Isolation and characterization of drought and ABA responsive promoter of a transcription factor encoding gene from rice.

Water deficit is a significant impediment to enhancing rice yield. Genetic engineering tools have enabled agriculture researchers to develop drought-tolerant cultivars of rice. A common strategy to achieve this involves expressing drought-tolerant genes driven by constitutive promoters such as CaMV35S. However, the use of constitutive promoters is often limited by the adverse effects it has on the growth and development of the plant. Additionally, it has been observed that monocot-derived promoters are more successful in driving gene expression in monocots than in dicots. Substitution of constitutive promoters with stress-inducible promoters is the currently used strategy to overcome this limitation. In the present study, a 1514 bp AP2/ERF promoter that drives the expression of a transcription factor was cloned and characterized from drought-tolerant Indian rice genotype N22. The AP2/ERF promoter was fused to the GUS gene (uidA) and transformed in Arabidopsis and rice plants. Histochemical GUS staining of transgenic Arabidopsis plants showed AP2/ERF promoter activity in roots, stems, and leaves. Water deficit stress and ABA upregulate promoter activity in transformed Arabidopsis and rice. Quantitative PCR for uidA expression confirmed induced GUS activity in Arabidopsis and rice. This study showed that water deficit inducible Os-AP2/ERF-N22 promoter can be used to overcome the limitations of constitutive promoters. Transformants overexpressing Os-AP2/ERF-N22 showed higher relative water content, membrane stability index, total chlorophyll content, chlorophyll stability index, wax content, osmotic potential, stomatal conductance, transpiration rate, photosynthetic rate and radical scavenging activity. Drought tolerant (N22) showed higher expression of Os-AP2/ERF-N22 than the susceptible (MTU1010) cultivar. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01246-9.

Implications of exposing mungbean (Vigna radiata L.) plant to higher CO2 concentration on seed quality.

Understanding the crop response to elevated carbon dioxide (e[CO2]) condition is important and has attracted considerable interest owing to the variability and crop-specific response. In mungbean, reports are available regarding the effect of e[CO2] on its growth, physiology and yield. However, no information are available on the germination and vigour status of seeds produced at e[CO2]. Therefore, in the present investigation, mungbean (Virat) was grown in the open top chamber during summer season of 2018 and 2019 to study the implications of e[CO2] (600 ppm) on quality of the harvested seeds (germination and vigour). The exposure of mungbean plant to e[CO2] had no major impact on seed quality as the percent viability (normal seedling + hard seeds) was not reduced. However, in one season (2018), the seed germination (normal seedling) was slightly reduced from 72 to 68%, attributed majorly to an increase in the hard seeds (from 13 to 19%), a predominant form of seed dormancy in mungbean. The changes in seed germination were apparent only in first year of the experiment. Accelerated ageing test (AAT) and storage studies revealed no differences in the vigour of seeds produced at ambient and e[CO2] environments. Also, the seeds from e[CO2] had low protein and sugar but recorded higher starch content than the seeds from ambient [CO2].

Effect of elevated carbon-dioxide on plant growth, physiology, yield and seed quality of chickpea (Cicer arietinum L.) in Indo-Gangetic plains.

In the present scenario of climate change with constantly increasing CO2 concentration, there is a risk of altered crop performance in terms of growth, yield, grain nutritional value and seed quality. Therefore, an experiment was conducted in open top chamber (OTCs) during 2017-18 and 2018-19 to assess the effect of elevated atmospheric carbondioxide (e[CO2]) (600 ppm) on chickpea (cv. JG 14) crop growth, biomass accumulation, physiological function, seed yield and its quality in terms of germination and vigour. The e[CO2] treatment increased the plant height, leaf and stem biomass over ambient CO2 (a[CO2]) treatment. The e[CO2] increased seed yield by 11-18% which was attributed to an increase in the number of pods (6-10%) and seeds plant-1 (8-9%) over a[CO2]. However, e[CO2] reduced the seed protein (7%), total phenol (13%) and thiobarbituric acid reactive substances (12%) and increased the starch (21%) and water uptake rate as compared to seeds harvested from a[CO2] environment. Exposing chickpea plant to e[CO2] treatment had no impact on germination and vigour of the harvested seeds. Also, the physical attributes, total soluble sugar and antioxidant enzymes activities of harvested seeds were comparable in a[CO2] and e[CO2] treatment. Hence, the experimental findings depict that e[CO2] upto 600 ppm could add to the growth and productivity of chickpea in a sub-tropical climate with an implication on its nutritional quality of the produce.

Molecular cloning and functional analysis of the promoter of γ-Tocopherol Methyl Transferase (γ-TMT) gene of soybean (Glycine max).

γ-Tocopherol methyl transferase (γ-TMT) (EC 2.1.1.95) is the key enzyme of the tocopherol biosynthetic pathway that determines the α-tocopherol concentration in plants. The overexpression of γ-TMT has been a successful approach for α-tocopherol enrichment of most plants including soybean. The typical soybean varieties are rich in γ-tocopherol (constitutes nearly 65-70% of its total seed tocopherol pool), while α-tocopherol, the biologically most active form among all tocopherols, constitutes only 10% of the total tocopherol content. The identification of soybean varieties that have seed α-tocopherol as high as > 20% of the total tocopherols has shifted attention towards the breeding based approach for α-tocopherol enrichment of this crop. Previous research on this aspect suggests that polymorphisms in γ-TMT promoter might be associated with the high α-tocopherol concentration of some soybean varieties. To understand the molecular basis of genetic variation for α-tocopherol concentration in Indian varieties of soybean we cloned the 1.4 kb upstream promoter region of γ-TMT from a high α-tocopherol containing soybean variety (Bragg) as well as from a low α-tocopherol containing variety (DS 2706). Cloning of each of these promoters in pORE R2 vector having GUS reporter gene and the subsequent GUS assay revealed a slightly high promoter activity of Bragg γ-TMT as compared to DS 2706 γ-TMT. On promoter sequence analysis, no sequence polymorphisms were observed in the core promoter region of this gene. However, seven single nucleotide polymorphisms (SNPs) were observed outside the core promoter region. Further study based on deletion construct analysis of this promoter will elucidate the significance of these SNPs in influencing the activity of γ-TMT promoter and the α-tocopherol concentration.

Molecular cloning, heterologous expression and functional characterization of gamma tocopherol methyl transferase (γ-TMT) from Glycine max.

γ-Tocopherol methyltransferase (γ-TMT) (EC 2.1.1.95) is the last enzyme in the tocopherol biosynthetic pathway and it catalyzes the conversion of γ-tocopherol into α-tocopherol, the nutritionally significant and most bioactive form of vitamin E. Although the γ-TMT gene has been successfully overexpressed in many crops to enhance their α-tocopherol content but still only few attempts have been made to uncover its structural, functional and regulation aspects at protein level. In this study, we have cloned the complete 909bp coding sequence of Glycine max γ-TMT (Gm γ-TMT) gene that encodes the corresponding protein comprising of 302 amino acid residues. The deduced Gm γ-TMT protein showed 74-87% sequence identity with other characterized plant γ-TMTs. Gm γ-TMT belongs to Class I Methyl Transferases that have a Rossmann-like fold which consists of a seven-stranded ß sheet joined by α helices. Heterologous expression of Gm γ-TMT in pET29a expression vector under the control of bacteriophage T7 promoter produced a 37.9 kDa recombinant Gm γ-TMT protein with histidine hexamer tag at its C-terminus. The expression of recombinant Gm γ-TMT protein was confirmed by western blotting using anti-His antibody. The recombinant protein was purified by Ni2+-NTA column chromatography. The purified protein showed SAM dependent methyltransferase activity. The α-tocopherol produced in the in-vitro reaction catalyzed by the purified enzyme was detected using reverse phase HPLC. This study has laid the foundation to unveil the biochemical understanding of Gm γ-TMT enzyme which can be further explored by studying its kinetic behaviour, substrate specificity and its interaction with other biomolecules.