Cold-induced Sweetening Research Articles

Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE.

Slowing down cold-induced sweetening (CIS) of potato (Solanum tuberosum) tubers is of economic importance for the potato industry to ensure high-quality products. The conversion of sucrose to reducing sugars by the acid invertase StvacINV1 is thought to be critical for CIS. Identification of the specific StvacINV1 inhibitor StInvInh2B and the α- and β-subunits of the interacting protein SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE from the wild potato species Solanum berthaultii (SbSnRK1) has led to speculation that invertase activity may be regulated via a posttranslational mechanism that remains to be elucidated. Using bimolecular fluorescence complementation assays, this study confirmed the protein complex by pairwise interactions. In vitro kinase assays and protein phosphorylation analysis revealed that phosphorylation of SbSnRK1α is causal for StvacINV1 activity and that its active form blocks the inhibition of StInvInh2B by SbSnRK1β, whereas its inactive form restores the function of SbSnRK1β that prevents StInvInh2B from repressing StvacINV1. Overexpression of SbSnRK1α in CIS-sensitive potato confirmed that SbSnRK1α has significant effects on acid invertase-associated sucrose degradation. A higher level of SbSnRK1α expression was accompanied by elevated SbSnRK1α phosphorylation, reduced acid invertase activity, a higher sucrose-hexose ratio, and improved chip color. Our results lend new insights into a subtle regulatory mode of invertase activity and provide a novel approach for potato CIS improvement.

Construction of efficient, tuber-specific, and cold-inducible promoters in potato

Promoter activity is crucial for precise gene expression. Previously, a synthetic tuber-specific and cold-inducible promoter, pCL, containing a C-repeat/dehydration-responsive element (CRT/DRE) cassette and a tuber-specific fragment, was constructed in order to regulate cold-induced sweetening (CIS) in potatoes. However, the utility of pCL is limited due to its low activity. To improve its inducibility in response to low temperatures, we modified the CRT/DRE and flanking sequences. In particular, promoter activity was significantly improved by site-specific mutation of flanking sequences next to the core element (CCGAC) of CRT/DRE. We also inserted a modified CRT/DRE cassette into pCL; although this enhanced activity, it was not more effective than mutation of the flanking sequences. Indeed, up to 20-fold enhanced pCL activity could be achieved by replacing the CRT/DRE cassette in pCL with tandem repeats of two mutated CRT/DRE cassettes. This improvement was due to an enhanced affinity between the CRT/DRE cassette(s) and the StCBF1 transcription factor. Together, these data suggest that altering the structure of CRT/DRE can enhance CBF-related transcription complex formation and thus improve the activity of this cold-inducible promoter.

Fine Screening for Resistance to Cold-Induced Sweetening in Potato Hybrids ContainingSolanum raphanifoliumGermplasm

Potato is an indispensable part of human food. Many wild and cultivated potato relatives have been screened to find the best germplasm to improve productivity and quality, but only a small sample of the available biodiversity has been exploited. Most wild relatives are self-incompatible diploids. Genetic variability exists within and among populations, even within a species. Therefore, it is necessary to carry out fine screening to identify individuals carrying traits of interest. This study was carried out to quantify phenotypic variability for resistance to cold-induced sweetening, an important processing trait. Five families were evaluated for potato chip (crisp) color following cold storage of tubers harvested from four greenhouse trials and one field trial. The families were generated by crossing a single diploid clone to five plants from one accession of the wild potato relativeSolanum raphanifolium. Analysis of variance revealed that resistance against cold-induced sweetening was dependent on family and trial. This study underscores the importance of fine screening to select individuals in potato accessions for use in potato improvement.

Sugar metabolism, chip color, invertase activity, and gene expression during long-term cold storage of potato (Solanum tuberosum) tubers from wild-type and vacuolar invertase silencing lines of Katahdin.

BackgroundStoring potato tubers at low temperatures minimizes sprouting and disease but can cause an accumulation of reducing sugars in a process called cold-induced sweetening. Tubers with increased amounts of reducing sugars produce dark-colored, bitter-tasting fried products with elevated amounts of acrylamide, a possible carcinogen. Vacuolar invertase (VInv), which converts sucrose produced by starch breakdown to glucose and fructose, is the key determinant of reducing sugar accumulation during cold-induced sweetening. In this study, wild-type tubers and tubers in which VInv expression was reduced by RNA interference were used to investigate time- and temperature-dependent changes in sugar contents, chip color, and expression of VInv and other genes involved in starch metabolism in tubers during long-term cold storage.ResultsVInv activities and tuber reducing sugar contents were much lower, and tuber sucrose contents were much higher, in transgenic than in wild-type tubers stored at 3-9°C for up to eight months. Large differences in VInv mRNA accumulation were not observed at later times in storage, especially at temperatures below 9°C, so differences in invertase activity were likely established early in the storage period and maintained by stability of the invertase protein. Sugar contents, chip color, and expression of several of the studied genes, including AGPase and GBSS, were affected by storage temperature in both wild-type and transgenic tubers. Though transcript accumulation for other sugar-metabolism genes was affected by storage temperature and duration, it was essentially unaffected by invertase silencing and altered sugar contents. Differences in stem- and bud-end sugar contents in wild-type and transgenic tubers suggested different compartmentalization of sucrose at the two ends of stored tubers.ConclusionsVInv silencing significantly reduced cold-induced sweetening in stored potato tubers, likely by means of differential VInv expression early in storage. Transgenic tubers retained sensitivity to storage temperature, and accumulated greater amounts of sucrose, glucose and fructose at 3°C than at 7-9°C. At each storage temperature, suppression of VInv expression and large differences in tuber sugar contents had no effect on expression of AGPase and GBSS, genes involved in starch metabolism, suggesting that transcription of these genes is not regulated by tuber sugar content.

Genome-Wide Identification of microRNAs and Their Targets in Cold-Stored Potato Tubers by Deep Sequencing and Degradome Analysis

Potato tubers, the economically important organ for food, processing raw materials, and seeds, are usually stored at different temperatures for various purposes. A general physiological process during storage is starch hydrolysis and accumulation of reducing sugars as breaking of tuber dormancy. The starch hydrolysis can be accelerated by low temperature. Valuable works have been reported on transcriptional and post-translational regulatory mechanisms that are involved in starch hydrolysis in stored potato tubers. However, little is known whether post-transcriptional events are also involved in this important process. In the present study, two small RNA libraries and two degradome libraries were constructed using 4- and 20 °C-stored potato tubers. Deep sequencing and whole genome-wide data analysis led to the identification of 53 known miRNAs and 60 novel miRNAs, largely increasing the number of miRNAs known in potato. Seventy genes were identified as miRNA targets by degradome analysis, indicating that miRNAs were active in gene expression regulation in the post-harvested tubers. The targets were potentially involved in various biological processes, such as stimulus response, signal transduction, transcriptional regulation, and metabolism. Further expression profiling revealed 11 miRNAs/miRNA*s and 34 targets that showed different patterns between two potato genotypes with distinct response to cold storage in terms of reducing sugar accumulation, implying a role of miRNAs in cold-induced sweetening. This study is the first genome-wide investigation of miRNAs and their targets in cold-stored potato tubers, thus broadening our perspectives on the roles of miRNAs in cold response, dormancy, and carbohydrate metabolism.

The potato amylase inhibitor gene SbAI regulates cold-induced sweetening in potato tubers by modulating amylase activity.

Potato cold-induced sweetening (CIS) is critical for the postharvest quality of potato tubers. Starch degradation is considered to be one of the key pathways in the CIS process. However, the functions of the genes that encode enzymes related to starch degradation in CIS and the activity regulation of these enzymes have received less attention. A potato amylase inhibitor gene known as SbAI was cloned from the wild potato species Solanum berthaultii. This genetic transformation confirmed that in contrast to the SbAI suppression in CIS-resistant potatoes, overexpressing SbAI in CIS-sensitive potatoes resulted in less amylase activity and a lower rate of starch degradation accompanied by a lower reducing sugar (RS) content in cold-stored tubers. This finding suggested that the SbAI gene may play crucial roles in potato CIS by modulating the amylase activity. Further investigations indicated that pairwise protein-protein interactions occurred between SbAI and α-amylase StAmy23, β-amylases StBAM1 and StBAM9. SbAI could inhibit the activities of both α-amylase and β-amylase in potato tubers primarily by repressing StAmy23 and StBAM1, respectively. These findings provide the first evidence that SbAI is a key regulator of the amylases that confer starch degradation and RS accumulation in cold-stored potato tubers.

Tuber starch amylose content is associated with cold-induced sweetening in potato.

Cold-induced sweetening (CIS) is the accumulation of reducing sugars in potato tubers at low storage temperatures. It is undesirable because it results in dark fry products. Our study evaluated the relationship between genetic resistance to CIS and two starch parameters, amylose content and starch granule size. We found that the amylose content in four CIS-resistant varieties was higher than that in five susceptible varieties. Amylose content was influenced not only by variety but also storage, production year, and field location. However, interactions between amylose content and environmental variables were not detected. In contrast, starch granule size was not associated with CIS resistance. No effect of storage on starch granule size was detected, and interactions among variety, production year, and field location were observed. Tuber starch amylose content should be considered a source of variability for CIS.

Amylase Analysis in Potato Starch Degradation During Cold Storage and Sprouting

As the fourth most important food crop, potato plays a key role in food safety and economic development of the world. Harvested potato tubers can be stored for a long time, but sprouting and cold-induced sweetening (CIS) can seriously affect the quality of tubers during storage. One of the key pathways involved in CIS is starch degradation, in which both α-amylase and β-amylase play important roles. However, each amylase belongs to an extensive gene family and it is not clear which genes are the key regulators. In this study, we identified genes most likely regulating starch degradation. We first selected candidate genes from the public potato genome database and then investigated their expression patterns associated with reducing sugars and amylase activities. The results showed that the activity of α-amylase was mainly caused by StAmy23 and the activity of β-amylase was mainly caused by StBAM1 and StBAM7. In addition, α-amylase and β-amylase may play important roles in starch degradation of the tubers stored at low temperature and during sprouting, and the amylase activity may be regulated by the amylase inhibitor in cold-stored tubers.

Comparative proteomic analysis of cold-induced sweetening in potato (Solanum tuberosum L.) tuber

Cold-induced sweetening is one of the major factors limiting the quality of fried potato products. To understand the mechanisms of protein regulation for cold-induced sweetening in potato tubers, a comparative proteomic approach was used to analyse the differentially expressed proteins both during control (25 °C, 30 days) and cold treatment (4 °C, 30 days) using two-dimensional gel electrophoresis. Quantitative image analyses indicated that there were 25 protein spots with their intensities significantly altered more than twofold. Of these proteins, 9 were up-regulated, 13 were down-regulated, 2 were absent, and 1 was induced in the cold-stored tubers. The MALDI-TOF/TOF MS analyses led to the identification of differentially expressed proteins that are involved in several processes and might work cooperatively to maintain metabolic homeostasis in tubers during low-temperature storage. The preponderance of metabolic proteins reflects the inhibition of starch re-synthesis and the accumulation of sugars in carbon fluxes, linking starch–sugar conversion. The respiration-related proteins suggest the transfer of respiratory activity from aerobic respiration to anaerobic respiration in the cold-stored tubers. The proteins associated with defence appear to protect the tuber cells from low-temperature stress. Some heat shock proteins that act as chaperones also displayed a differential expression pattern, suggesting a potentially important role in cold-stored tubers, although their exact contribution remains to be investigated. The proposed hypothetical model might explain the interaction of these differentially expressed proteins that are associated with cold-induced sweetening in tubers.

Interaction proteins of invertase and invertase inhibitor in cold-stored potato tubers suggested a protein complex underlying post-translational regulation of invertase

The activity of vacuolar invertase (VI) is vital to potato cold-induced sweetening (CIS). A post-translational regulation of VI activity has been proposed which involves invertase inhibitor (VIH), but the mechanism for the interaction between VI and VIH has not been fully understood. To identify the potential partners of VI and VIH, two cDNA libraries were respectively constructed from CIS-resistant wild potato species Solanum berthaultii and CIS-sensitive potato cultivar AC035-01 for the yeast two-hybrid analysis. The StvacINV1 (one of the potato VIs) and StInvInh2B (one of the potato VIHs), previously identified to be associated with potato CIS, were used as baits to screen the two libraries. Through positive selection and sequencing, 27 potential target proteins of StvacINV1 and eight of StInvInh2B were clarified. The Kunitz-type protein inhibitors were captured by StvacINV1 in both libraries and the interaction between them was confirmed by bimolecular fluorescence complementation assay in tobacco cells, reinforcing a fundamental interaction between VI and VIH. Notably, a sucrose non-fermenting-1-related protein kinase 1 was captured by both the baits, suggesting that a protein complex could be necessary for fine turning of the invertase activity. The target proteins clarified in present research provide a route to elucidate the mechanism by which the VI activity can be subtly modulated.

The potato protease inhibitor gene, St-Inh, plays roles in the cold-induced sweetening of potato tubers by modulating invertase activity

Reducing sugar accumulation is determined mainly by acid invertase activity in cold-stored potato tubers. The potato Kunitz-type protease inhibitor St-Inh reduces acid invertase activity in vitro, is linked with a quantitative trait locus for sugar content, and is therefore speculated to be involved in the cold-induced sweetening (CIS) of potato tubers. In this study, the expression profile of St-Inh in various organs of potato plants and stored tubers was characterized, and it was found that expression was highest in tubers and was strongly suppressed by low temperatures. This expression pattern was opposite to reducing sugar accumulation in the cold-stored tubers, suggesting a possible involvement of St-Inh in CIS in tubers. Overexpression of St-Inh in tubers resulted in lower acid invertase activities and reducing sugar contents in comparison with wild-type tubers, confirming the role of St-Inh in resistance to CIS. Interestingly, a greater reduction in potato tuber CIS was obtained after overexpression of the tobacco invertase inhibitor NtInvInh2, which belongs to the pectin methylesterase/invertase inhibitor family. The NtInvInh2 transgenic tubers had an even lighter chip color than did the St-Inh transgenic tubers. Both inhibitors are confirmed to be involved in reducing potato CIS, although protease inhibitors of the pectin methylesterase/invertase type may have a stronger capacity to inhibit acid invertase activity than Kunitz-type ones. These results provide novel clues to the mechanism by which potato CIS is regulated.

Profiling of StvacINV1 Expression in Relation to Acid Invertase Activity and Sugar Accumulation in Potato Cold-Stored Tubers

Reducing sugars (RS) accumulation in potato (Solanum tuberosum L.) tubers stored at low temperature, a phenomenon known as cold-induced sweetening, is a serious postharvest problem for the potato processing industry. RS accumulation is prevented by suppression of a gene encoding potato vacuolar acid invertase (StvacINV1), which catalyzes irreversible hydrolysis of sucrose into glucose and fructose. Six potato genotypes were selected for dynamic analysis of sugar accumulation and invertase expression pattern in tubers during 1 month of low-temperature storage. The significant increase of sucrose content was found to occur earlier than the remarkable rise of RS content in all the genotypes tested. RS accumulation continued from 15 to 30 days of storage while sucrose content was almost steady in this period. The transcripts of StvacINV1 increased rapidly in tubers exposed to cold treatment and the activity of soluble acid invertase increased accompanied with sugar accumulation. Correlation analysis across eight genotypes revealed that invertase activity was positively correlated with RS content while the correlation between soluble acid invertase activity and the level of StcacINV1 transcripts was weak. Our results suggest that sucrose cleavage is causal for RS accumulation in cold-stored tubers and the acid invertase activity is possibly modulated by post-translational mechanisms.

Promoter regions of potato vacuolar invertase gene in response to sugars and hormones

Potato vacuolar acid invertase (StvacINV1) (β-fructofuranosidase; EC 3.2.1.26) has been confirmed to play an important role in cold-induced sweetening of potato tubers. However, the transcriptional regulation mechanisms of StvacINV1 are largely unknown. In this study, the 5'-flanking sequence of StvacINV1 was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINV1 promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINV1 promoter was suppressed by sucrose, glucose, fructose, and cold, while enhanced by indole-3-acetic acid (IAA), and gibberellic acid (GA3). Further deletion analysis clarified that the promoter regions from-118 to-551,-551 to-1021, and-1021 to-1521 were required for responding to sucrose/glucose, GA3, and IAA, respectively. These findings provide essential information regarding transcriptional regulation mechanisms of StvacINV1.

A synthetic tuber-specific and cold-induced promoter is applicable in controlling potato cold-induced sweetening

Cold-induced sweetening (CIS) in potato seriously hinders the potato processing industry. It could be of great value for genetic improvement of potato CIS to have a target gene specifically expressed in cold stored tubers. In this study, we used a synthetic promoter, pCL, in potato transformation to drive an antisense expression of StvacINV1, the acid vacuolar invertase gene from Solanum tuberosum. The measurements of expression and enzyme activity of target gene showed that pCL promoter could efficiently govern target gene to express specifically and remarkably regulate the activity of acid vacuolar invertase in potato tubers at low temperature, furthermore, it had almost no effect in other tissues or the tubers under room temperature. The transgenic tubers showed decrease in reducing sugar content during storage atlow temperature and acceptable chip color without significant changes observed in plant morphology and tuberization between the nontransgenic and transgenic lines. This tuber-specific and cold-induced feature could maximally reduce the background expression of the target gene which might bring about potential negative or detrimental effects to plant development. The synthetic promoter confirmed here would be optimal for gene function research in potato tubers in response to low temperature.

StInvInh2 as an inhibitor of StvacINV1 regulates the cold‐induced sweetening of potato tubers by specifically capping vacuolar invertase activity

Reducing sugar (RS) accumulation in cold-stored potato tubers, known as cold-induced sweetening (CIS), is a crucial factor causing unacceptable colour changes and acrylamide formation of fried products. The activity of vacuolar invertase (StvacINV1) is proved important for the CIS process, and invertase inhibitors are speculated to play roles in the post-translational regulation of StvacINV1 activity. In our previous research, two putative inhibitors (StInvInh2A and StInvInh2B) of StvacINV1 were implied to be involved in potato CIS. Here, we further reported that StInvInh2A and StInvInh2B had similar function that specifically inhibited StvacINV1 activity in potatoes. The genetic transformation of these inhibitor genes in potatoes by overexpression in CIS-sensitive and RNAi-silenced in CIS-resistant genotypes showed that StvacINV1 activity was strongly regulated by alteration of the transcripts of the inhibitors without impacting on the expression of StvacINV1. A negative power relationship was found between the transcripts of the inhibitors and StvacINV1 activity, suggesting 1) a transcriptional determination of the inhibitory capacity of StInvInh2A and StInvInh2B and 2) a significant inhibitory role of these inhibitors in post-translational modulation of StvacINV1. The results also demonstrated that depression of StvacINV1 activity through overexpression of StInvInh2A and StInvInh2B weakened accumulation of RS and acrylamide in cold-stored tubers and consequently improved the chip quality. The present research strongly suggest that both StInvInh2A and StInvInh2B function as inhibitors of StvacINV1 and play similar roles in regulating potato CIS by capping StvacINV1 activity. These inhibitors could be novel genetic resources applicable for improving quality of potato processing products.

A novel RING finger gene, SbRFP1, increases resistance to cold-induced sweetening of potato tubers

The modulation of the activity of enzymes associated with carbohydrate metabolism is important for potato cold-induced sweetening (CIS). A novel RING finger gene SbRFP1 was cloned and its expression was found to be cold-inducible in potato tubers of the CIS-resistant genotypes. Transformation of SbRFP1 in potatoes confirmed its role in inhibiting β-amylase and invertase activity, which consequently slowed down starch and sucrose degradation and the accumulation of reducing sugars in cold stored tubers. These findings strongly suggest that SbRFP1 may function as a negative regulator of BAM1 and StvacINV1 to decelerate the accumulation of reducing sugars in the process of potato CIS.

Novel candidate genes influencing natural variation in potato tuber cold sweetening identified by comparative proteomics and association mapping

BackgroundHigher plants evolved various strategies to adapt to chilling conditions. Among other transcriptional and metabolic responses to cold temperatures plants accumulate a range of solutes including sugars. The accumulation of the reducing sugars glucose and fructose in mature potato tubers during exposure to cold temperatures is referred to as cold induced sweetening (CIS). The molecular basis of CIS in potato tubers is of interest not only in basic research on plant adaptation to environmental stress but also in applied research, since high amounts of reducing sugars affect negatively the quality of processed food products such as potato chips. CIS-tolerance varies considerably among potato cultivars. Our objective was to identify by an unbiased approach genes and cellular processes influencing natural variation of tuber sugar content before and during cold storage in potato cultivars used in breeding programs. We compared by two-dimensional polyacrylamide gel electrophoresis the tuber proteomes of cultivars highly diverse for CIS. DNA polymorphisms in genomic sequences encoding differentially expressed proteins were tested for association with tuber starch content, starch yield and processing quality.ResultsPronounced natural variation of CIS was detected in tubers of a population of 40 tetraploid potato cultivars. Significant differences in protein expression were detected between CIS-tolerant and CIS-sensitive cultivars before the onset as well as during cold storage. Identifiable differential proteins corresponded to protease inhibitors, patatins, heat shock proteins, lipoxygenase, phospholipase A1 and leucine aminopeptidase (Lap). Association mapping based on single nucleotide polymorphisms supported a role of Lap in the natural variation of the quantitative traits tuber starch and sugar content.ConclusionsThe combination of comparative proteomics and association genetics led to the discovery of novel candidate genes for influencing the natural variation of quantitative traits in potato tubers. One such gene was a leucine aminopeptidase not considered so far to play a role in starch sugar interconversion. Novel SNP’s diagnostic for increased tuber starch content, starch yield and chip quality were identified, which are useful for selecting improved potato processing cultivars.

M7 Germplasm Release: A Tetraploid Clone Derived from Solanum infundibuliforme for Use in Expanding the Germplasm Base for French Fry Processing

A new source of russet germplasm has been identified as a parent for processing and fresh market breeding programs. It was derived via bilateral sexual polyploidization following a cross between a diploid cultivated potato and the diploid wild species Solanum infundibuliforme. This clone, designated M7, is tetraploid and highly fertile, based on crossability to tetraploid germplasm. It produces large tubers with long shape and russet skin. M7 offspring produce tubers with a desirable appearance and some are resistant to cold-induced sweetening. In addition, M7 is resistant to common scab and soft rot.

Post‐translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold‐induced sweetening of potato tubers

Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.

Allele diversity for the apoplastic invertase inhibitor gene from potato

In planta the enzymatic activity of apoplastic and vacuolar invertases is controlled by inhibitory proteins. Although these invertase inhibitors (apoplastic and vacuolar forms) have been implicated as contributing to resistance to cold-induced sweetening (CIS) in tubers of potato (Solanum tuberosum L.), there is a lack of information on the structure and allelic diversity of the apoplastic invertase inhibitor genes. We have PCR-isolated and sequenced the alleles of the apoplastic invertase inhibitor gene (Stinh1) from three tetraploid potato genotypes: 1021/1 (a genotype with very high tolerance to CIS), 'Karaka' and 'Summer Delight' (two cultivars that are highly susceptible to CIS). In total, five alleles were identified in these genotypes, of which four (Stinh1-c, Stinh1-d, Stinh1-e, Stinh1-f) were novel. An analysis of allele diversity was conducted by incorporating previously published sequences of apoplastic invertase inhibitors from potato. Eight alleles were assessed for sequence polymorphism in the two exons and the single hypervariable intron. Contrary to the hypervariable intron, only 65 single nucleotide polymorphisms were observed in the exons, of which 42 confer amino acid substitutions. Phylogenetic analysis of amino acid sequences indicates that the alleles of the invertase inhibitor are highly conserved amongst members of the Solanaceae family.