Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Changes in cell wall components and polysaccharide-degrading enzymes in relation to differences in texture during sweetpotato storage root growth.

Sweetpotato has special texture characteristics, which directly affect the eating quality and post-production processing quality of sweetpotato. To investigate the texture change mechanism of sweetpotato during the growth process, this study selected two varieties with significant differences in texture from 35 varieties. The storage roots were sampled at 50, 80, 110, and 140 days after planting. Measure the texture parameters, the cell wall composition content, cell wall-related enzyme activities and the expression of expansin genes of sweetpotato storage roots. The results show that the hardness, adhesiveness and chewiness parameters of 'Yushu No 10' were significantly lower than those of 'Mianfen No 1', they have significantly different texture properties. In terms of cell wall composition, the soluble pectin content of 'Yushu No 10' was more than twice that of 'Mianfen No 1', whereas the insoluble pectin content was lower than that of 'Mianfen No 1', with the cellulose content of 'Yushu No 10' being significantly higher than that of 'Mianfen No 1'. In terms of cell wall-related enzymes, 'Yushu No 10' hardness gumminess and chewiness had a significant correlation with hemicellulose activity, and 'Mianfen No 1' had insignificant correlation with four cell wall-related enzymes. Expansin genes were also expressed differently during the various stages of root tubers expansin. The expressions of IbEXP1, IbEXP2 and IbEXPL1 were significantly correlated with the changes in cell wall component content, and were related to the qualitative structure changes. The research conclusion shows that the texture changes during the growth of sweetpotato are related to cell wall composition, cell wall-related enzyme activity changes, and the expression of expansin genes. This study provides theoretical guidance for in-depth study of texture changes of sweetpotato, post-harvest processing and utilization and quality improvement of storage roots.

Reactive oxygen species induce cyanide-resistant respiration in potato infected by Erwinia carotovora subsp. Carotovora.

Studies have shown that pathogenic bacteria infections induce the overproduction of reactive oxygen species (ROS) in plants. Cyanide-resistant respiration, an energy-dissipating pathway in plants, has also been induced by a pathogenic bacteria infection. However, it is unknown whether the induction of cyanide-resistant respiration under the pathogenic bacteria infection was caused by ROS. In this study, two pathogenic Erwinia strains were used to infect potato tuber, and membrane lipid peroxidation levels and the cyanide-resistant respiration capacity were determined. In addition, StAOX expression and regulation by ROS in potato tuber were analyzed. Moreover, the role of the Ca2+ pathway in regulating cyanide-resistant respiration was determined. The results showed that ROS induced cyanide-resistant respiration in potato tuber infected by Erwinia. Cyanide-resistant respiration inhibited the production of H2O2. Intracellular Ca2+ regulated the expression of calcium-dependent protein kinase (StCDPK1, StCDPK4, and StCDPK5) in potato, which indirectly controlled intracellular ROS levels. These results indicate that Ca2+ metabolism is involved in ROS-induced cyanide-resistant respiration.

A new insight into purification of polyphenol oxidase and inhibition effect of curcumin and quercetin on potato polyphenol oxidase.

In the literature, the polyphenol oxidase (PPO) enzyme has been purified a many times via Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. In order to study PPO purification efficiency, 2-aminophenol and 4-aminophenol were applied as a spacer arm to CNBr-activated Sepharose 4B. The effects of the spacer arm on specific activity, purification fold, and electrophoretic properties were investigated. The best performance with 11.7-fold purification and 23951 U/mg protein specific activity was achieved with the 4-aminophenol extension arm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with was done to check the purity of the potato PPO enzyme obtained from affinity columns. According to the results of SDS-PAGE and native PAGE, the molecular weight of the enzyme is 50 kDa. Furthermore, the inhibition effects of curcumin and quercetin on the enzyme activity were examined, and the IC50 and Ki values were computed for the mentioned substances. IC50 values were determined to be 0.018 and 0.029 mM for potato PPO with curcumin and quercetin inhibitors with catechol as a substrate, respectively. IC50 value was also determined to be 0.0086 mM for quercetin inhibitor with 4-methylcatechol as a substrate. Ki constant was 0.0753 ± 0.0085 mM for curcumin using catechol as a substrate. No inhibition effect was observed for curcumin with the 4-methylcatechol substrate. The Ki constant for quercetin was 0.0398 ± 0.00743 mM with the 4-methylcatechol substrate and 0.0109 ± 0.0021 mM with the catechol substrate.

New paths of cyanogenesis from enzymatic-promoted cleavage of ß-cyanoglucosides are suggested by a mixed DFT/QTAIM approach.

Cyanogenesis is an enzyme-promoted cleavage of ß-cyanoglucosides; the release of hydrogen cyanide is believed to produce food poisoning by consumption of certain crops as Cassava (Manihot esculenta Crantz). The production of hydrogen cyanide by some disruption of the plant wall is related to the content of two ß-cyanoglucosides (linamarin and lotaustralin) which are stored within the tuber. Some features about the mechanistic bases of these transformations have been published; nevertheless, there are still questions about the exact mechanism, such as the feasibility of a difference in the kinetics of cyanogenesis between both cyanoglucosides. In this work, we have performed a theoretical analysis using DFT and QTAIM theoretical frameworks to propose a feasible mechanism of the observed first step of the enzyme-catalyzed rupture of these glucosides; our results led us to explain the observed difference between linamarin and lotaustralin. Meanwhile, DFT studies suggest that there are no differences between local reactivity indexes of both glucosides; QTAIM topological analysis suggests two important intramolecular interactions which we found to fix the glucoside in such a way that suggests the linamarin as a more reactive system towards a nucleophilic attack, thus explaining the readiness to liberate hydrogen cyanide.

Effect of high oxygen pretreatment of whole tuber on anti-browning of fresh-cut potato slices during storage.

The surface browning usually occurs on fresh-cut potato during storage. The effect of short-time high oxygen pretreatment on anti-browning of fresh-cut potato slices was investigated. The whole potato tubers were firstly immersed in the oxygen concentration of 21%, 60% and 80% for 20 min. Then, the potatoes were peeled, cut and stored at 4 °C for 8 days. The results showed that the short-time 80% oxygen pretreatment possessed significantly anti-browning effect by retarding the increase of polyphenol oxidase (PPO) activity and the accumulation of malondialdehyde (MDA) content, maintaining the cell integrity. Meanwhile, the 80% oxygen treatment could increase the activities of phenylalanine ammonia lyase (PAL) and peroxidase (POD), and the total phenolic content. Importantly, the 80% oxygen treatment could effectively improve the antioxidant capacity. Overall, all results suggest that the short-time high oxygen pretreatment holds great promise on anti-browning of fresh-cut potato.

Darkening, damage and oxidative protection are stimulated in tissues closer to the yam cut, attenuated or not by the environment.

BACKGROUND: This study investigated how the activities of the enzymes and metabolites of oxidative metabolism are affected in different regions of cut tissue, associating these changes with the evolution of browning in fresh-cut yam. Samples were collected from yam at 0-5 and 5-10 mm from the cut site. Fresh-cut yams were stored at 5 or 26 °C for days or hours respectively, simulating commercialization, with or without packaging. RESULTS: The results demonstrated that the injury to the yam was a physical inducer of changes in the levels of lipid peroxidation and phenolic compounds and in the activities of superoxide dismutase, catalase, ascorbate peroxidase, polyphenol oxidase and peroxidase. These responses were significant in the tissue closest to the wound but also observed, although less intense, in the more distant tissue. The combined effects of wounding and dehydration during storage intensified the above responses. Conversely, refrigeration attenuated the transmission of the wounding response through the adjacent tissue. CONCLUSION: The results of this work provide the first evidence in cut yam roots that the membrane degradation products, enzymes involved in oxidative protection and enzymes that modulate the oxidation of phenolic compounds are intertwined mechanisms that cause tissue darkening. © 2018 Society of Chemical Industry.

Histochemical observations and gene expression changes related to internal browning in tuberous roots of sweet potato (Ipomea batatas).

The mechanism underlying internal browning (IB), or brown discoloration, of the central region of tuberous roots of sweet potato (Ipomoea batatas) was examined. IB disorder begins in roots from approx. 90 days after transplanting, and the severity increases significantly with time. IB damage initially occurs in cells around the secondary vascular tissue, and the area per cell occupied by starch grains in this region was larger than in the unaffected region. High levels of reducing sugars, polyphenol oxidase (PPO) activities, chlorogenic acid, and hydrogen peroxide (H2O2) were detected in cells from the IB damaged regions. The content of sugar and polyphenols was higher in disks (transverse sections) with larger amounts of damaged tissues than in disks of sound root. The transcript levels of acid invertase (IbAIV) tended to be higher with greater IB severity, whereas fluctuation patterns of ADP-glucose pyrophosphorylase (IbAGPase), granule bound starch synthase (IbGBSS), and starch branching enzyme 1 (IbSBE1) were lower with higher IB severity. These observations suggest that the incidence of IB disorder in sweet potato is largely dependent on the excessive generation of reactive oxygen species (ROS) in cells around the secondary vascular tissues due to the abundant accumulation of sugar and/or starch grains during the root maturation period.

Promising perspectives for ruminal protection of polyunsaturated fatty acids through polyphenol-oxidase-mediated crosslinking of interfacial protein in emulsions.

Previously, polyunsaturated fatty acids (PUFA) from linseed oil were effectively protected (>80%) against biohydrogenation through polyphenol-oxidase-mediated protein crosslinking of an emulsion, prepared with polyphenol oxidase (PPO) extract from potato tuber peelings. However, until now, emulsions of only 2 wt% oil have been successfully protected, which implies serious limitations both from a research perspective (e.g. in vivo trials) as well as for further upscaling toward practical applications. Therefore, the aim of this study was to increase the oil/PPO ratio. In the original protocol, the PPO extract served both an emulsifying function as well as a crosslinking function. Here, it was first evaluated whether alternative protein sources could replace the emulsifying function of the PPO extract, with addition of PPO extract and 4-methylcatechol (4MC) to induce crosslinking after emulsion preparation. This approach was then further used to evaluate protection of emulsions with higher oil content. Five candidate emulsifiers (soy glycinin, gelatin, whey protein isolate (WPI), bovine serum albumin and sodium caseinate) were used to prepare 10 wt% oil emulsions, which were diluted five times (w/w) with PPO extract (experiment 1). As a positive control, 2 wt% oil emulsions were prepared directly with PPO extract according to the original protocol. Further, emulsions of 2, 4, 6, 8 and 10 wt% oil were prepared, with 80 wt% PPO extract (experiment 2), or with 90, 80, 70, 60 and 50 wt% PPO extract, respectively (experiment 3) starting from WPI-stabilized emulsions. Enzymatic crosslinking was induced by 24-h incubation with 4MC. Ruminal protection efficiency was evaluated by 24-h in vitro batch simulation of the rumen metabolism. In experiment 1, protection efficiencies were equal or higher than the control (85.5% to 92.5% v. 81.3%). In both experiments 2 and 3, high protection efficiencies (>80%) were achieved, except for emulsions containing 10 wt% oil emulsions (<50% protection), which showed oiling-off after enzymatic crosslinking. This study demonstrated that alternative emulsifier proteins can be used in combination with PPO extract to protect emulsified PUFA-rich oils against ruminal biohydrogenation. By applying the new protocol, 6.5 times less PPO extract was required.

Inhibition of plastid PPase and NTT leads to major changes in starch and tuber formation in potato.

The importance of a plastidial soluble inorganic pyrophosphatase (psPPase) and an ATP/ADP translocator (NTT) for starch composition and tuber formation in potato (Solanum tuberosum) was evaluated by individual and simultaneous down-regulation of the corresponding endogenous genes. Starch and amylose content of the transgenic lines were considerably lower, and granule size substantially smaller, with down-regulation of StpsPPase generating the most pronounced effects. Single-gene down-regulation of either StpsPPase or StNTT resulted in increased tuber numbers per plant and higher fresh weight yield. In contrast, when both genes were inhibited simultaneously, some lines developed only a few, small and distorted tubers. Analysis of metabolites revealed altered amounts of sugar intermediates, and a substantial increase in ADP-glucose content of the StpsPPase lines. Increased amounts of intermediates of vitamin C biosynthesis were also observed. This study suggests that hydrolysis of pyrophosphate (PPi) by action of a psPPase is vital for functional starch accumulation in potato tubers and that no additional mechanism for consuming, hydrolysing, or exporting PPi exists in the studied tissue. Additionally, it demonstrates that functional PPi hydrolysis in combination with efficient ATP import is essential for tuber formation and development.

Cloning and functional characterization of a p-coumaroyl quinate/shikimate 3'-hydroxylase from potato (Solanum tuberosum).

Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) for CGA biosynthesis has not been isolated and functionally characterized in potato. In this work, we cloned StC3'H from potato and showed that it catalyzed the formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the expression of StC3'H proteins was blocked through antisense (AS) inhibition under the control of a tuber-specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest that StC3'H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in the core tissue of potato tubers is an intricately controlled process and that additional C3'H activity may also be involved in CGA biosynthesis in potato.

Suppression of the vacuolar invertase gene delays senescent sweetening in chipping potatoes.

BACKGROUND: Potato chip processors require potato tubers that meet quality specifications for fried chip color, and color depends largely upon tuber sugar contents. At later times in storage, potatoes accumulate sucrose, glucose, and fructose. This developmental process, senescent sweetening, manifests as a blush of color near the center of the fried chip, becomes more severe with time, and limits the storage period. Vacuolar invertase (VInv) converts sucrose to glucose and fructose and is hypothesized to play a role in senescent sweetening. To test this hypothesis, senescent sweetening was quantified in multiple lines of potato with reduced VInv expression. RESULTS: Chip darkening from senescent sweetening was delayed by about 4 weeks for tubers with reduced VInv expression. A strong positive correlation between frequency of dark chips and tuber hexose content was observed. Tubers with reduced VInv expression had lower hexose to sucrose ratios than controls. CONCLUSION: VInv activity contributes to reducing sugar accumulation during senescent sweetening. Sucrose breakdown during frying may contribute to chip darkening. Suppressing VInv expression increases the storage period of the chipping potato crop, which is an important consideration, as potatoes with reduced VInv expression are entering commercial production in the USA. © 2017 Society of Chemical Industry.

Chemical Constituents of Sweetpotato Genotypes in Relation to Textural Characteristics of Processed French Fries.

Sweetpotato French fries (SPFF) are growing in popularity, however limited information is available on SPFF textural properties in relation to chemical composition. This study investigated the relationship between chemical components of different sweetpotato varieties and textural characteristics of SPFF. Sixteen sweetpotato genotypes were evaluated for (1) chemical constituents; (2) instrumental and sensory textural properties of SPFF; and (3) the relationship between chemical components, instrumental measurements, and sensory attributes. Dry matter (DM), alcohol-insoluble solids (AIS), starch, sugar, and oil content, and also α- and ß-amylase activities were quantified in raw sweetpotatoes and SPFF. Peak force and overall hardness describing instrumental textural properties of SPFF were measured using a texture analyzer. Descriptive sensory analysis was conducted and 10 attributes were evaluated by a trained panel. Results showed that DM, AIS, and starch content in raw sweetpotatoes were significantly correlated (P < 0.05) with instrumental peak force and overall hardness (r = 0.41 to 0.68), and with sensory surface roughness, hardness, fracturability, and crispness (r = 0.63 to 0.90). Total sugar content in raw sweetpotatoes was positively correlated with sensory smoothness and moistness (r = 0.77), and negatively correlated with instrumental peak force and overall hardness (r = -0.62 to -0.69). Instrumental measurements were positively correlated with sensory attributes of hardness, fracturability, and crispness (r = 0.68 to 0.96) and negatively correlated with oiliness, smoothness, moistness, and cohesiveness (r = -0.61 to -0.91). Therefore, DM, AIS, starch, and total sugar contents and instrumental measurements could be used as indicators to evaluate sweetpotato genotypes for SPFF processing. PRACTICAL APPLICATION: In recent years, sweetpotato French fries (SPFF) have grown in popularity, but limited information is available on SPFF textural properties in relation to the differences in chemical constituents among sweetpotato varieties. This study demonstrated that sensory texture attributes of SPFF varied widely and were significantly correlated with chemical components such as dry matter, starch, and total sugar contents of raw sweetpotatoes and instrumental texture measurements of SPFF. The knowledge generated from this study will benefit the food industry and breeding programs with the selection of sweetpotato varieties for improved SPFF quality.

Quantitative trait loci for tuber blackspot bruise and enzymatic discoloration susceptibility in diploid potato.

Tuber tissue discolorations caused by impact (blackspot bruising) and enzymatic discoloration (ED) after tuber cutting are crucial quality traits of the cultivated potato. To understand the complex genetics of the traits, quantitative trait locus (QTL) analysis using diploid mapping population and diversity array technology (DArT) markers was performed. The phenotypic assessment included the complex evaluation of blackspot bruising susceptibility through two methods: rotating drum (B RD) and falling bolt (B FB) in combination with the evaluation of enzymatic discoloration. Because of observed in-practice relationship between bruising susceptibility and tuber starch content (TSC), analysis of starch content-corrected bruising susceptibility (SCB) was performed. QTLs for bruising were detected on chromosomes I, V with both test methods. The rotating drum method enabled the detection of additional QTLs on chromosomes VIII and XII. Analysis of SCB enabled the identification of the major QTL on chromosome V and two weaker QTLs on chromosomes VIII and XII, independently of starch content. The QTL for bruising detected on chromosome I overlapped with the most significant QTL for tuber starch content. This QTL was not significant for starch content-corrected bruising susceptibility, and the effect of the QTL on chromosome V was enhanced for this trait. The QTL analysis of ED revealed the contribution of seven QTLs for the trait, located on six chromosomes, including these detected for the first time: a major locus on chromosome V and minor QTLs on chromosomes VII and X, which were specific for the trait. The QTL for ED on chromosome VIII was co-localized with the marker for polyphenol oxidase (POT32). The phenotypic correlation between bruising and ED was confirmed in QTL analyses of both traits, and the QTLs detected for these traits overlapped on chromosomes I, V, and VIII. Our results should provide a basis for further studies on candidate genes affecting blackspot bruise susceptibility and enzymatic discoloration.

Cytosolic glyceraldehyde-3-phosphate dehydrogenases play crucial roles in controlling cold-induced sweetening and apical dominance of potato (Solanum tuberosum L.) tubers.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme that functions in producing energy and supplying intermediates for cellular metabolism. Recent researches indicate that GAPDHs have multiple functions beside glycolysis. However, little information is available for functions of GAPDHs in potato. Here, we identified 4 putative cytosolic GAPDH genes in potato genome and demonstrated that the StGAPC1, StGAPC2, and StGAPC3, which are constitutively expressed in potato tissues and cold inducible in tubers, encode active cytosolic GAPDHs. Cosuppression of these 3 GAPC genes resulted in low tuber GAPDH activity, consequently the accumulation of reducing sugars in cold stored tubers by altering the tuber metabolite pool sizes favoring the sucrose pathway. Furthermore, GAPCs-silenced tubers exhibited a loss of apical dominance dependent on cell death of tuber apical bud meristem (TAB-meristem). It was also confirmed that StGAPC1, StGAPC2, and StGAPC3 interacted with the autophagy-related protein 3 (ATG3), implying that the occurrence of cell death in TAB-meristem could be induced by ATG3 associated events. Collectively, the present research evidences first that the GAPC genes play crucial roles in diverse physiological and developmental processes in potato tubers.

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance.

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.

Isolation and Abiotic Stress Resistance Analyses of a Catalase Gene from Ipomoea batatas (L.) Lam.

As an indicator of the antioxidant capability of plants, catalase can detoxify reactive oxygen species (ROS) generated by environmental stresses. Sweet potato is one of the top six most important crops in the world. However, its catalases remain largely unknown. In this study, a catalase encoding gene, IbCAT2 (accession number: KY615708), was identified and cloned from sweet potato cv. Xushu 18. It contained a 1479 nucleotides' open reading frame (ORF). S-R-L, Q-K-L, and a putative calmodulin binding domain were located at the C-terminus of IbCAT2, which suggests that IbCAT2 could be a peroxisomal catalase. Next-generation sequencing (NGS) based quantitative analyses showed that IbCAT2 was mainly expressed in young leaves and expanding tuberous roots under normal conditions. When exposed to 10% PEG6000 or 200 mmol/L NaCl solutions, IbCAT2 was upregulated rapidly in the first 11 days and then downregulated, although different tissues showed different degree of change. Overexpression of IbCAT2 conferred salt and drought tolerance in Escherichia coli and Saccharomyces cerevisiae. The positive response of IbCAT2 to abiotic stresses suggested that IbCAT2 might play an important role in stress responses.

Concurrent Overexpression of Arabidopsis thaliana Cystathionine γ-Synthase and Silencing of Endogenous Methionine γ-Lyase Enhance Tuber Methionine Content in Solanum tuberosum.

Potatoes (Solanum tuberosum) are deficient in methionine, an essential amino acid in human and animal diets. Higher methionine levels increase the nutritional quality and promote the typically pleasant aroma associated with baked and fried potatoes. Several attempts have been made to elevate tuber methionine levels by genetic engineering of methionine biosynthesis and catabolism. Overexpressing Arabidopsis thaliana cystathionine γ-synthase (AtCGS) in S. tuberosum up-regulates a rate-limiting step of methionine biosynthesis and increases tuber methionine levels. Alternatively, silencing S. tuberosum methionine γ-lyase (StMGL), which causes decreased degradation of methionine into 2-ketobutyrate, also increases methionine levels. Concurrently enhancing biosynthesis and reducing degradation were predicted to provide further increases in tuber methionine content. Here we report that S. tuberosum cv. Désirée plants with AtCGS overexpression and StMGL silenced by RNA interference are morphologically normal and accumulate higher free methionine levels than either single-transgenic line.

Distinct cold responsiveness of a StInvInh2 gene promoter in transgenic potato tubers with contrasting resistance to cold-induced sweetening.

Potato (Solanum tuberosum L.) vacuolar invertase (ß-fructofuranosidase; EC 3.2.1.26) inhibitor 2 (StInvInh2) plays an important role in cold-induced sweetening (CIS) of potato tubers. The transcript levels of StInvInh2 were increased by prolonged cold in potato tubers with CIS-resistance but decreased in potato tubers with CIS-sensitivity. However, the transcript regulation mechanisms of StInvInh2 responding to prolonged cold are largely unclear in CIS-resistant and CIS-sensitive genotypes. In the present study, the 5'-flanking sequence of the StInvInh2 was cloned, and cis-acting elements were predicted. No informative differences in StInvInh2 promoter structure between resistant and sensitive-CIS potato genotypes were observed. Histochemical assay showed that the promoter of StInvInh2 mainly governed ß-glucuronidase (GUS) expression in potato microtubers. Quantitative analysis of GUS expression suggested that StInvInh2 promoter activity was enhanced by prolonged cold in CIS-resistant genotype tubers but suppressed in CIS-sensitive tubers. These findings provide essential information regarding transcriptional regulatory mechanisms of StInvInh2 in cold-stored tubers contrasting CIS capacity.

Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development.

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (ß-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.