DGGE-RAPD analysis as a useful tool for cultivar identification

Denaturing gradient gel electrophoresis-random amplified polymorphic DNA (DGGE-RAPD) was used to overcome the main drawbacks of RAPD (i.e., the low levels of reproducibility and polymorphism). As a model, six barley cultivars of known origin were tested for RAPD markers using DGGE methodology with 29 arbitrary primers. Among a total of 418 bands observed, as high as 99 were polymorphic. Comparison between agarose-RAPD and DGGE-RAPD revealed that the latter was highly reproducible and gave higher level of polymorphism and consequently more markers. The relationships among barley cultivars derived from this study based on DGGE-RAPD are consistent with the known lineage of these cultivars. In conclusion, we recommend the use of DGGE-RAPD as an alternative tool to the more costly DNA-based analysis in cultivar identification in laboratories with limited funds.Key words: Denaturing gradient gel electrophoresis-random amplified polymorphic DNA, artificial heteroduplex, dendogram, lineage

Ethylene responsive transcription factor ERF109 retards PCD and improves salt tolerance in plant

Semi-quantitative RT-PCR for tobacco VIGS lines of 13 knocked down TFs induced 2 h post oxalic acid treatment (20 mM) as compared to their WT and VIGS line with empty pTRV2 (V2) plants. Amplicon sizes of different genes and primers used are shown in Additional file 5: Table S3. The Nbactin gene was used as the house-keeping control. Gene codes refer to those indicated in Additional file 3: Table S2. (DOCX 684 kb

The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes

The influence of two binary vector systems, pGreen and pCAMBIA, on the Agrobacterium-mediated transformation ability of wheat and triticale was studied. Both vectors carried selection cassettes with bar or nptII driven by different promoters. Two cultivars of wheat, Kontesa and Torka, and one cultivar of triticale, Wanad, were tested. The transformation rates for the wheat cultivars ranged from 0.00 to 3.58% and from 0.00 to 6.79% for triticale. The best values for wheat were 3.58% for Kontesa and 3.14% for Torka, and these were obtained after transformation with the pGreen vector carrying the nptII selection gene under the control of 35S promoter. In the case of the bar selection system, the best transformation rates were, respectively, 1.46 and 1.79%. Such rates were obtained when the 35S::bar cassette was carried by the pCAMBIA vector; they were significantly lower with the pGreen vector. The triticale cultivar Wanad had its highest transformation rate after transformation with nptII driven by 35S in pCAMBIA. The bar selection system for the same triticale cultivar was better when the gene was driven by nos and the selection cassette was carried by pGreen. The integration of the transgenes was confirmed with at least three pairs of specific starters amplifying the fragments of nptII, bar, or gus. The expression of selection genes, measured by reverse transcriptase polymerase chain reaction (RT-PCR) in relation to the actin gene, was low, ranging from 0.00 to 0.63 for nptII and from 0.00 to 0.33 for bar. The highest relative transcript accumulation was observed for nptII driven by 35S and expressed in Kontesa that had been transformed with pGreen

Metabolomic Response of Calotropis procera Growing in the Desert to Changes in Water Availability

Water availability is a major limitation for agricultural productivity. Plants growing in severe arid climates such as deserts provide tools for studying plant growth and performance under extreme drought conditions. The perennial species Calotropis procera used in this study is a shrub growing in many arid areas which has an exceptional ability to adapt and be productive in severe arid conditions. We describe the results of studying the metabolomic response of wild C procera plants growing in the desert to a one time water supply. Leaves of C. procera plants were taken at three time points before and 1 hour, 6 hours and 12 hours after watering and subjected to a metabolomics and lipidomics analysis. Analysis of the data reveals that within one hour after watering C. procera has already responded on the metabolic level to the sudden water availability as evidenced by major changes such as increased levels of most amino acids, a decrease in sucrose, raffinose and maltitol, a decrease in storage lipids (triacylglycerols) and an increase in membrane lipids including photosynthetic membranes. These changes still prevail at the 6 hour time point after watering however 12 hours after watering the metabolomics data are essentially indistinguishable from the prewatering state thus demonstrating not only a rapid response to water availability but also a rapid response to loss of water. Taken together these data suggest that the ability of C. procera to survive under the very harsh drought conditions prevailing in the desert might be associated with its rapid adjustments to water availability and losses

Analysis of transcriptional response to heat stress in Rhazya stricta

Clusters of assembled transcripts of R. stricta SRA in mature leaves (A5-L8) at different time points of the day (A, morning; F-H, midday & L, dusk). Grey lines indicate expression patterns of individual transcripts in a given cluster. Blue lines indicate overall expression pattern across different transcripts of a given cluster. (PDF 397 kb

Rewiring carotenoid biosynthesis in plants using a viral vector

[EN] Plants can be engineered to sustainably produce compounds of nutritional, industrial or pharmaceutical relevance. This is, however, a challenging task as extensive regulation of biosynthetic pathways often hampers major metabolic changes. Here we describe the use of a viral vector derived from Tobacco etch virus to express a whole heterologous metabolic pathway that produces the health-promoting carotenoid lycopene in tobacco tissues. The pathway consisted in three enzymes from the soil bacteria Pantoea ananatis. Lycopene is present at undetectable levels in chloroplasts of non-infected leaves. In tissues infected with the viral vector, however, lycopene comprised approximately 10% of the total carotenoid content. Our research further showed that plant viruses that express P. ananatis phytoene synthase (crtB), one of the three enzymes of the heterologous pathway, trigger an accumulation of endogenous carotenoids, which together with a reduction in chlorophylls eventually result in a bright yellow pigmentation of infected tissues in various host-virus combinations. So, besides illustrating the potential of viral vectors for engineering complex metabolic pathways, we also show a yellow carotenoid-based reporter that can be used to visually track infection dynamics of plant viruses either alone or in combination with other visual markers.We thank Veronica Aragones and M. Rosa Rodriguez-Goberna for excellent technical assistance. This research was supported by Spanish Ministerio de Economia y Competitividad (MINECO) grants BIO2014-54269-R to J.-A.D., and BIO2014-59092-P and BIO2015-71703-REDT to M. R.-C. Financial support from the Generalitat Valenciana (PROMETEOII/2014/021), the Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (Ibercarot 112RT0445), and the Generalitat de Catalunya (2014SGR-1434) is also acknowledged. E.M. is the recipient of a pre-doctoral fellowship (AP2012-3751) from the Spanish Ministerio de Educacion, Cultura y Deporte. B.L. is supported by a postdoctoral fellowship (FPDI-2013-018882) from MINECO.Majer, E.; Llorente, B.; Rodríguez-Concepción, M.; Daros Arnau, JA. (2017). Rewiring carotenoid biosynthesis in plants using a viral vector. Scientific Reports. 7. https://doi.org/10.1038/srep41645S7O’Connor, S. E. Engineering of secondary metabolism. Annu. Rev. Genet. 49, 71–94 (2015).Sainsbury, F. & Lomonossoff, G. P. Transient expressions of synthetic biology in plants. Curr. Opin. Plant Biol. 19, 1–7 (2014).Gleba, Y. Y., Tusé, D. & Giritch, A. Plant viral vectors for delivery by Agrobacterium. Curr. Top. Microbiol. Immunol. 375, 155–192 (2014).Chen, Q., He, J., Phoolcharoen, W. & Mason, H. S. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum. Vaccin. 7, 331–338 (2011).Pogue, G. P., Lindbo, J. A., Garger, S. J. & Fitzmaurice, W. P. Making an ally from an enemy: plant virology and the new agriculture. Annu. Rev. Phytopathol. 40, 45–74 (2002).Peyret, H. & Lomonossoff, G. P. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol. J. 13, 1121–1135 (2015).Bedoya, L. C., Martínez, F., Orzáez, D. & Daròs, J. A. Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis. Plant Physiol. 158, 1130–1138 (2012).Majer, E., Daròs, J. A. & Zwart, M. P. Stability and fitness impact of the visually discernible Rosea1 marker in the Tobacco etch virus genome. Viruses 5, 2153–2168 (2013).Bedoya, L., Martínez, F., Rubio, L. & Daròs, J. A. Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector. J. Biotechnol. 150, 268–275 (2010).Kelloniemi, J., Mäkinen, K. & Valkonen, J. P. Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths. Virus Res. 135, 282–291 (2008).Carrington, J. C., Haldeman, R., Dolja, V. V. & Restrepo-Hartwig, M. A. Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo . J. Virol. 67, 6995–7000 (1993).Li, X. H. & Carrington, J. C. Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells. Proc. Natl. Acad. Sci. USA 92, 457–461 (1995).Fraser, P. D. & Bramley, P. M. The biosynthesis and nutritional uses of carotenoids. Prog. Lipid Res. 43, 228–265 (2004).Meléndez-Martínez, A. J., Mapelli-Brahm, P., Benítez-González, A. & Stinco, C. M. A comprehensive review on the colorless carotenoids phytoene and phytofluene. Arch. Biochem. Biophys. 572, 188–200 (2015).Rodríguez-Concepción, M. & Boronat, A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol. 130, 1079–1089 (2002).Giuliano, G. Plant carotenoids: genomics meets multi-gene engineering. Curr. Opin. Plant Biol. 19, 111–117 (2014).Cazzonelli, C. I. & Pogson, B. J. Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci. 15, 266–274 (2010).Ruiz-Sola, M. A. & Rodríguez-Concepción, M. Carotenoid biosynthesis in Arabidopsis: a colorful pathway. Arabidopsis Book 10, e0158 (2012).Nisar, N., Li, L., Lu, S., Khin, N. C. & Pogson, B. J. Carotenoid metabolism in plants. Mol. Plant 8, 68–82 (2015).Misawa, N. et al. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli . J. Bacteriol. 172, 6704–6712 (1990).Hasunuma, T. et al. Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. Plant J. 55, 857–868 (2008).Lu, Y., Rijzaani, H., Karcher, D., Ruf, S. & Bock, R. Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons. Proc. Natl. Acad. Sci. USA 110, E623–632 (2013).Mann, V., Harker, M., Pecker, I. & Hirschberg, J. Metabolic engineering of astaxanthin production in tobacco flowers. Nat. Biotechnol. 18, 888–892 (2000).Wurbs, D., Ruf, S. & Bock, R. Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J. 49, 276–288 (2007).Cordero, M. T. et al. Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana . Mol. Plant-Microbe Interact. doi: 10.1094/MPMI-11-16-0239-R (2016).Ye, X. et al. Engineering the provitamin A (b-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303–305 (2000).Ravanello, M. P., Ke, D., Alvarez, J., Huang, B. & Shewmaker, C. K. Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production. Metab. Eng. 5, 255–263 (2003).Fujisawa, M. et al. Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation. J. Exp. Bot. 60, 1319–1332 (2009).Ohara, K., Ujihara, T., Endo, T., Sato, F. & Yazaki, K. Limonene production in tobacco with Perilla limonene synthase cDNA. J. Exp. Bot. 54, 2635–2642 (2003).Gutensohn, M. et al. Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits. Plant J. 75, 351–363 (2013).Yamano, S., Ishii, T., Nakagawa, M., Ikenaga, H. & Misawa, N. Metabolic engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 58, 1112–1114 (1994).Bahieldin, A. et al. Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter. Plasmid 72, 18–28 (2014).Xie, W., Lv, X., Ye, L., Zhou, P. & Yu, H. Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metab. Eng. 30, 69–78 (2015).Li, Y., Cui, H., Cui, X. & Wang, A. The altered photosynthetic machinery during compatible virus infection. Curr. Opin. Virol. 17, 19–24 (2016).Tilsner, J. & Oparka, K. J. Tracking the green invaders: advances in imaging virus infection in plants. Biochem. J. 430, 21–37 (2010).Kumagai, M. H. et al. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc. Natl. Acad. Sci. USA 92, 1679–1683 (1995).Kumagai, M. H., Keller, Y., Bouvier, F., Clary, D. & Camara, B. Functional integration of non-native carotenoids into chloroplasts by viral-derived expression of capsanthin-capsorubin synthase in Nicotiana benthamiana . Plant J. 14, 305–315 (1998).Zhai, S., Xia, X. & He, Z. Carotenoids in staple cereals: metabolism, regulation, and genetic manipulation. Front. Plant Sci. 7, 1197 (2016).Zhang, H. et al. A Narcissus mosaic viral vector system for protein expression and flavonoid production. Plant Methods 9, 28 (2013).Nielsen, A. Z. et al. Redirecting photosynthetic reducing power toward bioactive natural product synthesis. ACS Synth. Biol. 2, 308–315 (2013).Sainsbury, F., Saxena, P., Geisler, K., Osbourn, A. & Lomonossoff, G. P. Using a virus-derived system to manipulate plant natural product biosynthetic pathways. Methods Enzymol. 517, 185–202 (2012).Geisler, K. et al. Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants. Proc. Natl. Acad. Sci. USA 110, E3360–3367 (2013).Kanagarajan, S., Muthusamy, S., Gliszczynska, A., Lundgren, A. & Brodelius, P. E. Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana . Plant Cell Rep. 31, 1309–1319 (2012).Mozes-Koch, R. et al. Expression of an entire bacterial operon in plants. Plant Physiol. 158, 1883–1892 (2012).Thole, V., Worland, B., Snape, J. W. & Vain, P. The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation. Plant Physiol. 145, 1211–1219 (2007).Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553 (2009).Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).Cunningham, F. X. Jr., Chamovitz, D., Misawa, N., Gantt, E. & Hirschberg, J. Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of b-carotene. FEBS Lett. 328, 130–138 (1993).Shivprasad, S. et al. Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255, 312–323 (1999).Schürer, H., Lang, K., Schuster, J. & Mörl, M. A universal method to produce in vitro transcripts with homogeneous 3′ ends. Nucleic Acids Res. 30, e56 (2002).Lu, R. et al. High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. EMBO J. 22, 5690–5699 (2003).Dickmeis, C., Fischer, R. & Commandeur, U. Potato virus X-based expression vectors are stabilized for long-term production of proteins and larger inserts. Biotechnol. J. 9, 1369–1379 (2014).Nakagawa, T. et al. Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci. Biotechnol. Biochem. 71, 2095–2100 (2007).Bedoya, L. C. & Daròs, J. A. Stability of Tobacco etch virus infectious clones in plasmid vectors. Virus Res. 149, 234–240 (2010).Sparkes, I. A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1, 2019–2025 (2006).Llorente, B. et al. Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light-dependent mechanism. Plant J. 85, 107–119 (2016)

Transcriptional analysis of Rhazya stricta in response to jasmonic acid

Background: Jasmonic acid (JA) is a signal transducer molecule that plays an important role in plant development and stress response; it can also efficiently stimulate secondary metabolism in plant cells. Results: RNA-Seq technology was applied to identify differentially expressed genes and study the time course of gene expression in Rhazya stricta in response to JA. Of more than 288 million total reads, approximately 27% were mapped to genes in the reference genome. Genes involved during the secondary metabolite pathways were up- or downregulated when treated with JA in R. stricta. Functional annotation and pathway analysis of all up- and downregulated genes identified many biological processes and molecular functions. Jasmonic acid biosynthetic, cell wall organization, and chlorophyll metabolic processes were upregulated at days 2, 6, and 12, respectively. Similarly, the molecular functions of calcium-transporting ATPase activity, ADP binding, and protein kinase activity were also upregulated at days 2, 6, and 12, respectively. Time-dependent transcriptional gene expression analysis showed that JA can induce signaling in the phenylpropanoid and aromatic acid pathways. These pathways are responsible for the production of secondary metabolites, which are essential for the development and environmental defense mechanism of R. stricta during stress conditions. Conclusions: Our results suggested that genes involved in flavonoid biosynthesis and aromatic acid synthesis pathways were upregulated during JA stress. However, monoterpenoid indole alkaloid (MIA) was unaffected by JA treatment. Hence, we can postulate that JA plays an important role in R. stricta during plant development and environmental stress conditions. How to cite: Hajrah, NH, Rabah SO, Alghamdi MK, et al. Transcriptional analysis of Rhazya stricta in response to jasmonic acid. Electron J Biotechnol 2021;50. https://doi.org/10.1016/j.ejbt.2021.01.00