Whispers of Nourishment: Unveiling the Role of Non-Coding RNA in Plant Nutrient Availability

Abstract

Micronutrients play a vital role in crop growth and development, as they are essential for various physiological processes and biochemical reactions within plants. Insufficient levels of micronutrients in the soil can lead to nutrient deficiencies in plants, resulting in stunted growth, reduced yields, and poor overall health. In the last decades, it has been seen that noncoding RNAs (ncRNAs) are involved in the regulation of micronutrient uptake, transport, and utilization in plants. They can modulate the expression of genes encoding transporters, chelators, and enzymes related to micronutrient acquisition and utilization. By fine-tuning gene expression, ncRNAs can help plants adapt to varying nutrient availability and optimize their nutrient uptake efficiency. Understanding the interplay between micronutrients and ncRNAs provides insights into the intricate molecular mechanisms underlying nutrient homeostasis and plant adaptation to nutrient stress. This study delves into the origin of various noncoding RNAs, such as miRNA, siRNA, and tsRNA, elucidating their pivotal role in maintaining micro- and macronutrient equilibrium within plant tissues. Overall, this research underscores the intricate interplay between micronutrients and noncoding RNAs in crop plants, shedding light on the intricacies of nutrient regulation and opening up new avenues for future investigation and potential applications in agriculture.

1. Introduction

Plants rely on a minimum of 14 vital phytonutrients, primarily obtained from the soil, to support normal growth, development, and life cycle completion. Investigating the acquisition and distribution of these nutrients is crucial, as their metabolic and biochemical functions play a fundamental role in various parts of plant physiology and functional biology [1]. It is important to carefully study the tissue and subcellular concentrations of these nutrient elements within a limited physiological range [2]. Consequently, the uptake and assimilation of nutrients must be well-coordinated, considering the intricate and controlled interaction between mineral supply and plant demand. Despite the significance of these mineral nutrients, mostly are inaccessible for plants to uptake [1]. As a result, substantial efforts are being followed toward enhancing plant nutrient efficiency in diverse areas.

Plants acquire mineral ions from the soil, employing both long-distance and short-distance transport mechanisms to allocate them in different plant compartments. The distinctive expression patterns of various transporters, which adapt to mineral availability, intricately regulate the molecular processes orchestrating the absorption, conveyance, and loading of mineral ions into storage organs [3]. In instances of inadequate mineral ions within the soil, plants elicit responsive signaling cascades, where the dynamic interplay between signaling molecules and transporter or carrier genes contributes to an enhanced facilitation of ion movement [4]. When a plant encounters a nutrient deficiency, swift signaling molecules promptly relay this information to the plant’s genetic material, ensuring the maintenance of nutritional equilibrium. The scarcity of nutrients triggers the activation of transporters or prompts alterations in root architecture and development, thus optimizing mineral absorption. This intricate process involves signaling molecules engaging in interactions with specific nucleotide sequences within messenger RNA (mRNA), thereby effecting modifications in gene expression [5].

Small regulatory RNAs, regarded as highly pervasive signaling molecules, wield significant influence over posttranscriptional gene expression modulation. These tiny RNA agents form precise bindings with mRNAs and engage in perfect base complementarities, which causes the attenuation of gene expression [6]. Small interfering RNAs (siRNAs) and micro-RNAs (miRNAs) are two of the most important types of small RNA in plant biology. They are closely linked to how genes are turned off. The former, which show up as small 20 to 24 nucleotide regulators, often come from endogenous genomic loci or are brought in from the outside as nucleic acid duplexes. A central role in this regulatory milieu is assumed by Dicer-like enzymes (referred to as DCL in Arabidopsis), adept at cleaving double-stranded duplexes, complete with hairpin loops, thereby yielding concise, impeccable duplexes. These are subsequently harnessed by the RNA-induced silencing complex (RISC). Within the RISC ensemble, the sense strand assumes the roles of recognition and binding to target genes or transcripts. This highly specific interaction leverages perfect nucleotide complementarities and consequently exerts regulatory control over gene expression, operating at either the posttranscriptional or transcriptional echelons [7].

Recent scientific inquiry has unveiled the presence of several novel miRNAs, a veritable frontier, which wield influence over the uptake and conveyance of nutrient minerals within the intricate web of plant biology [6]. The present comprehensive review delves into the contemporary understanding of diverse classes of nutrient-responsive miRNAs, delving deep into their functional roles within the intricate tapestry of nutrient homeostasis. This insightful exploration is poised to illuminate the role of miRNAs as paramount overseers, acting as maestros orchestrating the complex ballet of nutrient loading within plant systems. In this study, we tried to establish the interesting relation of different noncoding RNAs that might be able to control how transporter genes work. This opens the door to a new way of thinking that could have a lot of effects. This paradigm has the potential to revolutionize the landscape of crop biofortification, a process aimed at enhancing the nutritional profile of crops and thereby unraveling new vistas of mineral bioavailability within cereal grains, thereby elevating their intrinsic nutritional value. This, in turn, holds the promise of reshaping the future of agriculture and human nutrition.

2. Biogenesis of noncoding RNAs and its classifications

Various hypotheses are associated with the origins of noncoding RNAs (ncRNAs), exhibiting varieties in biological roles [8]. miRNAs, siRNAs, piRNAs, and lncRNAs are the main types of ncRNAs. These ncRNAs are generated through different mechanisms like duplication of genes, transcription of pseudogenes, from hairpin structure of double-stranded RNA, from heterochromatin regions of DNA and through transposable elements [9].

2.1 tsRNA

tsRNA (transfer RNA-derived small RNAs) is a class of ncRNA; 18–40 nucleotides long fragments originate from tRNA and can undergo site-specific cleavage by various endoribonuclease like Elac Ribonuclease Z 2 (ELAC2)/RNase Z, angiogenin, Dicer, and RNase L, which represent the variety pertaining to their sequence, length, and functional properties. 3’URF, 5′ or 3′ Trh, and 3′ or inter-tRFs are three main categories of tsRNA [10]. Majorly known tsRNA predominantly arise from tRNA, yet the exact mechanism of the biogenesis of tsRNA biogenesis remains uncertain. Nevertheless, some hypotheses have been suggested based on research. It is suggested that different enzymes like Arabidopsis S-like Ribonuclease 1 (RNS1)/DCL1 (Dicer-like protein) [11] take part in the synthesis of mature tsRNAs. tsRNA’s role has been validated in facilitating the acclimatization of plants to adverse environmental condition like stress by regulating target genes during both transcriptional and posttranscriptional phases.

2.2 miRNA

miRNAs, a significant class of ncRNA, are molecules of 20–22 nucleotides in length (Figure 1) [13]. Synthesis of miRNA begins with the miRNA gene, which transcribed product is processed with the assistance of RNA polymerase II followed by RNase cleaves pri-miRNA hairpin to yield a precursor-mi RNA (pre-miRNA). Subsequently, another cleavage event releases an miRNA/miRNA* duplex (Figure 2) [15]. Nuclear HUA ENHANCER 1 (HEN1) protein stabilizes miRNA complex (miRNA/miRNA*) through methylation process in its 3’ prime region [16]. The next step begins with ARGONAUTE 1 (AGO1) protein, which combines with processed miRNA in nucleus and transports into cytoplasm, where it function as posttranscriptional gene silencing [13, 17]. A diverse range of studies in different aspects of plant reveal that miRNA governs the fundamental developmental process like flowering time, leaf morphogenesis, as well as stress conditions such as biotic and abiotic stress [18].

Figure 1.

Flowchart demonstrating the classification of noncoding RNA (nc-RNA) and their length in nucleotides. Figure inspired from [12].

Figure 2.

Flowchart of miRNA biogenesis. Figure inspired from [14].

2.3 siRNA

Based on the mechanism of action and biogenesis, siRNA can be classified into three subtypes including heterochromatic siRNAs (hc-siRNAs), natural antisense siRNAs (nat-siRNA), and transacting siRNA (tasiRNAs). tasiRNA operates in a cis manner, distinct from its biogenesis site, and exhibits similar regulatory mechanism like miRNA, while hc-siRNA acts effectively in a cis manner within its origin or the region that is homologous to its origin [19]. nat-siRNA is further grouped into trans-nat-siRNAs and cis-nat-siRNA. Cis-nat-siRNA originates from the transcription of opposite strands of the same genomic location, while trans-nat-siRNA from the transcription of multiple genomic locations [20]. Synthesis of siRNA involves dsRNA, RNA-induced silencing complex with AGO protein, and DCL enzyme. RNA-induced silencing complex (RISC) governs the regulation at the posttranscriptional or transcription level [21]. DCL4, RNA-dependent RNA polymerase 6 (RDR6), and suppressor of gene silencing3 (SGS3) are proteins involved in the synthesis of tasiRNA [22], while nat-siRNAs synthesis depends on the activities of RDR6, DCL2, SGS3, DCL1, and NRPD1A (plant-specific RNA polymerase) [23]. DCL3, 5 s rRNA, Pol IV, NRPD1A, and RDR2 are involved in the production of hc-siRNAs [24]. siRNA’s role has been established in the developmental process, defense against bacteria, eukaryotic pathogen, and plant growth [25].

2.4 lncRNAs

lncRNAs (long noncoding RNAs) are similar to mRNAs like having poly A tail at the 3′ end and m⁷G cap at the 5′ end but have differences in length and exon number. lncRNA can be categorized into five distinct groups based on different transcription site by pol II, pol III, polIV, or polV. (1) sense lncRNAs are transcribed on the same strand as exons; (2) antisense lncRNAs are transcribed on the opposite strand of exons; (3) intronic lncRNAs are transcribed within introns; (4) intergenic lncRNAs are located between two separate genes; (5) enhancer lncRNAs originate from an enhancer region associated with protein-coding genes [12]. These lncRNAs exert control over target regulation through various mechanisms like chromatin remodeling [26], transcriptional repression, RNA splicing, as well as transcriptional enhancement [27]. Several studies support lncRNA’s important role in flowering time, abiotic stress, and reproduction [28]. Interestingly, lncRNAs can encode small peptides that are also important for numerous cellular process [29].

3. Role of nc-RNAs in soil: Plant-nutrients homeostasis

3.1 miRNAs in nitrogen (N) homeostasis

Among the array of macronutrients, nitrogen assumes a crucial role in the formation of vital cellular constituents like proteins, nucleic acids, and chlorophyll. This significance makes it indispensable for the comprehensive growth of plants [30]. Plants acquire nitrogen in various molecular forms from soil, encompassing ammonium, nitrate, and urea. Notably, nitrate (NO₃⁻) stands as the preferred form among these three, which subsequently undergoes for conversion into nitrite in plant cell and then into ammonium. These converting steps are facilitated by enzymes and culminate in the synthesis of amino acids [31]. So, scarcity of nitrogen in plants places the plant in a precarious situation. There is a wide range of approaches by which plants can overcome the nitrogen-limiting condition. But among all approaches, miRNAs are the efficient tools by which a plant changes internally at transcriptional and posttranscriptional levels to survive in harsh conditions.

The transcriptional patterns of diverse miRNA families have been detected in distinct agricultural plant species in the context of nitrogen. Examples includes soyabean, Arabidopsis thaliana, Bouteloua gracilis [32, 33, 34, 35, 36, 37, 38, 39, 40]. A comprehensive summary of various miRNAs participating in the response to nitrogen limitation and their target is listed in Table 1.

Sl.No.	miRNAs family	Target	Function	Plant species	Reference
1.	↑miR167	↓AUXIN RESPONSE FACTOR 8	Trigger lateral root outgrowth	Maize	[41]
2.	↑miR160 and miR171	↓ARF10/16/17	Lateral and adventitious root development	Maize	[42]
3.	miR171	SCARECROW-LIKE PROTEIN 6	Root development	Arabidopsis	[43]
4.	miR393	AUXIN-SIGNALING F-BOX PROTEIN 3 (AFB3)	Root system architecture	Maize	[44, 45]
5.	↓miRNA169	↑NUCLEAR FACTOR Y SUBUNIT A5 (NFYA5)	Downregulates N uptake	Maize	[46]
6.	↓miR164	↑NAC1	Lateral root production	Arabidopsis	[47]

Table 1.

Function and target gene of miRNA for nitrogen homeostasis in different plants.

↓ down regulation ↑ upregulation.

3.1.1 NcRNAs orchestrating nodulation and symbiosis in leguminous plant for nitrogen uptake

Higher plants demonstrate a range of adaptive strategies to enhance the capacity of roots to uptake nitrogen from soil. Among all forms of nitrogen available for uptake, ammonium is harnessed through the process of symbiotic nitrogen fixation. In this, ammonium is culminated via bacterial enzyme nitrogenase followed by conversion into accessible form [48]. Before the commencement of N₂ fixation, nodule development in the pulse crop follows different steps in succession starting from nodule organogenesis, bacterial infection, and the initiation of nitrogen fixation [49]. This process is triggered by the exchange of diffusible chemical signals between the host plant and symbiotic partner followed by bacterial penetration via infection into plant’s root hairs. Origination of nodule primordium is the attribute of infection thread, which divides into the plant cortex. Eventually, bacteria are liberated from the infection thread by endocytotic mechanism, leading to the formation of symbiosomes, where nitrogen fixation is executed [50]. In the favor of fixing nitrogen, bacteria receive carbohydrates as well as mineral nutrients.

Nodule development process is fully investigated and characterized at the molecular level [51]. On the other hand, miRNA also has a role in the nitrogen fixation process. Many miRNAs have been discovered till date who are involved in legume-rhizobia symbiosis [52]. Lelandais-Brière et al. [53] identified 100 conserved and novel miRNAs from nodules synthesized in Medicago truncatula roots 21–30 d postinoculation and suggested their role in the development of nodule. In soybean (Glycine max), sRNA library is sequenced and total 129 miRNAs generated in which 87 miRNA were novel from distinct developmental stages like root, seed, flower, and nodules [54]. In this pulse crop 15 small RNA libraries were sequenced in young, mature, and senescent nodules stages and 139 miRNAs were identified. Five parallel analysis of RNA ends (PARE) libraries sequenced and identified 533 miRNA targets including 8 nodule-specific genes. In Table 2, we have provided the function and list of miRNAs in the nodulation process.

S. No.	miRNA	Target	Function	Crop Species	Reference
1.	miR166	HD-ZIP III TF	Root nodule development inhibition	Medicago truncatula,; Glycine max	[55, 56]
2.	miR171c	NSP2 TF	Nodule infection	Legume lotus japonicas; M. truncatula	[57]
3	gma-miR171o, gma-miR171q	GmSSCL-6, GmNSP2	NIN,ENOD40 and ERN genes	Glycine max	[58]
4	miR396b	MtNSP2	Nodule formation	Medicago truncatula	[59, 60, 61]
5	miR393j-3p	Early Nodulin 93	Inhibition of nodule formation	Glycine max	[62]
6	miRNA169	Glyma10g10240 and Glyma17g05920	Inhibit HAP proteins	Soybean (Glycine max)	[63]
7	miR2118, miR2109, and miR1507	silencing of target NB-LRR genes post-transcriptional	Symbiotic interaction with rhizobia	Medicago truncatula	[64]

Table 2.

Role of different miRNAs in nodulation of leguminous crops for nitrogen uptake.

3.2 Role of miRNA in phosphorus (P) homeostasis

Phosphorus (P) stands as a pivotal element supporting an array of physiological and biochemical functions. It actively engages in a diverse spectrum of metabolic processes, notably in the synthesis of nucleic acids and the generation of energy within plants. It enters a wide range of metabolic processes, specifically, the synthesis of nucleic acids and energy generation of plants, which makes it hard for plants to grow under P starvation. This significance renders it challenging for plants to thrive in conditions of phosphorus scarcity.

Recent investigations have shed light on the role of miRNAs (small RNA molecules) in orchestrating the intricate regulation of phosphate-related gene expression across various plant species, such as Arabidopsis, rice, wheat, barley, maize, soybean, white lupin, and tomato [65]. In Arabidopsis, the detection of low phosphate levels by the shoots promptly triggers the synthesis of miR399a-f families. These miRNAs, particularly miR399, intricately bind to complementary sites within the phosphate over accumulator 2 (PHO2) transcript, positioned in the 5′ untranslated region (UTR) about 200–400 base pairs upstream. This binding event triggers mRNA cleavage. Notably, PHO2 serves as a pivotal transporter facilitating phosphate mobilization and is linked to the E2 ubiquitin conjugating enzyme. The downregulation of PHO2 assists in accumulating substantial phosphate levels within the shoots. Conversely, a long noncoding RNA known as induced by phosphate starvation1 (IPS1) plays a counteractive role. IPS1 inhibits the miR399-mediated regulation of the PHO2 gene, safeguarding the PHO2 transcript from degradation [66].

Remarkably, miR398a’s expression is subject to regulation not only by phosphorus availability but also by carbon (C) and nitrogen (N) limitations, hinting at its broader involvement in mineral homeostasis. Suppression due to carbon limitation has also been shown to contrast with its induction by sucrose [67]. Additionally, in the context of soybean roots, during phosphorus deficiency, miR159a experiences upregulation, while miR319a, miR396a, miR398b, and miR1507a undergo downregulation. This involves the modulation of miRNA expression in response to phosphorus starvation hinges on the prevalence of phosphorus-responsive motifs (P-responsive motifs) within the miRNA gene promoters. Notably, the cis-acting regions of miRNA genes are reported to contain a higher abundance of P-responsive motifs compared to their nonresponsive counterparts [68].

3.3 Role of miRNA in potassium homeostasis

Potassium (K) is a critical inorganic nutrient that is essential for plant growth, development, and yield formation in cereal crops [69]. Many agricultural soils around the world are K deficient, which limits sustainable crop development [70]. In Arabidopsis, the first upregulated miRNA, miR156, was characterized in the K deficiency condition. Later on, other miRNAs like miR169, miR395, and miR398 were also found to be responsive under K deficient conditions [71]. These miRNAs target genes like MADS-23, MADS-27a, MADS-27b, and MADS-57; synthesize a transcription factor; and ultimately endow the plant with nutrient deficient conditions [41]. Previous research has shown that the module miR319/TCP4 and the pathways facilitated by the module miR396/GRF play a significant role in enhancing barley’s ability to withstand low-K stress. Additionally, it has been found that ata-miR1432-5p functions as a regulator in the transduction pathways of Ca2+ signaling, which are triggered by low-K stress [42]. Furthermore, the involvement of stu-miR530_L-2R + 2 in potato has been shown in the modulation of responses to K treatments via the regulation of a specific gene that encodes a protein belonging to the Zinc Knuckle (CCHC-type) family, known as protein [43]. In barley, it has been reported that miR408 responds to low K through the blue copper protein (BCP) [44]. Collectively, these data suggest that microRNAs (miRNAs) have a role in facilitating plant responses to nutritional stress.

In wheat, an insightful exploration by Zhao [45] unearthed a noteworthy revelation. Among the 89 miRNA families investigated, a distinctive pattern of differential expression emerged under conditions of potassium (K) deficiency. This encompassed 68 families with known attributes, while an additional 21 families were newly identified. Notably, 11 specific miRNAs, namely, novel_17, miRNA319, miRNA531, miRNA9773, miRNA9670-3p, miRNA398, miRNA159a, miRNA9778, miRNA408, miRNA9776, and miRNA1133, demonstrated close associations with plant resilience in the face of K starvation. Delving deeper into these differentially expressed miRNAs through gene ontology and KEGG analysis, their roles were found to fall within the purview of ADP-binding activity and secondary metabolic pathways. A notable instance was the robust impact of tae-miR408 over expression, which not only enhanced plant morphology but also bolstered K-deficiency tolerance. This enhancement was achieved by elevating K uptake efficiency, stimulating photosynthetic pigment biosynthesis, and fostering a harmonious balance in ROS homeostasis.

3.4 Role of miRNA in calcium (Ca) homeostasis

Calcium, a fundamental macronutrient indispensable for plants, is required in significant quantities. Its pivotal role encompasses shaping plant cell structures and occupying a central position as the predominant secondary messenger in relaying responses to environmental stresses. According to some studies, cation channels and the apoplast facilitate the uptake of Ca²⁺ by plants from the soil. Following this absorption, the transpiration stream facilitates the subsequent transport of Ca²⁺ to the shoot through the xylem [46].

MicroRNAs (miRNAs) have significant influence across various dimensions of plant biology, spanning growth, development, stress responses, and epigenetic regulation. In a recent study on the orphan crop Eragrostis tef, researchers have found a strong link between the amount of miRNA in the root and shoot tissues and how much calcium (Ca) the plant took in [72]. This investigation unveiled noteworthy insights like E. tef plants enduring prolonged Ca²⁺ deficiency exhibited the identification of 1380 miRNAs, whereas control plants manifested the detection of 1495 miRNAs. In this comprehensive endeavor, a total of 509 miRNAs surfaced, comprising 161 miRNAs akin to those characterized in diverse plant species and 348 novel miRNAs. The remaining miRNAs remained enigmatic in their characterization. Furthermore, comprehensive predictions were made for the putative target genes and their roles, corresponding to both the known and newly identified miRNAs. Through gene ontology (GO) analyses, a diverse array of biological and molecular functions surfaced for the projected target genes, including roles in calcium absorption and transport. The discernment of differentially expressed miRNAs underscored distinct enrichments in either root or shoot tissues of plants exposed to low Ca²⁺ conditions. The potential of delving deeper into the intricacies of these miRNAs and their intertwined targets from this research venture stands to offer a heightened comprehension of how tef and similar underexplored crops respond to calcium deficiency.

In parallel, this paradigm extends to the area of peanut (Arachis hypogaea), warranting comparable in-depth exploration. A set of twelve distinct miRNA families specific to peanuts were identified, encompassing a group of 29 previously recognized miRNAs and 132 potential novel ones. According to the research by Chen [73], a total of 87 miRNAs exhibited varying expression patterns during the initial stages of embryonic development, both in conditions of insufficient calcium and at adequate calcium levels. The study further pinpointed 117 target genes that displayed differing levels of expression. An integrated investigation combining miRNA and transcriptome expression revealed that twenty miRNAs underwent expression changes, consequently impacting the expression of 52 target genes. By comparing gene chip analysis and transcriptome sequencing, unique targets with distinct expression patterns were identified. The study’s findings underscore the substantial role of microRNAs (miRNAs) in governing the expression of genes implicated in embryonic development. In particular, genes such as TCP3, AP2, EMB2750, GRFs, and cytochrome P450 (CYP707A1 and CYP707A3) involved in abscisic acid (ABA) signaling, along with BR1, which facilitates brassinosteroid (BR) transport, are actively influenced by miRNAs. Both miRNAs and their corresponding target genes are thought to participate in the process of peanut embryo abortion as a response to inadequate calcium. This study reveals the presence of miRNA-mediated regulatory systems involved in embryo abortion in peanuts, particularly when calcium is lacking [73].

3.5 Role of miRNA in magnesium (Mg) homeostasis

Magnesium (Mg), a core macronutrient involved in plant growth and development, has pivotal function in chlorophyll biosynthesis and acts as cofactor in carbon metabolism [55]. Magnesium deficiency conditions are prevalent in sandy, acidic soils where magnesium is very susceptible to leaching. Although the importance of magnesium in plants is well established, miRNA’s involvement in Mg homeostasis is relatively underexplored. Liang [56] conducted a study in Citrus sinensis to identify miRNAs that provide tolerance in Mg-deficiency conditions via Illumina sequencing. They identified 101 miRNAs were upregulated and 69 miRNAs downregulated in roots. In Mg-deprived conditions, miR158 was expressed, which repressed its target gene SPFH (stomatins, prohibitins, flotillins, and HflK/C). In Mg-starved conditions, miR1044, miR5029, miR6485, miR6190, and miR3437 control the expression of transport-related genes. Root development controlled by miR5261, miR6485, and miR158 in magnesium reduces conditions [56]. miR2919 inhibits root respiration by targeting phosphoenol pyruvate carboxylase 3 (PEPC3), which eventually induces low respiration and reduced accumulation [57]. Surprisingly, one more interesting finding suggested that disease resistance capacity elevated in Mg deprives plants by targeting NB-ARC domain-containing disease resistance protein through miR780 and miR6278 [56].

3.6 Role of miRNA in Sulfate (SO4) homeostasis

Sulfur, a crucial micronutrient for plant, is available in the form of carbohydrate, proteins, and sulfo-lipid. Inorganic sulfur is the most predominant form of sulfur that is translocated into different tissue. SULTR1;1 (sulfate transporter1;1) and SULTR1;2 (Sulfate transporter1;2), two vital S carriers, are responsible for sulfate uptake from topsoil [58]. SLIM1, a transcription factor, regulates sulfur utilization by controlling transporter like SULTR1;2 [59]; the enzyme APS, found in various isoforms enzyme (APS1, APS3, APS4), is a key player in S assimilation pathway [6]. APS1, APS4 within plastid facilitate sulfur reduction, leading to cysteine formation [60]. Sulfate-deprived condition triggers to activate SULTR1-2, SLIM1, and other S-deprivation response genes, which eventually upregulates miR395. Up regulation of miR395 lead to downregulation of three APS genes (APS1, APS3, APS4) and SULTR2;1 transcripts which eventually accumulate the sulfate in shoot and disrupt the transport of sulfate from mature leaves to young leaves respectively [61]. This cascade of pathways ultimately affects S uptake, facilitates phloem transport from roots to shoots, and also concurrently inhibits xylem-mediated shoot-to-root transfer [62]. Sulfur-deprived conditions change the expression of miR167, miR168, miR167, miR394, miR164, miR160, and miR156 in Brassica napus [63].

3.7 Role of miRNA in iron (Fe) homeostasis

Iron is an essential micronutrient, serves as cofactor in the electron-transport chains of both photosynthesis and respiration, and also functions in other cellular processes. Low availability of iron initiates a condition of stress in plants, as well as its high accumulation exerts detrimental impacts on plants by disruption lipid, protein, and DNA through Fenton catalytic reaction. miRNA responds in both situations, iron deficiency and iron toxicity. In an experiment, 8 miRNAs were cloned and small RNA library was constructed from Arabidopsis seedlings under Fe-deficiency condition, and it concluded that the expression of miR394b, miR173, miR172c, miR172d, miR169b, and miR169c families initially was higher followed by a lower expression, but miR394a and miR159a were upregulated at the end of trial during Fe-deficiency period [64].

Paul [74] proposed that significant difference in iron content between transgenic plants and their WT (wild type) counterpart was documented during the milk stage in an experiment, resulting in upregulation of some transporters in roots of transgenic plants by over-expressing soyFER1 gene. They also found a reciprocal association between low miRNA expression, specifically miR9, miR38, miR21, and miR28, and higher expression of target SAM-dependent carboxyl methyl transferase gene in root tissue. Notably, the study unveiled that expression level of OsNRAMP4 (iron transporter) was elevated in roots during the milk stage with lower expression of the four novel miRNAs: miR11, miR26, miR30, and miR31.In one study, miR399 and miR408 families were found to be significantly expressed in transgenic plant roots that enhance the expression of iron transporters by minimizing the activity of a repressor gene [75].

3.8 Role of miRNA in zinc (Zn) homeostasis

Zinc stands out as a highly essential micronutrient that serves a pivotal role as a catalytic, regulatory, or structural cofactor for a multitude of enzymes and regulatory proteins within both plant and animal systems [76]. The exploration of distinct miRNAs has yielded valuable insights into the intricate process of zinc ion uptake from soil. Through the utilization of miRNA microarrays, research focused on Sorghum bicolor has pinpointed eight miRNA families that exhibit responsiveness to zinc deficiency. Particularly, miR398 and miR528 were found to target two Cu/Zn superoxide dismutase genes, SbCSD1 and SbCSD2, respectively [77].

Numerous zinc-responsive microRNAs (miRNAs) play pivotal roles in regulating plant growth and development. MiR408 targets plantacyanin, influencing ROS signaling, defense, and reproduction. MiR166 regulates root development through HD ZIP proteins. MiR168 affects AGO1, while miR171 targets the SCL6 transcription factor. MiR319b modulates jasmonic acid production, and miR528 controls CSD expression [77]. Among them, miR171 and miR528 showed significant downregulation in leaves, while miR398, miR319, miR166, miR168, and miR399 were upregulated in roots. Resupplementation experiments validated their roles in phytohormone biosynthesis. Zinc deficiency influenced CSD expression differently for miR398 (downregulation) and miR528 (upregulation) [77].

In a similar vein, according to SHI [78] report, Brassica juncea has also demonstrated a variety of miRNAs that control zinc uptake. Within the spectrum of 21 conserved miRNA families, the spotlight falls on nine families displaying differential expression patterns. When B. juncea roots did not have enough Zn, 13 miRNAs were found to be upregulated, while miR399b and miR845a were downregulated. When the roots of B. juncea are exposed to abiotic stress, these miRNAs are changed. This changes how phytohormones react, how plants grow, and how they react to abiotic stress. In the case of rice, a full combination of miRNAome and transcriptome data showed that 12 different genes had different levels of expression and could be targets for 10 miRNAs that respond to Zn. Among these miRNAs are miR171g-5p, miR397b-5p, miR398a-5p, and miR528-5p [44].

3.9 Role of miRNA in manganese (Mn) homeostasis

Manganese (Mn) plays a significant role as a micronutrient in overseeing the progression and maturation of plants. It serves as the metallic element within several proteins and enzymes, including superoxide dismutase (SOD) and the oxygen-evolving complex within photosystem II. Furthermore, Mn plays a role in the creation of secondary metabolites [79]. It is an inorganic catalyst, facilitating the enzymatic processes necessary for plant growth. Mn plays a crucial role in various biochemical functions of plants, including photosynthesis, respiration, nitrogen assimilation, and pollen germination. It is indispensable for pollen tube growth, elongation of root cells, and shielding roots from pathogens [80]. However, limited research has delved into Mn toxicity in plants, necessitating further investigations into the potential modulation of Mn homeostasis by miRNAs and their target genes. As a result, a comprehensive understanding of posttranscriptional regulation regarding Mn homeostasis remains challenging to obtain.

Manganese (Mn) homeostasis has been observed to trigger changes in the expression of certain microRNAs (miRNAs). Notably, an excess of Mn leads to an increase in the expression of miR826. This miRNA specifically targets AOP2 and its related counterparts, AOP1 and AOP3. These genes, which are called AOPs as a group, make enzymes called 2-oxoglutarate-dependent dioxygenases. These enzymes help to make glucosinolates. It is reported that when miR826 is over-expressed in transgenic plants, the levels of glucosinolates and anthocyanins go down and the plants can handle less nitrogen (N) better. These findings suggest that excessive Mn disrupts nutrient balance by influencing the expression of miRNAs. Another miRNA, miR781, is associated with mini chromosome Maintenance 2 (MCM2), a gene known to influence root meristem function and play an essential role in embryonic development, as indicated by research by Sabelli [81]. The upregulation of miR781a and miR781b in Mn-treated seedlings corresponds to observable changes in root growth patterns. Moreover, miR5595 and miR5995b are predicted to target Methyl Esterase 7 (MES7) and MES9, genes involved in the hydrolysis of methyl-SA (MeSA) and systemic acquired resistance in plants [82]. Intriguingly, both miR5595a and miR5995b are upregulated in response to Mn treatment. These collective findings highlight the intricate relationship between miRNA expression and the effects of Mn homeostasis on various aspects of plant physiology and growth.

Researchers employed both miRNA microarray hybridization and qRT-PCR techniques to identify miRNAs responsive to Mn stress in common beans (P. vulgaris). The study revealed 37 miRNAs exhibiting distinct expression patterns under abiotic conditions and Mn stress. Notably, Mn toxicity induced the activation of 11 miRNAs while inhibiting 11 others. New Mn-responsive miRNAs, including miR1508, miR1515, miR1510/2110, and miR1532, were discovered. Key targets among the Mn-responsive miRNAs were found to be leucine-rich repeat-resistant proteins, receptor kinase proteins, and calcium-dependent protein kinases [83]. Furthermore, recent research focusing on Arabidopsis has demonstrated differential expression of miRNAs linked to cell growth, nutrient balance, and ion transport under Mn stress conditions [84].

3.10 miRNA in boron stress

Boron (B) plays a vital role in promoting proper plant growth and development by engaging in a range of physiological processes. These include supporting protein and nucleic acid metabolism, metabolizing lipids, facilitating cell division, and maintaining cell walls [85]. Research indicates that miRNAs contribute to responses against B-induced stress. For instance, barley’s B-stress responsive miRNA network and associated pathways have been investigated [86]. Moreover, a comparative study of miRNA profiles between root and leaf samples was conducted. Within barley, 31 established and three novel miRNAs were identified, with 25 of them showcasing responsiveness to B treatment. Specific miRNA expression patterns were observed in distinct tissues; for example, miR156, miR171, miR397, and miR444 were exclusively expressed in leaves. These miRNAs specifically targeted and degraded 934 transcripts in barley. Furthermore, in the context of elevated B levels, variations in miRNA expression were detected in French beans (Phaseolus vulgaris L.), and the impacts of miRNA targets on gene expression under B stress were explored. This investigation was corroborated by Gene Ontology (GO) analysis, confirming the involvement of plant miRNAs in various cellular processes, including the circadian rhythm and vegetative development [87].

Citrus trees frequently encounter a widespread issue of boron (B) deficiency. Several studies have revealed the presence of B-responsive miRNAs in citrus species. Through high-throughput Illumina sequencing (HTIS) of C. sinensis roots, it was uncovered that 52 miRNAs were upregulated and 82 were downregulated. This showcases the remarkable adaptability of roots, which aids plants in enduring B-depleted conditions [88]. Certain miRNAs were proposed to influence root adaptation to B deficiency in several ways: (a) enhancing miR474 while reducing miR782 and miR843 to counteract reactive oxygen species (ROS); (b) boosting miR394 expression while dampening miR5023 expression to heighten cellular sensitivity to B deficiency; (c) lowering miR830, miR5266, and miR3465 levels to enhance fluid transport within cells; (d) managing osmo-protection via miR474 and other metabolic reactions through miR5023 and miR821. The research revealed that other miRNAs, such as miR472 and miR2118, exhibited reduced expression as B deficiency progressed, subsequently diminishing the presence of disease resistance genes and reducing root disease resistance [88]. Likewise, an examination of trifoliate orange (Poncirus trifoliata) through RNA transcript analysis revealed a decrease in miR397 levels following excessive B treatment. This treatment led to elevated transcription of laccase7 (LAC7), a target of miR397. Moreover, the treatment notably heightened laccase activity, underscoring the pivotal role of LAC7 in protein biosynthesis [89].

It has been shown that too much boron (B) has a genetic effect on wheat, especially when it comes to the expression of jasmonate (JA), ethylene, and changes in the cell wall. Under hazardous B conditions, Arabidopsis miRNAs that target JA and ethylene-related transcription factors (miR172 and miR319) and laccase (miR397) were studied. For the analysis of mature miRNA expression, Kayihan [90] recommended stem-loop qRT-PCR. In response to mild B toxicity, high levels of miRNAs that target transcription factors involved in the metabolism of jasmonic acid (JA) and ethylene stood out. However, this response was notably absent under severe B-toxicity conditions (designated as condition 3B). Among the regulating factors, the predominant influence was attributed to the miR172 and miR319 genes. On the other hand, the expression of miR397 exhibited no statistically significant alteration upon exposure to B toxicity, indicating that laccase-induced changes in the cell wall are not subject to posttranscriptional control. Furthermore, Arabidopsis thaliana exhibits a multifaceted ability to perceive oxidative stress and counter boron toxicity. This is achieved through the activation of miRNA expression, specifically targeting transcription factors associated with JA and ethylene metabolism [90].

3.11 miRNA and copper stress

Copper is an indispensable micronutrient that participates in a diverse range of physiological processes. However, plastocyanin, copper/zinc superoxide dismutase (CSD), and cytochrome c oxidase (COX) are highly prevalent Cu-rich proteins [91]. Cu-deficient conditions in plants are responsible for loss in agriculture production. Various approaches for modulating Cu homeostasis within plant have emerged, which involve the upregulation of Cu-miRNAs, eventually target mRNAs, and induce the release of Cu from protein to fulfill other essential requirements in plant [92]. miR398, miR397, miR408, and miR857 are the most significant miRNAs that respond in Cu deficiency [93]. In this process, Cu-responsive transcription factor SPL7 interacts with GTAC motifs in the MIR398 promoter region; eventually turns on the degradation of Cu/Zn-SOD, CCS1, and COX5b-1 mRNAs; and facilitates the transfer of Cu from CSDs to PC; Cu/Zn-SODs biological process switches to Fe-SOD (FSD1) [94]. Moreover, in the Cu-deficient conditions, the miR398-dependent downregulation of CCS1 facilitates Cu release from CSDs [95]. Several miRNA have been studied that control transcription factor in Cu-deficient conditions such as miR156 regulate SPL 3 (SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE) transcription family member at posttranscriptional stage, activate in Cu-scarce condition, and act accordingly based on cues. RT-qPCR study for miR156, miR157 was conducted to study the expression level for different Cu concentration in media, and it was confirmed that miR156 and miR157 expressed more in shoot compared with in root [96].

4. Role of miRNAs in metal toxicity

Crop productivity is significantly hindered by metal toxicity, which encompasses both indispensable plant metals like copper, iron, zinc, and manganese, and nonessential metals such as cadmium (Cd), aluminum (Al), cobalt (Co), and mercury (Hg). An overarching consequence of elevated metal concentrations—be it aluminum, copper, cadmium, or mercury—is the suppression of root growth. The harmful impact of metal toxicity prompts the accumulation of reactive oxygen species, leading to detrimental effects on lipids, proteins, and DNA. The plant’s reaction to metal toxicity involves intricate biological mechanisms that demand precise regulation at both transcriptional and posttranscriptional tiers [97].

Examinations of small RNA expression patterns in plants exposed to metal toxicities have illuminated distinct miRNA expression patterns and their corresponding target variations, hinting at potential regulatory or signaling functions. A significant proportion of predicted targets for metal-responsive, evolutionarily conserved miRNAs are transcription factors, primarily associated with plant development. When a particular miRNA experiences upregulation leading to target degradation, it suggests that the targeted element might act as a negative modulator in the plant’s response to metal toxicity. Recent advancements in high-throughput genomic technologies and other genetic-genomic approaches have substantially expanded our understanding of miRNAs and their interactions with targets within signaling pathways concerning plant responses to metal toxicities. This growing body of knowledge has been gleaned from various plant species, further enhancing our insights into the intricate roles played by miRNAs in orchestrating responses to metal-induced stresses [98].

When it comes to miRNAs that respond to cadmium (Cd) toxicity, a set of conserved and new miRNAs have been found in rice seedlings that were exposed to Cd. Among the conserved miRNAs, miR160, miR164, and miR167, along with the novel Osa-miR602 and Osa-miR604, were discovered [99]. Notably, Osa-miR602 exhibited upregulation in rice roots after a 12-hour exposure to elevated Cd levels, with a xyloglucan endotransglucosylase/hydrolase as its predicted target. On the other hand, Osa-miR604, found to be upregulated in Cd-treated leaves for 6 hours, which led to the down regulation of a lipid transfer protein (LPT) [99]. In rice microarray data, the response to Cd stress revealed upregulation of miR528, while miR162, miR166, miR171, miR390, miR168, and miR156 families were downregulated [100]. Investigation into potential metal-responsive cis-acting elements highlighted the presence of a MRE-like sequence (5′-TGCGCNC-3′) in the promoter regions of most Cd-responsive miRNA genes [100].

In B. napus roots, Cd exposure for 8 hours led to downregulation of miR393, miR171, miR156, and miR396 [101]. Similarly, in the leaves of the model legume M. truncatula, Cd, Hg, and Al exposure triggered the upregulation of miR393, miR171, miR319, and miR529, while miR166 and miR398 were downregulated [102]. High-throughput small RNA sequencing unraveled the downregulation of miR159, miR160, miR319, miR396, and miR390 in response to aluminum (Al) toxicity [103]. More recently, a similar approach has identified miRNAs responsive to mercury (Hg) toxicity, including the upregulation of miR167, miR172, miR169, miR164, and miR395 families, while miR396, miR390, and miR171 were downregulated in this legume. Moreover, novel Hg-responsive miRNAs like miR2681, targeting transcripts coding for TIR-NBS-LRR disease resistance proteins, were uncovered in M. truncatula [102]. These findings underscore the intricate network of miRNA responses to diverse metal toxicities across various plant species.

5. Limitations of ncRNAs in plant-nutrient homeostasis

Nutrient uptake and metabolism are highly complex processes involving multiple genes, pathways, and regulatory mechanisms. Noncoding RNAs are just one layer of this complexity, and they may not be the primary regulators of nutrient uptake in many cases. Only ncRNAs do not act in isolation but are part of a larger regulatory network that includes transcription factors and other regulatory elements. Understanding how ncRNAs interact with these other components is crucial for a comprehensive understanding of nutrient uptake regulation. Our understanding of the role in specific ncRNAs in nutrient uptake is still evolving. The role of ncRNAs in regulation of nutrient transportation has not undergone comprehensive studies till date [104]. While some ncRNAs have been identified as regulators of nutrient-related genes, there is much we do not know about the intricacies of these regulatory networks.

miRNAs often target multiple genes, and a single miRNA can regulate the expression of several mRNAs. This lack of specificity can make it challenging to precisely control the expression of genes related to nutrient uptake without affecting other important cellular processes. Just for example, downregulation of miR169 in Arabidopsis thaliana affects the expression of multiple genes like nuclear transcription factor Y subunit-alpha (NFYA) genes, namely, NFYA8, NFYA5, and NFYA2 [37]. Similarly, over-expression of miR169a strongly correlates with the inhibition of nitrate transport system (NRT) genes, AtNRT2.1 and AtNRT1.1. So, this nonspecific binding of miRNA with multiple genes may create a problem for regulation of miRNA in nutrient uptake as well as transgenic development [37]. Secondly, redundancy creates a major problem for researchers for proper target identification. Many miRNAs have redundant functions, meaning that multiple miRNAs can regulate the same target genes. For example, in M. truncatula, multiple miRNAs, namely, miR2118, miR2109, and miR1507, act on NB-LRR genes for symbiotic interactions. This redundancy can compensate for the loss of one miRNA, making it difficult to observe significant phenotypic changes solely by manipulating a single miRNA [64]. In some studies, it was also observed that miRNA reduces the growth of lateral roots. miR167 serves to impede the accumulation of ARF8 (Auxin Response Factor 8) within the pericycle cell layer, which leads to reduction in the formation of lateral roots as well as N metabolic enzymes that are produced by the downstream of nitrification and absorption [105, 106]. Certain noncoding RNAs exhibit pleiotropic morphology effects on traits in transgenic situations, raising doubts about the precision of miRNA transformation for a specific event [107]. Conducting experiments to manipulate miRNAs and study their effects on nutrient uptake can be technically challenging. When miRNAs are subjected to cytoplasm and released from endosome, off-target effects play a major concern. These noncoding RNAs are produced to target various nutrient uptake pathways by imperfect hybridization with 3′ UTRs; thus, they might cause undesirable gene silencing of other genes. This off-target gene silencing can result in potential harmful effects and diminished therapeutic efficacy. The fact that a single miRNA could potentially affect multiple mRNA targets necessitates careful consideration, as it suggests the potential for unforeseen side effects [108].

6. Conclusion

Exploration into the realm of microRNAs (miRNAs) has illuminated their pivotal role in shaping the landscape of nutrient uptake within plants. The presented studies intricately elucidate the symbiotic interplay between miRNAs and diverse nutrient stresses, encompassing both micro- and macronutrient deficiencies. These revelations underscore the profound influence of miRNAs in orchestrating gene expression, thereby exerting a discernible impact on critical facets such as ion transport, metabolic adaptability, and acclimatization to nutrient-scarce conditions. The holistic comprehension of miRNA-mediated mechanisms beckons us toward promising avenues for augmenting crop productivity and bolstering resilience amidst the challenges posed by nutrient-related constraints. A deeper plunge into the intricate tapestry of miRNA-nutrient stress interactions harbors profound potential for devising innovative strategies that fine-tune nutrient uptake and utilization in plants. This endeavor aligns seamlessly with the objectives of sustainable agriculture and the global quest for food security. Furthermore, it is imperative to shift our focus toward unraveling the nuanced involvement of miRNAs in nutritional stress responses. This endeavor necessitates a keen observation of the phenotypic and physiological shifts induced by these regulatory processes. While the spotlight often falls on miRNAs, it is worth acknowledging that nutritional stress can also extend its reach to transposon-derived RNAs and tRNA-derived RNAs, with their precise physiological roles yet to be unraveled. In the pursuit of fortifying future food security, harnessing miRNA-centric strategies emerges as a vital linchpin. Based on the potential of miRNA-based interventions, it might be possible to make crop varieties that are more productive and resistant to both natural and man-made stresses.

Acknowledgments

We are sincerely grateful to the esteemed Vice Chancellor of G.B. Pant University of Agriculture and Technology (GBPUAT), Dean CBSH and Hrad MBGE, for their unwavering support and dedication in making this endeavor possible.

Conflict of interest

The authors declare no conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.