**2. Small noncoding RNAs**

The discovery of RNA interference (RNAi) in the late 1990s has been a tornado for the past decade in terms of surprising geneticists for it changed the earlier understanding of the RNA field and the complexity of posttranscriptional control and epigenetic regulation caused by small RNAs. In 1995, Guo and Kemphues used antisense RNA sequence to block the par-1 mRNA in *Caenorhabditis elegans* when they figured out par-1 mRNA is repressed by par-1 mRNA itself [1].

Posttranscriptional gene silencing (PTGS) by RNAs was first reported in plants in 1996. The lin-4 gene, which is known to be essential for the timing of larval development in *C. elegans*, is controlled by a short RNA, which is not translated to any proteins but has a partially comple‐ mentary sequence to the 3' region of lin-4 transcript that inhibited the translation of lin-4 to protein [2]. Hence, this discovery even changed the concept of PTGS mentioned in high school biology textbooks as it questioned the central dogma proposed by Francis Crick in 1956, which stated that RNAs carry the biological information encoded in DNA molecules and they subsequently provide the code for translation into proteins [3-4]. This discovery called so much attention that was introduced as the breakthrough of the year when it was published in the journal *Science* in 2002 by Couzin and changed the basic concepts about the gene expression and RNA functionality [5]. This was in agreement with the results published by three inde‐ pendent labs that discovered miRNAs in the model plant *Arabidopsis thaliana* [6-8]. They reported many miRNAs in plants, most of which have a very conserved sequence among different species. Small RNAs could be much more than what was thought. They can influence almost all the functions in a cell by targeting the transcription factors and key genes.

One of the main reasons that small RNAs called so much attention was that soon after their discovery, the target genes for these small RNAs were reported to be crucial in leaf or flower development, which was consistent with the previous reports [9-13]. They have been found to have an impact on almost all the biological processes in eukaryotic cells as they have a wide range of target genes, which are corresponded to some of the previously identified regulatory genes and transcription factors that proved to play key roles such as in controlling cellular metabolism, growth and differentiation, phase transition timing and leaf patterning, and defense mechanisms against biotic and abiotic stresses in case of plants. These 18–25-nucleo‐ tide (nt) RNAs are categorized into many different classes based on their size, their biogenesis pathway, and their mode of action.

sRNAs are short nucleic acid sequences that give rise to the assembly of protein–RNA complexes, which later are able to repress the expression of their identified target genes by sequence-specific base pairing. This silencing of the target sequence can occur through several ways by (1) reducing their rates of transcription, (2) reducing the stability of their mRNAs in the cell, or (3) reducing the translation of their mRNAs into protein.

Although much of the work on ncRNAs field has been focused on small RNAs of under 40 nucleotides long, there are larger ncRNAs called mRNA-like ncRNAs (or mlncRNAs)that have received much less attention and have been reported to play some roles in some of the plant functions such as phosphate starvation response and nodulation. The article by Rymarquis et al. explains about them [14].

The generation of sRNAs involves a set of evolutionary conserved proteins, such as Dicer (DCR) or Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR), which all together form the RNA silencing machinery in plants. The DCLs have been the most studied enzyme so far, which, in *Arabidopsis thaliana*, are classified into four groups: DCL1 acts during miRNA metabolism, DCL2 is responsible for the viral resistance, DCL3 triggers the transcriptional silencing, and DCL4 cooperates in posttranscriptional silencing and ta-siRNA metabolism (dissecting *Arabidopsis thaliana* dicer function in small RNA processing, gene silencing, DNA methylation patterning, and nature genetics). Plant genomes encode only one of the three known classes of AGO proteins, namely AGO1, which is involved in both miRNA and sRNA biogenesis.

The sRNAs are categorized into different classes based on their size, their biogenesis pathway, and their mode of action to at least six groups, including microRNAs (miRNAs), heterochro‐ matic small interfering RNAs (hc-siRNAs), trans-acting small interfering RNAs (ta-siRNAs), natural antisense small interfering RNAs (nat-siRNAs), repeat-associated small interfering RNAs (ra-siRNAs), and the piwi-interacting RNAs (piRNAs), which are found in metazoans.

#### **2.1. miRNAs**

tional, and posttranslational regulations. Although studies have been mostly focused on the transcriptional level of regulatory mechanisms so far, recent results lead us to the point that posttranscriptional events also play a very important role in gene expression regulation in major scenarios of a plant life, from developmental processes to stress responses. Small noncoding RNAs (sRNAs) are the important posttranscriptional regulatory factors in gene regulatory networks. They are involved in many important processes of plant development

sRNAs are roughly divided into different categories based on the genomic origins of their precursors: microRNAs (miRNAs), trans-acting small interfering RNAs (ta-siRNAs), and natural antisense small interfering RNAs (nat-siRNAs). These sRNAs are loaded into RNAinduced silencing complexes (RISC) and regulate the expression of their relative target genes negatively by affecting the mRNA levels, chromatin remodeling, and DNA methylation. Understanding of sRNA-guided stress regulatory networks should provide us with new tools and vision for the genetic improvement of plant stress tolerance and eventually developing

This chapter highlights the recent advances in understanding the crucial roles of sRNAs in plant responses to heat, drought, salinity, nutrient deficiency and low-temperature stresses, and proposes potential technologies and strategies used to identify abiotic stress-regulated sRNAs in addition to the recent advances and methods for validation and analysis of their

The discovery of RNA interference (RNAi) in the late 1990s has been a tornado for the past decade in terms of surprising geneticists for it changed the earlier understanding of the RNA field and the complexity of posttranscriptional control and epigenetic regulation caused by small RNAs. In 1995, Guo and Kemphues used antisense RNA sequence to block the par-1 mRNA in *Caenorhabditis elegans* when they figured out par-1 mRNA is repressed by par-1

Posttranscriptional gene silencing (PTGS) by RNAs was first reported in plants in 1996. The lin-4 gene, which is known to be essential for the timing of larval development in *C. elegans*, is controlled by a short RNA, which is not translated to any proteins but has a partially comple‐ mentary sequence to the 3' region of lin-4 transcript that inhibited the translation of lin-4 to protein [2]. Hence, this discovery even changed the concept of PTGS mentioned in high school biology textbooks as it questioned the central dogma proposed by Francis Crick in 1956, which stated that RNAs carry the biological information encoded in DNA molecules and they subsequently provide the code for translation into proteins [3-4]. This discovery called so much attention that was introduced as the breakthrough of the year when it was published in the journal *Science* in 2002 by Couzin and changed the basic concepts about the gene expression and RNA functionality [5]. This was in agreement with the results published by three inde‐ pendent labs that discovered miRNAs in the model plant *Arabidopsis thaliana* [6-8]. They reported many miRNAs in plants, most of which have a very conserved sequence among

and stress responses.

target genes.

mRNA itself [1].

more stress-resistant plants in future.

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

**2. Small noncoding RNAs**

Typically, miRNAs are derived from single-stranded RNA precursors that are transcribed by RNA polymerase II from MiRNA genes called primary microRNA transcript (pri-miRNA), which are capable of forming a self-complementary fold-back structure named hairpin or stem loop in which the mature miRNA could reside on the 3' or 5' end (Table 1). This imperfect double-stranded structure is further recognized and processed by DCL1 in association with other protein factors [15]. This gives birth to the miRNA/miRNA\* duplex which based on the thermodynamic features will have a different fate but usually the pre-miRNA strand is loaded onto an AGO1-containing, RNA-induced silencing complex (RISC) and the miRNA\* strand lives for a short time in the cell. Mature microRNAs (miRNAs), which are the so-called hairpinderived RNAs, are 20–24 nt long and single stranded while miRNA genes are 70–300 nt long. Mature miRNAs help the target recognition and cleavage in cooperation with AGO1 and miRISC. The first cleavage by DCL1 generates a stem-loop intermediate, called the precursor miRNA (pre-miRNA), and the second cleavage by DCL1 releases the miRNA duplex, one strand of which is known as mature miRNA and the other strand is known as miRNA\* (miRNA star).


**Table 1.** Small RNAs involved in plant response to abiotic stresses

Mammals use only one class of RNAse III enzyme, Dicer, to generate both miRNAs and siRNAs. In plants, there are a variety of specialized DCL endonucleases, which are classified into 10 categories. DCL1 is involved in miRNA biogenesis pathway while other DCLs participate in various aspects of sRNA-mediated generation or gene silencing pathway (Figure 1) [16].

All the information about the reported miRNAs and their sequences and annotations are stored in a database called miRBase (www.mirbase.org), which is updated on a regular basis with the new published data in the literature [17]. So far, there are 205 precursors and 384 mature miRNAs reported in the model plant *Arabidopsis thaliana*, which is about 1–2% of its genome. Theoretically, the perfect base pairing between miRNAs and complementary target mRNAs helps the process of finding target genes for each miRNA. By computational methods, there are a plenty of databases that are able to predict the potential target genes for each newly found miRNA.

### **2.2. siRNAs**

which are capable of forming a self-complementary fold-back structure named hairpin or stem loop in which the mature miRNA could reside on the 3' or 5' end (Table 1). This imperfect double-stranded structure is further recognized and processed by DCL1 in association with other protein factors [15]. This gives birth to the miRNA/miRNA\* duplex which based on the thermodynamic features will have a different fate but usually the pre-miRNA strand is loaded onto an AGO1-containing, RNA-induced silencing complex (RISC) and the miRNA\* strand lives for a short time in the cell. Mature microRNAs (miRNAs), which are the so-called hairpinderived RNAs, are 20–24 nt long and single stranded while miRNA genes are 70–300 nt long. Mature miRNAs help the target recognition and cleavage in cooperation with AGO1 and miRISC. The first cleavage by DCL1 generates a stem-loop intermediate, called the precursor miRNA (pre-miRNA), and the second cleavage by DCL1 releases the miRNA duplex, one strand of which is known as mature miRNA and the other strand is known as miRNA\* (miRNA

**Class Full Name Originating Loci Function Biogenesis**

through mRNA cleavage and translational repression

Silence repeats and transposons through RNA-dependent DNA methylation and chromatin

through mRNA cleavage

Stressed-induced nat-siRNA to repress target gene expression through mRNA cleavage

Germ-line-specific piRNA to suppress repeats and transposons

in flies and mammals

Mammals use only one class of RNAse III enzyme, Dicer, to generate both miRNAs and siRNAs. In plants, there are a variety of specialized DCL endonucleases, which are classified into 10 categories. DCL1 is involved in miRNA biogenesis pathway while other DCLs

modification

TAS loci Repress target gene expression

The fold-back structures of long ssRNA transcripts are

RDR-generated dsRNAs are cleaved by Dicers

TAS transcripts are cleaved by miRNAs, transcribed by RDR into dsRNA, and then processed by Dicers

The dsRNA derived from overlapping transcripts is cleaved by Dicers

ssRNA derived from transposons is cleaved by

PIWI protein

cleaved by Dicers

miRNA microRNA *MIRNA* genes Repress target gene expression

Repeats, transposons, and

(endogenous). Transgenes

Loci producing pairs of sense-antisense transcripts

Repeats, transposons, and

retroelements

**Table 1.** Small RNAs involved in plant response to abiotic stresses

retroelements

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

and viral RNAs (exogenous)

star).

siRNA short-

ta-siRNA trans-acting siRNA

nat-siRNA natural

piRNA piwi-

interfering RNA

antisense transcriptderived siRNA

interacting RNA

siRNAs were first identified in 1999 in plants [18], and later there were many reports about diverse sets of endogenous siRNAs in plants as well as in animals (Table 1) [6] [8][19][20-22]. Most of the plant siRNAs are around 24 nt in length that are excised from the long doublestranded RNA duplexes or transcripts generated from inverted repeat regions [23-24]. The sources of these double-stranded sequences that eventually trigger biogenesis of siRNAs could be endogenous or exogenous. Endogenous plant siRNAs can be classified into several categories, including miRNA-induced trans-acting siRNAs (tasiRNAs), natural antisense siRNAs (nat-siRNAs), cis-acting siRNAs (casiRNAs), heterochromatic siRNAs, and many other unclassified small RNAs [25]. In plants, ta-siRNAs are generated from the genomic loci named TAS genes, which are transcribed by RNA-pol II. The generation of ta-siRNAs is triggered by an miRNA, which cleaves a nonprotein-coding transcript of a tasiRNA gene [26-29]. In plants, there are eight TAS loci reported so far, which belong to four families (TAS1– 4). TAS1 and TAS2 families are cleaved by miR173 with the association of AGO1. TAS3 family transcripts are cleaved by the guidance of miR390 and AGO7 and usually target the auxin response factor (ARF) transcripts. TAS4 transcript is cleaved by miR828 guided together with AGO1 and they usually target myeloblastosis (MYB) transcription factors [30]. These cleaved RNAs are then processed by the suppressor of gene silencing 3 (SGS3) and copied into doublestranded RNAs by RNA-dependent RNA polymerase 6 (RDR6). DCL4 cleaves them in multiple rounds so that it finally gives rise to the 21-nt ta-siRNAs. ta-siRNAs are loaded onto AGO1 complex to degrade the target mRNAs [31].

The other class of siRNAs called nat-siRNAs are separated into two groups: cis-nat-siRNAs that are generated from two RNAs, which were transcribed from the same loci but opposite strands, and trans-nat-siRNAs, which were transcripts from different loci [32]. RDR6 and DCL2 are involved in generating 24-nt nat-siRNAs, and RDR6 and DCL1 are involved in generating 21-nt nat-siRNAs. trans-nat-siRNAs are transcripts from different loci but proc‐ essed by the same proteins (RDR6 and DCL2). ta-siRNAs cleave the target mRNAs by being partially or fully complementary with them.

Heterochromatic siRNAs mostly originate from transposable elements or repeats and their mode of action is slightly different from miRNAs and ta-siRNAs, as they modulate the histone modification at their homologous regions in the genome and inhibit the gene expression at the transcriptional level.
