- Email: [email protected]
Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses
Agricultural Sciences in China
September 2009
2009, 8(9): 1046-1052
Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses LU Shao-wei, LI Tian-lai and JIANG Jing The Key Laboratory of Protected Horticulture of Liaoning Province/College of Horticulture, Shenyang Agricultural University, Shenyang 110161, P.R.China
Abstract Changes in sucrose metabolism in response to salt (NaCl) and water (polyethylene glycol, PEG6000) iso-osmotic stresses were measured in tomato cultivar Liaoyuan Duoli (Solanum lycopersicum L.) and the objective was to provide a new evidence for the relationship between salt and osmotic stresses. The carbohydrate contents, as well as sucrose metabolizing enzymes activities and transcript levels were determined. The results indicated that soluble sugar and hexoses accumulated to higher levels and the contents of sucrose and starch were lower in mature fruit under the two stress treatments. Salt and water stresses can enhance the invertase and sucrose synthase activities of tomato fruit in a long period of time (45-60 days after anthesis), and elevate the expression of soluble acid invertase mRNA. It showed that two different stresses could also regulate the soluble acid invertase activity by controlling its gene expression. The activity of sucrose synthase was linked to the changes in soluble sugar levels but not with transcript levels. The effects of salt and water stress treatments on sucrose phosphate synthase activities were weak. Key words: tomato, salt stress, water stress, sucrose metabolism, gene expression
INTRODUCTION Salinity and drought are major abiotic stresses, affect almost every aspect of the physiology and biochemistry of plants, and significantly reduce yield. Great efforts have been devoted to understanding the physiological aspects of response to salinity and drought in plants as a basis for plant breeders to develop salinitytolerant and drought-tolerant genotypes (Cuartero et al. 2006). The yield and quality of tomato appear to be regulated by the net assimilation rate of the crop, the rate of import into individual fruit, and sink activity (Serge and Johnd 1988). High sink demand can significantly increase the quality of the tomato fruit by high accumulation of soluble solids, which is an important factor for Received 2 April, 2009
processing tomatoes. Sugars are the major components of the soluble solids in tomato mature fruit (Ninal and Johnd 1988). Since the water potential of the saline soil and saline water is lowered by the osmotic potential of the dissolved solutes, salt and water stresses produce similar plant responses (Perez-Alfocea et al. 1993). Deficit irrigation can improve strawberry and tomato fruit soluble sugar content, organic acid content, and sugar acid ratio (Shi and Tadashi 2001; Liu et al. 2001; Liu and Chen 2002; Qi et al. 2003). At the early stage of fruit development, salt stress can increase the ordinary tomato fruit soluble sugar accumulation (Balibrea et al. 1997, 2003). Under salinity and drought, the competition between different physiological processes and sink organs for the carbon supplies significantly
Accepted 15 July, 2009
Correspondence LI Tian-lai, Professor, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60312-0
Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses
affects overall plant growth and crop yield (Balibrea et al. 1999, 2000; Qi et al. 2003; Jiang et al. 2007). Carbohydrate content and composition are important factors determining tomato fruit quality and flavor. The composition of stored carbohydrate in tomato is associated with certain key enzymes responsible for sucrose metabolism. Invertase and sucrose synthase, catalyzing the breakdown of sucrose, are correlated with the reducing of sugar levels and the rate of carbon translocation into tomato fruit. It has been reported that soluble acid invertase is a major sucrolytic activity in cultivated tomato. This activity is considered to be involved in the composition of stored sugars (Klann et al. 1996; Qi et al. 2005). The sucrose synthase activity is to play an important function in the control of sucrose import and fruit growth parameters (D’Aoust et al. 1999). However, no clear relationship has been shown between salt and water stresses at carbohydrate accumulation and metabolism in tomato fruit. The objective of this work was to study the influences of sucrose metabolism in the different parts of developmental tomato fruit, and what strategies the plant developed to regulate sucrose metabolism, as well as the key enzyme activity and gene expression in sink organs under salt and water stresses during the fruiting period.
MATERIALS AND METHODS Plant materials and treatments
1047
and plants were trained to only one stem, eliminating all axilary buds. The salt stress and water stress treatments were prepared by adding NaCl (50 mmol L-1) and polyethylene glycol (PEG) 6000 iso-osmotic (Nicholas 1989; Michel and Kaufmann 1973) to the tanks (osmotic potential = -0.24 MPa, pH 6.5 ± 0.1). The nutrient solution was changed every 7 days and the levels adjusted every 3 days. The flowers were tagged at anthesis in the first trusses and stress treatments started at 20 days after anthesis (20 DAA). Yield and fruit number were controlled weekly from the beginning to the end of harvest period (about 2 months). Fruits were harvested at the start of ripening (45 DAA) or at the fully ripe stage (60 DAA). The sample fruits were then kept at -86°C until used for further analysis.
Carbohydrate determination Carbohydrate content of fruit was measured using a Waters 600E high-performance liquid chromatograph (HPLC). Carbohydrate column and 2410 refractive index monitor were used. The mobile phase was 75% acetonitrile and ultra water (75:25). The mobile rate was 1.0 mL min-1 and the temperature of the column was 35°C. Water Millennium software was used to handle date. The starch content was measured using perchloric acid hydrolyzed method. Total soluble sugar content was measured using anthrone method. Organic acid content was assayed with 0.1 mol L-1 NaOH titrate.
Enzyme extraction and assay The experiments were conducted in a solar greenhouse of science and research field of Horticultural Department of Shenyang Agricultural University, China. The cultivated tomato hybrid Liaoyuan Duoli (Solanum lycopersicum L.) seeds were sown and grown in plug containing a substrate composed of nutrient soil and vermiculite (2:1, v/v). When the seedlings started to develop the 6th leaf, 30 seedlings of uniform size were transferred to each of two tanks (2 m × 1.5 m × 0.1 m). The containers were filled with 200 L of full-strength Hoagland’s solution (pH 6.5 ± 0.1), where the plants were to be grown. The planting pattern was of 0.5 m between rows and 0.45 m between plants within rows
Enzyme extracts were prepared essentially as described as Minor and Schaffer (1991). Acid (AI; EC 3.2.1.25) and neutral (NI; EC 3.2.1.26) invertases activities were assayed in a final volume of 25 mL, containing 0.2 mL of dialyzed enzymatic extract, 0.8 mL of reaction solution (0.1 mol L-1 Na2HPO4-0.1 mol L-1 sodium citrate, 0.1 mol L-1 sucrose, and pH 4.8 or 7.2 for acid invertase and neutral invertase, respectively). The activities were measured by the quantity of reducing sugars released in the assay media with dinitrosalicylic acid. The reducing sugars were revealed by incubation at 100°C for 5 min and read at 520 nm in a Cary 100UV:VIS
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
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LU Shao-wei et al.
spectrophotometer (GBC Scientific Equipment Pty Ltd., Heareus, Germany). Sucrose synthase (SS; EC 2.4.1.13) activity was measured by using 0.4 mL reaction solution (0.05 mol L-1 fructose, 0.82% UDPG, 0.1 mol L-1 Tris, 10 mmol L-1 MgCl2) adding 0.2 mL enzyme at 37°C for 30 min and bathing for 1 min at 100°C, and a volume of 1 mL reaction products adding 0.1 mL 2 mol L-1 NaOH was placed in boiling water bath for 10 min and cooled in water, then adding 3.5 mL 30% HCl and 1 mL 0.1% resorcinol. Blank controls were obtained by adding the distilled water to the reaction medium containing resorcinol. The reducing sugars were revealed by incubation at 80°C for 10 min and read at 480 nm in a Cary 100UV:VIS spectrophotometer (GBC Scientific Equipment Pty Ltd., Heareus, Germany). Sucrose-phosphate synthase (SPS; EC 2.4.1.14) was assayed by measurement of sucrose produced from fructose 6phosphate plus UDP-glucose (Vassey and Sharkey 1989).
RNA isolation and hybridization RNA was extracted using the Trizol Regent (Invitrogen Life Technologies, Carlsbad, CA, USA). Fifteen microgram of total RNA were separated by electrophoresis through 1.2% (w/v) agarose formaldehyde gels and blotted onto a nylon membrane (Hybond-N + ; Amersham-Pharmacia Biotech, USA) by capillary transfer. Probes for the detection of AI (GenBank accession no. AF465613) and SS (GenBank accession no. AJ011535) transcripts were PCR-amplified with primers of AI2F (5´-AACTCCGCCTCTCGTTACACA3´) and AI2R (5´-TAGGATGGTAGCGGACCCTG-3´), SuS2F (5´-AACTTTAGCTGCTCACCGCAA-3´) and SuS2R (5´-TTGCCCTTATAATGGTGAGCG-3´). The gene-specific probes were labeled with DIG using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.
RESULTS Fruit growth and quality At the end of the stress period, NaCl and PEG treatments reduced the single fruit weight by 17 and 23% (as compared with control plants) in mature fruit (Table 1), respectively. This suggests that the discrepancy of differential stress on fruit growth was primarily due to water stress and ionic stress. The content of soluble sugar and titratable acids increased more under water stress than under NaCl stress, the treatments of 50 mmol L-1 NaCl had the highest ratio of brix-acid and the best flavor and quality in mature fruit. It is showed that a certain concentration of salt treatments may improve the quality of the fruit flavor and nutritional quality.
Hexose, sucrose, and starch contents As the fruit developed, the hexose (fructose and glucose) was the predominant sugars, and gradually increased with the highest level presenting at ripe stage (Qi et al. 2005). The salt and water stress also enhance the levels of hexose in the three tissues of tomato mature fruit, which accumulated more hexoses under PEG stress than under NaCl stress (Table 2). In the early stage of development, sucrose and starch were accumulated; but then declined sharply and remained at low level at ripe stage. The contents of sucrose and starch were lower in the three tissues of tomato mature fruit in both stresses than in the control. Salt and water stresses produced similar impact on the concentrations of sucrose and starch (Table 2).
Activities of sucrose-metabolizing related enzymes In order to determine whether part of the changes in
Table 1 Influence of NaCl and PEG6000 iso-osmotic stresses on tomato fruit weight and quality1) Treatment Control NaCl PEG
Soluble sugar (%)
Organic acid (%)
Brix-acid ratio
Fresh weight (mg FW g-1)
2.59 ± 0.21 Aa 3.92 ± 0.14 Bb 4.73 ± 0.18 Bc
0.71 ± 0.02 Aa 0.78 ± 0.05 Aa 0.98 ± 0.07 Ab
3.65 Aa 5.03 Bb 4.83 Bb
208.09 ± 28.17 Aa 172.71 ± 17.92 ABb 159.50 ± 25.72 Bb
Values are the mean ± SD of five replicate samples. A and a indicate significance at P 0.05 and P 0.01, respectively. The same as below.
1)
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses
carbohydrate content could be due to an increase in enzymes mobilizing sucrose or hexose, we measured the optimal activities of acid and neutral invertases, sucrose synthase and sucrose phosphate synthase at the start of ripening (45 DAA) and at the fully ripe stage (60 DAA) under PEG and NaCl treatments, respectively. The changes in invertase (acid invertase and neutral invertase) activity during the development of the three tissues of tomato fruit are shown in Fig.1. The invertase activity is generally increased toward the end of
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fruit development with ripening. The major differences among the three parts of fruit were found at the ripening fruit. The invertase activity of fruit was generally higher in the placental tissue than in the mesocarp and radial pericarp. NaCl and PEG treatments can slightly improve neutral invertase activity and significantly improve the acid invertase activity at ripen fruit. The invertase activity is higher under PEG stress than under NaCl stress, this tendency is coincident with that of the hexose content.
Table 2 Influence of NaCl and PEG6000 iso-osmotic stresses on tomato fruit carbohydrate content1) Tissue
Treatment
Mesocarp
Control NaCl PEG Control NaCl PEG Control NaCl PEG
Radial pericarp
Placental tissue
1)
Glucose (mg g-1)
Fructose (mg g -1)
Sucrose (mg g-1)
Starch (mg g-1)
21.93 ± 0.99 Aa 25.07 ± 0.74 Aa 31.73 ± 0.80 Aa 21.62 ± 0.65 Aa 22.67 ± 0.88 Aa 31.38 ± 0.74 Aa 15.40 ± 0.44 Aa 19.33 ± 0.97 Aab 24.70 ± 1.31 Ab
20.25 ± 0.53 Aa 25.16 ± 0.85 Aab 33.99 ± 0.75 Ab 23.06 ± 0.45 Aa 28.71 ± 0.35 Aab 33.13 ± 0.64 Ab 17.40 ± 0.24 Aa 26.68 ± 0.70 Aab 29.75 ± 0.43 Ab
3.16 ± 0.15 Aa 1.72 ± 0.05 Bb 1.89 ± 0.06 ABb 3.27 ± 0.13 Aa 1.39 ± 0.16 Bb 1.67 ± 0.02 Bb 2.11 ± 0.08 Aa 1.18 ± 0.03 Bb 1.43 ± 0.07 ABb
7.63 ± 0.15 Aa 4.66 ± 0.34 ABb 3.77 ± 0.16 Bb 10.41 ± 1.07 Aa 5.88 ± 0.13 Bb 5.35 ± 0.11 Bb 10.24 ± 1.29 Aa 6.62 ± 0.53 Bb 6.34 ± 0.66 Bb
Glucose, fructose, sucrose, and starch were determined in the same samples. Values are the mean ± SD of three replicate samples.
Fig. 1 Effect of NaCl and PEG6000 iso-osmotic stresses on sucrose-metabolizing related enzymes in tomato fruit. The activities of sucrose biosynthetic and mobilizing enzymes were determined in crude extracts in the same samples. Values are the mean ± SD of three replicate samples. AI, acid; NI, neutral; SS, sucrose synthase; SPS, sucrose-phosphate synthase.
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Sucrose synthase (SS) activity was lower at later stages of fruit development. At 45 DAA, SS activity was higher both under PEG and NaCl stresses in the mesocarp; PEG stress increased SS activity in the radial pericarp. SS activity was increased under NaCl and PEG stresses in mature fruit. The SS activity is higher under PEG stress than under NaCl stress, this tendency is coincident with that of the invertase activity. Sucrose phosphate synthase (SPS) was in the same range as the SS, and it also showed low activity at all stages with a small decline toward the end of fruit development increased with ripening. Activity was also greater both in the mesocarp and radial pericarp than in the placental tissue at 45 and 60 DAA. A slight increase in activity was observed under NaCl stress in the mesocarp and radial pericarp. SPS activity was lower under PEG stress than in control in the three tissues of tomato fruit at 45 DAA. The activity stayed approximately the same in the placental tissue under NaCl stress and in the three tissues under PEG stress at ripe fruit.
Gene expression of AI and SS The temporal and spatial expression patterns of acid invertase during tomato fruit development were investigated (Fig.2). In Northern blot analysis, transcriptions of AI were abundant in the placental tissue at 45 and 60 DAA. They were also increased towards the ripening of mesocarp and radial pericarp. After treatments with different stresses, acid invertase gene expression is more complex. There were no detection of gene expression in the mesocarp and radial pericarp at 45 DAA under normal condition and a higher expres-
Fig. 2 Northern blot analysis of total RNA from mesocarp, radial pericarp and placental tissue at 45 and 60 DAA using partial fragment of AI as probe.
LU Shao-wei et al.
sion at 45 DAA and 60 DAA under stress condition (as compared with the control). AI mRNA level was increased more under PEG stress than under NaCl stress. The highest expression activity of acid invertase was found by the PEG stress in the placental tissue at 60 DAA. The tendency is coincident with that of the acid invertase activity. SS transcripts were barely detectable (results not shown) and so were probably too low to show the expected increase of transcripts in tomato fruit in correlation with increased sugar levels and SS activity. The results point to the importance of posttranscriptional regulation.
DISCUSSION A survey was conducted on key sucrose metabolizing enzymes over development in tomato fruit under three different conditions, providing information on the enzymic bases for the accumulation of hexose. The results suggest that, under normal and stress conditions, there were differences in the activities of sucrose metabolism enzymes measured in the mesocarp and radial pericarp in comparison with the placental tissue. The placental tissue had increased consistently the activities of invertase in comparison with the mesocarp and radial pericarp. This is presumably because the different tissues serving different roles. The placental tissue acts as a conduit for nutrients going to the developing seeds, whereas the mesocarp and radial pericarp protect the seeds within the fruit. It is interesting to note that the invertase activity increased dramatically in the final stages of fruit development, especially in the placental tissue. This coincides with a reduction in the sucrose content (Konno et al. 1993). These results suggested that sucrose hydrolysis by invertase was important. It has been shown that, under in vitro conditions, SS was inhibited by high concentrations of fructose, and suggested that this also be the case in vivo (Schaffer and Peterikov 1997). Based on the inverse relationship between sucrose levels and the import rate, it has been suggested that diffusion along a sucrose concentration gradient is probably the driving force for unloading and translocation in the fruit. This sucrose gradient may be maintained by the metabolic conversion of sucrose to starch for storage in the plastid or to hexose for storage in the vacuole. Because hydrolysis of sucrose ar-
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses
riving in the sink is the initial step of sucrose metabolism, invertase and sucrose synthase, catalyzing the breakdown of sucrose, are expected to play a major role (Guan and Harry1991). The growth capacity of tomato plants under salinity and drought is related to the increase in sink activity of young leaves and fruits by the induction of vacuolar acid invertase and sucrose synthase activities (Balibrea et al. 2000; Qi et al. 2003). It has been suggested that invertase activity may play a major role in regulating the rate of carbon translocation in tomato fruit. In tomato, most of the invertase activity is attributable to soluble acid invertase, and the soluble neutral invertase and insoluble acid invertase are negligible (Serge et al. 1991; Husain et al. 2001). The major function of the high and constant invertase activity in red tomato fruit is to maintain the cellular hexose concentrations. This study found that acid invertase and neutral invertase activities were higher under water stress than under salinity and in control, while the SS and SPS activities were slightly different in compared with salinity in the later development stage of fruit. It may be appropriate to speculate that salt stress improved the fruit flavor of tomato which is due to invertase playing a major role. In further research, it showed that the gene expression of acid invertase changed significantly from 45 to 60 DAA. The enzyme activity didn’t coincide quite well with the gene expression. This is presumably because the gene of acid invertase had modification and regulation in the post-transcription. It had reported that fructose and glucose induced to participate in the apple fruit acid-invertase inhibitory post-translational regulation (Wang and Zhang 2002). This showed that the posttranslational modification might play an important role in regulation of the acid-invertase activity. Although only two representative time points in tomato fruit development have been considered in this study, the results showed that increase in fruit flavor and nutritional quality by salinity and water stresses in tomato fruits was related to the sucrolytic activities. But the identification of different isoforms, location, and regulatory mechanisms by endogenous factors, such as hormones or sugars, could be important in order to determine the role of these enzymes in maintaining sink capacity under salinity and drought. They should be also considered in the scope of the tomato sucrose
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metabolism in salinity and drought.
Acknowledgements The work was supported by the National Key Technologies R&D Program of China (2008BADA6B05).
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Liu M C, Chen D K. 2002. Effect of deficit irrigation on yield and quality of cherry tomato. China Vegetables, 6, 4-6. (in Chinese) Liu M C, Kojima T, Tanaka M, Chen H. 2001. Effect of soil moisture on plant growth and fruit properties of strawberry.
Qi H Y, Li T L, Zhang J, Wang L, Chen Y H. 2003. Effects on sucrose metabolism, dry matter distribution and fruit quality of tomato under water deficit. Agricultural Sciences in China, 11, 1253-1258. Schaffer A A, Petreikov M. 1997. Sucrose-to-strach metabolism
Acta Horticulture Sinica, 4, 307-311. (in Chinese) Michel B E, Kaufmann M R. 1973. The osmotic potential of
in tomato fruit undergoing transient starch accumulation. Plant Physiology, 113, 739-746.
polyethylene glycol 6000. Plant Physiology, 51, 914-916. Miron D, Schaffer A A. 1991. Sucrose phosphate synthase, su-
Serge Y, Johnd H. 1988. Sink metabolism in tomato fruit. III. Analysis of carbohydrate assimilation in a wild species. Plant
crose synthase and invertase activities in developing fruit of Lycopersicon esculentum Mill. and sucrose accumulation in
Physiology, 87, 737-740. Serge Y, Roger T C, Martin D, Joseph W D, Alan B B. 1991.
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Sink metabolism in tomato fruit. IV. Genetic and biochemical analysis of sucrose accumulation. Plant Physiology, 95,
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Vassey T L, Sharkey T D. 1989. Mild water stress of Phaseolus vulgaris plants leads to reduced starch synthesis and ex-
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243. (in Chinese) (Edited by ZHAO Qi)
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
September 2009
2009, 8(9): 1046-1052
Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses LU Shao-wei, LI Tian-lai and JIANG Jing The Key Laboratory of Protected Horticulture of Liaoning Province/College of Horticulture, Shenyang Agricultural University, Shenyang 110161, P.R.China
Abstract Changes in sucrose metabolism in response to salt (NaCl) and water (polyethylene glycol, PEG6000) iso-osmotic stresses were measured in tomato cultivar Liaoyuan Duoli (Solanum lycopersicum L.) and the objective was to provide a new evidence for the relationship between salt and osmotic stresses. The carbohydrate contents, as well as sucrose metabolizing enzymes activities and transcript levels were determined. The results indicated that soluble sugar and hexoses accumulated to higher levels and the contents of sucrose and starch were lower in mature fruit under the two stress treatments. Salt and water stresses can enhance the invertase and sucrose synthase activities of tomato fruit in a long period of time (45-60 days after anthesis), and elevate the expression of soluble acid invertase mRNA. It showed that two different stresses could also regulate the soluble acid invertase activity by controlling its gene expression. The activity of sucrose synthase was linked to the changes in soluble sugar levels but not with transcript levels. The effects of salt and water stress treatments on sucrose phosphate synthase activities were weak. Key words: tomato, salt stress, water stress, sucrose metabolism, gene expression
INTRODUCTION Salinity and drought are major abiotic stresses, affect almost every aspect of the physiology and biochemistry of plants, and significantly reduce yield. Great efforts have been devoted to understanding the physiological aspects of response to salinity and drought in plants as a basis for plant breeders to develop salinitytolerant and drought-tolerant genotypes (Cuartero et al. 2006). The yield and quality of tomato appear to be regulated by the net assimilation rate of the crop, the rate of import into individual fruit, and sink activity (Serge and Johnd 1988). High sink demand can significantly increase the quality of the tomato fruit by high accumulation of soluble solids, which is an important factor for Received 2 April, 2009
processing tomatoes. Sugars are the major components of the soluble solids in tomato mature fruit (Ninal and Johnd 1988). Since the water potential of the saline soil and saline water is lowered by the osmotic potential of the dissolved solutes, salt and water stresses produce similar plant responses (Perez-Alfocea et al. 1993). Deficit irrigation can improve strawberry and tomato fruit soluble sugar content, organic acid content, and sugar acid ratio (Shi and Tadashi 2001; Liu et al. 2001; Liu and Chen 2002; Qi et al. 2003). At the early stage of fruit development, salt stress can increase the ordinary tomato fruit soluble sugar accumulation (Balibrea et al. 1997, 2003). Under salinity and drought, the competition between different physiological processes and sink organs for the carbon supplies significantly
Accepted 15 July, 2009
Correspondence LI Tian-lai, Professor, E-mail: [email protected]
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(08)60312-0
Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses
affects overall plant growth and crop yield (Balibrea et al. 1999, 2000; Qi et al. 2003; Jiang et al. 2007). Carbohydrate content and composition are important factors determining tomato fruit quality and flavor. The composition of stored carbohydrate in tomato is associated with certain key enzymes responsible for sucrose metabolism. Invertase and sucrose synthase, catalyzing the breakdown of sucrose, are correlated with the reducing of sugar levels and the rate of carbon translocation into tomato fruit. It has been reported that soluble acid invertase is a major sucrolytic activity in cultivated tomato. This activity is considered to be involved in the composition of stored sugars (Klann et al. 1996; Qi et al. 2005). The sucrose synthase activity is to play an important function in the control of sucrose import and fruit growth parameters (D’Aoust et al. 1999). However, no clear relationship has been shown between salt and water stresses at carbohydrate accumulation and metabolism in tomato fruit. The objective of this work was to study the influences of sucrose metabolism in the different parts of developmental tomato fruit, and what strategies the plant developed to regulate sucrose metabolism, as well as the key enzyme activity and gene expression in sink organs under salt and water stresses during the fruiting period.
MATERIALS AND METHODS Plant materials and treatments
1047
and plants were trained to only one stem, eliminating all axilary buds. The salt stress and water stress treatments were prepared by adding NaCl (50 mmol L-1) and polyethylene glycol (PEG) 6000 iso-osmotic (Nicholas 1989; Michel and Kaufmann 1973) to the tanks (osmotic potential = -0.24 MPa, pH 6.5 ± 0.1). The nutrient solution was changed every 7 days and the levels adjusted every 3 days. The flowers were tagged at anthesis in the first trusses and stress treatments started at 20 days after anthesis (20 DAA). Yield and fruit number were controlled weekly from the beginning to the end of harvest period (about 2 months). Fruits were harvested at the start of ripening (45 DAA) or at the fully ripe stage (60 DAA). The sample fruits were then kept at -86°C until used for further analysis.
Carbohydrate determination Carbohydrate content of fruit was measured using a Waters 600E high-performance liquid chromatograph (HPLC). Carbohydrate column and 2410 refractive index monitor were used. The mobile phase was 75% acetonitrile and ultra water (75:25). The mobile rate was 1.0 mL min-1 and the temperature of the column was 35°C. Water Millennium software was used to handle date. The starch content was measured using perchloric acid hydrolyzed method. Total soluble sugar content was measured using anthrone method. Organic acid content was assayed with 0.1 mol L-1 NaOH titrate.
Enzyme extraction and assay The experiments were conducted in a solar greenhouse of science and research field of Horticultural Department of Shenyang Agricultural University, China. The cultivated tomato hybrid Liaoyuan Duoli (Solanum lycopersicum L.) seeds were sown and grown in plug containing a substrate composed of nutrient soil and vermiculite (2:1, v/v). When the seedlings started to develop the 6th leaf, 30 seedlings of uniform size were transferred to each of two tanks (2 m × 1.5 m × 0.1 m). The containers were filled with 200 L of full-strength Hoagland’s solution (pH 6.5 ± 0.1), where the plants were to be grown. The planting pattern was of 0.5 m between rows and 0.45 m between plants within rows
Enzyme extracts were prepared essentially as described as Minor and Schaffer (1991). Acid (AI; EC 3.2.1.25) and neutral (NI; EC 3.2.1.26) invertases activities were assayed in a final volume of 25 mL, containing 0.2 mL of dialyzed enzymatic extract, 0.8 mL of reaction solution (0.1 mol L-1 Na2HPO4-0.1 mol L-1 sodium citrate, 0.1 mol L-1 sucrose, and pH 4.8 or 7.2 for acid invertase and neutral invertase, respectively). The activities were measured by the quantity of reducing sugars released in the assay media with dinitrosalicylic acid. The reducing sugars were revealed by incubation at 100°C for 5 min and read at 520 nm in a Cary 100UV:VIS
© 2009, CAAS. All rights reserved. Published by Elsevier Ltd.
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spectrophotometer (GBC Scientific Equipment Pty Ltd., Heareus, Germany). Sucrose synthase (SS; EC 2.4.1.13) activity was measured by using 0.4 mL reaction solution (0.05 mol L-1 fructose, 0.82% UDPG, 0.1 mol L-1 Tris, 10 mmol L-1 MgCl2) adding 0.2 mL enzyme at 37°C for 30 min and bathing for 1 min at 100°C, and a volume of 1 mL reaction products adding 0.1 mL 2 mol L-1 NaOH was placed in boiling water bath for 10 min and cooled in water, then adding 3.5 mL 30% HCl and 1 mL 0.1% resorcinol. Blank controls were obtained by adding the distilled water to the reaction medium containing resorcinol. The reducing sugars were revealed by incubation at 80°C for 10 min and read at 480 nm in a Cary 100UV:VIS spectrophotometer (GBC Scientific Equipment Pty Ltd., Heareus, Germany). Sucrose-phosphate synthase (SPS; EC 2.4.1.14) was assayed by measurement of sucrose produced from fructose 6phosphate plus UDP-glucose (Vassey and Sharkey 1989).
RNA isolation and hybridization RNA was extracted using the Trizol Regent (Invitrogen Life Technologies, Carlsbad, CA, USA). Fifteen microgram of total RNA were separated by electrophoresis through 1.2% (w/v) agarose formaldehyde gels and blotted onto a nylon membrane (Hybond-N + ; Amersham-Pharmacia Biotech, USA) by capillary transfer. Probes for the detection of AI (GenBank accession no. AF465613) and SS (GenBank accession no. AJ011535) transcripts were PCR-amplified with primers of AI2F (5´-AACTCCGCCTCTCGTTACACA3´) and AI2R (5´-TAGGATGGTAGCGGACCCTG-3´), SuS2F (5´-AACTTTAGCTGCTCACCGCAA-3´) and SuS2R (5´-TTGCCCTTATAATGGTGAGCG-3´). The gene-specific probes were labeled with DIG using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.
RESULTS Fruit growth and quality At the end of the stress period, NaCl and PEG treatments reduced the single fruit weight by 17 and 23% (as compared with control plants) in mature fruit (Table 1), respectively. This suggests that the discrepancy of differential stress on fruit growth was primarily due to water stress and ionic stress. The content of soluble sugar and titratable acids increased more under water stress than under NaCl stress, the treatments of 50 mmol L-1 NaCl had the highest ratio of brix-acid and the best flavor and quality in mature fruit. It is showed that a certain concentration of salt treatments may improve the quality of the fruit flavor and nutritional quality.
Hexose, sucrose, and starch contents As the fruit developed, the hexose (fructose and glucose) was the predominant sugars, and gradually increased with the highest level presenting at ripe stage (Qi et al. 2005). The salt and water stress also enhance the levels of hexose in the three tissues of tomato mature fruit, which accumulated more hexoses under PEG stress than under NaCl stress (Table 2). In the early stage of development, sucrose and starch were accumulated; but then declined sharply and remained at low level at ripe stage. The contents of sucrose and starch were lower in the three tissues of tomato mature fruit in both stresses than in the control. Salt and water stresses produced similar impact on the concentrations of sucrose and starch (Table 2).
Activities of sucrose-metabolizing related enzymes In order to determine whether part of the changes in
Table 1 Influence of NaCl and PEG6000 iso-osmotic stresses on tomato fruit weight and quality1) Treatment Control NaCl PEG
Soluble sugar (%)
Organic acid (%)
Brix-acid ratio
Fresh weight (mg FW g-1)
2.59 ± 0.21 Aa 3.92 ± 0.14 Bb 4.73 ± 0.18 Bc
0.71 ± 0.02 Aa 0.78 ± 0.05 Aa 0.98 ± 0.07 Ab
3.65 Aa 5.03 Bb 4.83 Bb
208.09 ± 28.17 Aa 172.71 ± 17.92 ABb 159.50 ± 25.72 Bb
Values are the mean ± SD of five replicate samples. A and a indicate significance at P 0.05 and P 0.01, respectively. The same as below.
1)
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Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses
carbohydrate content could be due to an increase in enzymes mobilizing sucrose or hexose, we measured the optimal activities of acid and neutral invertases, sucrose synthase and sucrose phosphate synthase at the start of ripening (45 DAA) and at the fully ripe stage (60 DAA) under PEG and NaCl treatments, respectively. The changes in invertase (acid invertase and neutral invertase) activity during the development of the three tissues of tomato fruit are shown in Fig.1. The invertase activity is generally increased toward the end of
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fruit development with ripening. The major differences among the three parts of fruit were found at the ripening fruit. The invertase activity of fruit was generally higher in the placental tissue than in the mesocarp and radial pericarp. NaCl and PEG treatments can slightly improve neutral invertase activity and significantly improve the acid invertase activity at ripen fruit. The invertase activity is higher under PEG stress than under NaCl stress, this tendency is coincident with that of the hexose content.
Table 2 Influence of NaCl and PEG6000 iso-osmotic stresses on tomato fruit carbohydrate content1) Tissue
Treatment
Mesocarp
Control NaCl PEG Control NaCl PEG Control NaCl PEG
Radial pericarp
Placental tissue
1)
Glucose (mg g-1)
Fructose (mg g -1)
Sucrose (mg g-1)
Starch (mg g-1)
21.93 ± 0.99 Aa 25.07 ± 0.74 Aa 31.73 ± 0.80 Aa 21.62 ± 0.65 Aa 22.67 ± 0.88 Aa 31.38 ± 0.74 Aa 15.40 ± 0.44 Aa 19.33 ± 0.97 Aab 24.70 ± 1.31 Ab
20.25 ± 0.53 Aa 25.16 ± 0.85 Aab 33.99 ± 0.75 Ab 23.06 ± 0.45 Aa 28.71 ± 0.35 Aab 33.13 ± 0.64 Ab 17.40 ± 0.24 Aa 26.68 ± 0.70 Aab 29.75 ± 0.43 Ab
3.16 ± 0.15 Aa 1.72 ± 0.05 Bb 1.89 ± 0.06 ABb 3.27 ± 0.13 Aa 1.39 ± 0.16 Bb 1.67 ± 0.02 Bb 2.11 ± 0.08 Aa 1.18 ± 0.03 Bb 1.43 ± 0.07 ABb
7.63 ± 0.15 Aa 4.66 ± 0.34 ABb 3.77 ± 0.16 Bb 10.41 ± 1.07 Aa 5.88 ± 0.13 Bb 5.35 ± 0.11 Bb 10.24 ± 1.29 Aa 6.62 ± 0.53 Bb 6.34 ± 0.66 Bb
Glucose, fructose, sucrose, and starch were determined in the same samples. Values are the mean ± SD of three replicate samples.
Fig. 1 Effect of NaCl and PEG6000 iso-osmotic stresses on sucrose-metabolizing related enzymes in tomato fruit. The activities of sucrose biosynthetic and mobilizing enzymes were determined in crude extracts in the same samples. Values are the mean ± SD of three replicate samples. AI, acid; NI, neutral; SS, sucrose synthase; SPS, sucrose-phosphate synthase.
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Sucrose synthase (SS) activity was lower at later stages of fruit development. At 45 DAA, SS activity was higher both under PEG and NaCl stresses in the mesocarp; PEG stress increased SS activity in the radial pericarp. SS activity was increased under NaCl and PEG stresses in mature fruit. The SS activity is higher under PEG stress than under NaCl stress, this tendency is coincident with that of the invertase activity. Sucrose phosphate synthase (SPS) was in the same range as the SS, and it also showed low activity at all stages with a small decline toward the end of fruit development increased with ripening. Activity was also greater both in the mesocarp and radial pericarp than in the placental tissue at 45 and 60 DAA. A slight increase in activity was observed under NaCl stress in the mesocarp and radial pericarp. SPS activity was lower under PEG stress than in control in the three tissues of tomato fruit at 45 DAA. The activity stayed approximately the same in the placental tissue under NaCl stress and in the three tissues under PEG stress at ripe fruit.
Gene expression of AI and SS The temporal and spatial expression patterns of acid invertase during tomato fruit development were investigated (Fig.2). In Northern blot analysis, transcriptions of AI were abundant in the placental tissue at 45 and 60 DAA. They were also increased towards the ripening of mesocarp and radial pericarp. After treatments with different stresses, acid invertase gene expression is more complex. There were no detection of gene expression in the mesocarp and radial pericarp at 45 DAA under normal condition and a higher expres-
Fig. 2 Northern blot analysis of total RNA from mesocarp, radial pericarp and placental tissue at 45 and 60 DAA using partial fragment of AI as probe.
LU Shao-wei et al.
sion at 45 DAA and 60 DAA under stress condition (as compared with the control). AI mRNA level was increased more under PEG stress than under NaCl stress. The highest expression activity of acid invertase was found by the PEG stress in the placental tissue at 60 DAA. The tendency is coincident with that of the acid invertase activity. SS transcripts were barely detectable (results not shown) and so were probably too low to show the expected increase of transcripts in tomato fruit in correlation with increased sugar levels and SS activity. The results point to the importance of posttranscriptional regulation.
DISCUSSION A survey was conducted on key sucrose metabolizing enzymes over development in tomato fruit under three different conditions, providing information on the enzymic bases for the accumulation of hexose. The results suggest that, under normal and stress conditions, there were differences in the activities of sucrose metabolism enzymes measured in the mesocarp and radial pericarp in comparison with the placental tissue. The placental tissue had increased consistently the activities of invertase in comparison with the mesocarp and radial pericarp. This is presumably because the different tissues serving different roles. The placental tissue acts as a conduit for nutrients going to the developing seeds, whereas the mesocarp and radial pericarp protect the seeds within the fruit. It is interesting to note that the invertase activity increased dramatically in the final stages of fruit development, especially in the placental tissue. This coincides with a reduction in the sucrose content (Konno et al. 1993). These results suggested that sucrose hydrolysis by invertase was important. It has been shown that, under in vitro conditions, SS was inhibited by high concentrations of fructose, and suggested that this also be the case in vivo (Schaffer and Peterikov 1997). Based on the inverse relationship between sucrose levels and the import rate, it has been suggested that diffusion along a sucrose concentration gradient is probably the driving force for unloading and translocation in the fruit. This sucrose gradient may be maintained by the metabolic conversion of sucrose to starch for storage in the plastid or to hexose for storage in the vacuole. Because hydrolysis of sucrose ar-
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Tomato Key Sucrose Metabolizing Enzyme Activities and Gene Expression Under NaCl and PEG Iso-Osmotic Stresses
riving in the sink is the initial step of sucrose metabolism, invertase and sucrose synthase, catalyzing the breakdown of sucrose, are expected to play a major role (Guan and Harry1991). The growth capacity of tomato plants under salinity and drought is related to the increase in sink activity of young leaves and fruits by the induction of vacuolar acid invertase and sucrose synthase activities (Balibrea et al. 2000; Qi et al. 2003). It has been suggested that invertase activity may play a major role in regulating the rate of carbon translocation in tomato fruit. In tomato, most of the invertase activity is attributable to soluble acid invertase, and the soluble neutral invertase and insoluble acid invertase are negligible (Serge et al. 1991; Husain et al. 2001). The major function of the high and constant invertase activity in red tomato fruit is to maintain the cellular hexose concentrations. This study found that acid invertase and neutral invertase activities were higher under water stress than under salinity and in control, while the SS and SPS activities were slightly different in compared with salinity in the later development stage of fruit. It may be appropriate to speculate that salt stress improved the fruit flavor of tomato which is due to invertase playing a major role. In further research, it showed that the gene expression of acid invertase changed significantly from 45 to 60 DAA. The enzyme activity didn’t coincide quite well with the gene expression. This is presumably because the gene of acid invertase had modification and regulation in the post-transcription. It had reported that fructose and glucose induced to participate in the apple fruit acid-invertase inhibitory post-translational regulation (Wang and Zhang 2002). This showed that the posttranslational modification might play an important role in regulation of the acid-invertase activity. Although only two representative time points in tomato fruit development have been considered in this study, the results showed that increase in fruit flavor and nutritional quality by salinity and water stresses in tomato fruits was related to the sucrolytic activities. But the identification of different isoforms, location, and regulatory mechanisms by endogenous factors, such as hormones or sugars, could be important in order to determine the role of these enzymes in maintaining sink capacity under salinity and drought. They should be also considered in the scope of the tomato sucrose
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metabolism in salinity and drought.
Acknowledgements The work was supported by the National Key Technologies R&D Program of China (2008BADA6B05).
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