*2.2.1. nat-siRNAs (natural antisense siRNAs)*

Natural antisense transcripts (NATs) are small RNA molecules, which are endogenous and show partial or entire complementarity to other transcripts (Table 1). cis-NATs are categorized in the nat-siRNAs group and are transcribed from the same genomic loci but in the opposite strand of DNA as their sense transcripts. This class of NATs is very common in eukaryotes (l7–30% of the genes encode complementary cis-NATs in animals and plants) [33-37]. In animals, NATs are involved in alternative splicing, DNA methylation, RNA editing, and genomic imprinting [38-41]. In plants, several cis-NATs are involved in gene regulatory mechanisms [42-43]. There are already some reports about the identified cis-NATs in *Arabi‐ dopsis* and rice on the genome-wide scale [44-46].

### *2.2.2. ta-siRNA (trans-acting short interfering RNAs)*

ta-siRNAs are 21 nt in length and are reported to be found only in plants so far (Table 1). tasiRNAs originate from a noncoding RNA precursor, which is initially targeted to be cleaved by an miRNA molecule. RNA-dependent RNA polymerase converts the cleaved products into double-stranded RNA molecules, which are later cleaved again into 21-nt ta-siRNAs. Hence, the formation of these RNAs is determined by the presence of both miRNA (Dicer-Like1, Argonaute1, HYPONASTIC LEAVES1, and HUA ENHANCER 1) and siRNA (RNA-depend‐ ent RNA polymerase 6 and DCL4) biosynthesis pathways components. ta-siRNAs can guide cleavage of target mRNAs and regulate gene expression at the posttranscriptional level like plant miRNAs.

#### **2.3. Small RNAs in abiotic stress**

Abiotic stress is known to be one of the attention-calling factors globally, which causes a considerable yield loss each year. Hence, much effort has been made in understanding the complex stress-response mechanisms, especially in the identification of stress-responsive protein-coding genes. But, in recent years, after the discovery of small noncoding RNAs, they have been found to be involved in plant stress responses and indeed very functional players in these pathways. These small RNAs regulate the gene expression in different levels and hence are entangled within all the vital pathways in the plant development, metabolism, and stress response.

As sessile organisms, plants have evolved their specific adaptation and acclimation mecha‐ nisms in order to survive during the hard spell. To do the morphological and physiological adaptations to abiotic stresses, the plant needs to manage the complicated rearrangement of gene expression networks, which are controlled at transcriptional and post-transcriptional levels. The concern about future food shortages makes it imperative to better understand the genetic control of stress tolerance networks and pathways and to use this knowledge to increase the total tolerance of important crop species. As the first step, we have to understand the complex responses of the plants to stress, from changes in molecular level to physiological level.

modification at their homologous regions in the genome and inhibit the gene expression at the

Natural antisense transcripts (NATs) are small RNA molecules, which are endogenous and show partial or entire complementarity to other transcripts (Table 1). cis-NATs are categorized in the nat-siRNAs group and are transcribed from the same genomic loci but in the opposite strand of DNA as their sense transcripts. This class of NATs is very common in eukaryotes (l7–30% of the genes encode complementary cis-NATs in animals and plants) [33-37]. In animals, NATs are involved in alternative splicing, DNA methylation, RNA editing, and genomic imprinting [38-41]. In plants, several cis-NATs are involved in gene regulatory mechanisms [42-43]. There are already some reports about the identified cis-NATs in *Arabi‐*

ta-siRNAs are 21 nt in length and are reported to be found only in plants so far (Table 1). tasiRNAs originate from a noncoding RNA precursor, which is initially targeted to be cleaved by an miRNA molecule. RNA-dependent RNA polymerase converts the cleaved products into double-stranded RNA molecules, which are later cleaved again into 21-nt ta-siRNAs. Hence, the formation of these RNAs is determined by the presence of both miRNA (Dicer-Like1, Argonaute1, HYPONASTIC LEAVES1, and HUA ENHANCER 1) and siRNA (RNA-depend‐ ent RNA polymerase 6 and DCL4) biosynthesis pathways components. ta-siRNAs can guide cleavage of target mRNAs and regulate gene expression at the posttranscriptional level like

Abiotic stress is known to be one of the attention-calling factors globally, which causes a considerable yield loss each year. Hence, much effort has been made in understanding the complex stress-response mechanisms, especially in the identification of stress-responsive protein-coding genes. But, in recent years, after the discovery of small noncoding RNAs, they have been found to be involved in plant stress responses and indeed very functional players in these pathways. These small RNAs regulate the gene expression in different levels and hence are entangled within all the vital pathways in the plant development, metabolism, and stress

As sessile organisms, plants have evolved their specific adaptation and acclimation mecha‐ nisms in order to survive during the hard spell. To do the morphological and physiological adaptations to abiotic stresses, the plant needs to manage the complicated rearrangement of gene expression networks, which are controlled at transcriptional and post-transcriptional levels. The concern about future food shortages makes it imperative to better understand the genetic control of stress tolerance networks and pathways and to use this knowledge to increase the total tolerance of important crop species. As the first step, we have to understand

transcriptional level.

plant miRNAs.

response.

**2.3. Small RNAs in abiotic stress**

*2.2.1. nat-siRNAs (natural antisense siRNAs)*

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

*dopsis* and rice on the genome-wide scale [44-46].

*2.2.2. ta-siRNA (trans-acting short interfering RNAs)*

With the help of high-throughput gene expression analysis, there have been many reports about the modulated genes and different small RNAs under abiotic stresses. The difference in the expression level of these genes could indicate that it might be responsive to stress condition and, as a result, it can help the plant to survive the hard condition. Many studies have been published in this regard in various plant species, some of which having an economic impor‐ tance, like rice, wheat, legumes, barley, sugarcane, potato, and tomato as well as many other species. Some of the genes are induced after facing the stress conditions in these studies while some are downregulated, which is connected with the roles these genes play during the stress condition. Also, the respective miRNAs or other sRNAs that target these mRNAs show a different expression pattern during the stress condition. sRNAs, which are accumulated by stress, might downregulate their target genes and act as a negative regulator of stress tolerance; for instance, the genes involved in cell expansion and division should be downregulated as the plant needs to save energy in order to pass through the hard environmental condition. On the other hand, reduction of sRNA level might lead to upregulation of their target genes, mRNAs, which positively regulates the stress tolerance.

The molecular basis of plant tolerance to abiotic stresses and stress regulation of small RNAs has been studied using different methods to observe the altered expression of these molecules and their related target genes; for instance, the sequence analysis of small RNA libraries before and after the stress condition, microarray data analysis, mutagenesis, and RNAi. Their reports have identified numerous genes and sRNAs that are induced by applying different stress conditions, which is the material for the next step: making transgenic plants and check if these overexpressed transgenics could exhibit an improvement in stress tolerance. But the fact is that even though some of the genes and small RNAs show altered expression under stress, they do not play any role to make the plant more tolerant to the stress. And the reason is largely because of the complex genetic interactions underlying the plant tolerance toward stress, which are still to be understood.

From transcriptomic studies, we know that the stress conditions such as heat, drought, cold, and salt evoke the expression of an overlapping set of genes, suggesting that their signaling transduction pathways share common control points. Most of the genes, which are detected to be responsive to abiotic stresses, are usually the genes that regulate plant development and reproduction (as the plant faces the urge to save more energy for producing viable seeds rather than a high biomass), also senescence-related genes (as to recycle the nutrients from the old leaves to younger leaves and reproductive parts and wasting less water and energy for them), as well as the genes that are involved in the abscisic acid (ABA) pathway, which play a crucial role in plant growth and development pathway and redox pathway.

RNA interference technology is one of the potential reverse genetics tools for understanding the functional significance of these genes and their respective regulatory sRNAs. The infor‐ mation about stress-induced genes and sRNAs including the sequence and annotation could be found in the genome databases like National Centre for Biotechnology Information (NCBI) or stress complementary DNA (cDNA) databases. In addition to these stress-induced genes, the regulatory elements for these genes are also altered during stress condition including small regulatory RNAs. A number of these sRNAs, which are induced in different plant species and under different stress conditions, can be found in some recent review papers [47]. A vast amount of data has been published about the expression profiling of different sRNAs in various stress conditions. Although these expression-profiling experiments can provide us with some clues about the involvement of these sRNAs in gene regulation under those specific circumstances, to find the relevance of each of these sRNAs in imparting stress tolerance in plants can only be studied by functional genomic approaches like gene overexpression or downregulation. RNA interference technology using constructs transcribing self-complemen‐ tary hairpin RNA is one of the reverse genetics approaches to downregulate genes in plants.

Another powerful technique to learn about gene functions in a developmental or physiological context in plants is by mutagenesis and to isolate the corresponding mutants with altered phenotypes. Various mutagenic agents, including chemical and biological, have been widely used in this regard, each of them with its own advantages and inconveniences.

For *Arabidopsis thaliana*, the genome sequence is publicly available; hence, relying on reverse genetics to understand the relevant roles of genes is currently a common practice. There are specific screening methods used in order to measure the effect of each stress on the overall plant physiology after the treatment as well as the methods used for inducing the specific stress condition in the plant, which have been studied and reviewed many times.

### **2.4. Drought stress**

Drought stress is known as the most significant stress especially with regard to the climate change and global warming. It restricts plant growth and development severely, while tolerant plants are able to survive by several mechanisms such as consuming small amounts of water or keeping their stomata closed at a high rate under drought conditions.

Approximately two-thirds of the potential yield of major crops are lost every year due to the adverse growing environments [48]. A worldwide increase in arid areas, including the Mediterranean basin, has been predicted by the International Plant Protection Convention (IPPC) in 2001 and 2007. Water deficit also leads to salinity stress in many cases, which makes the growth situation even harder for the plants. Therefore, it is regarded as the most important abiotic stress and it is necessary to develop strategies toward sustainable use of water and improve plant-drought resistance [49]. Many genes have already been studied and reported to be involved in the drought-resistance response network in the plants. But in recent years, it has become clear that sRNAs play pivotal roles in stress responses as well in regulating the expression of resistance genes.

MiRNA-expression profiling under drought stress has now been performed in *Arabidopsis*, rice, and *Populus trichocarpa*, and many other plants under drought-stress conditions and some of the miRNAs were shown to be responsive toward this stress in different plants, some of which can be reviewed in the available literature [50-51].

Another group has worked on miRNA expression patterns of drought-resistant *Triticum turgidum* ssp*. Dicoccoides* in response to drought stress, using an miRNA microarray platform [52]. MiR474, which targets proline dehydrogenase (PDH), was upregulated during drought stress in Zea maize [53]. Zhao et al. worked on miR169g and miR393 under drought condition in rice, while miR393 was conversely induced by drought [54]. Many other recent reports used several different methods to study the expression pattern changes of miRNAs in different plants under the stress condition [47].

### **2.5. Salt stress**

the regulatory elements for these genes are also altered during stress condition including small regulatory RNAs. A number of these sRNAs, which are induced in different plant species and under different stress conditions, can be found in some recent review papers [47]. A vast amount of data has been published about the expression profiling of different sRNAs in various stress conditions. Although these expression-profiling experiments can provide us with some clues about the involvement of these sRNAs in gene regulation under those specific circumstances, to find the relevance of each of these sRNAs in imparting stress tolerance in plants can only be studied by functional genomic approaches like gene overexpression or downregulation. RNA interference technology using constructs transcribing self-complemen‐ tary hairpin RNA is one of the reverse genetics approaches to downregulate genes in plants. Another powerful technique to learn about gene functions in a developmental or physiological context in plants is by mutagenesis and to isolate the corresponding mutants with altered phenotypes. Various mutagenic agents, including chemical and biological, have been widely

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

used in this regard, each of them with its own advantages and inconveniences.

condition in the plant, which have been studied and reviewed many times.

or keeping their stomata closed at a high rate under drought conditions.

**2.4. Drought stress**

expression of resistance genes.

which can be reviewed in the available literature [50-51].

For *Arabidopsis thaliana*, the genome sequence is publicly available; hence, relying on reverse genetics to understand the relevant roles of genes is currently a common practice. There are specific screening methods used in order to measure the effect of each stress on the overall plant physiology after the treatment as well as the methods used for inducing the specific stress

Drought stress is known as the most significant stress especially with regard to the climate change and global warming. It restricts plant growth and development severely, while tolerant plants are able to survive by several mechanisms such as consuming small amounts of water

Approximately two-thirds of the potential yield of major crops are lost every year due to the adverse growing environments [48]. A worldwide increase in arid areas, including the Mediterranean basin, has been predicted by the International Plant Protection Convention (IPPC) in 2001 and 2007. Water deficit also leads to salinity stress in many cases, which makes the growth situation even harder for the plants. Therefore, it is regarded as the most important abiotic stress and it is necessary to develop strategies toward sustainable use of water and improve plant-drought resistance [49]. Many genes have already been studied and reported to be involved in the drought-resistance response network in the plants. But in recent years, it has become clear that sRNAs play pivotal roles in stress responses as well in regulating the

MiRNA-expression profiling under drought stress has now been performed in *Arabidopsis*, rice, and *Populus trichocarpa*, and many other plants under drought-stress conditions and some of the miRNAs were shown to be responsive toward this stress in different plants, some of

Another group has worked on miRNA expression patterns of drought-resistant *Triticum turgidum* ssp*. Dicoccoides* in response to drought stress, using an miRNA microarray platform Crops worldwide are threatened by excessive soil salinity due to the accumulation of salt delivered along with irrigation water and by coastal flooding and the high evapotranspiration rates caused by climate change. About 6% of the total arable land in the world is affected by excess salt [55] and it has been predicted to increase to about 30% of the world's arable land by 2025 and 50% by the year 2050 [56]. Several genes and pathways in plants are affected by salt stress [57]. Hence, the promising approach to address the problem of soil salinity is to increase the understanding of response of plants to salinity-related stress. These genes are mostly involved in signal transduction, activation of ion channels, and growth-factorregulated modification of plant architecture, and, in particular, root morphology.

Besides the genes, numerous differentially regulated miRNAs have also been identified in saltstressed plants. For instance, miR156, miR158, miR159, miR165, miR167, miR168, miR169, miR171, miR319, miR393, miR394, miR396, and miR397 were all reported to be overex‐ pressed in response to salt stress in *Arabidopsis*, while the accumulation of miR398 was downregulated [50].

miR169 was also reported to be induced by high salinity stress [46]. The authors found a cisacting ABA-responsive element (ABRE) in the upstream region of miR169n, which suggested that miR169n might be regulated by ABA. Another group used microarray experiments as a method to explore the miRNA profile of maize in different lines (salt-tolerant and saltsensitive); finally, it was reported that the expression levels of miR156, miR164, miR167, and miR396 family members were downregulated considerably, while it was increased in miR162, miR168, miR395, and miR474 families after salt-shock in root tissue [58].

#### **2.6. Cold stress**

Some plants increase their tolerance to cold in order to deal with the low temperatures. This phenomenon is known as cold acclimation. In recent years, many cold-regulated genes have been identified in plants under cold stress. The C-repeat binding factor (CBF) cold-responsive pathway was considered as the most known cold tolerance pathway in plants [59]. There are three CBF/DREB1 family members, including CBF1, CBF2, and CBF3 (DREB1b, DREB1c, and DREB1a, respectively), encoding the DNA-binding proteins of Apetala2/ethylene responsive factor (AP2/ERF) family [60]. Also, the expression of many miRNAs in cold stress has been examined in different plants including *Arabidopsis thaliana*. Several miRNAs belonging to different families were reported to be upregulated under cold-stress condition in *Arabidopsis thaliana* (miR165/166, miR393, miR396, and miR408), while some other miRNAs (miR156/157, miR159/319, miR164, miR394, and miR398) were shown to be either transient or mildly regulated under cold-stress treatment condition [19][50]. In another report, the expression 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].

#### **2.7. Heat stress**

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 essential regulatory loop for plant thermotolerance [62].

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 *HTT2* promoter regions and activate them directly. Finally, they proposed that HTT1 interacts with Hsp70-14 and Hsp40, nuclear factor Y, and subunit C2 complex.

Wheat miRNAs showed differential expression in response to heat stress; by using Solexa highthroughput sequencing, Xin et al. cloned the small RNAs from wheat leaves treated by heatstress gene [66]. Stief et al. also reported that miR156 is responsible for heat stress memory in *Arabidopsis* [67].

**Figure 1.** Biogenesis of miRNAs and ta-siRNAs
