Model Plant Tobacco Research Articles

Transcriptomics assisted proteomic analysis of Nicotiana occidentalis infected by Candidatus Phytoplasma mali strain AT.

Phytoplasmas are pathogenic bacteria within the class of Mollicutes, which are associated with more than 1000 plant diseases. In this study, we applied quantitative mass spectrometry to analyse affected pathways of the model plant tobacco (Nicotiana occidentalis) upon Candidatus Phytoplasma mali strain AT infection. Using tissue obtained from leaf midribs, 1466 plant-assigned proteins were identified. For 1019 of these proteins, we could reproducibly quantify the expression changes of infected versus noninfected plants, of which 157 proteins were up- and 173 proteins were downregulated. Differential expression took place in a number of pathways, among others strong downregulation of porphyrin and chlorophyll metabolism and upregulation of alpha-linolenic acid metabolism, which was consistent with observed increased levels of jasmonic acid, a key signal molecule of plant defence. Our data shed light on the molecular networks that are involved in defence of plants against phytoplasma infection and provide a resource for further studies.

Expression of a rice Zn transporter, OsZIP1, increases Zn concentration in tobacco and finger millet transgenic plants

Zinc (Zn) deficiency is very widespread both from the plant and human nutrition perspective. One of the approaches to improve Zn in crop plants is by over-expression of Zn transporters such that plants can uptake and accumulate additional Zn added to soil. The ZIP family transporters generally contribute to Zn homeostasis in plants by regulating Zn transport into the cell. We over-expressed OsZIP1 in finger millet and model plant tobacco under the control of constitutive (35S) and endosperm-specific (Bx17) promoters to study the improvement in Zn accumulation. The transcript analysis revealed the induction of ZIP1 in leaf and root tissue under Zn deprivation in finger millet cultivars. Ectopic expression of OsZIP1 in tobacco under CaMV35S (n35S) and Bx17 (nBx17) improved seed Zn concentration compared to untransformed wild-type plants. In addition, we successfully developed finger millet transgenic plants, f35S and fBx17, expressing the OsZIP1 under 35S (f35S) and Bx17 (fBx17) promoter, respectively. The transgenic plants accumulated significantly higher Zn in seeds compared to untransformed wild-type finger millet plants. Plants expressing the gene under Bx17 promoter accumulated more Zn in seed than 35S plants. Apart from Zn, finger millet transgenic plants also showed increased Mn content in seeds. Our results show the involvement of OsZIP1 in improving Zn concentration in tobacco and finger millet. Using endosperm-specific promoter, it is possible to improve the seed Zn concentration in the edible part of cereals.

GOverexpression of the Glyoxalase II Gene Leads to Enhanced Salinity Tolerance in Brassica Juncea

Engineering of salinity tolerance in agronomically important crop plants is required to increase their productivity by enabling them to grow in saline soils, which are otherwise left uncultivated. Since an increase in the enzymes of glyoxalase system has been shown to impart salinity tolerance in the model plant tobacco, we used the glyoxalase II gene for engineering salinity tolerance in an important oil yielding crop, Brassica juncea. The transgenic plants of B. juncea overexpressing the glyoxalase II gene showed higher salinity tolerance as compared to the untransformed control plants as observed by delayed senescence in leaf discs at 400 mM and 800 mM NaCl in T1 generation. The percentage of germination of the T2 transgenic seeds was higher at 150 mM and 200 mM NaCl as compared to the seeds of untransformed plants. This for the first time demonstrates the applicability of utilizing the glyoxalase II gene for enhanced salinity tolerance in an oilseed crop plant B. juncea.

Plant plastid engineering.

Genetic material in plants is distributed into nucleus, plastids and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is becoming more popular and an alternative to nuclear gene transformation because of various advantages like high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic crop plants have been generated which possess higher resistance to biotic and abiotic stresses and molecular pharming. In this mini review, we will discuss the features of the plastid DNA and advantages of plastid transformation. We will also present some examples of transplastomic plants developed so far through plastid engineering, and the various applications of plastid transformation.

Designing and Transgenic Expression of Melanin Gene in Tobacco Trichome and Cotton Fiber

In Streptomyces antibioticus, there are two genes TYRA and ORF438 required for the melanin biogenesis. To investigate whether expression of these two genes in cotton can change cotton fiber colour, we modified the TYRA and ORF438 genes to make their codon usage closer to the codon preference of cotton fiber genes. The resulting versions of these two genes were referred to as DTYRA and DORF438, respectively. Vacuolar targeting signals were also added to their ends. Under the cotton fiber specific LTP3 promoter, DORF438 and DTYRA were first transformed into model plant tobacco (Nicotiana tabacum). Molecular analyses showed that both the DORF438 and DTYRA genes were successfully expressed in transgenic plants, and the melanin deposition was observed in the trichomes of transgenic tobacco. Excitedly, when the same DORF438 and DTYRA expression cassettes were transformed into cotton (Gossypium hirsutum L.) by pollen tube pathway, the colour of cotton fiber changed from white to brown. Molecular analyses confirmed that both genes were transformed into cotton and expressed successfully. All these results indicate that the synthesized DOFR438 and DTYRA genes can work well in tobacco and cotton. Our study may provide a potential method for modifying the colour of cotton fiber.

Phenotypic subunit composition of the tobacco ( Nicotiana tabacum L.) vacuolar-type H +-translocating ATPase

The model plant tobacco ( Nicotiana tabacum L.) was chosen for a survey of the subunit composition of the V-ATPase at the protein level. V-ATPase was purified from tobacco leaf cell tonoplasts by solubilization with the nonionic detergent Triton X-100 and immunoprecipitation. In the purified fraction 12 proteins were present. By matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and amino acid sequencing 11 of these polypeptides could be identified as subunits A, B, C, D, F, G, c, d and three different isoforms of subunit E. The polypeptide which could not be identified by MALDI analysis might represent subunit H. The data presented here, for the first time, enable an unequivocal identification of V-ATPase subunits after gel electrophoresis and open the possibility to assign changes in polypeptide composition to variations in respective V-ATPase subunits occurring as a response to environmental conditions or during plant development.

Multiple pathways for Cre/lox-mediated recombination in plastids.

Plastid transformation technology involves the insertion by homologous recombination and subsequent amplification of plastid transgenes to approximately 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subsequent role in the transformed line. We report here a feasibility study in the model plant tobacco, to test the heterologous Cre/lox recombination system for antibiotic marker gene removal from plastids. To study its efficiency, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molecular assays, we show that transgene excision occurs completely from all plastid genomes early in plant development. Selectable marker-free transplastomic plants are obtained in the first seed generation, indicating a potential application of the Cre/lox system in plastid transformation technology. In addition to the predicted transgene excision event, two alternative pathways of Cre-mediated recombination were also observed. In one alternative pathway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in the plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, the resultant plastid mutations are silent, and their study provides the first insights into tRNA accumulation and trans-splicing events in higher plant plastids.