**2. Canonical small RNAs**

In plants, small RNAs exhibit unexpected complexity, but two main categories can be distin‐ guished based on their biogenesis and function and these are miRNAs (microRNAs) and siRNAs (short interfering RNAs). Baking yeast lack these two categories of small RNAs since their biogenesis and functions involve RNAi machinery, which is absent in *S. cerevisiae*.

#### **2.1. miRNAs**

balanced internal conditions for optimal growth and function. Industrial applications expose yeast to multiple unfavorable environmental conditions, therefore accurate stress response is key for yeast cells. When environmental conditions change abruptly, yeast cells rapidly adjust

The physiological and biochemical changes in plants under stress conditions are a result of alteration in abundance of many transcripts and therefore proteins [3] pinpointing the role of TGS (transcriptional gene expression) and PTGS (post-transcriptional gene expression) in the adaptation to environmental changes. To achieve precise and timely regulation of stressresponsive gene networks in addition to regulatory DNA sequences and different types of proteins, plants, as well as many other organisms, including yeast, have employed the gene

Differentially expressed RNAs are involved in regulation of plant and yeast metabolism pathways during develop‐ ment as well as stress conditions. Baking yeast are depleted of RNAi dependent molecules (miRNA and siRNA) that

**Figure 1.** Overview of different classes of ncRNAs associated with response to abiotic stress conditions in plants and

For many decades RNA was considered to be a passive intermediate in the flow of information from DNA to protein, apart from other well-known and described functions in translation (tRNAs, rRNAs) or splicing (UsnRNA). In 1998, its new role has been discovered and regula‐ tory capacity of RNA molecules immensely broadened our understanding of gene expression processes. Moreover, new sequencing technologies and high-resolution microarray analysis

its internal balance to that required for growth in the new conditions.

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

expression regulation by RNA molecules.

are present in plants.

yeast.

miRNAs are predominantly 21 nucleotides in length [6] and they are derived from 70–500 nucleotide long, imperfect, hairpin-like structured primary transcripts called pri-miRNAs by the activity of RNase III dicer-like (DCL) enzyme [7]. DCL1 processes primary miRNA transcript into an miRNA-miRNA\* duplex with 2nt overhangs at the 3' end [6]. With the help from hyponastic leaves (HYL1) and SERRATE proteins miRNA duplex is released and then Hua Enhancer 1 (HEN1) methylates 3' ends of the duplex and therefore stabilizes it by preventing uridylation and subsequent degradation. HASTY, the plant homolog of exportin 5, transports the miRNA duplex into the cytoplasm [8, 9]. The mature miRNA is loaded into RISC (RNA-induced silencing complex) and guides the translational inhibition or cleavage of target mRNAs through the action of AGO proteins in a sequence-specific manner [8–10].

To date, many stress-responsive miRNAs have been identified and the vast majority of their targets are transcriptional factors or proteins playing important roles not only in develop‐ mental and growth processes but also in biotic and abiotic stress responses [11]. There appears to exist common pathways of stress responses based on the induction/reduction of similar small noncoding RNA entities. It has been shown that different stress conditions trigger similar responses in various plant species; but on the other hand, the plant's response to similar stress condition could trigger the expression of different pools of miRNAs even in different varieties of the same plant species.

Drought and salinity are major external factors influencing crop productivity and seed quality worldwide. Understanding plant's responses to these stresses is therefore of high importance and one of the main research topics nowadays. Several studies showed differential expression profiles of many genes during water deficit conditions [12, 13]. Recent efforts have also led to the identification of many miRNAs altered in response to drought in many plant species such as cowpea [14], soybean [15], or *Triticum dicoccoides* [16]. Strikingly, the plant response varies in different species, depending on the habitat or plant family, resulting in majority of miRNAs having differential accumulation patterns; nevertheless, some miRNAs share common accumulation pattern as shown below. The differences observed are not only attributable to plant species or growth conditions but could also result from different genotypes in the same species. Soybean seedlings representing two different genotypes (drought-sensitive and tolerant) showed an increase in the level of five miRNAs (miR166-5p, miR169f-3p, miR1513c, miR397ab, and miR-seq13) [15]. In wild emmer wheat, 13 miRNAs were found to be differen‐ tially regulated upon drought (miR1867, miR896, miR398, miR528, miR474, miR396, miR894, miR156, miR1432, miR166, and miR171) [16]. Interestingly, some miRNAs share common accumulation pattern; for example, miR474 has also been shown to be upregulated in maize under water deficit [17]. Genome-wide approach allowed to identify 30 miRNA families significantly down- (16: miR156, miR159, miR168, miR170, miR171, miR172, miR319, miR396, miR397, miR408, miR529, miR896, miR1030, miR1035, miR1050, miR1088, and miR1126) or upregulated (14: miR159, miR169, miR171, miR319, miR395, miR474, miR845, miR851, miR854, miR896, miR901, miR903, miR1026, and miR1125) under the drought stress in *Oryza sativa*; strikingly 9 miRNAs (miR156, miR168, miR170, miR171, miR172, miR319, miR396, miR397, and miR408) showed opposite expression patterns to previously described in drought-stressed Arabidopsis [18]. Some miRNA gene families were identified in both down- and upregulated groups (miR171, miR319, miR896). One miRNA that is consistently being upregulated during drought is miR393. The increase in the level of this microRNA has been observed for rice, Arabidopsis, *Medicago truncatula*, and *Phaseolus vulgaris* [19–23].

It is established that somewhere between 6% to 20% of worldwide cultivated arable land is affected by excessive concentrations of salt [24–26]. Some cellular and metabolic processes observed in plants during salt stress conditions (as a result of the limitation of the plant's ability to uptake water) are similar to those observed during drought [24]. Nevertheless, there are many genes and pathways that discriminate these two stress conditions [12, 28]. In salt-tolerant and salt-sensitive *Zea mays* lines, microarray hybridization experiments led to the identification of 98 salt-responsive miRNAs representing 27 families. Several microRNAs showed differen‐ tial accumulation levels upon salinity in roots: members of miR156, miR164, miR167, and miR396 families were downregulated while miR162, miR168, miR395 and miR474 were upregulated [28]. In rice, other microRNAs appear to be involved in response to salt stress, these are miR169g, miR169n, miR169o, as well as miR393 [29, 30]. These microRNAs target the transcript of NF-YA gene encoding for a subunit A of a conserved transcription factor previously shown to be downregulated in drought-affected wheat [31]. Three microRNAs (miR414, miR164e, and miR408) have been established to be downregulated under salinity in rice [32]. Although, their target genes OsABP (ATP-binding protein), OsDBH (DEAD-box helicase), and OsDSHCT (DOB1/SK12/helY-like DEAD-box helicase), were for the first time validated and upregulated in response to salt stress their functions still need to be experimen‐ tally confirmed.

Drought and salinity are major external factors influencing crop productivity and seed quality worldwide. Understanding plant's responses to these stresses is therefore of high importance and one of the main research topics nowadays. Several studies showed differential expression profiles of many genes during water deficit conditions [12, 13]. Recent efforts have also led to the identification of many miRNAs altered in response to drought in many plant species such as cowpea [14], soybean [15], or *Triticum dicoccoides* [16]. Strikingly, the plant response varies in different species, depending on the habitat or plant family, resulting in majority of miRNAs having differential accumulation patterns; nevertheless, some miRNAs share common accumulation pattern as shown below. The differences observed are not only attributable to plant species or growth conditions but could also result from different genotypes in the same species. Soybean seedlings representing two different genotypes (drought-sensitive and tolerant) showed an increase in the level of five miRNAs (miR166-5p, miR169f-3p, miR1513c, miR397ab, and miR-seq13) [15]. In wild emmer wheat, 13 miRNAs were found to be differen‐ tially regulated upon drought (miR1867, miR896, miR398, miR528, miR474, miR396, miR894, miR156, miR1432, miR166, and miR171) [16]. Interestingly, some miRNAs share common accumulation pattern; for example, miR474 has also been shown to be upregulated in maize under water deficit [17]. Genome-wide approach allowed to identify 30 miRNA families significantly down- (16: miR156, miR159, miR168, miR170, miR171, miR172, miR319, miR396, miR397, miR408, miR529, miR896, miR1030, miR1035, miR1050, miR1088, and miR1126) or upregulated (14: miR159, miR169, miR171, miR319, miR395, miR474, miR845, miR851, miR854, miR896, miR901, miR903, miR1026, and miR1125) under the drought stress in *Oryza sativa*; strikingly 9 miRNAs (miR156, miR168, miR170, miR171, miR172, miR319, miR396, miR397, and miR408) showed opposite expression patterns to previously described in drought-stressed Arabidopsis [18]. Some miRNA gene families were identified in both down- and upregulated groups (miR171, miR319, miR896). One miRNA that is consistently being upregulated during drought is miR393. The increase in the level of this microRNA has been observed for rice,

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

Arabidopsis, *Medicago truncatula*, and *Phaseolus vulgaris* [19–23].

It is established that somewhere between 6% to 20% of worldwide cultivated arable land is affected by excessive concentrations of salt [24–26]. Some cellular and metabolic processes observed in plants during salt stress conditions (as a result of the limitation of the plant's ability to uptake water) are similar to those observed during drought [24]. Nevertheless, there are many genes and pathways that discriminate these two stress conditions [12, 28]. In salt-tolerant and salt-sensitive *Zea mays* lines, microarray hybridization experiments led to the identification of 98 salt-responsive miRNAs representing 27 families. Several microRNAs showed differen‐ tial accumulation levels upon salinity in roots: members of miR156, miR164, miR167, and miR396 families were downregulated while miR162, miR168, miR395 and miR474 were upregulated [28]. In rice, other microRNAs appear to be involved in response to salt stress, these are miR169g, miR169n, miR169o, as well as miR393 [29, 30]. These microRNAs target the transcript of NF-YA gene encoding for a subunit A of a conserved transcription factor previously shown to be downregulated in drought-affected wheat [31]. Three microRNAs (miR414, miR164e, and miR408) have been established to be downregulated under salinity in rice [32]. Although, their target genes OsABP (ATP-binding protein), OsDBH (DEAD-box helicase), and OsDSHCT (DOB1/SK12/helY-like DEAD-box helicase), were for the first time

Cold stress is one of the most severe abiotic stresses. The disorders triggered by low temper‐ atures depend on the duration of stress condition and the rate of temperature decrease. Cold stress includes chilling (above 0o C) that inhibits water uptake and freezing (below 0o C) that induces cellular dehydration and thus osmotic stress and hyper accumulation of reactive oxygen species [33]. There are several factors (genes and transcription factors) playing important roles in cold stress acclimatization [10, 34–37]. Post-transcriptional mechanisms of cold acclimation include pre-mRNA processing, mRNA stability, and export from the nucleus. During the last few years, an emerging role of microRNAs in cold stress acclimatization and responses began to unveil. Cold, as well as ABA, dehydration, and salt stress upregulate the expression of miR393, miR397b, and miR402. In accordance with the upregulation of miR393 under cold stress, its target, putative E3 ubiquitin ligase SCF complex F-box protein, appears to be downregulated by cold stress in Arabidopsis [38]. Thus, during cold acclimation, the cleavage of E3 ubiquitin ligase mRNA would lead to diminished proteolysis of its targets (probably positive regulators of cold stress). Moreover, one of the targets of miR393 encodes an F-box protein (At4g03190) [39, 40], which is similar to glucose repression resistance 1 (GRR1), a yeast protein involved in glucose repression. Plants can use sugar status as a signal to modulate growth and development in response to abiotic stresses since it has been observed that sugar metabolism is affected by various abiotic stresses. It has been speculated therefore that miR393 could integrate sugar signaling with cold stress responses [41]. Moreover, in sugarcane, it has been observed that miR319 is differentially expressed during cold treatment for different periods of time, in both shoots and roots [42]. These data are in accordance with previous reports showing that miR319 was upregulated in response to cold stress in Arabi‐ dopsis [39, 40]. Genome-wide experiments in *Prunus persica* led to the identification of miR156, miR159, miR164, miR167, miR172, miR393, miR396, miR414, miR2275, and miR5021 as cold responsive [43, 44]. They are regulating genes involved in flower and leaf developmental processes. In rice, 18 miRNAs have been shown to respond rapidly to cold stress conditions (miR156, miR166, miR166m, miR167a,b,c, miR168b, miR169e, miR169f, miR169h, miR171a, miR535, miR319a,b, miR1884b, miR444a.1, miR1850, miR1868, miR1320, miR1435, and miR1876). Most of them are downregulated, which suggests that their targets are being upregulated in adaptation to cold stress. Interestingly, there were four miRNAs (miR1435, miR1876, miR1320, and miR1884) present in rice, but not Arabidopsis, in response to cold stress, which suggests a species-specific mechanisms [10]. miR171 is a large family consisting of 12 known members. In Arabidopsis, miR171a was induced 6 h after cold treatment [40]. On the other hand, miR171a in rice (rice and Arabidopsis share the same mature sequence) was significantly downregulated. Moreover, the expression levels of the other three miRNAs (e, f, i) were also decreased in rice in response to cold. In contrast, miR171 c, d, and h were induced after 6 h [10]. The difference in expression pattern as in the case of miR171 could be reflective of divergence in response of particular plant species to cold stress.

miRNAs also appear to play crucial roles in regulation of nutrient homeostasis. There are 14 mineral elements essential for plant growth and development. Phosphate (Pi) is one of the most prominent ones; it is involved in phosphorylation reactions, synthesis of nucleic acids, membrane lipids, but also in energy delivery and often is a limiting nutrient for plant growth. The phosphate acquisition from soil particles requires the secretion of plant enzymes that release phosphate, then phosphate transport through Pht1 family of transporters (phosphate transporters protein family 1). Phosphate homeostasis in cells is regulated through the action of PHO1 and PHO2 proteins, transcription factors PHR1, WRK75, ZAT6, bHLH32, mYB62, in Arabidopsis and PTF1 in rice and microRNAs. MiR399, upregulated in low-phosphate stressed plants [45–47] targets two genes; a phosphate transporter PHO2 and a putative ubiquitin conjugating enzyme UBC24 transcript [6, 48] have been isolated from the phloem and their level increases in the phloem upon phosphate starvation, suggesting their role as phosphate starvation signals being translocated from shoots to roots where they promote phosphate uptake by downregulating PHO2 [49, 50]. Recently, miR-827-3p was shown to be upregulated in nitrogen and phosphorus deficiency conditions [51, 52]. In rice, it has been shown to target two genes encoding proteins containing SPX and MFS (major facilitator superfamily) domains. SPX-domain proteins are involved in Pi sensing and/or Pi transport [53]; whereas MFS-domain proteins are membrane proteins functioning in small molecule transport [54]. It has been suggested that SPX-MFS proteins might be involved in intracellular or intercellular Pi transport through regulating the expression of Pi transporter (PT) genes [55] and/or Pi storage or metabolism [56]. Similarly, in Arabidopsis, it targets the nitrogen/phosphate balance regulator Nitrogen Limitation Adaptation (NLA, AT1g02860) transcripts [57]. Overexpression of miR827 under Pi-starvation conditions causes the downregulation of NLA (together with downregulation of PHO2 through the action of miR399) leading to increased levels of phos‐ phate transporter 1 (PHT1) and elevated activity of Pi uptake at the plasma membrane.

Sulfur (S) is another fundamental macronutrient necessary for proper growth and develop‐ ment. For plants, it is available from the soil in sulfate form, it is taken up by the roots, and distributed within the whole plant by sulfate transporters. It is found in amino acids, peptides and proteins, iron-sulfur clusters, and several co-factors, but also in an oxidized form in polysaccharides, lipids, and modified proteins [58, 59]. Sulfate deprivation induces the accumulation of miR395 that targets ATP sulfurylases (APSs) and sulpfate transporter AST68 [6, 60], thus reducing their levels. In Brassica plants, deprived of sulfur, the level of miR395 increases in the root, stem, and leaf tissues, it is however strongest in the phloem [61, 62].

The regulation of copper (Cu) homeostasis is also managed through the action of microRNAs. This micronutrient is indispensable for photosynthesis and plant protection against reactive oxygen species [63, 64]. Under copper-deficient conditions, upregulation of miR398 leads to the downregulation of cytosol and chloroplastic Cu/Zn superoxidase dismutase transcripts level (CSD1 and CSD2) and copper chaperone for superoxide dismutase CCS1 [65, 67]. Cu/Zn SOD can be, therefore, replaced with FeSOD in chloroplasts [68]. The level of miR398 is elevated in Brassica under copper deprivation in leaf, stem, and root tissue but most impor‐ tantly in the phloem [62], such as in the case of sulfur deprivation, which is in accordance with other research suggesting that the phloem has a distinct set of microRNAs from leaves and roots and responds specifically to stress conditions [63]. Several other microRNAs have also been recently linked with copper-deficient conditions (miR397, miR408, and miR857). They are predicted to target Cu-containing proteins (laccases and plantacyanin) [66].

Under high, heavy metal concentrations, plants are subjected to cation imbalances that lead to alterations in plant physiology and biochemistry. It is not surprising, therefore, that in response to these kind of stresses a set of microRNAs is differentially regulated. It has been observed in rice [68], *M. truncatula* [69], *Brassica napus* [70], and Arabidopsis [71]. In *Brassica*, several microRNAs have been linked with cadmium exposure (miR156, miR160, miR164b, miR171, miR393, miR394a,b,c and miR396a,) [70, 72]. In rice, assorted microRNAs have been upregu‐ lated in roots (miR601, miR602, and miR603) while some were downregulated in leaves (miR602 and miR606) or in roots (miR604) [68]. The levels of miR171, miR319, miR393, and miR529 were all heightened in response to mercury (Hg), cadmium (Cd), and aluminium (Al) exposure in Medicago [69].

### **2.2. siRNAs**

membrane lipids, but also in energy delivery and often is a limiting nutrient for plant growth. The phosphate acquisition from soil particles requires the secretion of plant enzymes that release phosphate, then phosphate transport through Pht1 family of transporters (phosphate transporters protein family 1). Phosphate homeostasis in cells is regulated through the action of PHO1 and PHO2 proteins, transcription factors PHR1, WRK75, ZAT6, bHLH32, mYB62, in Arabidopsis and PTF1 in rice and microRNAs. MiR399, upregulated in low-phosphate stressed plants [45–47] targets two genes; a phosphate transporter PHO2 and a putative ubiquitin conjugating enzyme UBC24 transcript [6, 48] have been isolated from the phloem and their level increases in the phloem upon phosphate starvation, suggesting their role as phosphate starvation signals being translocated from shoots to roots where they promote phosphate uptake by downregulating PHO2 [49, 50]. Recently, miR-827-3p was shown to be upregulated in nitrogen and phosphorus deficiency conditions [51, 52]. In rice, it has been shown to target two genes encoding proteins containing SPX and MFS (major facilitator superfamily) domains. SPX-domain proteins are involved in Pi sensing and/or Pi transport [53]; whereas MFS-domain proteins are membrane proteins functioning in small molecule transport [54]. It has been suggested that SPX-MFS proteins might be involved in intracellular or intercellular Pi transport through regulating the expression of Pi transporter (PT) genes [55] and/or Pi storage or metabolism [56]. Similarly, in Arabidopsis, it targets the nitrogen/phosphate balance regulator Nitrogen Limitation Adaptation (NLA, AT1g02860) transcripts [57]. Overexpression of miR827 under Pi-starvation conditions causes the downregulation of NLA (together with downregulation of PHO2 through the action of miR399) leading to increased levels of phos‐ phate transporter 1 (PHT1) and elevated activity of Pi uptake at the plasma membrane.

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

Sulfur (S) is another fundamental macronutrient necessary for proper growth and develop‐ ment. For plants, it is available from the soil in sulfate form, it is taken up by the roots, and distributed within the whole plant by sulfate transporters. It is found in amino acids, peptides and proteins, iron-sulfur clusters, and several co-factors, but also in an oxidized form in polysaccharides, lipids, and modified proteins [58, 59]. Sulfate deprivation induces the accumulation of miR395 that targets ATP sulfurylases (APSs) and sulpfate transporter AST68 [6, 60], thus reducing their levels. In Brassica plants, deprived of sulfur, the level of miR395 increases in the root, stem, and leaf tissues, it is however strongest in the phloem [61, 62].

The regulation of copper (Cu) homeostasis is also managed through the action of microRNAs. This micronutrient is indispensable for photosynthesis and plant protection against reactive oxygen species [63, 64]. Under copper-deficient conditions, upregulation of miR398 leads to the downregulation of cytosol and chloroplastic Cu/Zn superoxidase dismutase transcripts level (CSD1 and CSD2) and copper chaperone for superoxide dismutase CCS1 [65, 67]. Cu/Zn SOD can be, therefore, replaced with FeSOD in chloroplasts [68]. The level of miR398 is elevated in Brassica under copper deprivation in leaf, stem, and root tissue but most impor‐ tantly in the phloem [62], such as in the case of sulfur deprivation, which is in accordance with other research suggesting that the phloem has a distinct set of microRNAs from leaves and roots and responds specifically to stress conditions [63]. Several other microRNAs have also been recently linked with copper-deficient conditions (miR397, miR408, and miR857). They

are predicted to target Cu-containing proteins (laccases and plantacyanin) [66].

siRNAs are derived from double-stranded RNAs that result from transcription of invertedrepeat sequences, natural cis-antisense transcript pairs, or by the action of RNA-dependent RNA polymerases (RDRs) that convert single-stranded RNA transcripts (e.g., virus replica‐ tion) into double-stranded RNAs [8]. They also derive from heterochromatic regions and DNA repeats and mediate the silencing maintenance of the regions from which they originate [73]. These are then cleaved by dicer-like (DCL) enzymes to produce 21-24-nucleotide long species, the size depends on the specific catalytic activity of the respective DCL protein. Similarly to miRNAs, siRNAs are then loaded into Argonaute (AGO) protein-containing RISC that guides target regulation at the transcriptional level via the so called RNA-directed DNA methylation (RdDM) or at post-transcriptional level [8, 26, 74]. Several classes of siRNA can be distinguish‐ ed, such as trans-acting siRNAs (tasiRNA), heterochromatic RNAs (hc-siRNA), and natural antisense siRNAs (nat-siRNA).

Tasi-RNAs are derived from specific genetic loci called TAS genes through a microRNAdependent pathway [75, 76]. Cleaved TAS transcripts are converted into dsRNAs by RNAdependent RNA polymerase 6 (RDR6), which are in turn cleaved into 21-nt siRNAs. Four families of TAS genes have been identified in Arabidopsis. TAS2 siRNAs target PPR mRNAs (pentatricopeptide repeat); TAS3 siRNAs target ARF2, 3 and 4 transcription factors; TAS4 siRNAs decrease the level of MYB transcription factors [78]. TAS1 and TAS2 are recognized by miR173, TAS3 by miR390 and TAS4 by miR828 [64]. TasiRNAs derived from TAS4 have been implicated in Pi deficiency conditions. They are directly involved in the biosynthesis of anthocyanins in response to low Pi [78]. A putative siRNA pta22 from *Pinus taeda* has been implicated in the response to pathogens, targeting transcripts of two genes encoding for disease resistance proteins [79]. The levels of TAS1, TAS2, and TAS3 tasiRNAs have been elevated in hypoxia-treated samples in Arabidopsis suggesting their role in stress responses [80]. These changes in tasiRNA levels have been proved to be correlated with the levels of TAStargeting miRNAs (miR173 and miR390). The decrease in PPR transcripts level can be associ‐ ated with the protection of mitochondria during hypoxia stress.

The usage of high-throughput sequencing methods allowed to identify thousands of genes in convergent overlapping pairs that can generate complementary transcripts in rice and *Arabidopisis* [41, 81, 82]. A natural cis-antisense transcript pair SRO5-P5CDH nat-siRNA plays an important role in osmoprotection and oxidative stress management that results from high salinity [83]. P5CDH (pyrroline-5-carboxylate dehydrogenase) and SRO5 (similar to RCD One) are both involved in regulation of proline metabolism. The downregulation of P5CDH transcripts causes accumulation of proline, which is an important mechanism involved in the increased tolerance to salt stress conditions. But the decrease in the level of P5CDH causes an increase in a toxic metabolite P5C and reactive oxygen species that are detoxified by SRO5 proteins in the mitochondria [83].

There has also been a report showing changes in 4 siRNA levels in wheat seedlings under cold, heat, salinity, and dehydration stresses. Two of them (named 002061\_0636\_3054.1 and 005047\_0654\_1904.1) were downregulated in heat, salinity, and dehydration. The latter one was also responding to the cold stress by being upregulated. The level of another one (080621\_1340\_0098.1) was decreased under heat but upregulated under cold stress. The fourth one (997927\_0100\_2975.1) was not differentiating only under heat stress, but downregulated when plants were treated with salinity, cold, and dehydration conditions [84].

Heterochromatic 24-nt siRNAs are essential components of RdDM pathway [85]. The pathway has been established to protect plants from invasive nucleic acids, such as transposable elements and repetitive sequences, through DNA methylation and chromatin modifications [86–88]. It has been shown that 21-nt and 24-nt hcsiRNAs are the most abundant class (80%) of all small RNAs in plants [60, 89, 90]. The role of hcsiRNAs in abiotic stress responses is not well understood; although in the 1980s, McClintock has already suggested that all kinds of stresses could potentially reshape a plant genome via transpose activation [91]. Global analysis in plants such as Arabidopsis and rice suggests that the vast majority of transposons are inactive, methylated, and targeted by siRNAs [92]. Recently, few researches suggested that hcsiRNAs could play an important role in the adaptation to stress conditions. In Arabidopsis seedlings subjected to heat stress, ONSEN (copia-type retrotransposon) levels have been shown to increase in mutants affected in RdDM pathway [93]. After the recovery period, the level of ONSEN transcripts decreased gradually suggesting a role for hcsiRNAs. What is interesting is that the insertion of new ONSEN copies has been observed during flower development but before gametogenesis. Surprisingly, ONSEN insertions were also able to confer heat stress responsiveness to nearby genes. Together, all these data suggest that hcsiRNAs can contribute to control of transgenerational retrotransposition triggered by abiotic stresses and can participate in new regulatory networks established to protect plants against stress conditions. The detection of mPing transposition into a rice homologue of flowering time gene-CONSTANS in stressed cultivars supported this intriguing hypothesis. It turns out that stress-activated transposons could positively contribute to genome adaptation to growth in colder climates (reviewed in [1]). Moreover, stress-mediated induction was shown for Tos17 (rice), Tto1 (tobacco), Tnt1 (tobacco), and BARE-1 (barley) retrotransposons. Low temperature treatment was shown to decrease methylation and increase the excision rate of Tam3 trans‐ poson by binding its transposase to GCHCG sites immediately after DNA replication, thus preventing de novo sequence methylation (reviewed in [1]).
