Non-Coding RNA and Its Prospective Utilization in Plant Breeding

Abstract

Non-coding RNA molecules are generally present in a dispersed manner throughout the genome. They may behave as long ncRNAs or convert into small RNAs of around 20–24 nts that are universally categorized using their size, function, or chromosomal position. ncRNAs are thought to play a vital role in regulating and modulating gene expression apart from their prospective role in several epigenetic mechanisms controlling specificity in biochemical pathways and phenotype development in clonal cells. They are also part of the natural defense system against viruses. ncRNA modulates genes by transcriptional and translational control of growth, development, and stress response alongside other RNA molecules. Some modes of action have unraveled in recent years. A lot more needs to be pondered upon for comprehending their involvement in the extremely intricate processes in a more wholesome manner. In this chapter, we will discuss the different ncRNA, their origin, classification, and their role in various physiological processes. Practical examples of the discovery of ncRNA in different crops and their functions have also been elucidated with the required details. The yield and quality enhancement, along with the better stress response being the aim of the crop improvement program, the prospective utilities of ncRNA are also explained in the subsequent part of the chapter.

1. Introduction

RNA is the ribonucleic acid, one of the earliest formed molecules to shape life on earth. Being single-stranded, these are known to have a shorter life span and integrity compared to DNA. But, from other angles, these properties make RNA a magical molecule with a unique ability to work inside and outside the nucleus, leading to diverse sorts of roles in structural to regulatory aspects. In the case of the prokaryotes, most of the transcribed RNA is translated because of the smaller size of the genome and the variety and load of work endowed on it for the organism’s survivability. But, in the case of the eukaryotes, as the genome size gets bigger, the actual coding part of the chromosomes becomes sparse and rather scattered, mostly. Secondly, the coding genes occupy only 1–2% of the genome. A very high percentage of eukaryotic genomes around up to 90% undergo transcription to produce RNA, nevertheless only a little portion of transcripts get actually translated into proteins [1].

Out of the total RNAs, non-coding RNA (ncRNA) pertains to active and functional RNA molecules that are not translated into proteins, although being transcribed from DNA. They encompass a wide range of RNA molecules having the potential to play in the regulation of gene expression [1]. They can act as long ncRNAs or be converted into s very smaller size RNA molecule proteins in eukaryotic cells. Mostly ncRNA is categorized worldwide based on its size, function, or genetic origin [1].

Based on the size of the molecule, their origin, functionality, and ncRNA are sorted into either the long non-coding RNAs (lncRNAs) having nucleotide lengths of more than 200 nucleotides or the small RNAs (sRNAs), which are comparatively quite smaller in size. Again the sRNAs are further classified into different types of RNA, such as miRNA, siRNA, piRNA, which will be disused further in detail with the various aspects of their origin, features, functions, and other aspects in the later section of the chapter.

ncRNAs are responsible for a wide range of biological functions. They control gene expression at the transcriptional, RNA processing, and translational levels by the particular structural aspects of RNA itself. Recent discoveries also show their role in various epigenetic phenomena affecting multiple physiological pathways and expression of particular phenotypes in a different situation. The fraction of the coding region of the protein genome varies considerably and is oppositely proportionate to the genome’s size and complexity [2]. Many regulatory ncRNAs do have relatively high specificity of the target, whereas others serve as a major modulator of extensive regulatory signaling networks by acting across the genome [3]. They keep alien nucleic acids out of genomes and safeguard the genome against them. Non-coding RNAs target a single gene and pathways involving multiple genes at the genome level through distinct molecular mechanisms. Hence, these regulatory ncRNAs could be potential breeding targets for advanced breeding programs in plants [4]. They can regulate the synthesis of DNA and also take part in the reorganization of the genome. The biological activity of ribozymes and riboswitches is served by several ncRNAs that use the power of base pairing to interact with other nucleic acids preferentially [5].

Non-coding RNAs (ncRNAs), which act as a natural defense mechanism against—attacking viruses, have also been found as effectors in RNA-mediated gene silencing and hence now utilized in crop genetic modification [6]. The role of ncRNA has been observed in RNA interference and other regulatory mechanisms in plants; these provide a huge scope for the use of advanced molecular biology tools on these for enhancing the production potential of plants and modulation of growth and development of a plant to a certain extent. These have also been reported to influence the genes and biochemical pathways involving important traits like floral growth, maturation of seed, various biotic and abiotic stresses, along with pest and disease resistance processes. The ncRNA and their detailed structural to functional aspects are narrated meticulously in the upcoming sections of the chapter.

2. History of non-coding RNA

The beginning of the era of RNA dates back to the discovery of nucleic acids by Friedrich Miescher in the late 1860s. Later on, rigorous research was carried out about its structure, mode of action, and expression. Soon it became clear that majorly three kinds of RNA were involved in the protein production process, translation where mRNA was the carrier of genetic information ultimately translated into protein, and the process is assisted by tRNA and rRNA. Secondly, with the advent of newer technologies in molecular biology, several new RNA molecules appeared in the picture, out of which some had some role in the regulatory pathways or physiological pathways, for some other function is still unknown. Since, among all these only, mRNA codes for the protein, all other RNAs are termed non-coding RNA.

The first non-coding RNA to be discovered was the tRNA and its role in transferring amino acids was first observed by Paul C Zamecnik and Mahlon Hoagland in a cell-free system when RNA molecules were radioactively labeled [7]. Furthermore, it was the first non-coding RNA to be sequenced [8]. Later, in the early twenty-first century, many types of non-coding RNA, such as siRNA, miRNA, and piRNA, were discovered namely which had a role in gene regulation. During post-transcriptional gene silencing, a 25-nucleotide antisense RNA complementary to the target RNA was detected. This short interfering RNA, in virus-induced gene silencing, suppresses the production of viral proteins on binding with the target viral mRNA. This is a type of defense mechanism based on RNA against RNA and DNA viruses [9].

Caenorhabditis elegans is a completely sequenced nematode used as a model organism for many research programs. Out of its four larval stages, L1, L2, L3, and L4, it was found that the gene lin-4, the first miRNA discovered, was crucial for the transition of the larva from L1 to L2 [10].

H19 and Enod40 were the first eukaryotic lncRNA to be discovered [11]. The first plant lncRNA was discovered by Crespi in 1994. Long non-coding RNAs were first described during the whole genome sequencing and several types of lncRNA, such as Xist, Airn, MALATI, HOTAIR were discovered [12]. Table 1 summarizes the different discovery events of the ncRNAs.

S. No.	Type of ncRNA	Year of discovery	Scientists discovered	Remark
1.	tRNA	Before the 1960s	Paul C Zamecnik and Mahlon Hoagland	An adaptor molecule that mediates translation
2.	rRNA	Before 1965		Ribosomal RNA
3.	snRNA	1966		Small nuclear RNA
4.	snoRNA	1968		Small nucleolar RNA—U3 species—first snoRNA discovered
5.	siRNA	1999	Hamilton and Baulcombe	Small interfering RNA
	phasiRNA		Chen et al. [13]
	tasiRNA	2004		Trans-acting siRNA
	easiRNA		Slotkin et al. [14]	Epigenetically-acting siRNA
	natsiRNA
6.	miRNA	1993	Lee et al. [10]	Micro RNA
7.	piRNA	2006	Aravin et al. [15]	PIWI: P-element Induced WImpy testis in Drosophila
8.	lncRNA		Pachnis et al. [11]	H19, XIST, and HOTAIR

Table 1.

List of ncRNA and their brief history.

3. Classification of non-coding RNAs

The major category of grouping non-coding RNAs is based on their origin, nature of biogenesis, as well as based on its mechanism of action. The non-coding RNA transcripts can either perform housekeeping or regulatory functions. Those non-coding RNAs dynamically involved in cellular and ribosomal functions include tRNAs, snRNAs, rRNAs, and snoRNAs, whereas the regulatory ncRNAs are actively involved in most of the plant growth and development processes dealing with biotic and abiotic stress responses and plant immunity [16]. These regulatory ncRNAs, such as miRNAs, piRNAs, siRNAs, and lncRNAs, are transcribed from DNA and are primarily involved in transcriptional and post-transcriptional gene regulations [17]. The basic classification of ncRNA is presented in Tables 2 and 3 described the brief details about the ncRNA. These regulatory ncRNAs cannot generally transcribe into proteins whereas housekeeping ncRNAs assist in protein translation [18].

Table 2.

Classification of ncRNA.

ncRNA		Remark
Housekeeping	tRNA	Actively involved in decoding genetic information from mRNA at the site of ribosome subunits during protein translation.
	rRNA	It is essentially a component of the ribosome, constituting small and large subunits of the ribosome. This ribozyme is RNA transcribed from ribosomal DNA (rDNA).
	snRNA	Small nuclear RNA (snRNA) is involved in the processing of pre-messenger RNA (hnRNA) in the nucleus, which is transcribed by either RNA polymerase II or RNA polymerase III, and the average length of snRNA is approximately 150 nucleotides.
	snoRNA	Small nucleolar RNAs have a role in tuning ribosomal and spliceosomal function by guiding ribose methylation and pseudouridylation at targeted nucleotide residues of ribosomal and small nuclear RNAs.
Regulatory	miRNA	microRNAs regulate gene expression post-transcriptionally. They generally bind to the 3’-UTR (untranslated region) of their target mRNAs and repress protein production by destabilizing the mRNA and translational silencing.
	siRNA	Small interfering RNA is sometimes known as short interfering RNA or silencing RNA is a class of double-stranded RNA, typically 20–s24 (normally 21) base pairs in length, and operating within the RNA interference (RNAi) pathway.
	i. hasiRNA	Trans-acting siRNAs are known to target complementary mRNAs for degradation and to function in development.
	ii.tasiRNA	Trans-acting siRNA, represses gene expression through post-transcriptional gene silencing in land plants.
	iii.easiRNA	Epigenetically activated small interfering RNAs (easiRNAs) from reactivated transposable elements triggered by miRNA.
	iii.natsiRNA	Natural antisense short interfering RNA. They are endogenous RNA regulators, which are between 21 and 24 nucleotides in length, and are generated from complementary mRNA transcripts, which are further processed into siRNA.
	piRNA	piwi-interacting RNA (piRNA) is the largest, expressed in animal cells. Mostly involved in the epigenetic and post-transcriptional silencing of transposable elements and other spurious or repeat-derived transcripts, but can also be involved in the regulation of other genetic elements in germ line cells.
	lncRNA	Long non-coding (lnc) RNAs are longer than 200 nt, which primarily interact with mRNA, DNA, protein, and miRNA and consequently regulate gene expression at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels in a variety of ways.
	i. Linear	Maintaining nuclear structure integrity and positively or negatively regulating genes in cis or trans by recruiting transcription factors or chromatin-modifying complexes to DNA targets in the nucleus.
	ii.Circular	Function as a sponge to recruit miRNAs or transcriptional effectors to regulate target gene expression.

Table 3.

Brief details about the ncRNA classification.

4. Biogenesis of non-coding RNA

Basically, ncRNAs are consequence of various processes, such as the process of duplication, modification of transposons during the evolutionary process, pseudogenization of actual coding sequences, doubling of RNA viruses, part of some hairpin structures, double-stranded RNAs from heterochromatin regions and DNA repeats [19]. Despite having a similar structural build, the sRNA varies greatly in their method of biogenesis.

4.1 miRNA

MicroRNAs are generated out of primary microRNAs (pri-miRNAs) by the action of RNA polymerase II. These precursor molecules have a double-stranded secondary structure which later on gets cleaved by DICER-LIKE 1 (DCL1) molecules to form single-stranded siRNA. These raw miRNAs is further processed in the nucleus before being exported to the cytoplasm [1]. Mature single miRNA that incorporates the RISC complex binds with other complementary mRNA sequences [20].

4.2 IsomiRNAs

IsomiRNAs (IsomiRNAs) are one of the variants of miRNAs that arise because of the inaccurate cleavage action performed through the RNase III enzyme. These may also be developed via the process of post-transcriptional RNA editing [21, 22].

4.3 siRNA

siRNAs are derived from long dsRNAs produced during varied mechanisms, such as folding in an inverted sequence, from a long non-coding RNA, hybridization of two fully or partially complementary sequences . This further gets processed by the action of DCL2, DCL3, and DCL 4 proteins, leading to the formation of 22 nt, 24 nt, and 21 nt siRNA, respectively [3]. RNA-dependent RNA polymerases 2 and 6 (RDR2, RDR6), and DNA-dependent RNA polymerases IV and V in plants also take part in the production of siRNAs [23].

4.4 phasiRNA and tasiRNA

In plants, phasiRNAs are the partially degraded product of mRNA as being passed through the RISC complex. DCL proteins are involved in this slicing process of dsRNAs to form a group of 21- or 24-nt siRNAs, termed phasiRNAs, Trans-acting siRNAs (tasiRNAs) are a class of DCL4-dependent 21-nt phasiRNAs generated from non-coding TAS transcripts [24, 25].

4.5 lncRNA

lncRNAs have appeared from intergenic, particularly regions giving rise to long intergenic ncRNAs (lincRNAs) and those developed from intronic regions giving intronic ncRNAs (incRNAs) [26]. lincRNAs and IncRNAs belong to conventional linear lncRNAs. Circular RNAs (circRNAs) generally develop from coding regions or intronic regions.

4.5.1 Linear-long non-coding RNAs

The lncRNAs, such as lincRNAs and IncRNAs are linear lncRNAs transcribed by Pol II. Because of having similar features as that of mRNA with a 5_ m7G cap and a 3_ poly (A) tail, they undergo similar modifications later on called mRNA mimics [27].

4.5.2 Circular long non-coding RNAs

Mostly these are circRNAs derived from back-splicing reactions of internal exons in pre-mRNAs and further move to the cytoplasm. Some other circRNA localized in the nucleus is produced from excised intron lariats that fail to be debranched [27].

5. Characteristic features of non-coding RNA

Non-coding RNAs are RNA molecules that are transcribed from DNA but cannot code for a protein. With the advances in transcriptomics and sequencing techniques, thousands of small and long non-coding RNAs were identified. They generally play a major role in gene regulation at the transcriptional or post-transcription level and some regulatory ncRNAs possess high target specificity and some ncRNAs are involved in epigenetic mechanisms too. A few major ones are discussed below.

5.1 tRNA (transfer RNA)

Next to mRNA which is the coding RNA, tRNA, and rRNA are considered more prominent. tRNA typically contains less than 100 nucleotides and, as their name indicates, their job is to carry an amino acid to the protein-synthesizing machinery. tRNA usually takes a clover leaf secondary structure, which forms a 3D L- shaped structure by stacking the helices. The structure of tRNA was found to be almost similar among different tRNA species. The secondary structure includes the acceptor arm, anticodon arm, T-arm and D-arm, and a variable arm. It was found that the size of the acceptor arm, anticodon arm, and T-arm were conserved whereas the D-arm and variable arm differ in their sizes [28]. This difference in the sizes of the variable arm led to the grouping of tRNAs into two classes. The major proportion of tRNA belongs to class I with less than 10 nucleotides in the variable loop; the class II tRNAs included tRNA^Ser, tRNA^Leu, and tRNA^Tyr with more than 10 nucleotides [8].

5.2 rRNA (ribosomal RNA)

It is one of the longest and most stable RNA molecules which form a major constituent of protein-synthesizing organelle comprising nearly 60% of ribosome’s mass. Ribosomes of both prokaryotes and eukaryotes are made of smaller and larger subunits and they form a complex during translation. The smaller subunit of prokaryotes constitutes an RNA molecule with a Svedberg coefficient of 16S and its sedimentation rate is 30S after combining with other proteins. The larger subunit has two RNA molecules (5S and 23S) and they form a 50S subunit by binding with other proteins. On the contrary, the eukaryotic ribosome consists of 60S and 40S subunits with the larger subunit containing two long RNA molecules (18S and 28S) and the smaller subunit containing two short RNAs molecules (5S and 5.8S). A unique rRNA component, such as an undescribed helical structure, was found in the small subunit near the mRNA exit channel of 80S ribosome of Trypanosoma cruzi, a protozoan that causes Chagas disease. This structure was most likely involved in the binding of the ribosome to the 5’end of the mRNA facilitating translation [29].

5.3 snRNA (small nuclear RNA)

One of the most important post-transcriptional modifications is the splicing of pre-mRNA which is carried out by an RNA-protein complex known as spliceosome. Small nuclear RNAs form a part of this spliceosome and catalyze splicing [30]. On account of their sub-nuclear localization, snRNAs are grouped into spliceosomal uridylate snRNA, which is the most conserved among eukaryotes, small nucleolar RNA (snoRNA), and small Cajal-body-specific RNA (scaRNA) [31]. UsnRNA comprises U1–U6 being the most abundant and U7–U14 being the low abundant ones. The five major types U1, U2, U4, U5, and U6 are involved in the splicing of mRNA, whereas U3, U8, U13, and U14 are involved in the processing of mRNA [32]. Small nuclear ribonucleoprotein complexes (snRNPs) are formed by the association of each snRNA with one or more proteins.

5.4 snoRNA (small nucleolar RNA)

snoRNAs are functional non-coding RNAs with a length of 60–300 nucleotides, which are usually found near nucleoli and are prevalent in all eukaryotic organisms. Like snRNPs, they also form snoRNPs in association with a set of proteins [33]. snoRNA are majorly categorized into C/D box, snoRNAs which contain two conserved sequences box C (RUGAUGA and box D (CUGA) and direct 2′-O-ribose methylation, whereas H(ANANNA)/ACA box snoRNAs directs pseudouridylation. This classification is based on conserved sequence motifs [34]. The binding of fibrillarin, Nop56p, Nop58p, and 15.5 kDa/Snu13p snoRNP proteins are directed by the box C/D motif and form a kink turn, which is the most prevalent motif found in various RNAs. Proteins like dyskerin/Cbf5p, Gar1p, Nhp2p, and Nop10p are associated with the box H/ACA snoRNAs [35].

5.5 Other small non-coding RNA

Many types of small non-coding RNA have emerged in the last decade, but it is mainly classified into si (short interfering RNA), miRNA (microRNA), and piRNA (piwi-interacting RNAs). These are small non-coding RNAs with a length of about 20–30 nucleotides and form a protein complex with the Argonaute protein family and are present only in eukaryotes [36, 37].

5.5.1 miRNA and siRNA

Both miRNA and siRNA are initially part of a double-stranded RNA molecule with a guided strand and passenger strand. Their size is around 20–24 nucleotides only. A unique feature of siRNA is the occurrence of di-nucleotide overhang at the 3’OH. Phased siRNAs, trans-acting siRNAs, epigenetically activated siRNAs, and natsiRNAs are some of the types of siRNAs that play a role in regulating gene expression. A similar type of small non-coding RNA is the miRNA which is a small single-stranded RNA transcribed from DNA sequences into primary miRNA and processed into precursor miRNAs and finally becomes a mature miRNA [38]. Both siRNA and miRNA are almost similar in their biogenesis where an enzyme of the RNAse III family cleaves dsRNA into siRNA and miRNA. Respective RNA-induced silencing complexes are formed with the association of siRNA and miRNA termed siRSC and miRSC and are involved in gene regulation [39].

5.5.2 piRNA

piRNAs are also single-stranded with a length of 23–36 nucleotides and are more prevalent in animals. They bind to PIWI proteins that belong to the Argonaute protein family. The binding to PIWI and the independence from Dicer distinguished piRNA from siRNA and miRNA. piRNA is grouped into transposon-derived piRNA, miRNA-derived piRNA, lnc-derived piRNA, and Caenorhabditis-specific piRNA [40].

5.6 lncRNA (long non-coding RNA)

As previously said, non-coding RNA is of two types, small and long. lncRNA due to its length of more than 200 nucleotides develops complicated secondary and tertiary structures. They are prevalent either in the nucleus or in the cytoplasm of the cell. To sustain their function, structural conservation is more common than nucleotide sequence conservation. Furthermore, when compared to other non-coding RNAs the conservation is found to be less and low prevalence adds to the challenge of identifying and understanding the mode of action. Long non-coding RNA contains linear and circular lncRNA. They are synthesized from pre-mRNA by alternate splicing, which consists of a 5′ cap and 3′ tail. In circular ncRNA, the 5’end and 3′ end are linked forming a circle-like structure [41]. It was found that the level of expression varies between different types of lncRNA, some are organ and tissue-specific and the rest are expressed after encountering different external or internal stresses [42, 43].

6. Role of ncRNA in different physiological pathways in plants

Recent development in molecular biology tools has led to advanced research in the area of ncRNA, which in turn gave rise to newer insights about the various roles of ncRNA in plants. The finding and reports about these roles are summarized in Table 4.

Type of ncRNA	Role	Example	Author
miRNA	The mutation of DCL1 in different plants leads to lethal embryos or to pleiotropic developmental effects, which are attributed mainly to the significant decrease in the miRNAs levels.	of Arabidopsis	Liu et al. [44]; Nodine and Bartel, [45]
	The transition of vegetative phase by regulating SPL genes in several angiosperm species.	Rice, maize, barley, soybean
	Regulate the epigenomic machinery by regulating the expression of genes involved in the rearrangement of chromatin.	—	Choi et al. [46]
	Monocot-specific miR444 controls tillering.	Rice	Guo et al. [47]
	Monocot-specific miRNA is induced by nitrogen luxury conditions and regulates lodging resistance by targeting the lignin biosynthesis genes ZmLACCASE 3 (ZmLAC3) and ZmLAC5.	Maize
	miRNA represses auxin-responsive factors, promoting the development of lateral root growth and development.	Rice	Marin et al. [48]
	Overexpression results in developmental defects characterized by dwarf-ism, serrated leaves, and early flowering.	Arabidopsis Rice	Wu et al. [49]
siRNA	Involved in PTGS	—	Xie and Yu [50]
	DNA methylation	—	Nuthiattu et al. [51]
natsiRNA	Pathogen resistance	—	Katiyar-Agarwal et al. [52]
	Salt tolerance	—	Borsani et al. [53]
	Cell wall biosynthesis	—	Held et al. [54]
	Alter gene expression under environmental stress conditions.	—	Zhang et al. [55]
easiRNA	Heritable transcriptional gene silencing	—
lncRNA	Response to environmental stimuli and stress	—	Wang et al. [56]
	Involved in protein and miRNAs hijack	—	Ariel et al. [57]
	Modulation of mRNA; stability and translation	—
	Modification of chromatin at diverse levels	—
	Stress-responsiveness is induced by the infection with powdery mildew and by heat.	Wheat	Xin et al. [58]
	Tissue- or stress-specific expression	Foxtail millet; Black cottonwood Chinese white; poplar; Soybean; Peach; Brassica rapa,	Qi et al. [59]; Wang et al. [60]; Yu et al. [61]; Shuai et al. [62]; Ye et al. [63]; Chen et al. [64]
	Photoperiod-sensitive male sterility (PSMS)	Rice	Ding et al. [65]

Table 4.

Different ncRNA and their role in different processes in plant.

6.1 ncRNAs determining plant yield and nutrition

The role of ncRNAs in various physiological traits and growth parameters is well studied, moreover also have an indirect influence on yield through these traits viz., tillering modulation and panicle branching related genes in rice through SPL transcription factors controlling, such as OsTB1 [66] and DEP1 [67] (regulatory non-coding article). Zhang et al. [68] reported overexpression of miR397 resulted in increased panicle branching with desired grain size suppressing the LACCASE gene. Apart from miRNA, several lncRNAs controlling photoperiod sensitive male sterility LDMAR (for long-day specific male-fertility-associated RNA) in rice are responsible for panicle development, floral organ development, sexual reproduction, and also control the premature programmed cell death of developing anthers.

It is interesting to understand the role of ncRNA that was earlier considered junk having a role in nutrient use efficiency as well as nutrient absorption efficiency. Both miRNAs and lncRNAs are well involved in phosphate metabolism and homeostasis. miR399 suppresses phosphate homeostasis genes PHO2 leading to increased uptake of phosphorous [69]. Similarly, lncRNAs in rice control the expression of the OsPHO1 gene family during phosphate-deprived conditions [70]. Thus, modulating nutrient-related traits thereby contributes toward good yield performance. Not only phosphorus but these ncRNAs are also reported to be involved in root nodulation thereby on nitrogen metabolic pathway [71].

6.2 ncRNAs in plant growth and development

Apart from their major role in stress response, these ncRNAs, such as miRNAs, are actively involved in the regulation of the transition of the vegetative phase by regulating SPL genes in several angiosperm species. For example, the vital role of miR172 in flowering control has been studied in rice, maize, barley, and soybean. In rice, the monocot-specific miR444 controls tillering [47] and participates in antiviral defense. miR528, a monocot-specific miRNA, is induced by nitrogen luxury conditions in maize and regulates lodging resistance by targeting the lignin biosynthesis genes ZmLACCASE 3 (ZmLAC3) and ZmLAC5 [72]. si RNAs, on the other hand, are involved in transcriptional gene silencing regulated by RNA-directed DNA methylation and also have a vital role in genome stability. siRNAs are also reported to monitor, genome compatibility and dosage during reproduction and hybridization [73].

7. Function of ncRNA concerning prospective areas of utilization of ncRNA in plant breeding

A major portion of eukaryotic transcriptomes comprised of ncRNAs, which were considered “junk” till the recent past, actually carry out significant roles in almost all the biological processes via regulation of gene expression at transcriptional and post-translational stages. Thus, these diverse ncRNA plays a vital role in plant development and environmental responses, which can be well employed in applied plant breeding and crop improvement. In drosophila, 30 UTR of mRNA is required for oogenesis [74]. Many studies also report the significant role of 30 UTR mRNAs in transmitting information required for cell proliferation as well as cell differentiation during cancers [75].

7.1 Abiotic and biotic stress response

These ncRNAs have considerable responsibility for plant stress response as well as plant immunity that can be better utilized in plant breeding for crop improvements. Jha et al. [76] have highlighted the differential expressions of ncRNAs in plants when they are ubjected to unfavorable conditions. Zhang et al. [68] identified that 17 microRNA were downregulated and 16 upregulated when subjected to drought stress at the seedling stage. A similar study was carried out on maize screened for drought conditions by Liu et al. [77]. When drought condition was induced, eight and seven miRNAs were enhanced in leaves and roots, respectively, whereas 13 and seven miRNAs were found to be suppressed in leaves and roots, respectively. In addition, a single miRNA can be upregulated and downregulated to express the same stress reaction. For example, in maize miR156, miR164, and miR171 undergo varying expressions when subjected to drought conditions. Similarly, differential expression was exhibited by miR156, and miR171 in rice was also reported [78, 79]. Table 5 depicts the use of various miRNA in stress responses.

Application	Crop	Gene	References
Viral diseases	Potato	HC-Pro (Potato virus-Y)	Waterhouse et al. [80]
		Silencing of viral coat protein	Missiou et al. [81]
	medicinally important papaya	Silencing of viral coat protein	Kertbundit et al. [82]
	Black gram	Hairpin construct of the non-coding intergenic region of mungbean yellow mosaic India virus	Pooggin et al. [83]
	Tobacco	Tobacco mosaic virus asRNA CP	Powell et al. [84]
	Pepper	PMMoV RNAi PMMoV replicase	Dalakouras et al. [85]
	Cassava	siRNAs designed to the replicase (Rep)-coding sequence of African cassava mosaic virus	Vanitharani et al. [86]
		Cassava brown streak disease (CBSD)	Patil et al. [87]
Bacterial diseases	Arabidopsis thaliana	Crown gall disease management strategy that targets the process of tumorigenesis (gall formation) by initiating RNAi of the iaaM and ipt oncogenes	Dunoyer et al. [88]
	Rice	Leaf blight RNAi OsSSI2	Younis et al. [89]
Fungal disease	Nicotiana tabacum	Downregulation of GST (glutathione S-transferases) enzyme against Phytophthora parasitica var. nicotianae	Hernández et al. [90]
	Apple	Apple scab fungus RNAi GFP & THN	Fitzgerald et al. [91]
	Wheat and Barley	Powdery mildew fungi Blumeria graminis	Nowara et al. [92]
	Rice	Sheath blight pathogen RNAi RPMK1-1/-2	Ila Mukul Tiwari et al. [93]
	Banana	Fusarium oxysporum f. sp. Cubense by RNAi-mediated knockdown of vital genes of fungus (velvet and Fusarium transcription factor 1)	Ghag et al. [94]
Insect resistance	Cotton	Cotton bollworm gut-specific-c cytochrome P450 gene CYP6AE14, which confers resistance to gossypol	Mao et al. [93]
	Corn	In plant expression of dsRNA against western corn rootworm larvae (Diabrotica virgifera)	Baum et al. [95]; Mao et al. [93]
	Tobacco	dsRNA against; EcR-USP (ecdysone receptor ultra-spiracle particle),; AChE (acetylcholinesterase) and HR3 involved in the regulation of molting and development in H. armigera	Zhu et al. [96]; Kumar [97]; Xiong et al. [98]
Nematode attack		Megalaima incognita mitochondrial ATP synthase b subunit) silencing of root-knot nematodes	Huang et al. [99]
Male sterility	Tobacco	Downregulation of anther-specific gene TA29 by RNAi	Nawaz-ul-Rehman et al. [100]
	Tomato	Male sterility RNAi SmTAF10/13	Toppino et al. [101]
	Rice	hairpin RNA for OsGEN-L (OsGEN-like) gene	Moritoh et al. [102]
	Tomato	S-Adenosylmethionine decarboxylase; (SAMDC), control of tapetal-specific A9 promoter using RNAi	Sinha and Rajam [103]
Quality	Maize	Downregulation of lysine-poor zein gene via RNAi	Angaji et al. [104]
	Rice	Increased 2-Acety-1-pyrroline production by silencing OSBADH2 gene	Khandagale et al. [105]
	Cotton	hpRNA-mediated gene silencing of two fatty acid desaturase genes, stearoyl-acylcarrier protein D9-desaturase and oleoylphosphatidylcholine u6-desaturase for the fatty acid composition of cottonseed oil	Liu et al. [106]
	Potato	Silencing the β-carotene hydroxylase gene (BCH) to enhance β-carotene content	Eck et al. [107]
	Brassica napus	Silencing of DE-ETIOLATED1 (DET1) for increased levels of lutein, β-carotene, and zeaxanthin	Wei et al. [108]
	Tomato	Suppressing an endogenous photomorphogenesis regulatory gene, DET1, both carotenoid and flavonoid contents were increased	Davuluri et al. [109]
	Cassava	Removing linamarin, using RNAi silencing CYP79D1/D2	Meena et al. [110]
	Wheat	Enhancing amylose content using asRNA, targeted gene: Sbe2a	Sestili et al. [111]
	Rice	Reduce cadmium RNAi OsPCS1	Li et al. [112]
	Brassica	Reduce erucic acid RNAi BnFAE1	Shi et al. [113]
	Wheat	Reduce glutinin RNAi ɣ-gliadins	Gil-Humanes et al. [114]
Secondary metabolites	Rice	hpRNA from an inverted repeat for glutelin, leading to lower glutelin	Kusaba et al. [115]
	Papaver somniferum	Reduce the levels of the gene encoding the morphine biosynthetic enzyme salutaridinol 7-O-acetyltransferase (SalAT) led to the accumulation of the intermediate compounds, salutaridine and salutaridinol	Kempe et al. [116]
	Cotton	RNAi construct of the d-cadinene synthase gene of gossypol synthesis fused to a seed-specific promoter caused seed-specific reduction of gossypol	Sunilkumar et al. [117]
Keeping quality	Tomato	Chimeric RNAi-ACS construct designed to target ACS homologs effectively repressed the ethylene production	Gupta et al. [118]
	Tomato	SlSGR1 (encoding a STAYGREEN protein, retention of firmness and sustained cell membrane integrity and resulting in delayed fruit senescence	Luo et al. [119]
	Banana	Enhanced shelf-life RNAi MaMADS1/S2	Elitzur et al. [120]

Table 5.

Application of RNAi in crop quality and stress breeding.

Sunar et al. [121] have found the role of siRNAs in response to abiotic stresses. Several studies of abiotic stress tolerance in wheat indicate that siRNA is upregulated when exposed to cold stress but are down-regulated when subjected to heat stress, NaCl and dehydration conditions [122]. Wang et al. [123] reported the contribution of lncRNA in abiotic stress response by utilizing two distinct mechanisms, either they block the miRNA interactions with their target by mimicking as competitive endogenous RNAs. This mechanism of abiotic stress tolerance was reported in rice. The alternative mechanism followed by lncRNAs is antisense lncRNAs interact with sense mRNAs, forming double-stranded RNAs thereby preventing the expression of the gene. Zhang et al. [124] mentioned such interactions in drought stress studies of maize.

7.2 Plant immunity

ncRNAs also regulate plant disease resistance by switching on downstream R-genes, as well as the genes responsible for pathogenesis-related proteins or phenolic compounds or phytoalexins, and several other phytohormones signals in response to pathogen attack. miRNAs are engaged in Resistance gene (R-gene) regulation, whose activation is essential at the time of invasion by the pathogen. miRNAs, such as miR482, are downregulated during pathogen infection in potato, whereas its overexpression may lead to hypersensitive reactions [125]. Tables 6 and 7 shows the crop-wise representation of different miRNA and other ncRNAs in maintaining plant immunity.

Crop	Function and target	References
Arabidopsis	Trigger phasiRNA production target is RPS5	Boccara et al. [126]
	Regulate immune receptor targeting PPRL	Katiyar-Agarwal et al. [52]
	As transcription factor targeting GRFs	Soto-Suarez et al. [127]
	Regulate receptor-like kinase targeting ARLPK1/ARLPK2	Niu et al. [57]
	ROS accumulation, targeting genes; PPR1/PPR2 and At5g38850/At3g04220	Park et al. [128]; Nie et al. [129]
	PR gene expression; MEMB12	Zhang et al. [130]
	Callose deposition; MET2	Salvador-Guirao et al. [131]
	Hormone; TIR1/AFB2/AFB3	Navarro et al. [132]
	miRNA biosynthesis pathway; SERRATE	Niu et al. [57]
Rice	Act as transcription factor for the targeted genes NF-YAs, NAC60 and IPA1	Li et al. [133]; Wang et al. [134]; Liu et al. [135]
	ROS accumulation; ASCORBATE OXIDASE	Wu et al. [136]; Yao et al. [137]
	PR gene expression; Nramp6	Campo et al. [138]; Sanchez-Sanuy et al. [139]
	Callose deposition; ARF16	Li et al. [140]
Tobacco	Trigger phasiRNA production, target is N-gene	Li et al. [141]; Deng et al. [142]
	Trigger phasiRNA production, target is EU713768	de Vries et al. [143]
Brassica	Regulate immune receptor, targets BraTNL1	He et al. [144]
Medicago	Trigger phasiRNA production, Medtr4g023400/ Medtr4g014580/ Medtr5g071220	Zhai et al. [145]
Soybean	Regulate immune receptor	Cui et al. [146]
Barley	Trigger phasiRNA production, MLA1	Liu et al. [147]

Table 6.

miRNA in plant immunity.

Crop	Function and target	References
Arabidopsis	Regulate immune receptor targeting TOE1/TOE2	Zou et al. [148]
Rice	Act as transcription factor for ST1	Zhang et al. [149]
Tomato	Regulate immune receptor	Jiang et al. [150]; Jiang et al. [151]
	Regulate receptor-like kinase	Hong et al. [152]
	ROS accumulation; SIGRX21/SIGRX22 and; RBOH	Cui et al. [153]; Cui et al. [154]
	PR-gene expression; miR168a	Hou et al. [155]

Table 7.

Other ncRNAs in plant immunity.

Other than stress breeding, the aspects of immunity, ncRNA, especially miRNA, have been explored in detail for their potential use in different attributes of crop breeding like enhancing the quality of the yield and its keeping quality, deposition of the secondary metabolites. It can also be used to induce male sterility in the plant, which is an important aspect of hybrid breeding. Table 5 encapsulates different reports of usage of miRNA in various crops in the above-mentioned aspects of crop breeding.

8. Different repositories and databases of ncRNA research

In the recent past, non-coding RNAs have grabbed the attention of many researchers for their role in gene regulation. Different databases for housekeeping non-coding RNA and regulatory ncRNA have been developed which provided the scientists with a lot of information for their functional study. The sequence of tRNA was the one first compiled and published in 1989 with 455 tRNA sequences and 981 tRNA genes [156]. The primitive database for non-coding RNA was ncRNAdb which had 30,000 sequences with no specificities [157] and later the amount of information drastically increased and individual databases for each non-coding RNA are developed, such as silva for rRNA, miRNase for miRNA, snOPY for small nucleolar RNA.

Countless novel techniques had been emerged in the past decade, which led to the discovery of several new ncRNAs and ncRNA genes that are functionally characterized by modern biotechnological tools. Eukaryotic sRNAs, such as miRNA and siRNA, are short sequenced RNA molecules with a length of a maximum of 25 nucleotides. Detection algorithms of sRNA from RNA seq data involve mapping of single or paired-end reads to a reference genome later converted into genome-wide distribution. This is feasible when the size of sRNA is small and where the distribution remains uniform throughout the transcript. However, in bacteria with a lengthier sRNA ranging from 50 to 350 nucleotides, algorithms have to be designed in such a way to overcome the challenges imposed due to the extremely variable number of small transcripts. One such tool is APERO (analysis of paired-end RNA-seq output), which is used to detect bacterial sRNAs from the sequence data of RNA neglecting the need of converting the reads to genome-wide coverage which leads to the loss of information. Instead, it is based on detecting the 5’end of the small transcripts and recognizing the extension of the transcript where the conserved information of sequenced fragments increases the accuracy [158]. Table 8 brings together different databases available for the advanced panel of research on ncRNAs.

S. No.	Database	Specification
1.	tRNAdb	tRNA sequences and tRNA genes
2.	miRTarBase	microRNA-target interactions database
3.	snoRNA	Archaeal snoRNAs
4.	RiboVision	Ribosomal annotations
5.	Rfam	Collection of non-coding RNA families
6.	piRBase	piRNA
7.	NONCODE	Non-coding RNAs
8.	lncRNAdb	Eukaryotic long non-coding RNAs
9.	GtRNAdb	tRNA gene predictions
10.	5SrRNAdb	5S ribosomal RNAs

Table 8.

Different databases and their specifications.

Recent years have witnessed the advancement in sequencing methodologies, such as long-end sequencing and optical mapping, for more accurate and faster sequencing at affordable rates. Cufflinks, and CIRCexplorer [159, 160] are some of the bioinformatics tools used for the discovery of ncRNA. Molecular approaches, such as cloning and hybridization techniques, were able to detect and characterize ncRNAs, but they came with a lot of false positives. Currently, the most reliable approach for predicting and functionally characterizing ncRNAs are NGS (Next Generation Sequencing) and CRISPR-Cas9 genome editing techniques [161]. The list of databases developed for non-coding RNA and specific ncRNA is given below. (https://rnacentral.org/expert-databases) [162].

9. Conclusion

Non-coding RNAs (ncRNAs) possess little or no protein-coding capacity yet are indeed functional. They make up a huge and significant percentage of eukaryotic transcriptomes. It modulates expression levels at various stages of protein synthesis, playing an important regulative involvement in practically all biological processes. MicroRNAs (miRNAs), small interference RNAs (siRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs) are the major non-coding RNAs. These can either operate as long ncRNAs or be converted into small RNAs. They are classed worldwide based on their size, function, and genetic origin. Non-coding modulates its targets via interacting with DNA, RNA, and proteins. These have a role in multiple epigenetic mechanisms controlling phenotypes, as well as the specification of various physiological pathways. MicroRNAs control the level of gene expression by increasing the disintegration of target mRNAs or inhibiting translation. They are involved in many aspects of plant growth and have the power to reconfigure responses to various biotic and abiotic stresses. The modulation of immunological responses in plants has been linked to non-coding RNAs, DNA and RNA methylation, along with other epigenetic changes. Regulatory ncRNAs in plants are being highlighted as potential targets for molecular breeding of agricultural trait improved crop plants, such as improved abiotic and biotic stress tolerance, herbicide resistance, yield, enhancement, and plants with amazing nutritional value with prospective high agricultural importance. Non-coding RNAs (ncRNAs) are also observed to work as a defense system against invading viruses as effectors molecules in RNA-mediated gene silencing and are being exploited in agricultural genetic modification. They also act as key moderators in the level of plant immunity and adaptation to different environments. Plant lncRNAs participate in a wide range of biological processes, including regulation of flowering time and morphogenesis of reproductive organs, as well as abiotic and biotic stress responses. Given the discoveries of these ncRNA in the above-discussed processes, be it physical or physiological, they show a new ray of light toward the use of them in crop breeding. In this regard, the areas, especially the quality breeding, stress breeding for abiotic and biotic stresses, have a huge potential. Along with that, looking at their role in changing the flowering and morphogenesis of plants, further research may be carried forward in the direction of their utilization in altering plant growth duration or producing genotypes for off-season breeding. The role of ncRNAs in epigenetics also can be further studied for their exact role in the inheritance pattern of different important traits. Over the last two decades, the research on non-coding RNAs has shown newer insights about their structure, properties, and possible utilities in different fields of life science. Further work is required to be expanded in newer areas to more agriculturally important crops to harness the wonders of ncRNAs.