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The potato protease inhibitor gene, St-Inh, plays roles in the cold-induced sweetening of potato tubers by modulating invertase activity
Postharvest Biology and Technology 86 (2013) 265–271
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The potato protease inhibitor gene, St-Inh, plays roles in the cold-induced sweetening of potato tubers by modulating invertase activity Xun Liu a,b,c , Shanhan Cheng a,d , Jun Liu a , Yongbin Ou a , Botao Song a , Chi Zhang a,e , Yuan Lin a , Xiu-Qing Li b , Conghua Xie a,∗ a Key Laboratory of Horticultural Plant Biology, Ministry of Education, National Centre for Vegetable Improvement (Central China), Huazhong Agricultural University, Wuhan 430070, China b Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, New Brunswick E3B 4Z7, Canada c College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China d College of Horticulture and Forestry, Hainan University, Haikou 570228, China e Institute of Root and Tuber Crops, Zhejiang Agriculture and Forestry University, Hangzhou, Linan 311300, China
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Article history: Received 22 March 2012 Accepted 1 July 2013 Keywords: Potato Protease inhibitor Cold-induced sweetening
a b s t r a c t 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. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Potato (Solanum tuberosum L.) is the fourth most important food crop in the world. Globally, consumers are eating potatoes more and more as value-added, processed products rather than as fresh produce. Two such processed products are French fries and potato chips, which are available in fast-food chains and snack bars worldwide. For an uninterrupted supply to the industry, potato tubers need to be stored at low temperatures throughout the year. However, low-temperature storage leads to the accumulation of
Abbreviations: AI, acid invertase; CIS, cold-induced sweetening; KPI, Kunitz-type protease inhibitor; PMEI, pectin methylesterase/invertase inhibitor; RS, reducing sugar. ∗ Corresponding author. Tel.: +86 27 87280969; fax: +86 27 87286939. E-mail addresses: [email protected], [email protected] (C. Xie). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.07.001
reducing sugars (RS), which is referred to as cold-induced sweetening (CIS). Reducing sugars negatively influence processing quality because they react with free amino acids during frying, resulting in dark-colored food products. Therefore, preventing RS accumulation during tuber storage is of economic importance to the potato processing industry. During tuber storage at low temperatures, RS accumulation varies in response to environmental and genetic factors. The activity of acid invertase (AI), which hydrolyzes sucrose to glucose and fructose, is positively associated with RS accumulation (Cheng et al., 2004b; McKenzie et al., 2005; Xu et al., 2009). Suppressing a cold-responsive AI gene, StvacINV1, was found to prevent RS accumulation and improve the quality of fried products (Bhaskar et al., 2010; Liu et al., 2011), whereas increasing the abundance of StvacINV1 mRNA in cold-stored tubers was reported to enhance AI activity (Liu et al., 2011). In addition, AI activity is likely also controlled post-translationally by invertase inhibitors (Pressey, 1966, 1967; Schwimmer et al., 1961). Although a great deal of attention
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had been paid to invertase inhibitor in potato tubers, it took until the late 1990s before purifying the inhibitor to apparent homogeneity yielded the information on protein or peptide sequences that was required to clone the corresponding cDNA; at that time, Greiner et al. (1998, 1999) was able to characterize two invertase inhibitor cDNAs from Nicotiana tabacum. Overexpression of one of the two tobacco invertase inhibitors, NtInvInh2, strongly reduced RS accumulation in potato tubers (Greiner et al., 1999). These inhibitors belong to the pectin methylesterase/invertase inhibitor (PMEI) family, which is referred as PMEI-RP (Hothorn et al., 2004). Subsequently, Glaczinski et al. (2002) purified, from potato tubers, soluble protease inhibitors that were completely inhibitory to AI activity in vitro. N-terminal amino acid sequencing of the purified proteins indicated that these proteins are members of a small tuber protein family known as Kunitz-type protease inhibitors (KPIs). Based on the N-terminal amino acid sequence of the proteins, a full-length cDNA of the KPI St-Inh was cloned from potato, and its recombinant protein from E. coli inhibited AI activity, indicating that St-Inh has a role as an invertase inhibitor (Cheng et al., 2004a). The St-Inh gene was mapped to its position as marker CP6 on potato chromosome III (Heibges et al., 2003a), which is linked with a quantitative trait locus for sugar content (Menéndez et al., 2002), suggesting that St-Inh could be a candidate gene for regulating CIS by its post-translational mechanism. However, the roles that St-Inh plays in CIS in tubers have not been clearly described because of the lack of evidence for gene function in vivo. The present study investigated the expression patterns of StInh in various organs of growing potato plants and in cold-stored potato tubers. Together with NtInvInh2, the contribution of St-Inh to CIS resistance was also assessed by overexpression in potato tubers. The results suggest that both NtInvInh2 and St-Inh play roles in slowing down CIS in tubers by modulating AI activity. NtInhInh2 appeared to have a stronger capacity for resistance to CIS than St-Inh did. 2. Materials and methods 2.1. Plant materials At the Agriculture and Agri-Food Canada (AAFC) Potato Research Centre in Fredericton, New Brunswick, Canada, plants of the potato cultivar Russet Burbank were grown in 24-cm pots in the greenhouse at 20–25 ◦ C with a 16-h photoperiod supplemented with artificial light (approximately 90 mol m−2 s−1 ). Flowers and floral buds, vegetative buds, sink leaves, source leaves, senescent leaves, stems, roots, stolons, immature tubers, and mature tubers were sampled from three plants 60 d after emergence. The samples were immediately frozen in liquid N2 and stored in a freezer at −80 ◦ C for RNA isolation as described by Liu et al. (2011). The CIS-sensitive cultivars AC Novachip and Yukon Gold and the CISresistant cultivars ND860-2, Snowden, and Atlantic were used to test the expression of the St-Inh gene in relation to CIS in the tubers. The tubers were harvested from the Potato Research Station’s Benton Ridge Potato Breeding Substation and were immediately stored at 21 ◦ C for two weeks (T0) to permit skin set. Subsequently, the tubers were subjected to one of the following temperature treatments: - 7C60d: Tubers stored at 7 ◦ C for 60 d. - R21C1d: Tubers stored at 7 ◦ C for 60 d and then transferred to 21 ◦ C for reconditioning for 1 d. - R21C21d: Tubers stored at 7 ◦ C for 60 d and then transferred to 21 ◦ C for reconditioning for 21 d. - 21C60d: Tubers maintained at 21 ◦ C for 60 d as the control.
The tubers were sampled at T0, 21C60d, 7C60d, R21C1d and R21C21d. The tubers were peeled and cut into small pieces, immediately frozen in liquid N2 , and stored in the freezer at −80 ◦ C until use. The frozen tissues were ground in liquid N2 . The total RNA was extracted using the method described previously by Yang et al. (2006), and the RNA was further purified with the RNeasy Plant Mini Kit (Qiagen, USA) according to the manufacturer’s instructions. The quality of the total RNA was checked using the OD260 /OD280 nm ratio and by 1% agarose gel electrophoresis. The total RNA (10 g) was treated with the TURBO DNA-Free Kit (Ambion, USA) to eliminate residual genomic DNA in accordance with the manufacturer’s protocol. For each RNA sample, quantitative PCR was carried out using the primers of the ef1˛ gene (AB061263) to verify that no genomic DNA contamination was present (Nicot et al., 2005). For the reverse transcription, DNAfree RNA (2 g) was converted to first-strand cDNA using TaqMan Reverse Transcription Reagents (Applied Biosystems, USA) with random hexamers, and then the cDNA was purified using the QIAquick PCR Purification Kit (Qiagen, USA) according to the manufacturer’s instructions. 2.2. qRT-PCR Quantitative reverse transcription PCR (qRT-PCR) was performed on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, USA). The gene-specific primer pairs (forward: 5 -GGAAACCTTCAATGCCCAAAC-3 ; reverse: 5 CCGACTCCGACTTACGAATGA-3 ) were designed with Primer Express 2.0 software (Applied Biosystems, USA). The PCR reactions were carried out in a 20-L system containing 1× Power SYBR Green PCR Master Mix (Applied Biosystems, USA), 1 L of cDNA corresponding to 20 ng of original RNA, and 0.6 mol L−1 each of the specific forward and reverse primers. The following amplification program was used: 50 ◦ C for 2 min, 95 ◦ C for 10 min, 40 cycles of 95 ◦ C for 15 s, and 60 ◦ C for 1 min. The specificity of the individual PCR amplifications was confirmed by dissociation curve analysis from 60 to 95 ◦ C following the final cycle of the qRT-PCR. Aliquots of the PCR reactions were electrophoresed on agarose gels to verify the specificity of the primers, and the amplified bands were cloned and sequenced to confirm that they were fragments of the target genes. Relative quantification of individual gene expression was performed using a comparative Ct method as described in ABI PRISM 7700 SDS Use Bulletin #2 (Applied Biosystems, USA). The potato gene ef1˛ (AB061263) was used as the control, because it is an appropriate reference for cold stress in potatoes (Nicot et al., 2005). The approximately equal efficiency in PCR amplification of the target and reference genes was verified by performing standard curves for each amplicon. 2.3. Gene constructs and plant transformation The complete coding sequence of St-Inh obtained by restriction with BamHI and SacI was subcloned in sense orientation into the binary vector pBI121 containing the CaMV35S promoter. The construct was introduced into Agrobacterium tumefaciens strain LBA4404 and transformed into potato CIS-sensitive cultivar E-potato 3 (Ep3) by Agrobacterium-mediated transformation as previously described (Si et al., 2003). The regenerated plants were designated as D lines. In addition, plants with overexpression of NtInvInh2 controlled by the CaMV35S promoter in Ep3 (designated as A lines) had been previously obtained in the laboratory of some of the present coauthors (Cheng, 2004). Tubers were harvested from the transgenic plants (D and A lines) and from wild-type plants grown at 20–25 ◦ C in the greenhouse at the National Centre for Vegetable Improvement (Central China), Huazhong Agricultural
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University, in Wuhan, China. After 10 d at 20 ◦ C for skin set, the tubers were divided into two sets, with one stored at 4 ◦ C for cold induction and the other maintained at 20 ◦ C for comparison. The tubers were sampled after 0, 15, 30, and 45 d of storage, peeled and cut into small pieces, immediately frozen in liquid N2 , and stored in a freezer at −80 ◦ C for further analysis. 2.4. Determination of soluble acid invertase activity, sugar content, and chip color The soluble sugars were extracted from the potato tubers, and the AI activity was assayed according to the method of Greiner et al. (1999). Sucrose and RS contents were measured as previously described (Liu et al., 2010). Tuber slices were fried for 3 min at 191 ◦ C or until bubbling ceased, and the chip color was visually determined using the Color Standards Reference Chart for Potato Chips (Snack Food Association, USA). Each sample consisted of 7–10 crushed potato chips. Two or three tuber samples from different plants were used for each experiment. Regression analysis of the data was conducted using the Microsoft Excel program (Microsoft Office XP, 2003). Data are means ± SE. The significance between the treatments was tested by ANOVA and LSD using SPSS13.0 software for Windows (SPSS Inc., USA). 3. Results 3.1. Expression patterns of St-Inh in various organs and stored tubers The full-length cDNA of St-Inh (NCBI Accession Number AY594178) had been cloned previously in the laboratory of some of the present coauthors (Cheng et al., 2004a). To understand the structure of the St-Inh gene, we amplified it from potato genomic DNA. The result showed identical sequences between the coding sequence of St-Inh and genomic DNA (data not shown), demonstrating that the St-Inh gene consists of a single exon. The expression of St-Inh was analyzed in various organs by qRTPCR (Fig. 1A). The St-Inh gene was found to be expressed mainly in tubers in terms of mRNA abundance, which increased progressively with tuber growth. This expression profile implies that St-Inh is involved mainly in tuber development. To assess the response of StInh to low temperatures in tubers, the relative expression of St-Inh during cold storage was analyzed (Fig. 1B). The results showed that the level of St-Inh transcripts decreased markedly at 7 ◦ C compared with the level in the samples stored at 21 ◦ C. This temperaturedependent regulation was further confirmed by a rapid and obvious increase in transcript abundance in all cultivars tested when the tubers were reconditioned at 21 ◦ C for 1 d. The gene’s mRNA abundance decreased again when the tubers were reconditioned at 21 ◦ C for 21 d. These results reveal that the expression pattern of St-Inh is opposite to RS accumulation in cold-stored tubers, suggesting that the gene may be involved in controlling the RS content in potato tubers. 3.2. Effects of St-Inh and NtInvInh2 on cold-induced sweetening in transgenic potato tubers To determine whether St-Inh has a role in regulating CIS in potato tubers, a CaMV35S::St-Inh construct was made and used to transform the potato cultivar Ep3 by Agrobacterium-mediated transformation. A total of 45 transgenic plants (D lines) were obtained. Eight D lines that showed higher transcript abundances of St-Inh than wild-type tubers during storage at 4 ◦ C for 30 d were chosen for further analysis (Fig. S1). In addition, five NtInvInh2 transgenic lines (A lines) that had been obtained previously
Fig. 1. Expression pattern of St-Inh in potato organs and stored tubers, as determined by qRT-PCR analysis. The relative expression levels of St-Inh were standardized to the expression level (1 0 0) of potato reference gene ef1˛ (AB061263) by qRT-PCR. (A) The various potato organs were sampled from three plants of the potato cultivar Russet Burbank grown in 24–cm pots in a greenhouse at 20–25 ◦ C with a 16-h photoperiod supplemented with artificial light 60 d after emergence. FFB, flowers and floral buds; VB, vegetative buds; SiL, sink leaves; SoL, source leaves; SeL, senescent leaves; Sm, stems; Rt, roots; Sl, stolons; IT, immature tubers; MT, mature tubers. (B) Tubers harvested from the CIS-resistant potato cultivars ND8602, Atlantic, and Snowden and the CIS-sensitive cultivars AC Norchip and Yukon Gold were immediately stored at 21 ◦ C for two weeks (T0) to permit skin set. Subsequently, the tubers were divided into two groups for the temperature treatment. The first group of tubers was stored at 7 ◦ C for 60 d (7C60d), and then some of those tubers were transferred to storage at 21 ◦ C for reconditioning for 1 d (R21C1d) or 21 d (R21C21d). The second group was maintained at 21 ◦ C for 60 d (21C60d) as the control. The tubers were sampled at T0, 21C60d, 7C60d, R21C1d, and R21C21d. Similar results were obtained for two tuber samples from different plants. Each sample was represented by one tuber.
(Cheng, 2004) were also chosen for a simultaneous analysis as the positive control. No visible phenotypic effects were observed for either of the St-Inh and NtInvInh2 transgenic lines under normal growth conditions. The tubers of the transgenic lines (A and D) and the wild-type plants were stored at 4 or 20 ◦ C for 30 d. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.postharvbio. 2013.07.001. The experiments were repeated two or three times in different years. In comparison with the wild-type Ep3 plants, the A and D lines had significantly reduced RS accumulation (Table 1), although there was some variation in amounts observed between the repeats. The RS accumulation in the A and D lines was reduced by 41.3–67.2% and by 27.8–35.1%, respectively. These results suggest that the expression of St-Inh and NtInvInh2 play roles in reducing CIS in potato tubers. Tuber slices were fried further, and the chip color was visually determined using the Color Standards Reference Chart for Potato Chips (Snack Food Association, USA). In comparison with the wild-type Ep3 plants, the chip color index decreased one to four levels in the transgenic tubers (Table 2), suggesting that potato processing quality was improved by overexpression of St-Inh and NtInvInh2 in the transgenic tubers.
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Table 1 Reducing sugar (RS) content of transgenic and wild-type tubers stored at 4 ◦ C for 30 d. Linea
2005
2006
RS content (g kg−1 ) Ep3 A06 A12 A20 A30 A31 D02 D03 D05 D26 D35 D51 D53 D60
15.18 ± 5.32 ± 5.40 ± 10.25 ± 9.64 ± 8.12 ± 11.24 ± 11.44 ± 11.31 ± – – – – –
Reduction (%)
0.94 0.27** 0.17** 1.01** 0.55** 0.20** 1.38* 1.54* 1.07*
65.0 64.4 32.5 36.5 46.5 26.0 24.6 25.5 – – – – –
2007
Average reduction (%)
RS content (g kg−1 )
Reduction (%)
RS content (g kg−1 )
9.94 ± 2.62 ± 4.36 ± 5.20 ± 5.21 ± 5.53 ± – 6.96 ± 7.16 ± 7.35 ± 6.58 ± 7.04 ± 7.22 ± 6.50 ±
73.6 56.1 47.7 47.6 44.4 – 30.0 28.0 26.1 33.8 29.2 27.4 34.6
10.60 4.03 5.66 5.96 5.69 6.29 7.47 7.44 7.41 6.76 7.13 6.25 7.17 7.92
0.93 1.38** 0.73** 0.41** 0.72** 1.03** 0.39* 0.42* 1.19* 0.17* 0.63* 0.33* 1.14*
± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.37 0.32** 0.51** 0.44** 0.12** 0.23** 0.87* 1.41* 0.44* 0.44* 1.08* 0.19** 0.22* 0.57*
Reduction (%) 62.9 46.6 43.8 46.3 40.7 29.5 29.8 30.1 36.2 32.7 41.0 32.4 25.3
67.2 55.7 41.3 43.5 43.9 27.8 28.1 27.9 31.1 33.2 35.1 29.9 30.0
Data are means ± SE of three tuber samples from different plants. The LSD values for each year are as follows: 2005, LSD0.05 = 3.18, LSD0.01 = 4.38; 2006, LSD0.05 = 2.58, LSD0.01 = 3.46; and 2007, LSD0.05 = 2.67, LSD0.01 = 3.61. a Ep3, non-transgenic control; A lines, lines overexpressing NtInvInh2; D lines, lines overexpressing St-Inh. * Significance of LSD0.05 . ** Significance of LSD0.01 .
3.3. Effects of St-Inh and NtInvInh2 on acid invertase activities in transgenic potato tubers To examine the effects of St-Inh and NtInvInh2 on invertase inhibitory activity, we determined the AI activities in transgenic and wild-type tubers in three years. Compared with the wild-type Ep3 tubers, the A and D lines showed significantly reduced AI activities (Table 3). The AI activities in the D and A lines were reduced by 54.7–72.5% and by 25.3–36.0%, respectively. Plotting the reduction in AI activities (x) in the transgenic tubers stored at 4 ◦ C against the corresponding reduction in RS accumulation (y) revealed a significant linear relationship (y = 0.616x + 12.03, R2 = 0.831, P < 0.01) (Fig. S2), reinforcing that RS accumulation in potato tubers is largely regulated by AI activity in the tubers when they are exposed to low temperatures. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.postharvbio. 2013.07.001. To further investigate the capacity of St-Inh and NtInvInh2 to regulate CIS, the AI activity, RS and sucrose contents, and chip color of
tubers of the transgenic lines A06, D51, and D60, the wild-type cultivar Ep3, and the chip cultivar Atlantic were measured after storage at 4 ◦ C for 0, 15, 30, and 45 d. The AI activity and RS content increased in A06, D51, D60, Ep3, and Atlantic during cold storage. As expected, Atlantic, a widely used variety suitable for chip processing, had the lowest AI activity and RS accumulation among the materials tested. In comparison with the wild-type Ep3, however, A06, D51, and D60 exhibited lower AI activity (Fig. 2A), lower RS content (Fig. 2B), and higher sucrose content (Fig. 2C) during cold storage. The results demonstrate that the slowdown in RS accumulation might have resulted from the inhibition of AI activity by overexpression of StInh and NtInvInh2 in the transgenic tubers. Consequently, Ep3 had
Table 2 Chip color index of transgenic and non-transgenic potato tubers after 30 d of storage at 4 or 20 ◦ C. Linea
Chip color index 4 ◦C
Ep3 A06 A12 A20 A30 A31 D02 D03 D05 D26 D35 D51 D53 D60
10.0 6.38 6.75 7.33 7.25 7.25 8.67 8.33 8.67 9.00 8.33 7.33 8.33 8.00
20 ◦ C ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.82** 0.66** 0.58** 0.50** 0.50** 0.58* 0.58** 0.58* 0.00 0.58* 0.17*** 0.58* 0.00**
4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Data are means ± SE. a Ep3, non-transgenic control; A lines, lines overexpressing NtInvInh2; D lines, lines overexpressing St-Inh. Each line provided three tuber samples from different plants, and each tuber provided 7–10 potato chips. LSD0.05 = 1.25; and LSD0.01 = 1.70. * Significance of LSD0.05 . ** Significance of LSD0.01 .
Fig. 2. Acid invertase (AI) activities and sugar contents of transgenic and nontransgenic potato tubers during cold storage. (A) AI activities, (B) reducing sugar (RS) contents, and (C) sucrose contents were analyzed for tubers of Ep3 (non-transgenic control), D51and D60 (overexpression of St-Inh), A06 (overexpression of NtInvInh2), and Atlantic (chip variety control) stored at 4 ◦ C and 85% relative humidity. Data are means ± SE of three tuber samples from different plants.
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Table 3 Acid invertase (AI) activities of transgenic and wild-type tubers stored at 4 ◦ C for 30 d. Linea
2005
2006
AI activity (mmol s−1 kg−1 ) Ep3 A06 A12 A20 A30 A31 D02 D03 D05 D26 D35 D51 D53 D60
795.0 ± 286.2 ± 301.8 ± 406.2 ± 363.6 ± 406.2 ± 526.2 ± 502.2 ± 528.6 ± – – – – –
93.6 31.2** 66.6** 24.0** 30.0** 90.0** 112.2* 69.0* 106.2*
Reduction (%)
64.0 62.0 48.9 54.3 48.9 33.8 36.8 33.5
2007
AI activity (mmol s−1 kg−1 ) 1221.0 148.2 406.2 489.6 222.6 229.8 870.6 846.6 952.2 742.2 898.8 646.2 871.2 857.4
± ± ± ± ± ± ± ± ± ± ± ± ± ±
217.2 19.8** 84.6** 64.8** 118.2** 72.6** 41.4* 130.2* 105.0 91.8** 67.2* 229.2** 187.2* 148.8*
Reduction (%)
87.9 66.7 59.9 81.8 81.2 28.7 30.7 22.0 39.2 26.4 46.4 28.7 29.8
Average reduction (%)
AI activity (mmol s−1 kg−1 ) 781.8 268.2 290.4 349.2 324.6 388.8 676.8 564.0 595.8 652.8 585.0 582.0 553.2 529.8
± ± ± ± ± ± ± ± ± ± ± ± ± ±
91.8 51.6** 86.4** 79.8** 47.4** 94.2** 89.4 142.2* 172.2* 84.6 119.4* 85.2* 57.6* 102.6*
Reduction (%)
65.7 62.9 55.3 58.5 50.3 13.4 27.9 23.8 17.3 25.2 25.6 29.2 32.2
72.5 63.9 54.7 64.9 60.1 25.3 31.8 26.4 28.3 25.6 36.0 29.0 31.0
Data are means ± SE of three tuber samples from different plants. The LSD values for each year are as follows: 2005, LSD0.05 = 254.4, LSD0.01 = 350.4; 2006, LSD0.05 = 271.2, LSD0.01 = 372.6; and 2007, LSD0.05 = 186.0, LSD0.01 = 252.0. a Ep3, non-transgenic control; A lines, lines overexpressing NtInvInh2; D lines, lines overexpressing St-Inh. * Significance of LSD0.05 . ** Significance of LSD0.01 .
Fig. 3. Chip color of transgenic and non-transgenic potato tubers. Chip color was examined in tubers of Ep3 (non-transgenic control), D51 and D60 (overexpression of St-Inh), A06 (overexpression of NtInvInh2), and Atlantic (chip variety control) stored separately at 20 or 4 ◦ C for 30 d. Similar results were obtained for three tuber samples from different plants. The results were represented by one tuber sample.
a dark brownish chip color after storage at 4 ◦ C for 30 d, whereas the transgenic lines A06, D51, and D60 had much lighter chip colors for the same storage period (Fig. 3). These results indicate that the expression of St-Inh and NtInvInh2 reduced CIS by reducing AI activity in potato tubers. 4. Discussion Protease inhibitors are a large, ubiquitous family of small proteins with diverse biological functions in plants. The first plant protease inhibitor was characterized in soybean (Glycine max L.) (Kunitz, 1945) and became a representative of the KPI family. The potato KPIs can be separated into the subfamilies A, B, and C based on their sequence similarity (Heibges et al., 2003a; Turrà et al., 2009). These inhibitors frequently accumulate in the storage organs of potato plants, where they take on a variety of presumed functions. These inhibitors could be involved in the response to biotic and abiotic stresses in addition to participating in signal transduction for growth and development (Hermosa et al., 2006; Odeny et al., 2010; Turrà et al., 2009). However, few studies have actually proven the functions of the plant protease inhibitors in vivo. Plant protease inhibitors perform their roles by interacting with their protease counterparts to form protein complexes that modulate enzyme activity and consequently the function of the
protease. Biological activities have been demonstrated for some potato KPIs in vitro (Heibges et al., 2003b). Specific KPI isoforms of subgroup C from potato tubers were found to inhibit soluble AI activity in vitro (Glaczinski et al., 2002). In a previous study by some of the present coauthors, a full-length cDNA, St-Inh, was cloned based on the amino acid sequences of subgroup C. The sequence analysis revealed that St-Inh had a classical signal sequence “NPIVLP” for vacuole localization, and its recombinant protein inhibited potato soluble AI activity (Cheng et al., 2004b). Soluble AI activity has been recognized as a critical factor controlling RS accumulation in cold-stored potato tubers (Bhaskar et al., 2010; Liu et al., 2011). To determine whether St-Inh participates in CIS in tubers, the St-Inh transcriptional profiles were analyzed in the cold-stored tubers with distinct CIS sensitivity. Five potato genotypes, namely three CIS-resistant and two CIS-sensitive ones, were employed. The results show that the transcriptional expression of St-Inh was suppressed (Fig. 1B), whereas RS accumulation increased in the cold-stored tubers, suggesting that St-Inh is potentially involved in controlling potato CIS. To further illuminate the role of St-Inh in CIS in tubers, the AI activity, RS accumulation, and chip color of cold-stored tubers of the transgenic plants were investigated. In comparison with the wild-type tubers, the St-Inh transgenic tubers had lower AI activity (Table 3), less RS accumulation (Table 1), and smaller chip color index (Table 2), strongly
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demonstrating that the overexpression of St-Inh slows down RS accumulation in cold-stored tubers and can improve chip quality by suppressing AI activity. Expression of NtInvInh2 was previously reported to reduce hexose accumulation by 75% (Greiner et al., 1999). Similar results were obtained in the present study, where overexpression of NtInvInh2 in the transgenic A06 tubers resulted in a 67.2% reduction in RS accumulation (Table 1) and a 72.5% reduction in AI activity (Table 3), confirming that NtInvInh2 can effectively suppresses AI activity to slow down RS accumulation. Because NtInvInh2 and St-Inh are members of the PMEI and KPI families, respectively, the present results indicate that both types of invertase inhibitors can reduce potato CIS. Plants have also evolved additional strategies to regulate protease activity, such as linking multiple factors to target one enzyme in various ways, or containing multiple inhibitory domains to target multiple enzymes simultaneously (Bateman and James, 2011). All of the plant protease inhibitors display an overall stability that helps minimize the entropy loss when the inhibitor binds to the enzyme. This structural stability is due to features such as disulfide bonds (Li et al., 2011), hydrogen bonds, compact size, and even the cyclization of the N- and C-termini (Bateman and James, 2011). In addition, KPIs and Bowman–Birk inhibitors and their involvement with proteolytic enzymes have also been investigated in terms of the control of pathophysiological processes. However, the effect of KPIs is weaker than that of Bowman–Birk inhibitors, because the former are unstable under environmental factors such as thermal treatment (Oliva et al., 2011). In the present study, St-Inh exhibited a less significant influence on CIS than NtInnInh2 did (Figs. 2 and 3). Their different capacities could have resulted from the structural difference between protein families. Recently, the crystal structure of Arabidopsis cell-wall invertase 1 (AtcwINV1) in complex with a tobacco cell-wall invertase inhibitor (NtInvInh1) became available. The structure identifies a small amino acid motif in NtInvInh1 that competes with sucrose and directly targets the invertase active site. The activity of AtcwINV1 and its interaction with NtInvInh1 are strictly pH-dependent and peak at about 4.5. At this pH, NtInvInh1 tightly binds its target with nanomolar affinity (Hothorn et al., 2010). Based on conserved residues among known invertase inhibitor sequences, it can be expected that the related vacuolar invertase inhibitors use essentially the same targeting mechanism to inhibit vacuolar isoenzymes (Hothorn et al., 2010). A previous study demonstrated that the vacuolar acid invertase gene StvacINV1 was the key AI gene in regulating CIS in potato tubers (Liu et al., 2011). Consistent with the study by Greiner et al. (1999), overexpression of NtInvInh2 also strongly prevented CIS in potato tubers in the present study (Tables 1 and 2), suggesting that StvacINV1 was strongly inhibited by NtInvInh2. Many KPIs acting on different classes of proteolytic enzymes present distinct structural and functional characteristics. Some protease inhibitors are bifunctional inhibitors of not only proteases but also ␣-amylases, the enzymes responsible for cleavage of ␣1,4-glycoside bonds from glycogen or starch. For example, the Ragi bifunctional inhibitor and corn Hageman factor inhibitor inhibit both ␣-amylases and serine proteases (Gomes et al., 2011). Soybean Kunitz trypsin inhibitor was first isolated from soybean meal in 1945 and was subsequently described as an ␣-chymotrypsin inhibitor, but this inhibition was weaker in comparison with the anti-trypsin effect (Gomes et al., 2011). A family of 28 KPI genes was identified in the potato genome (Potato Genome Sequencing Consortium, 2011). Kunitz-type protease inhibitor genes are frequently induced after pest and pathogen attacks and act primarily as inhibitors of exogenous proteases. Therefore, the KPI family may provide resistance to biotic stress for the vulnerable, newly evolved underground organ (Potato Genome Sequencing Consortium, 2011). However, some protease inhibitors may have
a role not only in plant defense against biotic stresses but also in the adaptation of storage organs to an abiotic stress, such as changing temperatures. More generally, this role is consistent with the notion that, to prevent unnecessary metabolic costs, inhibitors of endogenous enzymes are regulated in such a way as to ensure inhibition only when it is physiologically required (Bellincampi et al., 2004). In the present study, the expression patterns of St-Inh observed were very similar between the CIS-sensitive and CISresistant tubers in storage (Fig. 1B). The same expression pattern may indeed be caused by a more general cold-defense system present in both resistant and sensitive plants. The St-Inh gene may have a prior role in the adaptation of storage organs to changing temperatures. The present research suggests that both PMEIs and KPIs contribute to CIS in potato tubers by modulating invertase activity and that PMEIs may have a stronger capacity to counteract AI activity than KPIs. These results provide novel clues for further investigation of the mechanism by which potato CIS is regulated. It has therefore been proposed that the PMEI proteins from potato may be important factors regulating CIS in tubers. Four PMEI genes from potato have been identified (Liu et al., 2010). Analysis based on the activity of recombinant protein and protein–protein interaction strongly suggests that StInvInh2 and StvacINV1 are counterparts, and the levels of StInvInh2 transcripts are higher in CIS-resistant genotypes than in CIS-sensitive ones, indicating that StInvInh2 is an important candidate factor regulating CIS in potato tubers (Liu et al., 2010). In addition, the RNA alternative splicing of StInvInh2 may also contribute to CIS resistance by increasing the additional functional capacity (Brummell et al., 2011). The contribution of StInvInh2 to CIS resistance merits further investigation to gain a deeper understanding of the mechanism of CIS in potato tubers.
Acknowledgments This study was supported by the National Natural Science Foundation of China (31201258 and 30800754), the National High Technology Research and Development (863) Program of China (2009AA10Z103), the China Postdoctoral Science Foundation (20110491173), and A-base funding from Agriculture and AgriFood Canada.
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X. Liu et al. / Postharvest Biology and Technology 86 (2013) 265–271 Greiner, S., Krausgrill, S., Rausch, T., 1998. Cloning of a tobacco apoplasmic invertase inhibitor: proof of function of the recombinant protein and expression analysis during plant development. Plant Physiology 116, 733–742. Greiner, S., Rausch, T., Sonnewald, U., Herbers, K., 1999. Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers. Nature Biotechnology 17, 708–711. Heibges, A., Glaczinski, H., Ballvora, A., Salamini, F., Gebhardt, C., 2003a. Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.). Molecular Genetics and Genomics 269, 526–534. Heibges, A., Salamini, F., Gebhardt, C., 2003b. Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers (Solanum tuberosum L.). Molecular Genetics and Genomics 269, 535–541. Hermosa, M.R., Turrà, D., Fogliano, V., Monte, E., Lorito, M., 2006. Identification and characterization of potato protease inhibitors able to inhibit pathogenicity and growth of Botrytis cinerea. Physiological and Molecular Plant Pathology 68, 138–148. Hothorn, M., van den Ende, W., Lammens, W., Rybin, V., Scheffzek, K., 2010. Structural insight into the pH-controlled targeting of plant cell-wall invertase by a specific inhibitor protein. Proceedings of the National Academy of Sciences of the United States of America 107, 17427–17432. Hothorn, M., Wolf, S., Aloy, P., Greiner, S., Scheffzek, K., 2004. Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins. Plant Cell 16, 3437–3447. Kunitz, M., 1945. Crystallization of a trypsin inhibitor from soybean. Science 101, 668–669. Li, X.-Q., Zhang, T., Donnelly, D., 2011. Selective loss of cysteine residues and disulphide bonds in a potato proteinase inhibitor II family. PLoS One 6 (4), e18615. Liu, X., Song, B., Zhang, H., Li, X.-Q., Xie, C., Liu, J., 2010. Cloning and molecular characterization of putative invertase inhibitor genes and their possible contributions to cold-induced sweetening of potato tubers. Molecular Genetics and Genomics 284, 147–159. Liu, X., Zhang, C., Ou, Y., Lin, Y., Song, B., Xie, C., Liu, J., Li, X.-Q., 2011. Systematic analysis of potato acid invertase genes reveals that a cold-responsive member, StvacINV1, regulates cold-induced sweetening of tubers. Molecular Genetics and Genomics 286, 109–118.
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McKenzie, M.J., Sowokinos, J.R., Shea, I.M., Gupta, S.K., Lindlauf, R.R., Anderson, J.A.D., 2005. Investigations on the role of acid invertase and UDP-glucose pyrophosphorylase in potato clones with varying resistance to cold-induced sweetening. American Journal of Potato Research 82, 231–239. Menéndez, C.M., Ritter, E., Schäfer-Pregl, R., Walkemeier, B., Kalde, A., Salamini, F., Gebhardt, C., 2002. Cold sweetening in diploid potato: Mapping quantitative trait loci and candidate genes. Genetics 162, 1423–1434. Nicot, N., Hausman, J.-F., Hoffmann, L., Evers, D., 2005. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany 56, 2907–2914. Odeny, D.A., Stich, B., Gebhardt, C., 2010. Physical organization of mixed protease inhibitor gene clusters, coordinated expression and association with resistance to late blight at the StKI locus on potato chromosome III. Plant, Cell & Environment 33, 2149–2161. Oliva, M.L., da Silva Ferreira, R., Ferreira, J.G., de Paula, C.A.A., Salas, C.E., Sampaio, M.U., 2011. Structural and functional properties of Kunitz proteinase inhibitors from Leguminosae: a mini review. Current Protein and Peptide Science 12, 348–357. Potato Genome Sequencing Consortium, 2011. Genome sequence and analysis of the tuber crop potato. Nature 475, 189–195. Pressey, R., 1966. Separation and properties of potato invertase and invertase inhibitor. Archives of Biochemistry and Biophysics 113, 667–674. Pressey, R., 1967. Invertase inhibitor from potatoes: purification, characterization, and reactivity with plant invertases. Plant Physiology 42, 1780–1786. Schwimmer, S., Makower, R.U., Rorem, E.S., 1961. Invertase and invertase inhibitor in potato. Plant Physiology 36, 313–316. Si, H., Xie, C., Liu, J., 2003. An efficient protocol for Agrobacterium-mediated transformation with microtuber and the induction of an antisense class I patatin gene into potato. Acta Agronomica Sinica 29, 801–805. Turrà, D., Bellin, D., Lorito, M., Gebhardt, C., 2009. Genotype-dependent expression of specific members of potato protease inhibitor gene families in different tissues and in response to wounding and nematode infection. Journal of Plant Physiology 166, 762–774. Xu, C., Coleman, W.K., Meng, F.-R., Bonierbale, M., Li, X.-Q., 2009. Relationship between glucose accumulation and activities of acid invertase and its inhibitors in potatoes under simulated commercial conditions. Potato Journal 36, 35–44. Yang, J., Song, B., Li, Y., Liu, J., 2006. A simple and efficient method for RNA extraction from potato tuber. Journal of Agricultural Biotechnology 14 (2), 297–298.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
The potato protease inhibitor gene, St-Inh, plays roles in the cold-induced sweetening of potato tubers by modulating invertase activity Xun Liu a,b,c , Shanhan Cheng a,d , Jun Liu a , Yongbin Ou a , Botao Song a , Chi Zhang a,e , Yuan Lin a , Xiu-Qing Li b , Conghua Xie a,∗ a Key Laboratory of Horticultural Plant Biology, Ministry of Education, National Centre for Vegetable Improvement (Central China), Huazhong Agricultural University, Wuhan 430070, China b Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, New Brunswick E3B 4Z7, Canada c College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China d College of Horticulture and Forestry, Hainan University, Haikou 570228, China e Institute of Root and Tuber Crops, Zhejiang Agriculture and Forestry University, Hangzhou, Linan 311300, China
a r t i c l e
i n f o
Article history: Received 22 March 2012 Accepted 1 July 2013 Keywords: Potato Protease inhibitor Cold-induced sweetening
a b s t r a c t 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. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Potato (Solanum tuberosum L.) is the fourth most important food crop in the world. Globally, consumers are eating potatoes more and more as value-added, processed products rather than as fresh produce. Two such processed products are French fries and potato chips, which are available in fast-food chains and snack bars worldwide. For an uninterrupted supply to the industry, potato tubers need to be stored at low temperatures throughout the year. However, low-temperature storage leads to the accumulation of
Abbreviations: AI, acid invertase; CIS, cold-induced sweetening; KPI, Kunitz-type protease inhibitor; PMEI, pectin methylesterase/invertase inhibitor; RS, reducing sugar. ∗ Corresponding author. Tel.: +86 27 87280969; fax: +86 27 87286939. E-mail addresses: [email protected], [email protected] (C. Xie). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.07.001
reducing sugars (RS), which is referred to as cold-induced sweetening (CIS). Reducing sugars negatively influence processing quality because they react with free amino acids during frying, resulting in dark-colored food products. Therefore, preventing RS accumulation during tuber storage is of economic importance to the potato processing industry. During tuber storage at low temperatures, RS accumulation varies in response to environmental and genetic factors. The activity of acid invertase (AI), which hydrolyzes sucrose to glucose and fructose, is positively associated with RS accumulation (Cheng et al., 2004b; McKenzie et al., 2005; Xu et al., 2009). Suppressing a cold-responsive AI gene, StvacINV1, was found to prevent RS accumulation and improve the quality of fried products (Bhaskar et al., 2010; Liu et al., 2011), whereas increasing the abundance of StvacINV1 mRNA in cold-stored tubers was reported to enhance AI activity (Liu et al., 2011). In addition, AI activity is likely also controlled post-translationally by invertase inhibitors (Pressey, 1966, 1967; Schwimmer et al., 1961). Although a great deal of attention
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had been paid to invertase inhibitor in potato tubers, it took until the late 1990s before purifying the inhibitor to apparent homogeneity yielded the information on protein or peptide sequences that was required to clone the corresponding cDNA; at that time, Greiner et al. (1998, 1999) was able to characterize two invertase inhibitor cDNAs from Nicotiana tabacum. Overexpression of one of the two tobacco invertase inhibitors, NtInvInh2, strongly reduced RS accumulation in potato tubers (Greiner et al., 1999). These inhibitors belong to the pectin methylesterase/invertase inhibitor (PMEI) family, which is referred as PMEI-RP (Hothorn et al., 2004). Subsequently, Glaczinski et al. (2002) purified, from potato tubers, soluble protease inhibitors that were completely inhibitory to AI activity in vitro. N-terminal amino acid sequencing of the purified proteins indicated that these proteins are members of a small tuber protein family known as Kunitz-type protease inhibitors (KPIs). Based on the N-terminal amino acid sequence of the proteins, a full-length cDNA of the KPI St-Inh was cloned from potato, and its recombinant protein from E. coli inhibited AI activity, indicating that St-Inh has a role as an invertase inhibitor (Cheng et al., 2004a). The St-Inh gene was mapped to its position as marker CP6 on potato chromosome III (Heibges et al., 2003a), which is linked with a quantitative trait locus for sugar content (Menéndez et al., 2002), suggesting that St-Inh could be a candidate gene for regulating CIS by its post-translational mechanism. However, the roles that St-Inh plays in CIS in tubers have not been clearly described because of the lack of evidence for gene function in vivo. The present study investigated the expression patterns of StInh in various organs of growing potato plants and in cold-stored potato tubers. Together with NtInvInh2, the contribution of St-Inh to CIS resistance was also assessed by overexpression in potato tubers. The results suggest that both NtInvInh2 and St-Inh play roles in slowing down CIS in tubers by modulating AI activity. NtInhInh2 appeared to have a stronger capacity for resistance to CIS than St-Inh did. 2. Materials and methods 2.1. Plant materials At the Agriculture and Agri-Food Canada (AAFC) Potato Research Centre in Fredericton, New Brunswick, Canada, plants of the potato cultivar Russet Burbank were grown in 24-cm pots in the greenhouse at 20–25 ◦ C with a 16-h photoperiod supplemented with artificial light (approximately 90 mol m−2 s−1 ). Flowers and floral buds, vegetative buds, sink leaves, source leaves, senescent leaves, stems, roots, stolons, immature tubers, and mature tubers were sampled from three plants 60 d after emergence. The samples were immediately frozen in liquid N2 and stored in a freezer at −80 ◦ C for RNA isolation as described by Liu et al. (2011). The CIS-sensitive cultivars AC Novachip and Yukon Gold and the CISresistant cultivars ND860-2, Snowden, and Atlantic were used to test the expression of the St-Inh gene in relation to CIS in the tubers. The tubers were harvested from the Potato Research Station’s Benton Ridge Potato Breeding Substation and were immediately stored at 21 ◦ C for two weeks (T0) to permit skin set. Subsequently, the tubers were subjected to one of the following temperature treatments: - 7C60d: Tubers stored at 7 ◦ C for 60 d. - R21C1d: Tubers stored at 7 ◦ C for 60 d and then transferred to 21 ◦ C for reconditioning for 1 d. - R21C21d: Tubers stored at 7 ◦ C for 60 d and then transferred to 21 ◦ C for reconditioning for 21 d. - 21C60d: Tubers maintained at 21 ◦ C for 60 d as the control.
The tubers were sampled at T0, 21C60d, 7C60d, R21C1d and R21C21d. The tubers were peeled and cut into small pieces, immediately frozen in liquid N2 , and stored in the freezer at −80 ◦ C until use. The frozen tissues were ground in liquid N2 . The total RNA was extracted using the method described previously by Yang et al. (2006), and the RNA was further purified with the RNeasy Plant Mini Kit (Qiagen, USA) according to the manufacturer’s instructions. The quality of the total RNA was checked using the OD260 /OD280 nm ratio and by 1% agarose gel electrophoresis. The total RNA (10 g) was treated with the TURBO DNA-Free Kit (Ambion, USA) to eliminate residual genomic DNA in accordance with the manufacturer’s protocol. For each RNA sample, quantitative PCR was carried out using the primers of the ef1˛ gene (AB061263) to verify that no genomic DNA contamination was present (Nicot et al., 2005). For the reverse transcription, DNAfree RNA (2 g) was converted to first-strand cDNA using TaqMan Reverse Transcription Reagents (Applied Biosystems, USA) with random hexamers, and then the cDNA was purified using the QIAquick PCR Purification Kit (Qiagen, USA) according to the manufacturer’s instructions. 2.2. qRT-PCR Quantitative reverse transcription PCR (qRT-PCR) was performed on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, USA). The gene-specific primer pairs (forward: 5 -GGAAACCTTCAATGCCCAAAC-3 ; reverse: 5 CCGACTCCGACTTACGAATGA-3 ) were designed with Primer Express 2.0 software (Applied Biosystems, USA). The PCR reactions were carried out in a 20-L system containing 1× Power SYBR Green PCR Master Mix (Applied Biosystems, USA), 1 L of cDNA corresponding to 20 ng of original RNA, and 0.6 mol L−1 each of the specific forward and reverse primers. The following amplification program was used: 50 ◦ C for 2 min, 95 ◦ C for 10 min, 40 cycles of 95 ◦ C for 15 s, and 60 ◦ C for 1 min. The specificity of the individual PCR amplifications was confirmed by dissociation curve analysis from 60 to 95 ◦ C following the final cycle of the qRT-PCR. Aliquots of the PCR reactions were electrophoresed on agarose gels to verify the specificity of the primers, and the amplified bands were cloned and sequenced to confirm that they were fragments of the target genes. Relative quantification of individual gene expression was performed using a comparative Ct method as described in ABI PRISM 7700 SDS Use Bulletin #2 (Applied Biosystems, USA). The potato gene ef1˛ (AB061263) was used as the control, because it is an appropriate reference for cold stress in potatoes (Nicot et al., 2005). The approximately equal efficiency in PCR amplification of the target and reference genes was verified by performing standard curves for each amplicon. 2.3. Gene constructs and plant transformation The complete coding sequence of St-Inh obtained by restriction with BamHI and SacI was subcloned in sense orientation into the binary vector pBI121 containing the CaMV35S promoter. The construct was introduced into Agrobacterium tumefaciens strain LBA4404 and transformed into potato CIS-sensitive cultivar E-potato 3 (Ep3) by Agrobacterium-mediated transformation as previously described (Si et al., 2003). The regenerated plants were designated as D lines. In addition, plants with overexpression of NtInvInh2 controlled by the CaMV35S promoter in Ep3 (designated as A lines) had been previously obtained in the laboratory of some of the present coauthors (Cheng, 2004). Tubers were harvested from the transgenic plants (D and A lines) and from wild-type plants grown at 20–25 ◦ C in the greenhouse at the National Centre for Vegetable Improvement (Central China), Huazhong Agricultural
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University, in Wuhan, China. After 10 d at 20 ◦ C for skin set, the tubers were divided into two sets, with one stored at 4 ◦ C for cold induction and the other maintained at 20 ◦ C for comparison. The tubers were sampled after 0, 15, 30, and 45 d of storage, peeled and cut into small pieces, immediately frozen in liquid N2 , and stored in a freezer at −80 ◦ C for further analysis. 2.4. Determination of soluble acid invertase activity, sugar content, and chip color The soluble sugars were extracted from the potato tubers, and the AI activity was assayed according to the method of Greiner et al. (1999). Sucrose and RS contents were measured as previously described (Liu et al., 2010). Tuber slices were fried for 3 min at 191 ◦ C or until bubbling ceased, and the chip color was visually determined using the Color Standards Reference Chart for Potato Chips (Snack Food Association, USA). Each sample consisted of 7–10 crushed potato chips. Two or three tuber samples from different plants were used for each experiment. Regression analysis of the data was conducted using the Microsoft Excel program (Microsoft Office XP, 2003). Data are means ± SE. The significance between the treatments was tested by ANOVA and LSD using SPSS13.0 software for Windows (SPSS Inc., USA). 3. Results 3.1. Expression patterns of St-Inh in various organs and stored tubers The full-length cDNA of St-Inh (NCBI Accession Number AY594178) had been cloned previously in the laboratory of some of the present coauthors (Cheng et al., 2004a). To understand the structure of the St-Inh gene, we amplified it from potato genomic DNA. The result showed identical sequences between the coding sequence of St-Inh and genomic DNA (data not shown), demonstrating that the St-Inh gene consists of a single exon. The expression of St-Inh was analyzed in various organs by qRTPCR (Fig. 1A). The St-Inh gene was found to be expressed mainly in tubers in terms of mRNA abundance, which increased progressively with tuber growth. This expression profile implies that St-Inh is involved mainly in tuber development. To assess the response of StInh to low temperatures in tubers, the relative expression of St-Inh during cold storage was analyzed (Fig. 1B). The results showed that the level of St-Inh transcripts decreased markedly at 7 ◦ C compared with the level in the samples stored at 21 ◦ C. This temperaturedependent regulation was further confirmed by a rapid and obvious increase in transcript abundance in all cultivars tested when the tubers were reconditioned at 21 ◦ C for 1 d. The gene’s mRNA abundance decreased again when the tubers were reconditioned at 21 ◦ C for 21 d. These results reveal that the expression pattern of St-Inh is opposite to RS accumulation in cold-stored tubers, suggesting that the gene may be involved in controlling the RS content in potato tubers. 3.2. Effects of St-Inh and NtInvInh2 on cold-induced sweetening in transgenic potato tubers To determine whether St-Inh has a role in regulating CIS in potato tubers, a CaMV35S::St-Inh construct was made and used to transform the potato cultivar Ep3 by Agrobacterium-mediated transformation. A total of 45 transgenic plants (D lines) were obtained. Eight D lines that showed higher transcript abundances of St-Inh than wild-type tubers during storage at 4 ◦ C for 30 d were chosen for further analysis (Fig. S1). In addition, five NtInvInh2 transgenic lines (A lines) that had been obtained previously
Fig. 1. Expression pattern of St-Inh in potato organs and stored tubers, as determined by qRT-PCR analysis. The relative expression levels of St-Inh were standardized to the expression level (1 0 0) of potato reference gene ef1˛ (AB061263) by qRT-PCR. (A) The various potato organs were sampled from three plants of the potato cultivar Russet Burbank grown in 24–cm pots in a greenhouse at 20–25 ◦ C with a 16-h photoperiod supplemented with artificial light 60 d after emergence. FFB, flowers and floral buds; VB, vegetative buds; SiL, sink leaves; SoL, source leaves; SeL, senescent leaves; Sm, stems; Rt, roots; Sl, stolons; IT, immature tubers; MT, mature tubers. (B) Tubers harvested from the CIS-resistant potato cultivars ND8602, Atlantic, and Snowden and the CIS-sensitive cultivars AC Norchip and Yukon Gold were immediately stored at 21 ◦ C for two weeks (T0) to permit skin set. Subsequently, the tubers were divided into two groups for the temperature treatment. The first group of tubers was stored at 7 ◦ C for 60 d (7C60d), and then some of those tubers were transferred to storage at 21 ◦ C for reconditioning for 1 d (R21C1d) or 21 d (R21C21d). The second group was maintained at 21 ◦ C for 60 d (21C60d) as the control. The tubers were sampled at T0, 21C60d, 7C60d, R21C1d, and R21C21d. Similar results were obtained for two tuber samples from different plants. Each sample was represented by one tuber.
(Cheng, 2004) were also chosen for a simultaneous analysis as the positive control. No visible phenotypic effects were observed for either of the St-Inh and NtInvInh2 transgenic lines under normal growth conditions. The tubers of the transgenic lines (A and D) and the wild-type plants were stored at 4 or 20 ◦ C for 30 d. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.postharvbio. 2013.07.001. The experiments were repeated two or three times in different years. In comparison with the wild-type Ep3 plants, the A and D lines had significantly reduced RS accumulation (Table 1), although there was some variation in amounts observed between the repeats. The RS accumulation in the A and D lines was reduced by 41.3–67.2% and by 27.8–35.1%, respectively. These results suggest that the expression of St-Inh and NtInvInh2 play roles in reducing CIS in potato tubers. Tuber slices were fried further, and the chip color was visually determined using the Color Standards Reference Chart for Potato Chips (Snack Food Association, USA). In comparison with the wild-type Ep3 plants, the chip color index decreased one to four levels in the transgenic tubers (Table 2), suggesting that potato processing quality was improved by overexpression of St-Inh and NtInvInh2 in the transgenic tubers.
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Table 1 Reducing sugar (RS) content of transgenic and wild-type tubers stored at 4 ◦ C for 30 d. Linea
2005
2006
RS content (g kg−1 ) Ep3 A06 A12 A20 A30 A31 D02 D03 D05 D26 D35 D51 D53 D60
15.18 ± 5.32 ± 5.40 ± 10.25 ± 9.64 ± 8.12 ± 11.24 ± 11.44 ± 11.31 ± – – – – –
Reduction (%)
0.94 0.27** 0.17** 1.01** 0.55** 0.20** 1.38* 1.54* 1.07*
65.0 64.4 32.5 36.5 46.5 26.0 24.6 25.5 – – – – –
2007
Average reduction (%)
RS content (g kg−1 )
Reduction (%)
RS content (g kg−1 )
9.94 ± 2.62 ± 4.36 ± 5.20 ± 5.21 ± 5.53 ± – 6.96 ± 7.16 ± 7.35 ± 6.58 ± 7.04 ± 7.22 ± 6.50 ±
73.6 56.1 47.7 47.6 44.4 – 30.0 28.0 26.1 33.8 29.2 27.4 34.6
10.60 4.03 5.66 5.96 5.69 6.29 7.47 7.44 7.41 6.76 7.13 6.25 7.17 7.92
0.93 1.38** 0.73** 0.41** 0.72** 1.03** 0.39* 0.42* 1.19* 0.17* 0.63* 0.33* 1.14*
± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.37 0.32** 0.51** 0.44** 0.12** 0.23** 0.87* 1.41* 0.44* 0.44* 1.08* 0.19** 0.22* 0.57*
Reduction (%) 62.9 46.6 43.8 46.3 40.7 29.5 29.8 30.1 36.2 32.7 41.0 32.4 25.3
67.2 55.7 41.3 43.5 43.9 27.8 28.1 27.9 31.1 33.2 35.1 29.9 30.0
Data are means ± SE of three tuber samples from different plants. The LSD values for each year are as follows: 2005, LSD0.05 = 3.18, LSD0.01 = 4.38; 2006, LSD0.05 = 2.58, LSD0.01 = 3.46; and 2007, LSD0.05 = 2.67, LSD0.01 = 3.61. a Ep3, non-transgenic control; A lines, lines overexpressing NtInvInh2; D lines, lines overexpressing St-Inh. * Significance of LSD0.05 . ** Significance of LSD0.01 .
3.3. Effects of St-Inh and NtInvInh2 on acid invertase activities in transgenic potato tubers To examine the effects of St-Inh and NtInvInh2 on invertase inhibitory activity, we determined the AI activities in transgenic and wild-type tubers in three years. Compared with the wild-type Ep3 tubers, the A and D lines showed significantly reduced AI activities (Table 3). The AI activities in the D and A lines were reduced by 54.7–72.5% and by 25.3–36.0%, respectively. Plotting the reduction in AI activities (x) in the transgenic tubers stored at 4 ◦ C against the corresponding reduction in RS accumulation (y) revealed a significant linear relationship (y = 0.616x + 12.03, R2 = 0.831, P < 0.01) (Fig. S2), reinforcing that RS accumulation in potato tubers is largely regulated by AI activity in the tubers when they are exposed to low temperatures. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.postharvbio. 2013.07.001. To further investigate the capacity of St-Inh and NtInvInh2 to regulate CIS, the AI activity, RS and sucrose contents, and chip color of
tubers of the transgenic lines A06, D51, and D60, the wild-type cultivar Ep3, and the chip cultivar Atlantic were measured after storage at 4 ◦ C for 0, 15, 30, and 45 d. The AI activity and RS content increased in A06, D51, D60, Ep3, and Atlantic during cold storage. As expected, Atlantic, a widely used variety suitable for chip processing, had the lowest AI activity and RS accumulation among the materials tested. In comparison with the wild-type Ep3, however, A06, D51, and D60 exhibited lower AI activity (Fig. 2A), lower RS content (Fig. 2B), and higher sucrose content (Fig. 2C) during cold storage. The results demonstrate that the slowdown in RS accumulation might have resulted from the inhibition of AI activity by overexpression of StInh and NtInvInh2 in the transgenic tubers. Consequently, Ep3 had
Table 2 Chip color index of transgenic and non-transgenic potato tubers after 30 d of storage at 4 or 20 ◦ C. Linea
Chip color index 4 ◦C
Ep3 A06 A12 A20 A30 A31 D02 D03 D05 D26 D35 D51 D53 D60
10.0 6.38 6.75 7.33 7.25 7.25 8.67 8.33 8.67 9.00 8.33 7.33 8.33 8.00
20 ◦ C ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.82** 0.66** 0.58** 0.50** 0.50** 0.58* 0.58** 0.58* 0.00 0.58* 0.17*** 0.58* 0.00**
4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Data are means ± SE. a Ep3, non-transgenic control; A lines, lines overexpressing NtInvInh2; D lines, lines overexpressing St-Inh. Each line provided three tuber samples from different plants, and each tuber provided 7–10 potato chips. LSD0.05 = 1.25; and LSD0.01 = 1.70. * Significance of LSD0.05 . ** Significance of LSD0.01 .
Fig. 2. Acid invertase (AI) activities and sugar contents of transgenic and nontransgenic potato tubers during cold storage. (A) AI activities, (B) reducing sugar (RS) contents, and (C) sucrose contents were analyzed for tubers of Ep3 (non-transgenic control), D51and D60 (overexpression of St-Inh), A06 (overexpression of NtInvInh2), and Atlantic (chip variety control) stored at 4 ◦ C and 85% relative humidity. Data are means ± SE of three tuber samples from different plants.
X. Liu et al. / Postharvest Biology and Technology 86 (2013) 265–271
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Table 3 Acid invertase (AI) activities of transgenic and wild-type tubers stored at 4 ◦ C for 30 d. Linea
2005
2006
AI activity (mmol s−1 kg−1 ) Ep3 A06 A12 A20 A30 A31 D02 D03 D05 D26 D35 D51 D53 D60
795.0 ± 286.2 ± 301.8 ± 406.2 ± 363.6 ± 406.2 ± 526.2 ± 502.2 ± 528.6 ± – – – – –
93.6 31.2** 66.6** 24.0** 30.0** 90.0** 112.2* 69.0* 106.2*
Reduction (%)
64.0 62.0 48.9 54.3 48.9 33.8 36.8 33.5
2007
AI activity (mmol s−1 kg−1 ) 1221.0 148.2 406.2 489.6 222.6 229.8 870.6 846.6 952.2 742.2 898.8 646.2 871.2 857.4
± ± ± ± ± ± ± ± ± ± ± ± ± ±
217.2 19.8** 84.6** 64.8** 118.2** 72.6** 41.4* 130.2* 105.0 91.8** 67.2* 229.2** 187.2* 148.8*
Reduction (%)
87.9 66.7 59.9 81.8 81.2 28.7 30.7 22.0 39.2 26.4 46.4 28.7 29.8
Average reduction (%)
AI activity (mmol s−1 kg−1 ) 781.8 268.2 290.4 349.2 324.6 388.8 676.8 564.0 595.8 652.8 585.0 582.0 553.2 529.8
± ± ± ± ± ± ± ± ± ± ± ± ± ±
91.8 51.6** 86.4** 79.8** 47.4** 94.2** 89.4 142.2* 172.2* 84.6 119.4* 85.2* 57.6* 102.6*
Reduction (%)
65.7 62.9 55.3 58.5 50.3 13.4 27.9 23.8 17.3 25.2 25.6 29.2 32.2
72.5 63.9 54.7 64.9 60.1 25.3 31.8 26.4 28.3 25.6 36.0 29.0 31.0
Data are means ± SE of three tuber samples from different plants. The LSD values for each year are as follows: 2005, LSD0.05 = 254.4, LSD0.01 = 350.4; 2006, LSD0.05 = 271.2, LSD0.01 = 372.6; and 2007, LSD0.05 = 186.0, LSD0.01 = 252.0. a Ep3, non-transgenic control; A lines, lines overexpressing NtInvInh2; D lines, lines overexpressing St-Inh. * Significance of LSD0.05 . ** Significance of LSD0.01 .
Fig. 3. Chip color of transgenic and non-transgenic potato tubers. Chip color was examined in tubers of Ep3 (non-transgenic control), D51 and D60 (overexpression of St-Inh), A06 (overexpression of NtInvInh2), and Atlantic (chip variety control) stored separately at 20 or 4 ◦ C for 30 d. Similar results were obtained for three tuber samples from different plants. The results were represented by one tuber sample.
a dark brownish chip color after storage at 4 ◦ C for 30 d, whereas the transgenic lines A06, D51, and D60 had much lighter chip colors for the same storage period (Fig. 3). These results indicate that the expression of St-Inh and NtInvInh2 reduced CIS by reducing AI activity in potato tubers. 4. Discussion Protease inhibitors are a large, ubiquitous family of small proteins with diverse biological functions in plants. The first plant protease inhibitor was characterized in soybean (Glycine max L.) (Kunitz, 1945) and became a representative of the KPI family. The potato KPIs can be separated into the subfamilies A, B, and C based on their sequence similarity (Heibges et al., 2003a; Turrà et al., 2009). These inhibitors frequently accumulate in the storage organs of potato plants, where they take on a variety of presumed functions. These inhibitors could be involved in the response to biotic and abiotic stresses in addition to participating in signal transduction for growth and development (Hermosa et al., 2006; Odeny et al., 2010; Turrà et al., 2009). However, few studies have actually proven the functions of the plant protease inhibitors in vivo. Plant protease inhibitors perform their roles by interacting with their protease counterparts to form protein complexes that modulate enzyme activity and consequently the function of the
protease. Biological activities have been demonstrated for some potato KPIs in vitro (Heibges et al., 2003b). Specific KPI isoforms of subgroup C from potato tubers were found to inhibit soluble AI activity in vitro (Glaczinski et al., 2002). In a previous study by some of the present coauthors, a full-length cDNA, St-Inh, was cloned based on the amino acid sequences of subgroup C. The sequence analysis revealed that St-Inh had a classical signal sequence “NPIVLP” for vacuole localization, and its recombinant protein inhibited potato soluble AI activity (Cheng et al., 2004b). Soluble AI activity has been recognized as a critical factor controlling RS accumulation in cold-stored potato tubers (Bhaskar et al., 2010; Liu et al., 2011). To determine whether St-Inh participates in CIS in tubers, the St-Inh transcriptional profiles were analyzed in the cold-stored tubers with distinct CIS sensitivity. Five potato genotypes, namely three CIS-resistant and two CIS-sensitive ones, were employed. The results show that the transcriptional expression of St-Inh was suppressed (Fig. 1B), whereas RS accumulation increased in the cold-stored tubers, suggesting that St-Inh is potentially involved in controlling potato CIS. To further illuminate the role of St-Inh in CIS in tubers, the AI activity, RS accumulation, and chip color of cold-stored tubers of the transgenic plants were investigated. In comparison with the wild-type tubers, the St-Inh transgenic tubers had lower AI activity (Table 3), less RS accumulation (Table 1), and smaller chip color index (Table 2), strongly
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demonstrating that the overexpression of St-Inh slows down RS accumulation in cold-stored tubers and can improve chip quality by suppressing AI activity. Expression of NtInvInh2 was previously reported to reduce hexose accumulation by 75% (Greiner et al., 1999). Similar results were obtained in the present study, where overexpression of NtInvInh2 in the transgenic A06 tubers resulted in a 67.2% reduction in RS accumulation (Table 1) and a 72.5% reduction in AI activity (Table 3), confirming that NtInvInh2 can effectively suppresses AI activity to slow down RS accumulation. Because NtInvInh2 and St-Inh are members of the PMEI and KPI families, respectively, the present results indicate that both types of invertase inhibitors can reduce potato CIS. Plants have also evolved additional strategies to regulate protease activity, such as linking multiple factors to target one enzyme in various ways, or containing multiple inhibitory domains to target multiple enzymes simultaneously (Bateman and James, 2011). All of the plant protease inhibitors display an overall stability that helps minimize the entropy loss when the inhibitor binds to the enzyme. This structural stability is due to features such as disulfide bonds (Li et al., 2011), hydrogen bonds, compact size, and even the cyclization of the N- and C-termini (Bateman and James, 2011). In addition, KPIs and Bowman–Birk inhibitors and their involvement with proteolytic enzymes have also been investigated in terms of the control of pathophysiological processes. However, the effect of KPIs is weaker than that of Bowman–Birk inhibitors, because the former are unstable under environmental factors such as thermal treatment (Oliva et al., 2011). In the present study, St-Inh exhibited a less significant influence on CIS than NtInnInh2 did (Figs. 2 and 3). Their different capacities could have resulted from the structural difference between protein families. Recently, the crystal structure of Arabidopsis cell-wall invertase 1 (AtcwINV1) in complex with a tobacco cell-wall invertase inhibitor (NtInvInh1) became available. The structure identifies a small amino acid motif in NtInvInh1 that competes with sucrose and directly targets the invertase active site. The activity of AtcwINV1 and its interaction with NtInvInh1 are strictly pH-dependent and peak at about 4.5. At this pH, NtInvInh1 tightly binds its target with nanomolar affinity (Hothorn et al., 2010). Based on conserved residues among known invertase inhibitor sequences, it can be expected that the related vacuolar invertase inhibitors use essentially the same targeting mechanism to inhibit vacuolar isoenzymes (Hothorn et al., 2010). A previous study demonstrated that the vacuolar acid invertase gene StvacINV1 was the key AI gene in regulating CIS in potato tubers (Liu et al., 2011). Consistent with the study by Greiner et al. (1999), overexpression of NtInvInh2 also strongly prevented CIS in potato tubers in the present study (Tables 1 and 2), suggesting that StvacINV1 was strongly inhibited by NtInvInh2. Many KPIs acting on different classes of proteolytic enzymes present distinct structural and functional characteristics. Some protease inhibitors are bifunctional inhibitors of not only proteases but also ␣-amylases, the enzymes responsible for cleavage of ␣1,4-glycoside bonds from glycogen or starch. For example, the Ragi bifunctional inhibitor and corn Hageman factor inhibitor inhibit both ␣-amylases and serine proteases (Gomes et al., 2011). Soybean Kunitz trypsin inhibitor was first isolated from soybean meal in 1945 and was subsequently described as an ␣-chymotrypsin inhibitor, but this inhibition was weaker in comparison with the anti-trypsin effect (Gomes et al., 2011). A family of 28 KPI genes was identified in the potato genome (Potato Genome Sequencing Consortium, 2011). Kunitz-type protease inhibitor genes are frequently induced after pest and pathogen attacks and act primarily as inhibitors of exogenous proteases. Therefore, the KPI family may provide resistance to biotic stress for the vulnerable, newly evolved underground organ (Potato Genome Sequencing Consortium, 2011). However, some protease inhibitors may have
a role not only in plant defense against biotic stresses but also in the adaptation of storage organs to an abiotic stress, such as changing temperatures. More generally, this role is consistent with the notion that, to prevent unnecessary metabolic costs, inhibitors of endogenous enzymes are regulated in such a way as to ensure inhibition only when it is physiologically required (Bellincampi et al., 2004). In the present study, the expression patterns of St-Inh observed were very similar between the CIS-sensitive and CISresistant tubers in storage (Fig. 1B). The same expression pattern may indeed be caused by a more general cold-defense system present in both resistant and sensitive plants. The St-Inh gene may have a prior role in the adaptation of storage organs to changing temperatures. The present research suggests that both PMEIs and KPIs contribute to CIS in potato tubers by modulating invertase activity and that PMEIs may have a stronger capacity to counteract AI activity than KPIs. These results provide novel clues for further investigation of the mechanism by which potato CIS is regulated. It has therefore been proposed that the PMEI proteins from potato may be important factors regulating CIS in tubers. Four PMEI genes from potato have been identified (Liu et al., 2010). Analysis based on the activity of recombinant protein and protein–protein interaction strongly suggests that StInvInh2 and StvacINV1 are counterparts, and the levels of StInvInh2 transcripts are higher in CIS-resistant genotypes than in CIS-sensitive ones, indicating that StInvInh2 is an important candidate factor regulating CIS in potato tubers (Liu et al., 2010). In addition, the RNA alternative splicing of StInvInh2 may also contribute to CIS resistance by increasing the additional functional capacity (Brummell et al., 2011). The contribution of StInvInh2 to CIS resistance merits further investigation to gain a deeper understanding of the mechanism of CIS in potato tubers.
Acknowledgments This study was supported by the National Natural Science Foundation of China (31201258 and 30800754), the National High Technology Research and Development (863) Program of China (2009AA10Z103), the China Postdoctoral Science Foundation (20110491173), and A-base funding from Agriculture and AgriFood Canada.
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