Physiological basis of submergence tolerance in rice genotypes with reference to carbohydrate metabolism  

Sidhartha Banerjee , Nirmalya Ghosh , Chiranjib Mandal , Narottam Dey , Malay Kumar Adak
1. Plant Physiology and Molecular Biology Research Unit, Department of Botany, University of Kalyani, Kalyani-741 235, West Bengal, India
2. Department of Biotechnology, Visvabharati, Santiniketan-731 235, West Bengal, India
Author    Correspondence author
Plant Gene and Trait, 2015, Vol. 6, No. 2   doi: 10.5376/pgt.2015.06.0002
Received: 21 Feb., 2015    Accepted: 13 Apr., 2015    Published: 30 Apr., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Banerjee et al., 2015, Physiological basis of submergence tolerance in rice genotypes with reference to carbohydrate metabolism, Plant Gene and Trait, Vol.6, No.2 1-11 (doi: 10.5376/pgt.2015.06.0002)

Abstract

Rice genotypes viz., Swarna Sub1A, FR13A and Swarna were evaluated for their carbohydrate profiles on seven days of submergence. Swarna Sub1A showed minimum increase in proline content than FR13A and Swarna. The depletion of total carbohydrate in roots was recorded having maximum and minimum depletion in Swarna and FR13A respectively. In shoots the depletion was maximum in Swarna Sub1A and minimum in Swarna. Depletion of starch under submergence was maximum in Swarna Sub1A and minimum in Swarna. Sucrose was maximally consumed in Swarna Sub1A and minimally in Swarna in shoots. Invertase activity was increased in roots in Swarna and FR13A but it declined in Swarna Sub1A. In shoots, the activity got declined in Swarna and FR13A and increased in Swarna Sub1A. SPS activity in shoots was down regulated under submergence with maximum and minimum values in FR13A and Swarna respectively. Amylase activity was significantly upregulated in other two varieties but got declined in Swarna Sub1A. Cellulose in root and shoot was accumulated maximally and minimally in Swarna and FR13A respectively. Hemicellulose was accumulated with maximum and minimum values in Swarna Sub1A and in FR13A respectively in roots whereas it was maximum in Swarna Sub1A and minimum in Swarna in shoots.

Abbreviations FW, Fresh weight; DW, Dry weight; DNSA, 3,5 Dinitrosalicylic acid; DTT, Dithiothreitol; MOPS, 3-(N-morpholino) propanesulfonic acid; ATP, Adenosine triphosphate; GA, Gibberellic acid; ABA, Abscissic acid; ROS, Reactive Oxygen Species; SPS, Sucrose Phosphate Synthase.

Keywords
Rice; Starch; Structural carbohydrates; Submergence; Sucrose

Germination of seeds and its subsequent development into seedling fails to grow properly under anoxic conditions when exposed to prolonged period of submergence in rice. The later, being a semi aquatic plant could tolerate partial or complete submergence inducing anoxia for stipulated period. Direct seeding or broadcasting has been a recommended practice for rice cultivation under rain fed conditions. (Farooq et al. 2011). Development of seedlings under complete or partial submergence is constrained by poor crop establishment through lodging sensitivity. Breeding for better crop improvement with satisfactory yield under submerged condition has met limited success due to submergence injury more with oxidative exposure. This is more acute at the post submergence period with recede of water level that exposes the plant with sudden high oxygen tension (Adak et al. 2011). Therefore, analysis and identification of suitable physiological traits linked to submergence tolerance are essentially required for rice varieties. The reserved carbohydrate in the seeds and its subsequent utilization in seedling growth necessitate the required metabolism under anoxia of submergence. In general, hypoxia or even anoxia could be tolerated, however, for some period by the rice plants with more carbohydrate pool than other seeds enriched with fats. This is also supported with the views that anaerobically germinated rice seedlings express various genes specifically for the metabolism of carbohydrates. The deprivation of oxygen tension in the rice roots is thus adopted to utilize ATP production by several folds increase with fermentative (anaerobic) pathways (Bailey-Serres and Voesenek 2010). However, this adaptation is also varied with rice genotypes with respect to timing, duration, and quality of water under which plants are to thrive for a long period (Kende et al. 1998). Therefore, an increased rate of anaerobic carbohydrate metabolism is the primary compulsion to ensure submergence tolerance. Endurance to oxidative damages, a secondary impact to submergence is accomplished by more accumulation of structural carbohydrate in leaf sheath and culm. Therefore, rice genotype with higher deposition of structural carbohydrate on the tissue wall is set as a selective pressure of submergence tolerance (Nagai et al. 2010). In addition maintenance of stored carbohydrate in the seedlings at submergence period linked to moderate shoot elongation at internodes determines the quiescence strategy (Hattori et al. 2009). Thus, a rice variety maintaining higher stored structural carbohydrates as well as carbohydrate constituents in the stems is supposedly proved more stress tolerant, even under oxidative exposure also (Das et al. 2005; Nagai et al. 2010). Therefore, assessment and selection of genetic variations in rice in context to carbohydrate metabolism should be a thrust area for submergence tolerance. Rice exhibits a wider genetic variability in abiotic stress sensitivity and submergence being the predominant of those. Semi tall indica varieties are quite vulnerable to submergence stress with varying degrees of tolerance. (Nishiuchi et al. 2012) Thus, those varieties show a significant reduction in survival, growth and consecutive yield. In the present investigation, the commonly employed indica rice genotypes as cultivated in lowland fallows are discussed in light of carbohydrate metabolism. To materialize this objective, we have selected three indica rice genotypes: Swarna, the susceptible variety; FR13A, the low yielding landrace having submergence tolerant property and Swarna Sub1A, the most recommended promising cultivar developed through breeding. Swarna Sub1A is thought to restrict the elongation ability with some ethylene responsive character (Steffens et al. 2011). In addition, Swarna Sub1A also have two extra alleles, Sub1B and Sub1C in addition to Sub1A. On that perspective, the intolerant semi tall indica is deficit of Sub1A despite the presence of Sub1B and Sub1C. Swarna was initially constructed with the introgression of QTL from FR13A through marker assisted breeding programme. Moreover, submergence tolerance with this factor is directly related with the alternative carbohydrate metabolism by up/ down regulating few concerned enzymes (Xu et al. 2006). Therefore, the comparative assessment of carbohydrate profile and its metabolic aspects may highlight some information with respect to Swarna, naturally tolerant FR13A and introgressed tolerant Swarna Sub1A to submergence stress. To the best expectation, this will be a valid report for the pattern of carbohydrate metabolism when used as selective drive for other indigenous rice cultivars for verification of submergence tolerance.

1 Materials and Methods
1.1 Growth of seedlings and submergence
In order to fulfill the aforesaid objectives, an experiment was conducted in the laboratory of Plant Physiology and Plant molecular Biology Research Unit, Department of Botany, University of Kalyani, Kalyani- 741235, West Bengal, India. Seeds of three rice varieties (namely Swarna, Swarna Sub1A and FR13A) were collected from Rice Research Institute, Chinsurah, West Bengal, India. The seeds were first soaked in 0.1 % mercuric chloride for 15 minutes, followed by rinsing them in distilled water thrice. The seeds were then kept for germination on plastic trays at 37℃ for 5 days. Germinated seedlings of uniform appearance were selected and transplanted to earthen pots containing 5 kg of alluvial soil (containing 58.9, 4.5 and 64.7 mg kg-1 of N, P and K respectively in the form of urea, single super phosphate and muriate of potash respectively) with 7 seedlings per pot. Plants were grown in a green house and subjected to natural conditions of temperature (34-37 ℃), photoperiod (13/11 hour light/dark) with 70-75 % relative humidity. The entire work was carried out during the summer season (April-June 2013). Fourteen-days-old seedlings were submerged for 7 days in a cemented tank under 90 cm depth of water from the floor in order to submerge the plants completely (Panda and Sarkar 2012). After completion of 7 days of submergence, plants were taken out of the water, separated into roots and shoots and the estimation of different metabolites were done according to standard referred protocols.
1.2 Proline estimation
The proline content of the shoot was estimated following the procedure of Bates et al. (1973). Briefly, the shoot samples were extracted using 3 % aqueous sulphosalicylic acid. The supernatant obtained after extraction was mixed with glacial acetic acid and acid-ninhydrin mixture (1.25 g of ninhydrin was dissolved in 30 ml of glacial acetic acid and 20 ml of 6M phosphoric acid) and heated in a boiling water bath for one hour. The reaction was terminated by keeping the reaction mixture in ice bath, followed by the addition of toluene and stirring. The toluene layer was then aspirated and the reaction mixture was warmed to develop the red colour. The changes in absorbance were recorded at 520 nm. The results were obtained from a standard curve of different proline concentrations at 520 nm.
1.3 Estimation of Non structural carbohydrates
Non structural carbohydrates (NSC) include total carbohydrate, starch, sucrose. The total carbohydrate and starch content of the sample were estimated following the procedure of Yoshida et al. (1976). Briefly, for each of the estimation, the shoot and root samples of three plants were ground to a fine powder and extracted using 80 % ethanol (v/v). The extract so obtained was used for the analysis of total carbohydrate content by adding ice cold 2 % (v/v) anthrone reagent. The absorbance was taken at 630 nm using a spectrophotometer Cecil (CE 7200). The residue left after total carbohydrate extraction was dried and extracted using perchloric acid for the analysis of starch content using ice cold 2 % (v/v) anthrone reagent. The absorbance was taken at 630 nm. Using the standard curve of glucose, the total carbohydrate content and starch content was estimated and expressed as mg/g FW.
1.4 Estimation of structural carbohydrates
Structural carbohydrates referred in this experiment include cellulose and hemicellulose. Cellulose content of shoot and root samples was estimated following the procedure of Thimmaiah (1999). The dry powder of the sample was extracted with acetic nitric reagent (150 ml of 80 % acetic acid and 15 ml of conc. Nitric acid) and centrifuged at 12,000 rpm for 20 min. The pellet was saved discarding the supernatant. The pellet was repeatedly washed with distilled water for the detection of soluble sugar. Each time the run off liquid was checked with Dinitrosalicylic acid reagent and iodine solution for the presence of sugar residues. Finally, the washed pellet was mixed with 10 ml of 67 % sulphuric acid and was kept for 1 hour. Finally, the volume was diluted and the cellulose content was analyzed by adding ice cold 2 % (v/v) anthrone reagent. The absorbance was taken at 630 nm. The amount of cellulose was calculated from the standard curve of glucose and expressed as mg cellulose/g DW.
Hemicellulose content of shoot and root samples was estimated adopting the procedure of Loomis and Shull (1937). The dry powder of the sample was extracted in hot 80 % ethanol (v/v). The dried, sugar free residue so obtained, was hydrolyzed using 1(N) HCl and the reducing sugar was treated with ice cold 2 % (v/v) anthrone reagent. The absorbance was taken at 630 nm. The amount of sugar was calculated from the standard curve of glucose and the hemicellulose content was obtained by multiplying the amount of sugar by 0.9 as suggested by Loomis and Shull (1937). Hemicellulose content was expressed as mg hemicellulose/g DW.
1.5 Assay of carbohydrate metabolizing enzymes
The assay of invertase was based on the method of Hubbard et al. (1984). Properly diluted enzyme extract obtained from shoots and roots was reacted with 1.5 ml of assay mixture containing citrate buffer (pH 3.8), sucrose (200 mmol/L) at 37 ℃ of incubation for 90 min. The reaction was terminated with NaOH (1.5 mol/L, pH 6.5) at 100℃ for 30 min. Using the standard curve of glucose, invertase activity was expressed as µg/min/g FW.
Sucrose Phosphate Synthase (SPS) activity was assayed according to Saman et al. (1995). Properly diluted enzyme extract was reacted with 2 ml of reaction mixture containing 10mM UDP-glucose, 10 mmol/L fructose-6-phosphate, 50 mmol/L MOPS (pH 7.9), 15 mmol/L MgCl2, 2.5 mmol/L DTT for 10 min at 25 ℃. The reaction was terminated by adding 30 % KOH in the reaction mixture in a boiling water bath for 10 min. The mixture was treated with ice cold 2 % (v/v) cold anthrone reagent and the absorbance was taken at 620 nm. The enzyme activity was expressed as µmol/min/g protein. Sucrose was assayed according to the modified anthrone method of Van Handel (1968) and expressed as µg/g FW.
The assay of shoot amylase was done adopting the procedure of Peter Bernfield (1955). The shoot sample was extracted using ice cold calcium chloride (10mM) and centrifuged. The supernatant, so obtained, was used as the enzyme source and incubated with equal volume of starch solution at 27 ℃ for 15 min. The reaction was terminated using DNSA reagent and the absorbance was taken at 560 nm. Using the standard curve of maltose, the amylase activity was calculated and expressed as µg/min/g FW.
1.6 Statistical Analysis
All the observations were recorded with three replications (n=3) and data were expressed as mean ± SE. The statistical analysis was performed by one-way (ANOVA) followed by least significance difference (LSD) test with 5 % (p≤0.05) levels of significance (Gomez and Gomez 1984). The ANOVA was done using SPSS (version 10.0) software. Windows Microsoft Excel 2007 software was employed for computation, data analysis and graphics.
2 Results
The accumulation of proline was studied in the plant leaves to observe the submergence induced water deficit in the studied rice varieties. Though proline accumulation is found irrespective of the tissues under stress condition, however, in the present experiment it was recorded in the leaf samples of the seedlings. It showed that proline content was significantly induced by submergence, though variably in the varieties (Figure 1a). Thus, the maximum accumulation of this particular stress responsive amino acid was observed in the variety Swarna with 1.88 fold and minimum in that of Swarna Sub1A by 1.168 fold. FR13A had the intermediatory value by 1.182 fold. On account of proline consideration, Swarna Sub1A had the minimum increase of proline content and thereby assumed to be more sensitive to water deficit stress so developed under submergence. It is true that water deficit in plants is analyzed with the measurement of water potential of the concerned tissues, in real; however, in the present experiment the water potential indirectly measured through the assay of proline content out of non availability of the concerned instrument of water potential. On account of carbohydrate metabolism, we have measured both structural and non structural carbohydrates of the rice varieties under submergence in roots and shoots. A distinct variation of total carbohydrate depletion in roots and shoots under submergence irrespective of varieties was recorded. The variation of total carbohydrate depletion in the plants under submergence as compared to control was recorded maximum in case of shoot than root with 44.36 % and 40.56 % respectively. On varietal performance, the depletion in carbohydrate content was maximum in Swarna (63.6 %) followed by Swarna Sub1A (38.3 %) and FR13A (19.8 %) in case of root (Figure 1b). For shoot, the trend of decline in carbohydrate content was in order of Swarna Sub1A (65.8 %), FR13A (34.7 %) and Swarna (32.6 %) (Figure 1c). Therefore, Swarna Sub1A was evident to exercise more carbohydrate utilization from its shoot. However, the reserved carbohydrates in some varieties were not depleted in rapidity and perhaps making an access for its proper utilization at post submergence period while plants may recover. Starch accumulation recorded a significant down regulation in case of Swarna under submergence and that was 26.5 % less compared to control (Figure 1d). Similarly, the other two varieties namely Swarna Sub1A and FR13A observed the similar trend in depletion of starch content by 53.1 % and 44 % respectively. Therefore, the utilization of the starch for the shoot tissues was maximally recorded in the variety Swarna Sub1A. Sucrose, the most translocable solute is found to be gradually depleted irrespective of varieties under submergence (Figure 1e). On comparative basis, the maximum depletion of sucrose content was found in variety Swarna Sub1A which recorded 53 % less over control. The other two varieties namely Swarna and FR13A recorded 24 % and 44 % utilization of sucrose in shoot respectively. With the view of stored carbohydrate content and its metabolic conversion, plants were analyzed for invertase and amylase activity. Both the enzymes are required to play role in carbohydrate metabolism for a steady supply of reserved carbohydrates as well as transport of soluble sugars in shoots. The activity of invertase that makes an access of continued flow for soluble sugars to the growing tissues was found to be up regulated in roots for rice varieties except Swarna Sub1A (Figure 2a). For a sustained rate of energy yielding pathways, reducing sugar needs to be efficiently supplied to the growing tissues and thus Swarna and FR13A are more induced for invertase activity than Swarna Sub1A. Thus, for Swarna and FR13A, the level of up regulation was 1.90 fold and 1.68 fold respectively. Under similar condition, Swarna Sub1A had declined the activity by 11 % in roots. Invertase activity happens to be inductive for the demand of more reducing sugar for respiratory flux. The elevated level of energy demand may meet the viability of the tissues, specifically in leaf sheath and culm. Therefore, Swarna and FR13A had to compete more to consume soluble sugars for maintaining their growth under submergence than Swarna Sub1A. On the other hand, inductions of invertase activity for hydrolyzing sucrose in the sink tissues and its concomitant transport creates a thrust to produce more sucrose by photo assimilation in leafy shoots. Thus, the activity of invertase was curtailed by 71 % and 27.5 % in Swarna and FR13A respectively but it gained 1.93 fold activity in case of Swarna Sub1A (Figure 2b). Another important enzyme is the Sucrose phosphate synthase (SPS), the key enzyme for the biosynthesis of sucrose. SPS activity was assayed from the leaves and it recorded 33.63 % decrease under submergence irrespective of the varieties. On varietal basis, FR13A was maximally affected (48 % decrease) than Swarna Sub1A (35.3 % decrease) and Swarna (17.6 % decrease) (Figure 2c). The lesser declining trend of SPS activity may mark the distinct feature of Swarna to sustain the sucrose synthesizing capacity even under submergence. The differential behavior of varieties with regards to carbohydrate metabolism could be more illustrated when amylase activities were studied in all the varieties under submergence. Thus, except the Swarna Sub1A, there recorded a fair up regulation of amylase activities by 1.4 fold and 2.8 fold than control in Swarna and FR13A respectively under submergence (Figure 2d). On the contrary, a steady decline in the activity was recorded in Swarna Sub1A by 28 % less than the control. This is more important to observe that the variety Swarna Sub1A could consume lesser sugar in culm and leaf sheath by hydrolysis of starch. This could be taken as an adaptive feature by favoring more starch accumulation in culm for mechanical rigidity of the plants. Since cellulose is the most abundant polysaccharide on cell wall, submergence tolerance is more based on this compound favoring the culm strength. Figure 3a and Figure 3b shows a steady increase in cellulose content althrough the roots and shoots respectively, however, almost in equal extent. The cellulose accumulation was maximum in Swarna for shoots (2.15 fold) and roots (1.368 fold) where as it was minimum in FR13A for shoots (1.13 fold) and roots (1.12 fold). The variety Swarna Sub1A had an intermediatory accumulation of cellulose in shoot (1.29 fold) and root (1.15 fold). So, it appears that to maintain the mechanical strength of the root and shoot, the varieties were almost similarly induced. This also holds true that at early stages of growth, plants are more in favour of reservations of carbohydrate in the form of starch than to allocate into cellulose. Hemicellulose, another form of polysaccharide, constituting of heteropolymeric units are mostly xyloarabinose and xyloglucan. Hemicellulose accumulation draws its attention for its more flexible and easily hydrolysable in nature and thus, may provide more involvement for elongation ability of the plants, particularly under submerged condition. Admitting this, there recorded a steeper increase in hemicellulose content regardless the varieties induced under submergence. Thus, the hemicellulose distribution in roots were promoted under submergence and irrespective of varieties, it recorded 1.142 fold over control. On varietal basis, hemicellulose content was varied with the range of 1.125 fold (in FR13A) to 1.166 fold (in Swarna Sub1A) through 1.137 fold (in Swarna) under submergence (Figure 3c). Similarly, the variation in hemicellulose content among the varieties were significant and ranged from 1.115 fold (in Swarna) to 1.33 fold (in Swarna Sub1A) through 1.157 fold (in FR13A) in shoots (Figure 3d). The formation of cell wall micro fibrils complexed with lignin, may offer more rigidity to the cell wall. Under submergence, the viability of the tissues from degradation is delayed with this trait and more so displayed in the variety Swarna Sub1A in the present experiment.


Figure 1 Changes in proline content (a), total carbohydrate content (b) root and (c) shoot, starch content (d), and sucrose content (e) under 7 days of complete submergence of rice genotypes. The data presented herein with the mean ± S.E of three replicates (n=3). The different letters on each bar denotes the significant differences (p≤0.05). SN-Swarna under normal, SS- Swarna under submergence, SS1N- Swarna Sub1A under normal, SS1S- Swarna Sub1 under submergence, FN- FR13A under normal and FS- FR13A under submergence


Figure 2 Changes in invertase activity (a) root and (b) shoot, SPS activity (c) and amylase activity (d) under 7 days of complete submergence of rice genotypes. The data presented herein with the mean ± S.E of three replicates (n=3). The different letters on each bar denotes the significant differences (p≤0.05). SN-Swarna under normal, SS- Swarna under submergence, SS1N- Swarna Sub1 under normal, SS1S- Swarna Sub1A under submergence, FN- FR13A under normal and FS- FR13A under submergence


Figure 3 Changes in cellulose content (a) root and (b) shoot, hemicellulose content (c) root and (d) shoot under 7 days of complete submergence of rice genotypes. The data presented herein with the mean ± S.E of three replicates (n=3). The different letters on each bar denotes the significant differences (p≤0.05). SN-Swarna under normal, SS- Swarna under submergence, SS1N- Swarna Sub1 under normal, SS1S- Swarna Sub1A under submergence, FN- FR13A under normal and FS- FR13A under submergence

3 Discussion
Submergence tolerance in plants is more related to sustenance of metabolic activities in a steady state condition under anoxia or hypoxia, preliminary, and thereafter coping up with the ROS induced oxidative damages. In rice plants both these exert collectively for the loss of seedling under such condition (Fukao et al. 2011). The hypoxic or even anoxic condition so prevails under submergence, in fact, is the actual bottleneck for maintaining the metabolic activities of rice roots. However, the decaying of roots under anaerobic condition leads to loss of absorbing area of water. In fact, submergence also creates a sort of osmotic deficit of the tissue affected (Fukao et al. 2011). The water stress is related to the fall in root hydraulic conductance of the plants. The root hydraulic conductance, which functions for root membrane permeability to water absorption are either perturbed under submergence by the damage of membrane structures like channel proteins (aquaporins) or disruption of membrane potential (Blokhina et al. 2003). In the present experiment, the moisture stress so developed in rice varieties was documented through the accumulation of proline. A significant rise in the proline content irrespective of rice varieties could establish the osmotic adjustment for developed water stress in the leaf tissues. In our experiment, Swarna Sub1A had recorded minimum increase in proline content than Swarna and FR13A. Therefore, Swarna Sub1A was found to be inclined towards water stress under submergence. Proline, a compatible solute could mark its physiological relevance even under submergence that to encounter the water stress for crop plants. Proline has also been referred as a supportive index for assuming osmotic deficits out of submergence as studied earlier (Mostajeran and Rahimi-Eichi 2009). In addition, the fall in root hydraulic conductance that essentially communicates the shrinkage of leaves not maintaining proper water relation has also been featuring in rice varieties under submergence. Therefore, in the present experiment, the observed flaccid nature of the leaves of the rice varieties may be adjoining with the developed water deficit in the plants. This indirectly may reflect the changes in the proline content in the leaf tissues under submergence.
Submergence becomes more detrimental for plants when water level recedes and becomes a critical factor for seedling’s survival (Kawano et al. 2009). Carbohydrate status in culm is most important criteria for submergence tolerance. The depletion of presubmergence carbohydrate accumulation in the culm may support less elongation of the internodes of the tolerant rice varieties. More importantly, submergence is concerned to the rate of depletion of stored carbohydrate with very rapidity for susceptible varieties and thus makes the plants feeble to stress in subsequent duration. Thereby, the carbohydrate content in Swarna Sub1A, FR13A and Swarna were proportionately accumulated during the period of submergence and their enhanced rate of consumption might be contributory for their sustainability. The sustaining of more carbohydrate and their utilization in proportionate manner is established as a reliable physiological attribute. Since hypoxic condition seriously hampers normal respiration leading to decrease in ATP synthesis thereby taking advantage of anaerobic mode of respiration is the option for plants for their resumed metabolic activity (Ahmed et al. 2013). It is more urgent for the plant to preserve some carbohydrates for elongation and release the energy when the water level is receding to support and resume their further growth. Interestingly, one of the gene expression adhered to Sub1 locus (i.e., Sub1A) is induced by submergence to down regulate the elongation ability as well as accomplish the carbohydrate metabolic activities during desubmergence. The genes under this regulation serve some ABA mediated water stress recovery and inhibition of GA induced internodal elongation as quiescence strategies adopted by plants (Nishiuchi et al. 2012). The starch content and its utilization are indexed as a major contribution amongst the carbohydrate fractions. Starch content, however, represent, a non structural carbohydrate in the shoots has been the most fundamental to attribute rice seedlings tolerance under submergence for long days. To supplement the available energy from starch hydrolysis and subsequent catabolic reaction, starch accumulation in leaf sheath is considered for suitable criteria of selection of rice varieties under submergence. In the present experiment, the more accumulation of starch content in variety Swarna Sub1A under submergence advocates its potential to be more tolerant in comparison to other varieties. In connection of water deficit and to commit osmotic adjustment, plants often use reducing sugars also as compatible solutes (Guglielminetti et al. 2001). Sucrose and its hydrolyzed product by invertase activity and hexosephosphates are most readily offered to plants for such purposes. Sucrose is the most available soluble carbohydrate obtained as current photosynthates in plants. It contributes significantly for energy yielding substrate under submergence and even also the recovery of seedlings during post submergence period (Magneschi and Perata 2009). A significant depletion of sucrose from submerged tissues of rice plants undoubtedly suggests the more energy demand under anoxic condition to make thrive of the plants. The more accumulation and rapid hydrolytic fates of sucrose as a characteristic trait adhered to tolerant varieties had also corroborated in the present study. Thus, Swarna Sub1A was found with more sucrose depletion under submergence than other two varieties. Thus, we find the set of enzymatic activities to support the fermentative carbohydrate metabolism are significantly varied amongst the plants in the present experiment. Of those, invertase is found predominantly in vacuoles, cell wall, and other apoplastic spaces. In addition, invertase being privileged sitting on cell wall and other non cellular paths can easily transport the sucrose from phloem to apoplastic spaces. The increased activity of invertase in shoot draws attention in support of depressed photosynthetic carbon assimilation. A sustained hydrolytic activity on sucrose by invertase for energy yielding metabolism may readily support the demand for glycolytic pathways under submergence, however, through anaerobic reactions. In case of rice, when submergence creates a hypoxic or even anoxic condition, it is the varietals’ efficiency to provide energy for rapidly growing tissues more in internodes and leaf sheath. In the present experiment Swarna Sub1A, which is derived by introgression of Sub1 cuticle from FR13A unexpectedly promised to have more invertase activity. In general, FR13A should be the donor of all improved characters related to submergence tolerance than other land races and thereby this variety is universally regarded as the best submergence tolerance donor. In case of root, the activity of invertase was found to be upregulated in FR13A by several folds under submergence which was depleted in Swarna Sub1A. However, the decline in activity in root of swarna Sub1A under submergence is not statistically significant and thus it appears to be insensitive. On the contrary, Swarna Sub1A had just an opposite trend when invertase activity was assayed from root. A decline in activity though not significant as recorded may possibly be suggestive for other alternative basis. Rice roots could be more vulnerable for anoxic injuries than shoots and thus invertase activity appears to be depressed and thereby the behaviour of FR13A for sustaining more invertase activity promised its fitness under submergence (Panda and Sarkar 2013a). This could be reversed on supplementation of oxygen to the root tissues. Irrespective of any organ has been useful for more sucrose transportation particularly in roots to compensate the osmotic imbalances even under submergence (Dejardin et al. 1999). In addition, sucrose is also metabolized through another pathway which is more energy efficient in plants by Sucrose Phosphate Synthase (SPS). This enzyme biochemically adjoins UDP-glucose and fructose into sucrose phosphate in a reversible way and also establishes itself as a rate regulatory enzyme of current photosynthate biosynthesis in plants, particularly, more with cereal crops (Panda and Sarkar 2013a). The activity of SPS was found to be significantly subdued under submergence and it was maximum in FR13A followed by Swarna, while it was more restored in Swarna Sub1A. The maintenance of stable sucrose synthesis even under depleted photosynthetic rate is undoubtedly a varietal promise towards sustainability under submergence from carbohydrate metabolism. Carbohydrate utilization and its rapid transport are assumed to be over expressed to accommodate photo assimilates in excess from photosynthetic rate per se
(Ram et al. 2002). This also suggest that sucrose biosynthesis may not be bearing any direct correlation with current photosynthetic rate per se, but may be relevant to the biosynthetic pathways involving allocation of reduced carbon moieties into more complex carbohydrate residues through downstream reactions. A distinct correlation was recorded in rice where SPS activity was more close to turn over or hydrolysis of the sucrose into reducing sugars in the culm (or other sink tissues), at the limiting condition of photosynthetic surplus (Panda and Sarkar 2011). This possibly may suggest a high activating state of carbohydrate metabolizing enzymes are more in up regulation state to meet the demand of respiratory substrate under submergence also. Amylase activity is also thought as an added trait in relation to carbohydrate metabolism under submergence. Under oxygen deprivation of submergence stress, alpha amylase activity could be under expressed linking inadequate seed germination and even insufficient flooding tolerance in adult plants (Ismail et al. 2009). The rice varieties possessing Sub1 gene(s) displays a distinguishing flooding tolerance, (more in flash flood situation) by suppressing the activities of alpha amylase in the seeds and thereby can escape the flood exposure. The exceptional role of carbohydrate metabolism exhibited by Sub1 gene antagonizing the amylase activity might be followed up in the seedlings also. Thereby, a distinct fall in alpha amylase activity was recorded in Swarna Sub1A which got some opposite trend in FR13A and Swarna. The decreased activity of the enzyme might possibly be the regulator of starch hydrolysis as supported by other cereal also (Meheta et al. 2010). Under submergence, the down regulation of starch hydrolysis may be supportive for reservation of more carbohydrate in the stem that gives more durability. Irrespective of the rice genotypes, the possession of Sub1A locus along with Sub1B and Sub1C contributes more for carbohydrate metabolism than those of insensitive having only Sub1B and Sub1C (Perata and Voesenek 2007). Thereby, the over expressed activity of amylase in FR13A might be supportive of the fact for more submergence tolerance by existence of Sub1 loci. The storage carbohydrates predominantly refer the starch, cellulose and other allied polysaccharides, which are required for mechanical rigidity of the culm and leaf sheath. For this purpose (like starch accumulation) celluloses and hemicelluloses become the factors to contribute to the stiffness of culm. The hemicellulose is often found to be complexed with lignin for more compression of cell wall residues (Santiago et al. 2013). This possibly is the cause for an increase of cellulose and hemicellulose content both in roots and shoots, to avoid the cellular disintegration or tissue injuries from reactive oxygen species or allied chemical compounds irrespective of the varieties under stress. The cellulose and hemicellulose undergo chemically turned over into soluble residues and contribute in extension of complex carbohydrate polymers for elongation of culm (Panda and Sarkar 2013b). Therefore, the escape strategy of FR13A by elongating the stem under submergence is quite obvious for more accumulation of cellulose and hemicellulose as found in the present experiment and also supported by others. Contextually, Swarna Sub1A which follows the quiescence strategy reduces its internodal length possibly by lesser deposition of cellulose and hemicellulose. However, in other studies taking both tolerant and intolerant rice cultivars, the role of cellulose and hemicellulose for the sensitivity of submergence was clarified. The cellulose and hemicellulose complexed a major structural carbohydrate in addition to lignin showed an involvement for the elongation of the culm. It is quite accepted that FR13A so far has been the most promising and reliable submergence tolerant variety. From FR13A by the introgression of Sub1 QTL into Swarna, the variety Swarna Sub1A has been developed. In few cases, with reference to some parameters, Swarna Sub1A competes well with FR13A as observed in the present experiment. Still, more investigation is required for the proper validation of Swarna Sub1A and other varieties for its tolerance to submergence particularly, in natural field conditions.
4 Conclusion
Therefore, from the above discussion, it becomes prudent that accumulation of carbohydrates and its metabolism happens to be the most crucial in plants under complete inundation. Thus, plants are forced to adopt alternative pathways of carbohydrate metabolism through fermentative reactions. The decline in respiratory flux out of depleted gas exchange coupled with water dehydration stress compelled the plants to use storage carbohydrates. Thus, rice varieties in the present experiment have modulated the rate of carbohydrate utilization with altered enzyme activities of invertase, amylase, SPS etc. On the other hand, the induction and distribution pattern of storage carbohydrates were also effective in support to mechanical strength as well as to compensate the decline in photosynthetic carbon assimilates. All these physiological considerations are adopted by the rice plants to minimize the energy demand for maintaining the tissue viability (Das et al. 2009). Therefore, from the view of carbohydrate metabolism (as revealed from a few observations of the present experiment), submergence tolerance is manifested into plants by précised and controlled mechanism of energy production and its use. The rice varieties of the present experiment also behaved in a similar trend according to their genotypic potential to cope up with the submergence. However, under natural field condition, the situations might be more compounded with other edaphic factors to affect the plant’s survival under submergence.
Acknowledgement
Prof. G. Pal, Dean, Faculty of Science and Professor in Physiology, University of Kalyani, is sincerely acknowledged for his scientific advices to analyze the results. Authors are thankful to Dr. B. Adhikary, Principal Scientist, Rice Research Institute, Chinsurah, West Bengal for providing the seed materials. This work was financially supported by DST-PURSE programme, Department of Science and Technology, New Delhi.
References
Adak M.K., Ghosh N., Dasgupta D.K., and Gupta S., 2011, Impeded carbohydrate metabolism in rice plants under submergence stress, Rice Science, 18: 116-126
http://dx.doi.org/10.1016/S1672-6308(11)60017-6
Ahmed F., Rafii M.Y., and Ismail M.R., 2013, Waterlogging Tolerance of Crops: Breeding, mechanism of Tolerance Molecular Approaches and Future Prospects BioMed Research International, vol Article ID 963525. 10 pages. doi:10.1155/2013/963525
http://dx.doi.org/10.1155/2013/963525
Bailey-Serres J., and Voesenek L.A.C.J., 2010, Life in the balance: a signaling network controlling survival of flooding, Current Opinion in Plant Biology, 13: 489-494
http://dx.doi.org/10.1016/j.pbi.2010.08.002
Bates L.S., Waldren R.P., and Tears I.D., 1973, Rapid determination of free proline for water stress studies, Plant Soil, 39: 205-207
http://dx.doi.org/10.1007/BF00018060
Bernfeld P., 1955, Amylases α and β. Meth Enzymology, 1: 149-158
http://dx.doi.org/10.1016/0076-6879(55)01021-5
Blokhina O., Virolainen E., and Fagerstedt K.V., 2003, Antioxidants Oxidative Damage and Oxygen Deprivation Stress: a Review, Ann Bot, 91: 179-194
http://dx.doi.org/10.1093/aob/mcf118
Das K.K., Panda D., Sarkar R.K., Reddy J.N., and Ismail A.M., 2009, Submergence tolerance in relation to variable floodwater conditions in rice, Environ Exp Bot, 66: 425-434
http://dx.doi.org/10.1016/j.envexpbot.2009.02.015
Das K.K., Sarkar R.K., and Ismail A.M., 2005, Elongation ability and non-structural carbohydrate levels in relation to submergence tolerance in rice, Plant Sci, 168: 131-136
http://dx.doi.org/10.1016/j.plantsci.2004.07.023
Dejardin A., Sokolov L.N., and Kleczkowski L.A., 1999, Sugar/osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis, Biochem J, 344: 503-509
http://dx.doi.org/10.1042/0264-6021:3440503
Farooq M., Siddique K.H.M., Rehman H., Aziz T., Wahid A., and Lee D., 2011, Rice direct seeding: experiences. challenges and opportunities, Soil Till Res, 111: 87-98
http://dx.doi.org/10.1016/j.still.2010.10.008
Fukao T., Yeung E., and Bailey-Serres J., 2011, The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice, Plant Cell, 23: 412-427
http://dx.doi.org/10.1105/tpc.110.080325
Gomez K.A., and Gomez A.A., 1984, Statistical procedures for agricultural research, John Wiley New York
Guglielminetti L., Busilacchi H.A., Perata P., and Alpi A., 2001, Carbohydrate–ethanol transition in cereal grains under anoxia, New Phytologist, 151: 607-612
http://dx.doi.org/10.1046/j.0028-646x.2001.00218.x
Hattori Y., Nagai K., Furukawa S., Song X.J., Kawano R., Sakakibara H., Wu J., Matsuoka T., Yoshimura A., Kitano H., Matsuoka M., Mori H., and Ashikari M., 2009, The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water, Nature, 460: 1026-1030
http://dx.doi.org/10.1038/nature08258
Hubbard N.L., Huber S.C., and Pharr D.M., 1984, Sucrose phosphate synthase and acid invertase as determination of sucrose concentration in developing muskmelon fruits, Plant physiol, 91: 527-1534
Ismail A.M., Ella E.S., Vergara G.V., and Mackill D.J., 2009, Mechanisms associated with tolerance to flooding during germination and early seedling growth in rice (Oryza sativa), Ann Bot, 103: 197-209
http://dx.doi.org/10.1093/aob/mcn211
Kawano N., Ito O., and Sakagami J.I., 2009, Morphological and physiological responses of rice seedlings to complete submergence (flash flooding), Annals of Botany, 103: 161-169
http://dx.doi.org/10.1093/aob/mcn171
Kende H., Van der Knaap E., and Cho H.T., 1998, Deepwater rice: a model plant to study stem elongation, Plant Physiology, 118: 1105-1110
http://dx.doi.org/10.1104/pp.118.4.1105
Magneschi L., and Perata P., 2009, Rice germination and seedling growth in the absence of oxygen, Annals of Botany, 103: 181-196
http://dx.doi.org/10.1093/aob/mcn121
Loomis W.E., and Shull C.A., 1937, Methods in Plant Physiology, pp-290. Mc Graw – Hill publication in botanical science
Meheta P., Jajoo A., Mathur S., and Bharati S., 2010, Chlrophyll a fluorescence study revealing effect of high salt stress on photosystem II in wheat leaves, Plant Physiol Biochem, 48: 16-20
http://dx.doi.org/10.1016/j.plaphy.2009.10.006
Mostajeran A., and Rahimi-Eichi V., 2009, Effects of Drought Stress on Growth and Yield of Rice (Oryza sativa L.) Cultivars and Accumulation of Proline and Soluble Sugars in Sheath and Blades of Their Different Ages Leaves, American-Eurasian J Agric & Environ Sci, 5: 264-272
Nagai K., Hattori Y., and Ashikari M., 2010, Stunt or elongate? two opposite strategies by which rice adapts to floods, Journal of Plant Research, 123: 303-309
http://dx.doi.org/10.1007/s10265-010-0332-7
Nishiuchi S., Yamauchi T., Takahashi H., Kotula L., and Nakazono M., 2012, Mechanisms for coping with submergence and waterlogging in rice, Rice, 5: 2
http://dx.doi.org/10.1186/1939-8433-5-2
Patel S.S., Shah D.B., and Panchal H.J., 2014, Evolutionary studies in sub-families of leguminosae family based on matK gene, Plant Gene and Trait, 5: 1-9
Panda D., and Sarkar R.K., 2011, Non structural carbohydrate metabolism associated with submergence tolerance in rice, Genetics and Plant Physiology, 1: 155-162
Panda D., and Sarkar R.K., 2013a, Mechanism associated with non structural carbohydrate accumulation in submergence tolerant rice (Oryza sativa L.) cultivars, Journal of plant interaction, doi: 10.1080/17429145.2012.763000
http://dx.doi.org/10.1080/17429145.2012.763000
Panda D., and Sarkar R.K., 2013b, Structural Carbohydrates and Lignifications Associated with Submergence Tolerance in Rice (Oryza sativa L.), Journal of stress physiology and biochemistry, 9: 300-306
Perata P., and Voesenek A.C.J.L., 2007, Submergence tolerance in rice requires Sub1A an ethylene-response-factor-like gene, Trends in Plant Science, 12: 43-46
http://dx.doi.org/10.1016/j.tplants.2006.12.005
Ram P.C., Singh B.B., Singh A.K., Ram P., Singh P.N., and Singh H.P., 2002, Submergence tolerance in rainfed lowland rice: physiological basis and prospects for cultivar improvement through marker-aided breeding, Field Crops Research, 76: 131-152
http://dx.doi.org/10.1016/S0378-4290(02)00035-7
Santiago R., Barros-Rios J., and Malvar R.A., 2013, Impact of Cell Wall Composition on Maize Resistance to Pests and Diseases, Int J Mol Sci, 14: 6960-6980
http://dx.doi.org/10.3390/ijms14046960
Saman P., Seneweera A., Basra S., Edward W., Barlow J., and Conroy P., 1995, Diurnal regulation of leaf blade elongation in rice by CO2, Plant Physiol, 108: 1471-1477
Steffens B., Geske T., and Sauter M., 2011, Aerenchyma formation in the rice stem and its promotion by H2O2, New Phytologist, 190: 369-378
http://dx.doi.org/10.1111/j.1469-8137.2010.03496.x
Thimmaiah., 1999, Standard methods of biochemical analysis, Kalayni publisher, New Delhi
Van Handel E., 1968, Direct microdetermination of sucrose, Anal Biochem, 22: 280-283
http://dx.doi.org/10.1016/0003-2697(68)90317-5
Xu K., Xu X., Fukao T., Canlas P., Maghirang-Rodriguez R., Heuer S., Ismail A.M., Bailey-Serres J., Ronald P.C., and Mackill D.J., 2006, Sub1A is an ethylene responsive-factor like gene that confers submergence tolerance to rice, Nature, 442: 705-708
http://dx.doi.org/10.1038/nature04920

Yoshida S., Forno D.A., Cock J.H., and Gomez K.A., 1976, Laboratory manual for physiological studies of rice. pp. 14, 46. Philippines, IRR