转化酶抑制子调控番茄果实糖代谢的功能分析

Functional characterization of an invertase inhibitor gene involved in sucrose metabolism in tomato fruit 转化酶抑制子调控番茄果实糖代谢的功能分析 first-author †E-mail:zhangningsyau@163.comjj_syau@hotmail.com †E-mail:zhangningsyau@163.comjj_syau@hotmail.com zhangningsyau@163.com corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhangningsyau@163.comjj_syau@hotmail.com †E-mail:zhangningsyau@163.comjj_syau@hotmail.com zhangningsyau@163.com Key Laboratory of Protected Horticulture, Ministry of Education, College of Horticulture,Shenyang Agricultural University, Shenyang 110866, China Key Laboratory of Protected Horticulture, Ministry of Education, College of Horticulture,Shenyang Agricultural University, Shenyang 110866, China In this study, we produced tomato plants overexpressing an invertase inhibitor gene ( Sly-INH ) from tomato, using a simple and efficient transient transformation system. Compared with control plants, the expression of Sly-INH was highly upregulated in Sly-INH overexpressing plants, as indicated by real-time polymerase chain reaction (PCR). Physiological analysis revealed that Sly-INH inhibited the activity of cell wall invertase (CWIN), which increased sugar accumulation in tomato fruit. Furthermore, Sly-INH mediated sucrose metabolism by regulating CWIN activity. Our results suggest that invertase activity is potentially regulated by the Sly-INH inhibitor at the post-translational level, and they demonstrate that the transient transformation system is an effective method for determining the functions of genes in tomato. 目的 2 研究转化酶抑制子基因对番茄果实糖转运与积累的影响，并初步探讨其调控机制。 创新点 2 首次在番茄果实瞬时表达系统中证明转化酶抑制子可明显抑制细胞壁转化酶活性，并且此抑制作用是通过翻译后水平发挥作用。 方法 2 将Micro-Tom番茄分为对照组（果实未注射农杆菌及注射不含载体的空白农杆菌）和实验组（果实注射含有过表达转化酶抑制子基因的农杆菌），番茄果实分为萼片、中果肉、胶质胎座和心室隔壁四个部位，用实时定量聚合酶链式反应（qRT-PCR）技术检测转化酶抑制子和转化酶基因家族的表达变化，利用比色法分别检测了蔗糖代谢关键酶活性的变化，用高效液相色谱检测果实各部位果糖、葡萄糖和蔗糖的含量，利用高氯酸水解法测定淀粉的含量。 结论 2 利用农杆菌注射进行果实瞬时表达后果实各部位的细胞壁转化酶活性受到明显抑制，但转化酶基因家族的转录水平表达变化不大，果糖和葡萄糖含量下降，蔗糖含量有所升高。这一结果表明Sly-INH基因主要是通过在翻译后水平对番茄细胞壁转化酶进行调控，进而影响番茄果实糖的组成与含量。本研究从分子水平对转化酶抑制子及其调控的转化酶基因家族进行了系统性研究，这为利用栽培手段调控植物体内转化酶的表达和活性，调节库组织的蔗糖的输入速率以及同化产物的分配以改善品质提供依据与技术支持。 Invertase inhibitor Fruit development Transient transformation system Solanum lycopersicum Overexpression 转化酶抑制子 果实发育 瞬时表达体系 过表达 番茄 Introduction 1 Sucrose is an important transported sugar in higher plants, which is exported from the source tissues (leaves) via the phloem to various sink tissues (roots, stem, and reproductive organs). Cell wall invertase (CWIN) is an important enzyme in determining the apoplastic sucrose:glucose ratio and regulating related signaling pathways (Hanson and Smeekens, 2009 2009 ). Importantly, CWINs maintain sucrose concentration gradients, which are considered to be a driving force for sugar transport, partitioning, and storage between the photosynthetic source and sink tissues (Sturm, 1999 1999 ; Chourey et al., 2006 2006 ). The activity of CWIN is strictly controlled at both the transcriptional and post-transcriptional levels (Huang et al., 2007 2007 ). As a result of glycan decoration, the regulation of apoplasmic and vacuolar invertases may be highly dependent on post-translational mechanisms (Greiner et al., 2000 2000 ; Rausch and Greiner, 2004 2004 ; Tauzin et al., 2014 2014 ). Recent studies have suggested that invertase activity may be subject to post-translational suppression by its inhibitory protein (Hothorn et al., 2004 2004 ; Rausch and Greiner, 2004 2004 ). Biochemical characterization of plant invertase inhibitors was first performed in the 1960s (Schwimmer et al., 1961 1961 ; Pressey, 1966 1966 ), and the genes encoding these inhibitors were cloned three decades later (Greiner et al., 1998 1998 ). These inhibitors have been identified in a variety of species, such as Arabidopsis , potato, tomato, and tobacco (Weil et al., 1994 1994 ; Greiner et al., 1999 1999 ; Bate et al., 2004 2004 ; Link et al., 2004 2004 ; Reca et al., 2008 2008 ; Jin et al., 2009 2009 ; Kusch et al., 2009 2009 ; Brummell et al., 2011 2011 ). The specificity of plant invertase inhibitors was identified in vitro through the use of recombinant proteins and in vivo using transgenic plants (Bate et al., 2004 2004 ; Rausch and Greiner, 2004 2004 ; Brummell et al., 2011 2011 ). In tomato, silencing the expression of INH led to a 40% to 65% increase in apoplastic invertase activity in mature leaves, and the modified levels of invertase activity were specifically targeted to the apoplast (Jin et al., 2009 2009 ). Moreover, modifying invertase activity via specific inhibitors can potentially govern the senescence process in plants to help attain maximum yields and desirable crop quality. However, the realization of this potential will require a more thorough understanding of the involvement of invertase inhibitors in the regulation of growth and development in other plant tissues. Carbohydrate content and composition are important indicators of tomato fruit quality. Carbohydrates, which represent a major component of soluble solids, greatly contribute to tomato processing quality (Davies and Hobson, 1981 1981 ; Baxter et al., 2005 2005 ). The tomato is an ideal species for the study of metabolism related to soluble carbohydrate accumulation because of the natural genetic variation in tomato and the well-developed genetic and physiological information about Solanum lycopersicum and related species. Fruit quality has increasingly been linked to the activity of invertase inhibitors. The formation of the invertase inhibitor complex may be an important mechanism in the control of invertase activity in vivo , subsequently affecting carbon partitioning and fruit development (Fridman et al., 2004 2004 ; Hothorn et al., 2010 2010 ). Therefore, altering the activity of invertase inhibitors may serve as a strategy to increase the sucrose content and quality of fruit. The biological functionality of invertase inhibitors has largely been identified through in vitro assays using recombinant proteins (Greiner et al., 1998 1998 ; Bate et al., 2004 2004 ). In recent years, significant progress has been made in identifying and elucidating the involvement of invertase inhibitors in development and plant responses to various stimuli through the use of transgenic plants. Although some studies have examined the effects of silencing invertase inhibitor genes in various fruit tissues, few studies have examined the role(s) of these inhibitors through overexpressing invertase genes in vivo (McLaughlin and Boyer, 2004 2004 ; Jin et al., 2009 2009 ). Overexpressing endogenous invertase inhibitors in fruits will provide useful information about these proteins, which play important roles in the complex metabolic networks of plants, especially with regard to governing carbon allocation and fruit development. In this study, we selected tomato fruit as a model plant that undergoes fleshy fruit development. To study the role of invertase inhibitors in fruits, an invertase inhibitor complementary DNA (cDNA) from tomato, Sly-INH , was introduced into tomato via Agrobacterium mediated transient expression of a construct containing an expression carrier inhibitor. The results help to elucidate the effect of Sly-INH expression and invertase activity on tomato fruit development. Materials and methods 1 Plant materials 2 Tomato seeds (Micro-Tom) were surface sterilized with 0.5% (5 g/L) sodium hypochlorite, rinsed with water, and germinated for 3 d in the dark at 25 °C. Uniformly germinated seedlings were transferred to seedling trays containing growth medium comprising 1 part peat:1 part perlite:1 part vermiculite (v/v/v) and grown in a greenhouse under a 12-h day-night period with temperatures of 25 °C during the day and 15 °C at night and with an irradiance of 300 μmol photons/(m 2 ·s). The relative humidity varied, averaging 80%. Plasmid construction and plant transformation 2 A fruit-specific overexpression construct for Sly-INH was produced by cloning the respective full-length cDNA into pCAMBIA1300 downstream of the 2A11 promoter. Full-length Sly-INH cDNA was cloned by polymerase chain reaction (PCR) using the following primers: forward 5'-ATGAAAATTTT GATTTTCCC-3' and reverse 5'-TTACAATAAATT TCTTACAA-3'. Agroinjection was performed as described by Orzaez et al. ( 2006 2006 ). Tomato fruits ( S. lycopersicum cv. Micro-Tom) at 22 days after flowering (DAF) were injected with a maximum of 600 μl agroinjection solution into mature green tomatoes. When some drops of infiltration solution appeared in the hydathodes at the tips of sepals, the fruits were determined to be fully infiltrated. Completely infiltrated fruits were used in the experiments. Determination of soluble sugar and starch levels 2 The contents of fruit discs (0.5 cm 2 ) were extracted by incubating the discs in 1.0 ml 80% ethanol at 80 °C for 60 min and centrifuged at 4 °C for 5 min at 14 000 r/min. After they were transferred to new tubes, the cleared supernatants were evaporated to dryness at 40 °C. The dry residue was resolved in 250 μl of ultrapure water and used for soluble sugar analysis. Starch analysis was performed using the pellets derived from the centrifugation step. The materials were homogenized in 0.2 mol/L potassium hydroxide and incubated at 95 °C for 1 h. Then, the pH was adjusted to 5.5 with 1 mol/L acetic acid. Starch and soluble sugar assays were performed as described by Hajirezaei et al. ( 2000 2000 ). Enzyme extraction and activity assays 2 Fruit material samples (100 mg) were homogenized in 50 mmol/L 4-(2-hydroxyethyl)-1-pipera zineethanesulfonic acid (HEPES) buffer (10 mmol/L magnesium chloride, 2.5 mmol/L dithiothreitol, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L ascorbic acid, and 5% polyvinylpyrrolidone, pH 7.5). The homogenates were centrifuged for 20 min at 12 000 r/min at 4 °C. An aliquot of the resulting supernatant was desalted through Sephadex G-25 (medium), equilibrated and used for the various assays. Neutral invertase (NI), sucrose synthase (SS), and sucrose phosphate synthase (SPS) activities were determined as described by Zrenner et al. ( 1995 1995 ). CWIN and cytoplasmic invertase (CIN) activities were enzymatically assayed according to Gibon et al. ( 2004 2004 ) and Tomlinson et al. ( 2004 2004 ). RNA extraction and quantification 2 Total RNA (1 g) was extracted from specified fruit tissues using a TIANGEN RNAprep pure plant kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA synthesis of 1 μg of RNA in a final volume of 20 μl was performed using Moloney Murine Leukemia Virus Reverse Transcriptase, Point Mutant RNase H Minus (Promega, Beijing, China) following the manufacturer’s protocol using oligo(dT) 18 primer. The expressions of LIN5 , LIN6 , LIN7 , LIN8 , TIV1 , and INH were examined by real-time quantitative reverse transcription PCR (qRT-PCR) using the fluorescent intercalating dye SYBR Green in an iCycler detection system (Bio-Rad; http://www.bio-rad.com http://www.bio-rad.com ) following the protocol of Schaarschmidt et al. ( 2006 2006 ). The comparative C T method was performed for relative quantification of the target gene expression levels. The next primers used were as follows: for analysis of LIN5 transcript levels (GenBank accession No. AJ272304), forward 5'-AAAGGGATCTCAGCATCACAGG-3', reverse 5'-CGTCTTGGGCATATAGGTCAGC-3'; for LIN6 (GenBank accession No. AF506006), forward 5'-AT CAAGCCCGATAACAATCCA-3', reverse 5'-CCTCA CACTCCCAACCAATACTC-3'; for LIN7 (GenBank accession No. AF506006), forward 5'-TTTGGTGCT GGTGGAAAGACA-3', reverse 5'-GGCTCCGTTC CGTTGTTAAAC-3'; for LIN8 (GenBank accession No. AF506007), forward 5'-AAGGATGGGCGGG AATACA-3', reverse 5'-GGCCTGTGCTGGTGTGA TT-3'; for TIV1 (GenBank accession No. AF506007), forward 5'-AGGACTTTAGAGACCCGACTAC-3', reverse 5'-GCAGCACTCCATCCAATAGC-3'; for Sly-INH (GenBank accession No. AJ010943), forward 5'-GTATGCCAGAAGCATTAGAAGCA-3', reverse 5'-GCATCACCAGAAGAACCAACC-3'. To normalize gene expression levels (to eliminate differences in the efficiency of cDNA synthesis), the transcript levels of the constitutively expressed Actin gene in tomato (XM_004249818) were measured using the following primers: forward 5'-TGTCCCTATTTACGAGGGT TATGC-3'; reverse 5'-AGTTAAATCACGACCAG CAAGAT-3'. Statistical analysis 2 All agroinjection experiments were arranged in a completely randomized split-plot design with three replicates of 30 tomato plants each. Each extract was measured twice, and at least three replications were performed per analysis. For enzyme activity and sugar content assays, each biological replicate was composed of three technical replicates. Each experiment was repeated at least three times, and similar results were obtained. One representative set of data is described here from those that were carried out, and significant differences between the mean values among treatments were determined according to Tukey’s Least Significant Difference test ( P < 0.05) using analysis of variance (ANOVA; SPSS 11.0, Chicago, IL, USA). The standard errors (SEs) of the means were also calculated and are presented in the graphs as error bars. Results 1 Agroinjection of tomato fruit tissues 2 To assess the physiological role of Sly-INH in tomato fruit, we produced a Sly-INH overexpression construct harboring this gene driven by the fruit specific 2A11 promoter. We introduced this plasmid into tomato via Agrobacterium tumefaciens -mediated fruit injection, as described by Orzaez et al. ( 2006 2006 ), and measured the levels of Sly-INH transcript by qRT-PCR (Fig. 1 1 ). Data are expressed as mean±SE ( n =3). ** P < 0.01, compared with the control As shown in Fig. 1 1 , Sly-INH exhibited a tissue specific expression pattern in p1300-2A11- INH agroinjected tomato fruits, with maximum expression observed in the pericarp, followed by the pectinic placenta and dissepiments, perhaps because these structures form a diffusion barrier in the apoplastic network of the fruit. These results indicate that Sly-INH can be effectively expressed in fruit tissues via agroinjection. Notably, the relative expression of Sly-INH in sepals was significantly different in tomato fruit agroinjected with EHA105 containing p1300-2A11- INH plasmid (OE) plants versus the controls, and a significant reduction in INH expression was observed after 5 d, most likely because the infiltration solution in the hydathodes at the tips of sepals elicited Sly-INH gene silencing. Further studies are needed to determine whether the expression of Sly-INH is regulated via different routes. Activity of sugar metabolism-related enzymes 2 After agroinjection of tomato fruits with EHA105 bacterial solution, CWIN activity in each part of the fruit was altered. The activity of this enzyme was slightly higher than that of uninjected fruit, while CIN, NI, SS, and SPS activities were not significantly altered (Figs. 2 2 and 3 3 ). These results may be because Agrobacterium infection triggers a defense response in the plant, and CWIN activity is related to stress. By contrast, CWIN activity was observably reduced in plants transformed with p1300-2A11- Sly-INH compared with the control. A remarkable inhibition of CWIN activity was observed in Sly-INH OE tomato fruits, especially in sepals and dissepiments, at 3 d after injection, with CWIN activity decreasing by 58.8% and 30.4%, respectively. From 0 to 5 d after injection, CWIN activity decreased by 75.0% and 81.8% in sepals and dissepiments, respectively. In Sly-INH OE tomato fruits at 5 d after injection, CIN activity sharply increased in sepals, while CWIN activity decreased. It is worth noting that accurately comparing CWIN and tonoplast invertase (TIN) or CIN activity is difficult since CWIN activity is expressed based on cell-wall proteins whereas CIN and TIN activities are expressed on a soluble protein basis. (a) Cell wall invertase (CWIN) activity; (b) Cytoplasmic invertase (CIN) activity; (c) Neutral invertase (NI) activity. Data are expressed as mean±SE ( n =3). * P < 0.05, ** P < 0.01, compared with the control (a) SS activity; (b) SPS activity. Data are expressed as mean±SE ( n =3). * P < 0.05, compared with the control Analysis of the activities of other enzymes related to carbohydrate metabolism revealed an increase in the total activity of SS (when assayed in the direction of sucrose cleavage) and a decrease in the activity of SPS in the young fruits of plants most strongly overexpressing Sly-INH in the sepals (Fig. 3 3 ). This result suggests that injecting bacteria harboring a Sly-INH overexpression vector can reduce the activity of CWIN (Fig. 2 2 ). In Agrobacterium -mediated injected fruit, the expression of target genes can quickly be detected. A significant increase in the expression of the target gene in tomato fruit was detected at 3 d after injection. Relative expression levels and inhibition of sucrose invertase family genes 2 To demonstrate that the reduction in CWIN expression resulted from a specific reduction in invertase expression, we examined the relative expression levels of CWIN family genes in fruit (Fig. 4 4 ). The tomato CWIN gene family includes Lin5 , Lin6 , Lin7 , and Lin8 . In pectinic placenta, the expression level of Lin5 was high, while there was no significant expression in other parts of the fruit. Lin6 was mainly expressed in sepals, while Lin7 and Lin8 were not significantly expressed in any part of the tomato fruit examined. At 3 and 5 d after injection with bacteria harboring the Sly-INH overexpression vector, the expression of CWIN genes was not obviously affected. Interestingly, previous qRT-PCR analysis revealed no changes in the expression of Lin7 or the closest homolog Lin5 in transgenic tomato fruits subjected to agroinjection (Godt and Roitsch, 1997 1997 ; Fridman and Zamir, 2003 2003 ). Moreover, the expression of vacuolar invertase gene TIV1 in the Sly-INH overexpression lines was not obviously altered compared with the control (Fig. 5 5 ). (a) Relative expression value of LIN5 ; (b) Relative expression value of LIN6 ; (c) Relative expression value of LIN7 ;(d) Relative expression value of LIN8 . Data are expressed as mean±SE ( n =3). * P < 0.05, ** P < 0.01, compared with the control Data are expressed as mean±SE ( n =3). * P < 0.05, ** P < 0.01, compared with the control Carbohydrate composition and content 2 To investigate the consequences of reduced fruit CWIN activity on carbohydrate metabolism, we measured the levels of soluble sugars and starch in fruits in the three treatment groups. Tomato fruits primarily contain glucose, fructose, and sucrose, which largely affect the flavor and quality of the fruit. Starch is an important storage material in plants, as starch that accumulates during early fruit development is stored and serves as a carbon source for subsequent sugar synthesis. Developing fruits represent strong sinks for sugars during fruit development. After agroinjection of tomato fruits, reduced CWIN activity caused an approximately 50% decrease in starch contents compared with the control fruits, especially in sepals, pectinic placentas, and dissepiment (Fig. 6 6 ). While the amounts of glucose and fructose decreased slightly in injected fruits, sucrose levels increased approximately two-fold in injected fruits compared with controls. By contrast, there were no significant differences in carbohydrate contents between control fruits and fruits agroinjected with EHA105 (Fig. 6 6 ). (a) Fructose content; (b) Glucose content; (c) Sucrose content; (d) Starch content. Data are expressed as mean±SE ( n =3). * P < 0.05, ** P < 0.01, compared with the control Discussion 1 A. tumefaciens -mediated transient transformation is a simple, quick method that avoids the tedious process of tissue culture (Janssen and Gardner, 1990 1990 ; Kapila et al., 1997 1997 ). Most transferred DNA (T-DNA) that enters plant cells is not integrated into the plant genome but is instead temporarily stored in the nucleus, with a high copy number. Position effects and gene silencing have no effect on transient gene expression (Kapila et al., 1997 1997 ; Wroblewski et al., 2005 2005 ). In addition, traditional transformation systems require the use of antibiotics or other marker genes for initial screening; even so, false-positive plants are often selected, complicating analysis of test results. The agroinjection transformation system does not require the use of marker genes, avoiding the safety factor of antibiotics and transgenes and enabling the use of smaller vectors to transform larger fragments or more genes. Although agroinjection has many advantages, this method has certain limitations that should be considered when designing experiments. For example, its application is limited by the requirement to use only A. tumefaciens and similar species. Moreover, overexpression may affect the function and orientation of the gene product, and the stability is poor. In the current study, overexpression of Sly-INH has an inhibitory effect on apoplasmic invertase activity in tomato. The results suggest that at 3 to 5 d after Agrobacterium infection, high levels of expression of the transgenic product could be detected. Ripe fruits of commercial tomato variants are a good source of glucose and fructose (at equimolar concentrations), but they contain little sucrose (Klee and Giovannoni, 2011 2011 ) owing to the effects of cell wall-bound and vacuolar invertases on fruit sink strength (Yelle et al., 1991 1991 ; Klann et al., 1996 1996 ; Jin et al., 2009 2009 ; Zanor et al., 2009 2009 ). In the current study, in Micro-Tom tomato injected with blank bacterial solution (EHA105), invertase activity in all parts of the fruit increased slightly, perhaps because the defense response triggered by Agrobacterium infection increased CWIN activity. This result is consistent with previous findings (Roitsch et al., 2003 2003 ). After pathogen injection, the hexose content rapidly increases in plants, as hexose functions in the host defense response against pathogens (Herbers et al., 1996 1996 ; Biemelt and Sonnewald, 2006 2006 ). Scharte et al. ( 2005 2005 ) found that during the early defense process in tobacco leaves injected with tobacco mosaic virus, CWIN is rapidly induced. The induction of CWIN is assumed to favor sink development, enabling the pathogen to withdraw carbohydrates for its nutrition. In addition, the levels of hexoses formed via CWIN activity increase to support defense responses. In the current study, the reduced invertase activity in Sly-INH -overexpressing tomato tissues resulted in reduced hexose and starch levels and increased sucrose levels (Fig. 6 6 ). These findings demonstrate that the activation of metabolic carbohydrate fluxes maintains the balance between starch and sucrose metabolism. Recently, a number of related studies have shown that INH inhibits invertase activity in vitro (Greiner et al., 1998 1998 ). However, these studies, which used heterologous expression systems, did not elucidate the function of the endogenous INH gene. Jin et al. ( 2009 2009 ) found that inhibiting the expression of INH in tomato increased the activity of CWIN 40%–65% in mature leaves. In mature leaves, fruits, and seeds, inhibiting the expression of Sly-INH has an effect on the contents of INV mRNA. In the current study, CWIN activity decreased in sepals, pericarp, and pectinic placenta in tomato fruit after injection with Sly-INH , especially in sepals and dissepiment, whereas the activities of other sugar metabolism related enzymes remained unchanged. These findings indicate that overexpression of Sly-INH inhibits the activity of apoplasmic invertase in developing tomato fruit, which increases fruit sucrose levels. Emerging evidence indicates that CWIN activity is tightly controlled at both the transcriptional and post-transcriptional levels (Huang et al., 2007 2007 ). Invertase activity may be subject to post-translational suppression by its inhibitory protein (Hothorn et al., 2004 2004 ; Rausch and Greiner, 2004 2004 ; Jin et al., 2009 2009 ). Our results suggest that Agrobacterium -mediated fruit injection greatly affected the expression of the target gene Sly-INH but had no effect on the transcription levels of genes in the invertase gene family (Fig. 4 4 ). However, physiological analysis indicated that CWIN activity was inhibited in these fruits. Taken together, these results suggest that the inhibitor Sly-INH regulates the activity of CWIN at the post-translational level. This finding is in accordance with previous invertase structural analysis (Jin et al., 2009 2009 ; Hothorn et al., 2010 2010 ). Notably, the relative expression of Sly-INH was lower in sepals after injection compared with the control (Fig. 1 1 ), while reduced CWIN activity was evident in the sepals of OE plants (Fig. 2 2 ). These results raise the possibility that the reduced CWIN activity in 5-d sepals might have been due to different routes in the regulation of the inhibitory effect of Sly-INH against CWIN. For example, a recent site directed mutagenesis study showed that the presence of defective CWIN increased the inhibitory effect of INHs on CWIN activity (le Roy et al., 2013 2013 ; Palmer et al., 2015 2015 ). Conclusions 1 This study demonstrates that CWIN and CIN exist in glycosylated form and are relatively stable, suggesting that the activities of these two invertases largely depend on post-translational regulation. The results suggest that the expression of Sly-INH increased significantly at 3 d after injection, while CWIN activity decreased significantly. These results demonstrate that the invertase inhibitor Sly-INH has the potential to regulate CWIN activity at the post translational level. References NJ Bate , XP Niu , YW Wang , 等 . An invertase inhibitor from maize localizes to the embryo surrounding region during early kernel development . Plant Physiol , 2004 . 134 ( 1 ): 246 - 254 . DOI: 10.1104/pp.103.027466 http://doi.org/10.1104/pp.103.027466 . An invertase inhibitor from maize localizes to the embryo surrounding region during early kernel development C Baxter , F Carrari , A Bauke , 等 . Fruit carbohydrate metabolism in an introgression line of tomato with increased fruit soluble solids . Plant Cell Physiol , 2005 . 46 ( 3 ): 425 - 437 . DOI: 10.1093/pcp/pci040 http://doi.org/10.1093/pcp/pci040 . Fruit carbohydrate metabolism in an introgression line of tomato with increased fruit soluble solids S Biemelt , U Sonnewald . Plant-microbe interactions to probe regulation of plant carbon metabolism . J. Plant Physiol , 2006 . 163 ( 3 ): 307 - 318 . DOI: 10.1016/j.jplph.2005.10.011 http://doi.org/10.1016/j.jplph.2005.10.011 . Plant-microbe interactions to probe regulation of plant carbon metabolism DA Brummell , RK Chen , JC Harris , 等 . Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants . J. Exp. Bot , 2011 . 62 ( 10 ): 3519 - 3534 . DOI: 10.1093/jxb/err043 http://doi.org/10.1093/jxb/err043 . Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants PS Chourey , M Jain , QB Li , 等 . Genetic control of cell wall invertases in developing endosperm of maize . Planta , 2006 . 223 ( 2 ): 159 - 167 . DOI: 10.1007/s00425-005-0039-5 http://doi.org/10.1007/s00425-005-0039-5 . Genetic control of cell wall invertases in developing endosperm of maize JN Davies , GE Hobson . The constituents of the tomato fruit–the influence of environment, nutrition, and genotype . Crit Rev Food Sci Nutr , 1981 . 15 ( 3 ): 205 - 280 . DOI: 10.1080/10408398109527317 http://doi.org/10.1080/10408398109527317 . The constituents of the tomato fruit–the influence of environment, nutrition, and genotype E Fridman , D Zamir . Functional divergence of a syntenic invertase gene family in tomato, potato, and Arabidopsis . Plant Physiol , 2003 . 131 ( 2 ): 603 - 609 . DOI: 10.1104/pp.014431 http://doi.org/10.1104/pp.014431 . Functional divergence of a syntenic invertase gene family in tomato, potato, and Arabidopsis E Fridman , F Carrari , YS Liu , 等 . Zooming in on a quantitative trait for tomato yield using interspecific introgressions . Science , 2004 . 305 ( 5691 ): 1786 - 1789 . DOI: 10.1126/science.1101666 http://doi.org/10.1126/science.1101666 . Zooming in on a quantitative trait for tomato yield using interspecific introgressions Y Gibon , OE Blaesing , J Hannemann , 等 . A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness . Plant Cell , 2004 . 16 ( 12 ): 3304 - 3325 . DOI: 10.1105/tpc.104.025973 http://doi.org/10.1105/tpc.104.025973 . A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness DE Godt , T Roitsch . Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism . Plant Physiol , 1997 . 115 ( 1 ): 273 - 282 . DOI: 10.1104/pp.115.1.273 http://doi.org/10.1104/pp.115.1.273 . Regulation and tissue-specific distribution of mRNAs for three extracellular invertase isoenzymes of tomato suggests an important function in establishing and maintaining sink metabolism S Greiner , S Krausgrill , T Rausch . Cloning of a tobacco apoplasmic invertase inhibitor. Proof of function of the recombinant protein and expression analysis during plant development . Plant Physiol , 1998 . 116 ( 2 ): 733 - 774 . DOI: 10.1104/pp.116.2.733 http://doi.org/10.1104/pp.116.2.733 . Cloning of a tobacco apoplasmic invertase inhibitor. Proof of function of the recombinant protein and expression analysis during plant development S Greiner , T Rausch , U Sonnewald , 等 . Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers . Nat Biotechnol , 1999 . 17 ( 7 ): 708 - 711 . DOI: 10.1038/10924 http://doi.org/10.1038/10924 . Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers S Greiner , U Koster , K Lauer , 等 . Plant invertase inhibitors: expression in cell culture and during plant development . Aust J Plant Physiol , 2000 . 27 ( 9 ): 807 - 814 . DOI: 10.1071/PP99171 http://doi.org/10.1071/PP99171 . Plant invertase inhibitors: expression in cell culture and during plant development MR Hajirezaei , Y Takahata , RN Trethewey , 等 . Impact of elevated and apoplastic invertase activity on carbon metabolism during potato tuber development . J Exp Bot , 2000 . 51 ( Suppl. 1 ): 439 - 445 . DOI: 10.1093/jexbot/51.suppl_1.439 http://doi.org/10.1093/jexbot/51.suppl_1.439 . Impact of elevated and apoplastic invertase activity on carbon metabolism during potato tuber development J Hanson , S Smeekens . Sugar perception and signaling–an update . Curr Opin Plant Biol , 2009 . 12 ( 5 ): 562 - 567 . DOI: 10.1016/j.pbi.2009.07.014 http://doi.org/10.1016/j.pbi.2009.07.014 . Sugar perception and signaling–an update K Herbers , P Meuwly , WB Frommer , 等 . Systemic acquired resistance mediated by the ectopic expression of invertase: possible hexose sensing in the secretory pathway . Plant Cell , 1996 . 8 ( 5 ): 793 - 803 . DOI: 10.2307/3870282 http://doi.org/10.2307/3870282 . Systemic acquired resistance mediated by the ectopic expression of invertase: possible hexose sensing in the secretory pathway M Hothorn , S Wolf , P Aloy , 等 . Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins . Plant Cell , 2004 . 16 ( 12 ): 3437 - 3447 . DOI: 10.1105/tpc.104.025684 http://doi.org/10.1105/tpc.104.025684 . Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins M Hothorn , WV Ende , W Lammensc , 等 . Structural insights into the pH-controlled targeting of plant cell wall invertase by a specific inhibitor protein . PNAS , 2010 . 107 ( 40 ): 17427 - 17432 . DOI: 10.1073/pnas.1004481107 http://doi.org/10.1073/pnas.1004481107 . Structural insights into the pH-controlled targeting of plant cell wall invertase by a specific inhibitor protein LF Huang , PN Bocock , JM Davis , 等 . Regulation of invertase: a suite of transcriptional and post transcriptional mechanisms . Funct Plant Biol , 2007 . 34 ( 6 ): 499 - 507 . DOI: 10.1071/FP06227 http://doi.org/10.1071/FP06227 . Regulation of invertase: a suite of transcriptional and post transcriptional mechanisms BJ Janssen , RC Gardner . Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium . Plant Mol Biol , 1990 . 14 ( 1 ): 61 - 72 . DOI: 10.1007/BF00015655 http://doi.org/10.1007/BF00015655 . Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium Y Jin , DA Ni , YL Ruan . Posttranslational elevation of cell wall invertase activity by silencing its inhibitor in tomato delays leaf senescence and increases seed weight and fruit hexose level . Plant Cell , 2009 . 21 ( 7 ): 2072 - 2089 . DOI: 10.1105/tpc.108.063719 http://doi.org/10.1105/tpc.108.063719 . Posttranslational elevation of cell wall invertase activity by silencing its inhibitor in tomato delays leaf senescence and increases seed weight and fruit hexose level J Kapila , RD Rycke , M van Montagu . An Agrobacterium -mediated transient gene expression system for intact leaves . Plant Sci , 1997 . 122 ( 1 ): 101 - 108 . DOI: 10.1016/S0168-9452(96)04541-4 http://doi.org/10.1016/S0168-9452(96)04541-4 . An Agrobacterium-mediated transient gene expression system for intact leaves EM Klann , B Hall , AB Bennett . Antisense acid invertase ( TIV1 ) gene alters soluble sugar composition and size in tomato fruit . Plant Physiol , 1996 . 112 ( 3 ): 1321 - 1330 . DOI: 10.1104/pp.112.3.1321 http://doi.org/10.1104/pp.112.3.1321 . Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in tomato fruit HJ Klee , JJ Giovannoni . Genetics and control of tomato fruit ripening and quality attributes . Annu Rev Genet , 2011 . 45 ( 1 ): 41 - 59 . DOI: 10.1146/annurev-genet-110410-132507 http://doi.org/10.1146/annurev-genet-110410-132507 . Genetics and control of tomato fruit ripening and quality attributes U Kusch , K Harms , T Rausch , 等 . Inhibitors of plant invertases do not affect the structurally related enzymes of fructan metabolism . New Phytol , 2009 . 181 ( 3 ): 601 - 612 . DOI: 10.1111/j.1469-8137.2008.02688.x http://doi.org/10.1111/j.1469-8137.2008.02688.x . Inhibitors of plant invertases do not affect the structurally related enzymes of fructan metabolism K le Roy , R Vergauwen , T Struyf , 等 . Understanding the role of defective invertases in plants: tobacco Nin88 fails to degrade sucrose . Plant Physiol , 2013 . 161 ( 4 ): 1670 - 1681 . DOI: 10.1104/pp.112.209460 http://doi.org/10.1104/pp.112.209460 . Understanding the role of defective invertases in plants: tobacco Nin88 fails to degrade sucrose M Link , T Rausch , S Greiner . In Arabidopsis thaliana , the invertase inhibitors AtC/VIF1 and 2 exhibit distinct target enzyme specificities and expression profiles . FEBS Lett , 2004 . 573 ( 1-3 ): 105 - 109 . DOI: 10.1016/j.febslet.2004.07.062 http://doi.org/10.1016/j.febslet.2004.07.062 . In Arabidopsis thaliana, the invertase inhibitors AtC/VIF1 and 2 exhibit distinct target enzyme specificities and expression profiles JE McLaughlin , JS Boyer . Sugar-responsive gene expression, invertase activity, and senescence in aborting maize ovaries at low water potentials . Ann Bot , 2004 . 94 ( 5 ): 675 - 689 . DOI: 10.1093/aob/mch193 http://doi.org/10.1093/aob/mch193 . Sugar-responsive gene expression, invertase activity, and senescence in aborting maize ovaries at low water potentials D Orzaez , M Sophie , H Willemien , 等 . Agroinjection of tomato fruits: a tool for rapid functional analysis of transgenes directly in fruit . Plant Physiol , 2006 . 140 ( l ): 3 - 11 . DOI: 10.1104/pp.105.068221 http://doi.org/10.1104/pp.105.068221 . Agroinjection of tomato fruits: a tool for rapid functional analysis of transgenes directly in fruit WM Palmer , L Ru , Y Jin , 等 . Tomato ovary-to-fruit transition is characterized by a developmental shift of cell wall invertase and its inhibitor mRNAs from a dispersed expression to a phloem-specific pattern with the encoded proteins localized to sieve elements . Mol Plant , 2015 . 8 ( 2 ): 315 - 328 . DOI: 10.1016/j.molp.2014.12.019 http://doi.org/10.1016/j.molp.2014.12.019 . Tomato ovary-to-fruit transition is characterized by a developmental shift of cell wall invertase and its inhibitor mRNAs from a dispersed expression to a phloem-specific pattern with the encoded proteins localized to sieve elements R Pressey . Separation and properties of potato invertase and invertase inhibitor . Arch Biochem Biophys , 1966 . 113 ( 3 ): 667 - 674 . DOI: 10.1016/0003-9861(66)90246-3 http://doi.org/10.1016/0003-9861(66)90246-3 . Separation and properties of potato invertase and invertase inhibitor T Rausch , S Greiner . Plant protein inhibitors of invertases . Biochim Biophys Acta , 2004 . 1696 ( 2 ): 253 - 261 . DOI: 10.1016/j.bbapap.2003.09.017 http://doi.org/10.1016/j.bbapap.2003.09.017 . Plant protein inhibitors of invertases IB Reca , A Brutus , R D'Avino , 等 . Molecular cloning, expression and characterization of a novel apoplastic invertase inhibitor from tomato ( Solanum lycopersicum ) and its use to purify a vacuolar invertase . Biochimie , 2008 . 90 ( 11-12 ): 1611 - 1623 . DOI: 10.1016/j.biochi.2008.04.019 http://doi.org/10.1016/j.biochi.2008.04.019 . Molecular cloning, expression and characterization of a novel apoplastic invertase inhibitor from tomato (Solanum lycopersicum) and its use to purify a vacuolar invertase T Roitsch , ME Balibrea , M Hofmann , 等 . Extracellular invertase: key metabolic enzyme and PR protein . J Exp Bot , 2003 . 54 ( 382 ): 513 - 524 . DOI: 10.1093/jxb/erg050 http://doi.org/10.1093/jxb/erg050 . Extracellular invertase: key metabolic enzyme and PR protein S Schaarschmidt , T Roitsch , B Hause . Arbuscular mycorrhiza induces gene expression of the apoplastic invertase LIN6 in tomato ( Lycopersicon esculentum ) roots . J Exp Bot , 2006 . 57 ( 15 ): 4015 - 4023 . DOI: 10.1093/jxb/erl172 http://doi.org/10.1093/jxb/erl172 . Arbuscular mycorrhiza induces gene expression of the apoplastic invertase LIN6 in tomato (Lycopersicon esculentum) roots J Scharte , H Schon , E Weis . Photosynthesis and carbohydrate metabolism in tobacco leaves during an incompatible interaction with Phytophthora nicotianae . Plant Cell Environ , 2005 . 28 ( 11 ): 1421 - 1435 . DOI: 10.1111/j.1365-3040.2005.01380.x http://doi.org/10.1111/j.1365-3040.2005.01380.x . Photosynthesis and carbohydrate metabolism in tobacco leaves during an incompatible interaction with Phytophthora nicotianae S Schwimmer , RU Makower , ES Rorem . Invertase and invertase inhibitor in potato . Plant Physiol , 1961 . 36 ( 3 ): 313 - 316 . DOI: 10.1104/pp.36.3.313 http://doi.org/10.1104/pp.36.3.313 . Invertase and invertase inhibitor in potato A Sturm . Invertases: primary structures, functions, and roles in plant development and sucrose partitioning . Plant Physiol , 1999 . 121 ( 1 ): 1 - 8 . DOI: 10.1104/pp.121.1.1 http://doi.org/10.1104/pp.121.1.1 . Invertases: primary structures, functions, and roles in plant development and sucrose partitioning AS Tauzin , G Sulzenbacher , M Lafond , 等 . Functional characterization of a vacuolar invertase from Solanum lycopersicum : post-translational regulation by N -glycosylation and a proteinaceous inhibitor . Biochimie , 2014 . 101 39 - 49 . DOI: 10.1016/j.biochi.2013.12.013 http://doi.org/10.1016/j.biochi.2013.12.013 . Functional characterization of a vacuolar invertase from Solanum lycopersicum: post-translational regulation by N-glycosylation and a proteinaceous inhibitor KL Tomlinson , S McHugh , H Labbe , 等 . Evidence that the hexose-to-sucrose ratio does not control the switch to storage product accumulation in oilseeds: analysis of tobacco seed development and effects of overexpressing apoplastic invertase . J Exp Bot , 2004 . 55 ( 406 ): 2291 - 2303 . DOI: 10.1093/jxb/erh251 http://doi.org/10.1093/jxb/erh251 . Evidence that the hexose-to-sucrose ratio does not control the switch to storage product accumulation in oilseeds: analysis of tobacco seed development and effects of overexpressing apoplastic invertase M Weil , S Krausgrill , A Schuster , 等 . A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase . Planta , 1994 . 193 ( 3 ): 438 - 445 . DOI: 10.1007/BF00201824 http://doi.org/10.1007/BF00201824 . A 17-kDa Nicotiana tabacum cell-wall peptide acts as an in-vitro inhibitor of the cell-wall isoform of acid invertase T Wroblewski , A Tomczak , R Michelmore . Optimization of Agrobacterium -mediated transient assays of gene expression in lettuce, tomato, and Arabidopsis . Plant Biotechnol J , 2005 . 3 ( 2 ): 259 - 273 . DOI: 10.1111/j.1467-7652.2005.00123.x http://doi.org/10.1111/j.1467-7652.2005.00123.x . Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato, and Arabidopsis S Yelle , RT Chetelat , M Dorais , 等 . Sink metabolism in tomato fruit, genetic and biochemical analysis of sucrose accumulation . Plant Physiol , 1991 . 95 ( 4 ): 1026 - 1035 . DOI: 10.1104/pp.95.4.1026 http://doi.org/10.1104/pp.95.4.1026 . Sink metabolism in tomato fruit, genetic and biochemical analysis of sucrose accumulation MI Zanor , S Osorio , A Nunes-Nesi , 等 . RNA interference of LIN5 in tomato confirms its role in controlling Brix content, uncovers the influence of sugars on the levels of fruit hormones, and demonstrates the importance of sucrose cleavage for normal fruit development and fertility . Plant Physiol , 2009 . 150 ( 3 ): 1204 - 1218 . DOI: 10.1104/pp.109.136598 http://doi.org/10.1104/pp.109.136598 . RNA interference of LIN5 in tomato confirms its role in controlling Brix content, uncovers the influence of sugars on the levels of fruit hormones, and demonstrates the importance of sucrose cleavage for normal fruit development and fertility R Zrenner , M Salanoubat , L Willmitzer , 等 . Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants ( Solanum tuberosum L.) . Plant J , 1995 . 7 ( 1 ): 97 - 107 . DOI: 10.1046/j.1365-313X.1995.07010097.x http://doi.org/10.1046/j.1365-313X.1995.07010097.x . Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.) Compliance with ethics guidelines Ning ZHANG, Jing JIANG, Yan-li YANG, and Zhi-he WANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1400319 Project supported by the National Natural Science Foundation of China (Nos. 31372054 and 30971999) and the State Key Laboratory of Plant Physiology and Biochemistry Open Project of China (No. SKLPPBKF1404) 2014-11-19 2015-07-09 2015-06-08 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 16