability, and cis-Motifs of In silico Identified Phloem-Specific Promoters in Brassica juncea (L.).
ABSTRACT: Aphids, a hemipteran group of insects pose a serious threat to many of the major crop species including Brassica oilseeds. Transgenic strategies for developing aphid-resistant plant types necessitate phloem-bound expression of the insecticidal genes. A few known phloem-specific promoters, in spite of tissue-specific activity fail to confer high level gene-expression. Here, we identified seven orthologues of phloem-specific promoters in B. juncea (Indian mustard), and experimentally validated their strength of expression in phloem exudates. Significant cis-motifs, globally occurring in phloem-specific promoters showed variable distribution frequencies in these putative phloem-specific promoters of B. juncea. In RT-qPCR based gene-expression study promoter of Glutamine synthetase 3A (GS3A) showed multifold higher activity compared to others, across the different growth stages of B. juncea plants. A statistical method employing four softwares was devised for rapidly analysing stability of the promoter-activities across the plant developmental stages. Different statistical softwares ranked these B. juncea promoters differently in terms of their stability in promoter-activity. Nevertheless, the consensus in output empirically suggested consistency in promoter-activity of the six B. juncea phloem- specific promoters including GS3A. The study identified suitable endogenous promoters for high level and consistent gene-expression in B. juncea phloem exudate. The study also demonstrated a rapid method of assessing species-specific strength and stability in expression of the endogenous promoters.
Project description:Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1?, UBC, IF-4?, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.
Project description:We present here a modified, sonication-assisted transient transformation assay for rapid analysis of cis-regulatory elements. We tested promoter elements from MIR159B locus of Brassica juncea by generating stable transgenic lines and compared the transcriptional activity of GUS reporter with that of the transient assay method. To obtain reliable and repeatable results, and to omit false-positive data, we optimized several parameters including sonication duration and cycle and concentration of Agrobacterium tumefaciens measured as optical density (O.D.) at 600 nm. To the best of our knowledge, this is the first report of promoter characterization of MIR159B from Brassica juncea, and comparative analysis of stable and transient lines. Our analysis shows that the protocol described herein allows understanding promoter activity/transcriptional control in tissues other than leaf or protoplast which have remained the mainstay for transient analysis thus far. We tested reporter gene GUS under the control of constitutive promoter, CaMV 35S, and MIR159b from Brassica juncea. We optimized the duration of sonication (5-, 10- and 15-min cycle), bacterial density (measured as O.D at 600 nm = 0.6/0.8/1.0) and Agro-infection time (5, 10, 15 min), and co-cultivation (12-, and 24-h). Sonication cycle of 10-min, followed by Agro-infection and co-cultivation with Agrobacterium tumefaciens with O.D. 600 nm = 0.8 and for 12 h was found to be optimum. We could successfully express reporter genes in deep-seated tissues such as floral organs and pollen grains where it was previously not possible to perform transient assay. Constitutive GUS activity was observed when reporter was placed under control of the constitutive promoter of CaMV 35S. Reporter GUS when placed under transcriptional control of MIR159b promoter from Brassica juncea showed reporter activity in floral tissues, in mature pollen grains. Comparative analysis of reporter activity from stable transgenic lines at T2 generation with that of transient assay system reveals identical to near-identical reporter activity. Transient assay could be successfully performed in tissues collected not only from Arabidopsis thaliana, but also from Brassica juncea and Brassica nigra to demonstrate its wide applicability. Our modified method thus has the potential of quick and rapid analysis of promoter activity and allows us to record the developmental dynamics and spatio-temporal expression pattern driven by specific promoters. Suitable modification and controls should also allow analysis of hormonal regulation and identification of trans-factors via DNA-protein interactions. Furthermore, this method can also be extended to study promoters under various environmental conditions that otherwise do not allow growth and complete life cycle of healthy plants and can be modified to test reporter activity in other non-model plants or plants with long life cycle.
Project description:BACKGROUND: Metal hyperaccumulators, including various Thlaspi species, constitutively express the putative metal transporter MTP1 to high levels in shoots. Here we present data on the transcriptional regulation and localization of expression of the homologous gene BjMTP1 in Brassica juncea. Though B. juncea lacks the ability to hyperaccumulate metals, its relatively high biomass, rapid growth and relatedness to true metal hyperaccumulating plants makes it a promising starting point for the development of plants for phytoremediation. Our goal in this study is to determine the transcriptional regulation of MTP1 in order to start to better understanding the physiological role of MTP1 in B. juncea. RESULTS: Steady-state mRNA levels of BjMTP1 were found to be enhanced 8.8, 5.9, and 1.6-fold in five-day-old B. juncea seedlings after exposure to Ni2+, Cd2+ or Zn2+, respectively. This was also reflected in enhanced GUS activity in B. juncea seedlings transformed with BjMTP1 promoter::GUSPlus after exposure to these metals over a similar range of toxicities from mild to severe. However, no increase in GUS activity was observed after exposure of seedlings to cold or heat stress, NaCl or hydrogen peroxide. GUS expression in Ni2+ treated seedlings was localized in roots, particularly in the root-shoot transition zone. In four- week- old transgenic plants BjMTP1 promoter activity also primarily increased in roots in response to Ni2+ or Cd2+ in plants transformed with either GUS or mRFP1 as reporter genes, and expression was localized to the secondary xylem parenchyma. In leaves, BjMTP1 promoter activity in response to Ni2+ or Cd2+ spiked after 24 h then decreased. In shoots GUS expression was prominently present in the vasculature of leaves, and floral parts. CONCLUSION: Our studies establish that a 983 bp DNA fragment upstream of the BjMTP1 translational start site is sufficient for the specific activation by Ni2+ and Cd2+ of BjMTP1 expression primarily in roots. Activation of expression by both metals in roots is primarily localized to the xylem parenchyma cells. This study is the first to identify specific Ni2+ and Cd2+ transcriptional regulation and tissue localization of BjMTP1.
Project description:Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis.
Project description:BACKGROUND:Saccharum species such as sugarcane and energy cane are key players in the expanding bioeconomy for sugars, bioenergy, and production of high-value proteins. Genomic tools such as culm-regulated promoters would be of great value in terms of improving biomass characteristics through enhanced carbon metabolism for sugar accumulation and/or fiber content for biofuel feedstock. Unlike the situation in dicots, monocot promoters currently used are limited and mostly derived from highly expressed constitutive plant genes and viruses. In this study, a novel promoter region of Sugarcane bacilliform virus (SCBV; genus Badnavirus, family Caulimoviridae), SCBV21 was cloned and mapped by deletion analysis and functionally characterized transiently in monocot and dicot species and stably in sugarcane. RESULTS:In silico analysis of SCBV21 [1816 base pair (bp)] identified two putative promoter regions (PPR1 and PPR2) with transcription start sites (TSS1 and TSS2) and two TATA-boxes (TATAAAT and ATATAA), and several vascular-specific and regulatory elements. Deletion analysis revealed that the 710 bp region spanning PPR2 (with TSS2 and ATATAA) at the 3' end of SCBV21 retained the full promoter activity in both dicots and monocots, as shown by transient expression of the enhanced yellow fluorescent protein (EYFP) gene. In sugarcane young leaf segments, SCBV21 directed a 1.8- and 2.4-fold higher transient EYFP expression than the common maize ubiquitin 1 (Ubi1) and Cauliflower mosaic virus 35S promoters, respectively. In transgenic sugarcane, SCBV21 conferred a preferential expression of the ?-glucuronidase (GUS) gene in leaves and culms and specifically in the culm storage parenchyma surrounding the vascular bundle and in vascular phloem cells. Among the transgenic events and tissues characterized in this study, the SCBV21 promoter frequently produced higher GUS activity than the Ubi1 or 35S promoters in a manner that was not obviously correlated with the transgene copy number. CONCLUSIONS:The newly developed plant viral SCBV21 promoter is distinct from the few existing SCBV promoters in its sequence and expression pattern. The potential of SCBV21 as a tissue-regulated promoter with a strong activity in the culm vascular bundle and its storage parenchyma makes it useful in sugarcane engineering for improved carbon metabolism, increased bioenergy production, and enhanced stress tolerance.
Project description:Isolation of phloem-specific promoters is one of the basic conditions for improving the fiber development and resistance of ramie phloem using genetic engineering. In this study, we isolated a ramie endogenous promoter (named <i>P <sub><i>PSP1</i></sub> -BnPSP-1</i>) and analyzed the function of its truncated fragments in Arabidopsis. The results show that <i>P <sub><i>PSP1</i></sub> -BnPSP-1</i> can drive the GUS reporter gene to be specifically expressed in the veins of Arabidopsis. After hormone and simulated drought treatment of the independent Arabidopsis lines carrying <i>P <sub><i>PSP1</i></sub> -BnPSP-1</i> and its truncated fragments, only <i>P <sub><i>PSP</i>1-5</sub>-BnPSP-1</i> (-600 to -1 bp region of <i>P <sub><i>PSP1</i></sub> -BnPSP-1</i>) is stably expressed and exhibits phloem specificity. Our findings suggest that <i>P <sub><i>PSP</i>1-5</sub>-BnPSP-1</i> can be used as a phloem specific promoter for further research.
Project description:SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) is a master regulator of fibre secondary wall deposition in Arabidopsis thaliana (Arabidopsis), with homologs in other angiosperms and gymnosperms. However, it is poorly understood to what extent the fibre-specific regulation of the SND1 promoter, and that of its orthologs, is conserved between diverged herbaceous and woody lineages. We performed a reciprocal reporter gene analysis of orthologous SND1 promoters from Arabidopsis (AthSND1), Eucalyptus grandis (EgrNAC61) and Populus alba × P. grandidentata (PagWND1A) relative to secondary cell wall-specific Cellulose Synthase4 (CesA4) and CesA7 promoters, in both a non-woody (Arabidopsis) and a woody (poplar) system. ?-glucuronidase (GUS) reporter analysis in Arabidopsis showed that the SND1 promoter was active in vascular tissues as previously reported and showed interfascicular and xylary fibre-specific expression in inflorescence stems, while reporter constructs of the woody plant-derived promoters were partial to the (pro)cambium-phloem and protoxylem. In transgenic P. tremula × P. alba plants, all three orthologous SND1 promoters expressed the GUS reporter similarly and preferentially in developing secondary xylem, ray parenchyma and cork cambium. Ours is the first study to reciprocally test orthologous SND1 promoter specificity in herbaceous and woody species, revealing diverged regulatory functions in the herbaceous system.
Project description:To develop a new strategy that controls vascular pathogen infections in economic crops, we examined a possible enhancer of the vascular activity of XYLOGEN PROTEIN 1 promoter (Px). This protein is specifically expressed in the vascular tissues of Arabidopsis thaliana and plays an important role in xylem development. Although Px is predicted as vascular-specific, its activity is hard to detect and highly susceptible to plant and environmental conditions. The cauliflower mosaic virus 35S promoter (35S) is highly active in directing transgene expression. To test if 35S could enhance Px activity, while vascular specificity of the promoter is retained, we examined the expression of the uidA reporter gene, which encodes β-glucuronidase (GUS), under the control of a chimeric promoter (35S-Px) or Px by generating 35S-Px-GUS and Px-GUS constructs, which were transformed into tobacco seedlings. Both 35S-Px and Px regulated gene expression in vascular tissues. However, GUS expression driven by 35S-Px was not detected in 30- and 60-day-old plants. Quantitative real-time PCR analysis showed that GUS gene expression regulated by 35S-Px was 6.2-14.9-fold higher in vascular tissues than in leaves. Histochemical GUS staining demonstrated that 35S-Px was strongly active in the xylem and phloem. Thus, fusion of 35S and Px might considerably enhance the strength of Px and increase its vascular specificity. In addition to confirming that 35S enhances the activity of a low-level tissue-specific promoter, these findings provide information for further improving the activity of such promoters, which might be useful for engineering new types of resistant genes against vascular infections.
Project description:Establishment and maintenance of mutualistic plant-microbial interactions in the rhizosphere and within plant roots involve several root cell types. The processes of host-microbe recognition and infection require complex signal exchange and activation of downstream responses. These molecular events coordinate host responses across root cell layers during microbe invasion, ultimately triggering changes of root cell fates. The progression of legume root interactions with rhizobial bacteria has been addressed in numerous studies. However, tools to globally resolve the succession of molecular events in the host root at the cell type level have been lacking. To this end, we aimed to identify promoters exhibiting cell type enriched expression in roots of the model legume Lotus japonicus, as no comprehensive set of such promoters usable in legume roots is available to date.Here, we use promoter:GUS fusions to characterize promoters stemming from Arabidopsis, tomato (Lycopersicon esculentum) or L. japonicus with respect to their expression in major cell types of the L. japonicus root differentiation zone, which shows molecular and morphological responses to symbiotic bacteria and fungi. Out of 24 tested promoters, 11 showed cell type enriched activity in L. japonicus roots. Covered cell types or cell type combinations are epidermis (1), epidermis and cortex (2), cortex (1), endodermis and pericycle (2), pericycle and phloem (4), or xylem (1). Activity of these promoters in the respective cell types was stable during early stages of infection of transgenic roots with the rhizobial symbiont of L. japonicus, Mesorhizobium loti. For a subset of five promoters, expression stability was further demonstrated in whole plant transgenics as well as in active nodules.11 promoters from Arabidopsis (10) or tomato (1) with enriched activity in major L. japonicus root and nodule cell types have been identified. Root expression patterns are independent of infection with rhizobial bacteria, providing a stable read-out in the root section responsive to symbiotic bacteria. Promoters are available as cloning vectors. We expect these tools to help provide a new dimension to our understanding of signaling circuits and transcript dynamics in symbiotic interactions of legumes with microbial symbionts.
Project description:Chitinases are the hydrolytic enzymes which belong to the pathogenesis-related (PR) protein family and play an important role not only in plant defense but also in various abiotic stresses. However, only a limited number of chitinase genes have been characterised in B. juncea. In this study, we have characterised B. juncea class IV chitinase gene (accession no EF586206) in response to fungal infection, salicylic acid (SA), jasmonic acid (JA) treatments and wounding. Gene expression studies revealed that the transcript levels of Bjchitinase (BjChp) gene increases significantly both in local and distal tissues after Alternaria infection. Bjchitinase gene was also induced by jasmonic acid and wounding but moderately by salicylic acid. A 2.5 kb class IV chitinase promoter of this gene was isolated from B. juncea by Genome walking (accession no KF055403.1). In-silico analysis of this promoter revealed a number of conserved cis-regulatory elements related to defense, wounding and signalling molecules like SA, and JA. For validation, chitinase promoter was fused to the GUS gene, and the resultant construct was then introduced into Arabidopsis plants. Histochemical analysis of T2 transgenic Arabidopsis plants showed that higher GUS activity in leaves after fungal infection, wounding and JA treatment but weakly by SA. GUS activity was seen in meristematic tissues, young leaves, seeds and siliques. Finally investigation has led to the identification of a pathogen-inducible, developmentally regulated and organ-specific promoter. Present study revealed that Bjchitinase (BjChp) promoter is induced during biotic and environmental stress and it can be used in developing finely tuned transgenics.