Transgenic Approaches for Nutritional Enhancement of Potato

Abstract

Potatoes provide an excellent source of carbohydrates, minerals, vitamins, carotenoids, anthocyanins, and several other metabolites which play an important role in human nutrition. These bioactive compounds are effective in preventing diseases like cancer, diabetes, and heart-related issues. In addition to their industrial uses, potatoes are a major focus of genetic engineering programs for the modification of nutritional properties. Several important candidate genes operating in phenylpropanoid mechanism, ascorbic acid biosynthesis pathway, carbohydrate metabolism, steroidal glycoalkaloid biosynthesis pathway, and other-related metabolic steps have been cloned and characterized at the biochemical and molecular levels. Overexpression and down regulation of genes operating in these pathways has revealed important insights into improved nutritional quality. Expression of a transgene has successfully resulted in increasing carotenoids, anthocyanins, and vitamin content in transgenic tubers. Reduction in glycoalkaloid content, enzymatic browning, flesh color, and chipping quality has been achieved via modification of the genes involved in the respective biochemical pathway in potatoes. Transgenic approaches not only resulted in improved quality but also helped in understanding the biochemical and molecular mechanisms associated with the regulation of genes in these pathways. Although the commercialization of transgenic potatoes is still hindered by consumers approval and ethical restrictions, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system holds promise as a non-transgenic alternative for developing nutritionally enhanced potatoes.

1. Introduction

Potatoes form an integral component of human food diet and rank as the third most important staple food crop in the world after wheat and rice [1]. The tubers are important dietary source of carbohydrates, proteins, carotenoids, antioxidants, minerals, phenolics, anti-nutrients, and vitamins [2, 3, 4, 5]. These bioactive compounds are known to prevent and combat chronic diseases such as hypertension, cancer, diabetes, and heart disease [6, 7, 8, 9]. Potatoes also contains recommended amounts of minerals such as potassium, magnesium, iron, and bioactive compounds such as chlorogenic acid, the flavonoids apigenin, rutin, and kaempferol 3-O-rutinoside, polyamines and alkaloids such as calystegines, solanine, tomatine, and chaconine [10, 11]. It is widely used as a raw marketable product and in industry for making processed food stuffs such as chips and french fries etc. [12]. Although, they provide most of the calories and protein needed, potatoes are not nutritionally complete foods [13, 14]. To overcome the challenges of poverty and hunger worldwide, potato is considered as one of the promising crops for nutritional enhancement [15]. Traditional processing and preparation methods such as peeling, roasting, microwaving, boiling, frying, and baking alter nutritional quality of potatoes including loss of key micronutrients, adsorption of fat, and conversion of naturally resistant starch into highly digestible starch [16, 17]. Biotechnology could enhance micronutrient content and has the potential to reduce malnutrition especially among poor people from developing countries [13]. This approach will only be successful if clear advantages are demonstrated to both growers and consumers and the necessary safety precautions are addressed [18].

Potatoes are an excellent model crop for the genetic modification of metabolic pathways. Large-scale metabolomic studies have identified significant variation in nutrient content, minerals, and bioactive compounds in potatoes leading to the selection of specific cultivars for consumption of raw or processed potatoes [19, 20, 21]. Metabolite and mineral variation in raw and cooked potatoes can be used to predict the nutrient and bioactive content in cooked potato tuber for improved health traits [22]. Although conventional breeding efforts are underway for producing potato cultivars with altered nutritional properties [23, 24], the efforts are complicated by the tetrasomic inheritance and high level of heterozygosity of potatoes [25]. Transgenic technology and genome editing tools [26] offer significant opportunities for tailored improvement of nutritional quality traits in potato, such as starch and sugar content, chipping quality, flesh color, and taste, as well as ascorbic acid, anthocyanin, carotenoid, and glycoalkaloid content [27, 28, 29, 30, 31, 32].

Figure 1 outlines the important quality traits that have been modified by transgenic technologies or other genome editing tools in potato. These technologies include production of transgenic potatoes by overexpression or antisense repression of genes, RNA interference (RNAi) and gene editing tools such as Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) [26, 27, 31, 33, 34]. TALENs and CRISPR/Cas9 systems make site-specific gene modification by creating double-stranded DNA break. While TALEN recognizes the target site based on DNA protein interaction, the CRISPR system is based on site specific RNA protein interactions. CRISPR/Cas9 is a targeted mutagenesis technique to generate knockout mutations via non-homologous end-joining as well as gene targeting to edit an endogenous gene by homologous recombination [26, 27, 31, 33, 34]. CRISPR/cas9 avoids foreign DNA insertions in the plant genome, an important criterion in the development of crop varieties not subjected to the cumbersome GMO regulation process [26, 27, 28, 34]. Moreover, the availability of the potato genome sequence [35] has facilitated the development of comparative genomic analyses and functional studies of candidate genes to improve several important traits in potato [36]. In this review we discuss the past developments, and future perspectives of nutritional enhancement in potato using transgenic technologies.

Figure 1.

Important quality traits in potato. Figure highlights the important tuber quality traits. A very special thanks to Prof. Dr. David G. Holm, Department of Horticulture and Landscape Architecture, Colorado State University, USA and Dr. Sanjay Gupta, Department of Soil, Water and Climate, University of Minnesota, USA for providing the potato photographs used in the figure. Also, I would like to extend my thanks to https://www.idahopacific.com/potato-granules and Waltz (2015; 10.1038/nbt0115-12) from which the photographs have been obtained and modified.

2. Reducing the glycoalkaloid content in transgenic tubers

α-Chaconine and α-solanine are the two main glycoalkaloids that are toxic secondary metabolites present in the tubers of cultivated potato (Solanum tuberosum L.) [37, 38]. Symptoms of glycoalkaloid poisoning include abdominal pain, vomiting, and diarrhea in humans. Due to their toxic properties, 200 mg/kg fresh weight is the safety limit for total glycoalkaloid content in the tubers of released commercial potato cultivars [39, 40] because glycoalkaloids cannot be destroyed during food-processing treatments, such as boiling, baking or frying and even at high temperatures [41]. The general pathway of glycoalkaloid metabolism and important candidate genes used in the modification of glycoalkaloid content are highlighted in Figure 2 and Table 1).

Figure 2.

Glycoalkaloid biosynthetic pathway. The glycoalkaloid biosynthetic pathway starts from Acetyl-Co-A. The representative enzymes are HMGR1, 3-Hydroxy-3-methylglutaryl coenzyme A reductase; PVS1, vetispiradiene sesquiterpene cyclase; PSS1, squalene synthase; SMT1, sterol C24-methyltransferase type1; CH, cholestrole hydroxylase; SGT1, solanidine galactosyltransferase; SGT2, solanidine glucosyltransferas; SGT3, glycosterol rhamnosyltransferase; SMO, C-4 sterol methyl oxidase; SD, sterol C-5(6) desaturase; SSR, sterol side chain reductase; and GAME, glycoalkaloid metabolism genes. The figure is adapted and modified from Arnqvist et al. [27], Khan et al. [42], Sonawane et al. [43].

Gene name	Abbreviation	Quality trait	Reference
UDP-galactose:solanidine galactosyltransferase	SGT	Glycoalkaloid	McCue et al. [44], Shepherd et al. [45]
Cytochrome P450 monooxygenases	CYP72A208 and CYP72A188	Glycoalkaloid	Umemoto et al. [46]
Sterol side chain reductase 2	SSR2	Glycoalkaloid	Heftmann et al. [47], Sawai et al. [48], Itkin et al. [49], Nahar et al. [50]
Cytochrome P450 monooxygenase	CYP88B1	Glycoalkaloid	Akiyama et al. [51]
Dioxygenase	DOX	Glycoalkaloid	Nakayasu et al. [52]
Sterol 24-C-methyltransferase, sterol desaturase and C-4 sterol methyl oxidase	SMT1, SD and SMO	Glycoalkaloid	Arnqvist et al. [27], Kaminski et al. [53]
GLYCOALKALOID METABOLISM 9	GAME9	Glycoalkaloid	Cárdenas et al. [54], Ginzberg et al. [55]

Table 1.

Genes used in the modification of glycoalkaloid contents in transgenic potatoes.

The biosynthesis of γ-solanine is catalyzed by the enzyme UDP-galactose:solanidine galactosyltransferase (SGT) from galactose and solanidine [44]. Transgenic potato lines were produced using an antisense version of a cDNA encoding SGT under the control of Cauliflower Mosaic Virus 35S (CaMV35S) promoter or a tuber-specific granule bound starch synthase (GBSS) promoter. Transgenic lines produced from potato cv. ‘Lenape’ expressing antisense SGT exhibited significantly lower steroidal glycoalkaloids in the tubers. In another study [45], antisense suppression of the genes that encode the enzyme for the biosynthesis of γ-solanine from UDP-galactose and solanidine (SGT1), γ-chaconine from UDP-glucose and solanidine (SGT2), and α-solanine and α-chaconine from UDP-rhamnose, β-solanine and β-chaconine (SGT3) were down-regulated under the control of GBSS6 promoter. Down-regulation of SGT1 reduced the concentration of α-solanine without affecting the levels of α-chaconine. In contrast, down-regulation of SGT2 resulted in reduction of α-chaconine and increased levels of α-solanine. Down-regulation of SGT3 reduced concentrations of both α-chaconine and α-solanine [45]. Antisense manipulation of the SGT caused reduced glycoalkaloids content thereby decreasing toxicity in potato tubers.

RNAi, TALENs and CRISPR/Cas9-based systems have been used to reduce glycoalkaloid content [46, 52]. Glycoalkaloid biosynthesis is carried out by PGA1 and PGA2 encoding cytochrome P450 monooxygenases (CYP72A208 and CYP72A188) respectively. Transgenic lines using RNAi expressing either PGA1 or PGA2 showed very little steroidal glycoalkaloids accumulation and no effect on vegetative growth and tuber production [46]. Cholesterol and sterol side chain reductase 2 (SSR2) is a key enzyme in the biosynthesis of cholesterol and related steroidal glycoalkaloids [47, 56]. TALEN induced mutations in the SSR2 gene have lower levels of cholesterol and steroids without affecting the plant growth [48, 49, 50]. CRISPR/Cas9-edited StSSR2 resulted in a significant reduction of steroidal glycoalkaloids content [57]. CRISPR-Cas9-based knockout of CYP88B1, resulted in reduced levels of α-solanine and α-chaconine [51]. Likewise, knockout of dioxygenase St16DOX, responsible for steroid 16α-hydroxylation abolished St16DOX expression and prevented glycoalkaloids production [52]. These studies indicate that suppression or knockout of genes involved in glycoalkaloid biosynthesis is a viable strategy to manipulate the steroidal glycoalkaloid levels in potato.

In contrast to gene suppression strategies, other studies have shown that the overexpression of genes involved in steroidal glycoalkaloid biosynthesis pathway are also an effective strategy to reduce cholesterol and glycoalkaloid levels [27]. Steroidal glycoalkaloids are thought to be synthesized from cholesterol that is converted to solanidine, and then by two separate pathways to α-solanine and α-chaconine. Altered glycoalkaloid content has been associated with overexpression of genes such as sterol 24-C-methyltransferase (SMT1), sterol desaturase (SD) and C-4 sterol methyl oxidase (SMO) [53]. SMTs are involved in the biosynthesis of sterols and other products [58]. Overexpressing soybean SMT (GmSMT1) increased total sterols accompanied by a decrease in cholesterol and glycoalkaloids in leaves and tubers [27]. Glycoalkaloid metabolism9 (GAME9) is an APETALA2/Ethylene Response Factor associated with a major quantitative trait for steroidal glycoalkaloid content in tubers [54, 59]. Overexpression of GAME9 altered the levels of steroidal glycoalkaloids leaves and tuber skin [54, 55].

3. Genetic modification for vitamin C content

A whole, baked potato is an excellent source of vitamin C, vitamin B6, niacin and folate [60, 61, 62]. High consumption of potato tubers has been correlated with increased antioxidant level in blood and tissues and increased protection against oxidative stress [60]. However, the level of vitamin C is reduced if the potato is frozen, stored under refrigerated conditions, boiled or fried [63, 64, 65]. Thus far, there has been limited success in increasing vitamin C content in transgenic potato tubers [66, 67].

The pathway of ascorbic acid (vitamin C) production (Figure 3) includes the reduction of D-galacturonic acid to L-galactonic acid by D- galacturonic acid reductase (GalUR), followed by conversion to L-galactono-1,4-lactone by aldonolactonase. The L-galactono-1,4-lactone is then oxidized to ascorbic acid by L-galactono-1,4-lactone dehydrogenase (GALDH) [70]. Table 2 outlines the key genes used in the modification of ascorbic acid content. Overexpression of the strawberry GalUR resulted in increased ascorbic acid levels in potato [3]. Dehydroascorbate reductase (DHAR) plays an important role in maintaining the normal level of ascorbic acid by recycling oxidized ascorbic acid. Transgenic potato over-expressing the cytosolic DHAR significantly increased DHAR activity and ascorbic acid content in potato leaves and tubers, whereas chloroplastic DHAR overexpression only increased DHAR activity and ascorbic acid content in leaves [29]. Overexpression of the Arabidopsis GDP-_L-galactose phosphorylase (GGP) resulted in significantly enhanced ascorbic acid content. Overall studies suggest that genetic alteration of specific vitamin-related genes could be excellent targets for improving the nutritional content of potato.

Figure 3.

L-ascorbic acid biosynthesis pathway. The representative enzymes are GGP, GDP-_L-galactose phosphorylase; GalUR, D-galacturonic acid reductase; DHAR, dehydroascorbate reductase; GALDH, L-galactono-1,4-lactone dehydrogenase; and AL, aldono lactonase. The figure is adapted and modified from Hemavathi et al. [68], Venkatesh and Park [69].

Gene name	Abbreviation	Quality trait	Reference
D- galacturonic acid reductase	GalUR	Ascorbic acid/vitamin C	Hemavathi et al. [3]
L-galactono-1,4-lactone dehydrogenase	GALDH	Ascorbic acid/vitamin C	Linster et al. [70]
Dehydroascorbate reductase	DHAR	Ascorbic acid/vitamin C	Qin et al. [29]
GDP-L-galactose phosphorylase	GGP	Ascorbic acid/vitamin C	Bulley et al. [67]

Table 2.

Genes used in the modification of ascorbic acid content in transgenic potatoes.

4. Enhancing carotenoid content in transgenic potato

Carotenoids are yellow to red pigments that play an essential role in human nutrition, with the most important carotenoid being β-carotene, a major source of provitamin A. Deficiency of vitamin A is a major global micronutrient problem that causes blindness and weakens the immune system [71, 72]. Carotenoids enhance may improve the immune system, reduce cardiovascular disease and cancer and help prevent atherosclerosis [73, 74, 75]. For these reasons, there is considerable interest in developing potatoes with increased levels of carotenoids [76, 77, 78, 79].

Xanthophylls lutein, zeaxanthin, violaxanthin and neoxanthin are the major carotenoids present in the tubers of cultivated potato while that of β-carotene is found in low levels [80, 81]. The Candidate genes used in the modification of carotenoid content in transgenic potatoes are highlighted in Figure 4 and Table 3. Lycopene is produced from phytoene by phytoene desaturase (CRTI), and cyclized by lycopene β-cyclase (LCY-β) to form β-carotene. While α-carotene is produced by both LCY-β and LCY-ε). The hydroxylation of α-carotene yields lutein. Two subsequent hydroxylations of β-carotene, catalyzed by carotenoid β-hydroxylase (CHYB) produce zeaxanthin. Zeaxanthin can be epoxidized by zeaxanthin epoxidase (ZEP) to form violaxanthin, which can be used by violaxanthin de-epoxidase (VDE) to regenerate zeaxanthin. The final step in carotenoid biosynthesis is the conversion of violaxanthin to neoxanthin by neoxanthin synthase [92]. Introduction of various carotenogenesis-related genes has resulted in increased production of specific carotenoids and total carotenoid content using overexpression [84, 93], co-transformation with more than two genes [83], antisense [94], and RNAi technology [75, 87].

Figure 4.

Carotenoid biosynthesis pathway. The representative enzymes are PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTISO, carotenoids isomerase; LCY-e, lycopene e-cyclase; LCY-b, lycopene β-cyclase; CHB/BCH, β-carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NXS, neoxanthin synthase; CCD, carotenoid cleavage dioxygenase; Crt-ketolase. The figure is adapted and modified from Zhou et al. [31].

Gene name	Abbreviation	Quality trait	Reference
Phytoene synthase	PSY	Carotenoid	Cazzonelli and Pogson [82]
Phytoene synthase CrtB	CrtB	Carotenoid	Diretto et al. [83], Ducreux et al. [84]
Lycopene epsilon cyclase	LCY-e	Carotenoid	Diretto et al. [85]
β-carotene hydroxylases	CHY	Carotenoid	Diretto et al. [86]
β-carotene hydroxylase gene	CHB	Carotenoid	Van Eck et al. [75]
Carotenoid cleavage dioxygenases	CCDs	Carotenoid	Campbell et al. [87]
Ketolase crtO, crtW, bkt	crtO β-carotene, crtW, bkt	Carotenoid	Gerjets and Sandmann [88], Lu et al. [89]
Cauliflower Or	Or	Carotenoid	Lu et al. [89], Lopez et al. [90], Goo et al. [91]

Table 3.

Genes used in the modification of carotenoid content in transgenic potatoes.

Phytoene synthase (PSY) is the rate-limiting step in the carotenoid biosynthetic pathway [82] and manipulation of PSY expression resulted in enhanced carotenoid synthesis in tubers [84]. The hydroxylation of β-carotene is a second important regulatory step in carotenogenesis [95]. Tuber-specific overexpression of the bacterial phytoene synthase (CrtB) gene caused a 7-fold increase in total carotenoids [84]. Expression of three genes from the bacterium Erwinia herbicol encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene betacyclase (CrtY), under the tuber-specific patatin promoter resulted with deep yellow flesh and increased levels of β-carotene, α-carotene, lutein and violaxanthin [83]. Silencing of LCY-e resulted in increased carotenoid levels, with up to 14-fold more β-carotene in tubers [85]. Silencing of the genes encoding β-carotene hydroxylases CHY1 and CHY2 using the tuber-specific patatin promoter increased β-carotene levels along with increased levels of phytofluene, violaxanthin, neoxanthin, lutein and total carotenoids [86]. Both the overexpression and silencing of the major genes in carotenoid biosynthesis pathway produced increased carotenoids in transgenic potato tubers.

Zeaxanthin have become increasingly important due to their benefits in the prevention of degenerative diseases [96]. Zeaxanthin is an immediate biochemical derivative of β-carotene thus tubers that accumulate high levels of zeaxanthin produce β-carotene that subsequently serves as the substrate for zeaxanthin synthesis [75]. RNAi was used to silence the β-carotene hydroxylase gene (BCH/CHB), which converts β-carotene to zeaxanthin under the control of GBSS or CaMV35S promoters [75]. Transgenic lines with silenced CHB expression showed altered carotenoid profiles. Transformants derived from the GBSS promoter contained more β-carotene than CaMV35S transformants, demonstrating that silencing CHB has the potential to increase the content of carotenoids in potato for mitigating vitamin A deficiency [75]. Oxidative cleavage of carotenoids is catalyzed by carotenoid cleavage dioxygenases (CCDs). Down-regulation of CCD4 though RNAi resulted in a 2-5-fold higher carotenoid content due to elevated violaxanthin content. Down-regulation of zeaxanthin epoxidase under the control of GBSS promoter resulted in zeaxanthin-rich potato lines, increased total carotenoids, and reduced the amount of lutein [94]. Thus, RNAi is a useful strategy to improve the carotenoid content in potato tubers thereby alleviating the vitamin A deficiency.

Astaxanthin, is an important ketocarotenoid associated with the reduction in oral cancer and mammary tumor growth [97, 98] and increasing astaxanthin and other ketocarotenoids levels has been studied in potato [88]. Astaxanthin is derived from β-carotene by 3-hydroxylation and 4-ketolation at both ionone end groups [99]. The hydroxylation reaction is widespread in many organisms, but ketolation is restricted to a few bacteria, fungi, and some unicellular green algae [100]. Previous studies used the transgenic expression of ketolase genes to produce ketocarotenoids in potato [88] as well as other Solanaceae members such as tomato [101] and tobacco [102]. A transgenic potato cultivar that accumulates increased zeaxanthin due to inactivated zeaxanthin epoxidase was co-transformed with the ketolase (crtO β-carotene) gene from the cyanobacterium Synechocystis under the control of a constitutive promoter. The resulting transgenic potato plants accumulated more ketocarotenoids in leaves, as well as more 3′-hydroxyechinenone, 4-ketozeaxanthin and astaxanthin in the tuber [88]. Likewise, overexpression of the crtW gene from the marine bacterium Brevundimonas under the control of GBSS promoter resulted in enhanced astaxanthin content in transgenic potato tubers [101, 103]. These and other studies reveal that transgenic potato lines can be produced with increased carotenoid content using bacterial and algal genes.

In another approach, increased carotenoid content was observed in tubers when cauliflower Or gene was overexpressed in potato [89, 90]. Lu et al. [89] showed that the cauliflower Or gene encoding a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulation can be used to increase total carotenoid and β-carotene levels in potato tubers. Overexpression of the Or gene induced the formation of chromoplasts and resulted in high levels of carotenoids in transgenic tubers [90, 91, 103, 104]. This increase was found to be associated with the Or-regulated stability of PSY protein in these tubers, thus facilitating continuous carotenoid synthesis in the transgenic tubers [104]. Thus, overall studies indicated that genetic manipulation of Or genes can be an effective strategy to achieve increased carotenoid content and high quality potato tubers.

Overall, these studies indicate that the suppression and overexpression of various genes involved in carotenoid biosynthesis under different promoters resulted in altered carotenoid content in transgenic potato tubers. Although the transgenic strategy may be an effective way for increasing carotenoids production in potato, CRISPR/Cas9 gene editing might be challenging in tailoring efficient and non-transgenic potato cultivars with improved nutritional quality.

5. Manipulation for high levels of anthocyanins

Anthocyanins are water-soluble flavonoids that not only contribute to color of the fresh potatoes, but high anthocyanin content also enhances the antioxidant benefits on human health [105, 106, 107, 108]. The major anthocyanidins in purple potato are cyanidin, petunidin, pelargonidin, peonidin, and malvidin, while red potatoes contain cyanidin, pelargonidin, and peonidin [109, 110, 111]. Transcriptome analysis has identified 104 potentially important genes that may play an important role in anthocyanin biosynthesis in potato [112]. Several studies [113, 114] have investigated the impact of altered expression of some of the key genes in the anthocyanin biosynthesis pathway (Figure 5 and Table 4).

Figure 5.

Anthocyanin biosynthetic pathway. The representative enzymes are CHS, chalcone, synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase and UFGT, flavonoid 3-O-glucosyltransferase. The figure is adapted and modified from Mattoo et al. [115].

Gene name	Abbreviation	Quality trait	Reference
Dihydroflavonol 4-reductase	DFR	Anthocyanin	Stobiecki et al. [113], Zhang et al. [116]
Flavonoid 3’ ,5’ -hydroxylase	F3’5’H	Anthocyanin	Jung et al. [114]
Anthocyanidin 3-O-glucosyltransferase	3GT/UFGT	Anthocyanin	Yoshihara et al. [117]
UDP-glucose: flavonoid-3-O-glucosyltransferase	3GT	Anthocyanin	Wei et al. [30]
MYB transcription factor	MYB	Anthocyanin	Liu et al. [118], Kranz et al. [119]

Table 4.

Genes used in the modification of anthocyanin content in transgenic potatoes.

The biosynthesis of anthocyanins (Figure 5) initiates from 4-coumaroyl-CoA and malonyl CoA catalyzed by the enzyme chalcone synthase (CHS) to synthesize naringenin chalcone which is then converted to naringenin by chalcone isomerase (CHI). Naringenin is converted to dihydrokaempferol, catalyzed by flavanonone 3′-hydroxylase (F3H). Flavonoid 3′-hydroxylase (F3′H) thereafter hydroxylates dihydrokaempferol (DHK) into dihydroquercitin (DHQ) or to dihydro-myricetin (DHM) which is catalyzed by flavonoid 3′,5′ –hydroxylase (F3′5′H). All three dihydroflavonols DHK, DHM and DHQ are independently converted to colorless leucoanthocyanidins by the enzyme dihydroflavonol 4-reductase (DFR). The next enzymatic reaction involves the enzyme anthocyanidin synthase (ANS), which converts all three leucoathocyanidins to colored anthocyanidins [120]. Stobiecki et al. [113] demonstrated that overexpression of DFR gene under the control of the CaMV35S promoter showed increased tuber anthocyanin content along with a 4-fold increase in petunidin and pelargonidin derivatives in red skinned potato cv. ‘Desiree’. However, overexpression of DFR in the potato cv ‘Prince Hairy’, which has a white tuber, resulted in change in the flower color from light blue to purple, but no change in tuber color [116]. Likewise, the overexpression of F3′5′H in the cv. ‘Desiree’ resulted in plants with purple-colored tubers and stems [114].

In addition to genes involved in the biosynthesis of anthocyanins, several transcription factors have been associated with anthocyanin biosynthesis [118, 119, 121, 122]. Stushnoff et al. [121] studied the gene expression pattern associated with the accumulation of purple tuber anthocyanins using microarray. A total of 27 genes were identified those were differentially expressed in purple and white tuber tissues. One of these genes coded for a novel single-domain MYB transcription factor (StMYBA1) that has been shown to influence anthocyanin-pigment production in potato [121, 122]. StMYBA1 from potato was transformed into tobacco under the control of the CaMV 35S promoter and the resultant transformants showed anthocyanin accumulation in all tissues of transgenic tobacco lines [118]. In other studies, it has been demonstrated that there are two distinct classes of MYB transcription factors which negatively regulate anthocyanin accumulation: R3 MYB and R2R3 MYB repressors [119]. R2R3 MYB transcription factors; StAN1 and StbHLH1 are responsible for the coordinated regulation of the skin and flesh pigmentation, as well as anthocyanin biosynthetic pathway genes in white regions in potato [122, 123, 124]. Therefore, manipulation of specific transcription factor genes associated with anthocyanin synthesis through transgenic or CRISPR tools may be useful in enhancing nutritionally important traits of pigmented tuber flesh.

6. Improving the cooking and processing quality

Although potatoes are increasingly consumed in the form of processed food products such as chips, french fries, dehydrated products, etc., they do not deliver the same nutritional value as fresh potatoes [125, 126, 127]. Potatoes are rich in carbohydrates (starch, sucrose, glucose, and fructose) and non-starch polysaccharides from cell wall components [128]. Potato starch is composed of 20–30% amylose and 70–80% amylopectin [129, 130]. Both starch and sugars play an important role during growth and development (biosynthesis of starch) and during postharvest storage of potatoes (breakdown of starch) [131]. The amount of starch and sugars present in potato tubers is an important criterion for selection of potato cultivars for commercial purpose. Likewise, the amount of reducing sugars (RS) present in potato tubers affects the processing quality of fried products. The final color in fried potato products results from the heating reactions that occur between RS and amino acids such as asparagine [132]. Similarly, cooking and baking qualities that are important for consumers are related to color, texture, and flavor. Therefore, potatoes are selected with reduced sugar content, good processing quality, and with the absence of cooking defects such as enzymatic browning and stem-end blackening [133]. Engineering for modified starch and sugar content as well as alteration in cooking quality has resulted in enhanced nutritional quality of transgenic potato tubers [134, 135, 136, 137, 138].

6.1 Metabolic engineering for modifying starch content

Figure 6 and Table 5 highlights the information on important candidate genes used in the modification of starch content in transgenic potato tubers. ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS) and starch branching enzyme (SBE) are involved in the process of starch synthesis while amylases (AMY) and starch phosphorylase (SP) are responsible for its breakdown [152]. Amylose is synthesized by the granule bound starch synthase (GBSS), whereas soluble starch synthases (isoforms) and SBEI/SBEII, with various debranching enzymes (DBE), kinases and other enzymes are also involved in amylopectin synthesis [153, 154]. The modification of starch properties is important to food processing industries and the demand for amylose free potatoes wherein higher amylopectin content is desired [122, 155]. Food products containing high amylose content and long chains of amylopectin contribute to the formation of resistant starch that is responsible for a lower glycemic index after intake with enhanced health benefits by promoting the growth of healthy gut fora and lowering both the caloric intake and cholesterol levels in the blood [156, 157, 158].

Figure 6.

Simplified schematic overview of starch and sugar metabolism. The figure explains the simplified pathway of starch and sugar metabolism in potato tubers. Only the key enzymes involved in the pathway are shown in the figure. Starch synthesis involves AGPase, ADP-glucose pyrophosphorylase; SS, starch synthase; GBSS, granule bound starch synthase; and SBE, starch branching enzyme. This process involves more than one SS and SBE (not shown in figure). Further starch is degraded into Glucose-1-Phosphate (Glu-1-P) by Amy, amylase; SP, starch phosphorylase which forms Glucose-6-Phosphate (Glu-6-P) via PGM, phosphoglucomutase. Fructose-6-Phosphate (Fru-6-P) is formed from PHI, Glc-6-P via phosphohexoisomerase. Sucrose is formed in the cytoplasm from UDP-Glucose. SPS and S6S are sucrose phosphate synthase and sucrose 6 phosphate phosphatase. UGPase, UDP-glucose pyrophosphorylase; SPS, sucrose phosphate synthase; and S6P, sucrose 6-phosphate phosphatase are involved in the process. Sucrose is transported into the vacuole and converted by AI, acid invertase leading to the formation of RS glucose and fructose. AI is inhibited by vacuolar invertase inhibitor (INH) in the vacuole. NI, neutral invertase converts sucrose into glucose and fructose in the cytoplasm. The figure is adapted and modified from Sowokinos [139].

Gene name	Abbreviation	Quality trait	Reference
Granule bound starch synthase	GBSS	Starch	Visser [140], Kuipers et al. [141], Andersson et al. [142]
ADP glucose pyrophosphorylase	AGPase	Starch	Müller-Röber et al. [143]
Glucose-6-phosphate/phosphate translocator and the adenylate translocator (nucleotide translocator)	GPT and NTT	Starch	Claudia et al. [144]
R1 protein	R1 protein	Starch	Lorberth [145]
Starch branching enzyme	SBE	Starch	Tuncel et al. [146]
Vacuolar invertase	VI	Sucrose	Bhaskar et al. [147], Liu [148], Clasen [149]
RING finger	SbRFP1	Sucrose	Scheidig [150]
Vacuolar invertase inhibitor	INH2	Sucrose	Liu et al. [138], McKenzie et al. [151]

Table 5.

Genes used in the modification of carbohydrate metabolism and enzymatic browning in transgenic potatoes.

Transgenic studies focused on the modification of starch content and its properties using the genes involved starch biosynthesis in potatoes [140, 141, 143, 144, 145, 159, 160, 161, 162, 163]. AGPase is the key enzyme involved in the synthesis of starch in amyloplasts which consists of two regulatory subunits and two slightly smaller catalytic subunits. The function of the large subunit of AGPase is to modulate the regulatory properties of the small subunit (sAGP) and the function of sAGP is primarily catalysis [152, 159, 160]. An antisense inhibition of sAGP under the control of CaMV 35S promoter resulted in starch reduction and a lower amylose content in transgenic tubers [143].

Nutritional quality enrichment by starch modification can be efficiently performed using TALENs technology [164] as well as CRISPR/Cas9 system [142]. Site-specific mutations induced in GBSS gene (CaMv35S promoter) using Emerald-Gateway TALEN system resulted in 63 nucleotide deletion suggesting that the system can be utilized for nutritional enhancement in potato [164]. All four GBSS alleles were knockout by CRISPR/Cas9 technology in cv. ‘Kuras’ protoplasts resulted in complete loss of GBSS activity and amylose-free high amylopectin starch in regenerated potato microtubers [142]. In another study, overexpression of SBEII using hybrid cDNA/gDNA intragene construct containing a single intron increased short-chain branching of amylopectin and altered the physicochemical properties of starch in potato tuber [165]. CRISPR-Cas9 was used to crete mutations in the two SBEs (SBE1 or SBE2 alone or in combination) in potato. Results revealed that lines mutated in SBE1 did not have an altered starch structure, while tuber cells from SBE2 mutated lines displayed an increased number of starch granules. One line had a strong reduction in both SBEs, resulting in starch with an altered granule phenotype, longer amylopectin chains and reduction in a degree of branching [146]. The quality characteristics of potatoes for table and processing purposes are largely dependent on the starch, dry matter, sugar concentration and tubers free from any deformities such as enzymatic discoloration. Overall studies revealed that, transgenic, intragenic as well as CRISPR tools proved their usefulness in modification of various starch synthesizing enzymes using gene silencing or gene knockout approaches. Alteration in starch synthesis genes can lead to modification in starch properties without affecting the yield and dry matter content of the transgenic tubers.

6.2 Postharvest tuber quality for enhanced cold-induced sweetening

After harvest, storage of potato tubers under cold conditions (below 8–10°C) is required for year-round processing as well as to mitigate the possibility of sprouting and diseases. Stored tubers loose some of their starch content and accumulate high RS in a process known as cold-induced sweetening (CIS). Upon frying at high temperatures, these RS interact with asparagine to produce dark-colored fried products [166, 167]. Therefore, minimizing the accumulation of RS in tubers is of high importance to potato processing industry. The amount of RS in a tuber is regulated by a balance between the activity of invertases (cell wall invertase, vacuolar invertase and neutral invertase), which convert sucrose into RS and invertase inhibitors which limit the activity of invertase via protein-protein interactions [28, 136, 168]. Figure 6 and Table 5 highlights the information on important candidate genes used in the modification of sugar content in transgenic potatoes. Among various invertases, the acid invertase (AI) is the key enzymes involved in the conversion of sucrose into RS and transgenic approaches confirmed that overexpression of the vacuolar invertase inhibitor (INH2) gene reduces the expression of vacuolar invertase gene, AI activity and RS in transgenic potato tubers under the control of CaMV 35S or class I patatin promoter [136, 137, 138, 166]. In another study, overexpression of the vacuolar invertase inhibitor isoforms from potato resulted in decreased vacuolar invertase activity, low RS and low acrylamide content with improved chip quality in cold-stored transgenic potato [138, 151]. Suppression of the AI activity by silencing of the vacuolar invertase gene using RNAi or TALENS resulted in a very strong decrease in RS accumulation, light colored chips and low acrylamide in cold-stored transgenic tubers of different potato cultivars [147, 169]. Knockout of the vacuolar invertase was performed in ‘Ranger Russet’ potatoes using the TALENs technology. Five regenerated plants contained knockouts of all four invertase alleles with no detectable RS, light brown chip color and lower levels of acrylamide [148].

Other than, the vacuolar invertase and its inhibitor gene, Zhang et al. [170] hypothesized that RING finger gene (SbRFP1) could be a potential target for manipulation of the CIS in potato tubers. RING finger proteins constitute a large protein family in higher plants involved in cold response [170]. When a novel SbRFP1 was overexpressed (CaMV35S promoter) in potato microtubers (cv. ‘E-potato 3’), it resulted in inhibition of beta-amylase and invertase activity. As a result, starch and sucrose degradation slowed down and the accumulation of RS in cold stored tubers was prevented [170]. Study suggested that other potential genes could be involved in CIS in potato tubers and transgenic manipulation can be performed to overcome this persistent problem.

So far, much attention has been given to the manipulation of the acid invertase activity via suppression and overexpression of the vacuolar invertase and invertase inhibitor genes to control the RS content in transgenic potato tubers respectively. In contrast to the acid invertase, the potential involvement of neutral invertase which is involved in the conversion of sucrose into RS has not been demonstrated and well-studied in potato tubers. Datir and Regan [150] identified 8 neutral invertase genes from potato. Based on their expression pattern and enzymatic pattern in cold-stored potato tubers, they concluded that neutral invertases also may play a role in sucrose degradation. Therefore, genetic manipulation of neutral invertase may result in decreased RS along with improved processing quality of potato tubers.

6.3 Reducing the enzymatic browning

Enzymatic browning or discoloration of potato tubers occurs when phenolic compounds are oxidized by the enzyme polyphenol oxidase (PPO). This results in negative effects on color, taste, flavor, and nutritional value and forms undesired dark pigments that result in considerable economic losses to the potato food and processing industry [171, 172]. To prevent the quality loss and increase consumers acceptance, sulfiting agents (sulfur dioxide, sodium etc.) can be used to prevent the enzymatic browning, however, there are concerns about the health risks of sulfites [173, 174]. For this reason, there is a need to develop the alternative technologies for developing potatoes that are resistant to enzymatic browning [175]. Silencing of the PPO gene resulted in significantly reduced enzymatic browning in the tubers of transgenic lines [175, 176]. Two potato cultivars ‘Van Gogh’ and ‘Diamant’ transformed with antisense PPO placed under patatin and GBSS promoters abolished the expression of PPO in transgenic tubers [176]. In another study, the PPO activity was inhibited by expression of a sense as well as antisense PPO RNAs from a tomato PPO cDNA under the control of the CaMV 35S promoter in cv. ‘Russet Burbank’. Transgenic lines expressing PPO by sense and antisense approaches resulted in reduction in black spot susceptibility, decreased PPO activity and reduced enzymatic browning [175].

Recent studies have demonstrated that genome editing using CRISPR/Cas9 system can be successfully used to reduce the enzymatic browning in potato [177, 178]. The Cas9 nuclease guided by two RNA molecule/s (sgRNA/s) introduced a double stranded break in the PPO gene. The system that introduced the mutations in the PPO gene was delivered into the protoplasts of cv ‘Desiree’. 24% of CRISPR/Cas9-edited lines carried mutations in all four alleles of PPO without any off-target mutations in other PPO genes. Mutations induced in the four alleles of StPPO2 gene showed 69% reduction in tuber PPO activity and a 73% reduction in enzymatic browning, compared to the control [177]. These studies revealed that CRISPR/Cas9 system represents an important step towards the development of potato varieties that maintain the organoleptic, antioxidant and nutritional properties during harvest and post-harvest procedures, without the utilization of potentially harmful browning controlling agents.

7. Conclusions

Improvement in nutritional properties such as cooking, baking, and processing qualities such as dry matter content, vitamins, starch, sugar content, flavors, colors, and glycoalkaloids is one of the most important aspects of potato production. Genetic manipulation of these quality traits has significantly increased our understanding of the genes and their network involved in controlling these traits. However, transgenic potatoes still lack consumer and producer acceptance and are not widely used compared to other crops. Transgenics created doubts on the transformation processes and hence food safety evaluation tests are necessary to detect the any unintended effects in transgenic lines. Release of the Potato Genome Sequencing Consortium, genetic mapping, and genome-wide association-based studies, and the recent progress in CRISPR/Cas9-based tools have paved the way for development of potato cultivars with improved nutritional properties. Using intragenic or cisgenic and CRISPR-based approaches as well as proper assessment under field trials may alleviate the concerns by consumers, producers, and processors. Also, the unintended effects can be overcome by CRISPR/Cas9-based tools to produce transgene-free potatoes.

Acknowledgments

Authors are grateful to Prof. Dr. David G. Holm, Department of Horticulture and Landscape Architecture, Colorado State University, USA and Dr. Sanjay Gupta, University of Minnesota, USA for providing the potato photographs used in Figures 1–5. This research was supported by a Natural Science and Engineering Research Council grant to SR.

Conflict of interest

“The authors declare no conflict of interest.”

Acronyms and abbreviations

amiRNAs	artificial microRNAs
CaMV35S	cauliflower mosaic virus 35S
CRISPR	Clustered Regularly Interspaced Short Palindromic Repeats
QTL	quantitative trait loci
RNAi	RNA interference
TALENS	Transcription Activator-Like Effector Nucleases