Plants are continuously exposed to various biotic and abiotic stresses during their growth and development. In light of the challenges imposed by the global climate crisis, the need for improved plant varieties is increasing and urgent [1]. For the genetic improvement of crop plants, an in-depth understanding of regulators of growth, development and stress responses is key to improving agronomic traits [2]. microRNAs (miRNAs) are important post-transcriptional regulators of gene expression and play pivotal roles in various biological processes. The verified targets of miRNAs mostly encode transcription factors (TFs) and enzymes in signaling pathways, indicating that miRNAs play vital roles at the core of gene regulatory networks [3]. microRNA398 (miR398) is a highly conserved miRNA which is widespread in angiosperms [4]. As one of the earliest miRNAs cloned from Arabidopsis and rice ^[5][6][5,6], extensive studies of the mechanism of transcriptional regulation, biological function and environmental stress responses of miR398 have been carried out; the function of miR398 in plant stress responses has been reviewed by Zhu et al. [7].

2. Role of MiR398 and Its Targets in Abiotic Stress Responses

Crops and other plants in the field are subject to a combination of different abiotic stresses. The tolerance of plants to different stress combinations is an important area of research. 

2.1. Oxidative Stress

The miR398-CSD module is conserved across seed plant species, and its function was first identified in the adaptation of plants to oxidative stress ^[8][11]. High concentrations of ROS, including superoxide (O₂^•−), hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH^•), can oxidatively damage biomolecules (such as lipid proteins, nucleic acids, etc.). Environmental stress caused by heavy metals, high levels of light, salinity, drought, extreme temperature, air pollutants, ultraviolet-B (UV-B) radiation, pesticides and pathogen infections leads to a rapid, excessive accumulation of ROS in plant cells, and excess ROS induce oxidative stress ^[9][10][37,38]. SODs play a vital role in converting superoxide to H₂O₂ and molecular oxygen and thus reduce oxidative stress in plant cells. As two members of SODs, CSD1 and CSD2 function in the ROS scavenging system and protect plants from oxidative stress. The expression of both CSD1 and CSD2 is induced under oxidative stress conditions due to the down-regulation of miR398 ^[8][11]. In different species and under different environmental conditions that result in oxidative stress, the expression patterns and functions of miR398 and its target genes vary and will be discussed in detail below.

2.2. Heavy-Metal, Environmental-Pollutant, High-Light, Ozone and UV Stress

Heavy metals, such as Cu²⁺, Fe³⁺ and Ni²⁺, have the potential to generate hydroxyl radicals; miR398 is down-regulated in Arabidopsis, grapevine, castor bean (R. communis) and hickory (C. cathayensis) plants exposed to heavy metals. Correspondingly, the expression levels of the target genes, CSD1 and CSD2, are up-regulated in these species ^{[8][11][12][13]}[11,39,40,41]. The overexpression of these miR398 targets in grapevine—VvCSD1 and VvCSD2—enhances ROS scavenging systems and protects plants from metal-induced oxidative stress in tobacco (N. tabacum) ^[12][40]. Heavy metals cause land contamination, which adversely affects plant growth, while sulfur dioxide (SO₂) and phenanthrene are also common pollutants that can produce oxidative toxicity in plants. SO₂ and phenanthrene repress the expression of miR398 in Arabidopsis and wheat, and negative correlations between the levels of miR398 and its target mRNAs (CSD1 and CSD2) are observed ^[14][15][26,42]. High-light levels cause chloroplast damage, produce ROS and significantly repress miR398 expression in Arabidopsis and rice; this stimulates the expression of CSD1 and CSD2, thereby helping plants tolerate oxidative stress ^[8][16][11,43]. Ozone fumigation may result in another type of oxidative stress response which reduces miR398 levels and is accompanied by the up-regulation of CSD1 but not CSD2 ^[17][44]. miR398 expression is induced by UV-B radiation in Arabidopsis and Populus tremula plantlets ^[18][19][45,46]. It is interesting that miR398 is up-regulated by UV-B, which is contrary to its response to other types of oxidative stress. This indicates that there may be distinct machinery involved in the response to UV ^[19][46].

2.3. Salinity and Drought Stress

MiR398 is significantly down-regulated following NaCl treatment in Arabidopsis, cotton (G. hirsutum), wheat (T. aestivum) and tomato ^{[20][21][22][23]}[21,23,47,48]. The transgenic tomato lines with sly-miR398b overexpression show increased salt sensitivity, probably due to reduced activities of SODs, ascorbate peroxidase (APX) and catalase (CAT) ^[24][49]. In other species, miR398 may have different response modes to salt stress. In poplar, miR398 expression is induced at an early stage of salt treatment and is then suppressed 48 h later, while the expression of its target gene CSD1 shows an inverse correlation. In contrast, in Arabidopsis, miR398 is steadily and unidirectionally suppressed under similar conditions ^[21][23]. Even in the same plant, different tissues may have contrasting responses to salt stress: in response to salt stress, miR398 is down-regulated in sweet potato roots but up-regulated in leaves, suggesting that the miR398 regulation of abiotic stress responses is dependent on the plant developmental context ^[25][50]. Similarly, miR398 shows different responses to drought stress in different species. miR398 is down-regulated by drought in tomato ^[23][48] and cotton ^[26][51] and up-regulated in wild emmer wheat ^[27][52] and peanut ^[28][30], but it is unaltered in switchgrass ^[29][53]. miR398 overexpression lines are sensitive to drought stress in rice, indicating the negative regulation of miR398 in wild-type rice subjected to drought ^[16][43].

2.4. Water Deficit and Flood Stress

Water-related hazards such as water deficiency and flood are increasing because of climate change, and both cause stress in plants. In M. truncatula, miR398a and miR398b are up-regulated in both shoots and roots under water-deficit conditions, corresponding with the down-regulation of the miR398a target, COX5b (TC123882). As the COX5b protein forms part of the electron transport chain in mitochondria, these results highlight the involvement of miR398 in the regulation of mitochondrial respiration in response to water deprivation in M. truncatula ^[30][54]. In contrast, in pea (P. sativum) and common bean (P. vulgaris), miR398 accumulation is reduced, whereas CSD1 expression is enhanced upon water deficit ^[31][32][55,56]. In addition to water deficit, flooding (also known as waterlogging or submergence) is another major abiotic stress that restricts plant growth. Too much water causes the submergence of plants, which induces hypoxia: miR398 is down-regulated under these conditions in Arabidopsis, suggesting a role for miR398 in low-oxygen signaling ^[33][57].

2.5. Thermal Stress

Extreme temperatures induce ROS, and it appears that miR398 has different regulatory mechanisms for cold and heat stresses. Low temperature is a major abiotic stress affecting crop production in high-latitude areas, and cold stress suppresses miR398 expression in various species. MiR398 was first reported to be down-regulated during cold stress in Arabidopsis [6]. Similarly, in Chrysanthemum dichrum the expression level of miR398 is reduced upon freezing treatment, while the targets CSD1 and CSD2 show correspondingly elevated expression levels ^[34][58]. In winter turnip rape (Brassica rapa), which is also a dicotyledonous plant, miR398 is up-regulated in leaves under cold stress conditions ^[35][59], suggesting that the response of miR398 to cold stress is species-specific. In the monocot wheat (T. aestivum), the expression of miR398 also decreases in response to low temperatures, and, correspondingly, its target CSD1 shows the opposite expression pattern ^[20][36][21,28]. Cold stress may lead to cellular dehydration and membrane damage, thereby inducing ROS; the inhibition of miR398 and the up-regulation of CSD may be involved in ROS scavenging during cold adaptation. MiR398 is an ambient temperature-responsive miRNA ^[37][60]; it responds not only to cold stress but also to heat stress. Heat stress rapidly induces miR398 expression and reduces transcripts of its target genes CSD1, CSD2 and CCS1 in Arabidopsis. Although CSD and CCS1 act as antioxidants which enhance plant resistance to abiotic stresses, mutations in CSD1, CSD2 and CCS1 improve heat-tolerance in Arabidopsis. In contrast, transgenic plants that overexpress miR398-resistant forms of CSD1, CSD2 or CCS1 are more sensitive to heat stress due to reduced activities of heat-stress TFs and heat-shock proteins ^[38][39][61,62]. In monocot maize, miR398 is up-regulated, with a similar response pattern to that of the dicotyledonous Arabidopsis ^[38][61]. In Chinese cabbage (B. rapa ssp. chinensis), which also belongs to the Brassicaceae family, bra-miR398a and bra-miR398b are down-regulated in response to heat stress, and their target gene BracCSD1 is up-regulated correspondingly ^[40][63]. Heat stress can induce miR398 to different levels depending on species or tissue. Thus, miR398 is only slightly down-regulated under heat stress conditions in Populus tomentosa and switchgrass ^[29][41][53,64], and in Helianthus annuus, miR398 is up-regulated in leaves but down-regulated in roots ^[42][65].

2.6. MiR398 in Nutrient Homeostasis

MiRNAs are seen as emerging targets for biotechnology-based biofortification programs and can be utilized to combat micronutrient malnutrition ^[43][66]. MiR398 plays a key role in plant responses to imbalances of major nutrients, including copper (Cu), zinc (Zn), phosphorus (P) and nitrogen (N). The transition element Cu is important for photosynthetic and respiratory electron transport, oxidative stress protection, cell wall metabolism and ethylene perception in plants, but it is toxic when present in excess ^[44][67]. Plastocyanin (PC), Cu/Zn SODs and cytochrome c oxidase (COX) are major copper proteins in plant cells, and PC is essential for electron transfer: mutants with insertions in PC genes are seedling-lethal, while Cu/Zn SODs are relatively dispensable ^[45][24]. Based on the metal cofactors the enzyme binds, SODs can be classified into three types: Cu/Zn-SOD, Mn-SOD and Fe-SOD. In a situation of Cu deficiency, in order to preserve Cu for more important life processes, the Cu-responsive TF SPL7 binds to GTAC motifs in the MIR398 promoter region and induces its expression, as mentioned previously. Moreover, miR398 directs the degradation of Cu/Zn-SOD, CCS1 and COX5b-1 mRNAs, allowing for the efficient delivery of Cu from CSDs to PC; Cu/Zn-SODs activities are then replaced by Fe-SOD (FSD1) ^[45][46][47][24,25,68]. Since CCS1 supplies Cu to CSDs, the miR398-dependent down-regulation of CCS1 further promotes Cu release from CSDs under Cu-deficient conditions ^[48][69]. This mechanism allows plants to save Cu for the most essential functions when the metal is in limited supply. Zn is an essential micronutrient for plant growth and development. Cu/Zn SODs not only play a role in the regulation of Cu homeostasis but also respond to Zn deficiency in maize ^[49][70]. CSDs participate in Zn delivery, while Zn depletion depresses CSD function by the up-regulation of miR398 in Sorghum bicolor, suggesting that miR398 is an important regulator of the response to Zn deficiency in plants ^[50][71]. Inorganic phosphate (Pi) and N deficiency are known to be limiting factors for plant growth and agricultural productivity. Pri-miR398a and mature miR398 are down-regulated during P starvation and N limitation in Arabidopsis ^[51][72]. Pi starvation induces miR398 accumulation in tomato, N. benthamiana and alfalfa (Medicago sativa), implying a different Pi deficiency-response mechanism between Arabidopsis and these crops ^[52][53][54][16,73,74]. However, as in Arabidopsis, miR398 is down-regulated under nitrate-limiting conditions in maize and potato ^[55][56][75,76].

3. Role of MiR398 and Its Targets in Biotic Stress Responses

The expression level of miR398 is also regulated by various biotic stresses, including bacterial, fungal and viral infection, and the miR398-CSD module is involved in these disease resistance responses.  The model bacterial pathogen Pseudomonas syringae pv. tomato DC3000 infiltrates Arabidopsis leaves, which then exhibit the down-regulation of miR398 expression. CSD1—but not CSD2—mRNA levels are up-regulated in response to this biotic stress ^[17][44]. The overexpression of miR398b enhances the susceptibility of Arabidopsis to DC3000 and flg22, a conserved peptide derived from P. syringae flagellin, indicating that miR398b negatively regulates Arabidopsis disease resistance ^[57][77]. Flg22 suppresses miR398b accumulation, and, consistent with this, the expression of the miR398 target genes COX5b-1, CSD1 and CSD2 is increased ^[57][77]. The down-regulation of miR398 is also observed in citrus plants infected with harmful bacteria of the genus Candidatus Liberibacter ^[58][78]. In contrast to its negative role in defense against bacteria in Arabidopsis, miR398b is reported to positively regulate rice immunity against the blast fungus Magnaporthe oryzae ^[59][79]. Transgenic rice plants overexpressing miR398b display enhanced resistance to M. oryzae, which is associated with the reduced mRNA levels of miR398b targets in rice, i.e., CSD1, CSD2, SODX and CCSD ^[59][60][79,80]. The underlying mechanism by which miR398b regulates rice blast resistance involves boosting total SOD activity and increasing H₂O₂ concentration, thereby improving disease resistance ^[60][80]. In contrast, the transient expression of common bean miR398b in N. benthamiana leaves results in enhanced lesions caused by the fungus, Sclerotinia. sclerotiorum ^[61][17]. Powdery mildew is another major disease that reduces crop yields and is usually caused by the ascomycete fungus Blumeria graminis f. sp. hordei. MiR398 is involved in the defense against powdery mildew by regulating the expression of SOD1 in barley: the accumulation of SOD1 enhances plant resistance ^[62][81]. Interestingly, the expression patterns of miR398 vary with different types of biotic interaction. In the roots of bread wheat (T. aestivum), miR398 expression increases during the early response to Fusarium culmorum inoculation ^[63][82]. A down-regulation of miR398b and an up-regulation of CSD1 and the bean-specific miR398 target gene Nod19 occur in common bean (P. vulgaris) leaves challenged with S. scleortiorum ^[61][17]. It is likely that pathogens can manipulate miR398 levels to facilitate their infection. Studies of many species have shown that virus-infected plants exhibit elevated miR398 accumulation. Tobacco mosaic virus (TMV) and oilseed rape mosaic tobamovirus induce the expression of miR398 in Arabidopsis ^[64][65][83,84]. Tomato leaf curl virus (TolCNV)-infected tomato ^[66][85], potato virus X (PVX)- and potato virus Y (PVY)-infected N. benthamiana ^[67][86], Papaya meleira virus (PMeV)-infected Carica papaya ^[68][87] and potato spindle tuber viroid (PSTVd)-infected tomato ^[69][88] show miR398 accumulation. Whether CSD transcript levels are altered by viral infection may depend on the host–virus combination. Thus, PVX and PVY infection results in higher NbCSD transcript levels in tobacco, whereas ToLCNV infection reduces CSD1 and CSD2 expression in tomato. Bamboo mosaic virus (BaMV) infection increases the level of miR398, but this up-regulation of miR398 does not have an anti-BaMV effect, instead promoting the manifestation of virus-infection symptoms by increasing the levels of ROS ^[70][89]. In N. benthamiana, beet necrotic yellow vein virus infection induces miR398, and Liu et al. speculated that miR398 enhances plant resistance against viruses by targeting umecyanin ^[71][90].

4. Role of MiR398 in Plant Growth and Development

Both miR398 and its targets CSD1/2 show spatial and temporal expression patterns, implying a role of miR398 and its target genes in the regulation of plant growth and development ^[8][11]. However, compared with research on the role of miR398 in stress-response regulation, there are relatively few studies on its function in growth and development. Nevertheless, based on the reports to date, it appears that miR398 regulates growth via target genes other than the various CSDs. In the Arabidopsis ccs knockout mutant, almost all Cu/Zn SOD activity is lost; yet, these plants are phenotypically similar to the wild type under normal growth conditions ^[72][36]. MiR398 overexpression lines do not exhibit any visible growth phenotype despite a severe reduction in activity of both CSD1 and CSD2 ^[45][24]. A more detailed phenotypic characterization showed that MIR398c overexpression lines have reduced fertility and abnormal female gametophytes, although a mutant line with a T-DNA insertion in the MIR398c locus, resulting in reduced MIR398c expression, exhibits no obvious defects in ovule development ^[73][20]. The role of miR398 in female gametophyte development involves AGL51, AGL52 and AGL78, which belong to the MADS-box gene family. Female gametophyte development and ovule morphogenesis are regulated by the temporal and spatial control of miR398 biogenesis: mature miR398 is sequestered by AGO10 in the female gametophyte to ensure the expression of its targets AGL51/52/78, which are essential for female gametophyte development as well as integument growth ^[73][20]. In Arabidopsis, miR398 also regulates age-dependent leaf senescence by targeting APX6 ^[74][19]. In crops, miR398 is reported to regulate rice growth and yield. The overexpression of miR398 can increase panicle length, grain number and grain size in rice, and miR398 suppression in transgenic short tandem target mimic (STTM398) lines shows a significant decrease in grain length, width and 1000-grain weight ^[75][91]. Transgenic lines carrying a resistant version of one of the miR398 targets, a rice CSD2 homolog, show wild-type phenotypes for seed length, seed width and 1000-grain weight. The phenotype of yield-related traits in miR398 overexpression and STTM398 lines indicates that the manipulation of miR398 is a promising strategy that may lead to important yield improvements ^[75][91].