Differences in Root Exudation Among Phosphorus-Starved Genotypes ...

JOURNAL OF PLANT NUTRITION Vol. 26, No. 12, pp. 2391–2401, 2003

Differences in Root Exudation Among Phosphorus-Starved Genotypes of Maize and Green Gram and Its Relationship with Phosphorus Uptake Bhupinder Singh1,* and Renu Pandey2 1

Nuclear Research Laboratory and 2Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi, India

ABSTRACT Availability of phosphorus (P) in soil and its acquisition by plants is affected by the release of high and low molecular weight root exudates. A study was carried out to ascertain the qualitative and quantitative differences in root exudation among the genotypes of maize (Zea mays L.) and green gram (Vigna radiata L.) under P-stress. Results showed that both inter- and intra-species differences do exist among maize and green gram in terms of root exudation, P uptake, and shoot and root P content. In general, green gram, a legume crop, had greater root exudation compared to maize. However, the amino acid content of the total root exudates in maize was two-fold as compared to green gram. The maize and green gram genotypes possessed genetic variability in root exudation.

*Correspondence: Bhupinder Singh, Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi 110 012, India; E-mail: [email protected]. 2391 DOI: 10.1081=PLN-120025467 Copyright # 2003 by Marcel Dekker, Inc.

0190-4167 (Print); 1532-4087 (Online) www.dekker.com

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Singh and Pandey Irrespective of the species or genotypes, a positive relationship was found among P uptake rates, total root exudation, and shoot and root 32P content. The amount of sugars and amino acid present in the root exudates of P-starved seedlings also add to the variation in P uptake efficiency of genotypes. Key Words: Phosphorus-uptake; Zea mays L.; Vigna radiata L.; Root exudation; Free amino acid.

INTRODUCTION The possibility of exploiting genotypic differences for improving nutrient efficiency of crop plants has received increased attention in recent years.[1–3] Phosphorus (P) is an important macronutrient next to nitrogen. Although, its deficiency can be corrected through the use of phosphatic fertilizers, a higher dose of P fertilizer is, generally, required for sustaining crop yields due to high P-fixation capacity of problem soils. Long-term use of such high P dose not only caused irreparable damage to the soil and imbalance in the availability of secondary and micronutrients but also lead to environmental degradation. Development of P efficient genotypes is, therefore, desired for maintaining high productivity in low P soils and under low P input conditions. Genotypic differences in P efficiency have been reported.[4–6] These differences in P efficiency are related to differences in efficiency of acquisition by roots or in utilization by plant, or both. Phosphorus acquisition efficiency is influenced by size and distribution of root system,[4,7] root hairs,[8] kinetic uptake parameters,[9] root induced pH change,[5,10] root exudation,[11,12] and soil moisture.[13] Rhizosphere modification through root exudates not only regulates P-availability in the soil but also its acquisition by the plant. Root exudates contain both high (e.g., mucilage, ectoenzymes) and low molecular weight fractions (e.g., organic acids, sugars, phenolics and amino acids, and phytosiderophores). Besides P, they also mobilize other micronutrients (e.g., zinc, iron, and manganese).[12] These exudates alter nutrient dynamics in the rhizosphere and affect the nutrient status of a plant by mobilizing sparingly soluble inorganic phosphates into the soil solution. When plants are nutrient deficient the amount of exudates released by the root often increases.[14] Enhanced release of organic acids has been reported under P deficiency in dicots in general and legumes in particular.[15] The release of large amount of citric acid from P-deficient white lupin roots was found to be one of the efficient strategies for chemical mobilization of sparingly available P sources

Root Exudation in Maize and Green Gram

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in the rhizosphere.[16] A relationship between acid phosphatase activity, a high-molecular weight (HMW) constituent of exudates and P-stress has also been reported.[17] The objectives of this study were to measure the differences in root exudation of maize (Zea mays L.) and green gram (Vigna radiata L.) seedlings grown under P-stress and to assess the extent of genotypic variability in terms of root exudation and its relationship with plant P-uptake.

MATERIALS AND METHODS Plant Material An experiment was conducted in pot culture with maize (cv. Ganga-11 and Deccan-103) and green gram (cv. PS-16 and Pusa-105) genotypes. Seeds were procured from the Directorate of Maize Research and Pulse Research Laboratory, Indian Agricultural Research Institute, New Delhi, respectively. The maize genotypes were raised in sand supplied with full strength Hoagland solution without P from 8th day after sowing, while for green gram, the Hoagland solution was supplied from 10th day after sowing. Plants were irrigated with nutrient solution and distilled water on alternate days. A total of 12 pots with one plant each were maintained for each genotype following completely randomized design. Twenty-day-old seedlings of both the genotypes of maize and green gram were used for the experiment.

Root Exudation Experiment For root exudation studies, fully developed leaf selected at random, were supplied with 10 mL of 14C-sucrose (specific activity 580.0 mCi=mmole, activity 50 mCi=mL) after abrading the leaf surface. Spillage of radioactivity was avoided by lanolin smearing on the dorsal side. Ringing the area of treatment on the ventral surface was done to ensure solution penetration and absorption. Label was applied around late noon to reduce evaporation loss.[18] The treatment was triplicated and in each replicate, the radioactivity was applied on identified leaves at different positions of the main axis. After 24 h, pots were irrigated with double distilled water and the drainage was collected until no activity was traced in the water. The 14C activity in the drainage water was estimated using liquid scintillation counter (Packard–Tricarb). Free amino acid and reducing sugar content in the drainage water were determined following the method of Rosen[19] and Nelson,[20] respectively.

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P Uptake Experiment

For ascertaining P uptake differences among the genotypes, P-starved seedlings were uprooted and their roots were thoroughly washed with double distilled water. Roots were then immersed in 15 mM phosphate solution. To this, 32P phosphoric acid was added as a tracer to give an activity of 2000 Bq=mL to allow solution P concentration to be monitored radiometrically.[21] Solutions were aerated and 32P activity in the samples was analyzed after every 20 min up to 3 h using a liquid scintillation counter. Rates of P-uptake were calculated from depletion of P from the solution. 32P content of root and shoot was also determined.[21] Data were subjected to analysis of variance using ANOVA following the method of Cochran and Cox.[22]

RESULTS AND DISCUSSION Results indicate the differences in root exudation between maize and green gram (Table 1). The average amount of root exudation by green gram, based on 14C activity measurements, was almost 3-fold higher as compared to maize. These results are in contrast to those reported by Ae et al.[23] for pigeon pea, a leguminous plant, which was shown to possess high levels of P mobilizing capacity from P-deficient soils despite low root exudation. However, in this experiment maize exudates contained a higher level of free amino acids and reducing sugars (Table 1).

Table 1. Root exudation and free amino acid and reducing sugar contents in genotypes of maize and green gram. Root exudation (14C released, dpm= mg root dry wt.)

Free amino acids (mg leucine equiv.= g root dry wt.)

Reducing sugars (mg glucose equiv.= g root dry wt.)

106.26 7.21 65.11 5.14

0.49 0.053 0.37 0.012

3.50 0.75 2.70 0.39

Mean

84.19 6.17

0.43 0.032

3.20 0.57

Green gram PS-16 Pusa-105

174.50 11.2 321.17 17.0

0.16 0.03 0.21 0.02

1.70 0.27 2.30 0.19

Mean

247.84 14.1

0.19 0.025

2.00 0.23

Genotype Maize Ganga-11 Deccan-103

Note: Means SE, n ¼ 9.


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Studies with 14CO2 demonstrated that photosynthates are rapidly transported to roots from where a certain proportion is released into the rhizosphere.[24] These results also showed that about 98% of 14C was released by the roots as exudates. Reducing sugars constituted only 1.54% of the total exudate whereas amino acid represented the smallest fraction (0.18%) of the total root exudation. These results confirm those of Kraffczyk et al.[14] In studying root exudates a certain re-uptake of released LMW compounds should be taken into account for amino acid as suggested by Miller and Schmidt.[25] Accordingly, the detected quantities represent solely the difference between the amounts of amino acid that were released and reabsorbed. A significant difference among the genotypes of both maize and green gram for the amount of root exudation was also observed. Among the maize genotypes, the amount of exudation for Ganga-11 was almost twice of that of Deccan-103. Ganga-11 also revealed higher amino acid and sugar content in the root exudates than Deccan-103. Similarly, between the two green gram genotypes, Pusa-105 exuded almost 2-fold over that of PS-16. Pusa-105 contained higher amount of sugars and amino acid content as compared to PS-16. Inter-specific differences in terms of 32P-uptake, shoot and root P content were also significant (Table 2). Green gram had a 1.4 times higher P-uptake rate as compared to maize. The genotypic mean value for shoot P content in maize was higher than that of green gram. However, root P content was recorded

Table 2. gram.

32

P uptake and shoot and root

32

P activity in genotypes of maize and green

32 P uptake (dpm mg1 root dry wt. h1)a

Shoot 32P activity [103 dpm (32P) g1 dry wt.]

Root 32P activity [103 dpm (32P) g1 dry wt.]

19.00 0.89 18.80 0.57

4.22 1.03 3.78 0.89

0.85 0.02 0.97 0.02

Mean

18.90 0.73

4.00 0.96

0.91 0.02

Green gram PS-16 Pusa-105

21.30 1.11 29.60 0.98

2.10 0.04 3.30 0.05

1.00 0.04 1.50 0.02

Mean

25.45 1.04

2.70 0.04

1.25 0.03

Genotype Maize Ganga-11 Deccan-103

Note: Means SE, n ¼ 9. a 32 P uptake over the total time period of the experiment.

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higher in green gram. Moreover, the rate of 32P-uptake was significantly higher for the green gram genotype Pusa-105 than PS-16 (Fig. 1) whereas in maize, genotype Deccan-103 showed higher uptake rate especially during initial stages, until 2 h of incubation (Fig. 2). The uptake pattern for the genotypes showed a reverse trend thereafter, which continued until the end of the observation period. Phosphorus-uptake, averaged over the total time period, also varied for the two genotypes with Ganga-11 showing significantly higher 32P uptake compared to Deccan-103. Further, Ganga-11 also recorded higher shoot 32P content than Deccan-103, while the reverse was observed for root 32P content (Table 2). Similarly, between the two green gram genotypes, Pusa-105 recorded high mean rate of 32P uptake and higher 32P content in both shoot and root as compared to PS-16. Relatively higher and lower radioactivity in root and shoot, respectively, of these genotypes may be related to differential rates of translocation of absorbed P. These differences may be related to the differential kinetic parameters associated with P-translocator proteins in the two genotypes. Irrespective of the species or genotypes, a positive relationship

Figure 1. Difference in 32P uptake among the two green gram genotypes. Means SE, n ¼ 9.


Figure 2. Difference in n ¼ 9.

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P uptake among the two maize genotypes. Means SE,

was found between P uptake rates, total root exudation and shoot and root P (32P) content. The amount of sugars and amino acid, which constitute LMW components of root exudates, also supported the variation in P-uptake efficiency of genotypes. Differences in root exudation have also been reported for other crop species.[16,26,27] Amino acid content of maize genotypes was higher than those reported for legumes.[28] This may be related to the strategy adopted by graminaceous species, which show an enhanced release of non-proteinogenic amino acid in response to nutrient deficiency especially that of Fe.[29,30] These non-proteinogenic amino acids may be responsible for the higher free amino acid pool recorded in exudates of maize genotypes. Bowen[31] and Graham et al.[32] found a 2-fold and 3-fold increase, respectively, in the quantities of amino acid released from P stressed plants. Graham et al.[32] found a correlation between increased exudation and membrane permeability, using potassium-rubidium efflux and concluded that root exudation is directly related to the permeability of the membrane which is controlled largely by the phospholipid content of the root.[33] Lipton et al.[15]

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suggested that a depressed phospholipid content in the P-stressed alfalfa plants accounted for the increased release of citrate.

CONCLUSIONS These results indicate the existence of variability for root exudation and P uptake efficiency among the investigated maize and green gram genotypes. Also, this study found that legumes released higher amounts of total root exudates as compared to cereals. Results from this research indicate a positive relationship between exudation and P-acquisition by roots. The release of organic compounds from the roots under P-stress, therefore, may be an adaptive mechanism by which the plant can alter its micro-environment and subsequently affect nutrient availability in the rhizosphere. Although genetic studies have been done on manipulating root size and morphology and P uptake,[34] no studies have been reported so far on heritability of P efficiency mechanisms associated with root exudation and solubilization of P complexes considered plant-unavailable.[34] However, genetic variability in root exudation has been reported for several species.[27,35] Hence, more research into the genetic basis of qualitative and quantitative differences in root exudation is warranted.

ACKNOWLEDGMENT We thank the Project Director, Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi for providing the necessary facilities to conduct the experiment.

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5. Gahoonia, T.S.; Classen, N.; Jungk, A. Mobilization of phosphorus in different soils by ryegrass supplied with ammonium and nitrate. Plant Soil 1992, 143, 241–248. 6. Pandey R.; Singh, B.; Nair, T.V.R. Genotypic variability for P acquisition among wheat genotypes under P stress. In Proceedings of the 13th International Symposium of the International Scientific Center of Fertilisers; Gaziosman Pasa University, Takat, Turkey, 2002; 63–74. 7. Noordwijk van, M.; Willigen de, P.; Ehlert, P.A.I.; Chardon, W.J. A simple model of P uptake by crops as a possible basis for P fertilizer recommendations. Neth. J. Agric. Sci. 1990, 38, 317–332. 8. Gahoonia, T.S.; Care, D.; Nielsen, N.E. Root hairs and acquisition of phosphorus by wheat and barley cultivars. Plant Soil 1997, 191, 181–188. 9. Nielsen, N.E.; Barber, S.A. Differences among genotypes of corn in the kinetics of P uptake. Agron. J. 1978, 70, 695–698. 10. Gahoonia, T.S.; Nielsen, N.E. The root induced pH change on the depletion of inorganic and organic phosphorus in the rhizosphere. Plant Soil 1992, 143, 183–189. 11. Bar-Yosef, B. Root excretions and their environmental effects: influence on availability of phosphorus. In Plant Root: The Hidden Half; Waisel, Y., Eshel, A., Kafkafi, U., Eds.; Marcel Dekker, Inc.: New York, 1991; 529–557. 12. Marschner, H. Mineral Nutrition in Higher Plants; Academic Press Ltd.: London, UK, 1995. 13. Gahoonia, T.S.; Raza, S.; Nielsen, N.E. Phosphorus depletion in the rhizosphere as influenced by soil moisture. Plant Soil 1994, 159, 213–218. 14. Kraffczyk, I.; Trolldenier, G.; Beringer, H. Soluble root exudates of maize: influence of potassium supply and microorganisms. Soil Biol. Biochem. 1984, 16, 315–322. 15. Lipton, D.S.; Blanchar, R.W.; Blevins, D.G. Citrate, malate and succinate concentration in exudates from P-sufficient and P-stressed Medicago sativa L. seedlings. Plant Physiol. 1987, 85, 315–317. 16. Neumann, G.; Massoneau, A.; Martinoia, E.; Romheld, V. Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin. Planta 1999, 208, 373–382. 17. Sachay, J.E.; Wallace, R.L.; John, A. Phosphate stress response in hydroponically grown maize. Plant Soil 1991, 132, 85–90. 18. Singh, B.; Kaim, M.S. Use of isotopes in carbon translocation and rhizosphere modification. In Proceedings of Summer School on Use of Isotopes and Radiation in Soil Plant Relationship; Nuclear Research Laboratory, IARI: New Delhi, India, 1998; 287–289.

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19. Rosen, H. A modified ninhydrin colorimetric analysis for aminoacids. Arch. Biochem. Biophys. 1957, 67, 10–15. 20. Nelson, N. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 1944, 153, 375–380. 21. Jungk, A.; Asher, C.J.; Edwards, D.G.; Meyer, D. Influence of phosphate status on phosphate uptake kinetics of maize and soybean. Plant Soil 1990, 124, 175–182. 22. Cochran, W.G.; Cox, G.M. Experimental Designs, 2nd Ed.; Wiley & Sons: New York, 1957. 23. Ae, N.; Arihara, J.; Okada, K.; Yoshihara, T.; Johansen, C. Phosphorus uptake by pigeon pea and its role in cropping systems of Indian subcontinent. Science 1990, 248, 477–480. 24. McDougall, B.M. Movement of 14C-photosynthate into the roots of wheat seedlings and exudation of 14C from intact roots. New Phytol. 1970, 69, 37–46. 25. Miller, R.H.; Schmidt, E.L. Uptake and assimilation of amino acids supplied to the sterile soil: root environment of the bean plant (Phaseolus vulgaris). Soil Sci. 1965, 100, 323–330. 26. Ohwaki, Y.; Hirata, H. Differences in carboxylic acid exudation among P-starved leguminous crops in relation to carboxylic acid content in plant tissues and phospholipid levels in roots. Soil Sci. Plant Nutr. (Tokyo) 1992, 38, 235–243. 27. Subbarao, G.V.; Ae, N.; Otani, T. Genotypic variation in iron- and aluminium-phosphate solubilizing activity of pigeon pea root exudates under P deficient conditions. Soil Sci. Plant Nutr. 1997, 43, 295–305. 28. Singh, B. Difference in root exudation among green gram genotypes in relation to P-uptake J. Nuclear Agric. Biol. 2000, 29 (2), 104–108. 29. Romheld, V. The role of phytosiderophores in acquisition of iron and other micronutrients in graminaceous species: an ecological approach. Plant Soil 1991, 130, 127–134. 30. Von Wiren, N.; Marschner, H.; Romheld, V. Uptake kinetics of iron phytosiderophores in two maize genotypes differing in iron efficiency. Physiol. Plant. 1995, 93, 611–616. 31. Bowen, G.D. Nutrient status effects on loss of amides and amino acids from pine roots. Plant Soil 1969, 30, 139–142. 32. Graham, J.H.; Leonard, R.T.; Menge, J.A. Membrane-mediated decrease in root exudation responsible for inhibition of vesicular-arbuscular mycorrhiza formation. Plant Physiol. 1982, 68, 549–552. 33. Ratnayake, R.T.; Leonard, R.T.; Menge, J.A. Root exudation in relation to supply of phosphorus and its possible relevance to mycorrhizal formation. New Phytol.1978, 81, 543–552.


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34. Rengel, Z. Physiological mechanisms underlying differential nutrient efficiency of crop genotypes. In Mineral Nutrition of Crops: Fundamental Mechanisms and Implications; Rengel, Z., Ed.; FPP, The Haworth Press, Inc.: New York, 1999; 227–265. 35. Caradus, J.R. Genetic control of phosphorus uptake and phosphorus status in plants. In Genetic Manipulation of Crop Plants to Enhance Integrated Nutrient Management in Cropping Systems. 1. Phosphorus; Proceedings of an FAO=ICRISAT Expert Consultancy Workshop, March 15–18, 1994; Johansen, C., Lee, K.K., Sharma, K.K., Subbarao, G.V., Kueneman, E.A., Eds.; ICRISAT, Patencheru: Andhra Pradesh, India, 1995; 55–74.