Morpho-Physiological Changes in Roots of Rice Seedling upon Submergence

Liem T. Bui, Evangelina S. Ella, Maribel L. Dionisio-Sese, Abdelbagi M. Ismail

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Morpho-Physiological Changes in Roots of Rice Seedling upon Submergence

Liem T. Bui1, Evangelina S. Ella2, Maribel L. Dionisio-Sese3, Abdelbagi M. Ismail2

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Submergence is a serious environmental condition that causes large loss in rice production in rainfed lowland and flood affected area. This study evaluated morphological and physiological responses of rice roots to submergence using two tolerant rice genotypes FR13A and Swarna-Sub1 and two sensitive ones Swarna and IR42. The tolerant genotypes had higher survival rate and less shoot elongation but greater root elongation during submergence than the sensitive ones. After submergence, the tolerant genotypes also had higher root dry weight and more active roots than the sensitive ones. Tolerant genotypes exhibited less root injury, with less malondialdehyde production and slower electrolyte leakage after submergence. Tolerant genotypes also maintained higher concentrations of soluble sugar and starch in roots and shoots and higher chlorophyll retention after submergence than the sensitive ones. Our data showed that root traits such as root activity and root growth are associated with survival rate after submergence. This is probably accomplished through higher energy supply, and membrane integrity is necessary to preserve root function and reduce injury during submergence. These root traits are important for submergence tolerance in rice.

peroxidase; root activity; submergence;rice seedling;gene

Submergence is a serious problem affecting rice production in rainfed lowlands and flood-prone areas worldwide, and this problem is further worsened with climate change, increasing flood risks especially in areas affected by monsoon rains in Asia. Submergence can be caused by direct rain or overflow of rivers, and sometimes, by tidal inundation (Mohanty and Chaudhary, 1986; Sairam et al, 2008). Modern rice varieties can be severely damaged by complete submergence due to oxygen shortage. Under submerged conditions, gaseous diffusion is limited and the depletion of O2can create anoxia in plant tissues (Armstrong, 1979; Setter et al, 1997; Das and Uchimiya, 2002; Ram et al, 2002; Jackson and Ismail, 2015). Moreover, oxygen deprivation causes shift from aerobic to anaerobic metabolism, and this affects the growth and development of rice plants as anaerobic respiration requires high energy supply. Submergence also affects nutrient and water uptake. Changes in metabolism from aerobic to anaerobic respiration injure cells and tissues due to toxic products such as alcohol, aldehydes and lactic acid (Drew, 1997; Setter et al, 1997). Under submergence conditions, oxygen-dependent pathways are suppressed especially the energy generating systems, and this disturbs the functional relationships between organ assimilation and photosynthate utilization (Sarkar et al, 2006).

Since the energy generated by anaerobic respiration is much lower than that generated under aerobic condition, how plants use energy when oxygen is low also relates to flooding tolerance. Plant survival of flooding is also dependent on the amount of carbohydrate stored in plant tissues, and the rate of underwater photosynthesis and utilization of non-structural carbohydrates (NSC; soluble sugars and starch) for growth and maintenance metabolism during submergence (Ella et al, 2003a; Jackson and Ram, 2003; Sarkar et al, 2006; Winkel et al, 2014). Flood-tolerant rice varieties use energy reserves more efficiently and retain higher NSC concentrations in stems and leaves compared with flood-sensitive ones (Das et al, 2005). Also, anaerobic respiration, as alternative energy generating pathway, is induced more strongly in tolerant rice genotypes (Ram et al, 2002; Jackson and Ram, 2003). Submergence-tolerant rice genotypes show slower shoot elongation to conserve energy reserves needed for survival and recovery after desubmergence(Setter and Laureles, 1996). Conversely, shoot elongation is triggered by submergence in sensitive genotypes, causing energy exhaustion, which adversely affects recovery after desubmergence (Setter and Laureles, 1996; Jackson and Ram, 2003; Das et al, 2005; Sarkar et al, 2006; Singh et al, 2009).

Reactive oxygen species (ROS) produced when plants are exposed to oxidative stress after desubmergence, triggers membrane lipid peroxidation and adversely affects membrane functions, causing leakage of electrolytes (Drew, 1997; Rawyler et al, 2002). Effective control of ROS during and following submergence is associated with submergence tolerance in rice (Ella et al, 2003a). Some reports described cytoplasmic acidosis as a cause of cell death in plants exposed to oxygen deficiency (Drew, 1997; Vartapetian and Jackson, 1997; Felle, 2005). Rice genotypes, tolerant of complete submergence, were identified before. These genotypes retain their chlorophyll and adopt a slow-growth strategy depicted by limited elongation when submerged. This enables plants to maintain sufficient carbohydrate reserves to sustain metabolism during submergence and also recovery once the floodwater recedes (Setter and Laureles, 1996; Ella et al, 2003a,b; Das et al, 2005; Fukao et al, 2006; Sarkar et al, 2006; Xu et al, 2006; Perata and Voesenek, 2007). Tolerant rice genotypes carrying the submergence-tolerance alleleon chromosome 9 can endure complete submergence for up to two weeks by limiting leaf extension underwater (Xu and Mackill, 1996; Septiningsih et al, 2009; Singh et al, 2009). The physiological mechanisms, by whichregulates growth, have been thoroughly investigated based on shoot responses (Jackson, 1985; Jackson et al, 1987; Ella et al, 2003a,b; Fukao and Bailey-Serres, 2008a,b; Bailey-Serres et al, 2010; Schmitz et al, 2013).

Studies describing the responses of rice root development to submergence are limited, as compared to shoot development. This study aimed to assess the morphological and physiological changes in roots of rice genotypes contrasting in their submergence tolerance during submergence and the relationship between these changes to shoot growth and submergence tolerance.

Materials and methods

Experimental design

The experiments were conducted in greenhouse and the plant physiology laboratory at Crop and Environmental Science Division (CESD) of the International Rice Research Institute (IRRI), Los Ba?os, the Philippines.

Two separate experiments were set up to analyze several parameters which could not be measured at the same time, and the experiments were conducted using a randomized complete block design with three replications. Experiment I was used for analysis of survival rate,elongation ability, carbohydrate, chlorophyll and malondiadehyde (MDA) concentrations and electrolyte leakage; while Experiment II was used for analysis of root morphology, root tip viability and root peroxidase activity. Plant sampling and measurement of parameters were described detail in analysis methods.

Growth and submergence conditions

Four rice (L) genotypes varying in submergence tolerance were used, two tolerant (FR13A and Swarna-Sub1) and two sensitive (IR42 and Swarna). Seeds were soaked overnight in water then incubated for two more days at 30 oC in the dark. Uniformly germinated seeds were sown in trays containing 6 L soil (2 L sand mixed with4 L garden soil), fertilized with 6 g (NH4)2SO4as N source, 3 g solophosas P2O5source, and 3 g muriate of potash as K2O source with 80 seedlings of each genotype per tray (50cm ×30 cm×15cm). Twenty-one-day-old seedlings (days after soaking, DAS) were submerged in tapwater for 12–14 d in concrete tanks (1m in depth, 6m in width, and 10m in length), and then de-submerged when the sensitive genotype IR42 showed visual symptoms of injury (shoot-root junction starts to become soft). The seedlings were allowed to recover following de-submergence, and survival rate was recorded after 14 d of recovery, with seedlings having at least one new leaf recorded as surviving.

Environmental management and measurement

Weather conditions in the greenhouse and floodwater condition in the concrete tank were monitored daily. These included incident light (photosynthetically active radiation, PAR), relative humidity, and air temperature inside the greenhouse before and after submergence; and light, O2, pH and temperature of floodwater at 5, 50 and 75cm depths in the concrete tanks during submergence. Dissolved O2and floodwater temperature and pH were measured using a dual temperature and O2/pH meter (ORION 230A, Beverly, MA, USA) while incident light was measured using a light meter (LI-COR 250, Lincoln, USA).

Morphological and biochemical analysis

Shoot and root lengths were measured before and after submergence using the longest root. Elongation rates of shoot and root during submergence were calculated. Number of seedlings before submergence and 14 d after desubmergence were used to calculate survival rate.

The viability of root tip cells was determined via rapid staining in a solution of 2,7-dichlorofluorescein diacetate at 200 mg/L acetone, soon after de-submergence (Lascaris and Deacon, 1991; Noland and Mohammed, 1997). Seedlings [before submergence (at 21-day-old), and after submergence of 7 and 12 d] were sampled, and 20 random root tips, each 0.5-cm-long, were excised and wrapped in moistened paper towel, then incubated in the staining solution at room temperature for 15 min in the dark. Samples were subsequently rinsed three times with deionized water and examined for viability using a fluorescent microscope (ZEISS AXIOPLAN2, Oberkochen, Germany) under 450–490 nm blue light (long-pass suppression filter 520 nm). Root tips were examined for fluorescence with ranges from no fluorescence (dying/dead) to very bright and uniform yellow-green fluorescent color over the entire surface of the root tips (viable). Root tips were examined within 15 min after removal from the staining solution. Only viable cells with an intact plasmalemma and functioning esterase enzymes would have cytoplasm that accumulates enough fluorescein to fluoresce brightly (Rotman and Papermaster, 1966).

Leaf, stem and root samples of 21-day-old seedlings (before submergence) and 33-day-old seedlings were harvested and freeze-dried, and dry weights wererecorded before analysis. Chlorophyll and carbohydrates (ethanol-soluble sugars and starch) concentrations were measured before and after submergence. Chlorophyll degradation during submergence was used as a measure of leaf senescence and known to affect plant recovery after de-submergence (Krishnan et al, 1999; Ella et al, 2003b). Chlorophyll concentration was determined on a subsample of leaves harvested before submergence and soon after de-submergence, following the method of Mackinney (1941) and Arnon (1949). Briefly, 20 mg freeze-dried leaf samples were extracted with 20 mL of 80% acetone following 24 h of incubation in the dark. Absorbance of acetone extracts was read at 663, 652 and 645 nm (663,652and645, respectively) then the concentrations of chlorophyll a (a), chlorophyll b (b) anda+bwere determined using the formulae:

a= 12.70×663– 2.69×645

b= 22.90×645– 4.68×663

a+b= (652×1000)/34.5 = 28.99 ×652

Ethanol-soluble sugar and starch concentration in leaf, root and stem tissues were determined before and after submergence. Samples were harvested and frozen in liquid nitrogen and freeze-dried and weighed to obtain their dry weights. The dried samples were extracted in 80% ethanol and used for soluble carbohydrate analysis with anthrone reagent (Fales, 1951). Dry sample (200 mg) was added to 7 mL of 80% ethanol and incubated for 10 min at 80 oC. The mixture was centrifuged for 5 min at 1000 ×, and the supernatant was transferred to volumetric tubes. Incubation was repeated with 7 mL of 80% ethanol. The residue was then washed with 5 mL of hot 80% ethanol and all extracts were combined and filled up to volume of 25 mL with 80% ethanol. The extracts and residues were used for analysis of soluble sugar and starch, respectively.

Soluble sugars were analyzed by adding 0.5 mL extract and 5 mL anthrone reagent. The reaction mixture was rapidly boiled for 10 min and equilibrated to room temperature after cooling on an ice bath for about 5 min, followed by vortexing, and the absorbance of the mixture was read at 620 nm. The residue was dried and used for starch analysis following the method of Setter et al (1989). Approximately 20 mg ethanol-insoluble residue was placed into pyrex tube and boiled for 3 h after adding 2 mL acetate buffer. Another 2 mL acetate buffer with 1 mL amyloglucosidase was added into the pyrex tube and incubated for 24 h at 37 oC. The samples were centrifuged and the supernatant (hydrolysate) was transferred into 25 mL tubes, then the residues were washed two more times with 3 mL distilled water each time. Extracts were combined and filled up to volume of 25 mL. Samples were assayed colorimetrically by reading the absorbance at 450 nm in a reaction of 0.6 mL aliquot of hydrolysate and 3 mL glucose oxidase/peroxidase following incubation in the dark for 30 min as described by Kunst et al (1988).

Injury to the membrane disrupts membrane’s selective permeability and may cause electrolyte leakage. Under submergence, the extent of membrane leakage in roots was assessed by electrical conductivity (EC) of the leachate using an EC meter (HANNA HI 9835 N, Romania) as described by Dionisio-Sese and Tobita (1998). Root samples (200 mg) were cut into pieces(5 mm in length), washed and then placed in test tubes containing 20 mL distilled deionized water. The tubes were covered with plastic caps and placed in a water bath (32 oC). After 2 h, the initial EC of the medium (EC1) was measured. The samples were then autoclaved at 121 oC for 20 min and the final EC (EC2) was measured. The electrolyte leakage (EL) was expressed following the formula: EL (%) = (EC1/EC2) × 100.

MDA is one of the products of lipid peroxidation and was determined by using the modified thiobarbituric acid (TBA) method (Stewart and Bewley, 1980; Dionisio-Sese and Tobita, 1998; Ella et al, 2003a). MDA concentration in roots was determined after 12 d of submergence. About 0.5 g root sample was ground to fine powder with liquid nitrogen. The fine powder was homogenized for 2 min in a total volume of 3 mL with ice-cold 50 mmol/L potassium phosphate buffer (pH 7.0) and centrifuged at 4 oC for 30 min at 22000×. The supernatant was used as the crude extract. An equal volume of 0.5% TBA solution containing 20% trichloroacetic acid (TCA) was added to the crude extract sample. The mixture was heated at 95oC for 30 min and the reaction was stopped by quickly cooled in an ice-bath. The cooled mixture was then centrifuged at 10000 ×for 10 min, and the absorbance of the supernatant was determined at 532 and 600 nm. After subtracting the non-specific absorbance at 600 nm, the MDA concentration was determined using its extinction coefficient of 155 mmol-1·cm-1.

Peroxidase activity in rice roots was tested before and after submergence using α-naphthylamine method (Matsunaka, 1960). About 0.5 g cleaned root was cut into 2 cm pieces (max of 4 cm from the root tip) and placed into a clean dry 50 mL Erlenmeyer flask covered with aluminum foil. About 25 mL reaction reagent (0.1 mol/L phosphate buffer containing 20 mmol/L α-naphthylamine) was added into the flask with excised roots. The flask was swirled slowly at room temperature for 10 min and then shaken in the dark for 3–6 h. An aliquot of 1 mL of the reaction solution was added into a test tube containing 5.0 mL deionized water, 0.5 mL of 1% sulfanilic acid and 0.5 mL of 100 mmol/L sodium nitrite. The amount of α-naphthylamine in the mixture was measured by reading the absorbance at 510 nm.

Statistical analysis

Analysis of variance (ANOVA) of all data,-test and Duncan’s multiple range test were used to analyze non-structural carbohydrate concentrations. Correlation analysis among parameters of survival rate, shoot and root elongation, root MDA concentration, root electrolyte leakage and root peroxidase activity were conducted using R software version 3.4.2.

Results

Greenhouse and floodwater conditions

Weather data in the greenhouse were recorded daily at 11:00 AM during the trials. Average irradiance was 812.0μmol/(m2·s), rangingfrom 630.5 to 1177.8μmol/(m2·s). Light intensity decreased to about 600.3, 286.0 and 172.5 μmol/(m2·s), respectively, with water depths at 5, 50 and 75 cm from water surface. Maximum and minimum air temperatures were 38.2 oC and 36.5 oC, respectively, and water temperatures at 5, 50 and 75 cm depths averaged about 34.7 oC, 33.8 oC and 33.5 oC, respectively. The warm temperature increased algal growth which reduced light penetration with water depth. pH of floodwater also varied slightly (range of 8.25–8.45) with day time and water depth.

Fig. 1. Survival rate and elongation rate during submergence.

A, Plant survival rate after recovery from 12 d of complete submergence. B, Shoot elongation and root elongation during submergence.

Within groups, means followed by different letters are significantly different at the 0.05 level using the Duncan’s multiple range test.Bars are standard errors (= 3).

Survival rate and plant growth after submergence

Significant differences in survival rate were observed between tolerant and sensitive genotypes (Fig. 1-A). Survival rate after submergence in all the genotypes decreased compared to the control, with the greatest reduction in sensitive genotypes. Survival rate was the lowest in IR42 (9%), followed by Swarna (17%), Swarna-Sub1 (78%), and the highest in FR13A (85%). The average temperatures in air and water were warmly,which might reduce the submergence tolerance in all the genotypes, and the plant death appeared earlier.

Shoot elongation rate and root elongation rate during submergence were noted in all the genotypes. The tolerant genotypes FR13A and Swarna-Sub1 had less shoot elongation rate but greater root elongation rate than the sensitive ones Swarna and IR42 (Fig. 1-B).

Fig. 2. Photomicrographs of rice root tips stained with 2,7-dichlorofluorescein diacetate.

Root tips with brighter light are more active than darker ones.

Viability of root tips

All genotypes showed reduced root activity when submerged and the root tips viability decreased after 7d and 12d submergence. Before submergence, all genotypes had similar root tip viability, which then decreased upon flooding. Root tips of the tolerant genotypes relatively maintained viability even after 12d submergence (Fig. 2). Roots of the sensitive genotypes were browner at 7d and 12d submergence, whereas roots of the tolerant genotypes were whiter (data not shown), indicating that most roots of the tolerant genotypes were still functional. Evidently, the tolerant genotypes had more and longer functional roots. Thus, FR13A and Swarna-Sub1 maintained root growth as reflected by an increase in root length and number of active roots, whereas root elongation of the sensitive genotypes remained slow and there were fewer active roots, which could contribute to their reduction in survival rate after 12d submergence.

Dry matter accumulation

Submergence affected growth and dry matter accumulation in all the genotypes. Before submergence, the shoot dry weights of FR13A and IR42 were higher than those of Swarna-Sub1 and Swarna. After 12d submergence, the shoot dry weight of FR13A increased significantly whereas those of the others generally decreased (Fig. 3-A). Root dry weight increased significantly in all the genotypes during submergence (Fig. 3-B). Percentages of shoot and root biomass did not differ much among the genotypes before submergence (Table 1). However, after 12d submergence, all the genotypes showed reduction in percentage of shoot biomass, whereas increase in percentage of root biomass. Changes in percentage of shoot and root biomass were always the highest in IR42 and the lowest in FR13A.

Fig. 3. Physiological parameters before and after submergence.

A, Shoot dry weight before and after 12d submergence. B, Root dry weight before and after 12d submergence. C, Total chlorophyll concentration before and after 12d submergence. D, Effect of submergence on malondialdehyde (MDA) concentration in roots before and after 12d submergence. E, Effect of submergence on electrolyte leakage (EL) in roots before and after 12d submergence. F, Effect of submergence on root peroxidase (POD) activity before submergence and after 7d and 12d of submergence.Within groups, means followed by the samelowercase letters are not significantly different at< 0.05 using the Duncan’s multiple range test.Data are Mean ± SE (= 3).

Table 1. Percentage of shoot and root biomass of four rice genotypes before and after 12 d submergence.

BS, Before submergence; AS, After 12 d submergence. *, Significant at0.05.

Chlorophyll concentration

Chlorophyll concentration decreased significantly under submergence in all the genotypes (Fig. 3-C). Before submergence, Swarna and Swarna-Sub1 had significantly higher chlorophyll concentration than FR13A and IR42. Flooding caused severe damage to leaves especially for the sensitive genotypes. Chlorophyll concentration decreased much more in leaves of the sensitive genotypes especially in IR42, whereas Swarna-Sub1 and FR13A maintained relatively higher concentrations. This reflected faster leaf senescence in the sensitive genotypes than in the tolerant ones after submergence.

Carbohydrate reserves

FR13A and IR42 showed higher carbohydrate than Swarna-Sub1 and Swarna, and carbohydrate decreased only in IR42 roots after 12d submergence(Table 2). Both Swarna-Sub1 and FR13A maintained significantly higher carbohydrate in roots after 12 d submergence than the two sensitive ones. In shoots, all genotypes expressed the reduction of carbohydrate substantially after 12d submergence, but remained relatively higher in the tolerant genotypes (Table 2)

Table 2. Non-structural carbohydrate (starch and soluble sugar) concentrations in rice roots, shoots and seedlings. mg/g.

BS, Before submergence; AS, After 12 d of submergence.

Means in column with the same letters are not significantly different (< 0.05).

Total carbohydrate concentration (starch and soluble sugar) was significantly higher in FR13A seedlings than in the other genotypes before submergence, suggesting that carbohydrate concentration before submergence is not necessarily associated with submergence tolerance conferred by.The tolerant genotypes showed higher carbohydrate concentration than the sensitiveones after submergence (Table 2). Thus, tolerant genotypes seemed to maintain higher carbohydrate concentrations after 12d submergence than the sensitive genotypes.

Roots of FR13A had higher starch concentration before submergence than in the other genotypes (data not shown). Interestingly, starch concentration seemed to increase in roots after 12d submergence, particularly in the tolerant genotypes, with the increase significant in the case of Swarna-Sub1. The increase in starch concentration in roots during submergence might suggest that transport into roots is probably not affected as much as the breakdown of stored starch into sugars under low oxygen stress.

Malondialdehyde and electrolyte leakage in roots

After submergence, the MDA concentrations of roots increased significantly in the sensitive genotypes Swarna and IR42 and also in the tolerant genotype Swarna-Sub1, but not in the tolerant FR13A (Fig.3-D). However, the root MDA concentrations of sensitivegenotypes were significantly higher than the othersafter submergence, suggesting a negative relationship between tolerance and the extent of root injury.

Electrolyte leakage from roots was low and similar in all the genotypes before submergence, but it increased substantially after 12d submergence (Fig.3-E). Similar to the observed trend in MDA concentration, the sensitive genotypes showed significantly higher electrolyte leakage than the tolerant ones after 12d submergence.

Root peroxidase activity

Root peroxidase activity was reduced significantly during submergence in all the genotypes, although it was consistently significantly higher in the two tolerant genotypes especially after 7d submergence (Fig.3-F). After 12d submergence, the tolerant genotypes still maintaining relatively higher root peroxidase activity than the sensitive ones,however, the difference became smaller.The significant reduction in root activity during submergence indicated greater damage in the sensitive genotypes.

Fig. 4. Correlation between seedling survival rate with elongation rate during submergence (A), root peroxidase activity at 7d and 12 d of submergence (B), membrane damage measured as relative electrolyte leakage (C), androot MDA and root electrolyte leakage at 7d of submergence (D).

MDA, Malondialdehyde; EL, Electrolyte leakage.

Correlation between plant survival rate and morpho-physiological parameters

Correlation between seedling survival rate and shoot elongation rate was negative and significant (Fig.4-A). Conversely, correlation of seedling survival rate with root elongation rate was positive and highly significant.

Seedling survival rate also correlated strongly and positively with root peroxidase activity, with higher correlation coefficient when the activity was measured after7 d submergence compared with after 12dsubmergence (Fig.4-B). However, survival rate was reduced over time of flooding and was limited by root peroxidase activity.

Seedling survival rate showed significant negative correlations with MDA concentration (Fig.4-C). In turn, root MDA concentration and electrolyte leakage were correlated significantly and positively (Fig.4-D), suggesting electrolyte leakage is a consequence of increasing root MDA concentration or root injury.

Discussion

Numerous studies have evaluated the morpho-physiological processes associated with rice shoot responses to submergence (Das and Uchimiya, 2002; Ram et al, 2002; Ella et al, 2003b; Jackson and Ram, 2003; Sarkar et al, 2006; Fukao and Bailey-Serres, 2008a,b; Kawano et al, 2009). However, little information is available on root responses under flooded conditions. Roots are more prone to hypoxic or even anoxic stress conditions when the soil becomes waterlogged, particularly at significant flood depths.

Morphological changes

Rice genotypes that exhibit limited elongation rate during submergence are usually more tolerant to submergence (Setter and Laureles, 1996; Ram et al, 2002; Jackson and Ram, 2003; Sarkar et al, 2006). The two tolerant genotypes FR13A and Swarna-Sub1 had less shoot elongation rate during submergence compared with the sensitive ones, Swarna and IR42. This slow elongation rate was associated with higher survival rate. Rice genotypes that undergo rapid root extension growth use more energy, which is usually associated with the depletion of carbohydrate reserves and lower survival rate after submergence.

QTL has been known to suppress shoot elongation under submerged conditions for energy preservation. A negative correlation between shoot elongation rate and survival rate has been reported in numerous studies (Das et al, 2005; Ella and Ismail, 2006; Gautam et al, 2014).

The ability of tolerant genotype to maintain better root growth under flooded conditions is important for their survival, as roots play a vital role in metabolic adaptation, particularly in nutrient uptake and for anchorage. The positive correlation of survival rate with root elongation rate, root tip viability and root peroxidase activity in this study is consistent with the results of Singh et al (2014),which strongly highlights the importance of root traits for survival under submergence conditions.

Our evidence showed thatgenotypes balance between shoot growth and root growth, which enhances plant survival rate under submergence. These results thus highlighted that root growth and functional metabolic responses are also needed to unravel the associations between shoot and root in submergence tolerance.

Physiological changes

Tolerant rice genotypes show a slower growth under flooded conditions, which helps to avoid depletion of energy reserves (Setter and Laureles, 1996; Ram et al, 2002; Jackson and Ram, 2003; Bailey-Serres and Voesenek, 2008). Shoot elongation was expressed under submergence conditions in both sensitive and tolerant genotypes, however,shoot biomass did not increase. This process is not real growth in terms of dry matter accumulation and the biomass lost was due to cellular leakage and tissue decay. In contrast, the root elongation was much less than shoot elongation, but root biomass still accumulated during submergence.

In fact, submergence suppresses dry matter accumulation in both sensitive and tolerant genotypes, however, data on dry weight from FR13A and the other genotypes did not explain its contribution to plant survival rate after submergence. This might be due to additional QTLs, aside from, which contribute to the enhanced submergence tolerance of FR13A. The significant increases in root dry weights of tolerant FR13A and Swarna-Sub1 during submergence also suggest the importance of root growth for submergence tolerance in rice to ensure a sufficient nutrient uptake.

Reductions in chlorophyll concentrations in the leaves of both sensitive and tolerant genotypes during submergence have been reported with relatively greater effects on sensitive genotypes (Krishnan et al, 1999; Ella et al, 2003b). Ella and Ismail (2006) found that an enhancement in chlorophyll concentration before submergence by the addition of higher amounts of N fertilizer in both tolerant and sensitive genotypes does not improve survival rate, but conversely enhance mortality because of excessive growth and energy depletion before submergence. In this study, sensitive genotypes under submergence showed increased levels of leaf chlorosis which reduced photosynthesis underwater. Shoot elongation during submergence enhanced by gaseous ethylene also triggered leaf senescence associated with chlorophyll degradation. Maintaining chlorophyll concentration under submergence conditions is essential for plant survival and recovery because it ensures photosynthesis under water and continues plant growth recovery after de-submergence (Ella and Ismail, 2006; Singh et al, 2014). A loss of chlorophyll concentration also contributed to a reduction in seedling survival rate after submergence in this study,which indicated thatmight also be involved in chlorophyll catabolism under submergence.

High concentrations of carbohydrates, particularly after submergence, play a crucial role in seedling survival when floodwater has receded (Ram et al, 2002; Jackson and Ram, 2003; Das et al, 2005; Sarkar et al, 2006; Das et al, 2009; Gautam et al, 2016). In this study, carbohydrate concentration in roots increased during submergence. Conversely, the carbohydrateconcentration decreased substantially in the shoots of all genotypes but tolerant genotypes still maintained more carbohydrate reserves than sensitive ones after desubmergence. This suggests that tolerant rice genotypes store more carbohydrates in roots, which could be essential to maintain root growth and the active uptake of nutrients for further growth during the recovery stage. The high soluble sugar and starch concentrations in the tolerant genotypes following submergence suggest the importance of energy reserves during the recovery period, thus resulting in a higher survival rate. These results are in accordance with Das et al (2005, 2009). The carbohydrate dynamics data indicatedis a master regulator which regulates carbohydrate consumption and storage sufficiently for both shoot and root growth during and after submergence.

has been reported to be a key player in conferring submergence tolerance which adopts a ‘quiescence’ strategy under flooding (Perata and Voesenek, 2007; Bailey-Serres and Voesenek, 2008; Septiningsih et al, 2009; Singh et al, 2014).is involved in several cellular regulations such as carbohydrate metabolism, oxidative stress protection during and after submergence (Ella et al, 2003a; Das et al, 2009; Panda and Sarkar, 2012a, b; Gautam et al, 2016). Non-structural carbohydrate (soluble sugar and starch) showed a reduction under submergence conditions which was greater in sensitive genotypes especially at the time of de-submergence. This could be explained by energy conservation regulated by. Shoot carbohydrate metabolism confirmed that the tolerant genotypes (with) preserve energy resources more efficiently than the sensitive ones, whichis consistent with Panda and Sarkar (2012b). In contrast, root carbohydrates concentrations increased after submergence in both tolerant and sensitive genotypes except for IR42, but carbohydrate concentrations were higher in tolerant genotypes. This could contribute to the preservation and mobilization of carbohydrate resources at the whole plant level.

Under oxygen deficiency, plants shift from aerobic respiration to anaerobic fermentation pathways to provide minimum energy sources for survival (Davies, 1980; Drew, 1997; Ram et al, 2002; Jackson and Ram, 2003; Bailey-Serres and Voesenek, 2008; Ismail et al, 2009). This process can produce toxic substances as fermentative products or its by-products such as aldehydes, alcohols and lactic acids (Dennis et al, 2000; Ram et al, 2002; Gibbs and Greenway, 2003). Toxic substances together with other reactive oxygen species generated during stress can damage the endomembrane systems, resulting in the loss of the cellular integrity and compartmentation required for vital processes. A loss of membrane selectivity might trigger a leakage of cellular electrolytes associated with cell death during stress. Dionisio-Sese and Tobita (1998) and Ella et al (2003a, 2010) used MDA as an indicator of lipid peroxidation and membrane damage during stress. An increased lipid peroxidation, as indicated by root MDA concentration clearly showed oxidative stress induction under flooded conditions in this study. Submergence resulted in an increase in MDA concentration in the root tissues of all genotypes, but to a greater extent in the sensitive ones, Swarna and IR42. This implies that tolerant genotypes have better detoxification mechanisms, which protect cellular membranes from the toxic products generated during stress. This is further manifested by the reduced electrolyte leakage from the roots of tolerant genotypes after submergence. In addition, there was a strongly negative correlation of survival rate with root MDA concentration and electrolyte leakage, whereas a positive correlation was observed between root MDA concentration and electrolyte leakage. The tolerant genotypes had more active and functional roots, less MDA concentration and electrolyte leakage compared to the sensitive ones. These observations indicated that root electrolyte leakage was the consequence of root injury and an increase in MDA content. The tolerant genotypes seem to have better protective mechanisms, which help maintain the integrity of cellular membranes under stress.

Peroxidase (EC1.11.1) is known to regulate growth in various ways, as it is associated with cell elongation processes and responses that restrict growth (Maksimovic et al, 2008). Peroxidase activity has been associated with root activity and survival rate, and is also closely related with root respiration. A higher peroxidase activity is indicative of a higher respiratory activity of the roots. The roots of sensitive genotypes under submergence have shown lower peroxidase activity, and shoot elongation rate and peroxidase activity is negatively associated (Lee and Lin, 1996). Shoot elongation rate is mediated by the ethylene generated during submergence through processes involving the modulation of gibberellin and abscisic acid balances (Bailey-Serres and Voesenek, 2008; Fukao and Bailey-Serres, 2008b; Jackson, 2008). Ethylene enhances shoot elongation, while peroxidase suppresses this process.

In this study,the tolerant genotypes FR13A and Swarna-Sub1 showed limited shoot elongation rate during submergence, but enhanced root growth and activity. This suggests that the growths of root and shoot are regulated differently under submergence in these tolerant genotypes. The inhibition of shoot elongation coupled with high peroxidase activity is associated with higher survival rate.

Ella et al (2003a) and Panda and Sarkar (2012a) highlighted the important role of antioxidants and the ROS scavenging system when rice is subjected to submergence.genotypes improved photosynthesis activities under water and maintained higher levels of antioxidant enzymes, which protect cellular components from ROS submergence-induction. Our data on root activity and injury support the evidence thatQTL may also be involved in these processes.genotypes showed better root tip viability and respiration, and less tissue damage by a lower MDA content and electrolyte leakage.

Conclusions

Tolerant genotypes showed better root traits such as longer, whiter and more viable roots compared with the sensitive ones. Tolerant genotypes also used more available carbohydrate resources for essential metabolic processes such as growth maintenance under submergence, as in the case of FR13A which still accumulated dry matter, whereas the other genotypes showed negative growth. Although the root biomass of all the genotypes still increased during submergence, seedling survival rate was lower in sensitive genotypes. This could be due to the depletion of carbohydrates for recovery as well as the functional loss of defense systems which triggered tissue injuries in both the shoots and roots of sensitive genotypes. Carbohydrate resources may also be used for protective activities and thus tolerant genotypes had better protective mechanisms to reduce root injury through the protection of the cellular membrane from lipid peroxidation. Tolerant genotypes also exhibited higher root activities, such as root tip viability and root peroxidase, thus resulting in less damage in both the roots and shoots. Overall, our results highlighted the crucial role ofQTL as a key player in conferring submergence tolerance in rice.not only regulates the shoots but also the root physiology under flooded conditions. This study highlighted the importance of continued root growth and activity such as elongation, biomass and viability for submergence survival in rice.

Acknowledgements

We thank Lamberto Licardo and James Afuang Egdanc for their technical assistance, Blue lab staff for their guidance in using equipment. This work was partially supported by German Federal Ministry from Economic Cooperation and Development (BMZ).

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