**3. Perspective**

levels of miR168 and miR477 family members were increased after the cold-stress treatment, while miR156, miR475, and miR476 members were downregulated in *Populus* plants [55][19].

The average temperature of our planet is rising year by year because of the climate change. As a result, changes in the patterns of rainfall, droughts, and submergence stress are induced to the natural environments. Heat stress even alters the distribution and productivity of impor‐ tant crops negatively throughout the earth. A temperature rise of −5°C above the plant's optimum temperature is considered as a heat stress. It disrupts normal functions of cellular processes, may lead to delay in plant growth and development, and it might even result in death of the plant, but, usually, high temperatures result in water deficiency, which eventually leads to increase in salt concentration. Recent studies indicate that the projected global warming in the upcoming years will negatively affect the yield of important crops; hence, the necessity of focusing on gene networks and their regulatory components becomes obvious.

A major component with regard to responding to heat stress is the induction of heat shock proteins (HSPs), which get activated by heat shock transcription factors (HSFs). There are five classes of HSPs based on their molecular weights: HSP100, HSP90, HSP70, HSP60, and small heat shock proteins (sHSPs, 15–30 kDa). On the other hand, HSFs recognize heat stress elements on the promoter of heat stress-responsive genes (HSE: 5′-GAAnnTTC-3′). Plant HSFs are categorized into three classes based on their oligomerization domains (A, B, and C) [61].

However, the more upstream regulators of HSFs remain to be identified. Guan et al. have reported that miR398 is rapidly induced by being subjected to heat stress while its target genes (CSD1, CDS2, and CCS) are downregulated. They further reported that the expression levels of HSF and HSP genes in *csd1-*, *csd2-*, and *ccs-*mutant plants are increased under heat stress, and *csd1*, *csd2*, and *ccs* plants are more tolerant to heat stress than wild-type plants. They identified two HSFs, which act upstream of miR398, suggesting that this pathway is an

Based on deep sequencing experiments, Wang et al. suggest that there is a new class of small RNAs that originate from the chloroplast genome, which are responsive to heat stress [63]. They performed RNA sequencing (RNA-seq) and found 1031 cis-NATs in *Brassica rapa* based on the homology with *Arabidopsis* and 303 conserved cis-NATs, which correspond to the ones in *Arabidopsis* [64]. TAS1 (trans-acting siRNA precursor 1) targets, derived from small inter‐ fering RNAs named heat-induced TAS1 target1 (*HTT1*) and *HTT2*, are involved in thermotol‐ erance [65]. *HTT1* and *HTT2* genes were highly upregulated in *Arabidopsis thaliana* seedlings in response to heat shock based on their microarray analysis. TAS1a has a trans-acting small interfering RNA, which targets the *HTT* genes. Overexpression of TAS1a accelerated the expression of TAS1-siRNAs and decreased the expression levels of *HTT* genes that eventually led to weaker thermotolerance. Conversely, stronger expression of *HTT1* and *HTT2* genes upregulated various *Hsf* genes, helping the plants to achieve a stronger thermotolerance. In HsfA1a-overexpression transgenic plants, which present a higher tolerance to heat stress, the *HTT* genes were upregulated. In the meantime, HsfA1a was shown to bind to the *HTT1* and

essential regulatory loop for plant thermotolerance [62].

72 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

**2.7. Heat stress**

Physiological responses to stress are controlled by expression of a large number of genes, many of which are regulated by microRNAs. At the molecular level, identification of stress-respon‐ sive genes is an initial step toward understanding plant stress response as pyramiding of different genes in the same plant is an option for achieving better stress tolerance. Although finding genes and sRNAs, which show induction by stress, is an important step toward stress tolerance improvement, most of the studies in which they use transgenics only show the importance of the introduced transgene and not the overall metabolic effects that the transhost gets exposed to. On the other hand, the new stress-tolerant transgenic lines should have no or few undesired phenotypic changes plus a minimal yield penalty. In stress-tolerant transgenics, which are introduced so far, a constitutive promoter has been used for expressing the transgene in most of the cases. These transgenes must be utilized to overcome the problem of yield penalty and growth retardation in these experiments. Admittedly, most reports published on stress-tolerant transgenic plants are based on the limited characterization of the stress condition as well as the tolerant phenotypes. Adequate assays for phenotyping of the stress-tolerance trait must be undertaken under natural stress conditions. Overall, there is a lack of uniformity in the stress induction regimes applied by various research groups, which makes the comparisons of the responses among different reports difficult and this fact must be taken into consideration.
