Potato Genomics, Transcriptomics, and miRNomics under Abiotic Stressors

Abstract

Potato (Solanum tuberosum L.) is one of the essential non-cereal crops with noticeably greater production and consumption rates across the world. Because of the diverse range of utilization of nutritious tubers, potatoes can be used as an alternative food to address regional, national, and global food security issues compromised by global climate change. Since potato yield and quality are dramatically affected by abiotic stress conditions, the development of potato plants showing greater performance after being exposed to individual or combined stress treatments has become intriguing for the past decade. In this book chapter, recent studies and/or approaches associated with potato genomics, transcriptomics, and identification of miRNAs are summarized to discuss the response of potatoes to abiotic stress factors in different regulatory levels. Such a summary should encompass the importance and identification of factors for the development of potato plants under current and potential exacerbating effects caused by abiotic stress.

1. Introduction

Global climate change stimulates significant threats to sustainable agricultural production by altering climate patterns, causing large-scale detrimental impacts around the globe such as excessive heat, prolonged drought, severe hurricanes, and melting glaciers [1]. As a result, the frequency of unfavorable environmental conditions seriously undermining plant growth, development, and productivity increases each and every year [2]. Unfortunately, detrimental consequences caused by abiotic stresses on plant productivity and survival are likely to intensify as greenhouse gas emissions continue over a long period of time worldwide [1, 3]. Scientists have anticipated that ambient CO₂ concentrations will double by the end of the twenty-first century to 700 ppm, accompanied by a surge in global air temperatures of 0.3–4.8°C [1]. Hence, the expected rise in air temperature may exacerbate the evapotranspiration effect, resulting in water scarcity in the soil and agricultural drought [4, 5]. Additionally, the adverse influences of climate change on soil content are expected to boost soil salinity because of increasing sea levels and saltwater intrusion, particularly along coastal regions [6, 7]. While the estimated surge in atmospheric CO₂ levels is suggested to improve productivity in some crops including wheat, maize, rice, and soybean [8], yield losses resulting from high temperatures and water scarcity may outweigh the positive effect of the CO₂ increase [9]. A multitude of factors have also intensified the burden on plants including the growing increase rate in the world’s population, the reduced arable area caused by soil degradation, urbanization, and the increased need for animal feeding which entails the development of tolerant plants adapted to projected scenarios in the future and improved yields of food crops to fulfill individual, regional, national, and global food security requirements [10, 11].

Cultivated in more than a hundred countries and consumed by over a billion people worldwide in 2021, potato (Solanum tuberosum) is one of the major non-cereal staples with the highest harvested area (Figure 1) and production quantity in China (Figure 2) [12]. Although China, India, and Ukraine have a significant role in potato production, Kuwait, New Zealand, and the United States of America rank in the top three places based on yield in the world (Figure 3).

Figure 1.

Harvested area of potato around the world (Ha) [12]. Figure constructed by Orkun GENCER.

Figure 2.

Production quantity of potato around the world (Tonnes) [12]. Figure constructed by Orkun GENCER.

Figure 3.

Yield of potato around the world (Tonnes/Ha) [12]. Figure constructed by Orkun GENCER.

Along with a shallow root system, potato is known as a cool season crop that grows well in temperate locations under mild temperatures. Due to the high nutritional content of potato tubers including carbohydrates, vitamins, proteins, antioxidants, and minerals, attempts toward increasing potato crop productivity may help to fulfill the nutritional needs of the growing worldwide population under the adverse effects of climate change [13]. Physiological processes during potato tuber initiation and maturation that substantially shapes the quality and productivity of potato include carbohydrate synthesis via the photosynthesis process in the leaves, the transport of sucrose to the tubers, and the conversion of sucrose to starch in the tubers [14]. Depending upon the cultivar, duration, type, and mode of stress (either as individual and/or combined), the potato has been considerably influenced at morphological, physiological, and molecular levels as well as post-harvest storage.

Abiotic stresses, often known as drought, heat, salinity, heavy metals, and nutrient deficiency, that interfere with metabolic processes in potatoes are most likely to damage tuber productivity and quality by impairing during plant growth, development, and tuberization stages [15, 16]. Potato yield across the world is anticipated to fall markedly by 2055 as a consequence of the greenhouse effect and frequent occurrence of drought stress [17]. In addition to those, another study has predicted that global potato production would fall by 18–32% between 2040 and 2069 resulting from abiotic stresses linked to global climate change [18]. Although the severity of yield loss resulting from abiotic stress is dependent on several factors including the type of genotype, stress type/period, and developmental stage of the potato [19], the earlier exposure to drought stress, for example, indicates the more detrimental impacts during tuber formation, bulking and maturation due to a lower rate of carbon assimilation and then reduced partitioning of photosynthesis products to tubers [20]. Similar to drought stress, the severity of salinity influences on potato plants differs depending upon cultivar and salt concentration. The negative impacts of salt stress are analogous to those of drought based on morphological and physiological aspects including prolonged emergence of seedlings, dehydration/yellowing of leaves, accelerating leaf senescence, decreased tuber size/dry weight, reduced photosynthesis/transpiration rate, reduction in stomatal conductance, and less relative water content in leaves [21, 22] because both cause water stress, which forces stomatal aperture to shrink, K⁺ and Ca²⁺ mineral deficiency in soil and inhibition of plant growth/development [23, 24]. Salt stress has also been shown to increase Na⁺ and Cl⁻ concentration, intensify lipid peroxidation, and lower the rate of callus generation in potatoes [25, 26]. Likewise, heat stress considerably limits tuber formation and quality by limiting sucrose production and subsequent transfer to the stolon, resulting in a reduction in dry matter, malformations in tuber shape, and abnormal secondary growth [15, 16]. The sensitivity of potatoes to heat stress is also shown to be heavily influenced by cultivar type [27], developmental stage, and stress period [28]. The optimum temperature demands for above-ground biomass during potato development and below-ground tuber production and maturation differ; the former thrives in the 20–25°C range, while the latter thrives in the 15–20°C range [29]. A recent study has also suggested the greater impacts of heat stress on potato development and productivity when applied earlier during tuber formation as well as inhibition of tuber production temperatures over 25°C using six potato cultivars [30].

Considering the various negative direct or indirect impacts on potato quality and yield as aforementioned above, it is therefore highly essential to obtain potato lines with high tolerance to abiotic stress. Thus, the first step to achieve this is to identify the factors that confer improved tolerance to abiotic stress factors at the level of the genome, transcriptome, and microRNAs that are involved in either transcriptional and/or post-transcriptional regulation. Although a great number of studies on other plants may provide preliminary information for the discovery of stress tolerance mechanisms, the potato-specific factors that possess crucial roles under abiotic stresses need to be highlighted as potato production is mostly achieved by vegetative propagation through tubers.

2. Potato genomics on abiotic stress response/tolerance

Genomics is involved in the discovery of genetic variation underpinning changes in phenotypes, the uncovering of new sources for traits and diversity, and the elucidation of molecular pathways regulated by stress conditions. Over time, new breeding targets have been specified due to advances in genomic technologies including third-generation sequencing to obtain reference genomes with a greater quality, investigation of non-coding parts of the genome to illuminate the function and utilization of pangenome for the analysis of the structural variation at species-wide level [31]. Besides, some other approaches have also contributed to genomic studies such as machine learning, short breeding, and high-throughput phenotyping.

Globally, the most extensively grown potato varieties are Solanum tuberosum subsp. tuberosum, but there are also other potato species with different ploidy levels, including diploids, triploids, pentaploids, and hexaploids [32, 33]. Along with polyploid species, tetraploid cultivars are self-compatible, whereas diploids are often self-incompatible [34]. Due to complicated genetic inheritance and highly heterozygous characteristics of the S. tuberosum subsp. Tuberosum, diploids developed from tetraploid genotypes by anther/pollen culture or generated by interspecific hybridization with specific genotypes are widely utilized as parents for mapping analyzes [35]. Yet, the self-incompatibility nature of diploid potatoes hinders the production of pure lines, necessitating the use of F1-hybrid populations to employ genome mapping in potatoes as opposed to typical homozygous mapping approaches [36].

The Potato Genome Sequencing Consortium (PGSC), composed of 26 international institutes from 14 countries, successfully sequenced the doubled monoploid (DM) potato genome of S. tuberosum group Phureja, with the identification of 39,031 protein-coding genes [34]. Subsequently, another study refined the DM potato assembly more precisely by arranging scaffolds and pseudomolecules in the genome [37]. Lately, a chromosome-scale long-read reference assembly was created in potatoes [38]. More than 100 different potato species have now been sequenced or re-sequenced, primarily with the help of Illumina platforms including the wild S. commersonii, tuber-bearing Solanum species, Solanum chacoense “M6”, a somatic hybrid produced from Solanum pinnatisectum as well as cultivated potato taxa using Illumina and long-read PacBio platforms [39, 40, 41, 42, 43]. The identification of novel genes, markers, and haplotypes has been stimulated by the ongoing improvement of sequencing technologies and bioinformatic tools to develop our understanding of potato biology [44].

The genotyping-by-sequencing (GBS) method has recently become one of the most popular approaches as it attempts to illuminate genome-wide association studies and genetic diversity in a variety of crops, including potatoes [45, 46]. GBS has been successfully used in potatoes for the construction of physical mapping related to tuber yield and starch content/yield [47], to identify and characterize single nucleotide polymorphisms (SNPs) particularly linked to simple traits [48], genetic variation as well as population structure studies [49]. On the other hand, an SNP array-based high-throughput genotyping system has been developed and utilized in a wide variety of genomic studies in potatoes including the determination of novel genes and SNPs against late blight resistance (Phytophthora infestans) [50], identification/characterization of the genetic diversity and the construction of population structure for Andigenum germplasm [51], determination of haplotype-specific SNPs against wart resistance [52], and deciphering QTLs against common scab resistance in Canadian potato germplasm [53]. Moreover, QTL mapping has been effectively applied to identify genomic areas under abiotic stress, including drought [54], heat [55], and salinity [56]. One study has explored 28 QTLs specifically related to drought treatment, 17 QTLs under recovery treatment, and two QTLs in control conditions using a diploid potato population [57]. A few years later, it was suggested a total of 45 QTLs were linked to morphological, physiological, and agronomical traits upon exposure to terminal drought and well-watered application, of which 26 presumably attributed to drought tolerance [58]. Similarly, the identification of QTLs related to heat stress has revealed that allelic variation in the promoter region of heat shock cognate 70 (HSC70) corresponds with high-temperature tolerance by shielding photooxidative damage against PSII across a diverse variety of wild potato relatives [59].

Along with genomic QTLs, numerous additional classes of QTLs, including expression QTL (eQTL), proteomic QTL (pQTL), phenomic QTL (phQTL), and Meta-analysis QTL (MQTL) are also considered important tools in potato breeding. An eQTL attempts to uncover genetic variations that influence the expression of a particular gene(s) [60], whereas a pQTL correlates protein content and amount with genetic variability [61]. Besides, phQTL are chromosomal areas that include loci conferring to genetic variability in phenotypes [62]. Meta-analysis of QTLs (MQTL) involves QTL data from a wide range of studies, in various locations, years and genetic backgrounds, providing identification of stable and reliable QTLs [62]. Recent modern NGS-driven QTL mapping strategies namely, QTL-seq, individual population resequencing, and MutMap, which uses bi-parental mapping and mutant populations, could be utilized for effective detection of major QTLs controlling agronomic traits in potatoes. Consequently, with the progressive advances in the QTL mapping approach and sequencing technology such as high-throughput genotyping (HTG) and high-throughput digital phenotyping (HTP) as well as the development of specific software such as GWASpoly designed for tetraploid potato [63], more precise and accurate marker-trait association studies for traits including biotic/abiotic stress, quality, and productivity pave the way for the speeding up potato breeding cycles from over 10 years to around 4 years [64].

3. Recent transcriptomic studies of potato under abiotic stress

Transcriptomics is the investigation of the transcriptome, the overall set of RNA transcripts created by the genome, under certain conditions or in a single cell via high-throughput technologies such as RNA sequencing (RNA-seq) and microarray [65]. Transcriptome analysis facilitates the determination of genes that express differentially in particular cell types or reactions to different treatments. While genomics uncovers the entire set of genetic information such as genome structure and mapping, transcriptomics reveals all RNA-related information including their expression levels, roles, locations, and transports/degradations to understand the relationship between a given phenotype and transcriptome.

A great number of attempts at transcriptomic analysis have been undertaken in potatoes upon abiotic stress treatments including heat [66, 67], drought [68, 69], heat combined with drought [70], salt [71], a combination of drought and salt [72], and nitrogen deficiency [73, 74]. Another attempt has constructed cDNA libraries from normal potatoes grown at 20°C day/18°C night and those exposed to 3 days of heat treatment at 35°C day/28°C night [66]. A total of 1420 differentially expressed genes (DEGs) were discovered, of which 771 genes were considerably upregulated, whereas 649 were substantially downregulated. The expression profiles of 12 selected genes also coincided with the sequencing results. As shown by gene ontology analysis, these DEGs were grouped into 49 distinct Gene Ontology (GO) categories, revealing the functional variety of the high-temperature stress-responsive genes. Although StHsp26-CP and StHsp70 levels were significantly increased upon 3 days of heat application, the majority of potato heat transcription factors (StHsfs) and heat shock proteins (StHsps) were not effectively expressed according to the expression analyzes of DEGs. In a comprehensive study examining the potato transcriptome and metabolome, researchers have revealed the upregulation of 448 genes and downregulation of 918 genes when the heat-treated group was compared with the control plants [67]. After the application of short stress (6 h), a total of 160 genes have been upregulated and 538 genes have been downregulated, while 130 genes have been upregulated and 94 genes have been downregulated in response to extended heat stress (3 days). Differentially expressed genes have been closely associated with photosynthesis, protein denaturation, RNA processing, and cell wall damage under heat stress. Moreover, differentially expressed compounds related to amino acid production and accumulation of secondary metabolites including L-proline/tyrosine production and synthesis of flavonol-derivative molecules have largely been upregulated in response to heat stress, indicating that these compounds may have a remarkable role in improved heat tolerance. Meanwhile, it has been suggested to reduce some secondary metabolites such as stevioside under short heat stress and suppress amino acid production such as histidine as well as jasmonic acid (JA) hormone synthesis under prolonged heat stress. A study that utilized EMS-mutagenized potato plants showed an increased tolerance to drought treatment than the wild-type (WT) Desiree plants [68]. Moreover, alterations in transcripts induced by drought treatment in drought-tolerant (DR) and WT plants were analyzed using the de novo assembly approach. Total RNA was extracted from one-week-old WT and DR plants after being exposed to polyethylene glycol-8000 at different time intervals including 0, 6, 12, 24, and 48 h for RNA sequencing, respectively. Sixty one thousand one hundred transcripts and 5118 DEGs showing up or downregulation were discovered following the pairwise analyzes of WT and DR plants. Under the drought condition, transcriptome results have suggested a total of 909, 977, 1181, 1225, and 826 DEGs between WT and DR plants at 0, 6, 12, 24, and 48 h. As a result of KEGG enrichment analysis, photosynthetic-antenna protein and endoplasmic reticulum protein processing have been suggested to have major functions in the drought tolerance mechanism of the DR plant. With drought-tolerant potato landrace, scientists have examined the transcriptome response to drought, rehydration, and re-dehydration stress under consecutive treatments of dehydration, rehydration, and re-dehydration [69]. While, drought-responsive genes have been primarily associated with photosynthesis, regulation of lipid and sugar metabolism, signal transduction, wax formation, rigidity of cell wall, and osmoregulation. A total of 178 transcription factors were differentially expressed in drought-tolerant landrace JSY potato following drought stress, representing 4.7% of the drought-response genes (3764) and mainly related to 11 gene families. The majority of them were upregulated apart from the bHLH family, which was downregulated under drought stress. It has also been shown that the recovery of potato by regulating the gene expressions are closely associated with tuberin production, flavonoid, lipid, and sugar metabolism, detoxification mechanism as well as the reverse expression of the majority of drought-responsive genes. Moreover, upregulation and downregulation of transcriptional factor families, such as increased expression of the majority of ethylene responsive factor (ERF) transcription factor families, have been found more upon re-drought as compared to initial drought, indicating the previous drought treatment might induce a favorable response to the upcoming drought by drought hardening effect. In addition to drought-responsive genes regulating cell structure and components, drought hardening triggers the intensification of gene expressions that play important roles in photosynthesis, sugar metabolism, proteases and protease inhibitors, flavonoid production, transporters, and signal transduction. Instead of the individual stress treatment, researchers have tested the effects of drought, heat, and simultaneous drought and heat in a growing chamber using five different potato lines [70]. Interestingly, drought and combined stress have triggered an increase in chlorophyll content as well as typical responses including reduced leaf size and plant height, disruption in the membrane structure and decreasing relative water content (RWC) under abiotic stress conditions. They have also suggested that the concurrent treatment of drought and heat stress most likely had a higher impact on the physiological traits, causing wilting earlier and severe effects on growth rate as compared to a single treatment. A study has examined salt stress-responsive genes in Longshu No.5, a salt-tolerant tetraploid potato genotype [71]. A total of 5508 DEGs have been found in response to salt stress. As a result of GO and KEGG analysis, DEGs were found to be involved in the groups of nucleic acid binding, transporter function, ion movement, binding, kinase function, and oxidative phosphorylation. The highly differential expression of genes that function in the ion transport signaling, in particular, implies that modulation of this signaling cascade is crucial upon salt treatment in potatoes. In another recent study, researchers have predicted the effects of drought, salinity, and simultaneous drought and salinity on agricultural traits using maize and potato using a remote sensing approach in a wide range of geographical regions in the Netherlands [72]. They have discovered that both stressors (individual and co-occurring) had a considerable impact on the physiological traits including leaf area index, chlorophyll content, fraction of vegetation cover, relative water content, and amount of photosynthetically active radiation absorbed by plants in both groups, with drought being more pronounced effect than salt. Although, it has been revealed aggravating effects on the traits under combined stress conditions, the degree of impact level was substantially dependent on the growth and development season.

The number of studies is quite limited related to the simultaneous treatment of heat and salt in potatoes, however, its effects at morphological, physiological, and molecular levels have been analyzed to understand cross-talking mechanisms in tomato [75]. It has been found that the combination of stresses has affected profoundly in shoots and roots as well as dry weight compared to individual stress. Similarly, tomato plants exposed to salt (100 mM NaCl) or combined stress (42°C; 4 h/day) have shown a significant rise in Na⁺ levels in both roots and shoots, of which under combined stress (21 days of co-exposure) being more impacted than exposed to salinity stress alone. Moreover, inhibition of nutrient absorption proteins caused by heat stress has stimulated decreased uptake of Ca²⁺ and K⁺, imbalancing salt overly sensitive (SOS) pathway accompanied by the increased risk of toxicity which ultimately leads to considerable damage to plant development. Proline has been suggested to have important roles under a combination of heat and salt stress due to osmoprotectant and ROS scavenging function as well as energy source molecule under nutrient deficiency conditions, producing 30 ATP equivalents per oxidation of proline. Concerning the overall findings, it has been shown that activation of many alternative oxidases (AOX) enzymes and increased amount of antioxidant metabolites have overcome oxidative damage and build-up of reactive oxygen species upon stress treatments.

4. MiRNomics in potato

MiRNAs are small, single-stranded, and non-coding RNAs that modulate gene expression at either the transcriptional or post-transcriptional stages in plants by perfectly binding to the messenger RNA (mRNA) molecule of the target gene (Figure 4).

Figure 4.

General miRNA biogenesis pathway in plants. Figure constructed by Orkun GENCER.

In general, miRNAs have a length of 20–24 nucleotides and are classified as species-specific or conserved between species in plants. They have been involved in a wide range of processes such as the flowering period, growth development, hormone regulation, signaling pathways, homeostasis maintenance, and in response to biotic/abiotic stresses in plants [76]. As a result of the emerging studies, it has been suggested that miRNAs show different expression profiles depending on the type and duration of stress and the type of specific tissue in plant organisms [77, 78, 79]. Therefore, it is thought that miRNA-based molecular editing studies can be utilized in enhanced tolerance to abiotic stress in plants as well as an increase in yield and quality of agricultural products (Figure 5).

Figure 5.

Abiotic stress-responsive miRNAs in potato. Figure constructed by Orkun GENCER.

4.1 Identification of conserved and novel miRNAs in potato

Attempts to identify miRNAs and their target genes associated with potato growth/development and in response to abiotic stress, have begun in the last decade. As such, scientists have identified 48 potential miRNAs in Solanum tuberosum using an in silico approach by comparing known miRNAs from other organisms such as Arabidopsis thaliana and rice with potato expressed sequence tag (EST), genome survey sequence (GSS), and nucleoid databases [80]. As a result of bioinformatic analysis, they have reported that these miRNAs could regulate 186 potential target genes involved in above-ground structures e.g., flower, leaf, and stem development, root development, signal transduction, metabolic pathways, and stress response. RT-PCR analyzes have been conducted for 12 randomly selected out of 48 candidate miRNAs to validate the presence of potato miRNAs predicted by the bioinformatics approach [80]. Altogether, it has been discovered that while certain miRNAs have been expressed in all plant tissues, others have displayed temporal or spatial expression during development stages. Similarly, another in silico approach was used to predict miRNAs in potatoes where 71 putative miRNAs from 48 families have been identified [81]. It is noteworthy that 65 of the 71 miRNAs were predicted for the first time. Only 7 of the 71 potential miRNAs have been chosen for verification, and their expression levels in multiple potato tissues have been investigated using real-time PCR. The findings reveal that all seven miRNAs have been effectively amplified and their expression patterns have varied among tissues [81]. It has been postulated the highly variable expression level of each miRNA in various tissues might be linked to miRNA roles in the modulation of the vegetative development in potatoes. Another study has identified a total of 202 candidate miRNAs corresponding to 78 families using a newly modified comparative genome comparison approach in potatoes [82]. It has been discovered that 54 of the 78 families were novel in potatoes as well as 24 miRNA families reported in earlier studies. This group has analyzed the expressions of 12 miRNAs in young leaf, immature flower, and mature flower tissues. As a result, they have observed that a vast majority of the miRNAs have been expressed in all three tissues and one has no expression in young leaf tissue. Ref. [82] have estimated that the identified miRNAs target a total of 1094 genes, encoding transcription factors, regulating stress response, signal transduction, and other metabolic stages. According to GO analysis, these targets participate in 545 biological processes, 28 of which are associated with potato defensive systems, and biological metabolisms including carbon, starch, lipid, and primary/lateral root formation. Pathway enrichment study using KEGG indicated that the identified miRNAs are involved in 98 metabolic pathways including sucrose, fatty acid, amino acid metabolism, and carbon fixation as well as plant hormone synthesis. In another study to predict miRNAs and target genes in various agricultural plants belonging to the Solanaceae family, 22 miRNAs and 221 target genes were found by analyzing ESTs in potatoes [83]. Researchers continued to have identified 259 miRNAs belonging to 159 miRNA families in potatoes using next-generation and potato genome sequencing [84, 85]. Researchers have reported that only 28 of these miRNA families have been classified as conserved miRNA families in other plants, while the others have been suggested to be potato-specific confirmed by RNA gel blot hybridization. It has been revealed that the putative target genes are transcription factors and genes involved in defense mechanisms, signal transduction, ion regulation, flowering, and tuber production. In another study, 89 conserved miRNAs from 33 families involving 147 potato-specific and 112 potato-specific candidate miRNAs were discovered using samples from three distinct tissues and four different phases of tuber formation [84]. The results obtained suggested that the majority of conserved miRNAs target various important transcription factor families including SBP, GRAS, YA, NAC, and ARF highlighting the importance of conserved miRNAs in critical biological processes. According to the GO analyzes, the great majority of those have been implicated in oxidation-reduction activities, metabolism-related processes, defensive responses, and transportation as well as signal transduction, implying that the majority of potential targets of potato-specific miRNA have not overlapped with conserved miRNA targets. Furthermore, the expression analyzes by qRT-PCR results have indicated that certain miRNAs have been expressed based on tissue type, while the expression level of others has differed markedly depending upon the developmental stage of the potato, such as tuberization. In another study, a scientist identified 120 new miRNAs belonging to 110 families with a comparative genomics approach [86]. It has been shown that the expression of only 10 randomly selected miRNAs by quantitative RT-PCR. According to the findings, 433 putative genes targeted by 120 miRNAs are involved in signal transduction, modulation of growth/development, transcription factors, structural functions, and biotic stress response. Recently, a study group has identified the differentially expressed miRNAs and corresponding target genes that function in the build-up of anthocyanin during purple potato development by small RNA and degradome sequencing. A total of 275 differentially expressed miRNAs have been revealed from sRNA libraries. It has been found through interaction analysis that 37 miRNAs and 23 target genes were involved in anthocyanin-responsive miRNA-mRNA modules. Various miRNAs control the major enzymes involved in anthocyanin production by regulating structural and regulatory genes in purple potatoes. In addition, it has been proposed that identified miRNAs are crucial in the coloring and accumulation of anthocyanins in potatoes [87].

4.2 Identification of conserved and novel miRNAs under abiotic stress

miRNA studies in potato in response to abiotic stress conditions has a quite recent history compared to agriculturally essential crops including wheat [88], rice [89], and maize [90]. Lately, a growing number of studies have identified known and novel miRNAs under abiotic stresses to elucidate their functions in a multitude of metabolic pathways, which ultimately confer the development of potato plants with enhanced tolerance to individual or combined stress conditions. The majority of stress treatments on potatoes are related to the identification of drought-responsive miRNAs, while the studies on the identification of heat, cold, or salt-responsive miRNAs are quite limited.

4.2.1 Drought-responsive miRNAs in potato

Under drought stress, a study has identified three drought-responsive miRNAs in potatoes including stu-miR396, stu-miR156a, and stu-miR157a by comparative deep-sequencing, and sequence alignment [91]. They have found that the expression of each miRNA has differentially changed when exposed to 15% polyethylene glycol (PEG-6000) or an air-drying method over time. It has been suggested that stu-miR156a and stu-miR157a have 68 and 71 potential mRNA targets which have significant functions in the developmental process or cell differentiation, respectively. The same group has discovered drought-responsive miRNAs in potatoes, including stu-miR171a, stu-miR171b, and stu-miR171c that target GRAS family transcription factors, as well as their expression patterns and target mRNAs under drought stress. It has also been suggested that drought-induced miRNAs in potatoes also contain families of miR159, miR164, miR166, miR390, miR395, miR397, miR398, miR408, and miR482 [92]. In another study, researchers have shown altered expression of the miRNAs stu-miR172c, stu-miR172d, and stu-miR172e, and their targets by adding 15% polyethylene glycol (PEG 6000) or air-drying approach [93]. Contrary to stu-miR172e which shows a lack of transcription factor target, 8 of 13 identified targets for stu-miRNA172c and 6 of 11 putative stu-miRNA172d targets belong to transcription factor gene families that have vital functions in the developmental pattern or cell differentiation. A total of 11 miRNAs grouped into six different miRNA families have been predicted in the modulation of the gen expressions responsible for the build-up in proline amount in potatoes under drought stress [94]. Ten of 11 anticipated miRNAs have effectively been identified using qRT-PCR, encompassing nine downregulated miRNAs and one upregulated miRNA. It was discovered that miR172, miR396a, miR396c, and miR4233 may control the pyrroline-5-carboxylate synthase (P5CS) gene expression, whereas miR2673 and miR6461 may modulate the pyrroline-5-carboxylate reductase (P5CR) and proline dehydrogenase (PRODH) gene expressions, as suggested by expression and functional analyzes. Ref. [95] have specifically discovered three potato miR159s including stu-miR159a, stu-miR159b, and stu-miR159c, and corresponding target genes including StGAMyb-like1, StGAMyb-like2.1, and StGAMyb-like2.2. As a result of the expression analysis, the reduced expression of stu-miR159s and increased expression of GAMyb-like transcription factors in potatoes suggest that the potato adaptation might be improved by overexpression of GAMyb-like transcription factor family genes induced by stu-miR159s under drought stress treatment. Another study to investigate potato miRNAs implicated in the regulation of drought tolerance discovered 458 known and 674 new miRNAs in control samples, and 471 known and 566 new miRNAs in drought-treated samples [85]. Drought treatment has been performed using 15% PEG treatment once a day for 20 days. Conserved miRNAs whose expression varies more than twice under drought stress were selected, and a decrease in the expression of 100 miRNAs and an increase in the expression of 99 miRNAs have been explored in this study. As a consequence of PEG treatment, it has been found that downregulation of 100 and upregulation of 99 miRNAs, whereas upregulation of 119 novel miRNAs and downregulation of 151 novel miRNAs have been observed based on differential expression analyzes. Four miRNAs including miR811, miR814, miR835, miR4398, and their targets MYB transcription factor, hydroxyproline-rich glycoprotein, aquaporin, and WRKY transcription factor have been suggested to be involved in the modulation of drought-related genes based on expression analysis, target prediction, and annotation.

4.2.2 Potato cold responsive miRNAs

Scientists, for the first time, have identified genome-wide level miRNAs and corresponding targets in potatoes exposed to cold stress [96]. In this regard, small RNA and degradome libraries have been created by potato tubers held at 4 and 20°C for 23 days. As a result of deep sequencing and whole-genome sequence analysis, 53 known and 60 novel miRNAs have been explored. Degradome analyzes have revealed 70 miRNA targets, demonstrating that miRNAs have been involved in the modulation of gene expression in post-harvest tubers. The targets have notable functions in a multitude of biological processes such as regulation of carbohydrate metabolism, stimulus-response, transcriptional control, and signal transduction. Subsequent expression profiling has displayed unique expression patterns of 11 miRNAs/miRNA*s and 34 targets for two particular potato genotypes to cold storage treatment regarding the decrease in sugar deposition, indicating a potential role for miRNAs in cold-induced sweetening. Recently, another study group has reported the profiling of miRNA and target mRNAs in potato plants, Favorita var., using high-throughput sequencing under low temperature stress (0°C 12 h) by creating two RNA libraries and six mRNA libraries [97]. There were 294 known and 211 novel miRNAs identified, where 24 miRNAs showed higher expression in the low temperature-treated group than controls, and 80 miRNAs displayed lower expression in treated plants compared to control plants. Furthermore, it has been suggested that 3298 DEGs with 1629 upregulated and 1669 downregulated expression levels function in response to cold and drought response, and regulation of secondary metabolism. It has been also speculated that contrasting patterns of expression of two miRNAs have contributed to improved cold tolerance in potatoes by modulating peroxisome metabolism. Similar to this study, another study has investigated the impacts of low temperatures on miRNA profiling in potatoes exposed to various low temperatures, with freezing (−2°C), chilling (0°C), and critical growth temperature (2°C) [98]. Three hundred seven known miRNAs from 73 sRNA families and 211 novel miRNAs have been characterized in the study. The majority of miRNAs function under low temperature, and drought as well as disease stress where certain conserved miRNAs, such as stu-miR530, stu-miR156d, and stu-miR167b, have been discovered in response to low temperature in potatoes for the first time. It has been shown that 442 miRNA target genes showed differential expression most of which function in the carotenoid synthesis, regulation of circadian rhythm, and signal transduction prompting that numerous low temperature responsive pathways are possible; however, abscisic acid along with gibberellin has been suggested to have a coordinating role in a multitude of metabolism pathways under low temperature stress.

4.2.3 Potato heat-responsive miRNAs

Although heat stress severely disturbs potato yield and quality as compared to drought or cold stress, attempts to reveal heat stress-responsive miRNAs in potato is still meager. Over the past 15 years, one study has revealed 202 putative potato miRNAs belonging to 78 families by improved comparative genome method as well as 1094 target genes that are involved in the modulation of signal transduction, and a wide range of metabolic activities such as carbohydrate and lipid metabolisms, and stress response [82]. Of these families, miR414, miR1132, miR1435, miR1530, and miR1533 have been suggested in response to heat stress in potatoes by targeting genes encoding heat-shock proteins that function in the chloroplast or nucleus. A recent conducted study has analyzed the various roles of unknown function (DUF221) proteins in potatoes [99]. It has also been shown that DUF22 genes are associated with miRNA families upon heat treatment in potatoes. Among the explored 10 StDDP (Solanum tuberosum DUF domain containing) genes, StDDP4 has been targeted by 10 members of the stu-miR395. Three members of miR319-3p, stu-miR319a-3p, and stu-miR319b have targeted StDDP1 and StDDP5 genes, respectively. StDDP7 has been targeted by stu-miR172a-5 while StDDP2 and StDDP1 have been targeted by stu-miR172b-5p and stu-miR172d-3p, respectively. Moreover, more than one miRNA has been predicted to target the StDDP5, StDDP4, StDDP2, and StDDP1 genes along with StDDP6 targeted by stu-miR8033-3p. Additionally, it has been shown that high-temperature stress elevated the expression of StDDP2, StDDP5, and StDDP7 in potatoes yet the expressions of corresponding miRNAs have not been validated in this study.

4.2.4 Salt/alkaline responsive miRNAs in potato

After the identification of miR319 upregulation on the improved performance under salinity stress [100, 101], researchers have shown that transgenic potato plants constructed by overexpression of miR319 have displayed enhanced water conservation and cell membrane stability as compared with control plants under salt stress [102]. It has also been observed that accumulation of Na + in transgenic plants has been less than as compared to control plants upon salt treatment. Another similar study has used parallel screening of both noncoding and coding RNA transcriptomes to identify and characterize salt stress-responsive miRNA and corresponding target genes in the Andigena potato cultivar, Sullu. An inverse association was revealed between salinity stress-regulated miRNAs including miR166 targeting HD-ZIP-Phabulosa/Phavulota network and miR159 targeting Myb101 genes [103]. They have also postulated that the miR159-Myb101 pair could be critical for managing the stress-induced premature switch to the reproductive stage as well as regulating vegetative development whereas, miR166-HD-ZIP-Phabulosa/Phavulota network might be closely related to growth control under salt stress through modulating vegetative dormancy via interacting with defense-related pathways. Recently, a study group has analyzed alkali stress-responsive miRNAs and their targets in potatoes which differ from salt stress [104]. The findings of the miRNA sequencing study revealed 168 differentially expressed miRNAs upon alkali stress treatment, of which 21 known miRNAs and 110 novel miRNAs were constructed by 12 small RNA libraries. Besides, the mRNA sequencing data revealed 5731 differentially expressed mRNAs using the same number of mRNA libraries. MiRNA-mRNA integrated analysis yielded 33 miRNA-target gene pairings made up of 20 differentially expressed miRNAs and 33 differentially expressed mRNAs. It has been suggested that some mechanisms function under alkali stress in potatoes including the phenylpropanoid biosynthesis pathway, regulation of starch/sucrose pathway, and hormone-mediated signal transduction. Additionally, the negative impacts of miR4243-x and novel-m064-5p on shikimate O-hydroxycinnamoyltransferase (HCT) and sucrose-phosphate synthase (SPS) genes have been linked to the potato response under alkaline conditions.

4.2.5 Potato combined abiotic stress-responsive miRNAs

A recent study to identify miRNAs in contrasting potato cultivars under a simultaneous treatment of drought and heat stresses has shown 447 conserved miRNAs and 315 novel miRNAs from 10 libraries using similarity and structural analysis [79]. It has been indicated that miRNA expression profile varies based on the stress treatment alone or combined, and the type of cultivar, tolerant or sensitive (tolerant-Unica/sensitive-Russet Burbank cultivar). For example, a total of 156 miRNAs with differential expression in sensitive cultivars have been observed, 22 of which were unique to heat and drought combination stress. Besides, 111 miRNAs were differentially expressed in tolerant cultivars under drought stress, with 16 miRNAs being drought-specific based on multiple comparison analyzes. They have also analyzed eight miRNAs that function in either specific or common modulation under drought combined with heat stress. Six miRNAs were suggested downregulated in both potato cultivars under simultaneous heat and drought treatment and individual heat stress, whereas two were found to be upregulated in this study. Upon drought stress, however, four miRNAs were downregulated, whereas one was upregulated in both cultivars. These miRNAs have depicted in the modulation of metabolic processes including a total of 200 genes regulating ribonucleotide, purine ribonucleotide, adenyl nucleotide, carbohydrate derivative binding, purine nucleotide, 236 genes for nitrogen compound metabolism, more than 135 genes for ATP binding in sensitive cultivar under drought stress. Moreover, 213 genes have been closely linked to small molecules and anion binding as well as nucleotide phosphate binding in sensitive cultivars exposed to heat treatment. Upon heat and drought treatment, 200 genes have been found participating in the cyclic compound formation and the heterocycle metabolic process in the tolerant cultivar.

Following the identification/characterization of miRNAs upon abiotic stress conditions, the functions of those miRNAs were analyzed through overexpressing in potato plants when exposed to individual or combined stress treatment. For instance, a study has explored the upregulation of miR160a-5p and downregulation of auxin response factor (ARF16) under a combination of heat and drought stress in potato plants (Unica-tolerant and Russet Burbank-sensitive cultivar) using overexpression approach [105]. They have also indicated that transgenics have greater morphological (leaf number/size and stem size), physiological (gaseous exchange traits, chlorophyll content, and canopy temperature), and biological traits (less accumulation of H₂O₂) than control plants. In addition to these findings, researchers have proposed improved the physio-biochemical performances of transgenic potato plants obtained by overexpressing Novel_105 miRNA targeting E3 ubiquitin-protein ligase gene (XBAT35), which presumably results in the inhibition of cell death to some extent under single or combined stress condition [106]. Similarly, the role of miR172b-3p has been analyzed using two contrasting potato cultivars under drought combined with heat stress [107]. It has been reported that overexpression of miR172b-3p in transgenic lines inhibited ERTF RAP2-7-like expression, resulting in increased carbon fixation performance under a combination of drought and heat treatment.

4.3 Other studies to identify miRNAs in potato

Other than known stress conditions, there are also some studies for the identification of potato miRNAs to specific environmental or growth conditions. For example, a study has identified miRNAs in plants that are sensitive to a biocompatible stress resistance inducer which is a potential approach focused on generating induced resistance by using ecologically friendly chemicals [108]. As a result of next-generation sequencing (NGS) analysis on potato leaves exposed to potassium phosphite chemical, it has been discovered 25 differentially expressed miRNAs, 14 of which have already marked in miRBase and 11 of which have been mapped to the potato genome as possible novel miRNAs. Based on miRNA target prediction analysis, it has been implied that those genes are involved in disease resistance, transcription factors, and in response to oxidative stress. In addition to these studies, researchers have characterized miRNAs in potato-grown aeroponic environments under the application of high and low nitrogen regimes [109]. Along with 119 conserved miRNAs from 41 miRNA families, a total of 1002 potential new miRNAs have been identified in potatoes. It has been observed that 52 conserved and 404 putative novel miRNAs, have been differently expressed in roots exposed to low levels of nitrogen application, while 54 conserved miRNAs and 628 putative novel miRNAs have been differentially expressed in shoots. GO study revealed the vast majority of the 34,135 putative targets were linked to biological activities, followed by molecular functions and cellular components. The roles of miR397 and miR398 in potatoes under low nitrogen stress have been displayed followed by target validations.

5. Conclusion

Improved stress tolerance in plants against dynamic environmental conditions is a major focus in agriculture due to the adverse effects of climate change on plant growth, development, and yield. Because potatoes are a susceptible plant to various abiotic stress conditions, the application of one or more stress treatments results in negative impacts on potato morphology and physiology during developmental stages as well as yield and tuber quality during/after harvesting.

Classical breeding methods have been used for many years in potato breeding. Moreover, genomics, transcriptomics, and miRNomics have significantly contributed to the efficiency of potato breeding efforts. Utilization of genomic studies includes the use of both cultivated and wild potato genotypes as a reservoir of desirable quality traits/allelic variety, as well as the ability to modify whole chromosomal sets, providing sexual hybridization a potent technique for producing novel and desirable genotypes. Therefore, several parameters that impede potato genetic advancement such as tetrasomic inheritance, a high incidence of heterozygosity, and incompatibility in potatoes might be partially overcome in potato breeding studies. Over time, genomics provides all genetic information in potatoes, such as genome structure and mapping while transcriptomics reveals all RNA-related information including stress-responsive genes depending upon expression levels/functions/locations in order to comprehend the link between a particular phenotype and transcriptome. Moreover, miRNA-based editing can be used as an alternative approach to advance potato tolerance under such individual or combined abiotic stresses since miRNAs have not only vital but also diversified functions in a multitude of metabolic processes and under assorted abiotic stress factors such as drought, cold, heat, salinity, and mineral deficiency. Because of the unique roles of miRNAs resulting from spatiotemporal ability and type of stress treatment, identification/characterization of miRNAs under individual and combined abiotic stress might be the first step in understanding the function of miRNAs in a wide range of metabolic pathways in potatoes to confer improved tolerance against the ever-mounting effects of abiotic stress in the future. These advancements in potato genomic, transcriptomic and miRNomics are highly crucial to keep up with the high demand for potatoes across the world due to the ever-growing human population and the reduction of potato production areas.

Conflict of interest

The authors declare no conflict of interest.

Funding

This study received no external funding.