The increase in global temperatures has shown negative influence on the agricultural production all over the world. Developing thermal resistance in crops has become a great challenge for plant biology researchers and in agriculture. The capacity of the plant to tolerate heat stress can be attained and enhanced by altering the genes involved in thermal resistance. Scavenging of reactive oxygen species, membrane stability maintenance, accumulation of solutes, heat shock protein mediated chaperone signaling, the production of antioxidants, transcriptional activation are the mechanisms developed by the plant to deal with heat stress. The genes that were identified to be responsible for causing heat resistance when transferred into plants are heat shock factors, ascorbate peroxidase, glutaredoxin, HSPs, and down-regulated FAD7 (omega-3 fatty acid desaturase).
The mechanism of temperature sensing is present in the plasma membrane of the cell. Membrane fluidity alterations are triggered by high temperatures and results in the entry of calcium ions. Cyclic nucleotide gated ion channels in the plasma membrane are involved in thermo sensing, Arabidopsis thermo tolerance and HSPs (heat shock proteins) expression. Proteins in the plasma membrane play important roles in HSRs.
Brassica napus thermal resistance gene1 (BnTR1) is the gene that is influenced by heat stress. BnTR1 is found to be membrane oriented RINGv or really interesting new gene variant E3 ligase. The homologues of BnTR1 are wide spread in dicots and monocots. The expression of BnTR1 at a modest level will reduce bad influence of environment and confers resistance to high temperatures in various plant species without influencing their normal development. BnTR1 is an important gene that is involved in the conserved thermal resistance mechanism in plants.
Results of the study
BnTR1 is a gene induced by temperature
The genes in Brassica napus that are responsive to enhanced temperatures were identified by comparing the transcriptional variations between 15-day aged leaves growing under heat stress (35 degree C) with transcriptional variations under normal conditions (22 degree C). This was done with the help of mRNA differential display technique. There were 26 up-regulated and 9 down-regulated transcripts identified. The transcript of the gene that was up regulated by the enhanced temperature was cloned by amplification of 5' cDNA end and it is called as BnTR1.
The reading frame of the gene BnTR1 consists of 861 base pairs that encode for a polypeptide of 286 amino acid residues. BnTR1 is a highly conserved protein and it shares 88 percent of its sequence with its homologue in Arabidopsis thaliana, 62 percent with that of Ricinus communis, 57 percent with that of Oryza sativa and 54 percent with that of Zea mays. The neighbor-joining (NJ) bootstrap is a phylogenetic tree analysis through which BnTR1 is identified as a member of a novel gene family conserved in plants.
BnTR1 is a functional E3 ligase
BnTR1 protein consists of the RING-variant domain (Really interesting new gene variant), which is a C4HC3zinc-finger like sequence that is found to be present in many of the viral and cellular proteins. As some of these proteins act like ubiquitin E3 ligase, BnTR1 is known to have E3 ligase activity. To confirm these findings, ligase activity of BnTR1 was measured using in vitro ubiquitination assay. The BnTR1 fused to GST was initially expressed in E. coli and purified. The recombinant protein BnTR1 was mixed with E1 (human ubiquitin activating enzyme) and E2 (human ubiquitin conjugating enzyme) and then incubated. Ubiquitination was seen in the presence of BnTR1-GST and not in the GST control. Therefore, it is understood that BnTR1 comprises of E3 ligase activity.
BnTR1 is influenced by heat stress
It is experimentally proved that BnTR1 protein was found in large amounts in the membrane. In Silico analysis reported that BnTR1 gene has a heat stress responsive element present in the upstream of transcriptional beginning from -242 to -251 nucleotides (AAACAATTTC). To study the impact of heat stress on BnTR1 transcription, a luciferase gene that was controlled by BnTR1 promoter was cloned into a construct which was transfected into Arabidopsis protoplasts for observing the transient analysis. When the protoplasts are moved from normal temperatures to higher temperatures, there was 2 to 3 fold increase in the luciferase activity. Hence, it is found that BnTR1 is a heat responsive gene.
Plant resistance to heat stress was enhanced by BnTr1
Transgenic lines of B. napus and O. sativa over-expressing BnTR1 were created to check the impact of the gene expression on heat resistance of the plant. The transgenic plants that were subjected to heat stress appeared normal while the non-transgenics showed yellow spots and slowly reduced in size. The transgenic plants with up regulated BnTR1 were able to survive for a minimum of 9 days, while the non-transgenics could not survive after 6 days.
The O.sativa transgenic plants with inserted BnTR1 could yield in terms of total grains per plant as 8 to 17 percent more than that of non-transgenic plants. The number of primary branches per panicle of the transgenic plants with BnTR1 gene and the grains per plant were higher than those observed in non-transgenic plants. The yield of BnTR1 transgenic pants in terms of grains per plant and primary branches per panicle was 19 to 44 percent greater than that of non-transgenic.
Zhi-Bin Liu, Jian-Mei Wang, Feng-Xi Yang, Liang Yang, Yu-Fei Yue, Jun-Bei Xiang, Mei Gao, Fang-Jian Xiong, Dong Lv, Xian-Jun Wu, Ning Liu, Xun Zhang,Xu-Feng Li and Yi Yang. A novel membrane-bound E3 ubiquitin ligase enhances the thermal resistance in plants. Plant Biotechnology Journal (2014) 12, pp. 93-104.
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