Development and Functional Analysis of Novel Genetic Promoters Using DNA Shuffling, Hybridization and a Combination Thereof

BACKGROUND: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications. METHODOLOGY: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter. SIGNIFICANCE/CONCLUSION: Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants

Nutritional diversity and food potential of indigenous pigmented rice landraces from Koraput regions of Eastern Ghats

Abstract Compressive nutritional, nutraceuticals and mineral profiling was carried out in eight diverse pigmented rice landraces originated from Koraput and compared them with improved variety (IR 64). The proximate compositions such as moisture content varied from 8.23 to 11.65 g 100 g−1, ash 0.68–1.46 g 100 g−1, fat 1.07–2.23 g 100 g−1, protein 7.00–9.63 g 100 g−1, carbohydrate 76.37–80.66 g 100 g−1, fiber 0.11–1.69 g 100 g−1 and energy 346.3–362.11 kcal 100 g−1 in the studied rice lines. These landraces are rich in phenol, flavonoid, and antioxidant concentrations and varied from 3.0 to 9.0 mg g−1, 0.150 to 0.950 mg 100 g−1, and 10.8 to 40.20%, respectively. Principal component analysis explained 47.2% of the overall variation and reflected huge difference between explored genotypes. The heritability and genetic advance varied from 30.22–99.90% and 2.5–111.5%, respectively. In compared to improved IR 64 variety, rich in energy content was recorded in Paradhan, Bhatamali and Haladiganthi indicated its nutritional superiority. Further, exceptional rich in phenol, flavonoid, vitamin C, vitamin E and antioxidant capacity was recorded in Kalachudi, Bedagurumukhi and Kandulakanthi, which may create opportunities for its large-scale commercialization and cultivation. These nutrition rich landraces also hold great potential for future crop improvement programs aimed at enhancing quality

Rice under combined salt and flooding stress

Not AvailableChlorophyll a fluorescence (ChlF) parameters measured with fluorescence imaging techniques were used to investigate the combined effect of salt and partial submergence stress to understand photosynthetic performance in rice (Oryza sativa L.). ChlF parameters such as maximal fluorescence (Fm), variable fluorescence (Fv = Fm –F0), the maximal photochemical efficiency of PSII (Fv/Fm) and the quantum yield of nonregulated energy dissipation of PSII (Y(NO)) were able to distinguish genotypes precisely based on their sensitivity to stress. Upon analysis, we found the images of F0 were indistinguishable among the genotypes, irrespective of their tolerance to salt and partial submergence stress. On the contrary, the images of Fm and Fv/Fm showed marked differences between the tolerant and susceptible genotypes in terms of tissue greenness and the appearance of dark spots as stress symptoms. The images of effective PSII quantum yield, the coefficient of nonphotochemical quenching (qN) and the coefficient of photochemical quenching (qP) captured under different PAR were able to distinguish the tolerant and susceptible genotypes, and were also quite effective for differentiating the tolerant and moderately tolerant ones. Similarly, the values of electron transport rate, qN, qP and Y(NO) were also able to distinguish the genotypes based on their sensitivity to stress. Overall, this investigation indicates the suitability of chlorophyll fluorescence imaging technique for precise phenotyping of rice based on their sensitivity to the combined effect of salt and partial submergence

Differential expression analyses of host genes involved in systemic infection of Tomato leaf curl New Delhi virus (ToLCNDV)

Tomato leaf curl viruses (ToLCV) infect tomato plants and eventually cause several phenotypic defects, notably in the leaves in the form of upward curling. The entry of virus triggers plants basal defense responses which eventually introduce temporal changes in the transcriptome to evade the pathogen attack. In this study, we have identified about 20 tomato ESTs using subtractive hybridization that were induced in tomato leaves upon agro-infection with the constructs bearing the dimers of Tomato leaf curl New Delhi virus (ToLCNDV) DNA-A and DNA-B components. The induced ESTs belonged to the class of genes that play crucial roles in innate immunity, plants metabolism and ethylene signaling. The expression of few of these ESTs was validated by northern blot analysis and two out of six selected genes expressed exclusively in the infected leaf tissues. Besides leaves, the expression status of selected genes was checked in a wide variety of tissues (flower, fruit, stem and root) of both healthy and infected plants by RT-PCR. These results suggest that the flower and fruit tissues, similar to leaves, exhibited induction of most of the genes while the stem and root tissues suffered from down-regulation. Overall, these results indicate that the hosts transcriptome undergoes considerable changes in response to viral infection

Map of the mutation observed in two shuffled promoters: LhsFuasFScp-1 and LhsFuasFScp-18.

<p>(a) Two shuffled promoters LhsFuasFScp-1 and LhsFuasFScp-18 were aligned with the hybrid FuasFScp using ClustalW2 tool (<a href="http://www.ebi.ac.uk/Tools/nsa/clustalw" target="_blank">www.ebi.ac.uk/Tools/nsa/clustalw</a>). A square box marks mutation or deletion of important <i>cis</i>-elements. The oval shaped box marks TATA elements in these promoter sequences. (b) Free energy profile (helical stability) of each nucleotide present in FuasFScp, LhsFuasFScp-1 and LhsFuasFScp-18 promoter sequences was shown. (c) Diagrammatic representation of location of different <i>cis</i>-elements in hybrid promoter FuasFScp. Also point mutations, insertions, deletions in case of two shuffled promoters LhsFuasFScp-1 and LhsFuasFScp-18 were shown.</p

Transgenic analysis of promoter constructs.

<p>(<b>a</b>) <b>Comparative stable expression analysis of parent and hybrid promoter-GUS constructs in transgenic tobacco plants.</b> Promoter activities of parent and hybrid promoters were monitored in 21-days-old tobacco (<i>Nicotiana tabacum</i> cv. Samsun NN) seedlings (R1 progeny, 2nd generation, Kan^R) grown aseptically on an MS-agar medium in presence of kanamycin (300 µg/ml) and 3% sucrose. Soluble protein extracts (5 µg) from whole seedlings were used for the GUS assay. The data presented in the histogram as an average of three independent experiments for each construct with respective standard deviation (SD). The statistical analysis revealed a <i>P</i> value of 0.001 implying highly significant. In the histogram, GUS constructs: (1) Untransformed control tissue extract from the wild type <i>Nicotiana tabacum</i> cv Samsun NN (2) pKYLXGUS, with CaMV35S promoter, (3) pKFScpGUS, with FScp promoter; (4) pKFSGUS, with FS promoter; (5) pKFGUS, with F promoter; (6) pKFcpGUS, with Fcp promoter; (7) pKFSuasGUS, with FSuas promoter; (8) pKFuasGUS, with Fuas promoter; (9) pKFuasFScpGUS, with FuasFScp promoter; (10) pKFSuasFcpGUS, with FSuasFcp promoter; were shown. (<b>b</b>) <b>Comparative expression analysis of transgenic plants expressing GUS constructs of parent and hybrid promoters by qRT-PCR assay.</b> For each construct, 21-days-old seedlings (R1 progeny, 2nd generation, Kan^R) from independent transgenic lines were selected. Estimation of relative <i>GUS</i> transcript accumulation in transgenic plants developed using GUS constructs driven by CaMV35S, FScp, FS, F, Fcp, FSuas, Fuas, FuasFScp and FSuasFcp promoters was performed by qRT-RCR as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The data presented in the histogram were average fold difference of <i>GUS</i> transcript ± SD of two independent experiments carried out using cDNA derived from two RNA samples extracted from two different plants expressing individual promoter constructs. In the histogram, each bar represents number of fold increase in transcript level of <i>GUS</i> gene in plants compared to CaMV35S (taken as 1.0). Histograms (2) pKYLXGUS; (3) pKFScpGUS; (4) pKFSGUS; (5) pKFGUS; (6) pKFcpGUS; (7) pKFSuasGUS; 8: pKFuasGUS; (9) pKFuasFScpGUS; (10) pKFSuasFcpGUS were shown.</p

A schematic map of the parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp) and DNA shuffling strategy.

<p>(a) At the top, the coordinates of the respective promoters <i>Figwort mosaic virus</i> (FMV) full-length transcript promoter (F, −249 to +64), FMV sub-genomic transcript promoter (FS, −270 to +31), and two hybrid promoters (FuasFScp, −343 to +31; and FSuasFcp, −449 to +64), the relative position of the TATA box, transcription start site (TSS, +1), upsteam activation sequence (uas) and core-promoter (cp) regions marked with arrow were shown. (b) A schematic presentation of creating promoter libraries by DNA shuffling of single (F or FS), multiple (F and FS) and hybrid promoters (FuasFScp, FSuasFcp) was presented. The construction strategies of generating hybrid promoters (FuasFScp and FSuasFcp), the single shuffled libraries (LssF and LssFS), multiple shuffled libraries (LmsFFS and LmsFSF), and hybrid promoter shuffled libraries (LhsFuasFScp and LhsFSuasFcp) were described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>” section.</p

Comparative expression analysis of promoters and promoter fragments fused with reporter genes (GFP and GUS) using CLSM in tobacco transient protoplast assay.

<p>(a) GFP constructs of CaMV35S promoter, parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp), promoter fragments (FScp, Fcp, FSuas and Fuas) were created as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The GFP fluorescence intensity (in gray scale unit) was measured in protoplast transient assay using CLSM as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The average GFP intensity ± SD of two replicates of each construct was presented in the histogram. Error bar shows the 95% confidence intervals of the mean. Statistical (one-way analysis of variance, ANOVA) analysis showed an extremely significant <i>P</i> value of <0.01. Empty vector ‘Control’ with no GFP gene was shown. (b) GUS constructs of CaMV35S promoter, parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp), and promoter fragments (FScp, Fcp, FSuas and Fuas) were generated as described in“<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The GUS activity (n mole MU/min/mg protein) was measured in protoplast transient assay using CLSM as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The average GUS activity ± SD of two replicates of each construct was presented in the histogram. Error bar shows the 95% confidence intervals of the means. Statistical (one-way analysis of variance, ANOVA) analysis showed an extremely significant <i>P</i> value of <0.02. Empty vector ‘Control’ with no GUS gene was shown.</p

Comparative expression analysis (in Agro-infiltration assay) of selected shuffled promoters screened in protoplast transient assay.

<p>In histogram shown, twenty six shuffled promoters giving good activity in transient tobacco protoplast assay selected from six libraries: 4 from LssF, 3 from LssFS, 2 from LmsFFS, 6 from LmsFSF, 4 from LhsFSuasFScp and 7 from LhsFuasFScp; were taken for further comparative expression analysis along with the CaMV35S promoter (35S), parent promoters (F and FS) and hybrid promoters (FuasFScp and FSuasFcp) in Agro-infiltration experiment using whole tobacco plants (<i>Nicotiana tabacum</i> samsun NN) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031931#s2" target="_blank">Materials and Methods</a>”. The average GUS activity (n mole MU/min/mg protein ± SD) of three replicates of each construct was presented in the histogram. Error bar shows the 95% confidence intervals of the mean. Statistical (one-way analysis of variance, ANOVA) analysis showed an extremely significant <i>P</i> value of <0.001. Empty vector with no GUS gene was treated as ‘Control’.</p

Free energy Profile and GC content of shuffled and hybrid promoters.

<p>Free energy Profile and GC content of shuffled and hybrid promoters.</p