Plant Biotechnology Journal (2014) 12, pp. 984–993

doi: 10.1111/pbi.12221

The potato amylase inhibitor gene SbAI regulates cold-induced sweetening in potato tubers by modulating amylase activity Huiling Zhang1,2, Jun Liu1, Juan Hou1, Ying Yao1, Yuan Lin1, Yongbin Ou1, Botao Song1,* and Conghua Xie1 1

Key Laboratory of Horticultural Plant Biology (HAU), Ministry of Education, National Centre for Vegetable Improvement (Central China), Huazhong Agricultural

University, Wuhan, China 2

College of Forestry, Henan University of Science and Technology, Luoyang, China

Received 18 February 2014; revised 29 May 2014; accepted 30 May 2014. *Correspondence (Tel +86 27 87287382; fax +86 27 87286939; email [email protected])

Keywords: potato, cold-induced sweetening, SbAI, amylase activity, starch degradation.

Summary 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 a-amylase StAmy23, b-amylases StBAM1 and StBAM9. SbAI could inhibit the activities of both a-amylase and b-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.

Introduction The potato (Solanum tuberosum L.) is the fourth most important food crop in the world. To reduce sprouting, water loss and pathogenesis, potato tubers are often stored at low temperatures. However, low temperatures can lead to the accumulation of reducing sugars (RS), which is known as cold-induced sweetening (CIS). RS can react with the a-amino acid groups of nitrogenous compounds from nonenzymatic Maillard reactions during frying, resulting in dark-coloured food products and, more worrying, generating the carcinogen acrylamide (Shepherd et al., 2010). The carbohydrate metabolic pathways of plants have been clearly elucidated over the past few decades, and the genes that encode part of the enzymes involved in various pathways have been investigated for their CIS functions in potato tubers (Bhaskar et al., 2010; Borovkov et al., 1995; Solomos and Mattoo, 2005). Previous research into improving potato CIS has primarily focused on two strategies, in which one is to enhance the carbohydrate flux from hexoses to starch, and the other is to prevent starch and sucrose from undergoing hydrolysis to reduce RS production. The sucrose synthesis and sucrose cleavage processes have been extensively investigated in relation to potato CIS, with specific approaches such as the antisense RNA transformation of UDP-glucose pyrophosphorylase (Borovkov et al., 1995; Spychalla et al., 1994) and sucrose phosphate synthase (Krause et al., 1998), the RNAi suppression of acid invertase (Bhaskar et al.,

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2010) and the overexpression of invertase inhibitor genes (Liu et al., 2013; Mckenzie et al., 2013), and the regulation of starch metabolism is thought to be of great potential for improving potato CIS. The primary enzymes for starch biosynthesis are adenosine diphosphate glucose pyrophosphorylase (AGPase, EC 2.7.7.27), starch synthases (SS, EC 2.4.1.21) and branching enzymes (SBE, EC 2.4.1.18), which control the three basic steps required for starch formation. Unlike SS and SBE, for which the functions have not been elucidated in potato CIS, AGPase, a plastidial enzyme that catalyzes a rate-limiting reaction in starch biosynthesis, has attracted great attention. Expressing a mutant Escherichia coli (E. coli) AGPase gene called glgc-16 in potato plants increased the starch content by more than 30% when localizing the enzyme to plastids (Stark et al., 1992). Our previous research involved the overexpression of the small AGPase subunit gene from potato, which is called sAGP (GenBank: AY186620), and it exhibited a higher AGPase activity and lower RS accumulation in cold-stored potato tubers; the reverse was true following the antisense suppression of this gene (Song et al., 2005). The plastidic metabolite transporters supply amyloplasts with glucose 6-phosphate and ATP, the substrates for AGPase. Phosphate transporters such as adenylate and hexose phosphate transporters can influence starch biosynthesis. Accumulated starch increased in potato tubers when overexpressing the ATP/ADP transporter and it decreased in the RNA interference transgenic tubers (Tjaden et al., 1998).

ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd

The potato amylase inhibitor gene SbAI 985 Starch degradation can occur phosphorolytically or hydrolytically. Approximately 0.5% of the glucose residues in the storage starch of potato tubers are phosphorylated (Mikkelsen et al., 2006). Two classes of enzymes bind to starch and modulate the phosphate content of amylopectin and glucan, namely water dikinases (GWD) for adding phosphate groups, and phosphoglucan phosphatases for removing these phosphate groups. This reversible glucan phosphorylation is considered to be essential for normal starch degradation (Silver et al., 2014; Zeeman et al., 2010). When the GWD gene expression level decreased, there was a reduction in the phosphate content and the starch degradation and it moderately delayed the RS accumulation in cold-stored transgenic tubers (Lorberth et al., 1998). The functional dissection of a second GWD-like enzyme, or phosphoglucan water dikinase (PWD/GWD3), led to the speculation that GWD and PWD acted together to create a pattern of amylopectin phosphorylation to attack degradative enzymes at the starch granule surface (Smith et al., 2005). The hydrolytic pathway involves enzymes such as a-amylase (EC 3.2.1.1) and b-amylase (EC 3.2.1.2), and the phosphorolytic pathway involves starch phosphorylase (EC 2.4.1.1) (Preiss, 1982; Solomos and Mattoo, 2005). Physiological research demonstrates that the activities of starch-degrading enzymes increased during the potato CIS process (Cottrell et al., 1993), although there are some arguments for the predominant pathway (Morrell and Rees, 1986) or incompatible results between potato genotypes (Claassen et al., 1993; Hill et al., 1996). However, a functional analysis of potato a-amylase gene StAmy23 showed that the reducing sugar content was reduced in RNAi-suppressed tubers that were stored at 4 °C for 15 days in comparison with the untransformed control, but there was no difference in tubers that had been stored for 30 days (internal communication), implying that StAmy23 may play roles in starch hydrolysis on occasion during potato CIS. Although variations in the starch content of cold-stored potato tubers have also been addressed by many publications, the contribution of starch degradation to potato CIS still remain unanswered because the key enzymes and their regulators are unknown. The roles of a-amylase inhibitors in starch degradation have been reported in several species. a-amylase inhibitors contain an array of eight cysteine residues that form four highly conserved disulphide bonds (Lay and Anderson, 2005), and most of them inhibit amylase by forming a complex that blocks the active site or modifies the conformation of enzymes, ultimately leading to a reduction in its catalytic activity (Kumari et al., 2012). The purified a-amylase inhibitor from Colocasia corms was found to inhibit the activity of human salivary a-amylase and also to inhibit potato a-amylase and reduce the sugar content of treated potato slices (Kumari et al., 2012). The common bean a-amylase inhibitor-1 gene a-AI1 was transformed into the coffee plant, and the inhibitory in vitro assays of transgenic seed extracts showed up to 88% enzyme activity inhibition (Barbosa et al., 2010). The inhibitory roles of the a-amylase inhibitor protein VuD1 from Vigna unguiculata on amylases from different resources showed that VuD1 was highly inhibitory against a-amylases in gut extracts from the insect pests Acanthoscelides obtectus and Zabrotes subfasciatus, whereas it exhibited low inhibitory activity against mammalian a-amylases from porcine pancreas and human saliva, and it also had no effect on the a-amylase of Callosobruchus maculatus or the pathogenic fungus Aspergillus fumigatus (Pelegrini et al., 2008). These results suggest that there are specific interactions between individual amylases and amylase

inhibitors. However, the potato amylase inhibitor gene and its roles with regards to amylase have not been elucidated. In our previous research, the higher expression of putative amylase inhibitor gene SbAI was observed in cold-stored CISresistant potato tubers relative to CIS-sensitive ones, establishing a significant negative correlation between SbAI transcript abundance and RS content. Zhang (2013) speculated that SbAI could be an important regulator of starch degradation in potato CIS. In this study, we first cloned the full-length SbAI gene from the potato, and we further investigated the roles of SbAI in potato CIS by complementary function dissection and clarified its counterparts by protein–protein interaction assays, aiming to provide a theoretical and applicable basis for regulating starch hydrolysis to improve potato CIS.

Results SbAI cloning and characterization According to the EST C20-3-E15 sequence (GenBank: HS989783), 799-bp full-length cDNA was amplified from the cDNA of S. berthaultii tubers that had been stored at 4 °C for 5 days by the rapid-amplification of cDNA ends (RACE) method and by employing information from the Potato Genome Sequencing Consortium (PGSC). This sequence consists of a 621-bp open reading frame (ORF) and a 178-bp 30 -untranslated region (Figure 1a). The ORF encodes a deduced protein of 206 amino acids. The amino acid sequence analysis revealed that the deduced protein contains a ‘Tryp_alpha_amyI’ domain located at 30–108 aa. Genes containing ‘Tryp_alpha_amyI’ belong to the alpha-amylase inhibitor (AAI), lipid transfer protein (LTP) and seed storage (SS) protein family (Marchler-Bauer et al., 2011), and thus, the sequence was denoted SbAI (S. berthaultii amylase inhibitor) and submitted to the GenBank database (GenBank: JX523606). The deduced protein was analysed by Signal 4.1 (http://www.cbs.dtu.dk/services/SignalP/), and the results showed that SbAI is a secreted protein, which contains a signal peptide sequence of 1–23 aa. The subcellular localization showed that SbAI is possibly located in the cytoplasm (Figure S1). A phylogenetic analysis of proteins containing the ‘Tryp_alpha_amyI’ domain of potato, tomato and wheat clearly classified them into three groups. SbAI was assigned to the same group along with two potato proteins (St1 and St2), two tomato proteins (Sl1 and Sl2) and five wheat proteins (Tr1, Tr2, Tr3, Tr4 and Tr5) (Figure 1b). Three (Tr1, Tr3 and Tr5) of the five wheat proteins were demonstrated to have an amylase inhibitor function (de la Hoz et al., 1994; Kashlan and Richardson, 1981; Miyazaki et al., 1994), suggesting that SbAI may perform similar roles in potato tubers.

The SbAI function in potato CIS To explore the SbAI function in starch degradation when the tubers were stored at a low temperature, the overexpressing (OE) vector was transformed into a CIS-sensitive variety called E-potato 3 (E3) and the RNAi (RI) vector was transformed into a CISresistant diploid clone called AC142-01. The transgenic lines showed normal plant morphology and tuber development relative to their corresponding untransformed control (Figure S2A–D). Three OE-transgenic lines exhibited an increase in SbAI transcripts, and three RI-transgenic lines showed a reduction in transcripts that were selected for the SbAI function test. The tubers from the transgenic lines were stored at 4 °C for 30 days to investigate the association between SbAI transcrip-

ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 984–993

986 Huiling Zhang et al. (a)

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Figure 1 Characterizing SbAI cDNA. (a) The nucleotide and deduced amino acid sequences of SbAI. The deduced amino acid sequence is shown underneath the corresponding nucleotide sequence. The putative ‘Tryp_alpha_amyI’ domain is underlined. (b) The phylogenetic relations of genes containing the ‘Tryp_alpha_amyI’ domain. The amino acid sequences come from Solanum tuberosum (St1, PGSC0003DMP400055466; St2, PGSC0003DMP400028420; St3, PGSC0003DMP400034938; St4, PGSC0003DMP400023778), Solanum lycopersicum (Sl1, GenBank: BAB39169; Sl2, GenBank: CAA78466; Sl3, GenBank: CAA80273) and Triticum aestivum L. (Tr1, GenBank: 1HSS_D; Tr2, GenBank: AFJ45097; Tr3, GenBank: 0810252A; Tr4, GenBank: Q2A783; Tr5, GenBank: S51811).

tion and starch degradation. One set of tubers was also stored at 20 °C for 30 days for comparison; however, the transgenic tubers exhibited a similar variation to that of the tubers before storage (0 day of Figures 3 and 4) in terms of the starch, sucrose or reducing sugar contents (Figure S3). Therefore, the data sets obtained at 0 day in this study were used to elucidate the impacts of SbAI on CIS. By taking the SbAI transcripts in wild-type tubers before cold storage (0 day) as 1, the gene expression was stimulated by the low temperature (Figure 2). Along with storage at 4 °C, the SbAI expression progressively increased in the OE-transgenic tubers and decreased in the RItransgenic tubers. In a comparison between transgenic and control tubers stored at 4 °C for 30 days, the OE-transformation enhanced SbAI expression between twofolds (OE-12) and 185-folds (OE-11) (Figure 2a) and the RI-transformation suppressed the gene expression between 51% (RI-2) and 98% (RI11) (Figure 2b). The amylase activity, starch content, RS content and sucrose content were measured for the same tubers. SbAI overexpression had little effect on a-amylase activity when compared with untransformed E3, except for OE-11, which experienced a 19.3% reduction in the tubers before cold storage (Figure 3a). However, b-amylase activity and starch degradation were significantly influenced. The b-amylase activity was inhibited by 22%– 39.7% in OE-transgenic tubers before cold storage and by 50%–54% when the tubers were stored at 4 °C for 30 days (Figure 3c). The starch content was significantly increased in (a)

some OE-transgenic tubers (Figure 3e), but the reverse was not observed in RI-transgenic tubers (Figure 3f). According to the starch content before and after cold storage, the starch degradation showed a similar pattern as that of b-amylase activity and it was decreased by 9.5%–61.8% in the OE-transgenic tubers after cold storage (Figure 3g). By contrast, suppressing SbAI resulted in a significant increase in the activity of both a-amylase and b-amylase and the starch degradation rate in most cases compared with the AC142-01 control (Figure 3b,d,f,h). These results demonstrated that SbAI could influence starch degradation by regulating the amylase activity of potato tubers during cold storage. The starch content of transgenic leaves was also examined. The results showed that the starch content increased in the OE leaves (Figure S4A) and decreased in the RI leaves during the light period when compared with the untransformed control (Figure S4B), showing a similar trend in variation to that in the tubers. The sugar content was measured and the chip colour was evaluated for the tubers before and after cold storage. The sucrose content of transgenic tubers exhibited no significant difference with their corresponding control (Figure 4a,b). The RS content, however, exhibited a significant reduction in all the OE-transgenic tubers relative to the control (Figure 4c), and all the RI-transgenic tubers showed a significant increase (Figure 4d) before being exposed to a low temperature. A similar status was maintained after storage at 4 °C for 30 days, even though the RS level was higher in all stored tubers than that before storage.

(b) Figure 2 SbAI transcripts in the transgenic tubers. The relative expression of the SbAI gene in transgenic tubers stored at 4 °C for 0 days and 30 days is presented as the fold change to that of the control at 0 days (E3 and AC142-01, untransformed control of OE- and RI-transgenic lines, respectively), which is taken as 1. The columns represent the mean values  SD (n = 3). ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 984–993

The potato amylase inhibitor gene SbAI 987

Figure 3 The amylase activity, starch content and starch degradation ratio for transgenic tubers stored at 4 °C for 0 day and 30 days. (a and b) a-amylase activity of OE- and RI-transgenic tubers, respectively. (c and d) b-amylase activity of OE- and RI-transgenic tubers, respectively. (e and f) starch content of OE- and RI-transgenic tubers, respectively. (g and h): starch degradation rate of OE- and RI-transgenic tubers stored at 4 °C for 30 days, which is calculated by the formula (ab)/ a 9 100% (a and b represent the starch content before and after storage, respectively). The columns represent the mean values  SD (n = 3) (*P < 0.05; **P < 0.01).

Figure 4 The sugar content and chip colour of transgenic tubers stored at 4 °C for 0 day and 30 days. (a and b): sucrose content of OE- and RI-transgenic tubers, respectively. (c and d) reducing sugar (RS) content of OE- and RI-transgenic tubers, respectively. (e) chip colour of transgenic tubers. The columns represent the mean values  SD (n = 3) (*P < 0.05; **P < 0.01).

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Consequently, in comparison with the corresponding control, the chip colour was lighter in OE-transgenic tubers and darker in RI-transgenic tubers after they were stored at low temperatures (Figure 4e). These results strongly demonstrated that the SbAI gene plays important roles in potato CIS.

The SbAI regulatory mechanism for amylases Three potato amylase genes (StAmy23, StBAM1 and StBAM9) have been shown to be cold-inducible in potato tubers (Zhang, 2013). To explore the mechanism underlying how SbAI regulates potato CIS

ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 984–993

988 Huiling Zhang et al. first, the influence of SbAI on the transcriptional expression of StAmy23, StBAM1 and StBAM9 were investigated. The results showed that after 30 days at 4 °C, the expression of these genes was repressed in SbAI overexpressing tubers and promoted in SbAIsuppressed tubers (Figure 5). All the changes in transcript abundance for the amylase genes were less than twofolds in comparison with the untransformed control. However, the b-amylase activity was inhibited very significantly in OE-transgenic tubers (Figure 3c) and enhanced in RI-transgenic tubers except for RI-2, which was not significantly expressed in cold-stored tubers (Figure 3d). These results imply that amylase activity may be largely regulated by SbAI at the post-translational level. The protein–protein interactions between SbAI and each of the amylases were subsequently dissected by direct visualization in the tobacco BY-2 cells by bimolecular fluorescence complementation (BiFC). The fluorescence signal was observed when SbAIYFPN was co-expressed with StAmy23-YFPC, StBAM1-YFPC and StBAM9-YFPC (Figure 6a1–a3), whereas control cells that were transformed with empty vectors produced no fluorescence (Figure 6a4–a7). To reconfirm these interactions, SbAI and the three amylase proteins were further evaluated by Gal4-based yeast two-hybrid (Y2H) assay and selected by X-a-Gal assay. All three pairwise combinations showed an interaction signal as the positive control and a very weak signal was detected in the negative control (Figure 6b), verifying the high reliability of the BiFC results. These findings strongly suggest that SbAI interacts with StAmy23, StBAM1 and StBAM9. The crude protein of cold-stored potato tubers was extracted to determine the inhibitory effects of SbAI on amylases, and the SbAI, StAmy23, StBAM1 and StBAM9 proteins were expressed and purified. SbAI (Figure 7a) and StBAM1 (Figure 7c) were purified from E. coli, and StAmy23 (Figure 7b) was purified from yeast. The a-amylase activity and b-amylase activity in potato tubers were decreased as the SbAI concentration subsequently increased, and a similar SbAI inhibitory rate for a-amylase and bamylase was presented (Figure 7e). SbAI could also inhibit the recombinant proteins StAmy23 and StBAM1, and in a similar pattern, the residual activities of these two proteins were reduced

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rapidly as the SbAI concentration increased to 100 ng/mL and then progressively declined as a higher concentration of SbAI (up to 400 ng/mL) was applied (Figure 7f). Unfortunately, the StBAM9 protein that was purified from E. coli (Figure 7d), and the eukaryotic system did not have b-amylase activity; so, we were unable to evaluate the SbAI effects. Nevertheless, our results compellingly reveal that SbAI is an amylase inhibitor and associates with potato CIS through its impacts on amylase activities, primarily those of StAmy23 and StBAM1. At the same time, the specificity of SbAI for amylases from different sources was investigated and the results showed that SbAI exhibited a strong inhibitory role on the activity of amyloglucosidase from Aspergillus niger (ANA) by as much as 52%, but it had no remarkable effects on the a-amylase (MA) and b-amylase (MB) from malt, a-amylases from human saliva (HSA) and porcine pancreas (PPA) (Figure S5).

Discussion A major drawback of potato tubers stored at a low temperature is the CIS, which affects the commercial value of the processed products. Our previous research showed that starch degradation, sucrose decomposition and glycolysis were considered to be the key pathways in potato CIS (Chen et al., 2012). In the present research, we first cloned the amylase inhibitor gene SbAI from S. berthaultii and elucidated its function in potato CIS, which was achieved by inhibiting amylase activity to slow down starch hydrolysis in cold-stored potato tubers. The tuber starch content generally decreases along with the storage time (Figure 3e,f). This starch turnover can occur occasionally during the diurnal cycle in response to changes in sinksource transitions during development (Geigenberger and Stitt, 2000), or in postharvest tubers that can be triggered by sucrose depletion (Hajirezaei et al., 2003). However, our results demonstrated that overexpressing SbAI in CIS-sensitive potatoes decreased the starch degradation rate, which was accompanied by a reduction in b-amylase activity, while suppressing it in the CIS-resistant potato that produced the opposite results in cold-

Figure 5 The expression of amylase genes in transgenic tubers stored at 4 °C for 30 days. (a, c and e) gene expression in OE-transgenic tubers. (b, d and f) gene expression in RI-transgenic tubers. The columns represent the mean values  SD (n = 3). ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 984–993

The potato amylase inhibitor gene SbAI 989 (a)

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Figure 6 Interactions for StAmy23, StBAM1, StBAM9 and SbAI proteins. (a) interaction of proteins in tobacco BY-2 cells according to bimolecular fluorescence complementation. (a1–a3) transient co-expression of SbAI-YFPN and StAmy23-YFPC, StBAM1-YFPC and StBAM9-YFPC. All figures show YFP signals in the cells, which represent the interaction between two co-expressed proteins. (a4–a7) individual genes were co-expressed with an empty vector to serve as a negative control. No YFP signals were detectable in the cells. Bars indicate the image magnification. (b) results of the X-a-Gal assay. The survival and colour of the clones on QDO/X-a-Gal (SD/-Leu/-Trp/-His/-Ade/X-a-Gal) selecting plates represent the interaction between SbAI and the amylase proteins assayed with the BD Matchmaker Screening Kit (Clontech). (b1) positive control. (b2–b4) interactions between SbAI and StAmy23, StBAM1 and StBAM9, respectively. All results exhibited a positive signal; (b5) negative control. The positive and negative controls are represented by pGBK-53/pGADRecTb and pGBK-Lam/pGAD-RecTb, respectively. The experiment was repeated three times, with one shown here as a representative.

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Figure 7 The inhibitory effects of SbAI protein on amylase activity. (a) An SDS-PAGE of SbAI protein as expressed in E. coli cells. Lane 1, molecular marker; Lane 2, before being treated with IPTG; Lane 3, after being treated with IPTG; Lane 4, proteins from the soluble fraction; Lane 5, proteins not combined with the cartridge; Lane 6, proteins washed from the cartridge; Lane 7, purified SbAI protein (arrow). (b) An SDS-PAGE of StAmy23 protein expressed in yeast. Lane 1, molecular marker; Lane 2, before being treated with methanol; Lane 3, StAmy23 proteins (arrow) from the soluble fraction when treated with methanol. (c) An SDS-PAGE of StBAM1 protein expressed in E. coli cells. Lane 1, molecular marker; Lane 2, before being treated with IPTG; Lane 3, after being treated with IPTG; Lane 4, purified recombined pET-StBAM1 (arrow). (d) An SDS-PAGE of StBAM9 protein as expressed in E. coli cells. Lane 1, molecular marker; Lane 2, before being treated with IPTG; Lane 3, after being treated with IPTG; Lane 4, proteins from the soluble fraction; Lane 5, purified recombined pGEX-StBAM9 (arrow). (e) the inhibitory effects of SbAI on a-amylase and b-amylase activity when isolated from cold-stored tubers. (f) the inhibitory effects of SbAI on the activity of purified StAmy23 and StBAM1. The error bar indicates the standard error of the mean for three biological replicates. ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 984–993

990 Huiling Zhang et al. stored tubers (Figure 3). As a consequence, the reducing sugar content and chip colour of the tubers showed corresponding responses that enhanced the expression of SbAI favourites for improving potato CIS (Figure 4). This complementary function dissection reveals that the modulation of starch degradation associated with SbAI significantly affects the potato CIS process. It is interesting that, when transgenic plants were kept at 12-h light/ 12-h dark, the starch content of their mature leaves showed a circadian change. The starch accumulation started to increase as light was applied, and this accumulation reached its maximum at the end of the light period. The higher starch content was noticeably accompanied with SbAI OE leaves (Figure S4A), and a lower content was observed in the RI leaves during the light period relative to the untransformed control (Figure S4B), showing a similar variation trend to that of the tubers. These results suggest that SbAI participates wherever starch metabolism occurs in potato tissues. Genes encoding the enzymes that catalyze different steps of carbohydrate metabolism in potato CIS have been identified and functionally tested, suggesting that the post-translational modulation of the key enzymes may be critical for diverse resistance levels of potato varieties to CIS. However, only a few have been dissected between CIS-resistant and CIS-sensitive potato genotypes (Bhaskar et al., 2010). A post-translational regulation of potato invertase activity is revealed by identifying the specific acid invertase inhibitors, which play roles in regulating sucrose cleavage by capping acid invertase activity (Liu et al., 2013; Mckenzie et al., 2013). Because no obvious relation was established between the SbAI transcript abundance and the amylase activity of cold-stored potato tubers, we supposed that the starch hydrolysis could also be primarily regulated at the post-translational level, and it was supported by pairwise protein–protein interaction assays in the present research. Our results not only visually demonstrated the interactions between SbAI and the cold-inducible amylases StAmy23, StBAM1 and StBAM9 (Figure 6), but it also biochemically explained the inhibitory effects of SbAI on these amylase proteins (Figure 7). It is reasonable to conclude that SbAI has functions for repressing the cold-inducible amylases by slowing down starch hydrolysis and reducing sugar accumulation in cold-stored potato tubers. The starch degradation process in potato tubers primarily occurs in the amyloplasts. However, SbAI is a deduced secreted protein, and it is most likely located in the cytoplasm of tobacco cells as visualized by a fusion with the GFP protein (Figure S1). The interactions of SbAI and amylases StAmy23 and StBAM1 were also detected in the cytoplasm by BiFC (Figure 6). However, the techniques used for fluorescence detection in the present research were unable to distinguish the plastids in the cytoplasm, and it is worth further investigation to explain whether SbAI is expressed in amyloplasts, or the SbAI-amylase complex is imported into amyloplasts as a single unit. Changes were observed in the transcript abundance of amylase genes (Figure 5) in SbAI transgenic tubers. SbAI overexpression resulted in the down-regulation of amylase genes, more for b-amylase genes StBAM1 and StBAM9 than a-amylase gene StAmy23, which was accompanied by a significant reduction in the reducing sugars of the tubers, whereas SbAI repression slightly up-regulated the amylase gene expression with a remarkable increase in the RS content (Figures 4 and 5). This alteration in amylase gene expression may be influenced by sugar feedback effects, implying that reducing sugars may be signals that trigger amylase gene expression in potato tubers. As

reviewed by Koch (1996), sugar depletion can enhance the expression of a-amylase genes in rice and barley, and sugar abundance increases b-amylase gene expression in sweet potatoes; this trend was defined as a ‘feast and famine’ response at the gene expression level, although the author did not indicate the difference in expression patterns of plant species or tissues. Changes in the b-amylase activity of transgenic tubers were also observed (Figure 3c,d). However, the a-amylase activity of OE-transgenic tubers (except OE-11) exhibited similar levels with that of E3 (Figure 3a), and those in RI-transgenic tubers increased significantly in comparison with that of AC142-01 (Figure 3b). A possible explanation could be that the interaction between SbAI and a-amylase may have reached a threshold in OEtransgenic tubers, overexpressing the inhibitor, and it could not have had further impact on the a-amylase activity. A similar observation was reported by Park et al. (2011) regarding the overexpression of late embryogenesis abundant 14 gene (LEA14) in sweet potato; the lignin contents were increased significantly, and the RNAi lines showed similar levels of lignin accumulation as those of the controls. Moreover, purified SbAI could inhibit the a-amylase activity of potato tuber extracts (Figure 7e), but the a-amylase activity in OE-transgenic tubers did not experience significant declines (Figure 3a). This conflict may also have resulted from the difference in the reaction systems, and the in vivo interaction of SbAI and a-amylase in plant cells may involve other regulatory factors that are absent from the in vitro system with purified proteins, but they require further investigation. Previous research showed that amylase inhibitors from common beans and cowpeas could promote plant resistance against seed weevils in peas by inhibiting the a-amylase activity of seed weevils (Barbosa et al., 2010; Pelegrini et al., 2008). We also investigated the specificity of SbAI to other amylases from different sources. As shown in Figure S5, SbAI could significantly inhibit the ANA activity by as much as 52%, but it did not markedly influence the MA and MB activity of malt, HSA and PPA. In accordance with a previous conclusion that the a-amylase inhibitor protein VuD1 was highly inhibitory against a-amylases in gut extracts from the insect pests A. obtectus and Z. subfasciatus, this protein exhibited low inhibitory activity against mammalian a-amylases from porcine pancreas and human saliva, and it also had no effect on the a-amylase of C. maculatus or the pathogenic fungus A. fumigatus (Pelegrini et al., 2008). Our results suggest that SbAI may have a specific inhibitory function on the amylases, possibly possessing specific forms or committing peculiar biological actions, one example being the cold-inducible amylases of potatoes. We also observed that suppressing both StBAM1 and StBAM9 resulted in a lower starch degradation than suppressing only StBAM1 (unpublished), suggesting an added contribution by StBAM9. This finding is very similar to that of the b-amylase gene BAM4 in Arabidopsis thaliana. When expressed as a recombinant protein in E. coli, BAM4 did not have b-amylase activity. However, BAM4 could regulate starch breakdown independently of BAM1 and BAM3 (Fulton et al., 2008). Therefore, we speculate that StBAM9 may play roles in starch degradation independently of StBAM1, and this possibility is worthy of further investigation. The present investigators sought insight into the regulatory mechanism of starch hydrolysis at the post-translational level in coldstored potato tubers through the cloning and functional dissection of potato amylase inhibitor gene SbAI, uncovering its counterparts to be the cold-inducible amylases StAmy23, StBAM1 and StBAM9,

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The potato amylase inhibitor gene SbAI 991 and using a genetic transformation to potato genotypes with distinct CIS-resistance levels. To our knowledge, this is the first report to provide a theoretical and applicable basis for improving potato CIS by modulating starch degradation associated with SbAI.

Experimental procedures A cloning and bioinformatics analysis of SbAI By the rapid-amplification of cDNA ends (RACE) method, a fulllength SbAI cDNA was cloned from the single-stranded cDNA that was synthesized from the RNA of S. berthaultii tubers that had been stored at 4 °C for 5 days. Primer 50 -CTTGTCCAACGG CAACGGAACGG -30 (forward, for 30 -RACE) was designed, and the 30 -fragment was obtained using a SMARTTM RACE cDNA Amplification Kit (Clontech, Mountain View, CA) according to the EST C20-3-E15 (GenBank: HS989783) sequence, which was identified from the cDNA microarray. The PCR products were gelpurified and cloned into the pMD18-T vector (TaKaRa, Osaka, Japan) and transformed into E. coli DH5a. The recombinant plasmid DNA that was isolated from transformed DH5a was sequenced (BGI, Wuhan, China). Using the 30 -fragment, the homology sequence was searched in the potato Genome Sequencing Consortium (PGSC) database. Based on this sequence, the full-length cDNA of SbAI was obtained with the 50 -CGGGATCCATGGCTTTTCATTACTCTATTTCTTTCC-30 (forward) primer and the 50 -CATGAGCTCCTAATACAATATAAAAGCAAAT GCAGCAA-30 (reverse) primer. The protein conserved domains were predicted with Motif Scan (http://hits.isb-sib.ch/cgi-bin/PFSCAN). The amino acid sequences of the genes from potato, tomato and wheat that contained a ‘Tryp_alpha_amyI’ domain were obtained from the National Center of Biotechnology Information (NCBI, http://www.ncbi. nlm.nih.gov/) database and the Potato Genome Sequencing Consortium (PGSC, http://www.potatogenome.net/index.php/ Main_Page) database. Their predicted protein sequences were used to classify the evolutionary relations of SbAI, and a phylogenetic tree was constructed with CLUSTAL W (2.0) (Tompson et al., 1997) with the neighbour-joining method with 1000 bootstrap samples. The resulting tree was viewed with TREEVIEW software (version 1.6.6).

Constructing expression vectors and transforming potatoes The overexpression (OE) vector was constructed by subcloning the open reading frame (ORF) of SbAI that had been obtained by a restriction digestion with BamHI and SacI in the sense orientation into the binary vector pBI121, which contained the CaMV35S promoter. To construct the RNAi (RI) vector, a 375-bp fragment of SbAI containing the ORF and the 30 -untranslated region was amplified from the SbAI cDNA with specific primers (forward 50 -AAAAAGCAGGCTGCTCCTACTCC TTCTCCTAGTCC-30 and reverse 50 -AGAAAGCTGGGTAAAAAAT TCAAAGAGTCCTTAGTGT-30 ), which was subcloned into a pHellsGate8 vector by recombination (Helliwell et al., 2002). The recombinant plasmids were confirmed by sequencing (BGI, Wuhan, China) and then introduced into Agrobacterium tumefaciens strain LBA4404. The OE vector was transformed into the CIS-sensitive potato variety E3, and the RI vector was transformed into the CISresistant potato dihyploid clone AC142-01 as previously described (Si et al., 2003) with minor modifications in the transformation to AC142-01, that is, the 0.5 lM 6-benzylade-

nine (6-BA) used in the medium for shoot regeneration was replaced with 1.0 lm 6-BA. Regenerated shoots were rooted on MS medium containing 50 lg/mL kanamycin and 400 lg/mL carbenicillin. The transgenic plants together with untransformed controls were grown at 20–25 °C in 24-cm-diameter plastic pots in the greenhouse at Huazhong Agricultural University (Wuhan, China) with 16 h of light per day as supplemented with mercury lamps. Two months after planting, the mature leaves were sampled at the following time points: 0, 6, 12, 18 and 24 h (Figure S4). The sampled leaves were immediately frozen in liquid nitrogen and stored at 70 °C until use. When the leaves senesced naturally, the mature tubers were harvested and kept in the dark at 20 °C for 10 days for skin set, and they were then divided into two sets as follows: one set was stored at 4 °C and another at 20 °C for comparison. The tuber samples were taken at time points of 0 and 30 days of storage. Three to five tubers of similar sizes (~5 cm in diameter) were sampled from each treatment. Each tuber was peeled and cut into two parts. One part was used for chipping, and the other was frozen in liquid nitrogen and stored at 70 °C for molecular and biochemical analyses.

RNA isolation and real-time qRT-PCR RNA isolation, reverse transcription and qRT-PCR were performed as previously described (Liu et al., 2013). The ef1a potato gene (GenBank: AB061263) was used as a control because it was reported to be relatively stable in terms of its expression (Nicot et al., 2005). The qRT-PCR primers were designed with Primer express 2.0 software (Applied Biosystems, Carlsbad, CA). The primers for the SbAI gene were 50 -CCATCAGCAACGAATTCCG-30 (forward) and 50 -AATGCAGCAATGAGGACCAAT-30 (reverse). The amylase genes (StAmy23, StBAM1 and StBAM9) involved in potato CIS were selected according to our previous research and the same primers were used as before (Zhang et al., 2013).

Assessing the enzyme activity, starch and sugar contents and chip colour The a-amylase and b-amylase activities in leaves and tubers were determined using assay kits from Megazyme (Bray, Ireland) and by the method described by Scheidig et al. (2002). The starch and reducing sugar amounts were determined as previously detailed (Liu et al., 2010; Song et al., 2005), and the starch yield per plant was defined by the following formula: the starch yield per plant is equal to the starch content of the tuber times the tuber yield per plant. Chipping was performed according to Liu et al. (2011). The data were presented as means of three biological repeats, and the percentages were inverse-sine-transformed for the significance test using SAS 8.1 (SAS Institute, Gary, NC).

Bimolecular fluorescence complementation To generate the vector system for Bimolecular fluorescence complementation (BiFC) analysis, the full-length cDNA fragments of SbAI, StAmy23, StBAM1 and StBAM9 without their stop codons were cloned and inserted into the BamHI (XhoI for StBAM1) and KpnI restriction sites of BiFC vectors pUC-SPYCE/ pSPYCE-35S and pUC-SPYNE/pSPYNE-35S (Walter et al., 2004). SbAI was cloned into the pFF19GFP vector, resulting in pFF19SbAI-GFP for subcellular localization. Each fusion construct was verified by sequencing. The vectors were then transformed into the BY-2 cells by particle bombardment as previously described (von Arnim, 2007). The transformed cells

ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 984–993

992 Huiling Zhang et al. were incubated at 26 °C for 24 h in the dark. Fluorescence detection was performed using a confocal laser scanning microscope (LSM510 Meta, Zeiss, Germany). Fluorescence signals for the yellow fluorescence protein (excitation 514 nm) and green fluorescence protein (excitation 488 nm) of the cells were detected and recorded by LSM Image Examiner software (Zeiss, Germany).

Yeast two-hybrid assays SbAI was inserted into the NcoI/BamHI restriction sites of the pGBKT7 vector, and StAmy23, StBAM1 and StBAM9 were individually inserted into the NdeI/XhoI restriction sites of the pGADT7 vector. Afterwards, pGBKT7-SbAI and pGADT7-StAmy23, pGADT7-StBAM1 and pGADT7-StBAM9 were transformed into yeast stain AH109 as described in the BD Matchmaker Screening Kit protocols. The positive clones were selected as previously described (Lin et al., 2013).

The expression and activity analyses of recombinant proteins The cDNA of SbAI without a 69-bp signal peptide sequence was subcloned into expression vector pPAL7 (Bio-Rad, Hercules, CA). The pPAL7-SbAI plasmid was transformed into E. coli BL21 (DE3). The plasmid was expressed at 37 °C under induction with 0.2 M IPTG for 2.5 h. The SbAI protein purification was performed by following the Gravity Column Purification protocol. By the same methods, the recombinant proteins pET-StBAM1 (expression vector pET28a was selected) and pGEX-StBAM9 (expression vector pGEX-6p-1 was selected) were purified from E. coli. StAmy23 was expressed in the eukaryotic system containing a pPIC9K vector and a GS115 strain, and the protein was purified according to the manual (Invitrogen, Carlsbad, CA). The amylase inhibitory activity of SbAI was tested using assay kits from Megazyme (Bray, Ireland). SbAI was assayed against StAmy23 and StBAM1 and crude potato protein from E3, also against the amylases of porcine pancreas (Sigma), human saliva (Sigma), malt (Bray, Ireland) and amyloglucosidase from A. niger (Sigma), in 0.1 M citric acid buffer, pH 5.6. SbAI and the enzymes were pre-incubated for 30 min at 40 °C, and then 20 lL (5 U/ mL) Blocked p-nitrophenyl-a-D-maltoheptaoside (the substrate for a-amylase) or p-nitrophenyl-b-D-maltoheptaoside (the substrate for b-amylase) was added and incubated for 15 min at 40 °C. The reaction was stopped by adding 2% Tris base to the stop solution, and the absorbance was measured at 405 nm.

Acknowledgements We thank Prof. Dr. Uwe Sonnewald for the useful discussion, Dr. Xun Liu, Dr. Meng Li and Hui Fang for their assistance with the sugar measurements and Tengfei Liu for assistance with the yeast two-hybrid assay. We also thank Stephen Reid for language editing. This research was supported by grants from the National Science Foundation of China (31171602) and the National High Technology Research and Development Program of China (2009AA10Z103).

References von Arnim, A. (2007) Subcellular localization of GUS-and GFP-tagged proteins in onion epidermal cells. In Arabidopsis: A Laboratory Manual (Weigel, D. and Glazebrook, J., eds), pp. 252–262. New York: Cold Spring Harbor Laboratory.

Barbosa, A., Albuquerque, E.V.S., Silva, M.C.M., Souza, D.S.L., Oliveira-Neto, O.B., Valencia, A., Rocha, T.L. and Grossi-De-Sa, M.F. (2010) alpha-Amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits alpha-amylases from the coffee berry borer pest. BMC Biotechnol. 10, 44–51. Bhaskar, P.B., Wu, L., Busse, J.S., Whitty, B.R., Hamernik, A.J., Jansky, S.H., Buell, C.R., Bethke, P.C. and Jiang, J. (2010) Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato. Plant Physiol. 154, 939–948. Borovkov, A.Y., Mcclean, P.E., Sowokinos, J.R., Ruud, S.H. and Secor, G.A. (1995) Effect of expression of UDP-glucose pyrophosphorylase ribozyme and antisense RNAs on the enzyme activity and carbohydrate composition of field-grown transgenic potato plants. J. Plant Physiol. 147, 644–652. Chen, X., Song, B., Liu, J., Yang, J., He, T., Lin, Y., Zhang, H. and Xie, C. (2012) Modulation of gene expression in cold-induced sweetening resistant potato species Solanum berthaultii exposed to low temperature. Mol. Genet. Genomics, 287, 411–421. Claassen, P.A.M., Budde, M.A.W. and Van Calker, M.H. (1993) Increase in phosphorylase activity during cold-induced sugar accumulation in potato tubers. Potato Res. 36, 205–217. Cottrell, J., Duffus, C., Paterson, L., Mackay, G., Allison, M. and Bain, H. (1993) The effect of storage temperature on reducing sugar concentration and the activities of three amylolytic enzymes in tubers of the cultivated potato, Solanum tuberosum L. Potato Res. 36, 107–117. Fulton, D.C., Stettler, M., Mettle, T., Vaughan, C.K., Li, J., Francisco, P., Gil, M., Reinhold, H., Eicke, S., Messerli, G., Dorken, G., Halliday, K., Smith, A.M., Smith, S.M. and Zeeman, S.C. (2008) Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts. Plant Cell, 20, 1040–1058. Geigenberger, P. and Stitt, M. (2000) Diurnal changes in sucrose, nucleotides, starch synthesis and AGPS transcript in growing potato tubers that are suppressed by decreased expression of sucrose phosphate synthase. Plant J. 23, 795–806. €rnke, F., Peisker, M., Takahata, Y., Lerchl, J., Kirakosyan, A. Hajirezaei, M.-R., Bo and Sonnewald, U. (2003) Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum). J. Exp. Bot. 54, 477–488. Helliwell, C.A., Wesley, S.V., Wielopolska, A.J. and Waterhouse, P.M. (2002) High-throughput vectors for efficient gene silencing in plants. Funct. Plant Biol. 29, 1217–1225. € Hill, L.M., Reimholz, R., SchrODer, R., Nielsen, T.H. and Stitt, M. (1996) The onset of sucrose accumulation in cold-stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose-phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase. Plant, Cell Environ. 19, 1223– 1237. de la Hoz, P.S., Castagnaro, A. and Carbonero, P. (1994) Sharp divergence between wheat and barley at loci encoding novel members of the trypsin/ a-amylase inhibitors family. Plant Mol. Biol. 26, 1231–1236. Kashlan, N. and Richardson, M. (1981) The complete amino acid sequence of a major wheat protein inhibitor of a-amylase. Phytochemistry, 20, 1781–1784. Koch, K.E. (1996) Carbohydrate-modulated gene expression in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 509–540. Krause, K.P., Hill, L., Reimholz, R., Nielsen, T.H., Sonnewald, U. and Stitt, M. (1998) Sucrose metabolism in cold-stored potato tubers with decreased expression of sucrose phosphate synthase. Plant, Cell Environ. 21, 285–299. Kumari, B., Sharma, P. and Nath, A.K. (2012) a-Amylase inhibitor in local Himalyan collections of Colocasia: Isolation, purification, characterization and selectivity towards a-amylases from various sources. Pestic. Biochem. Physiol. 103, 49–55. Lay, F. and Anderson, M. (2005) Defensins-components of the innate immune system in plants. Curr. Protein Pept. Sci. 6, 85–101. Lin, Y., Liu, J., Liu, X., Ou, Y., Li, M., Zhang, H., Song, B. and Xie, C. (2013) Interaction proteins of invertase and invertase inhibitor in cold-stored potato tubers suggested a protein complex underlying post-translational regulation of invertase. Plant Physiol. Biochem. 73, 237–244. Liu, X., Song, B., Zhang, H., Li, X.-Q., Xie, C. and Liu, J. (2010) Cloning and molecular characterization of putative invertase inhibitor genes and their

ª 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 984–993

The potato amylase inhibitor gene SbAI 993 possible contributions to cold-induced sweetening of potato tubers. Mol. Genet. Genomics, 284, 147–159. Liu, X., Zhang, C., Ou, Y., Lin, Y., Song, B., Xie, C., Liu, J. and Li, X.Q. (2011) Systematic analysis of potato acid invertase genes reveals that a cold-responsive member, StvacINV1, regulates cold-induced sweetening of tubers. Mol. Genet. Genomics, 286, 109–118. Liu, X., Lin, Y., Liu, J., Song, B., Ou, Y., Zhang, H., Li, M. and Xie, C. (2013) StInvInh2 as an inhibitor of StvacINV1 regulates the cold-induced sweetening of potato tubers by specifically capping vacuolar invertase activity. Plant Biotechnol. J. 11, 640–647. Lorberth, R., Ritte, G., Willmitzer, L. and Kossmann, J. (1998) Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening. Nat. Biotechnol. 16, 473–477. Marchler-Bauer, A., Lu, S., Anderson, J.B., Chitsaz, F., Derbyshire, M.K., DeWeese-Scott, C., Fong, J.H., Geer, L.Y., Geer, R.C. and Gonzales, N.R. (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 39, 225–229. Mckenzie, M.J., Chen, R.K., Harris, J.C., Ashworth, M.J. and Brummell, D.A. (2013) Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers. Plant, Cell Environ. 36, 176–185. Mikkelsen, R., Suszkiewicz, K. and Blennow, A. (2006) A novel type carbohydrate-binding module identified in a-glucan, water dikinases is specific for regulated plastidial starch metabolism. Biochemistry, 45, 4674–4682. Miyazaki, T., Morimoto, T., Fukuyama, K. and Matsubara, H. (1994) Crystallization and preliminary X-ray diffraction studies of the a-amylase inhibitor coded 0.19 from wheat kernel. J. Biochem. 115, 179–181. Morrell, S. and Rees, T.A. (1986) Control of the hexose content of potato tubers. Phytochemistry, 25, 1073–1076. Nicot, N., Hausman, J.F., Hoffmann, L. and Evers, D. (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J. Exp. Bot. 56, 2907–2914. Park, S.-C., Kim, Y.-H., Jeong, J.C., Kim, C.Y., Lee, H.-S., Bang, J.-W. and Kwak, S.-S. (2011) Sweetpotato late embryogenesis abundant 14 (IbLEA14) gene influences lignification and increases osmotic-and salt stress-tolerance of transgenic calli. Planta, 233, 621–634. Pelegrini, P.B., Lay, F.T., Murad, A.M., Anderson, M.A. and Franco, O.L. (2008) Novel insights on the mechanism of action of alpha-amylase inhibitors from the plant defense in family. Proteins, 73, 719–729. Preiss, J. (1982) Regulation of the biosynthesis and degradation of starch. Annu. Rev. Plant Physiol. 33, 431–454. Scheidig, A., Frohlich, A., Schulze, S., Lloyd, J.R. and Kossmann, J. (2002) Downregulation of a chloroplast-targeted b-amylase leads to a starch-excess phenotype in leaves. Plant J. 30, 581–591. Shepherd, L.V.T., Bradshaw, J.E., Dale, M.F.B., McNicol, J.W., Pont, S.D.A., Mottram, D.S. and Davies, H.V. (2010) Variation in acrylamide producing potential in potato: segregation of the trait in a breeding population. Food Chem. 123, 568–573. Si, H., Xie, C. and Liu, J. (2003) An efficient protocol for Agrobacterium-mediated transformation with microtuber and the introduction of an antisense class I patatin gene into potato. Acta Agronomica Sinica, 29, 801–805. €tting, O. and Moorhead, G.B.G. (2014) Phosphoglucan Silver, D.M., Ko phosphatase function sheds light on starch degradation. Trends Plant Sci. http://dx.doi.org/10.1016/j.tplants.2014.01.008.

Smith, A.M., Zeeman, S.C. and Smith, S.M. (2005) Starch degradation. Annu. Rev. Plant Biol. 56, 73–98. Solomos, T. and Mattoo, A.K. (2005) Starch-sugar metabolism in potato (Solanum tuberosum L.) tubers in response to temperature variations. In: Genetic Improvement of Solanaceous Crops (Razdan, M.K. and Mattoo, A.K., eds), pp. 209–234. Plymouth: Science Publishers, Inc. Song, B., Xie, C. and Liu, J. (2005) Expression of potato sAGP gene and its effects on contents of starch and reducing sugar of transgenic potato tubers. Sci. Agric. Sin. 38, 1439–1446. Spychalla, J.P., Scheffler, B.E., Sowokinos, J.R. and Bevan, M.W. (1994) Cloning, antisense RNA inhibition, and the coordinated expression of UDP-glucose pyrophosphorylase with starch biosynthetic genes in potato tubers. J. Plant Physiol. 144, 444–453. Stark, D.M., Timmerman, K.P., Barry, G.F., Preiss, J. and Kishore, G.M. (1992) Regulation of the amount of starch in plant tissues by ADP glucose pyrophosphorylase. Science, 258, 287–292. €hlmann, T. and Kampfenkel, K. (1998) Altered plastidic ATP/ Tjaden, J., Mo ADP-transporter activity influences potato (Solanum tuberosum L.) tuber morphology, yield and composition of tuber starch. Plant J. 16, 531–540. Tompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. €tze, K., Batistic, O., Weckermann, K., N€ake, C., Walter, M., Chaban, C., Schu Blazevic, D., Grefen, C., Schumacher, K. and Oecking, C. (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438. Zeeman, S.C., Kossmann, J. and Smith, A.M. (2010) Starch: its metabolism, evolution, and biotechnological modification in plants. Annu. Rev. Plant Biol. 61, 209–234. Zhang, H. (2013) Expression patterns of the genes related to Cold-induced sweetening and function dissection of two starch degradation genes in potato. Dissertation, Huazhong Agricultural University. Zhang, H., Liu, X., Liu, J., Ou, Y., Lin, Y., Li, M., Song, B. and Xie, C. (2013) A novel RING finger gene, SbRFP1, increases resistance to cold-induced sweetening of potato tubers. FEBS Lett. 587, 749–755.

Supporting information Additional Supporting information may be found in the online version of this article: Figure S1 The subcellular localization of SbAI. Figure S2 The morphology of transgenic plants. Figure S3 The starch and sugar content of the transgenic tubers that were stored at 20 °C for 30 days. Figure S4 The starch contents of the leaves. Figure S5 An enzyme assay for SbAI against amylases from different sources: a-amylase of human saliva (HSA), a-amylase of porcine pancreas (PPA), a-amylase of malt (MA), b-amylase of malt (MB) and amyloglucosidase from A. niger (ANA).

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