Gene expression profiling quantifies mRNA levels at certain circumstances, revealing the pattern of genes expressed by a cell at the transcription level.
Expression analysis of DREB3 gene under osmotic stress
As a fundamental family of the transcription factor, DREB assumes a significant part in countering abiotic stresses (Niu et al.33). The expression profiling of the wheat DREB3 gene was first examined after PEG treatment for various timespans such as 0, 2, 4, 6, and 12 h. In drought-tolerant wheat genotypes (NR-499, NARC-2009, and Pakistan-2013), the expression of the DREB3 gene was incline significantly at 2 h (> fourfold), peaked at 4 h (6–7 folds) and then started to decline after 6 h of moisture stress (Fig. 1). In any case, it generally diminished after 6 h and recorded the most negligible and least expression at 12 h during this study. These results suggest that DREB3 is an early drought-responsive gene.
DREB3 expression levels in drought-tolerant and sensitive wheat genotypes. (Asterisk indicates P < 0.05).
Among drought-tolerant wheat genotypes, Pakistan-2013 showed the most elevated expression pattern of the DREB3 gene followed by NR-499 and NARC-2009. Although, drought-sensitive genotypes showed similar increase–decrease trend, but the significant change (increase) in expression of DREB3 was only observed in Borlaug-2016 (twofold) and NR-514 (1.8-fold) after 4 h of osmotic stress while NR-516 did not show any significant change in DREB3 expression (Fig. 1). In this way, moderately higher expression of the DREB3 gene in drought-tolerant genotypes suggests that the DREB3 is involved in PEG-induced dehydration resilience in bread wheat. In addition, it also shows that drought-tolerant wheat genotypes have the hereditarily genetic potential to endure dry spells. Several previous studies also described that different abiotic stresses trigger the expression pattern of TaDREB3 and dehydrin genes as a counter mechanism, as observed in this study in case of TaDREB333,39.
Expression analysis of TaARGOS genes under osmotic stress
ARGOS genes are engaged with different formative and stress reactive mechanisms in plants16,18. Earlier findings showed that the ARGOS genes of Arabidopsis and maize alleviate disease resistance and can be set off by different chemicals and hormones. In wheat, the expression of TaARGOS genes have been determined for in organ improvement and stress response16. Subcellular restriction of ARGOS-D protein indicated that it is limited to the endoplasmic reticulum16. The expression level of wheat TaARGOS genes was concentrated after osmotic pressure at various time stretches to determine whether ARGOS genes influence to improve resilience against PEG-induced dehydration in distinguished wheat lines. Distinctive expression levels were portrayed for TaARGOS-A, TaARGOS-B, and TaARGOS-D, articulating that these homeologs can perform important functions at various phases of growth and development.
Expression analysis of TaARGOS-A gene under osmotic stress
TaARGOS-A is the first homoeologous gene of the TaARGOS gene family, located on chromosome 4A in bread wheat16. In present study, initially up to 4 h of osmotic stress the expression of TaARGOS-A increased significantly, followed by a continuous decline till 12 h of osmotic stress in all the studied genotypes (Fig. 2). Importantly, all three drought tolerant genotypes showed markedly higher expression of TaARGOS-A than drought sensitive genotypes. For instance, the highest mRNA transcription of the TaARGOS-A gene was observed in Pakistan-2013, that showed 5.9 fold increase in gene expression at 4 h, followed by NARC-2009 and NR-499 at the same time point. In contrast, the drought sensitive genotypes did not show any significant change (except slight increase or decrease across the timepoint) in TaARGOS-A expression, except the significant increase after 4 h of osmotic stress which was highest in Borlaug-2016, followed by NR-516 and NR-514. These outcomes depicted that TaARGOS-A is a drought-responsive gene and engaged in dehydration resilience in wheat by aggregating in greater extents in the drought-tolerant genotypes under water limiting conditions. Similar findings were documented in Arabidopsis, bread wheat and maize, respectively2,16,19.
TaARGOS-A expression levels in drought-tolerant and sensitive wheat genotypes. (Asterisk indicates P < 0.05).
Expression analysis of TaARGOS-B gene under osmotic stress
The expression pattern of TaARGOS-B homeolog was likewise concentrated in drought-tolerant and sensitive wheat genotypes. Outcomes publicized that the expression level of TaARGOS-B was relatively lower in wheat genotypes under moisture deficient conditions contrasted with TaARGOS-A and TaARGOS-D. The expression of the TaARGOS-B gene was identified distinctly in drought-tolerant wheat genotypes; however, among drought-sensitive genotypes, no indication of gene expression was identified (Fig. 3). The explanation could be because of the mutation in the promoter region of this gene in such drought-sensitive genotypes, as detailed beforehand in wheat16. Since it was expressed distinctly only in three drought-tolerant wheat genotypes, therefore, this gene could have a significant feature in terms of its improved drought tolerance potential in wheat. Further research to discover the SNP variation for the advancement of TaARGOS-B allied markers would enormously help contrive quick and fast molecular markers for expedient crop breeding40,41.
TaARGOS-B expression levels in drought-tolerant and sensitive wheat genotypes. (Asterisk indicates P < 0.05).
Results illustrated that the expression pattern of the TaARGOS-B gene was actuated at 2 h following moisture stress and crested at the most elevated at 4 h of osmotic stress. Subsequently, mRNA accretion dynamically diminished, as it commenced to decrease at 6 h and reached at least expression fold at 12 h. The wheat genotypes Pakistan-2013 and NR-499 displayed the most remarkable expression of the TaARGOS-B by around two-folds compared to NARC-2009 (Fig. 3). Moreover, TaARGOS-B unveiled no or zero-fold expression at all studied timepoints among all three drought sensitive wheat genotypes (Fig. 3).
Expression analysis of TaARGOS-D gene under osmotic stress
Finally, the expression pattern of the TaARGOS-D gene in drought-tolerant and sensitive genotypes was investigated in increasing osmotic stress. The TaARGOS-D gene expression was determined in drought-tolerant genotypes to be considerably higher than in the sensitive group of genotypes, indicating the genetic potential of a tolerant group of genotypes (Fig. 4). The expression was higher at 2 and 4 h, but it began to decline afterward, indicating that sufficient protein was accumulated to cope with certain stress levels16. In addition to the drought-tolerant genotypes, Pakistan-2013 showed the greatest transcription by 6.5 folds of the TaARGOS-D gene at 4 h, followed by NR 499 and NARC-2009.
TaARGOS-D expression levels in drought-tolerant and sensitive wheat genotypes. (Asterisk indicates P < 0.05).
Conversely, the expression of the TaARGOS-D gene was stimulated 3–4 folds in drought sensitive genotypes, substantially implying that the transcription of TaARGOS-D articulated drought response in wheat (Fig. 4) substantially. In terms of the expression pattern of TaARGOS-D homolog, the drought sensitive genotypes showed non-significant variabilities. The TaARGOS-D gene is responsive to different abiotic stresses, notably drought and salt tolerance, and plays a significant role in many plants growth and developmental processes by activating auxin-based regulatory pathways, as previously described16. As a result, it might be useful in developing drought-tolerant wheat cultivars for arid to semi-arid climatic conditions.
Genes × genotypes cluster-based heatmapping
A heatmap was graphed to showcase the comparative levels and overall performance of all target genes and wheat genotypes examined at different periods. Predicted gene expression profiles as a heatmap has recently appeared as an excellent and popular technique to expediently visualize the expression pattern11,15,42.
Furthermore, clustering in the heatmap further enables the grouping of genes and samples with similar expression patterns much easier15. As a result, cluster analysis-based heatmapping for gene × genotypes association was used to simultaneously display the trend and tendencies of both genes and genotypes1. Corresponding to the color scale illustrated by the color strip, the positive darker scale represents drought-tolerant genotypes at a given osmotic stress timepoint. In contrast, the negative darker color strip represents low-expressing vulnerable genotypes (Fig. 5). Likewise, when the color strength diminishes, the specific strip-line exhibits a subtle and modest expression pattern on both positive and negative sides.
Expression heatmap and cluster analysis for DREB3 and TaARGOS genes among wheat genotypes.
The expression-heatmap, along with cluster analysis, identified two main clades and five sub-clades among the six wheat genotypes examined at certain time intervals (Fig. 5). The first clade was visualized individually, with three drought-tolerant wheat genotypes, i.e., Pakistan-2013, NARC-2009, and NR-499, only at 4 h of timepoint with darker positive color-strip. It suggested that all of the studied genes publicized significantly greater expression for such genotypes at a particular timepoint by contrast with the rest of the genotypes and timepoints.
The second clade includes control samples from drought-tolerant and drought-sensitive genotypes. All four studied genes portrayed a decreased expression pattern in this group. At 2, 6, and 12 h, the other sub-groups of the second clade revealed the existence of drought-tolerant genotypes. All genes in this group have expression profiles that vary from low to medium. The wheat variety Pakistan-2013 most efficiently outperformed in terms of mRNA transcription for TaARGOS-D, TaARGOS-A, and DREB3 at 4 h, followed by NARC-2009 and NR-499 for TaARGOS-A at a similar timepoint.
Furthermore, the cluster analysis revealed that the TaARGOS-A and TaARGOS-D genes cluster together and provide almost identical results. Because of the distinct expression pattern, the DREB3 gene was placed in a different category than the other three TaARGOS homoeologous genes. The TaARGOS-B gene expression pattern was also found to be divergent from the TaARGOS-A and TaARGOS-D genes for a tolerant group of wheat genotypes. It was found to be the least expressive gene in the tolerant group of genotypes. Contrastingly, no expression was detected in drought sensitive genotypes. Due to this, the TaARGOS-B gene in the cluster-based heatmap was separated from the other two homologs of TaARGOS.
These findings, when considered collectively, give important information on valuable selection indicators for wheat dehydration resistance. Drought tolerance in wheat is associated with several critical agronomic and physiological characteristics for efficient selection, as reported by many former researchers2,10. Additionally, according to the findings, drought tolerance in wheat genotypes under study is also influenced by the DREB3 and TaARGOS genes at the molecular level. Finally, based on the seedling stage, it can be concluded that Pakistan-2013, NR-499, and NARC-2009 have proved to be the most drought-tolerant wheat genotypes. These identified bread wheat genotypes may further be employed as drought-tolerant parental lines. Additionally, molecular characterization of ARGOS homoeologous genes in other elite and commercial cultivars of bread wheat and other crops, that can be most proficient approach for various breeding programs to mitigate intensifying climatic variability for arid and semi-arid regions of the world.
