Functional genomic approaches to improve crop plant heat stress tolerance

Heat stress as a yield limiting issue has become a major threat for food security as global warming progresses. Being sessile, plants cannot avoid heat stress. They respond to heat stress by activating complex molecular networks, such as signal transduction, metabolite production and expressions of heat stress-associated genes. Some plants have developed an intricate signalling network to respond and adapt it. Heat stress tolerance is a polygenic trait, which is regulated by various genes, transcriptional factors, proteins and hormones. Therefore, to improve heat stress tolerance, a sound knowledge of various mechanisms involved in the response to heat stress is required. The classical breeding methods employed to enhance heat stress tolerance has had limited success. In this era of genomics, next generation sequencing techniques, availability of genome sequences and advanced biotechnological tools open several windows of opportunities to improve heat stress tolerance in crop plants. This review discusses the potential of various functional genomic approaches, such as genome wide association studies, microarray, and suppression subtractive hybridization, in the process of discovering novel genes related to heat stress, and their functional validation using both reverse and forward genetic approaches. This review also discusses how these functionally validated genes can be used to improve heat stress tolerance through plant breeding, transgenics and genome editing approaches.


Introduction
Abiotic stresses have numerous adverse effects on crop plants, which further lead to yield and quality losses ( Figure 1). To feed the whole world in the scenario of the changing climate, new and better heat tolerant varieties of various crops is needed 1 . The understanding of various physiological, molecular and biochemical pathways can facilitate the development of superior heat tolerant varieties 2 . However, previous efforts, aimed at improving plant heat stress tolerance, have had limited success 3,4 because of the poor understanding of the genetics of heat tolerance. Fortunately, nowadays reference genomes of major food crops and model plant species are available publicly, which provide a solid platform for crop improvement. Moreover, wild species and various landraces of various crops have unknown heat tolerant genes that should be identified and incorporated to high yielding modern cultivars 5 . The functional genomic approaches such as genome wide association studies (GWAS) and gene expression profiling using microarrays can catalyse the discovery of novel genes associated to heat stress 6-8 . In addition, suppression subtractive hybridization (SSH) is another effective and productive technique used for the screening and cloning of the genes/ESTs that express differentially under heat stress 9,10 . Reverse genetic techniques can improve the understanding of their expression patterns under heat stress. The plant breeding strategies and new biotechnological tools including genome editing techniques can use these validated genes to enhance heat stress tolerance in crop plants ( Figure 2).

Figure 2.
A systematic flow chart depicting the approaches used for the mining of genes associated with heat stress, for the functional validation of candidate genes and approaches that can take advantage of functionally validated genes to increase heat stress tolerance.

Mining of stress linked genes
Present crop varieties have limited heat tolerance because earlier domestication, green revolution and conventional breeding were focused to increase yield and qualitative traits 11 . However, the knowledge of genes/markers/QTL regions associated to heat tolerance is now required to improve thermo tolerance. Previous studies suggested that vast genetic diversity still exists in the germplasms of various crops 12-14 . GWAS emerged as a powerful tool to identify the genetic basis behind complex phenotypic traits 15,16 , and it provides high mapping resolution compared with conventional genetic mapping 17,18 . So far this approach has been applied to major food crops, including wheat 7,19,20 , rice 21 , maize 22 , sorghum 23 and Brassica napus L. 24 , to identify the natural variation associated with heat stress and to understand this genetic basis. Another way to identify and understand the key molecular mechanisms in response to heat stress is a transcriptomic study 25,26 ; plants respond to heat stress by inducing various heat responsive genes, thus transcriptomic studies provide an effective screening of heat responsive candidate genes 6 . For example, microarray studies allow the screening of genes on the basis of their expression patterns under stressed conditions at a particular plant developmental stage 6,26,27 . Singh et al. (2015) 6 investigated the heat responsive genes for potato tuberization and Ginzberg et al. 28 identified the candidate heat responsive genes for potato periderm formation using microarrays. In addition, SSH is an easy and efficient approach for the identification of genes/ESTs with differential expression under heat stress. This technique is preferred when the genome sequence information is not available 9 . It can identify the tissue specific differentially expressed transcripts. To identify heat responsive ESTs cDNA libraries can be generated from plants grown under heat stressed conditions 29 . For example, SSH library of potato skin present 108 candidate genes for suberin and periderm formation 30 . To investigate the genes/ESTs involved in heat tolerance at the stage of grain filling in wheat, SSH library was constructed by using the leaf RNA samples from heat stressed plants 9,29 . The results of these studies provided many heat responsive genes/ESTs, which can be used to develop thermo tolerant wheat varieties.

Validating the stress responsive genes
The above approaches can identify potential candidate genes linked to heat stress tolerance. However, the functions of the candidate genes must be validated before incorporating them into present cultivars. Both forward and reverse genetic approaches can be employed for functional validation of genes (see examples in Table 1). Forward genetics detect variations in the nucleic acid sequence responsible for a given phenotype 31 , while reverse genetics detect the gene's functionality by observing the change in the phenotype due to alterations in known genetic sequence 32 . In addition, the genome-editing techniques such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) and clustered regularly interspace short palindromic repeat (CRISPR) can also be used as reverse genetic approaches to study the target gene function. TALENs using sequence specific nucleases (SSNs) became a powerful genome editing technique, which can also be applied as a reverse genetic approach to understand the function of a target gene.

Conclusion
Heat stress affects crop production significantly. Plants respond to heat stress by activating complex molecular networks, such as signal transduction, metabolite production and expressions of heat stress-associated genes. With the developments in plant functional genomics techniques, many novel genes related to heat stress tolerance have been identified and are being used to improve stress tolerance with the help of advanced biotechnological approaches. Next generation sequencing and genome-editing techniques will play crucial roles in crop improvement. In the near future, the scientists will have a better understanding of plant heat tolerant mechanisms and farmers will be able to grow better high yielding heat tolerant crop varieties in the fields.

Data availability
No data is associated with this article. During review process, I appreciated this study. Authors discuss new approach that is well written. However, I found following points to be check for further improvement in manuscript: Abstract: Please correct "in this area" instead of "in this era".
Introduction: Please replace "tolerant varieties" instead of "new and better heat tolerant varieties".
The conclusion need to be ameliorate because it is very brief. The review discussed very well and comprehensively the context of current literature. Validating the stress responsive genes I found this section is very interesting.
Approaches to enhance heat tolerance Do not write the gene name; AmDREB2C in Italics as rest of the gene names in text are not written in Italics.

Conclusion
Summarized the article in a coherent way.
Overall, the article is logically structured and well-organised, and provides a useful compilation of subject matter related to addressed topic. It is an important contribution and I highly recommend it.

Is the review written in accessible language? Yes
Are the conclusions drawn appropriate in the context of the current research literature? Yes No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Plant Genetics and Genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.