Waterlogging-induced increase in sugar mobilization, fermentation, and related gene expression in the roots of mung bean (Vigna radiata)

ARTICLE IN PRESS Journal of Plant Physiology 166 (2009) 602—616

www.elsevier.de/jplph

Waterlogging-induced increase in sugar mobilization, fermentation, and related gene expression in the roots of mung bean (Vigna radiata) Raj K. Sairama,, Kumutha Dharmara, Viswanathan Chinnusamyb, Ramesh C. Meenaa a

Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi-110 012, India Water Technology Centre, Indian Agricultural Research Institute, New Delhi-110 012, India

b

Received 4 July 2008; received in revised form 5 September 2008; accepted 5 September 2008

KEYWORD Alcohol dehydrogenase; Gene expression; Mung bean; Sucrose synthase; Waterlogging

Summary The objective of this study was to examine the role of root carbohydrate levels and metabolism in the waterlogging tolerance of contrasting mung bean genotypes. An experiment was conducted with two cultivated mung bean (Vigna radiata) genotypes viz., T44 (tolerant) and Pusa Baisakhi (PB) (susceptible), and a wild Vigna species Vigna luteola under pot-culture to study the physiological and molecular mechanism of waterlogging tolerance. Waterlogging resulted in decrease in relative water content (RWC), membrane stability index (MSI) in root and leaf tissues, and chlorophyll (Chl) content in leaves, while the Chl a/b ratio increased. Waterlogginginduced decline in RWC, MSI, Chl and increase in Chl a/b ratio was greater in PB than V. luteola and T44. Waterlogging caused decline in total and non-reducing sugars in all the genotypes and reducing sugars in PB, while the content of reducing sugar increased in V. luteola and T44. The pattern of variation in reducing sugar content in the 3 genotypes was parallel to sucrose synthase (SS) activity. V. luteola and T44 also showed fewer declines in total and non-reducing sugars and greater increase in reducing sugar and SS activity than PB. Activity of alcohol dehydrogenase (ADH) increased up to 8 d of waterlogging in V. luteola and T44, while in PB a marginal increase was observed only up to 4 d of treatment. Gene expression studies done by

Abbreviations: ADH, Alcohol dehydrogenase; AEC, Adenylate energy charge; Chl, Chlorophyll; DMSO, Dimethyl sulfoxide; DTT, Dithiothreitol; EDTA, Ethylene diamine tetra-acetic acid di-sodium salt; HEPES, N-2-hydroxyethyl piperazine-N-2-ethanesulphonic acid; MSI, Membrane stability index; PB, Pusa Baisakhi; PMSF, Phenyl methane sulphonyl fluoride; RWC, Relative water content; SS, Sucrose synthase. Corresponding author. Tel.: +91 11 25842815; fax: +91 11 25738766. E-mail address: [email protected] (R.K. Sairam). 0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.09.005

ARTICLE IN PRESS Waterlogging-induced sugar metabolism and genes in mung bean roots

603

RT-PCR in 24 h waterlogged plants showed enhanced expression of ADH and SS in the roots of V. luteola and T44, while in PB there was no change in expression level in control or treated plants. PCR band products were cloned and sequenced, and partial cDNAs of 531, 626, and 667; 702, 736, and 744 bp of SS and ADH, respectively were obtained. The partial cDNA sequences of cloned SS genes showed 93–100 homologies among different genotypes and with D10266, while in case of ADH the similarity was in the range of 97–100% amongst each other and with Z23170. The results suggest that the availability of sufficient sugar reserve in the roots, activity of SS to provide reducing sugars for glycolytic activity and ADH for the recycling of NADH, and for the continuation of glycolysis, could be one of the important mechanisms of waterlogging tolerance of V. radiata genotype T44 and wild species V. luteola. This was reflected in better RWC and Chl content in leaves, and membrane stability of leaf and root tissue in V. luteola and T44. & 2008 Elsevier GmbH. All rights reserved.

Introduction Waterlogging is a serious problem affecting crop growth and productivity. Waterlogging blocks the oxygen supply to the roots thus inhibiting root respiration, resulting in a severe decline in energy status of root cells affecting important metabolic processes of plants. Plants react to an absence of oxygen by switching from an oxidative to a solely substrate-level phosphorylation of ADP to ATP; the latter reactions predominantly involve glycolysis and fermentation. The overall yield of ATP produced during fermentation is only two molecules of ATP per glucose molecule as against 38 molecules of ATP produced during oxidative phosphorylation. The anaerobic response of maize roots cells studied using two-dimensional electrophoresis revealed a set of about 20 anaerobic proteins, which were synthesized during low oxygen treatment, while synthesis of the normal aerobic proteins was drastically repressed (Sachs et al., 1980). Many of these induced proteins were subsequently identified as enzymes of the glycolytic and fermentation pathways (Kelley, 1989; Dolferus et al., 2003). The identified ANP include sucrose synthase (SS), phosphohexose isomerase, fructose-1,6-diphosphate aldolase, pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH) (Johnson et al., 1994; Chung and Ferl, 1999; Zeng et al., 1999). Ethanol has been shown by a number of research workers to be the major product of fermentation in rice seedlings (Menegus et al., 1993; Ricard et al., 1994). The dominance of alcoholic fermentation is further supported by rapid increases in the activity of ADH in germinating seed of rice (Reggiani et al., 1986; Rivoal et al., 1989) when oxygen supply was severely restricted.

SS, which catalyzes the reversible conversion of sucrose and UDP to UDP-glucose and fructose, plays a crucial role in providing an adequate sugar supply during anoxic stress (Springer et al., 1986). Meristem death of maize seedlings was significantly alleviated in the presence of glucose in the anoxic incubation medium, indicating that an increase in glycolytic flux alone was sufficient to restore root tip viability (Ricard et al., 1998). Guglielminetti et al. (1995) reported that during the anoxic germination of rice invertase activity was depressed and that of SS enhanced, and suggested that SS was the enzyme mainly responsible for sucrose breakdown under anoxia. The essentiality of SS for anoxic tolerance has further been demonstrated in maize using double mutants of the enzyme (Ricard et al., 1998). Mung bean, an important summer–rainy season pulse crop, is exposed to waterlogging condition during germination and early vegetative growth phases. In spite of the seriousness of the problem, very little physiological and molecular studies have been done on the effect of waterlogging in legumes, except for soybean and pea. Since glycolytic–fermentation pathway is the only source of energy, which in turn yield only 2 ATP per mole of glucose as against 38 via glycolysis–Krebs Cycle– Electron transfer chain, it would thus seem that a species or its genotype with greater carbohydrate concentration in roots, and an efficient metabolic mechanism associated with carbohydrate mobilization and fermentation pathway will be more suited to face oxygen deprivation. The present investigation, therefore, has been planned to validate the role of carbohydrate metabolism and the underlying molecular mechanism in imparting hypoxia tolerance by using one tolerant and one susceptible mung bean genotypes, and one wild Vigna species (V. luteola) at an early vegetative stage.

ARTICLE IN PRESS 604 Materials and methods Plant material and growth conditions An experiment was conducted with two cultivated mung bean [Vigna radiata (L.) Wilczek] genotypes, viz., T44 (tolerant) and Pusa Baisakhi (PB) (susceptible), and V. luteola (a highly tolerant wild Vigna sps.) under potculture to study their response to waterlogging stress. Plant material was procured from Division of Genetics, Indian Agricultural Research Institute, New Delhi, India, Indian Institute of Pulse Research, Kanpur, (UP), India and National Bureau of Plant Genetic Resources, New Delhi, India. Sowing was done in 30  30 cm (h  dia) earthen pots filled with clay-loam soil and farm yard manure in 3:1 ratio during the summer–rainy season. Pots were supplied with basal dose of 60 kg ha1 each of phosphorus and potassium. Before sowing seeds were treated with the required Rhizobium culture. Initially four plants were sown in each pot, which were thinned to 2 plants per pot after 20 d. Waterlogging treatment was given by placing pots with 25 d old plants in plastic troughs measuring 100  70  35 cm (L  B  H), and filled with water to a height just 1–2 cm below the soil level in pots. Treatments consisted of control, 2, 4, 6, and 8 d of waterlogging, and recovery after 4 d of termination of treatment. Because 8 d waterlogged plants of susceptible genotypes PB showed more than 75% mortality during recovery, therefore, recovery was uniformly studied in all the genotypes for 6 d waterlogged plants only. At each stage, samples were collected in quadruplicate from four pots. Observations were recorded on relative water content (RWC), membrane stability index (MSI), chlorophyll (Chl) content, total, reducing and non-reducing sugars, and activity of SS and ADH. In case of enzymes and sugar estimations each sample was assayed twice (n ¼ 8), while for RWC, MSI, and Chl content the number of observations were four only. The design of the experiment was completely randomized and data was analyzed by the factorial RBD. Physiological parameters Leaf RWC was estimated by recording the turgid weight of 0.5 g fresh leaf samples by keeping in water for 4 h, followed by drying in hot air oven till constant weight is achieved (Weatherley, 1950). RWC ¼ ½ðFresh wt:  dry wt:Þ=ðturgid wt:  dry wt:Þ  100

MSI was estimated by taking two sets of 200 mg of leaf or root material in test tubes containing 10 mL of doubledistilled water (Sairam et al., 1997). One set was heated at 40 1C for 30 min in a water bath, and the electrical conductivity of the solution is recorded on a conductivity meter (C1). Second set is boiled at 100 1C on a boiling water bath for 10 min, and its conductivity (C2) is measured as above. MSI is calculated as MSI ¼ ½1  ðC1 =C2 Þ  100 Chl content was estimated by extracting 0.05 g of the leaf material in 10 mL dimethyl sulfoxide (DMSO) (Hiscox

R.K. Sairam et al. and Israelstam, 1979). Samples were heated in an incubator at 65 1C for 4 h, and then after cooling to room temperature the absorbance of extracts were recorded at 665 and 645 nm and chl content was estimated as per standard method (Arnon, 1949). Biochemical estimations For estimation of sugars, powdered and oven-dried root samples (0.5 g dry weight) were extracted by boiling with 20 mL of 95% ethanol in glass vials. For extraction of sugars, the supernatant (alcohol in which the leaf material is boiled) is decanted into a beaker. The extraction is repeated 3–4 times by boiling the sample with 20 mL of 80% (v/v) ethanol in water each time and decanting the supernatant to the same beaker (McCready et al., 1950). The combined sugar extract is made up to a final volume of 100 mL with distilled water in a volumetric flask. For clarification, 50 mL aliquot of the above sugar extract is evaporated in a water bath taking care not to let the liquid dry out completely. The sample is then treated with one mL saturated solution of lead acetate (to precipitate colloidal substances) and then filtered into a beaker containing 3.0 mL of saturated di-sodium hydrogen phosphate. Lead is precipitated as lead phosphate. After 2–3 washings, the contents of beaker are filtered into a 50 mL volumetric flask and made up to a final volume of 50 mL. An aliquot of this solution is used for determining the reducing sugars by improved copper reagent method (Nelson, 1944; Somogyi, 1952). Reducing sugars were estimated by heating 2.0 mL of sugar extract with 1 mL Somogyi copper reagent in a boiling water bath for 12 min. After cooling, the samples in running tap water, 1 mL of arsenomolybdate reagent was added and final volume made up to 10 mL with distilled water. Absorbance was measured at 530 nm in a UV–visible spectrophotometer (model Specord Bio-200, AnalytikJena, Germany). A blank (distilled water) and two freshly prepared glucose standards are included with each set of samples. Total sugars were estimated by hydrolyzing 5 mL of sugar extract by boiling with 2.5 mL of 0.5 N HCl for 30 min in a water bath, and later the pH was adjusted to 6.5 with 0.5 N NaOH. The final volume was made up to 10 mL with distilled water and solution was used for determining the total sugars in the same way, as described for reducing sugars. Non-reducing sugars were calculated by subtracting the reducing sugars from the total sugar content. Enzyme assays SS assay was based on the synthesis of sucrose from UDP-glucose and fructose and its estimation by anthrone reagent after the removal of untreated fructose by heating at 100 1C (Zeng et al., 1998). Extraction buffer consisted of 200 mM HEPES containing+1 mM DTT+5 mM magnesium chloride+1 mM EDTA+20 mM sodium ascorbate +1 mM PMSF+10% (w/v) polyvinyl polypyrrolidone, and pH

ARTICLE IN PRESS Waterlogging-induced sugar metabolism and genes in mung bean roots was adjusted to 7.5. One gram root material was ground to fine powder in liquid nitrogen in a chilled mortar pestle followed by extraction with chilled extraction buffer (buffer to tissue ratio 10:1). Extract is centrifuged at 14,000 g at 4 1C for 10 min in a Sigma refrigerated centrifuge (model 3K-30, Osterode, Deutschland). Supernatant is dialyzed at 4 1C for 24 h against extraction buffer diluted 1:40, which was changed at least three times during dialysis. Dialyzed extract is used as an enzyme. The reaction mixture in 3 mL contained 50 mM HEPES–NaOH buffer (pH 7.5), 15 mM magnesium chloride, 10 mM fructose, 5 mM UDP-glucose, 50 mL enzyme extract and water to make final volume 3.0 mL. Assay is conducted at 30 1C for 30 min in a shaking water bath incubator, and reaction is terminated by the addition of 1 mL of 30% KOH. Controls were terminated at 0 min. The un-reacted fructose is removed by subsequent heating at 100 1C for 10 min. After cooling, each assay mixture is incubated with 1 mL of 0.14% anthrone in H2SO4 at 40 1C for 20 min and absorbance was recorded at 620 nm in UV–visible spectrophotometer. ADH activity was assayed by the reverse reaction i.e., oxidation of ethanol by ADH with the help of NAD, resulting into the synthesis of acetaldehyde and NADH (Chung and Ferl, 1999). The extraction buffer consisted of 50 mM Tris HCl+15 mM DTT, pH 8.0. Root tissue (0.5 g) was first pulverized with liquid nitrogen and then homogenized with 5.0 mL of extraction buffer. The extract was centrifuged at 12,000 g for 15 min at 4 1C in refrigerated centrifuge, and the supernatant was used as a source of enzyme. The 3 mL reaction mixture contained 50 mM Tris buffer, 0.867 mM NAD, 20% ethanol, 50 mL enzyme, and water to make final volume of 3 mL. Reaction mixture except NAD is prepared in test tubes, and each sample can be used as blank to adjust zero. NAD is added to initiate the reaction, and increase in absorbance at 340 nm was recorded for 1 min in UV– visible spectrophotometer. Amount of NADH formed is computed by drawing a standard curve of NADH at 340 nm, and activity is expressed as nmol NADH formed per mg protein per min.

605

following conditions: initial PCR activation step: 15 min at 95 1C, reverse transcription: 30 min at 50 1C, denaturation: 1 min at 94 1C, annealing: 1 min at 60 1C, extension: 1 min at 72 1C, and final extension: 10 min at 72 1C. The amplification products were electrophoresed on 1.2% agarose gel at 120 V in TBE buffer (0.4 M Tris–borate, 0.001 M EDTA, pH 8.0) using known concentration DNA ladders. Gels were stained with ethidium bromide and visualized on Uvi Pro Gel Documentation system (Uvitec, England). Primer sequences for various genes are as follows: For ADH coding region, primers were designed from the database available on Pisum sativum, and for SS and tubulin from the V. radiata.

Gene Psadh

Primers F1: TGT AGT CGG TCG GGG TTG ATG TG F2: ATG TGT GTA TTT GTC ACT AYG ACT R: TCT GAC CAA CTG TGT TCG ACA TG VrSS1 F: CGT GTT CAC AGT CTC CGT GAG AG R: CCA ATC TCC TGG AAC TTG TGC TC VrTUB F: CTT GAC TGC ATC TGC TAT GTT CAG R: CCA GCT AAT GCT CGG CAT ACT G

Gene sequencing RT-PCR amplified cDNAs were fractionated on agarose gel and purified. The purified cDNAs for each gene were cloned into pTz57R/T vector and transformed into E. coli (strain DH5a) cells. DH5a cells transformed with recombinant plasmid were selected based on antibiotic resistance as well as a-complementation method. Ampicillin-resistant putative recombinants were selected for further analysis. Plasmid were isolated from the confirmed colonies and restriction analysis was carried out by using Kpn I and Hind III enzymes flanking the cloning site of the vector pTz57R/T, to confirm the presence of cloned insert cDNA. Cloned insert cDNA in the pTz57R/T vector was sequenced by the dideoxy chain termination method (Sanger et al., 1977) using T7 and SP6 primers.

Gene expression by RT-PCR For gene induction studies, 25 d old plants were subjected to waterlogging treatment only for 24 h, as 48 h waterlogging treatment resulted in decline in gene expression of both SS and ADH in all the genotypes. Root samples were harvested from control and treated plants. Total RNA from root tissue was extracted using Trizol reagent (GibcoBRL), as per the recommendations of the manufacturer.DNA contamination was removed from the RNA samples using DNase I (Qiagen Science, Maryland, USA). One microgram of total RNA was reverse transcribed using gene-specific degenerate primers and Qiagen onestep RT-PCR kit. PCR conditions were standardized using gene-specific primers for tubulin. Linear amplification for semi-quantitative RT-PCR was obtained with 35 cycles. Reactions were conducted using My Genie 32 Thermal Block PCR (Bioneer, Korea) under the

Results Physiological parameters RWC decreased under waterlogging condition in all the genotype and the decline was greater in PB than V. luteola and T44. By 8th d PB has suffered 54.0% decline, while the decline in V. luteola and T44 was only 16.4% and 24.28% (Table 1). Recovery studied in 6 d waterlogged plants was better in V. luteola and T44. MSI in both leaves and roots decreased with the duration of waterlogging, and the lowest values were observed on the 8th d. The decline in MSI was greater in PB i.e., 43.31% and 45.11% in leaves

ARTICLE IN PRESS

Crop recovery was studied in 6 days waterlogged plants after 4 days of termination of treatment, as more than 75% of Pusa Baisakhi failed to recover after 8 days to flooding. a

2.380 7.110 1.236 3.616 1.011 2.710 1.865 5.287 SEm7 CD at 5%

1.145 3.287

0.719 2.080 0.467 1.342 0.467 1.342

0.931 2.690

Treatment (T) V  T Variety (V) Variety (V) Treatment (T) V  T Variety (V)

Treatment (T) V  T

79.9672.26 58.3172.82 49.8272.47 38.3673.58 34.1871.70 43.8972.04 50.75 81.9672.41 74.4173.37 70.0671.92 63.1872.31 60.4373.54 70.3671.08 70.07 84.1972.21 80.1771.82 74.6972.96 68.3372.19 65.5473.16 74.6271.12 74.59 81.0071.95 70.0672.82 53.6272.47 41.3673.47 30.1171.78 45.9272.44 53.68 80.1872.41 75.0673.18 70.0871.92 62.1972.31 60.8173.56 71.1171.08 69.91 0 2 4 6a 8 4 days after recoverya Mean

82.1772.68 78.1971.88 72.9172.80 66.1572.19 63.5273.06 73.1871.12 72.67 81.6071.95 69.6073.02 59.8771.40 46.2070.96 37.5371.01 52.7772.44 59.93 83.5071.99 77.8771.63 72.0770.21 67.4070.83 63.2370.53 79.5372.46 73.93 85.2071.06 83.7070.79 80.0070.22 74.9370.63 71.2371.05 81.3771.07 79.41

Pusa Baisakhi Vigna luteola T44 Pusa Baisakhi Vigna luteola T44 Vigna luteola T44

MSI (%) leaves

MSI (%) roots

Pusa Baisakhi

R.K. Sairam et al.

Days of waterlogging/recovery RWC (%)

Table 1.

Effect of waterlogging treatment on relative water content in leaves (%) and membrane stability index (MSI) in leaves and roots of mung bean genotypes

606

and roots, respectively, than V. luteola and T44, which suffered 10.84 and 11.31; 11.37% and 14.15% decline in leaves and roots, respectively (Table 1). Recovery was faster in V. luteola and T44 than PB. Total chl content decreased under waterlogging condition in all the genotypes. In case of V. luteola and T44, the decline was 14.13% and 18.00%; 7.93% and 12.50% at 6th and 8th d, while in case of PB the decline was 46.48% and 59.24% at 6th and 8th d, respectively (Table 2). Recovery was also quick and better in V. luteola and T44. Chl a/b ratio increased with the duration of waterlogging, and the highest increase was observed on 8th d of waterlogging in PB (65.08%), while V. luteola and T44 showed an increase of 31.4% and 24.8%, respectively (Table 2). During recovery, the Chl a/b ratio came down to near normal values in V. luteola and T44, being only 5.79% and 3.20% higher than control plants, while in PB Chl a/b ratio was still 39.68% higher than the control plants.

Sugars contents Levels of total and non-reducing sugars declined under waterlogging and reached a lowest value on the 8th d of treatment (Fig. 1A and B). However, V. luteola and T44 retained higher contents of both the fractions of sugars than PB. During recovery, the sugars levels increased in all the genotypes. Reducing sugars content were doubled on 2nd d of waterlogging in V. luteola and T44, slightly increased on 4th d and remained almost constant on the 6th d, and thereafter decreased on the 8th d, though the values were still much higher than prestress level. PB showed continuous decline in reducing sugars under waterlogging (Fig. 1C). Recovery studied 4 d after the termination of treatment in 6 d waterlogged plants showed increase in reducing sugar content only in PB over 4, 6, and 8 d waterlogged plants. V. luteola and T44 did not show increase in reducing sugars during recovery, however, the values were higher than PB.

Enzyme activities SS activity was affected differentially by waterlogging in the three genotypes (Fig. 2A). V. luteola and T44 recorded a continuous increase in SS activity up to 6th d of treatment, when the values were 2.38 and 2.35 times higher, while on 8th d 1.93 and 1.74 times higher than the pre-stress level. PB recorded a continuous decrease under waterlogging, and the decline was 2.71 times on

ARTICLE IN PRESS Waterlogging-induced sugar metabolism and genes in mung bean roots

607

Table 2. Effect of waterlogging treatment on total chlorophyll content and chlorophyll a/b ratio in the mung bean genotypes Days of waterlogging/recovery Chlorophyll content (mg g1 fr. wt.)

0 2 4 6* 8 4 days after recoverya Mean

SEm7 CD at 5%

Chlorophyll a/b ratio

Vigna luteola T44

Pusa Baisakhi Vigna luteola T44

Pusa Baisakhi

3.6170.04 3.4970.04 3.3570.08 3.1070.09 2.9670.02 3.2070.03 3.30

3.7670.01 3.7170.05 3.6570.01 3.4670.01 3.2970.01 3.4870.03 3.55

3.6870.03 3.5170.03 2.8970.05 2.0870.04 1.5070.05 1.8370.04 2.58

1.2670.01 1.3970.03 1.6670.01 1.8970.03 2.0870.05 1.7670.04 1.68

Variety (V) 0.021 0.078

1.2170.01 1.2670.04 1.3670.07 1.4470.09 1.5970.02 1.2870.03 1.36

1.2570.02 1.2870.05 1.3870.02 1.4970.01 1.5670.01 1.2970.05 1.38

Treatment (T) V  T

Variety (V)

Treatment (T) V  T

0.025 0.070

0.011 0.025

0.012 0.035

0.060 0.181

0.041 0.070

a

Reducing sugars (mg g-1 dry wt.)

Non-reducing sugars (mg g-1 dry wt.)

Total soluble sugars (mg g-1 dry wt.)

Crop recovery was studied in 6 days waterlogged plants after 4 days of termination of treatment, as more than 75% of Pusa Baisakhi failed to recover after 8 days to flooding.

70 60 50 40 30 20 10 0 60

Vigna luteola

T 44

Pusa Baisakhi

50 40 30

ADH activity also increased under waterlogging (Fig. 2B). In V. luteola and T44 the activity increased up to 8th d of treatment, and was about 20 and 15 times more than the pre-stress level. PB, however, recorded an increase in ADH activity only up to the 4th d of treatment, and thereafter, it decreased. The ADH activity level in PB was significantly lower than V. luteola and T44 at all stages of waterlogging. ADH activity declined after termination of treatment in all the genotypes.

SS and ADH gene expression

20 10 0 30 20 10 0 0 2 4 Days after waterlogging

6

8 4 Days after recovery

Fig. 1. Effect of waterlogging on total sugars (A), nonreducing sugars (B), and reducing sugars (C) in root tissues in V. luteola, and tolerant (T44) and susceptible (PB) genotypes of V. radiata. LSD significant (Pp0.05). Vertical bars show7SE of mean.

8th d of waterlogging over control plants. During recovery, PB showed slight increase in SS activity over 8 d waterlogged plants, while in V. luteola and T44 activity decreased, though remaining higher than PB.

RT-PCR analysis of SS gene yielded an amplicons of about 700 bp. SS gene showed constitutive as well as induced expression in case of V. luteola. However, the induced expression was significantly higher than constitutive expression. There was very little constitutive expression in T44, however, the induced expression was very profound and more than the V. luteola. PB had very little constitutive as well as induced expression of SS gene (Plate 1A). In case of ADH gene, there was very little constitutive expression in the three genotypes examined. However, 24 h waterlogging treatment induced ADH gene expression only in V. luteola and T44, while there was very little waterlogging-induced expression in PB (Plate 1B). The b-tubulin expression was almost constant in all the genotypes, and did not change under control and waterlogging conditions (Plate 1C).

SS and ADH gene sequences SS gene-specific primers yielded partial sequences of 667, 531, and 626 bp in V. luteola,

ARTICLE IN PRESS 608

R.K. Sairam et al. 4.5 Vigna luteola T 44 Pusa Baisakhi

Sucrose synthase activity (µmol mg-1 protein min-1)

4 3.5 3 2.5 2 1.5 1 0.5

Alcohol dehydrogenase activity (µmol NADH mg-1 protein min-1)

0 6 5 4 3 2 1 0 2 4 Days after waterlogging

0

6

8

4 Days after recovery

Fig. 2. Effect of waterlogging on the activity of SS (A) and ADH (B) in root tissues in V. luteola, and tolerant (T44) and susceptible (PB) genotypes of V. radiata. LSD significant (Pp0.05). Vertical bars show7SE of mean.

M

1

2

3

4

5

6

A 900 800 700 600

B

900 800 700 600

SS 691 bp

721 bp ADH

C 600 500 400 300

Tubulin 422 bp

Plate 1. Expression analysis of sucrose synthase (SS), alcohol dehydrogenase (ADH), and tubulin genes under 24 h waterlogging stress and control conditions. Gene expression was determined by RT-PCR utilizing gene specific primer sets for each gene. b-Tubulin gene expression was used as an internal control. (M-1 Kb ladder, 1-Control V. luteola, 2-Treated V. luteola, 3Control T44, 4-Treated T44, 5-Control PB, and 6-Treated PB).

T44 and PB, respectively. Partial nucleotide sequences for SS of the three genotypes are given below V. luteola GTCTCCGTGAGAGGCTTGATGAAACCCTGTCTGCGAG CAGGAACGAAATTCTGGCCCTTCTGTCAAGGATCGAA GGCAAGGGCAAGGGAATCTTGCAACACCATCAGGTGA TTGCGGAGTTTGAGGAAATCCCCGAGGAGAGCAGACA GAAGCTTACTGATGGTGCCTTTGGAGAAGTTCTGAGA TCTACTCAGGAAGCCATAGTTTTGCCACCATGGGTTGC TCTGGCCGTTCGTCCAAGGCCTGGTGTGTGGGAGTAT CTGAGAGTGAATGTGCATGCTCTAGTCGTTGAGGTGT TGCAACCTGCTGAGTACCTGCACTTCAAGGAAGAACTT GTTGATGGAAGTTCTAATGGAAACTTTGTGCTTGAGTT GGACTTTGAACCCTTCACCGCATCCTTCCCCCGCCCAA CTCTTAACAAGTCAATTGGAAATGGTGTTCAGTTCCTC AACCGTCACCTTTCTGCCAAACTCTTCCATGACAAGGA GAGCTTACACCCGCTTTTGGAGTTCCTCAGGCTTCACA GCGTCAATGGAAAGACTTTGATGTTGAATGACAGAATT CAAAACCCGGATGCTCTACAACATGTTCTGAGGAAAGC TGAGGAGTATCTGGGCACAGTGCCTGCTGAAACCCCC TACTCAGCATTTGAGCACAAGTTCCAGGA T44 GCGAACAGGACGAAATTCTGGCCCTTCTGTCAAGGATC GAAGGCAAGGGCAAGGGAATCTTGCAACACCATCAGG TGATTGCGGAGTTTGAGGAAATCCCCGAGGAGAGCAG

ARTICLE IN PRESS Waterlogging-induced sugar metabolism and genes in mung bean roots ACAGAAGCTTACTGATGGTGCCTTTGGAGAAGTTCTG AGATCTACTCAGGAAGCCATAGTTTTGCCACCATGGGT TGCTCTGGCCGTTCGTCCAAGGCCTGGTGTGTGGGAG TATCTGAGAGTGAATGTGCATGCTCTAGTCGTTGAGGT GTTGCAACCTGCTGAGTACCTGCACTTCAAGGAAGAAC TTGTTGATGGAAGTTCTAATGGAAACTTTGTGCTTGAG TTGGACTTTGAACCCTTCACCGCATCCTTCCCCCGCCC AACTCTTAACAAGTCAATTGGAAATGGTGTTCAGTTCCT CAACCGTCACCTTTCTGCCAAACTCTTCCATGACGAGG AGAGCTTACACCCGCTCTTGGAGTTCCTCAGGCTTCACA GCGTCATGGAAAGACATTGATGTTGAATGACAGAATTCA PB TTCTGGCCCTTCTGTCAAGGATCGAAGGCAAGGGCAAG GGGATTTTGCAACACCATCAGGTCATCGCCGAGTTTGA GGAAATCCCCGAGGAGAGCAGACAGAAGCTTACTGATG GTGCCTTTGGAGAAGTTTTGAGGTCTACTCAGGAAGC CATAGTTTTGCCACCATGGGTTGCTCTGGCCGTTCGTC CAAGGCCTGGTGTGTGGGAGTACCTGAGAGTGAATGT GCACGCTCTAGTTGTTGAGGTGTTGCAACCTGCTGAG TACCTGCGCTTCAAGGAGGAACTTGTTGATGGAAGTTC TAATGGCAACTTTGTGCTTGAGTTGGACTTTGAACCCT TTACCGCATCCTTCCCCCGCCCAACTCTTAACAAGTCAA TTGGAAATGGCGTGCAGTTCCTCAACCGTCACCTTTCT GCCAAACTCTTCCATGACAAGGAGAGCTTGCACCCGCT TTTGGAATTCCTCAGGCTTCACAGCGTCAGGGAAAGAC TTTGATGTTGAATGACAGAATTCAAACCCGGATGCTCTT CAACATGTTCTGAGGAAAGCTGAGGAGTATCTGGGCAC AGTGCCTCCTGAAACCCTCCTACTCAGCATTTGAGCAC AAGTACCGGGAATTGGGAA All the genotypes showed 96% to 98% similarity with the SS complete CDS of V. radiata (D10266). V. luteola and T44 showed 99% similarity, V. luteola and PB showed 94%, and PB and T44 showed 93% similarity. ADH gene-specific primers yielded partial gene coding sequences of 744, 702, and 736 bp in V. luteola, T44 and PB, respectively. Partial nucleotide sequences for ADH of the three genotypes are given below V. luteola CTTCACCTCCCTTTGTCACACTGATGTTTACTTCTGGG AAGCCAAGGGCCAGACTCCATTGTTTCCTCGTATATTT GGTCATGAGGCTGGAGGGATTGTGGAGAGCGTAGGT GAGGGTGTGACTCATCTGAAACCCGGAGACCACGCCC TTCCTGTGTTTACAGGGGAGTGTGGGGATTGTGCACA TTGTAAGTCAGAGGAGAGCAACATGTGTGATCTGCTG AGGATCAACACTGATCGTGGTGTCATGATCCATGATAG CAAAACAAGATTTTCTATAAAGGGACAACCTATTTACCA TTTTGTTGGTACCTCTACATTCAGTGAATACACTGTTG TTCATGCTGGATGTGTTGCCAAAGTCAACCCTGCTGCC CCACTTGACAAAATTTGTGTTCTCAGTTGTGGAATATG CACAGGTCTTGGGGCTACTGTCAATGTTGCAAAACCG AAACCTGGTTCTTCTGTTGCCATTTTTGGACTTGGAGC TGTTGGCCTTGCTGCTGCTGAAGGGGCAAGGATTTCT GGTGCATCAAGAATCATTGGGGTTGATTTAGTTTCAAG CCGATTTGAAGAAGCTAAGAAGTTTGGGGTCAATGAAT

609

TTGTGAACCCAAAAGACCATGACAAACCCGTACAACAG GTAATTGCTGAAATGACAAATGGGGGTGTGGATCGTG CTGTGGAATGTACTGGCAGCATCCAGGCCATGGTCTCA GCATTCGAATGTGTCCACGATGGTTGGGG T44 CCTCTCTTTGTCACACTGATGTTTACTTCTGGGAAGCC AAGGGCCAGACTCCATTGTTTCCTCGTATATTTGGTCA TGAGGCTGGAGGGATTGTGGAGAGCGTAGGTGAGGG TGTGACTCATCTGAAACCCGGAGACCACGCCCTTCCT GTGTTTACAGGGGAGTGTGGGGATTGTGCACATTGTA AGTCAGAGGAGAGCAACATGTGTGATCTGCTGAGGAT CAACACTGATCGTGGTGTCATGATCCATGATAGCAAAA CAAGATTTTCTATAAAGGGACAACCTATTTACCATTTTG TTGGTACCTCTACATTCAGTGAATACACTGTTGTTCATG CTGGATGTGTTGCCAAAGTCAACCCTGCTGCCCCACT TGACAAAATTTGTGTTCTCAGTTGTGGAATATGCACAG GTCTTGGGGCTACTGTCAATGTTGCAAAACCGAAACCT GGTTCTTCTGTTGCCATTTTTGGACTTGGAGCTGTTG GCCTTGCTGCTGCTGAAGGGGCAAGGATTTCTGGTGC ATCAAGAATCATTGGGGTTGATTTAGTTTCAAGCCGAT TTGAAGAAGCTAAGAAGTTTGGGGTCAATGAATTTGT GAACCCAAAAGACCATGACAAACCCGTACAACAGGTAA TTGCTGAAATGACAAATGGGGGTGTGGATCGTGCTGT GGAATGTACTGGCAGCATCCAGGCCATG PB CCTCTCTTTGTCACACTGACGTTTACTTCTGGGAAGCC AAGGGCCAAACTCCATTGTTTCCTCGCATATTTGGTCA TGAGGCTGGAGGGATTGTGGAGAGCGTAGGTGAGGG TGTGACTCATCTGAAACCAGGGGACCATGCTCTTCCTG TGTTTACAGGGGAGTGTGGGGAATGTGCGCATTGTAA GTCAGAGGAGAGCAACATGTGTGATCTGCTGAGGATC AACACTGATCGTGGTGTCATGATCCATGATAGTCAAAC AAGATTTTCTATAAAGGGACAACCTATTTACCATTTTGT CGGTACCTCTACATTCAGTGAATACACTGTGGTTCATG CTGGATGTGTTGCCAAAGTCAACCCTGCTGCCCCACT TGACAAAATTTGTGTTCTCAGTTGTGGAATATGCACAG GTCTTGGGGCTACTGTCAATGTTGCAAAACCAAAACCT GGTTCTTCTGTTGCCATTTTTGGACTTGGAGCTGTTG GCCTTGCTGCTGCTGAAGGGGCAAGGATTTCCGGTGC ATCAAGAATCATTGGGGTTGATTTAGTTTCAAGCCGAT TTGAAGAAGCTAAGAAGTTTGGGGTCAATGAATTTGT GAACCCAAAAGACCATGACAAACCCGTACAACAGGTAA TTGCTGAAATGACAAATGGGGGAGTGGATCGTGCTGT TGAATGTACTGGCAGCATCCAGGCCATGGTGTCAGCAT TCGAATGTGTCCACGATGGTTG The three genotypes showed 97% to 98% similarity with the ADH complete CDS of Phaseolus acutifolius (Z23170). V. luteola and T44 showed 99% similarity, V. luteola and PB showed 97%, and PB and T44 showed 97% similarity.

Discussion In tropical and subtropical regions, severe crop losses are caused by prolonged seasonal rainfall.

ARTICLE IN PRESS 610 Excess water produces hypoxic soil condition within a few hours, and prolonged waterlogging results in anoxia. Plant roots, consequently, suffer hypoxia or anoxia. As a result of 8 d of waterlogging mung bean genotypes T44 and PB, and wild species V. luteola suffered loss in leaf area, dry matter (data not reported), RWC, MSI both in roots and leaves, leaf Chl, and increase in Chl a/b ratio. The decline in RWC, MSI both in roots and leaves and Chl was less in V. luteola and T44 than PB, which also suffered about 75% mortality during recovery (data not reported). Min and Bartholomew (2005) reported decrease in RWC during flooding, which further declined with the duration of flooding stress. Various workers have also reported waterlogginginduced decrease in leaf water potential (Naidoo, 1983; Else et al., 1995). Wilting under excess of water is due to the higher resistance to mass flow of water through the roots (Jackson and Drew, 1984). Membrane disintegration is one of the consequences of oxygen deprivation (Rawyler et al., 2002), resulting in more than 40 times increase in solute leakage from 4 d waterlogged pea plants (Jackson et al., 1982). Decrease in Chl content under waterlogging has been reported in wheat (Huang et al., 1994; Collaku and Harrison, 2002), maize (Younis et al., 2003; Prasad et al., 2004) and V. sinensis (Younis et al., 2003). Increase in Chl a/b ratio under flooding signify greater damage to Chl b than Chl a. Significantly greater damage to Chl b than Chl a have been reported under flooding (Ashraf and Arfan, 2005) and water stresses (Wentworth et al., 2006). Loss in Chl b, an accessory pigment of light harvesting complex, results in reduced efficiency of photochemical reactions as well as photoinhibition. Decline in Chl b under waterlogged conditions may have significant effect on LHCII complexes containing almost Chl b in the mature thylakoid membranes (Green, 1988). Carbohydrate starvation has been shown as one of the possible reasons of hypoxia/anoxia induced injuries (Crawford and Braendle, 1996; Schluter and Crawford, 2001). Kennedy et al. (1992) reported that the tolerant species Echinochloa phyllopogon maintained active carbohydrate and ATP levels under anoxia than susceptible E. cruspavonis. In this context greater concentration of total, non-reducing and reducing sugars in tolerant genotypes (V. luteola and T44) and also increase in reducing sugars under waterlogging condition, as well as greater and increasing activity of SS compared to susceptible genotype, PB suggest a carbohydrate based tolerance mechanism in V. luteola and T44. SS is a key enzyme responsible for the hydrolysis of sucrose to fructose and glucose (UDP-glucose) under oxygen deprivation (Ruan

R.K. Sairam et al. et al., 2003; Noguchi, 2004). Both glucose and fructose (reducing sugars) are substrate for glycolytic pathway, which is the major source of energy under hypoxia in the absence of oxidative phosphorylation (Gibbs and Greenway, 2003; Greenway and Gibbs, 2003). The essentiality of SS in anoxic tolerance has been demonstrated in maize using double mutant of the enzyme (Ricard et al., 1998). Hence, the greater increase in the SS activity under waterlogged condition in T44 and wild V. luteola resulted into an increased availability of reducing sugars, which sustained their energy requirement. Activity of ADH was significantly greater in V. luteola and T44, which further increased under waterlogging condition, while susceptible genotype PB showed very little activity even under waterlogging condition. ADH has a pivotal role in recycling of NADH to NAD, which is essential for continuation of glycolytic pathway (Ismond et al., 2003; Keyhani and Keyhani, 2004). The importance of ADH in flooding tolerance has been emphasized in the study of maize mutant that has a deficiency in one of its ADH genes and, therefore, unable to produce a functional ADH enzyme. Consequently this mutant was very sensitive to flooding injury than wild type and died within 3 d of submergence. It can thus be suggested that greater ADH activity in V. luteola and T44 is a factor contributing to their better survival under waterlogged condition. Gene expression studies conducted to examine the effect of 24 h waterlogging treatment on SS and ADH gene expression using gene-specific primers in the case of wild genotype V. luteola and V. radiata genotypes T44 (tolerant) and PB (susceptible) showed significant degree of SS-mRNA expression even in aerobically grown plants of T44, which further increased under waterlogging, while V. luteola showed little expression in control plants and highest under waterlogging; susceptible genotype PB has very little SS-mRNA expression under both the conditions. This suggests that the basic machinery for the synthesis of glycolytic substrate, viz., glucose and fructose is lacking in susceptible genotype at the gene level itself (Plate 1A). The SS gene expression study further confirmed the SS activity results, which showed highest waterlogging-induced activity in V. luteola, followed by T44, and a decline in PB. Partial SS gene sequences of 667, 531, and 626 bp were obtained in V. luteola, T44 and PB, respectively. These sequences were compared with V. radiata complete CDS for SS, D10266 (Arai et al., 1992) using CLUSTAL W (1.83) multiple alignment tool (Fig. 3a and b). The comparison results confirmed that the obtained PCR band were of the SS gene. V. luteola, T44 and PB showed more

ARTICLE IN PRESS Waterlogging-induced sugar metabolism and genes in mung bean roots

611

VLSS T44SS D10266 PbSS

----------GTCTCCGTGAGAGGCTTGATGAAACCCTGTCTGCGAGCAGGAACGAAATT ------------------------------------------GCGAACAGGAACGAAATT CGTGTTCACAGTCTCCGTGAGAGGCTTGATGAAACCCTGTCTGCCAACAGGAACGAAATT ----------------------------------------------------------TT ********************************** ***************

50 17 60 2

VLSS T44SS D10266 PbSS

CTGGCCCTTCTGTCAAGGATCGAAGGCAAGGGCAAGGGAATCTTGCAACACCATCAGGTG CTGGCCCTTCTGTCAAGGATCGAAGGCAAGGGCAAGGGAATCTTGCAACACCATCAGGTG CTGGCCCTTCTGTCAAGGATCGAAGGCAAGGGCAAGGGGATT TTGCAACACCATCAGGTC CTGGCCCTTCTGTCAAGGATCGAAGGCAAGGGCAAGGGGATT TTGCAACACCATCAGGTC ************************************** ** *****************

110 77 120 62

VLSS T44SS D10266 PbSS

ATTGCGGAGTTTGAGGAAATCCCCGAGGAGAGCAGACAGAAGCTTACTGATGGTGCCTTT ATTGCGGAGTTTGAGGAAATCCCCGAGGAGAGCAGACAGAAGCTTACTGATGGTGCCTTT ATTGCCGAGTTTGAGGAAATCCCCGAGGAGAGCAGACAGAAGCTTACTGATGGTGCCTTT ATCGCCGAGTTTGAGGAAATCCCCGAGGAGAGCAGACAGAAGCTTACTGATGGTGCCTTT ** ** ******************************************************

170 137 180 122

VLSS T44SS D10266 PbSS

GGAGAAGTTCTGAGATCTACTCAGGAAGCCATAGTTTTGCCACCATGGGTTGCTCTGGCC GGAGAAGTTCTGAGATCTACTCAGGAAGCCATAGTTTTGCCACCATGGGTTGCTCTGGCC GGAGAAGTTT TGAGATCTACTCAGGAAGCCATAGTTTTGCCACCATGGGTTGCTCTGGCC GGAGAAGTTT TGAGGTCTACTCAGGAAGCCATAGTTTTGCCACCATGGGTTGCTCTGGCC ********* **** *********************************************

230 197 240 182

VLSS T44SS D10266 PbSS

GTTCGTCCAAGGCCTGGTGTGTGGGAGTAT CTGAGAGTGAATGTGCAT GCTCTAGTCGTT GTTCGTCCAAGGCCTGGTGTGTGGGAGTAT CTGAGAGTGAATGTGCAT GCTCTAGTCGTT GTTCGTCCAAGGCCTGGTGTGTGGGAGTACCTGAGAGTGAATGTGCACGCTCTAGTT GTT GTTCGTCCAAGGCCTGGTGTGTGGGAGTACCTGAGAGTGAATGTGCACGCTCTAGTT GTT ***************************** ***************** ******** ***

290 257 300 242

VLSS T44SS D10266 PbSS

GAGGTGTTGCAACCTGCTGAGTACCTGCACTTCAAGGAAGAACTTGTTGATGGAAGTTCT GAGGTGTTGCAACCTGCTGAGTACCTGCACTTCAAGGAAGAACTTGTTGATGGAAGTTCT GAGGTGTTGCAACCTGCTGAGTACCTGCGCTTCAAGGAGGAACTTGTTGATGGAAGTTCT GAGGTGTTGCAACCTGCTGAGTACCTGCGCTTCAAGGAGGAACTTGTTGATGGAAGTTCT **************************** ********* *********************

350 317 360 302

VLSS T44SS D10266 PbSS

AATGGAAACTTTGTGCTTGAGTTGGACTTTGAACCCTTCACCGCATCCTTCCCCCGCCCA AATGGAAACTTTGTGCTTGAGTTGGACTTTGAACCCTTCACCGCATCCTTCCCCCGCCCA AATGGCAACTTTGTGCTTGAGTTGGACTTTGAACCCTTT ACCGCATCCTTCCCCCGCCCA AATGGCAACTTTGTGCTTGAGTTGGACTTTGAACCCTTT ACCGCATCCTTCCCCCGCCCA ***** ******************************** *********************

410 377 420 362

VLSS T44SS D10266 PbSS

ACTCTTAACAAGTCAATTGGAAATGGT GTT CAGTTCCTCAACCGTCACCTTTCTGCCAAA ACTCTTAACAAGTCAATTGGAAATGGT GTT CAGTTCCTCAACCGTCACCTTTCTGCCAAA ACTCTTAACAAGTCAATTGGAAATGGCGTGCAGTTCCTCAACCGTCACCTTTCTGCCAAA ACTCTTAACAAGTCAATTGGAAATGGCGTGCAGTTCCTCAACCGTCACCTTTCTGCCAAA ************************** ** ******************************

470 437 480 422

VLSS T44SS D10266 PbSS

CTCTTCCATGACAAGGAGAGCTTACACCCGCTTTTGGAGTTCCTCAGGCTTCACAGCGTC CTCTTCCATGACGAGGAGAGCTTACACCCGCTCTTGGAGTTCCTCAGGCTTCACAGCGTC CTCTTCCATGACAAGGAGAGCTTGCACCCGCTTTTGGAATTCCTCAGGCTTCACAGCGTC CTCTTCCATGACAAGGAGAGCTTGCACCCGCTTTTGGAATTCCTCAGGCTTCACAGCGTC ************ ********** ******** ***** *********************

530 497 540 482

VLSS T44SS

AAT GGAAAGACTTTGATGTTGAATGACAGAATTCAAAACCCGGATGCTCTACAACATGTT 590 AAT GGAAAGACATTGATGTTGAATGACAGAATTCA------------------------- 531

D10266 PbSS

AAGGGAAAGACTTTGATGTTGAATGACAGAATTCAAAACCCGGATGCTCTT CAACATGTT 600 AAGGGAAAGACTTTGATGTTGAATGACAGAATTCAAAACCCGGATGCTCTT CAACATGTT 540 ** ******** ************************************** *********

VLSS

CTGAGGAAAGCTGAGGAGTATCTGGGCACAGTGCCTGCTGAAACCCCCTACTCAGCATTT 650

T44SS D10266 PbSS

-----------------------------------------------------------CTGAGGAAAGCTGAGGAGTATCTGGGCACAGTGCCTCCTGAAACCCCCTACTCAGCATTT 659 CTGAGGAAAGCTGAGGAGTATCTGGGCACAGTGCCTCCTGAAACCCCCTACTCAGCATTT 600 ************************************ ***********************

VLSS T44SS D10266 PbSS

GAGCACAAGTTCCAGGA---------667 -------------------------GAGCACAAGTTCCAGGAGATTGG---683 GAGCACAAGTACCCCAATTCCCAA--626 ************************

Fig. 3. (a) Clustal W (1.83) multiple sequence alignment and comparison of partial coding sequences of SS in root tissues in V. luteola, and tolerant (T44) and susceptible (PB) genotypes of V. radiata with V. radiata complete CDS (D10266). (b) Clustal W (1.83) multiple sequence alignment and comparison of deduced amino acid sequences of SS in root tissues in V. luteola, and tolerant (T44) and susceptible (PB) genotypes of V. radiata with V. radiata complete CDS (D10266).

ARTICLE IN PRESS 612

R.K. Sairam et al.

PbSS D10266 VLSS

---------------------------LALLSRIEGKGKGILQHHQVIAEFEEIPEESRQ 33 MATDRLTRVHSLRERLDETLSANRNEILALLSRIEGKGKGILQHHQVIAEFEEIPEESRQ 60 -----------LRERLDETLSASRNEILALLSRIEGKGKGILQHHQVIAEFEEIPEESRQ 49

T44SS

----------------------EQDEILALLSRIEGKGKGILQHHQVIAEFEEIPEESRQ 38 *********************************

PbSS D10266 VLSS T44SS

KLTDGAFGEVLRSTQEAIVLPPWVALAVRPRPGVWEYLRVNVHALVVEVLQPAEYLRFKE KLTDGAFGEVLRSTQEAIVLPPWVALAVRPRPGVWEYLRVNVHALVVEVLQPAEYLRFKE KLTDGAFGEVLRSTQEAIVLPPWVALAVRPRPGVWEYLRVNVHALVVEVLQPAEYLH FKE KLTDGAFGEVLRSTQEAIVLPPWVALAVRPRPGVWEYLRVNVHALVVEVLQPAEYLH FKE ********************************************************:***

93 120 109 98

PbSS D10266 VLSS T44SS

ELVDGSSNGNFVLELDFEPFTASFPRPTLNKSIGNGVQFLNRHLSAKLFHDK ESLHPLLE ELVDGSSNGNFVLELDFEPFTASFPRPTLNKSIGNGVQFLNRHLSAKLFHDK ESLHPLLE ELVDGSSNGNFVLELDFEPFTASFPRPTLNKSIGNGVQFLNRHLSAKLFHDK ESLHPLLE ELVDGSSNGNFVLELDFEPFTASFPRPTLNKSIGNGVQFLNRHLSAKLFHDEESLHPLLE ***************************************************:********

153 180 169 158

PbSS D10266 VLSS T44SS

FLRLHSVKGKTLMLNDRIQNPDALQHVLRKAEEYLGTVPPETPYSAFEHK---------- 203 FLRLHSVKGKTLMLNDRIQNPDALQHVLRKAEEYLGTVPPETPYSAFEHKFQEIGLERGW 240 FLRLHSVN GKTLMLNDRIQNPDALQHVLRKAEEYLGTVPAETPYSAFEHKFQ-------- 221 FLRLHSVMER H------------------------------------------------- 169 ******* :

PbSS D10266 VLSS T44SS

-----------------------------------------------------------GDNAERVLESIQLLLDLLEAPDPCTLETFLGRIPMVFNVVILSPHGYFAQDNVLGYPDTG 300 -----------------------------------------------------------------------------------------------------------------------

Fig. 3. (Continued)

than 96–98% similarity with V. radiata complete gene sequence for SS. The asterisk marked regions in the partial cDNA sequence show conserved regions, while the underlined bases denote polymorphism. The regions of polymorphism showed that V. luteola and T44 have identical bases (99% similarity), while at these points PB and D10266 (V. radiata) have different bases than V. luteola and T44, but were having 98% similarity with each other. Search of conserved domains for the partial amino acid sequence of SS revealed that the obtained fragment belongs to, Sucrose_synth (pfam00862), SS. SSs catalyze the synthesis of sucrose from UDP-glucose and fructose. This family includes the bulk of the SS protein. However, the carboxyl terminal region of the SSs belongs to the glycosyl transferase family (pfam00534), which was not present in the deduced partial amino acid sequence obtained in this study. Under low-oxygen condition in Arabidopsis, microarray and qRT-PCR studies identified the over-expressing genes to be involved in sugar utilization, notably SS and ADH (Liu et al., 2005). Similarly Gonzali et al. (2005) used microarrays to study the anaerobic responses in Arabidopsis and revealed that ADH and SS are reliable markers of anoxic metabolism. Waterlogging induced ADH-gene expression was observed only in V. luteola and T44. In untreated plants very little mRNA expression was observed in the three genotypes. The susceptible genotype PB failed to express ADH-mRNA both in control and

waterlogged condition. This could be the reason for very little ADH activity in susceptible genotype. The waterlogging-induced increase in ADH activity in V. luteola and T44 is thus due to an up-regulation of the ADH transcript in tolerant genotypes. As there was no increase in ADH transcripts in susceptible genotype PB; it was manifested in a decline in ADH activity. Rapid increases in activity of ADH in rice seedlings (Reggiani et al., 1986; Rivoal et al., 1989) have been reported under O2 deprivation. RT-PCR amplified ADH cDNAs of V. luteola, T44 and PB were sequenced and about 744, 702, and 736 bp of partial coding and 247, 234, and 277 amino acid sequences were obtained, which were compared with Phaseolus acutifolius complete CDS for ADH, Z23170 (Garvin et al., 1994) by CLUSTAL W (1.83) multiple sequence alignment tool (Fig. 4a and b). The comparison results confirmed that the obtained PCR band were of the ADH gene. All the three genotypes showed more than 97% similarity with Phaseolus acutifolius full coding sequence of ADH gene. T44 and V. luteola showed 99% similarity in cDNA sequence. Conserved domains were identified using ‘PROSITE’ and the partial amino acid sequence of ADH showed two conserved domains: (1) ADH_Zn, zinc containing ADH signature (PS 00059) (amino acid residues 26–40 in V. luteola; 24–38 in T44 and PB), where H (histidine) is the zinc ligand, which is the 2nd ligand of the catalytic zinc atom (Fig. 4b). (2) ADH_N, ADH GroES-like domain (amino acid residues 83–96 in V. luteola; 80–93 in

ARTICLE IN PRESS Waterlogging-induced sugar metabolism and genes in mung bean roots

613

VL-ADH T44ADH Pb-ADH Z23170

--------CTT CACCTCCCTTTGTCACACTGAT GTTTACTTCTGGGAAGCCAAGGGCCAG -------------CCTCTCTTTGTCACACTGAT GTTTACTTCTGGGAAGCCAAGGGCCAG -------------CCTCTCTTTGTCACACTGACGTTTACTTCTGGGAAGCCAAGGGCCAA AAGATCCTCTACACCTCTCTTTGCCACACTGAT GTTTACTTCTGGGAAGCCAAGGGCCAG **** ***** ******** **************************

52 47 47 180

VL-ADH T44ADH Pb-ADH Z23170

ACTCCATTGTTTCCTCGTATATTTGGTCATGAGGCTGGAGGGATTGTGGAGAGCGTAGGT ACTCCATTGTTTCCTCGTATATTTGGTCATGAGGCTGGAGGGATTGTGGAGAGCGTAGGT ACTCCATTGTTTCCTCGCATATTTGGTCATGAGGCTGGAGGGATTGTGGAGAGCGTAGGT ACTCCATTGTTTCCTCGTATATTTGGTCATGAGGCTGGAGGGATTGTGGAGAGCGTAGGT ***************** ******************************************

112 107 107 240

VL-ADH T44ADH Pb-ADH Z23170

GAGGGTGTGACTCATCTGAAACCCGGAGACCACGCCCTTCCTGTGTTTACAGGGGAGTGT 172 GAGGGTGTGACTCATCTGAAACCCGGAGACCACGCCCTTCCTGTGTTTACAGGGGAGTGT 167 GAGGGTGTGACTCATCTGAAACCAGGGGACCAT GCT CTTCCTGTGTTTACAGGGGAGTGT 167 GAGGGTGTGACTCATCTGAAACCCGGGGACCACGCCCTTCCTGTGTTTACT GGGGAGTGT 300 *********************** ** ***** ** ************** *********

VL-ADH T44ADH Pb-ADH Z23170

GGGGATTGTGCACATTGTAAGTCAGAGGAGAGCAACATGTGTGATCTGCTGAGGATCAAC 232 GGGGATTGTGCACATTGTAAGTCAGAGGAGAGCAACATGTGTGATCTGCTGAGGATCAAC 227 GGGGAATGTGCGCATTGTAAGTCAGAGGAGAGCAACATGTGTGATCTGCTGAGGATCAAC 227 GGGGAGTGTGCACATTGTAAGTCAGAAGAGAGCAACATGTGTGATT TGCTGAGGATCAAC 360 ***** ***** ************** ****************** **************

VL-ADH T44ADH Pb-ADH Z23170

ACTGATCGTGGTGTCATGATCCATGATAGCAAAACAAGATTTTCTATAAAGGGACAACCT 292 ACTGATCGTGGTGTCATGATCCATGATAGCAAAACAAGATTTTCTATAAAGGGACAACCT 287 ACTGATCGTGGTGTCATGATCCATGATAGTC AAACAAGATTTTCTATAAAGGGACAACCT 287 ACTGATCGGGGTGTCATGATCCATGATAGTC AAACAAGATTTTCTATAAAGGGACAACCT 420 ******** ******************** *****************************

VL-ADH T44ADH Pb-ADH Z23170

ATTTACCATTTTGTTGGTACCTCTACATTCAGTGAATACACTGTT GTTCATGCTGGATGT ATTTACCATTTTGTTGGTACCTCTACATTCAGTGAATACACTGTT GTTCATGCTGGATGT ATTTACCATTTTGTCGGTACCTCTACATTCAGTGAATACACTGTGGTTCATGCTGGATGT ATTTACCATTTTGTTGGTACCTCTACATTCAGTGAATACACTGTGGTTCATGCTGGATGT ************** ***************************** ***************

352 347 347 480

VL-ADH T44ADH Pb-ADH Z23170

GTTGCCAAAGTCAACCCTGCTGCCCCACTTGACAAAATTTGTGTTCTCAGTTGTGGAATA GTTGCCAAAGTCAACCCTGCTGCCCCACTTGACAAAATTTGTGTTCTCAGTTGTGGAATA GTTGCCAAAGTCAACCCTGCTGCCCCACTTGACAAAATTTGTGTTCTCAGTTGTGGAATA GTTGCCAAAGTCAACCCTGCTGCCCCACTTGAT AAAATTTGTGTTCTCAGTTGTGGAATA ******************************** ***************************

412 407 407 540

VL-ADH T44ADH Pb-ADH Z23170

TGCACAGGTCTTGGGGCTACTGTCAATGTTGCAAAACCGAAACCTGGTTCTTCTGTTGCC TGCACAGGTCTTGGGGCTACTGTCAATGTTGCAAAACCGAAACCTGGTTCTTCTGTTGCC TGCACAGGTCTTGGGGCTACTGTCAATGTTGCAAAACCAAAACCTGGTTCTTCTGTTGCC TGCACAGGTCTTGGGGCTACTGTCAATGTTGCAAAACCGAAACCTGGTTCTTCTGTTGCT ************************************** *********************

472 467 467 600

VL-ADH T44ADH Pb-ADH Z23170

ATTTTTGGACTTGGAGCTGTTGGCCTTGCTGCTGCTGAAGGGGCAAGGATTTCTGGTGCA ATTTTTGGACTTGGAGCTGTTGGCCTTGCTGCTGCTGAAGGGGCAAGGATTTCTGGTGCA ATTTTTGGACTTGGAGCTGTTGGCCTTGCTGCTGCTGAAGGGGCAAGGATTTCCGGTGCA ATTTTTGGACTTGGAGCTGTTGGCCTTGCTGCTGCTGAAGGGGCAAGGATTTCTGGTGCA ***************************************************** ******

532 527 527 660

VL-ADH T44ADH Pb-ADH Z23170

TCAAGAATCATTGGGGTTGATTTAGTTTCAAGCCGATTTGAAGAAGCTAAGAAGTTTGGG TCAAGAATCATTGGGGTTGATTTAGTTTCAAGCCGATTTGAAGAAGCTAAGAAGTTTGGG TCAAGAATCATTGGGGTTGATTTAGTTTCAAGCCGATTTGAAGAAGCTAAGAAGTTTGGG TCAAGAATCATTGGGGTTGATTTAGTTTCAAGCCGATTTGAAGAAGCTAAGAAGTTTGGG ************************************************************

592 587 587 720

VL-ADH

GTCAATGAATTTGTGAACCCAAAAGACCATGACAAACCCGTACAACAGGTAATTGCTGAA 652

T44ADH Pb-ADH Z23170

GTCAATGAATTTGTGAACCCAAAAGACCATGACAAACCCGTACAACAGGTAATTGCTGAA 647 GTCAATGAATTTGTGAACCCAAAAGACCATGACAAACCCGTACAACAGGTAATTGCTGAA 647 GTCAATGAATTTGTGAACCCAAAAGACCATGACAAACCT GTACAAGAGGTAATTGCTGAA 780 ************************************** ****** ************** ATGACAAATGGGGGTGTGGATCGTGCTGTGGAATGTACTGGCAGCATCCAGGCCATGGTC 712 ATGACAAATGGGGGTGTGGATCGTGCTGTGGAATGTACTGGCAGCATCCAGGCCA----- 702 ATGACAAATGGGGGAGTGGATCGTGCTGTT GAATGTACTGGCAGCATCCAGGCCATGGTG 707 ATGACAAATGGGGGTGTGGATCGTGCTGTT GAATGTACTGGCAGCATCCAGGCT ATGATC 840 ************** ************** *********************** ******

VL-ADH T44ADH Pb-ADH Z23170

VL-ADH T44ADH Pb-ADH Z23170

TCAGCATTCGAATGTGTCCACGATGGTTGGGG---------------------------- 744 -----------------------------------------------------------TCAGCATTCGAATGTGTCCACGATGGTTG------------------------------- 736 TCAGCATTCGAATGTGTTCATGATGGTTGGGGTGTTGCTGTACTTGTTGGAGTGCCAAAC 900 ************************************************************

Fig. 4. (a) Clustal W (1.83) multiple sequence alignment and comparison of partial coding sequences of ADH in root tissues in V. luteola, and tolerant (T44) and susceptible (PB) genotypes of V. radiata with Phaseolus acutifolius complete CDS (Z23170). (b) Clustal W (1.83) multiple sequence alignment and comparison of deduced amino acid sequences of ADH in root tissues in V. luteola, and tolerant (T44) and susceptible (PB) genotypes of V. radiata with Phaseolus acutifolius complete CDS (Z23170).

ARTICLE IN PRESS 614

R.K. Sairam et al.

Z23170 Pb-ADH T44ADH VL-ADH

MSSTAGQVIKCKAAVAWEAGKPLVMEEVEVAPPKAGEVRLKILYTSLCHTDVYFWEAKGQ ---------------------------------------------SLCHTDVYFWEAKGQ ---------------------------------------------SLCHTDVYFWEAKGQ -------------------------------------------FTSLCHTDVYFWEAKGQ ***************

60 15 15 17

Z23170 Pb-ADH T44ADH VL-ADH

TPLFPRIFGHEAGGIVESVGEGVTHLKPGDHALPVFTGECGECAHCKSEESNMCDLLRIN TPLFPRIFGHEAGGIVESVGEGVTHLKPGDHALPVFTGECGECAHCKSEESNMCDLLRIN TPLFPRIFGHEAGGIVESVGEGVTHLKPGDHALPVFTGECGDCAHCKSEESNMCDLLRIN TPLFPRIFGHEAGGIVESVGEGVTHLKPGDHALPVFTGECGDCAHCKSEESNMCDLLRIN *********************************:****************** ********

120 75 75 77

Z23170 Pb-ADH T44ADH VL-ADH

TDRGVMIHDSQTRFSIKGQPIYHFVGTSTFSEYTVVHAGCVAKVNPAAPLDKICVLSCGI TDRGVMIHDSQTRFSIKGQPIYHFVGTSTFSEYTVVHAGCVAKVNPAAPLDKICVLSCGI TDRGVMIHDSK TRFSIKGQPIYHFVGTSTFSEYTVVHAGCVAKVNPAAPLDKICVLSCGI TDRGVMIHDSK TRFSIKGQPIYHFVGTSTFSEYTVVHAGCVAKVNPAAPLDKICVLSCGI **********:*************************************************

180 135 135 137

Z23170 Pb-ADH T44ADH VL-ADH

CTGLGATVNVAKPKPGSSVAIFGLGAVGLAAAEGARISGASRIIGVDLVSSRFEEAKKFG CTGLGATVNVAKPKPGSSVAIFGLGAVGLAAAEGARISGASRIIGVDLVSSRFEEAKKFG CTGLGATVNVAKPKPGSSVAIFGLGAVGLAAAEGARISGASRIIGVDLVSSRFEEAKKFG CTGLGATVNVAKPKPGSSVAIFGLGAVGLAAAEGARISGASRIIGVDLVSSRFEEAKKFG ************************************************************

240 195 195 197

Z23170 Pb-ADH T44ADH VL-ADH

VNEFVNPKDHDKPVQEVIAEMTNGGVDRAVECTGSIQAMISAFECVHDGWGVAVLVGVPN VNEFVNPKDHDKPVQQVIAEMTNGGVDRAVECTGSIQAMVSAFECVHDG----------VNEFVNPKDHDKPVQQVIAEMTNGGVDRAVECTGSIQAM--------------------VNEFVNPKDHDKPVQQVIAEMTNGGVDRAVECTGSIQAMVSAFECVHDGW---------***************:**********************************

300 244 234 247

Z23170 Pb-ADH T44ADH VL-ADH

KDDAFKTHPVNFLNERTLKGTFYGNYKPRTDLPLVVEQYMNGELELDKFITHTVPFSEIN 360 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Z23170 Pb-ADH T44ADH VL-ADH

KAFDYMLKGESIRCIIRMGE 380 --------------------------------------------------------------------------

Fig. 4. (Continued)

T44 and PB). Mung bean genotype PB showed an amino acid replacement at the 86th position by Q (glutamine), where as other genotypes had K (lysine) in that position. This may be one of the reasons for the less expression of ADH in PB under waterlogged condition than V. luteola and T44. The expression of b-tubulin did not vary under the control or waterlogged condition in all the three genotypes. b-Tubulin, is component of a heterodimeric protein composed of two closely related 55 kDa proteins called a and b-tubulin. The sequences of these genes are highly conserved throughout the eukaryotic kingdom. The expression of tubulin is not affected by environmental conditions, and, therefore, b-tubulin was used as an internal control. The results suggest that the availability of sufficient sugar reserve in the roots, activity of SS to provide reducing sugars for glycolytic activity and ADH for the recycling of NADH, which is essential for the continuation of glycolysis, the major source of energy under hypoxia may constitute one of the important mechanism of waterlogging tolerance of wild V. luteola and V. radiata genotype T44. The increase in the activity of the two key enzymes in V. luteola and T44 is associated

with hypoxia-induced expression of SS and ADH genes. This was reflected in better RWC and Chl content in leaves and MSI in V. luteola and T44, while PB showed heightened injury in the form of low membrane stability of leaf and root tissues, and decline in Chl content and RWC due to poor metabolic adaptation, which in turn was due to its failure to trigger molecular events, as is clear from the lack of SS- and ADH-mRNA expression under waterlogging condition.

Acknowledgment Authors are thankful to the Head, Division of Plant Physiology for providing the necessary facilities. D. Kumutha is also thankful to University Grant commission, New Delhi for providing Senior Research Fellowship during the course of the study.

References Arai M, Mori H, Imaseki H. Expression of the gene for sucrose synthase during growth of mung bean seedlings. Plant Cell Physiol 1992;33:503–6.

ARTICLE IN PRESS Waterlogging-induced sugar metabolism and genes in mung bean roots Arnon DI. Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol 1949;24:1–15. Ashraf M, Arfan M. Gas exchange characteristics and water relations in two cultivars of Hibiscus esculentus under waterlogging. Biol Plant 2005;49:459–62. Chung HJ, Ferl RJ. Arabidopsis alcohol dehydrogenase expression in both shoots and of roots is conditioned by root growth environment. Plant Physiol 1999;121:429–36. Collaku A, Harrison SA. Loses in wheat due to waterlogging. Crop Sci 2002;42:444–50. Crawford RMM, Braendle R. Oxygen deprivation stress in a changing environment. J Exp Bot 1996;47:145–59. Dolferus R, Klok EJ, Delessert C, Wilson S, Ismond KP, Good AG, Peacock WJ, Dennis ES. Enhancing the anaerobic response. Ann Bot 2003;91:111–7. Else MA, Davies WS, Malone M, Jackson MS. A negative hydraulic message from oxygen-deficient roots of tomato plant? (Influence of soil flooding in leaf water potential, leaf expansion and synchrony between stomatal conductance and root hydraulic conductivity). Plant Physiol 1995;109:1017–24. Garvin DF, Weeden NF, Doyle JJ. The reduced stability of a plant alcohol dehydrogenase is due to the substitution of serine for a highly conserved phenylalanine residue. Plant Mol Biol 1994;26:643–55. Gibbs J, Greenway H. Mechanisms of anoxia tolerance in plants. I. Growth, survival and anaerobic catabolism. Funct Plant Biol 2003;30:1–47. Gonzali S, Loreti E, Novi G, Poggi A, Alpi A, Perata P. The use of microarrays to study the anaerobic response in Arabidopsis. Ann Bot 2005;96:661–8. Green BR. The chlorophyll–protein complexes of higher plant photosynthetic membranes. Photosynth Res 1988;15: 3–32. Greenway H, Gibbs J. Mechanisms of anoxia tolerance in plants. II. Energy requirements for maintenance and energy distribution to essential processes. Funct Plant Biol 2003;30:999–1036. Guglielminetti L, Perata P, Alpi A. Effect of anoxia on carbohydrate metabolism in rice seedlings. Plant Physiol 1995;108:735–41. Hiscox JD, Israelstam GF. A method for extraction of chloroplast from leaf tissue without maceration. Can J Bot 1979;57:1332–4. Huang B, Johnson JW, Nesmith D, Bridges DC. Growth, physiological and anatomical responses of two wheat cultivars to waterlogging and nutrient supply. J Exp Bot 1994;271:193–202. Ismond KP, Dolferus R, De Pauw M, Dennis ES, Good AG. Enhanced low oxygen survival in Arabidopsis through increased metabolic flux in the fermentative pathway. Plant Physiol 2003;132:1292–302. Jackson MB, Drew MC. Effects of flooding on growth and metabolism of herbaceous plants. In: Kozlowski TT, editor. Flooding and plant growth. Orlando, Florida: Academic Press; 1984. p. 47–128. Jackson MB, Herman B, Goodenogh A. An examination of the importance of ethanol in causing injury to flooded plants. Plant Cell Environ 1982;5:163–72.

615

Johnson JR, Cobb BG, Drew MC. Hypoxic induction of anoxia tolerance in roots of Adh null Zea mays. Plant Physiol 1994;105:61–7. Kelley PM. Maize pyruvate decarboxylase mRNA is induced anaerobically. Plant Mol Biol 1989;13: 213–22. Kennedy RA, Rumpho ME, Fox T. Anaerobic metabolism in plants. Plant Physiol 1992;100:1–6. Keyhani E, Keyhani J. Hypoxia/anoxia as signaling for increased alcohol dehydrogenase activity in saffron (Crocus sativus L.) corm. Ann NY Acad Sci 2004;1030: 449–57. Liu F, Van Toai T, Moy LP, Bock G, Linford LD, Quackenbush J. Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis. Plant Physiol 2005;137:1115–29. McCready RM, Guggloz J, Silviera V, Owens HS. Determination of starch and amylose in vegetables. Anal Chem 1950;22:1156–8. Menegus F, Cattaruzza L, Molinari H, Ragg E. Rice and wheat seedlings as plant models of high and low tolerance to anoxia. In: Rosenthal M, van den Tillart G, editors. Surviving hypoxia: metabolism of adaptation and control. Boca Raton, Florida: CRC Press; 1993. p. 53–64. Min XJ, Bartholomew DP. Effects of flooding and drought on ethylene metabolism, titratable acidity and fruiting of pineapple. Acta Hortic 2005;666:135–48. Naidoo G. Effects of flooding on leaf water potential and stomatal resistance in Bruguiera gymporrhiza (L.) Lam. New Phytol 1983;93:369–76. Nelson N. A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 1944;153:375–80. Noguchi HK. Sugar utilization and anoxia tolerance in rice roots acclimated by hypoxic pretreatment. J Plant Physiol 2004;161:803–8. Prasad S, Ram PC, Uma S. Effect of waterlogging duration on chlorophyll content, nitrate reductase activity, soluble sugars and grain yield of maize. Ann Plant Physiol 2004;18:1–5. Rawyler A, Arpagaus S, Braendle R. Impact of oxygen stress and energy availability on membrane stability of plant cells. Ann Bot 2002;90:499–507. Reggiani R, Brambilla I, Bertani A. Effect of exogenous nitrate on anaerobic metabolism in excised rice roots III. Glycolytic intermediates and enzymatic activities. J Exp Bot 1986;37:1472–8. Ricard B, Couee I, Raymond P, Saglio PH, Saint-Ges V, Pradet A. Plant metabolism under hypoxia and anoxia. Plant Physiol Biochem 1994;32:1–10. Ricard B, Van Toai T, Charley P, Saglio P. Evidence for the critical role of sucrose synthase for anoxic tolerance of maize roots using a double mutant. Plant Physiol 1998;116:1323–31. Rivoal J, Ricard B, Pradet A. Glycolytic and fermentative enzyme induction during anaerobiosis in rice seedling. Plant Physiol Biochem 1989;27:43–52. Ruan YL, Llewellyn DJ, Furbank RT. Suppression of sucrose synthase gene expression represses cotton

ARTICLE IN PRESS 616 fiber cell initiation, elongation and seed development. Plant Cell 2003;15:952–64. Sachs MM, Freeling M, Okomoto R. The anaerobic proteins of maize. Cell 1980;20:761–7. Sairam RK, Deshmukh PS, Shukla DS. Tolerance to drought and temperature stress in relation to increased antioxidant enzyme activity in wheat. J Agron Crop Sci 1997;178:171–7. Sanger F, Nickler S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977;74:5463–7. Schluter U, Crawford RMM. Long term anoxia tolerance in leaves of Acorus calamus L. and Iris pseudacorus L. J Exp Bot 2001;52:2213–25. Somogyi M. Notes on sugar determination. J Biol Chem 1952;195:19–23. Springer B, Werr W, Starlinger P, Bennett DC, Freeling M. The shrunken gene on chromosome 9 of Zea mays L. is expressed in various plant tissues and encode an anaerobic protein. Mol Gen Genet 1986;220: 461–8.

R.K. Sairam et al. Weatherley PE. Studies in water relations of cotton plants. I. The field measurement of water deficit in leaves. New Phytol 1950;49:81–7. Wentworth M, Murchie EH, Gray JE, Daniel Villegas D, Claudio-Pastenes C, Pinto M, Horton P. Differential adaptation of two varieties of common bean to abiotic stress. II. Acclimation of photosynthesis. J Exp Bot 2006;57:699–709. Younis ME, El-Shahaby OA, Nemat Alla MM, Bastawisy ZM. Kinetin alleviates the influence of waterlogging and salinity on growth and affects the production of plant growth regulators in V. sinensis and Zea mays. Agronomie 2003;23:277–85. Zeng Y, Wu Y, Avigne WT, Koch KE. Differential regulation of sugar sensitive sucrose syntheses by hypoxia and anoxia indicate complementary transcriptional and post transcriptional responses. Plant Physiol 1998;116:1573–83. Zeng Y, Avigne WT, Koch KE. Rapid repression of maize invertase by low oxygen. Invertase/sucrose synthase balance, sugar signaling potential and seedling survival. Plant Physiol 1999;121:599–608.