Based on the results fetched from the Bio-Rad gel docking system for both the genes responsible for curcuminoid biosynthesis in turmeric, a significant difference was observed in the expression levels of the treatments, which were down regulated compared to the controls. As known, the amplicon sizes of both the genes were 700bps for DCS and 589bps for MAPK 1 (Table 3). The results clearly showed that there was a maximum down regulation of both the genes caused by salinity stress in their gene expression levels.
Both the turmeric cultivars Vallabh Sharad and New Selection 1 showed reduced gene expression under salt stress conditions, indicating curcuminoid biosynthesis. This indicates that there is a change in curcuminoid biosynthesis with respect to both the genes DCS and MAPK1 under salinity stress conditions, which helps in deciding the suitable turmeric cultivar recommended for the areas affected by severe salinity stress in between 50–100 mM concentrations.
The structure of both the genes selected for gene expression work were obtained from the gene structure prediction tool (online), where the entire sequence shows no traces of introns and completely exon sequences for both the MAPK1 and DCS genes.
Gene expressions analysis in different stages of salinity stress in turmeric cultivars:
STAGE 1:
This is the primary stage in which more importance is given to the foliage than to underground rhizomes. This increase in the foliage growth would later affect the growth of entire plants, including the underground rhizomes. This stage is crucial not only for rhizome production but also for an increase in the foliage of the turmeric plants. The curcuminoid biosynthesis was observed to be initiated in the leaves at this stage, followed by the rhizomes.
Two varieties, Vallabh Sharad and New Selection 1, were chosen for the observation of curcuminoid biosynthesis under salinity stress in turmeric in Stage 1 (one month after salinity treatment) using selected genes. These two varieties were known for their production with respect to the quality and quantity of rhizomes along with curcuminoid biosynthesis under normal growth conditions.
Therefore, in our study, these two varieties were checked for curcuminoid biosynthesis in Stage 1 (one month after salinity treatment) and it was observed that the gene expression levels in the control were maximum compared to the treatments (50 mM and 100 mM) due to stress conditions, which indicates that these genes under 50 mM and 100 mM concentrations were gradually down regulated.
With respect to the band intensities observed under the Bio-Rad gel dock system, there was down regulation in the expression levels of both the genes in both the treatments (50 mM and 100 mM) at this stage and it was believed to be due to salinity stress.
STAGE 2:
This is the stage at which salinity stress is observed in plants two months after salt treatment. This is the most critical stage because the turmeric cultivars' growth and development were known to be rapid at this stage, and the accumulation of curcuminoids was greatest. During this stage, the rhizome development starts underneath the soil, intimating the growth of the plant, which is triggered by the leaves.
The gene expression analysis for MAPK 1 and DCS genes for curcuminoid biosynthesis in Stage 2 (two months after salinity treatment) were observed be down regulated compared to the control plants. Therefore, in our experiment it was observed that the expression levels of DCS and MAPK1 genes were increased slightly under control conditions due to the stage of plant growth for curcuminoid production. This rhizome production in this stage was initiated but, there was a gradual down-regulation in the gene expression levels of both the genes in 50mM and 100mM treatments for both the cultivars compared to their controls. This observation of band intensities was done by gel documentation system which indicated that thick bands were observed in controls compared to treatments due less production of curcuminoids.
STAGE 3:
This stage is known to be the most crucial in the development of turmeric plants. At this stage, the development of the plants will be reduced and more focus will be diverted to the initiation and development of the rhizomes. During this stage, young rhizomes grow along with the production of curcuminoids. In this stage, the curcuminoid production was maximum in the case of leaves, and in the case of rhizomes, the curcuminoid production was just initiated. The young cells developed in the rhizomes multiply together and trigger the production of curcuminoids.
The gene expression levels of both the genes DCS and MAPK1 were known to be down-regulated compared to the controls in the case of treatments (50 mM and 100 mM concentrations) for curcuminoid biosynthesis under salinity stress. This stage, which was expected to provide maximum curcuminoid biosynthesis in the case of leaves, as the curcuminoid production was maximum in the leaves, whereas the curcuminoid production in the rhizomes was just initiated. Therefore, the gene expression levels in the treatments were observed to be low compared to the controls in both the varieties and for both the genes.
Band intensity/ thickness values from gel documentation software:
Table 4: Band intensities for the selected genes under salinity stress depicted in form of a heat map to observe the expression levels of genes (MAPK1 and DCS)
It was observed that the band intensities gradually decreased at 50 mM followed by 100 mM compared to controls in both the cultivars and genes, indicating maximum down regulation in treatments, especially at 100 mM concentration in each and every stage.
The band intensity levels were increased gradually in control plants from stage 1 to stage 3. Band intensity was highest in 50mM and 100mM from Stage 1 to Stage 3 in all plants, not just control plants. This increase in the values of band intensity was observed due to the initiation of curcuminoid production in the leaves first and then in rhizomes (Table 4). However, salt stress conditions reduced gene expression in 50 mM followed by 100 mM compared to controls.
Even though there was an increase in the band intensity values from stage 1 to stage 3, which indicates the presence of cDNA, both the selected genes were down regulated in the treatments compared to the controls in each individual stage due to salinity stress.
Therefore, the availability of mRNA or curcuminoid biosynthesis increases at this stage as there is maximum production of curcuminoids 3–4 months after sowing. Although there is a gradual increase in the values in stage 3, it does not indicate the up-regulation of the genes but indicates the presence of more curcuminoid expression in that particular stage.
In conclusion of our experiment, it was observed that plants exhibit changes due to salinity stress not only morphologically, biochemically, and physiologically but also for gene expressions in which down-regulation of both the genes was observed in the case of treatments compared to controls with respect to DCS and MAPK 1, which were mainly affected by the salinity stress.