Differential gene expression analysis under salinity stress in the selected turmeric (Curcuma longa L.) cultivars for curcuminoid biosynthesis

Curcuminoids are the phenolic compounds found exclusively in turmeric. Their presence is known to increase immunity and resistance against certain cancers and neurological disorders in humans also, protecting the plant itself against salinity stress. In this experiment, we studied the expression levels of MAPK1 and DCS genes, their curcuminoid biosynthesis under salinity stress conditions so that the impact of individual genes can be understood using semi- quantitative PCR. The expressions of the genes with respect to curcuminoid biosynthesis showed fluctuations in their band intensity values due to the production of curcuminoids, which is initiated first in the leaves followed by the rhizomes. Not all the genes responsible for the curcuminoid biosynthesis show positive regulation under salt stress conditions which is observed in response to the severity of the stress imposed on the cultivars. In our findings, both the genes MAPK1 and DCS were down-regulated for curcuminoid biosynthesis compared to their controls in both the cultivars Vallabh Sharad and Selection 1.


Introduction
Curcuminoids have gained much importance in the recent years due to their beneficial, therapeutic properties towards mankind.These curcuminoids, are known to promote useful anti-inflammatory, anti-cancerous, anti-diabetic, and antiarthritic properties.A daily intake of prescribed curcuminoids helps in improving digestion and is known to detoxify the liver, proving its good health [1].Also, these curcuminoids are known to be responsible for boosting the immune system, aiding protection against several viruses, bacterial and fungal infections.Not only this, the current beauty industry has improved their products by adding turmeric powder to their creams due to the presence of curcuminoids, which promotes healthy and glowing skin.These are the lesser-known facts about curcuminoids, whereas, many other benefits like wound healing and weight loss are also a great asset by the consumption of turmeric powder, which is rich in curcuminoids [2].
Turmeric plant, which is also a miracle herb with lots of beneficial properties, is known to provide maximum goodness for industrial application and consumption.India, is known as the world's top producer of turmeric, with an annual turn-over of nearly 400 tons, or 6500 kg/ha in 2022 [3].Therefore, the importance and need for turmeric production is increasing rapidly due to the presence of highly valuable compounds called curcuminoids [4].
These curcuminoids include curcumin, bis-dimethoxy curcumin and dimethoxy curcumin.Out of all the three different curcuminoids, curcumin is the most abundant and highly beneficial phenolic compound [5].Scientists believe that this curcumin is able to control many ailments in the human body and can treat several disorders and diseases.It is an interesting fact that the intake of curcumin has proven to improve mental disorders, anxiety, stress and helps in relaxing the muscles [6].Therefore, curcumin being the most abundant and very important phytochemical compound, has many advantages and benefits in improving human health by keeping neurological problems, arthritis, cancers, and immunological syndromes at bay [7].Not only curcumin, but also its derivates are tested for their phenolic activities and are proven to be beneficial as well.These curcuminoids together help in improving anti-oxidant properties in humans, hence they are termed as anti-oxidant rich compounds which are used as natural preservatives [8,9].
According to the research, the curcumin content is highest in freshly ground turmeric powder, with a teaspoon of turmeric powder containing nearly 200 mg of curcumin, which is highly beneficial [10].Therefore, it is suggested that a daily intake of 500 mg of curcuminoids will provide more benefits to the human body in improving immunity and resistance against several diseases [11].But, the presence of curcumin and its quantity are greatly affected by different biotic, abiotic, and environmental factors [12].Though, many factors influence the growth and development of turmeric plants as well as their quality, abiotic stress has been identified as a key factor in the reduction of curcuminoid content.Based on the current research, salinity stress has caused significant havoc in the developing turmeric cultivars, at nearly 100 mM of salt concentrations in the soils [13].
Salinity stress, which is sometimes not considered as a major problem in some parts of the world, is often threatening in certain areas near to the waterbodies and mangroves.Salinity stress is likely to occur in almost all crops grown near the sea, particularly those located near stagnant waters or rivers [14].Not only for this reason, but improper agronomical practices along with severe use of chemical fertilizers leave residues in soils and encourage the build-up of many salts in such areas.It is a well-known fact that these stresses reduce the plant's growth not only physically but also affect the rhizome quality, growth, turmeric powder quantity, and importantly the amount of curcuminoids [15].Certain studies revealed that the increased salt concentrations about 50 mM and ranges between 75 and 100 mM in the soils under turmeric cultivation resulted in the death of many cultivars.It is observed that, salinity stress also has an effect on the curcuminoids in the plants internally thereby, affecting the regulation of the genes involved in curcuminoid production [16].
Therefore, our research aims at the study of salt-treated turmeric cultivars, their physical growth and, their curcuminoids which helps in identifying the important genes responsible for the production of curcuminoids [17].The genes suggested and selected for this work are MAPK1 and DCS, which are the major classes of genes helpful in promoting maximum curcuminoid biosynthesis in many crops.DCS gene has been reported previously in rice under imposed salinity stress conditions [18].This gene was previously known to be a pre-cursor for the synthesis of CURS genes useful in curcumin production.Whereas, the MAPK cascade has been reported in Rice, Wheat and Arabidopsis for salinity stress.This cascade involves a total of 12 MAPKs, out of which only a few are reported to be up-regulated under salinity stress.So far, there is no evidence of DCS and MAPK1 gene regulation in turmeric under salinity stress conditions [19].Accordingly, a few turmeric cultivars that are never been reported for salinity stress, and are able to cope with the abiotic stresses were known to produce maximum yields are selected for the experiment [20].Along with these, the detailed expressions of DCS and MAPK1 genes are carefully studied and observed in such cultivars that are able to tolerate salt stress with respect to their physical appearance and morphological characteristics [21].The salinity treatment is given in a range of 50 mM and 100 mM to both of these cultivars and allowed to grow up to a certain stage of young rhizome development.The gene expression analysis is carried out in three different stages in all the treated and control plants (for comparison) [22].The leaf samples for RNA isolation are collected every 30 days, 60 days, and 90 days after salinity treatment to observe the changes in the levels of the expressions of DCS and MAPK 1 genes with respect to curcuminoid biosynthesis.
These samples are collected carefully and wrapped in an aluminum foil with the identification number and name and dipped immediately into liquid nitrogen to maintain temperature conditions of -196 for RNA isolation, cDNA synthesis and gene expression analysis under laboratory conditions [23].Hence, that being the case of imposed salinity stress in the selected turmeric cultivars, the curcuminoids' production is observed and tolerant cultivars are recommended for further use in highly saline areas.Therefore, the purpose behind this study is to identify the suitable turmeric cultivar that could cope with the salinity stress with maximum curcuminoid production by applying molecular biology and biotechnology methods [24,25].

Experimental conditions
Two turmeric cultivars, namely Vallabh Sharad and Selection 1, were selected for the gene expression analysis based on their outstanding performance under salt stress conditions with respect to their morphological characters (Supplementary Fig. 2).These varieties were able to cope up with the salt stress.
These newly emerged seedlings were allowed to grow for 2 months from the date of sowing so that they can withstand the imposed salt stress conditions.After 2 months of sowing, the NaCl solution was prepared in the required proportions, i.e., 50 mM and 100 mM, with which each cultivar was treated.After the salinity treatment, these cultivars were allowed to absorb the salt water without irrigation for 2 days and irrigated immediately after the second day.These plants were regularly watered for a month.Immediately, 1 month after the salinity treatment, the leaf samples were collected from both the cultivars, including control samples, 50 mM samples, and 100 mM samples [26] and this is continued up to 3 months i.e., 90 days after the salt treatment.
This method of sample collection was carried out in three stages, Stage 1: One month after salinity treatment Stage 2: Two months after salinity treatment Stage 3: Three months after salinity treatment

Isolation of RNA and cDNA synthesis
The TRIzol method was an easy, yet effective to isolate large quantities of mRNA from the fresh plant tissues (turmeric leaves) [27].The quantification and the purity of the obtained RNA was checked using the Nanodrop 2000 instrument, which indicates the extent of pure RNA in the absorbance range of A260/280, ratio between 1.9 and 2.1.From this pure RNA, the cDNA was obtained by reverse transcribing the mRNA with poly A tail with the help of oligo dT primers through the process of reverse transcription.

The cDNA was synthesized for 20 µl reaction using
The required ingredients for cDNA synthesis were taken carefully, and the nuclease free water was taken along with pure RNA for about 2 µl, cDNA buffer of 4 µl, dNTP mixture of 2 µl, oligo dt primer, RT enhancer and verso enzyme mix of 1 µl, individually were added to the mixture to make up the final volume of 20 µl.This mixture was carefully vortexed until all the ingredients were mixed well for cDNA synthesis [28,29].A 30 min cycle of polymerase enzyme activity at 42 °C was involved in the cDNA synthesis protocol, which is followed by a 2 min additional step for inactivation of the enzyme (Sigma cDNA synthesis Kit) [30].

Primers and gene expression analysis by semi-quantitative PCR
Two genes were selected to carry the gene expression work in order to understand the levels of curcuminoid biosynthesis in the turmeric germplasms under salt stress.Gene-specific primers were designed for both the selected genes by checking their amplicon sizes and lengths.These primers help in understanding the expression patterns and the levels of both the genes for curcuminoid biosynthesis under salt stress in turmeric.The modified sequences of these selected genes were taken as the respective primers for the amplification at their respective loci.
A total of 25 µl reaction mixture was taken which includes all the ingredients (Supplementary table 1) used in amplifying converted cDNA to check the expression analysis carried out by these two genes DCS and MAPK1 in two different cultivars with different treatments (Table 1, Supplementary Table 1).

Results
Based on the results fetched from the Bio-Rad gel docking system for both the genes that were responsible for curcuminoid biosynthesis in turmeric, a significant difference was observed in the expression levels of the treatments, where the treated plants were down regulated compared to their controls.As known, the amplicon sizes of both the genes were 700bps for DCS and 589bps for MAPK 1 (Supplementary Table 2).The results clearly showed that there was a maximum down regulation of both the genes caused by salinity stress in their gene expression levels compared to their controls.
Both the turmeric cultivars Vallabh Sharad and Selection 1 showed reduced gene expressions under salt stress conditions, indicating low levels of curcuminoid biosynthesis.This represents a change in the curcuminoid biosynthesis with respect to both the genes DCS and MAPK1 under salt stress conditions, which helps in deciding the suitable turmeric cultivar recommendable for the areas affected by severe salinity stress ranging between 50 and 100 mM salt concentrations.The structures of both the genes selected for gene expression work were obtained from the gene structure display server 2.0 web tool, where the entire sequence shows no traces of introns and completely exon sequences for both the MAPK1 and DCS genes (Supplementary Fig. 1).

STAGE 1
This is the primary stage in which more importance is given to the foliage growth than the development of the underground rhizomes.This increase in the foliage growth would later affect the growth of the entire plant, 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 Selection 1, were chosen for the observation of curcuminoid biosynthesis under salinity stress in turmeric in Stage 1 (1 month after salinity treatment) using the selected genes.These two varieties were known for their curcuminoid production under normal growth conditions.
Therefore, in our study, these two varieties were checked for curcuminoid biosynthesis in Stage 1 (1 month after salinity treatment) and it was observed that the gene expression levels in the controls were maximum compared to their treatments (50 mM and 100 mM) due to the salt stress conditions (Figs. 1, 2), 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 occur as a result of salinity stress.

STAGE 2
This is the stage where; salinity stress was observed in the plants 2 months after the salt treatment.This is the most critical stage because the turmeric growth and development was increasing at this stage, and the accumulation of curcuminoids was at its peak.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 at Stage 2 (2 months after salinity treatment) was observed to be down regulated compared to their 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 the 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 50 mM and 100 mM treatments for both the cultivars compared to their controls (Figs. 3, 4).This observation of band intensities was done by gel documentation system which indicated that thick bands were observed in the controls compared to treatments due to the less production of curcuminoids.

STAGE 3
This stage is known to be the most crucial in the development of the turmeric plants.At this stage, the development of the leaves and curcuminoid production in them is reduced gradually and more focus will be diverted to the growth and development of the rhizomes.During this stage, the growth of young rhizomes was observed and the curcuminoid production was maximum in the case of leaves (Table 2).
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 showed maximum curcuminoid biosynthesis in the leaves (Figs. 5, 6).Therefore, the gene expression levels in the treatments were observed to be low compared to their controls in both the varieties and for both the genes.

Band intensity/thickness values from gel documentation software
It was observed that the band intensities gradually decreased from 50 mM followed by 100 mM compared to the controls in both the cultivars in the case of MAPK 1 and DCS genes, indicating maximum down regulation in the treatments, especially at 100 mM concentration in each and every stage (Table 2).However, salt stress conditions reduced the gene expressions in the plants treated with 50 mM salt concentration followed by 100 mM salt concentration compared to their controls in all the three stages.
In spite of the fact that, the band thickness from stage 1 to stage 3, indicates the presence of cDNA, both the selected genes were down regulated in the treatments compared to their controls in each individual stage due to salinity stress.
In conclusion, it was observed that the plants exhibit changes due to salinity stress not only morphologically, biochemically, and physiologically but also in their gene expression levels where the down-regulation of both the genes was observed in case of the treatments compared to their controls

Discussions
Turmeric has gained much importance in the recent years due to its very useful phytochemical properties which treats several diseases like cancers, neurological disorders, kidney problems, skin allergies etc. Salinity has caused much disturbance in the cropping patterns and yields of turmeric, thereby reducing the phytochemical production in the turmeric rhizomes.These phytochemicals, i.e., the curcuminoids, play a major role in improving human health, which also makes this crop a very important medicinal herb.The production of these phytochemicals was greatly affected by the salinity of the soils.Also, there were certain evidences that this soil salinity can be occurred by many other factors like poor agricultural practices, varied environmental conditions, biotic and abiotic stresses which effects the yields and curcuminoids production in the turmeric plants.
In our study, the gene expression levels of DCS and MAPK1 were checked for the curcuminoid biosynthesis.The greater number of curcuminoids in the rhizomes of turmeric increases the medicinal value of this herb.Curcuminoids, not only improving human health but also maintains the growth of the plants against several biotic factors.Certain investigations revealed that the saline conditions reduce the rhizome yields and the curcumin content in the rhizomes.Saline soils would halt the growth of the plants to a greater extent when the salinity in the soils is less than 50 mM.Also, maximum salinity in the soils i.e., between 50 to 100 mM would affect the growth of the plants by making them weak and ultimately leading to the death of the plants.
Therefore, our study was planned accordingly to check the curcuminoid biosynthesis in Stage 1 (1 month after salinity treatment), Stage 2 (2 months after salinity treatment), and Stage 3 (3 months after salinity treatment).The expressions of the two genes (MAPK1 & DCS) were down regulated with respect to curcuminoid production under salt stress conditions in all the three stages compared to their respective controls [31,32] which was observed from the readings obtained from Bio-Rad gel documentation system.
Our investigations revealed that the expression levels of both the genes in all the stages were maximum in Control, reduced in 50 mM and least in 100 mM treatments proving down-regulation compared to the controls due to salt stress conditions.Therefore, both the cultivars Vallabh Sharad and Selection 1 showed outstanding performance with respect to their growth and development compared to the other cultivars.However, the production of curcuminoids was greatly affected in all the treatments compared to their controls due to salt stress conditions in all the three stages.But, these two cultivars can be improved using gene manipulation techniques to improve their growth along with the curcuminoids production under unfavorable conditions.

Conclusions
Turmeric has several important therapeutic and medicinal properties, especially for treating cancers and other ailments.All of this was achieved due to the presence of important polyphenolic compounds called curcuminoids.In the recent years, turmeric was known to experience loss in the powder quality and yields due to salinity stress.The curcuminoids production was low in salt-treated plants compared to the control plants, of the same cultivar as an indication that the salinity stress effects the plants in their growth, development and curcuminoid production.
In our experiment, two turmeric cultivars, Vallabh Sharad and Selection 1 were selected for curcuminoid biosynthesis under salt stress conditions.Also, MAPK1 and DCS genes were chosen to observe the expression levels of curcuminoid biosynthesis as these genes were previously known to promote curcuminoid biosynthesis.The treatment was conducted by taking one control (untreated) and two treatments (50 mM and 100 mM) salt concentrations in three different stages of salinity treatment.
The expressions of MAPK1 and DCS genes were greatly affected in treated plants compared to their controls due to salinity stress indicating down-regulation of both the genes in both the treatments (50 mM and 100 mM).Both the cultivars, Vallabh Sharad and Selection 1 were proven to have maximum curcuminoid synthesis without salinity stress which was observed in their control samples making them wonderful and recommendable cultivars for maximum curcuminoid production.But, the salt stress resistance of these two cultivars can be achieved by gene manipulations and breeding techniques.
Therefore, the gene expressions were negatively regulated towards curcuminoid biosynthesis and curcumin yield under salt stress conditions.Proper gene regulation studies and genetic engineering in suitable, robust cultivars may increase the expression levels of both the genes and promote salt stress tolerance.It was clear from the literature that the curcuminoids production promotes several medicinal properties that contribute to a healthy lifestyle in humans, conducting such experiments is beneficial to many fields like beauty industry, agriculture, medicine etc. believing that in future safe cropping patterns would lead to the curcuminoids production by promoting salt tolerance in the turmeric plants.

Fig. 1 Fig. 2
Fig. 1 Gene expression analysis in Vallabh Sharad under salt stress in stage 1

Fig. 3 Fig. 4
Fig. 3 Gene expression analysis in Vallabh Sharad under salt stress conditions in stage 2

Table 1
Primers selected for the work