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 ß-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 SbSnRK1ß, whereas its inactive form restores the function of SbSnRK1ß 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.

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, ß-amylases StBAM1 and StBAM9. SbAI could inhibit the activities of both α-amylase and ß-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.

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.

Strength comparison between cold-inducible promoters of Arabidopsis cor15a and cor15b genes in potato and tobacco.

The cold-inducible promoter is ideal for regulating ectopic gene expression in plants to cope with the cold stress. The promoters of two cold-regulated genes, cor15a and cor15b, were cloned from Arabidopsis thaliana and their strengths were assayed in potato and tobacco. Although the cis-element composition and cold-inducible property were similar between the two promoters, the cor15b promoter showed significantly higher activity than the cor15a promoter in both potato and tobacco. In order to elucidate the factors determining this discrepancy, cor15a and cor15b promoters were separately truncated from 5'-end to construct short promoters with similar size containing a single C-repeat/dehydration-responsive element (CRT/DRE). Subsequently, two synthetic promoters were constructed by swapping the flanking sequences of CRT/DRE in the truncated promoters. The promoter strength comparison demonstrated that the flanking sequence could affect the promoter strength. These findings provide a potential regulatory mechanism to control the promoter strength without impact on other properties.

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

Potato vacuolar acid invertase (StvacINV1) (ß-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 ß-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 at low 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 ß-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.

A new isoform of thioredoxin h group in potato, SbTRXh1, regulates cold-induced sweetening of potato tubers by adjusting sucrose content.

UNLABELLED: In order to study the molecular mechanism of the cold-induced sweetening (CIS) of potato tubers, a novel isoform of thioredoxin h group, SbTRXh1, which was up-regulated early in the 4 °C storage of CIS-resistant potato (Solanum berthaultii) tubers, was cloned in present research. The genetic transformation of over-expression (OE) and RNA interference (RNAi) of SbTRXh1 into potato cv. E-Potato 3 (E3) was carried out to clarify its function in CIS regulation. The results showed that the transcripts of SbTRXh1 in either OE- or RNAi-tubers were strongly induced in 4 °C storage and quantitatively related to the reducing sugar (RS) accumulation, indicating that SbTRXh1 is involved in the CIS process of potato tubers. Regression analysis between the transcripts and protein contents of SbTRXh1 showed a very significant logarithmic relationship implying that the expression of SbTRXh1 may be mainly regulated at transcriptional level. Further monitoring the variation of the sugar contents in cold-stored tubers demonstrated a linear relationship between RS and sucrose (Suc). Thus, it can be inferred that SbTRXh1 may function in the Suc-RS pathway for CIS regulation of potato tubers. KEY MESSAGE: SbTRXh1 is primarily demonstrated to be involved in the regulation of cold-induced sweetening (CIS) of potato tubers, and it may function in the Suc-RS pathway for CIS regulation.

Systematic analysis of potato acid invertase genes reveals that a cold-responsive member, StvacINV1, regulates cold-induced sweetening of tubers.

Acid invertase is believed to play a regulatory role during plant developmental processes and to respond to environmental stimuli. The expression profiles of the entire acid invertase family are not yet available for potato. By searching existing databases, it was determined that there are at least six acid invertase genes in potato, including four cell-wall invertase genes and two vacuolar invertase genes. They were subjected to comparative expression profiling in various organs of potato plants and in stored tubers to exploit their potential functions. The results revealed that each gene exhibited a unique expression pattern, which differed in transcript abundance or showed organ-specific features, pointing to the possible involvement of individual genes in plant development. The vacuolar invertase gene StvacINV1 had the highest expression level among three genes detected in the potato tubers. Further storage experiments showed that StvacINV1 was strongly induced by low temperatures, which is consistent with glucose accumulation in cold-stored tubers. Suppression of StvacINV1 by the antisense transformation in potato confirmed that lower StvacINV1 transcript abundance in transgenic tubers is related to lower reducing sugar content and lighter chip color in comparison with the wild type. The evidence strongly suggests that StvacINV1 is a gene involved in regulation of cold-induced sweetening of potato tubers. This provides an avenue for studying the mechanism involved in the regulation of the cold-induced sweetening trait and for agronomic enhancement.

Construction and functional characteristics of tuber-specific and cold-inducible chimeric promoters in potato.

The improvement of processing quality of potato products (fries and chips) demands less accumulation of reducing sugars (glucose and fructose) in cold-stored potato (Solanum tuberosum) tubers. Control of gene expression to achieve this requires promoters with specificity to tubers as well as inducible activity under low temperatures. Here we use overlapping extension PCR to construct two chimeric promoters, pCL and pLC, to control gene expression in a tuber-specific and cold-inducible pattern. This combined different combinations of the LTRE (low-temperature responsive element) from Arabidopsis thaliana cor15a promoter and the TSSR (tuber-specific and sucrose-responsive sequence) from potato class I patatin promoter. The cold-inducible and tuber-specific activities of the chimeric promoters were investigated by quantitative analysis of GUS activity in transgenic potato cultivar E3 plants. The results showed that the cis-elements, LTRE and TSSR, played responsive roles individually or in combination. pCL with the TSSR closer to the TATA-box showed substantially higher promoter activity than pLC with the LTRE closer to the TATA-box at either normal (20 degrees C) or low temperature (2 degrees C), suggesting that the promoter activity was closely associated with the position of the two elements. The chimeric promoter pCL with tuber-specific and cold-inducible features may provide valuable tool for controlling the expression of gene constructs designed to lower the formation of reducing sugars in tubers stored at low temperature and to improve the processing quality of potato products.