Transformation of Sucrose to Starch and Protein in Rice Leaves and Grains under Two Establishment Methods

Available online at www.sciencedirect.com

ScienceDirect Rice Science, 2016, 23(5): 255–265

Transformation of Sucrose to Starch and Protein in Rice Leaves and Grains under Two Establishment Methods Manisha KUMARI, Bavita ASTHIR (Department of Biochemistry, Punjab Agricultural University, Ludhiana 141004, Punjab, India)

Abstract: Six rice varieties, PR120, PR116, FengAiZan, PR115, PAU201 and Punjab Mehak 1 were raised under aerobic and transplanting conditions to assess the effects of planting conditions on sucrose metabolising enzymes in relation to the transformation of free sugars to starch and protein in flag leaves and grains. Activities of sucrose synthase, sucrose phosphate synthase and acid invertase increased till flowering stage in leaves and mid-milky stage (14 d after flowering) in grains and thereafter declined in concomitant with the contents of reducing sugar. Under aerobic conditions, the activities of acid invertase and sucrose synthase (cleavage) significantly decreased in conjunction with the decrease in non-reducing sugars and starch content in all the varieties. Disruption of starch biosynthesis under the influence of aerobic conditions in both leaves and grains and the higher build up of sugars possibly resulted in their favoured utilization in nitrogen metabolism. FengAiZan, PR115 and PR120 maintained higher levels of sucrose synthase enzymes in grains and leaves and contents of metabolites (amino acid, protein and non-reducing sugar) under aerobic conditions, while PR116, Punjab Mehak 1 and PAU201 performed better under transplanting conditions, thus showing their adaptation to environmental stress. Yield gap between aerobic and transplanting rice is attributed primarily to the difference in sink activity and strength. Overall, it appear that up-regulation of sucrose synthase (synthesis) and sucrose phosphate synthase under aerobic conditions might be responsible in enhancing growth and productivity of rice varieties. Key words: aerobic; rice; acid invertase; protein; amino acid; starch; sucrose synthase; sucrose phosphate synthase; water soluble carbohydrate; sugar

Rice (Oryza sativa L.) is one of the most important food crops in the world. Almost half of the world’s population depend on rice as their staple diet, so demand for rice production is still rising because of the continuous increase in population. To sustain present food self-sufficiency and meet future food requirements, rice productivity has to be increased by 3% per annum (Ke et al, 2009; Pyngrope et al, 2013). Rice system consumes 4 × 1011 L water which results in sharp decline in fresh water resources in the world (Tuong and Bouman, 2003). Owing to increased water scarcity and labour cost, a shifting trend towards less water demanding rice i.e aerobic cultivation is the need of the hour. Alternatively, aerobic rice which is established directly from seeds without up-holding of

water offers a promising approach for higher water conservation with reasonable crop productivity (Bernier et al, 2008; Farooq et al, 2011). Water soluble carbohydrates (WSC) mobilises from the leaf during grain filling stages can become an important source of assimilate for grain yield in rice under water deficit conditions (Li et al, 2006). Leaf WSC accumulation is influenced by environmental factors (Winder et al, 1998; Heidary et al, 2007). However, considerable genotypic variation in leaf WSC concentration has been documented and positive relationships between leaf WSC concentration at flowering and wheat grain weight under certain waterlimited environmental conditions, have been observed (Xue et al, 2008). Therefore, high WSC concentration

Received: 17 November 2015; Accepted: 5 May 2016 Corresponding author: Bavita ASTHIR ([email protected]) Copyright © © 2016, 2016, China China National National Rice Rice Research Research Institute. Institute. Hosting Hosting by by Elsevier Elsevier B B.V. Copyright V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer review review under under responsibility responsibility of of China China National National Rice Rice Research Research Institute Institute Peer http://dx.doi.org/10.1016/j.rsci.2016.08.003 http://dx.doi.org/10.1016/j.rsci.2016

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is considered to be a potentially useful trait for improving grain weight and yield in water limited environments. Apparently, senescence induced by water deficits shortens grain-filling period and can result in reductions in grain weight and grain yield (Saeedipour, 2011). Protein content is known to be influenced by many factors, including the genotype and fertilizers, but is mainly influenced by the planting conditions (Meena et al, 2012). Proteins accumulated under stress conditions may provide a storage form of nitrogen that is re-utilized in poststress recovery and also play a role in osmotic adjustments (Danai-Tambhale et al, 2011). Sucrose metabolism plays pivotal roles in development, stress response and yield formation, mainly by generating a range of sugars as metabolites to fuel growth and synthesize essential compounds (Li et al, 2006; Tang et al, 2009). Although sucrose is produced primarily in mature leaves, it can be resynthesized in sink tissues. Sucrose synthesis and breakdown are central to energy sustainability. Sucrose is enzymatically degraded into hexoses to power and support the growth of sinks. There is a compelling evidence that sucrose metabolism is among the key regulatory systems conferring tolerance to abiotic stress (Bala et al, 2010; Ruan et al, 2010). Upon translocation through the phloem to sinks, sucrose is degraded by either invertase or sucrose synthase into hexoses or their derivatives, which are then used in diverse ways. Invertase hydrolyzes sucrose into glucose and fructose whereas sucrose synthase degrades sucrose in the presence of uridine diphosphate (UDP) into UDP-glucose and fructose (Ruan et al, 2014). Although several studies were carried out on aerobic rice system, work regarding the comparative role of carbohydrate metabolism at different developmental stages under both planting conditions was lacking. So biochemical consequences of aerobic conditions mediated by changes in carbohydrate mechanism are worthy of investigation. Therefore, this study aimed to evaluate the changes of sucrose metabolising enzymes in six rice varieties under both aerobic and transplanting conditions.

MATERIALS AND METHODS Rice materials, cultivation and sampling procedure A field study was conducted at the Punjab Agricultural University (PAU), Ludhiana, India (30o56′ N, 75o52′ E, 247 m above the sea level). The

climate is characterised by hot summers and very cold winters. The crop was raised in triplicates under two cropping/water management systems i.e. dry directseeded aerobic (aerobic) and conventional puddled transplanted (transplanting) rice in puddled soils followed by flood irrigations with alternate wetting and drying (flooded) in loamy soil (pH 7.7), which is low in organic carbon and available N, medium in available P and K in plot area of 1 m × 1 m (random block design). Six rice varieties, PR120, PR116, FengAiZan, PR115, PAU201 and Punjab Mehak 1, were raised under two different conditions, i.e. aerobic and transplanting conditions. PAU201 and PR120 have wider adaptability in the region with respect to high yield potential, while PR115, FengAiZan and Punjab Mehak 1 are early vigor and short duration varieties. PR115, PR116, PR120, PAU201 and Punjab Mehak 1 are pure lines developed by PAU whereas FengAiZan is a Chinese line. For aerobic rice, land was prepared with two plowings with a disc harrow and one planking. The aerobic rice was seeded directly at 2–3 cm depth in moist seed bed using 50 kg/hm2 in rows spaced at 20 cm. The aerobic condition was maintained by applying flash irrigation (5 cm) every time when the soil moisture reaches -15 kPa at 15 cm depth. The sowing of transplanted crop was done at 7 d before the direct sowing and 30-day old nursery was transplanted which ensured the same climatic conditions for both the planting systems. Land preparation for conventional transplanted rice consisted of one dry plowing, followed by irrigation and two harrowings to puddle the soil. Transplanting was done in the puddled field in rows with space of 20 cm × 15 cm. The field was ponded for the first 15 d and thereafter, it was repeatedly flood irrigated 2 d after the water infiltrated in the soil until two weeks before harvesting. During the reproductive stage, the irrigation was applied between -10 kPa soil moisture tension. Nitrogen at 120 kg/hm2 (260 kg urea) was applied in three equal splits i.e. 40 kg/hm2 each at puddling, 21 d and 42 d after transplanting. All aerobic direct seeded rice treatments were sprayed twice with 1% ferrous sulfate (250 L/hm2) at 35 and 42 d after transplanting. For the control of annual weeds, Butachlor 50 EC at 3.0 L/hm2 was applied 2 d after transplanting. All other management practices were done as per the recommendations of the Package and Practices of PAU. All enzymatic and nonenzymatic determinations were performed on flag leaf at tillering, flowering, 7, 15 and 30 d after flowering

Manisha KUMARI, et al. Transformation of Sucrose to Starch and Protein in Rice Leaves and Grains

(DAF) and in developing grains at 7, 14, 21, 28 and 35 DAF raised under aerobic and transplanted conditions in triplicates. Samples were wrapped in aluminium foil and stored in plastic bags at -20 ºC. Extraction and assay of enzymes Flag leaves and grains (0.5 g) were homogenized at 0 ºC to 4 ºC in 50 mmol/L HEPES-NaOH buffer (pH 7.5) containing 5 mmol/L MgCl2, 1 mmol/L sodium EDTA, 2.5 mmol/L dithiothreitol, 0.5 mg/mL bovine serum albumin and 0.05% Triton × 100. Homogenates were centrifuged at 15 000 r/min for 20 min and the pellets were re-suspended in extraction buffer and centrifuged. Supernatant was pooled and dialyzed overnight in extraction buffer (3–4 times dilute) using dialyzing tubes. Dialysate was used for enzyme assay. Soluble acid and neutral invertase were assayed from the supernatant according to Schaffer et al (1989) with some modifications. Reaction mixture containing 0.2 mL of 250 mmol/L sucrose, 0.2 mL enzyme preparation and 0.6 mL of 0.2 mol/L sodium acetate buffer (pH 4.8) was incubated at 37 °C for 20 min and the reaction was terminated by addition of 1 mL Nelson reagent C. The amount of reducing sugars released from sucrose was determined by reaction with arsenomolybdate reagent i.e. reagent D (Nelson, 1944) and the concentration of hydrolysed sucrose was calculated by multiplying the reducing sugar concentration by the factor 0.95. The procedure for the assay of soluble neutral invertase was the same as used for soluble acid invertase except that in this case 0.2 mol/L sodium phosphate buffer (pH 7.5) was used in place of sodium acetate buffer (pH 4.8). Sucrose synthase (cleavage) activity was assayed from the supernatant according to Hubbard et al (1989) with some modifications. Reaction mixture (1.0 mL) containing 0.2 mL of 250 mmol/L sucrose, 0.2 mL enzyme extract, 0.1 mL of 20 mmol/L UDP and 0.5 mL of 0.2 mol/L HEPES-NaOH buffer (pH 4.8) was incubated at 37 °C for 30 min and the reaction was terminated by addition of 1 mL Nelson reagent C. The content of reducing sugars released from sucrose was determined by reaction with arsenomolybdate reagent D (Nelson, 1944). The concentration of sucrose hydrolysed was calculated by multiplying the reducing sugar concentration by a factor of 0.95. Sucrose synthase/phosphate synthase activity was assayed from the supernatant according to Hubbard et al (1989) with some modifications. The reaction

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mixture for sucrose synthase (synthesis) contained 150 mmol/L fructose, 20 mmol/L uridine diphosphate glucose (UDPG) in 40 mmol/L HEPES-NaOH buffer (pH 7.5) and 0.2 mL enzyme extract. After incubation at 37 °C for 30 min, 0.1 mL of 30% KOH was added. The contents were heated for 20 min in boiling water bath to destroy free fructose. One millilitre resorcinol (0.15 glacial acetic acid + 0.25 g thiourea) and 3 mL of 30% HCl were added. The contents were mixed thoroughly and incubated at 80 °C for 10 min and absorbance was read at 490 nm. Sucrose phosphate synthase was assayed essentially by the same method used for sucrose synthase (synthesis), except that in this case fructose-6-phosphate was used in place of fructose. Extraction and assay of free amino acids and total soluble proteins Soluble proteins were extracted in 0.1 mol/L NaOH, precipitated with trichloroacetic acid and estimated by the method of Lowry et al (1951). Supernatant was used for the total free amino acid determination as described by Lee and Takahashi (1966). Extraction and assay of free sugars and starch Flag leaves and developing grains were oven dried at 70 °C for 12 h, homogenised in 80% ethanol and placed in water bath at 80 °C for 20 min, then free sugars were extracted sequentially with 80% and 70% ethanol. The extracts containing sugars were concentrated by evaporating off the ethanol under vacuum. The total sugars were estimated calorimetrically using phenol sulphuric acid method described by Dubois et al (1956), and reducing sugars were determined colorimetrically by the Nelson-Somogyi method as described by Roe (1934). Reducing sugar was calculated according to Loomis and Shull (1937) where non-reducing sugar was calculated by subtracting reducing sugar from the total sugars. From free residue of sugars, starch was determined as per Yoshida et al (1976). Sediment of the extract that filtered in sugar content dried, weighed and boiled with deionised water. The starch was estimated from the test extracts with phenol-sulphuric acid reagent method of Dubois et al (1956). Statistical analysis Means ± SD (n = 3) were statistically analysed by Microsoft Excel 2007 and multifactor ANOVA (CPCS1) at the 0.05 level.

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Table 1. Yield attributes and yields of the studied rice varieties. Plant height (cm) Aerobic Transplanted PR120 71 95 PR115 75 100 PR116 67 104 FengAiZan 75 86 PAU201 62 107 Punjab Mehak 1 64 97 CD (5%) Variety 0.69 Condition 0.15 Variety × Condition 1.29 Variety

No. of tillers per m2 Aerobic Transplanted 54 60 52 63 46 67 57 65 48 67 50 65

No. of grains per panicle 1000-grain weight (g) Aerobic Transplanted Aerobic Transplanted 54 60 24.8 26.9 52 63 20.2 24.3 46 69 22.7 28.1 57 51 25.5 24.1 49 67 21.5 26.3 50 65 23.2 25.2

0.65 0.13 1.27

RESULTS Changes in yield attributes and yield Significant decreases in plant height, tiller number, grain number per panicle, 1000-grain weight and yield were observed under aerobic conditions in all the rice varieties except for FengAiZan (Table 1). Under transplanted conditions, PAU201 showed the maximum plant height and tiller number, while PR116 showed the maximum tiller number, grain number per

0.65 0.13 1.27

Grain yield (t/hm2) Aerobic Transplanted 10.9 11.9 11.4 12.2 10.3 13.5 11.6 11.7 9.7 13.1 8.9 12.7

0.47 0.50 1.20

36 76 153

panicle, 1000-grain weight and yield. Under aerobic conditions, FengAiZan showed the maximum values for these yield related traits and yield. Changes in content of chlorophyll, total amino acid, soluble protein and starch Chlorophyll, amino acid, soluble protein and starch contents in leaves increased from tillering to flowering stages and then progressively decreased in all the varieties under both the two conditions (Table 2). In both flag leaves and grains, under transplanting conditions, the higher contents of amino acid and

Table 2. Variation in chlorophyll, amino acid, protein and starch contents in rice flag leaves. Aerobic T F 7 DAF 15 DAF 30 DAF T Chlorophyll PR120 2.55 3.64 3.19 2.45 1.15 2.71 (mg/g) PR115 2.73 3.81 2.97 2.61 1.22 2.99 PR116 2.39 3.37 2.91 2.35 1.05 2.84 FengAiZan 2.47 3.90 3.51 2.79 1.65 2.54 PAU201 2.22 3.22 2.81 2.19 0.94 3.41 Punjab Mehak 1 2.08 3.01 2.65 1.57 0.81 3.05 CD (5%) V (0.28), C (0.21), S (0.47), V × C (0.48), V × S (0.55), C × S (0.09) Amino acid PR120 0.53 0.83 0.68 0.43 0.34 0.28 (%) PR115 0.46 0.76 0.61 0.35 0.24 0.26 PR116 0.39 0.69 0.54 0.29 0.20 0.34 FengAiZan 0.49 0.79 0.64 0.40 0.31 0.24 PAU201 0.44 0.74 0.59 0.37 0.28 0.40 Punjab Mehak 1 0.34 0.65 0.49 0.33 0.26 0.31 CD (5%) V (0.51), C (0.94), S (0.47), V × C (0.08), V × S (0.05), C × S (0.02) Protein PR120 5.21 5.84 4.61 3.40 1.60 3.21 (%) PR115 5.01 5.61 4.42 3.18 1.21 3.11 PR116 4.31 5.06 3.72 2.52 1.50 3.71 FengAiZan 4.75 5.31 4.10 2.93 1.42 2.81 PAU201 3.93 4.54 3.24 2.20 1.03 3.52 Punjab Mehak 1 4.54 5.11 3.92 2.73 0.80 3.87 CD (5%) V (0.08), C (0.09), S (0.67), V × C (0.07), V × S (0.04), C × S (0.08) Starch PR120 7.7 20.3 17.6 13.6 10.5 8.3 (%) PR115 7.1 21.8 18.9 14.8 11.6 7.6 PR116 4.2 14.4 12.5 8.9 8.4 8.6 FengAiZan 6.5 19.1 16.5 12.4 10.2 7.2 PAU201 5.8 17.5 15.2 10.8 9.6 9.1 Punjab Mehak 1 5.8 17.5 15.2 10.8 9.6 8.7 CD (5%) V (0.28), C (0.21), S (0.47), V × C (0.48), V × S (0.55), C × S (0.09) T, Tillering stage; F, Flowering stage; DAF, Days after flowering; V, Variety; C, Condition; S, Stage. Trait

Variety

F 4.01 4.23 4.45 3.97 4.69 4.18

Transplanted 7 DAF 15 DAF 30 DAF 3.41 2.58 1.24 3.08 2.75 1.40 4.10 3.01 2.21 3.75 2.95 1.71 4.31 3.52 2.10 3.94 3.31 1.61

0.50 0.54 0.64 0.58 0.68 0.61

0.36 0.39 0.50 0.42 0.55 0.45

0.21 0.20 0.26 0.24 0.30 0.28

0.20 0.23 0.18 0.22 0.26 0.24

3.72 3.20 4.31 3.50 3.91 4.50

2.20 2.53 3.51 2.72 3.04 3.72

1.91 1.72 2.36 1.84 2.01 2.21

1.24 0.92 1.01 1.14 0.91 0.71

21.4 22.8 25.4 19.7 24.0 27.1

19.1 20.6 21.7 17.4 23.4 24.1

14.5 16.1 16.4 13.1 17.4 16.8

12.5 13.4 13.5 12.1 14.1 13.8

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protein were found in PAU201, Punjab Mehak 1 and PR116 while under aerobic conditions these traits were the maximum in PR120 (Table 2 and Fig. 1). Aerobic conditions led to higher increases in amino acid and protein contents while chlorophyll and starch contents predominated more under transplanting conditions in all the six rice varieties (Table 2). In grains, protein content increased steadily upto 14 DAF and the total amino acid upto 21 DAF under both planting conditions (Fig. 1) and thereafter showed decrease in their contents.

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Changes in sugar content In leaves, reducing and non-reducing sugar contents peaked at flowering stage and then declined towards maturity in all the varieties under both planting conditions (Fig. 2). Under aerobic conditions, FengAiZan, PR120 and PR115 showed higher concentration of reducing and non-reducing sugar whereas under transplanting conditions PAU201, Punjab Mehak 1 and PR116 revealed higher contents of sugars. Invariably, content of non-reducing sugar was higher under transplanting conditions and reducing sugar content was higher

Fig. 1. Variation in contents of protein, amino acid, starch, reducing sugar and non-reducing sugar in rice grains under aerobic and transplanted conditions.

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Fig. 2. Variation in reducing and non-reducing sugar contents in flag leaves of rice varieties under aerobic and transplanted conditions. DAF, Days after flowering.

under aerobic conditions in all varieties. In grains, non-reducing sugars progressively declined during grain development stages in all genotypes under both planting conditions, however, reducing sugars peaked at 14 DAF stage and thereafter decreases towards maturity (Fig. 1). The highest concentrations of reducing sugar, non-reducing sugar and starch were observed in PAU201, Punjab Mehak 1 and PR116 under transplanting conditions and in FengAiZan, PR120 and PR115 under aerobic conditions. Changes in sucrose synthase (cleavage), acid and neutral invertase activity Sucrose synthase (cleavage) and soluble acid invertase activities revealed sharp increase from tillering to flowering stages in leaves and thereafter declined with further leaf developmental stages of all studied varieties under both aerobic and transplanting conditions (Fig. 3). In contrast, neutral invertase activity increased till 7 DAF and thereafter decreased. Aerobic conditions had lower levels of all sucrolytic enzymes as compared to transplanting conditions. In grains, the highest activities of acid invertase and sucrose synthase (cleavage) were recorded at the actively metabolising stage of grain development i.e. at 14 DAF in PAU201, Punjab Mehak 1 and PR116 under transplanting conditions and in FengAiZan, PR120 and PR115 under aerobic conditions (Fig. 4).

Neutral invertase activity revealed its peak at 21 DAF under both planting conditions. Changes in sucrose synthase (synthesis) and sucrose phosphate synthase activity Activities of sucrose synthase (synthesis) and sucrose phosphate synthase increased upto flowering stage in flag leaves and then declined during further development stages in all six rice varieties under both cultivation conditions (Fig. 3). Aerobic conditions led higher upregulation of sucrose synthase/sucrose phosphate synthase activities as compared to transplanted conditions at all stages of leaf development. In developing grains, peak activity of sucrose synthesising enzymes and acid invertase was observed during actively metabolising stage of grain development i.e. mid-milky stage (14 DAF) under both planting conditions in all the varieties (Fig. 4). FengAiZan, PR120 and PR115 showed higher activities of sucrose phosphate synthase and sucrose synthase (synthesis) under aerobic conditions while PAU201, Punjab Mehak 1 and PR116 under transplanting conditions.

DISCUSSION Cultivation of aerobic rice offers an alternative approach to conserve water and reduce labour cost over transplanting ones. Haryanto et al (2008) indicated

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Fig. 3. Variation in activities of sucrose phosphate synthase, sucrose synthase (cleavage), sucrose synthase (synthesis), acid invertase and neutral invertase in flag leaves of rice varieties under aerobic and transplanted conditions. DAF, Days after flowering.

that carbon metabolism is significantly affected under water stress conditions which results in significant yield loss as also observed in aerobic condition. However, plant employs numerous strategies to control water status and resist water loss. For instance, sugars particularly non-reducing sugars and their metabolisms might increase tolerance to water deficit but their

relative contributions may vary genotypically along with different growing conditions at different stages. Although their exact mechanism of action and their association during grain filling under transplanting condition are known, their differential responses under aerobic and transplanting conditions need to be explored. Plant height, tiller proliferation and grain number

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Fig. 4. Variation in activities of sucrose phosphate synthase, sucrose synthase (cleavage), sucrose synthase (synthesis), acid invertase and neutral invertase in grains of rice varieties under aerobic and transplanted conditions.

per panicle are water stress affected traits. In this study, there was a significant reduction in the yield and yield related parameters under aerobic conditions. However, reduction of these parameters was relatively less in FengAiZan, Punjab Mehak 1 and PR115. A significant reduction in plant height may be due to limitation in cell elongation resulting in reduction of internodal length. It is also noticeable that aerobic

conditions reduced the development of tillers and also lead to shortening of grain filling duration which ultimately resulted in decreased yield and quality by decreasing grain weight. Yield gap between aerobic and flooded rice was also attributed primarily to the difference in sink formation, differences in panicle number per plant and 1000-grain weight. Similar decreases in plant height, tiller number, grain number

Manisha KUMARI, et al. Transformation of Sucrose to Starch and Protein in Rice Leaves and Grains

per panicle and 1000-grain weight under stress conditions in rice were reported by Zhao et al (2007), Patel et al (2010) and Vanitha and Mohandass (2014). The transplanting conditions produced higher grain yield particularly in PR116, PAU201 and Punjab Mehak 1. Such effect may result from the reduction of CO2 fixation, decrease of assimilate translocation from leaves and reduction in the expression of starch and protein synthesis genes (Emam et al, 2014). Carbohydrate, which represents one of the major component of the dry matter, was affected under aerobic conditions, with decreases in non-reducing sugar, starch and protein contents of the leaves and grains. Invariably, decline in chlorophyll content lowered photosynthetic rate and net synthesis of sugars in leaves which ultimately decreased starch content in grains. Reduction in chlorophyll content under aerobic condition is due to the suppression of enzymes required for chlorophyll synthesis. Chaum et al (2007) reported that reduction of chlorophyll content in rice plant under salt stress is accompanied by decrease in photosynthetic products. However, the total amino acids and soluble protein got enhanced under aerobic conditions in both leaves and grains that apparently caused a shift in the balance of carbon and nitrogen metabolism by inhibiting starch biosynthesis and so generate carbon skeleton for amino acid biosynthesis. Our results are in the agreement with the earlier reports in rice where increases in protein and amino acid contents under drought conditions was observed (Tang et al, 2008; Meena et al, 2012; Liao et al, 2014). A major perturbation of plant protection under aerobic stress is ascribed to carbon metabolism which is manifested in premature cessation of starch deposition which is not compensated by higher rate of plant growth (Yang et al, 2003; Singh et al, 2008). PAU201, Punjab Mehak 1 and PR116 showed significant reduction in starch synthesis in leaves inspite of sufficient level of non-reducing sugars under aerobic conditions, indicating that supply of assimilate was not responsible for less accumulation of starch but its utilization hindered its deposition. In grains, declines in non-reducing sugars and starch during stress conditions further indicated more utilisation of carbohydrates to overcome stress. The non-reducing sugars in form of sucrose could be a critical sugar conferring water stress tolerance under aerobic conditions both in leaves and grains. This observation is further supported by activity changes of sucrose synthase/sucrose phosphate synthase and invertases, which are key enzymes controlling sucrose

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metabolism (Hubbard et al, 1989). Similar to leaves, more reduction in starch content was observed in grains under aerobic conditions especially in PAU201, Punjab Mehak 1 and PR116, indicating that sucrose metabolising enzymes got significantly disrupted in these varieties. Singh et al (2012) also reported that the drought tolerant varieties had higher starch content than the susceptible varieties around the vascular bundles and periphery region of roots after following water stress conditions. Sucrose synthase (cleavage) activity predominated over invertase in both leaves and grains at all stages under both planting conditions, indicating its pivotal role of the former enzyme in providing carbon for actively growing tissue. Sucrose synthase may also be involved in sucrose synthesis, but the equilibrium is usually in the direction of degradation because of overall lower energy costs (Jayashree et al, 2008; Liu et al, 2012). An appreciable activity of invertases (acidic and neutral) was recorded, and invariably, the activity of acid invertase was higher than that of neutral invertase, indicating active role of acid invertase. The activities of acid invertase and sucrose synthase (cleavage) showed peak values at flowering stage as acid invertase enzyme is related with cell division, and when significant cells are formed, the activity of acid invertase decreased and neutral invertase activity became functional. Thus, the function of invertases in these tissues is to hydrolyze sucrose under condition when there is a high demand for hexoses from source to sink tissues (grains). A similar situation seems to prevail in the wheat leaves under water stress conditions (Saeedipour, 2011). Activities of sucrolytic enzymes decreased significantly under aerobic conditions at all stages of plant growth in all the varieties. FengAiZan, PR120 and PR115 exhibited higher activities of sucrose catabolising enzymes under aerobic conditions, indicating their superior tolerance mechanism under stress conditions (Ruan, 2014). Sucrose synthase (synthesis) and sucrose phosphate synthase activities also revealed a decreasing trend after flowering till leaf and grain maturity which might be due to continuous turnover of sugars to starch and other storage products, and this is in agreement with Liang et al (2001) who reported a decrease in activity of sucrose synthase/sucrose phosphate synthase in developing rice grains. Aerobic conditions led to up-regulation of sucrose synthase (synthesis) in leaves and grains, indicating down-regulation of the genes involved in sucrose utilization (Chen et al, 2005). FengAiZan, PR120 and PR115 showed significantly

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higher activity of sucrose synthase/phosphate synthase under aerobic conditions, which may be correlated to stress tolerant behaviour of these varieties. On the contrary, the activities of sucrose synthase/ phosphate synthase decreases in parallel with decline in non-reducing sugars accumulation which reflects conversion of sugars to starch synthesis. A number of studies have demonstrated that increased sucrose phosphate synthase activity and reduced activity of sucrose hydrolytic enzymes were observed in plants under stress conditions (Fresneau et al, 2007; Reguera et al, 2013). Invariably, content of non-reducing sugars was predominant over reducing sugars in all the varieties at all stages of plant development under both planting conditions. Under aerobic conditions, reducing sugars significantly increased while starch and nonreducing sugars decreased, however, alteration in content of these metabolites was comparatively less in FengAiZan, PR120 and PR115. Soluble sugars allow plants to maximise sufficient carbohydrate storage under transplanting conditions, however, under water stress facilitate vitrification and thus avoid the damage to cells (Parvaiz and Satyawati, 2008). Sugar accumulation is probably associated with the balance between the breakdown and synthesis reaction and is governed by several enzymes. Results of the present study highlight inter-relationship of sucrolysis to grain carbohydrate metabolism for improved grain/sink enhancement whereas aerobic conditions enhanced build up of disaccharide (non-reducing sugars) for maintaining osmotic balance under stress conditions. FengAiZan, PR120 and PR115 seem to be relatively more adapted to aerobic conditions while PAU201, Punjab Mehak 1 and PR116 under transplanting conditions. To conclude, under aerobic conditions, upregulation of sucrose synthase/ phosphate synthase in relation to protein and amino acid contents contributes to adaptation to water stress. However, in transplanted rice, there is a marked enhancement in sucrose synthase cleavage and invertases (acidic and neutral) in parallel with increased levels of sugars and starch which helps in plant establishment. In addition, these varieties can be used as an inbred line to develop more tolerant varieties to various abiotic stress so as to overcome future incoming problems like water scarcity and increasing demand of food.

REFERENCES Bala S, Asthir B, Bains N S. 2010. High temperature response leads to altered membrane permeability in conjunction with

carbon utilization in wheat. Seed Sci Biotech, 4(1): 10–14. Bernier J, Atlin G N, Serraj R, Kumar A, Spaner D. 2008. Breeding upland rice for drought resistance. J Sci Food Agric, 88(6): 927–939. Chaum S, Supaibulwatana K, Kirdmanee C. 2007. Glycinebetaine accumulation, physiological characterizations and growth efficiency in salt-tolerance and salt-sensitive lines of indica rice (Oryza sativa L.) in response to salt stress. J Agron Crop Sci, 193(3): 157–166. Chen S, Hajirezaei M, Bornke F. 2005. Differential expression of sucrose-phosphate synthase iso enzymes in tobacco reflects their functional specialization during dark-governed starch mobilization in source leaves. Plant Physiol, 139(3): 1163–1174. Danai-Tambhale S, Kumar V, Shriram V. 2011. Differential response of two scented indica rice (Oryza sativa) cultivars under salt stress. J Stress Physiol Biochem, 7(4): 387–397. Dubois M, Gilles K A, Hamilton J K, Rebers P A, Smith F. 1956. Colorimetric method for the determination of sugars and related substances. Anal Chem, 28(3): 350–356. Emam M M, Khattab H E, Helal N M, Deraz A E. 2014. Effect of selenium and silicon on yield quality of rice plant grown under drought stress. Aust J Crop Sc, 8(4): 596–605. Farooq M, Siddique K H M, Rehman H, Aziz T, Lee D J, Wahid A. 2011. Rice direct seeding: Experiences, challenges and opportunities. Soil Till Res, 111(2): 87–98. Fresneau C, Ghashghaie J, Gabriel C. 2007. Drought effect on nitrate reductase and sucrose-phosphate synthase activities in wheat (Triticum durum L.): Role of leaf internal CO2. J Exp Bot, 58(11): 2983–2992. Haryanto T A D, Suwarto S, Yoshida T. 2008. Yield stability of aromatic upland rice with high yielding ability in Indonesia. Plant Prod Sci, 11(1): 96–103. Heidary R, Heidary M, Jameiy R. 2007. Study on tolerance to salinity and drought of 4 barley cultivar in germination phase. Res Recon Mag, 4: 34–42. Hubbard N L, Huber S C, Pharr D M. 1989. Sucrose phopshate synthase and acid invertase as determinants of sucrose concentration in developing muskmelon (Cucumis melo L.) fruits. Plant Physiol, 91(4): 1527–1534. Jayashree B, Pradeep R, Kumar A, Gopal B. 2008. Correlation between the sucrose synthase protein subfamilies variations in structure and expression in stress-derived expressed sequence tag datasets. J Proteomic Bioinform, 1(8): 408–423. Ke Y Q, Han G Q, He H Q, Li J X. 2009. Differential regulation of proteins and phosphoproteins in rice under drought stress. Biochem Biophys Res Commun, 379(1): 133–138. Lee Y P, Takahashi T. 1966. An improved colorimetric determination of amino acids with the use of ninhydrin. Anal Biochem, 14(1): 71–77. Li X J, Wang H G, Li H B, Zhang L Y, Teng N J, Lin Q Q, Wang J, Kuang T Y, Li Z S, Li B, Zhang A M, Lin J X. 2006. Awns play a dominant role in carbohydrate production during the grainfilling stages in wheat (Triticum aestivum L.). Physiol Plant, 127: 701–709. Liang J S, Zhang J H, Cao X Z. 2001. Grain sink strength may be related to the poor grain filling of indica-japonica rice (Oryza

Manisha KUMARI, et al. Transformation of Sucrose to Starch and Protein in Rice Leaves and Grains sativa) hybrids. Physiol Plant, 112(4): 470–477. Liao J L, Zhou H W, Zhang H Y, Zhong P A, Huang Y J. 2014. Comparative proteomic analysis of differentially expressed proteins in the early milky stage of rice grains during high temperature stress. J Exp Bot, 65(2): 655–671. Liu Y H, Cao J S, Li G J, Wu X H, Wang B G, Xu P, Hu T T, Lu Z F, Patrick J W, Ruan Y L. 2012. Genotypic differences in pod wall and seed growth relate to invertase activities and assimilate transport pathways in asparagus bean. Ann Bot, 109: 1277–1284. Loomis W E, Shull C A. 1937. Methods in Plant Physiology. Mund W. Sinnot. Mc Graw-Hill Publication in Bbotanical Science: 290. Lowry O H, Rosebrough N J, Farr A L, Randall R J. 1951. Protein measurement with the folin phenol reagent. J Biol Chem, 193(1): 265–275. Meena R K, Verulkar S B, Chandel G. 2012. Nutrient characters analysis in rice genotypes under different environmental conditions. Bull Environ Pharmacol Life Sci, 1: 61–64. Nelson N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem, 153: 375–380. Parvaiz A, Satyawati S. 2008. Salt stress and phyto-biochemical responses of plants: A review. Plant Soil Environ, 54(3): 89–99. Patel D P, Das A, Munda G C, Ghosh P K, Bordoloi J S, Kumar M. 2010. Evaluation of yield and physiological attributes of high yielding rice varieties under aerobic and flood irrigated management practices in mid-hills ecosystem. Agric Water Manage, 97(9): 1269–1276. Pyngrope S, Kumari B, Dubey R S. 2013. Oxidative stress, protein carbonylation, proteolysis and antioxidative defense system as a model for depicting water deficit tolerance in indica rice seedlings. Plant Growth Regul, 69(2): 149–165. Reguera M, Peleg Z, Abdel-Tawab Y M, Tumimbang E B, Delatorre C A, Blumwald E. 2013. Stress-induced cytokinin synthesis increases drought tolerance through the coordinated regulation of carbon and nitrogen assimilation in rice. Plant Physiol, 163(4): 1609–1622. Roe J H. 1934. A colorimetric method for the determination of fructose in blood and urine. J Biol Chem, 107: 15–22. Ruan Y L, Jin Y, Yang Y J, Li G J, Boyer J S. 2010. Sugar input, metabolism, and signaling mediated by invertase: Roles in development, yield potential, and response to drought and heat. Mol Plant, 3(6): 942–955. Ruan Y L. 2014. Sucrose metabolism: Gateway to diverse carbon use and sugar signaling. Annu Rev Plant Biol, 65: 33–67. Saeedipour S. 2011. Effect of drought at the post-flowering stage on remobilization of carbon reserves in two wheat cultivars

265

differing in senescence properties. Afr J Biotech, 10(18): 3549–3557. Schaffer A A, Rylski L, Fogelman M. 1989. Carbohydrate content and sucrose metabolism in developing Solanum muricatum fruits. Phytochem, 28(3): 737–739. Singh S, Asthir B, Bains N S. 2008. High temperature tolerance in relation to carbohydrate metabolism in barley. Ecol Environ Cons, 14: 55–59. Singh N, Kaur R, Sharma N, Mahajan G, Bharaj T S. 2012. Changes in yield and grain quality characteristics of irrigated rice (Oryza sativa) genotypes under aerobic conditions. Ind J Agric Sci, 82: 589–595. Tang R S, Zheng J C, Jin Z Q, Zhang D D, Huang Y H, Chen L G. 2008. Possible correlation between high temperature-induced floret sterility and endogenous levels of IAA, GAs and ABA in rice (Oryza sativa L.). Plant Growth Regul, 54(1): 37–43. Tang T, Xie H, Wang Y X, Lu B, Liang J S. 2009. The effect of sucrose and abscisic acid interaction on sucrose synthase and its relationship of grain filling of rice (Oryza sativa L.). J Exp Bot, 60(9): 2641–2652. Tuong T P, Bouman B A M. 2003. Rice production in water scarce environments. In: Kijne J W, Barker R, Molden D J. Water Productivity in Agriculture: Limits and Opportunities, for Improvement. Colombo, Sri Lanka: International Water Management Institute: 53–67. Vanitha K, Mohandass S. 2014. Drip fertigation could improve source-sink relationship of aerobic rice (Oryza sativa L.). Afr J Agric Res, 9(2): 294–301. Winder T L, Sun J D, Okita T W, Edwards G E. 1998. Evidence for the occurrence of feedback inhibition of photosynthesis in rice. Plant Cell Physiol, 39(8): 813–820. Xue G P, Mclntyre C L, Jenkins C L D, Glassop D, van Herwaarden A F, Shorter R. 2008. Molecular dissection of variation in carbohydrate metabolism related to water-soluble carbohydrate accumulation in stem of wheat. Plant Physiol, 146: 441–454. Yang J C, Zhang J H, Wang Z Q, Zhu Q S, Liu L J. 2003. Activities of enzymes involved in source-to-starch metabolism in rice grains subjected to water stress during filling. Field Crops Res, 81(1): 69−81. Yoshida S, Forno D A, Cock J H, Gomez K A. 1976. Laboratory Manual for Physiological Studies of Rice. Los Banos, the Philippines: International Rice research Institute: 46–49. Zhao D L, Bastiaans L, Atlin G N, Spiertz J H J. 2007. Interaction of genotype × management on vegetative growth and weed suppression of aerobic rice. Field Crops Res, 100: 327–340. (Managing Editor: LI Guan)