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Carbon and nitrogen assimilation, and growth of moongbean (Vigna radiata [L.]Wilczek) cultivars grown under sulfur regimesNaser A. Anjum a; Shahid Umar b; Muhammad Iqbal b; Nafees A. Khan a

a Aligarh Muslim University, Aligarh, India b Hamdard University, New Delhi, India

Online Publication Date: 01 April 2009

To cite this Article Anjum, Naser A., Umar, Shahid, Iqbal, Muhammad and Khan, Nafees A.(2009)'Carbon and nitrogen assimilation,and growth of moongbean (Vigna radiata [L.] Wilczek) cultivars grown under sulfur regimes',Archives of Agronomy and SoilScience,55:2,207 — 215

To link to this Article: DOI: 10.1080/03650340802350626

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Carbon and nitrogen assimilation, and growth of moongbean

(Vigna radiata [L.] Wilczek) cultivars grown under sulfur regimes

Naser A. Anjuma*, Shahid Umarb, Muhammad Iqbalb and Nafees A. Khana

aAligarh Muslim University, Aligarh, India; bHamdard University, New Delhi, India

(Received 7 June 2008; final version received 17 July 2008)

Sulfur (S) has become an essential macronutrient for plants but its deficiency inagricultural soil is increasing worldwide. In a greenhouse pot-experiment theinfluence of S availability (0 and 40 mg S kg71 soil) on some growth andphysiological attributes and their relationships was determined in two cultivars(Pusa 9531 and PS 16) of moongbean (Vigna radiata [L.] Wilczek) at 20 and 40days after sowing (DAS). Sulfur deficiency translated as decreased plant dry mass(PDM), leaf area (LA), net photosynthesis (PN), nitrate reductase activity (NRA),contents of chlorophyll (Chl), soluble protein and leaf sulfate (SO4

27). Thesechanges were in correspondence with significant increase in contents of leaf nitrate(NO3

7) and free amino acids being more pronounced in cultivar PS 16 than Pusa9531. The relationship of NRA with plant dry mass and PN was worked out inboth cultivars. Pusa 9531 significantly maintained the coordination between Cand N assimilation and hence, the plant growth under S-deficient condition. Theinformation may be useful in breeding moongbean cultivars.

Keywords: carbon and nitrogen assimilation; moongbean; nitrate reductaseactivity; photosynthesis; sulfur regimes

Introduction

Sulfur (S) is an essential macronutrient of plants and plays important role in plantgrowth and in the regulation of plant development (Ernst 1998; Ahmad and Abdin2000; Anjum et al. 2008a, 2008b; Nazar et al. 2008). Sulfur is required for proteinsynthesis and being a structural constituent of several coenzymes and prostheticgroups such as ferredoxin, it is also significant for nitrogen (N) assimilation (Petrovicand Kastori 1994; Marschner 1995). Nitrogen is consumed in the greatest abundanceand also limits the growth and productivity of plants as well. The assimilatorypathways of S and N have been considered functionally convergent and wellcoordinated as the availability of one element regulates the other (Reuveny et al.1980; Schnug et al. 1993) and that C assimilation pathway is closely linked to nitrateassimilation in plants (Ahmad and Abdin 2000).

Sulfur deficiency in the soil is increasing globally due either to the use of high-analysis low-S containing fertilizers, the decreasing use of S-containing fungicides andpesticides, high yielding varieties, intensive agriculture and/or to the reduction of SO2

emission from industrial sources (Scherer 2001; Eriksen et al. 2004). Thus, the

*Corresponding author. Email: dnaanjum@gmail.com

Archives of Agronomy and Soil Science

Vol. 55, No. 2, April 2009, 207–215

ISSN 0365-0340 print/ISSN 1476-3567 online

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DOI: 10.1080/03650340802350626

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identification and cultivation of S efficient cultivars that could use available soil-Sefficiently may be a realistic alternative to S fertilizer application in S-deficient soil.Moongbean, a leguminous crop requires S in a large quantity, probably because of itshigh protein content and shows considerable changes in Nmetabolism, particularly thebiosynthesis of amino acids (Anjum 2006). The C and N assimilation, and growth havenot been studied in moongbean grown under S regimes. The present study hence, aimsto determine the response of moongbean cultivars when grown under S regimes, and tounderstand the involvement of changes in the coordination between C and Nassimilation, and growth. To accomplish the aims moongbean cultivars namely Pusa9531 and PS 16 were tested for PDM accumulation, LA, PN, Chl content, NRA,contents of leaf SO4

27 and NO37. NRA was correlated with PN and PDM in both

cultivars.

Materials and methods

Plant cultivation and treatments

Seeds of moongbean (Vigna radiata [L.] Wilczek) cultivars Pusa 9531 (highphotosynthetic capacity) and PS 16 (low photosynthetic capacity) (Anjum et al.2008c) were sown in 40 cm diameter earthen pots filled with 10 kg soil. The soil wassandy loam in texture, deficient in available S with pH 7.8, E.C. 3.8 dsm, organiccarbon 0.43% and available potassium (K) was 70 ppm. Sufficient amounts ofnitrogen (N), phosphorus (P) and K at the rate of 120, 30 and 80 mg kg71 soil,respectively, were applied at the time of sowing in the form of urea, single superphosphate and muriate of potash. Two treatments: þS (40 mg S kg71 soil) and 7S(0 mg S kg71 soil) were designed. The treatments were arranged in a randomizedblock design and each treatment was replicated three times. After seedlingemergence, three plants per pot were maintained and irrigated when needed. Thepots were kept in naturally illuminated green house (photosynthetically activityradiation [PAR] 4 960 mmol m2 s71, day/night temperature 25/20 + 48C andrelative humidity of 70 + 5%) in the Department of Botany, Hamdard University,New Delhi, India (288380 N, 778110 E and 228 m altitude) during a summer season.All the measurements were performed at 20 and 40 days after sowing (DAS).

Measurements

Plant dry mass, leaf area, net photosynthetic rate and chlorophyll content

Plant dry mass was recorded after drying the plants for 48 h to a constant weight at808C. Leaf area was measured by a leaf area meter (LI-3000A, Nebraska). Netphotosynthetic rate (PN) was measured on fully expanded third leaf from top usinginfra red gas analyzer (LI-6400, Nebraska) at 09:00–11:00 h at light saturatingintensity. The content of chlorophyll (a þ b) was estimated by using dimethylsulphoxide (DMSO), following the protocol described byHiscox and Israelstam (1979).

Nitrate reductase activity, soluble protein and free amino acids contents

Nitrate reductase activity and contents of soluble protein and free amino acids weremeasured by the methods of Klepper et al. (1971), Bradford (1976) and Lee andTakahashi (1966), respectively.

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Leaf SO427 and NO3

7 contents

Dried leaf powder (100 mg) was digested in a mixture of concentrated HNO3 and60% HClO4 (85:1, v/v) and the content of SO4

27 was estimated using theturbidimetric method of Chesnin and Yien (1951) while, the leaf NO3

7 content wasmeasured by spectrophotometry at 410 nm as performed by Cataldo et al. (1975).Both SO4

27and NO37 content in leaf were expressed in mg g71 dry weight (DW).

Statistical analysis

Data were analyzed statistically and given as mean + standard error (SE). Analysisof variance (ANOVA) was performed by SPSS (Statistical Procedure for SocialSciences) (Version 10.0 Inc., Chicago, USA) and least significant difference (LSD)was calculated to identify significant differences between treatments. The treatmentsmean was separated using Duncan’s Multiple Range Test (DMRT) at p � 0.05.

Results

Overall, S deprivation caused a dramatic reduction in moongbean growth, measuredas plant dry mass (PDM) and leaf area (LA) (Table 1). However, a difference in thegrowth inhibition caused by S deprivation was found between the two cultivars.Thus, cultivar PS 16 showed a significant reduction in both PDM and LA whengrown under S deprivation compared to control (40 mg S kg71 soil). With Sapplication (40 mg S kg71 soil) the maximum values of PDM and LA occurred infor Pusa 9531 and minimum for PS 16. The effect of S deprivation on chlorophyll(Chl) content and net photosynthetic rate (PN) also varied significantly withcultivars. Cultivar PS 16 showed a significant reduction in both Chl content and PN

when grown under S deprivation compared to control (40 mg S kg71 soil). With theapplication of S (40 mg S kg71 soil) increase the content of Chl and PN; beinggreater in Pusa 9531 than PS 16 (Table 1).

Table 1. Changes in plant dry mass (PDM), leaf area (LA), net photosynthesis (PN) andchlorophyll (Chl) content in moongbean cultivars as affected by S availability at 20 and 40days after sowing (DAS). Values are means + SE (n ¼ 3). Data followed by the same letterare not significantly different at p � 0.05 level as determined by DMRT.

Cultivars

S levels(mg kg71

soil)PDM

(g plant71) LA (cm2)PN (mmol CO2

m72 s71)Chl content(mg g71 FW)

20 DASPusa 9531 0 1.18b + 0.03 117.41b + 2.94 12.20b + 0.31 1.10ab + 0.03

40 1.47a + 0.04 147.11a + 3.68 13.41a + 0.34 1.21a + 0.03PS 16 0 1.04c + 0.03 104.22c + 2.61 9.89c + 0.25 0.98c + 0.02

40 1.19b + 0.03 123.05b + 3.08 10.78c + 0.27 1.07bc + 0.03

40 DASPusa 9531 0 8.36b + 0.21 312.85b + 7.82 18.07a + 0.45 1.31ab + 0.03

40 10.06a + 0.25 360.26a + 9.01 18.98a + 0.47 1.43a + 0.04PS 16 0 5.25d + 0.13 210.07d + 5.25 14.10b + 0.35 1.14c + 0.03

40 6.03c + 0.15 247.32c + 6.18 14.69b + 0.37 1.23bc + 0.03

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The activity of nitrate reductase (NR) increased by 40.5 and 25.4% with Saddition at 20 and 40 DAS, respectively in Pusa 9531. While in PS 16, the activity ofNR increased by 25.5 and 21% with the application of S (40 mg kg71 soil),respectively at 20 and 40 DAS. A similar pattern of leaf soluble protein and SO4

27

contents was found: with the addition of S the content of soluble protein increasedby 21.1 and 16.9%, respectively, at 20 and 40 DAS in Pusa 9531. In PS 16, thecontent of soluble protein increased by 16.2 and 11.3%, respectively, at 20 and 40DAS due to S application. With the addition of S to soil increased the leaf SO4

27

content by 75.2 and 198.0% at 20 and 40 DAS, respectively, in Pusa 9531; while inPS 16, the leaf SO4

27 content increased by 39.9 and 26.7% with S addition at 20and 40 DAS, respectively. With the addition of S Pusa 9531 showed the maximumleaf NO3

7 and free amino acids accumulation decreased significantly. Leaf NO37

content decreased by 28.4 and 15.8% with S addition at 20 and 40 DAS,respectively in Pusa 9531. Contrarily to this, in PS 16, leaf NO3

7 contentsignificantly increased by 13.1 and 7.2% at 20 and 40 DAS, respectively. However,the accumulation of free amino acids decreased with S addition in both cultivars butthe extent of decrease was greater in Pusa 9531 than PS 16. In Pusa 9531, free aminoacids accumulation decreased by 37.7 and 29.5 at 20 and 40 DAS, respectively;while decrease of 8.5 and 4.4% were noted at 20 and 40 DAS with S addition in PS16 (Tables 2 and 3).

A strong positive correlation (p � 0.05) of NRA with PDM and PN was found(Figure 1).

Discussion

Sulfur is an essential macronutrient that plays a vital role in the regulation of plantgrowth and development (Ernst 1998; Anjum et al. 2008a, 2008b; Nazar et al. 2008).In the present study, the capacity of moongbean cultivars to survive in an S-deprivedsituation was traced out through measuring changes in C and N, and growthcharacteristics and it was found that it is partially linked to their efficiency in the

Table 2. Changes in leaf nitrate reductase activity (NRA), soluble protein content and freeamino acid content in moongbean cultivars as affected by S availability at 20 and 40 days aftersowing (DAS). Values are means + SE (n ¼ 3). Data followed by the same letter are notsignificantly different at p � 0.05 level as determined by DMRT.

CultivarsS levels

(mg kg71 soil)NRA (mmol NO2

h71 g71 FW)

Soluble proteincontent

(mg g71 FW)

Free aminoacid content(mg g71 FW)

20 DASPusa 9531 0 2.17b + 0.05 3.12b + 0.08 6.88a + 0.17

40 3.05a + 0.08 3.78a + 0.09 4.28b + 0.11PS 16 0 1.89c + 0.05 2.16d + 0.05 7.05a + 0.18

40 2.41b + 0.06 2.51c + 0.06 6.45a + 0.16

40 DASPusa 9531 0 4.63b + 0.12 4.01b + 0.10 9.56a + 0.24

40 5.81a + 0.15 4.69a + 0.12 6.74b + 0.17PS 16 0 3.19d + 0.08 3.11c + 0.08 10.23a + 0.26

40 3.86c + 0.10 3.46c + 0.09 9.78a + 0.24

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maintenance of coordination between C and N assimilation, growth. A higher LAresulted from the application of S (40 mg S kg71 soil), which indicates better LAexpansion in turn helped in subsequent interception and efficient utilization of solarradiation. An increased rate of PN due to S application (40 mg S kg71 soil) could beexplained well with the other findings of this paper that S (40 mg S kg71 soil)-induced improvement in NRA, protein synthesis and maintenance of high Chlcontent. The S nutrition of a plant strongly influences PDM production. Thus, theimprovement in LA and PN due to S application (40 mg S kg71 soil) translated ashigher PDM due to production of more photosynthates. These findings are in closeconformity with Zhao et al. (1997), Ahmad and Abdin (2000) and Ahmad et al.(2005a, 2005b).

In the present study, S deprivation resulted in inhibition of soluble proteincontent and increased accumulation of free amino acids. There is a concomitant Sdeprivation-induced decline in PN and total Chl content which at least in part, issufficient to support the decrease in content of soluble protein and increase in thecontent of free amino acids (Table 1). This is in close conformity with the findings ofKlapheck et al. (1982) and Gilbert et al. (1997) who found reduced synthesis ofprotein and subsequent accumulation of free amino acids due to S starvation inNicotiana tabacum and Triticum aestivum, respectively. Besides, S starvation hasbeen shown to mainly affect NRA, the rate limiting step in N assimilation in plants(Reuveny et al. 1980). In fact, NO3

7 assimilation results in to the production ofprincipal nitrogenous compounds (amino acids and proteins) (Barneix and Causin1996). The S deprivation-induced reductions in S-containing amino acids (cysteineand methionine) could result in increase in proteolysis and/or decline in the rate ofprotein synthesis (Brunold 1993) which in turn lead to high accumulation of freeamino acids. Besides, S deprivation also led to increase in leaf NO3

7 which isassimilated by NRA (Table 2). In this way S deprivation-induced inhibition of NRAcould also explain the accumulation of free amino acids, leaf NO3

7 and decrease inprotein content. These findings have strong supports from studies of Klapheck et al.(1982), Barney and Bush (1985), Brunold (1993), Stuiver et al. (1997) and Ruizet al. (2005). Contrary to this, free amino acids accumulation in S-deprived

Table 3. Changes in leaf nitrate (NO37) and leaf sulfate (SO4

72) in moongbean cultivars asaffected by S availability at 20 and 40 days after sowing (DAS). Values are means + SE(n ¼ 3). Data followed by the same letter are not significantly different at p � 0.05 level asdetermined by DMRT.

CultivarsS levels

(mg kg71 soil)Leaf NO3

7 content(mg g71 DW)

Leaf SO472 content

(mg g71 DW)

20 DASPusa 9531 0 2.85b + 0.07 2.19d + 0.05

40 2.04c + 0.05 3.77c + 0.09PS 16 0 3.67a + 0.09 5.45b + 0.14

40 3.09b + 0.08 6.53a + 0.16

40 DASPusa 9531 0 2.97c + 0.07 1.98d + 0.05

40 3.36b + 0.08 2.77c + 0.07PS 16 0 3.88a + 0.10 3.17b + 0.08

40 4.16a + 0.10 3.70a + 0.09

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cultured Zea mays cells was suggested as a consequence of de-novo synthesis(Amancio et al. 1997). However, S deprivation-induced decrease in NRA wassuggested as an important factor for the observed changes (Reuveny et al. 1980;Migge et al. 2000).

The increase in leaf SO427, NRA with decrease in leaf NO3

7 with S (40 mg Skg71 soil) as obtained in our study, thus, may be due to metabolic coupling betweenS and N metabolism. Nitrate reductase catalyzes the rate limiting step of nitrateassimilatory pathway and provides reduced N for use in the synthesis of free aminoacids and proteins while the C assimilation pathway provides C for the same(Ahmad and Abdin 1999a, 1999b, 2000). A possible role of applied S in the

Figure 1. Correlation of nitrate reductase activity (NRA) with plant dry mass (PDM) andnet photosynthesis (PN) in two cultivars of Vigna radiata.

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regulation of NRA has been found in various crop species (Reuveny et al. 1980;Barney and Bush 1985; Takahashi and Saito 1996; Ahmad et al. 1999a, 1999b;Ahmad and Abdin 2000).

To substantiate the validity of these findings, NRA was correlated with thePDM and PN in both cultivars during changes in leaf SO4

27 (Figure 1). Fromminimum reductions in C, N assimilation, and PDM and LA due to Sdeprivation and positive relationship of NRA with PN and PDM in Pusa 9531showed that S deprivation perturbs less to growth by reducing the leaf SO4

27,increased leaf NO3

7 mainly by less disruption of the coordination between Cand N metabolism than cultivar PS 16.

Conclusion

Sulfur deficiency was differentially translated as decreased PDM, LA, PN, NRAcontents of Chl, soluble protein and leaf SO4

27 in moongbean cultivars. Betweenmoongbean cultivars, Pusa 9531 was found very efficient in the maintenance ofcoordination between C and N assimilation and hence, the plant growth under S-deficient condition. When supplied with S (40 mg S kg71 soil) greater increases inleaf SO4

27, NRA, PN, LA, PDM, contents of Chl and soluble protein with decreasesin leaf NO3

7and free amino acids contents were noticed in Pusa 9531 than PS 16,thus, showing efficient uptake and assimilation of available soil-SO4

27 and itssubsequent utilization in the reduction of NO3

7 by Pusa 9531. Besides, the studyalso shows the existence of metabolic coupling between S and N metabolism, and Cassimilation and also speculates the presence of different types of S-transporterssystems in roots of moongbean cultivars.

Acknowledgements

The first author is deeply indebted to Hamdard National Foundation (HNF), New Delhi,India, and to the American Foundation for Muslims from India (AFMI) for financialassistance for the work. Authors wish to thank Dr Altaf Ahmad, for critical reading of themanuscript and helpful discussion and Drs Naresh Chandra and JL Tickoo, scientists atPulses Breeding Section, Division of Genetics of Indian Agriculture Research Institute (IARI),New Delhi, India, for providing authentic moongbean seeds.

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