The roles of cell wall invertase inhibitor in regulating chilling tolerance in tomato
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Hexoses are important metabolic signals that respond to abiotic and biotic stresses. Cold stress adversely affects plant growth and development, limiting productivity. The mechanism by which sugars regulate plant cold tolerance remains elusive.
We examined the function of INVINH1, a cell wall invertase inhibitor, in tomato chilling tolerance. Cold stress suppressed the transcription of INVINH1 and increased that of cell wall invertase genes, Lin6 and Lin8 in tomato seedlings. Silencing INVINH1 expression in tomato increased cell wall invertase activity and enhanced chilling tolerance. Conversely, transgenic tomatoes over-expressing INVINH1 showed reduced cell wall invertase activity and were more sensitive to cold stress. Chilling stress increased glucose and fructose levels, and the hexoses content increased or decreased by silencing or overexpression INVINH1. Glucose applied in vitro masked the differences in chilling tolerance of tomato caused by the different expressions of INVINH1. The repression of INVINH1 or glucose applied in vitro regulated the expression of C-repeat binding factors (CBFs) genes. Transcript levels of NCED1, which encodes 9-cisepoxycarotenoid dioxygenase (NCED), a key enzyme in the biosynthesis of abscisic acid, were suppressed by INVINH1 after exposure to chilling stress. Meanwhile, application of ABA protected plant from chilling damage caused by the different expression of INVINH1.
In tomato, INVINH1 plays an important role in chilling tolerance by adjusting the content of glucose and expression of CBFs.
KeywordsAbscisic acid Cell wall invertase inhibitor Chilling tolerance C-repeat binding factors Solanum lycopersicum Sugar signaling
C-repeat binding factors
In higher plants, sucrose is the major transport form of carbohydrates. Cleavage of sucrose is catalyzed by either sucrose synthase (EC 184.108.40.206) or invertase (EC 220.127.116.11). The products (hexoses) are not only substrates of respiration and biosynthesis, but also important metabolic signals in plant response to abiotic and biotic stresses [1, 2, 3, 4, 5].
Invertases irreversibly hydrolyze sucrose into glucose and fructose. Based on their pH optima, solubility characteristics and subcellular localization, invertases are categorized as vacuolar, neutral/alkaline and cell wall invertases [6, 7, 8, 9]. Both vacuolar and neutral/alkaline invertases are soluble, with an acidic pI, while cell wall invertases are insoluble, with a mostly basic pI. Unlike the enzymes in vacuoles or cell walls, neutral/alkaline invertases are not glycosylated and possess an optimal pH of 7.0–7.8 [8, 9]. Neutral/alkaline invertases, localized in cytoplasm and mitochondria , are essential for normal plant growth, development and stress responses. Vacuolar invertases, with an optimal pH of 4.7–5.5, correlate with the sugar accumulation in sink tissues [11, 12] and cell expansion [13, 14]. Cell wall invertases, with an optimal pH of 4.3–5.5, hydrolyze sucrose to maintain the sucrose concentration gradient between source and sink tissues . Much progress has been made in understanding the role of cell wall invertase in sink tissue (seed & fruit) development [16, 17, 18, 19, 20], in fruit-set under heat stress  or during water deficit , and in leaf senescence .
The protein of cell wall invertase is intrinsically stable because of their glycosylated nature [6, 23]. Thus, the activity of cell wall invertase is largely regulated at the protein level. Inhibitors directly target the invertase active site and compete with sucrose, the substrate of invertase, for the same binding site . After the initial biochemical characterization , a group of small proteins (<20 KD) were observed to inhibit the activity of invertase in tobacco , maize , tomato [18, 28], potato , soybean  and Arabidopsis .
Transgenic approaches have led to some progress in understanding the role of invertase inhibitors in plants. Overexpression of an invertase inhibitor in potato prevented cold-induced sweetening of potato tubers [12, 32, 33]. Suppressing the expression of cell wall invertase inhibitor led to an increase of seed weight in soybean  and seed germination in Arabidopsis . Jin et al.  demonstrated that INVINH1 (a cell wall invertase inhibitor) could regulate the activity of the cell wall invertase in vivo, and silencing the expression of INVINH1 in tomato resulted in enlarged seed size, increased sugar content in fruit and delayed leaf senescence. These studies focused on the function of INVINH1 in sink tissues rather than in vegetative organs, although INVINH1 was highly expressed in root, stem and leaf during the vegetative period.
Interestingly, the expression of the cell wall invertase inhibitor was induced by abscisic acid [18, 23, 30], which is involved in the response to various biotic and abiotic stresses, including chilling stress . Low temperature is an important factor which affects the growth and development of plants. Plants adjust the delicate balance between multiple pathways, including transcription factor, DNA modification, hormones, secondary messengers, phosphatases and protein kinases among others to get acclimatized . The content of sugar, known to have an osmoprotective function increased during cold treatment . Glucose induced the expression of cold response genes . The ectopic expression of tomato GDP-L galactose phosphorylase gene in tobacco enhanced tolerance to chilling stress . These above suggested that sucrose metabolism may be involved in the regulation of chilling tolerance. However little was known about the function of cell wall invertase activity in chilling stress tolerance. This study aimed to explore the roles of INVINH1 in tomato cold tolerance and our data highlight the function of INVINH1 in plant tolerance to cold stress and provide a possible mechanism of plant cold tolerance.
Plant material, growth conditions and cold treatments
Tomatoes (Solanum lycopersicum XF-2) were grown in the greenhouse with 16 h of light at 25–27°C and 8 h of darkness at 22°C. Under this conditions the visible flower buds appear 65–75 days after germinate, so we chose 45 days old and 60 days old plants in this study. The first mature leaf was used for RT-PCR, proline content and the peroxidase (POD) activities measurement. For plants grown in vitro, seeds were surface sterilized and germinated on half-strength MS medium without sugar at 25°C with a 16-h photoperiod. For cold treatments, plants were transferred from 25°C to 4°C and were maintained under the same photoperiod as previously described.
Gene constructs and plant transformation
To construct Rbcs3a:INVINH1, the full-length Rbsc3a promoter  was digested with XbaI and HindIII and cloned into vector pCAMBIA1300 (Cambia), upstream of INVINH1.
Tomato plants were transformed with Rbcs3a:INVINH1 constructs according to Jin et al. . PCR analysis was used to monitor the incorporation of the transgene. Plants transformed with Rbcs3a:INVINH1 were analyzed using the following primer pair:5′-GCCTCTAGATATTGCTTTCTAGTCTCT-3′ and 5′-GAATTCCAATAAATTTCTTACAAT-3′. Twenty-four primary transgene (T0) lines were generated. Among them, three were PCR-positive for the transgene.
Semi-quantitative and real-time RT-PCR analysis
Leaves were collected, immediately frozen in liquid N2 and stored at −80°C. A total RNA kit (Invitrogen) was used to isolate total RNA from the stored leaves, which was then treated with RNase-free DNase (Promega) to remove genomic DNA. M-MLV reverse transcriptase (Takara) was used to synthesize first-strand cDNA. A tomato ACTIN fragment amplified with Actin-RT primers was used as an internal control. The primer sequences are listed in Additional file 1: Table S1.
POD activity assays
Leaves (0.3 g), which were harvested from plants after cold treatment, were ground with 9 ml of ice-cold 20 mM KH2PO4 buffer. The homogenates were centrifuged at 4000 g at 4°C for 15 min and supernatants were used to determine enzymatic activity. POD activity was assayed by measuring the increase in absorbance at 470 nm for 3 min.The assay mixture (3 ml final volume) comprised 100 mM potassium phosphate buffer (pH 6.0), 3.7 mM H2O2, 5.0 mM guaiacol and 1 ml enzyme extract . POD activity 1 U means “the change of OD470nm per minute per gram fresh weight”. Each value is mean ± SE of at least ten biological replicates.
Proline content measurement
Proline contents were determined according to , with some modifications. Leaves (approximately 0.3 g) were heated for 5 min in 5 mL 3% (v/v) aqueous acetylsalicylic acid. After cooling, the homogenate was filtered. The filtrate was mixed with glacial acetic acid, deionized water (2 ml each) and acid-ninhydrin agent (4 mL) and heated for 1 h at 100°C. The reaction mixture was extracted with 4 mL toluene. Absorbance at 520 nm was used to calculate the proline content. 1 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL purified proline were used as standards to produce calibration curve. Each value is mean ± SE of at least ten biological replicates.
Enzyme assay and sugar measurement
Cell wall, vacuolar, and cytoplasmic invertase activities and sugar levels were assayed as described by Jin et al. . Each value is mean ± SE of at least three biological replicates.
ABA content measurement
An Abscisic acid immunoassay detection kit (Sigma PGR1) was used to quantitate the levels of ABA in tomato. Each value is mean ± SE of at least four biological replicates.
Cell wall invertase activity correlates with tomato cold tolerance
Silencing INVINH1 in tomato enhances chilling tolerance
INVINH1 overexpression tomatoes are more sensitive than wildtype plants to chilling stress
To further examine the role of INVINH1 in tomato cold tolerance, we transformed tomato plants with an INVINH1 overexpression construct. In our earlier experiment, we introduced a 35S:INVINH1 overexpression construct into tomato. Unfortunately, all transgenic seeds aborted and no T1 progeny were obtained . To solve this problem, the Rbsc3A promoter, which does not have transcriptional activity in tomato seeds , was used to replace the 35S promoter in the overexpression construct. The Rbsc3A:INVINH1 overexpression construct was introduced into tomato through Agrobacterium tumefaciens-mediated transformation. Three primary transgenic lines (T0) were identified. The T0 plants were self-pollinated for seeds. T1 generations were analyzed to identify the presence or absence of the transgene by PCR . Three T1 lines were used for detailed analysis along with their non-transgenic segregants, which were analogous to the wildtype.
Consistent with the previous results for INVINH1 RNAi plants, 4°C treatment induced proline accumulation and peroxidase (POD) activities in both INVINH1 overexpression and wildtype plants. After 24 h of cold treatment, the levels of proline and activities of POD were significantly higher in wildtype compared with the INVINH1 overexpression plants (Fig. 5c & d). In comparison with untreated leaves, transforming with INVINH1 overexpression construct increased the proline content by an average of 57.7% and POD activity by 13.7%. In contrast, the wildtype plant increased its proline content by an average of 61.7% and its POD activity by 42.8%. These results indicated that INVINH1 overexpression plants are more sensitive than wildtype plants to low temperature.
Regulation of Lin6 & INVINH1 gene expression and sugar content in INVINH1 overexpression and silenced plants under chilling stress
Sucrose increases in wildtype and INVINH1 RNAi under cold stress (Fig. 6d). Sugar measurement revealed increases in glucose and fructose levels after treatment at 4°C for 24 h in both transgenic (INVINH1 overexpression and silencing) and wildtype. The hexoses content increased when the expression of INVINH1 was silenced, before and after cold treatment. Consistently, the glucose and fructose levels decreased if the expression of INVINH1 increased, before and after cold treatment (Fig. 6d).
External glucose rescued the different phenotype caused by the alteration expression of INVINH1 under chilling stress
The increase of sugar (glucose, fructose and sucrose) content by chilling stress, the increase or decrease in glucose and fructose content by silencing or overexpression of the cell wall invertase inhibitor INVINH1 and the resultant impact on cold tolerance in tomato indicated that tomato cold resistance is sensitive to sugar levels in vivo. If this is the case, exogenously applied sugars might have a positive impact on tomato cold tolerance. In consideration of glucoses not only act as sugars but also as signaling factor  and when external glucose supplied, the internal glucose, fructose and sucrose content accumulated in the same proportion , we hypothesized that exogenously applied glucose might contribute to chilling stress tolerance.
Alteration of CBFs expression in INVINH1 overexpression and silenced plants under chilling stress
Glucose applied in vitro masked the differences in chilling tolerance of tomato caused by the different expressions of INVINH1 (see above) indicate that the transcription levels of CBFs genes may be regulated by exogenously applied hexoses. To this end, we examined expression of CBFs with or without 2% glucose in chilling tolerance, 2% mannitol was used as an osmotic control [50, 51]. Mannitol treatment did not affect the expression of CBFs (Fig. 8a–c and d–e). Figure 8d, e & f showed that the transcripts of these three CBFs genes were significantly up-regulated after a 2-h chilling stress with 2% glucose or mannitol. However, no difference was detected in their mRNA levels between the transgenic plants and wildtype after exposure to 4°C for 2 h with 2% glucose. These results show that INVINH1 regulated the expression of CBFs gene by adjust the contents of glucose. Interestingly compared with mannitol, glucose only induced the expression of CBF3. These suggest that except by adjusting the contents of glucose, INVINH1 may have another inhibitory effect on cold tolerance.
INVINH1 suppressed endogenous ABA synthesis
ABA is an important signal in molecule plants’ response to stresses, including cold, drought and salinity . The application of ABA led to a strong increase of cell wall invertase inhibitor NtCIF mRNA in tobacco [23, 53]. ABA up-regulate the expression of cell wall invertase inhibitor INVINH1 and down-regulated the expression of cell wall invertase gene lin6 in tomato . Notably, exogenous glucose specifically increased the expressions of ABA synthesis and signaling genes . These observations prompted us to examine whether INVINH1 affects cold stress tolerance by regulating ABA synthesis.
Cell wall invertase inhibitor INVINH1 is involved in tomato chilling tolerance
Invertase inhibitor, which post-translationally regulates the activity of invertase, plays important roles in controlling fruit and seed development [18, 21, 28, 30, 31], leaves senescence , cold-induced sweetening of potato tubers [12, 29, 22, 33, 56], fruit set under heat stress  and the plant defense response . However, to the best of our knowledge, little evidence has been presented showing the involvement of the expression of cell wall invertase inhibitor or the activity of cell wall invertase in plant chilling tolerance.
Our results demonstrate that high cell wall invertase activities, which are regulated by cell wall invertase inhibitor (INVINH1), play important roles in chilling tolerance of tomato. First, cold treatment affected the transcript level of cell wall invertase and its inhibitor, and induced the activity of cell wall invertase (Figs. 1 & 6). We hypothesized that repression of INVINH1 may be required for chilling tolerance. Second, the transformation of tomato with INVINH1 RNAi constructs led to enhanced chilling tolerance of the transgenic tomatoes (Fig. 2). These data demonstrated that the presence of the INVINH1 RNAi transgene is the causal basis of the observed enhanced chilling tolerance phenotype. By contrast, overexpression of INVINH1 in tomatoes made them more sensitive to cold stress compared with the wildtype plants (Fig. 5).These data confirmed the role of INVINH1 in tomato cold tolerance. Finally and importantly, the degree of INVINH1 expression correlated well with the level of plant chilling tolerance (Figs. 6 & 7a). These results demonstrate that the tomato chilling tolerance is highly sensitive to changes in INVINH1 expression.
INVINH1 regulated chilling tolerance by adjusting the expression of CBFs genes and ABA synthesis
During cold tolerance, plants reprogram their gene expression through transcriptional, posttranscriptional and posttranslational mechanisms . Among various factors, the CBF-dependent transcriptional pathway induces or is involved in cold tolerance in plants such as Arabidopsis , rice , wheat  and tomato . In tomatoes, overexpression of Arabidopsis CBF1 gene increased chilling tolerance . The underlying molecular mechanism, however, remains unclear. The expressions of CBFs were suppressed by INVINH1 after 4°C treatments (Fig. 8); therefore, we hypothesized that the repression of INVINH1 may be required for the CBF-dependent cold tolerance pathway. Silencing the expression of INVINH1 induced the expression of CBF genes, even before cold treatment (Fig. 8), indicating that the expression of INVINH1 is a prerequisite for CBF-dependent cold tolerance.
ABA accumulates during cold stress and is involved in chilling tolerance . Our analyses revealed that the expression of INVINH1 affected the transcript level of the ABA synthesis gene NCED1 (Fig. 9). The above analyses concur with previous findings  that environmental stress regulation of ABA biosynthesis primarily occurs at the level of transcription; however, the expression of INVINH1 suppressed the expression of endogenous ABA synthesis (Fig. 9).
INVINH1 controls cell wall invertase activity, which contributes to chilling tolerance by regulating the hexose content
The glucose and fructose levels were induced after cold treatment in both wildtype and transgenic plants (Fig. 6d). These data suggested that the glucose and fructose contents need to reach optimum levels to protect the plant from the cold stress. This may be achieved by adjusting the activity of cell wall invertase, because the expression of a cell wall invertase gene (Lin6) was induced (Fig. 6b) and the cell wall inhibitor gene was depressed by cold treatment (Fig. 6a).
Cell wall invertase, whose activity is tightly regulated by its inhibitor in planta at the protein level, maintains the apoplastic glucose and fructose content at an optimum level . Apoplastic glucose and fructose not only provide carbon nutrients, but also play major roles in sugar signaling. For example, in maize, mutation of a cell wall invertase (INCW2) resulted in a miniature seed phenotype [20, 63].In tobacco, silencing the expression of cell wall invertase led to pollen abortion . Interestingly, the phenotypes above, which were caused by cell wall invertase deficiency, could be partially, but not completely, recovered by exogenous supply of hexoses [23, 65]. However, unlike the phenotype caused by cell wall invertase deficiency in reproductive tissue with rapid mitosis, in our study, the differences in chilling tolerance of tomatoes that were caused by different expressions of INVINH1 were totally blocked by the application of 2% glucose in vitro (Fig. 7).
Cell wall invertase inhibitor INVINH1 plays an important role in regulation of chilling tolerance in tomato by adjusting the sugar content, the expression of CBFs genes.
This work was supported by the National Natural Science Foundation of China (No. 31200192), the self-determined research funds of CCNU from the colleges’ basic research and operation of MOE (No. CCNU11A0122), and the project of Hubei Key Laboratory of Genetic Regulation and Integrative Biology (No. GRIB201306). The funding bodies did not play a role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript, but just provide the financial support.
Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
WY and YJ designed research. XX and YJ carried out most of the experiments. QH performed quantitative RT-PCR. XX, QH, WY and YJ analyzed data. YJ wrote the paper. WY revised the manuscript. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
Tomato (Solanum lycopersicum XF-2) seeds “zhongshusihao” were purchased from the institute of vegetables and flowers Chinese academy of agricultural sciences. The tomato seeds have obtained the permission of Ministry of Agriculture of the People’s Republic of China, which the phytosanitary certificate number is 130,922,006,820,110,062.
Consent for publication
The authors declare that they have no competing interests
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