The effect of phosphorus deficiency on nutrient uptake, nitrogen ...

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Feb 28, 2008 - David A. Lightfoot Æ Kounosuke Fujita ... Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2008. Abstract ...
Acta Physiol Plant (2008) 30:537–544 DOI 10.1007/s11738-008-0152-8

ORIGINAL PAPER

The effect of phosphorus deficiency on nutrient uptake, nitrogen fixation and photosynthetic rate in mashbean, mungbean and soybean Muhammad Iqbal Chaudhary Æ Joseph J. Adu-Gyamfi Æ Hirofumi Saneoka Æ Nguyen Tran Nguyen Æ Ryuichi Suwa Æ Shynsuke Kanai Æ Hany A. El-Shemy Æ David A. Lightfoot Æ Kounosuke Fujita

Received: 14 December 2007 / Accepted: 6 February 2008 / Published online: 28 February 2008 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2008

Abstract Phosphorous (P) fertilization is the major mineral nutrient yield determinant among legume crops. However, legume crops vary widely in the ability to take up and use P during deficiency. The aim here was to compare P uptake and translocation, biological nitrogen fixing ability and photosynthetic rate among mashbean (Vigna aconitifolia cv. ‘Mash-88’), mungbean (Vigna radiata cv. ‘Moong-6601’) and soybean (Glycine max L. cv. ‘Tamahomare’) during deficiency in hydroponics. Two treatments, the withdrawal of P from the solution (Pdeprivation) and continued P at 160 lM (P sufficient) were effected at the pod initiation stage. Plants were grown for 20 days. Short-term labeling with 32P showed the uptake and distribution of P into plant parts. Withdrawal of P from the solution reduced biomass, photosynthetic activity, and

nitrogen fixing ability in mungbean, and mashbean more than in soybean. P deprivation decreased P accumulation more than N accumulation. The decrease was more severe in mungbean and mashbean than soybean. More P was translocated and distributed into leaves in soybean than in mungbean and mashbean. Leaf P amount was more correlated to leaf area than to photosynthetic rate per unit leaf area among all three legume species. The results indicate that selection for increased efficiency of P utilization and leaf area may be used to improve leguminous crops. Keywords BNF  Leaf area development  Mashbean  Mungbean  Soybean  32P-labelled P

Introduction

Communicated by G. Klobus. M. I. Chaudhary  H. Saneoka  N. T. Nguyen  R. Suwa  S. Kanai  H. A. El-Shemy  K. Fujita Graduate School of Biosphere Sciences, Hiroshima University, 1-4-4 Kagamiyama, Higashi Hiroshima 739-8528, Japan J. J. Adu-Gyamfi International Atomic Energy Agency (IAEA), Wagramer Strasse 5, 400 Vienna, Austria H. A. El-Shemy (&) Department of Biochemistry, Faculty of Agriculture, Cairo University, Giza 12613, Egypt e-mail: [email protected] H. A. El-Shemy  D. A. Lightfoot Center for Excellence in Soybean Research, Teaching and Outreach, Southern Illinois University at Carbondale, Carbondale, IL 62901, USA

Plants display a diverse array of physiological adaptations to low phosphorus (P) availability (Marschner 1995). A better understanding of the underlying physiological mechanisms is a prerequisite for a successful deployment of plant traits in crop improvement. Phosphorus limitation directly reduces photosynthesis through effects on leaf area development and photosynthetic ability per unit leaf area for example in cotton (Gossypium hirsutum L., Radin and Eidenbock 1984). In legumes the direct effects are compounded by indirect effects that reduce N2-fixation, nodule biomass and nitrogenase activity, for example pigeon pea (Cajanus cajan L., Adu-Gyamfi et al. 1989), and common bean (Phaseolus vulgaris L., Lynch et al. 1991). Phosphorus deficiency in those legumes reduced leaf area, decreased the number of leaves and the number of nodes, the number of branches, and slowed the relative leaf appearance rate. Radin and Eidenbock (1984) reported that low P limited leaf expansion and decreased the hydraulic

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conductance of the root system. Lynch and Ho (2005) observed that common bean genotypes with superior low P adaptation expressed traits that reduced the respiratory burden on root growth. The reduction in photosynthesis under low P conditions was attributed to a reduction in the export of triose-P from the chloroplast to the cytosol via a Pi translocator (Leegood et al. 1985) and a reduction in RuBP (Rao and Terry 1995). Conversely, phosphorus fertilization increases symbiotic dinitrogen fixation by stimulating host plant growth rather than exerting a direct influence on nodule initiation, growth, development and function (Graham and Rosas 1979; Robson et al. 1981; Jacobsen 1985). The P status of several legumes solely dependent on symbiotic N2-fixation has been reported to increase tissue N concentration as well as overall host plant growth (Israel 1987; Singleton et al. 1985). Greater stimulation of symbiotic N2-fixation than of host plant growth with improvement in P nutrition was associated with an enhancement in the specific nitrogenase activity of the nodule (Bethlenfalvay and Yoder 1981; Jacobsen 1985; Sa and Israel 1991). However, the effect varied among crop species. Identifying the physiological bases of adaptation of legume crops to low P conditions through leaf development may accelerate the selection during breeding of crops with increased P use efficiency and high productivity in low-P environments. In this study, P deficient mashbean, mungbean and soybean were compared for (a) dry matter production in relation to photosynthesis and N2-fixing abilities, and (b) the efficiency of translocation and utilization of P.

Materials and methods Seeds of mashbean (Vigna aconitifolia cv. ‘Mash-88’) and mungbean (Vigna radiata cv. ‘Moong-6601’) were obtained from Ayub Agricultural Research Institute, Faisalabad, Pakistan, and seeds of soybean (Glycine max L. cv. ‘Tamahomare’) were available commercially. Experiment 1 The whole experiment was repeated for 2 years. The first replicate was carried out between June and September 1996. The second replication was carried out between June and October 1997. Seeds were surface sterilized with 0.7 L L-1 ethanol for 3 min, washed and rinsed with deionized water and then germinated on a moist filter paper at 25°C and 78% relative humidity in an incubator. The germinated seeds were cultivated in containers inside a glasshouse of the Graduate School of Biosphere Sciences, Hiroshima University, Japan. The inoculation of the germinated seeds was achieved by applying small amount of

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soil including an effective rhizobia for mashbean, mungbean, and Bradyrhizobium japanicum (strain-J-10) for soybean, respectively, into a seed bed including a mixture of soil and coarse vermiculite (1:4 v/v). Seedlings were grown in soil for 3 weeks. Nodulated 3-week old seedlings were transferred to each 70-L plastic pot that contained 60-L of nutrient solution composed of (lM) 160 P, 512 K, 374 Ca, 412 Mg, 4.47 Fe, 0.91 Mn, 23.1 B, and 0.08 Zn, 0.08 Cu, 0.05 Mo, and 0.09 Co. The nutrient solution was continuously aerated and renewed every 3–4 days. The plants were grown under natural light, and maximum and minimum temperatures were 32 and 20°C. At the pod initiation stage (15 days after transplanting), 2 treatments consisting of withdrawal of P from the solution culture (P-deprivation) and maintaining 160 lM P (P-sufficiency) were applied. The treated plants were allowed to grow for another 20 days. Three plants of each species from respective P treatments were harvested at 0, 10, and 20 days after the treatment (DAT). Photosynthetic rate (Po) and dinitrogen-fixing activity estimated by the acetylene reduction assay (ARA) were measured. The harvested plants were separated into leaf blades, stems with petioles, pods, roots and nodules, and the respective plant parts were dried at 70°C for 72 h for 3 days, weighed and ground. Measurement of photosynthetic rate Photosynthetic rate of the terminal leaflets of second and third trifoliated leaves from the uppermost position in each legume species were measured at 0, 10, and 20 DAT with a portable infrared gas analyzer (Model LI-6200 Li-Cor Co, Ltd, Lincoln, NE, USA) under natural sunlight. The measurement was made during 2 h starting from 11 AM on the sunny day and air temperature was in the range of 25– 30°C. The photosynthetically active radiation during measurement was above 1,700 lmol m-2 s-1 and the plant reached a steady photosynthesis. The leaf area was determined by an auto leaf area meter (Model AAM-5, Hayashi Denko Co, Ltd, Osaka, Japan). All the measurements were recorded three times and the differences between the measurements were negligible. Measurement of dinitrogen-fixing activity The dinitrogen-fixing activity was estimated by the ARA (Hardy et al. 1973). After detachment of shoots, the nodulated roots were sealed in a 100 mL bottle and incubated for 10 min in an atmosphere of 100 mL L-1 acetylene at room temperature. Two 0.5 mL gas samples per the nodulated root were analyzed for ethylene using a gas chromatograph, Hitachi 163, equipped with a flame ionization detector. All the measurements were made in three replicates.

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Nitrogen and phosphorus determination An aliquot of ground powder of respective plant parts was used for determination of total nitrogen by the semi-micro Kjeldahl method. Another aliquot of the powdered sample was digested with concentrated H2SO4:HNO3 (1:1 v/v) and P concentration was measured by the molybdenum blue methods (Murphy and Riley 1962).

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For mean separation, treatment sum of squares was partitioned by the method of orthogonal contrasts. The coefficients of variation for all response variables were below 10%.

Results Experiment 1

Experiment 2 Plant growth The experiment was repeated two times. The first replicate was carried out between June and September 1997. The second replication was carried out between July and October 1997. Seeds of mashbean and soybean were germinated in a seed bed containing of vermiculite mixed with a small amount of soil previously cultivated with these crops to serve as inoculants. Plants were grown for 25 days. Nodulated seedlings were transplanted ten to each 70-L plastic container. Each container contained 60 L nutrient solution described in Experiment 1. Two P treatments of no-P (P deficient) and 160 lM P (P sufficient) were initiated 25 DAT and allowed to grow for 15 days. Labeling of plants with

32

P

The 32P-labeling was conducted at 65 DAT in a growth chamber at day temperature of 25°C and night temperature of 20°C with relative humidity (RH) of 70%. Prior to 32Plabeling, four uniform seedlings from each treatment of each species were transferred to 1.5-L flask covered with aluminum foil containing the same nutrient solution as described in Experiment 1. The seedlings were cultured overnight. Label was added to each flask with 37 MBq32PO4 at specific activity of 1.7 GBq mL-1 and incubated for 24 h. Two plants from each species at the respective P concentrations were sampled at 3 and 24 h after the initiation of labeling. The plants were separated into leaf blades, stems with petioles, pods, roots and nodules. Plant parts were dried at 70°C for 72 h in an airforced oven, weighed and ground. The ground samples of each plant part were wet-digested in a (1:2 v/v) solution of H2O2 and HClO4. The digests were diluted with distilled water and the radioactivity was measured by liquid scintillation spectrometry (LSC Aloka 700 type, Kyoto, Japan) using a fluor solution consisting of 1 mL of plant part digests and 9 mL of Aquasol-2 (NEN research product).

P-deprivation caused a significant (P \ 0.01) decrease in whole plant leaf area relative to P sufficiency at DAT 10 and DAT 20. The decrease was greatest in mungbean (to 31% of P sufficient control leaf area), then mashbean (50%) and least affected was soybean (61%; Table 1). Only measured at DAT 20 were leaf weight and leaf thickness. Leaf weight was significantly (P \ 0.01) decreased by P deprivation in all species. However, leaf thickness was not significantly reduced in soybean but was significantly (P \ 0.05) reduced in both mungbean and mashbean. The reduction of whole plant biomass (Table 2) followed a similar trend as the whole plant leaf area. Among the plants parts, stems with petioles, and leaf blades weight were the least affected by P-deprivation among all three species. The total nodule weights of mashbean and mungbean were drastically reduced, whereas nodules weight of soybean was not affected. In contrast, root weight was significantly increased in mungbean and soybean. Therefore, only in mashbean were root weight and nodule weight both significantly reduced.

Table 1 Effect of P-deprivation on leaf area, leaf weight and leaf thickness at 20 days after treatment (DAT) in soybean, mashbean and mungbean plants DAT

Soybean +P

-P 2

Mashbean

Mungbean

+P

+P

-P

-P

-1

Leaf area (cm plant ) 0

519

519

73

73

63

63

10

527

417**

90

54**

101

30**

20

1,359

828**

370

184**

304

95**

85*

156

56*

0.45**

0.51

0.59**

-1

Leaf weight (dry mass mg plant ) 20

544

388 NS

123

Leaf thickness (dry mass mg cm-2) 20

0.40

0.47**

0.33

Statistical analysis

Pulses had been grown hydroponically for 20 days in either complete nutrient solution (+P) or in nutrient solution containing no added P (-P)

All the experiments were statistically analyzed for the effect of treatments on the measured traits according to the expected mean squares method given by McIntosh (1983).

NS Non-significant * Significant at P \ 0.05 ** Significant at P \ 0.001

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Table 2 Effect of P-deprivation on whole plant biomass and its partitioning (%) in various plant parts of mashbean, mungbean and soybean at 20 DAT

Table 4 Effect of P-deprivation on biological nitrogen fixation (BNF), specific nodule activity (SNA) and nodule weight in mashbean, mungbean and soybean

Species

Plant parts

DAT

Mashbean

Whole plant biomassa

Mungbean

Soybean

+P (% w/w)

-P (% w/w)

Leaf blades

60.0

64.0 NS

Stem plus petioles Roots

26.0 10.0

28.0 NS 6.0*

Nodules

4.0

2.0*

Whole plant biomassa

1.53

0.46**

-P

Mashbean

Mungbean

+P

-P

+P

-P

BNF (lmol C2H4 plant-1 h-1) 0

39

39

13

13

11

11

10

38

26**

7

3**

6

4**

20

58

25**

13

4**

5

1** 550

SNA (lmol C2H4 g-1 nodule h-1)

Leaf blades

55.8

48.2*

0

186

186

433

433

550

Stem plus petioles

19.4

26.3**

10

79

79 NS

100

50**

100

133**

Roots

20.5

24.6**

20

79

45**

144

80**

42

100**

Nodules

4.3

0.9**

Whole plant biomassa

8.10

5.68**

0.02

Nodule weight (g dry weight plant-1) 0

0.21

0.21

0.03

0.03

0.02

Leaf blades

36.0

35.4 NS

10

0.48

0.33**

0.07

0.07 NS

0.06

0.03**

Stem plus petioles

43.5

41.6 NS

20

0.73

0.56**

0.09

0.09 NS

0.12

0.01**

Roots

15.6

17.8*

Nodules

4.9

5.2 NS

Pulses had been grown hydroponically for 20 days in either complete nutrient solution (+P) or in nutrient solution containing no added P (-P) NS Non-significant a

+P

0.73**

1.16

Soybean

Pulses had been grown hydroponically for 20 days in either complete nutrient solution (+P) or in nutrient solution containing no added P (-P). Nodulated roots was sealed in a container and production of C2H4 was determined by acetylene assay NS Non-significant * Significant at P \ 0.05

Dry weight (g plant-1)

** Significant at P \ 0.001

* Significant at P \ 0.05 ** Significant at P \ 0.001

compared to 0 and 10 DAT due to lower light intensity on the day of measurement at 20 DAT?

Photosynthetic rate Photosynthetic rate per unit leaf area (Po) was decreased by P-deprivation compared to the control at both 10 and 20 DAT. At both times the decrease was more pronounced in mungbean (94% reduction), mashbean (65% reduction) than in soybean (18% reduction; Table 3). However, photosynthetic rate was decreased in all treatments at 20 DAT Table 3 Effect of P-deprivation on photosynthetic rate in upper developed leaves of mashbean, mungbean and soybean DAT

Soybean +P

-P

Mashbean

Mungbean

+P

+P

-P -2

Photosynthetic rate (lmol CO2 dm

N and P accumulation in plant parts

h )

25.0

25.0

27.0

27.0

17.0

10 20

27.0 11.7

17.0** 8.4**

22.0 16.7

15.0* 5.8**

21.0 9.3

17.0 9.0** 0.6**

Pulses had been grown hydroponically for 20 days in either complete nutrient solution (+P) or in nutrient solution containing no added P (-P). Photosynthetic rate of the terminal leaflets of upper trifoliated leaves in each legume species were measured with a portable infrared gas analyzer ** Significant at P \ 0.001

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The biological nitrogen fixation rate estimated by acetylene reduction was reduced by P deficiency at both 10 and 20 DAT in all three species. Most seriously affected by P deprivation was mungbean among the three legumes studied. The reduction was due to a drastic decrease in total nodule weight in soybean and munbean but not mashbean (Table 4). In mashbean the whole plant was smaller during P deficiency but nodules were not proportionally decreased. Specific nodule activity was decreased by P deficiency to a greater extent in mashbean than other leguminous plants.

-P

-1

0

* Significant at P \ 0.05

Nitrogen fixation rate

P deprivation led to a decrease in whole plant N accumulation in all organs of all species (Table 5). The decrease was more pronounced in mungbean and less in soybean. Among various plant parts, N concentration was highest in nodules and lowest in stems with petioles irrespective of the treatments (Table 5). Stems with petiole N concentrations were the least affected by P deficiency. In contrast, P concentrations in the plant parts were reduced by P deprivation in all species. However, the reduction was

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541

Table 5 Effect of P-deprivation on percentage and amount of N in various parts of mashbean, mungbean and soybean at 20 DAT

Table 6 Effect of P-deprivation on percentage and accumulation of P in various parts of mashbean, mungbean and soybean at 20 DAT

Plant parts

Plant parts

Soybean

Mashbean

Mungbean

+P

+P

+P

-P

-P

-P

N content (mg N g dry weight-1)

Soybean

Mashbean

Mungbean

+P

+P

-P

+P

-P

-P

P content (mg P g dry weight-1)

Leaf blade

27

25*

40

30**

36

25**

Leaf blades

1.1

0.7*

1.7

1.1*

3.5

1.0 **

Stem plus petioles

7

7 NS

17

16 NS

13

12*

Stem plus petioles

8.6

7.2*

2.7

2.8 NS

2.5

6.7**

Roots

15

17**

24

23 NS

21

22*

Roots

11.1

2.0 **

6.1

3.0 **

11.0

4.4**

Nodules

42

40**

63

52**

57

45**

Nodules

16.0

3.0 **

5.9

0.9**

23.0

5.1**

N amount (mg N plant-1)

P amount (mg P plant-1)

Leaf blade

14.7

9.7**

4.9

2.5**

5.6

1.4**

Leaf blades

6.1

2.6**

6.6

4.0 **

5.8

1.4**

Stem plus petioles

4.5

3.2**

0.9

0.6**

0.7

0.4**

Stem plus petioles

9.7

2.1**

17.0

11.5**

1.3

1.3 NS

Roots

3.5

3.3*

0.8

0.5**

1.2

0.6**

Roots

6.4

0.6**

14.2

7.5**

3.2

0.6**

Nodules Whole plant

3.1 25.8

2.2** 18.4**

0.6 7.2

0.3** 3.9**

0.7 8.2

0.1** 2.5**

Nodules Whole plant

1.3 23.5

0.04** 5.3**

4.0 41.8

0.5** 23.5**

1.3 11.6

0.4** 3.7**

Proportion (%)

100

23**

100

56**

100

32**

Pulses had been grown hydroponically for 20 days in either complete nutrient solution (+P) or in nutrient solution containing no added P (-P). An aliquot of dried ground sample was used for determination of N content by semi-micro Kjeldahl method NS Non-significant

Pulses had been grown hydroponically for 20 days in either complete nutrient solution (+P) or in nutrient solution containing no added P (-P). An aliquot of dried, ground sample of each plant part was digested and determined P content by molybdenum blue method

* Significant at P \ 0.05

* Significant at P \ 0.05

** Significant at P \ 0.001

** Significant at P \ 0.001

more pronounced in nodules than the other parts (Table 6). Total P amount in soybean and mungbean was lower than in mashbean.

hydroponics. The shift of biomass allocation to more leaves by soybean compared to mungbean and mashbean could be a candidate mechanism for better adaptation to low P. The 32P tracer experiment revealed that recently absorbed P tended to accumulate in the roots and stems of P deprived mashbean during the early hours after labeling, whereas there was more uptake and more translocation of the 32P-labeled P from roots to leaf blades by P sufficient mashbean and P deficient soybean (Table 7). Therefore, when P-supply is limited, soybean may accumulate more dry matter through an induced high efficiency P absorption and distribution mechanism directed toward leaves and petioles. Similar results were reported by Adu-Gyamfi et al. (1989) for the adaptation to low-P by pigeon pea cultivars of different growth duration. Pigeon pea cultivars that distributed more P to the stem instead of the leaves were less tolerant to low-P than cultivars that translocated more P to the growing leaves developing leaf areas (Adu-Gyamfi et al. 1989). Barber et al. (1967), Bieleski (1973), Loneragan (1978) and Adu-Gyamfi et al. (1991), have suggested that P-deficient plant species have a higher ability to retranslocate P from inactive to the active sites. Thus, the sequential translocation of recently absorbed P from stem through petioles to leaf blades could be used as an indicator for selecting P-efficient species (Adu-Gyamfi 2002). For instance, in soybean, petioles appear to serve as more

Experiment 2 The 32P specific activity was highest in roots, and lowest in leaf blades indicating the high affinity for P by roots compared to the other plant parts (Table 7). Under P-deprivation conditions, the total 32P activity was higher in soybean than in mashbean after 3 h of feeding (Table 7). Total 32 P activity were highest in roots of P deprived soybean after 3 h of feeding and sixfold higher than P sufficient soybean roots (Table 7). In contrast, P deprived mashbean roots had taken up about half 32P than P sufficient roots. By 24 h the 32P in roots was decreased, in contrast 32P-labeled P in shoots increased. Translocation percentage of 32P-labelled P from roots to the shoot was slightly higher in mashbean than in soybean at the P adequate conditions, and increased more in mashbean at the P-deprivation conditions (data not shown).

Discussion Physiological adaptations to low-P availability varied widely among soybean, mungbean and mashbean in

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Table 7 Effect of P-deprivation on specific activity and total activity of 32P in various parts of mashbean and soybean at 65 DAT Hours after P feeding

Plant parts

Soybeana

Mashbeana

+P

+P

32

-P

-P

32

P specific activity (lmol kg dry weight-1)

3

Leaf blades Stem plus petioles Roots

2 17 637

Nodules 24

3

1** 10** 2,885** 2**

49 140 3,360 7

1** 7** 2,282** 3**

Leaf blades

13

19**

55

7**

Stem plus petioles

33

65**

93

153**

Roots

865

Nodules

13

3,235**

2,596

2,174**

117**

22

31**

2

1**

44

1**

13

6**

43

2**

32

P total activity (lmol plant part-1)

3

Leaf blades Stem plus petioles Roots

205

Nodules 24

1

1,318** 0*

Whole plant

221

Leaf blades

11

28**

26

61**

Stem plus petioles Roots Nodules Whole plant

1,324**

1,525 1 1,612

688** 1** 691**

54

9**

42

134**

612 2

1,196** 116**

1,485 2

664** 62**

652

1,405**

1,582

869**

32

Pulses were exposed to 37 MBq PO4 for 3 and 24 h. Three plants from each species at the respective P concentration were sampled at 3 and 24 h after the initiation of feeding a Pulses had been grown hydroponically for 20 days in either complete nutrient solution (+P) or in nutrient solution containing no added P (-P) * Significant at P \ 0.05 ** Significant at P \ 0.001

active sink for P than the stem, whereas the reverse was observed for the early maturity cultivar of pigeon pea, as evidenced from lower specific radioactivity in stem and petioles (Adu-Gyamfi et al. 1991). Sinclair and Vadez (2002) reported that a possibility for sustaining high mass accumulation under low N and P conditions is to concentrate the available N and P into the leaves at the top of the leaf canopy. Leaf area development is one of the more sensitive responses of crop growth to P deficiency and the options of decreasing the sensitivity of leaf area development to P stress would be quite beneficial. The poor performance of mungbean and mashbean to P-deprivation may be attributed to the severe reduction in leaf area relative to control compared to soybean. The reduction in leaf area development at P-deprivation tended to occur earlier than the depression of photosynthetic rate, and subsequently the latter was more deeply depressed in mashbean and mungbean (Tables 1, 3), implying that suppression of leaf area

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development is reflected in the suppression of photosynthetic rate under suboptimal P supply conditions. These results suggest that more susceptibility of mungbean and mashbean to P deficiency than soybean is partly due to more susceptibility of leaf development to low-P conditions. It was reported that expansion of leaves under P stress was limited by the number of cell divisions, which would imply control of cell division by a common regulatory factor (Chiera et al. 2002). Among the plant part, nodules were the most severely affected by the P-deprivation treatment. There was a sharp decrease in P concentration and the biological nitrogen fixing ability of nodules by P deprivation. The decrease was less severe in soybean than in mungbean and mashbean. The high requirement for P in legumes was consistent with the involvement of P in the energy transfer that must take place in the nodule. P deficiency might be directly involved in inhibiting the active loading of ureides to the xylem. Severe decreases in nodule mass affects nodule ability to produce ureides (Vadez et al 1997). In the uptake experiment the high 32P specific activity in nodules of soybean compared to mashbean (Table 7) suggested that soybean could translocate more P for nitrogenase activity than mungbean. Data shown in Table 2 suggested that when supplied with insufficient P, the photosynthetic and nitrogen-fixing abilities were more severely affected than other functions (Adu-Gyamfi et al. 1989; Adu-Gyamfi 2002; Sa and Israel 1995). The factors controlling tolerance to low-P based on the whole plant photosynthetic rates (Pn), estimated by whole plant leaf area (Po) and N2-fixing abilities among species were compared. Table 2 showed that depression of biomass production of mashbean by P-deprivation was due to a more significant decrease in Pn, and a concomitant reduction of N2-fixing ability. Although the depression of Pn by P-deprivation was attributed to decreases in both Po and leaf area in all the species examined, the reduction in Po was more substantial in mashbean and mungbean compared with soybean (Tables 1, 3). Such phenomenon was observed in extreme soil P deficient conditions when fertilizer P was not applied. Lynch et al. (1991) reported that P availability affected common bean growth primary through effects on leaf appearance and biomass partitioning between photosynthetic and respiring organs, rather than through effects on leaf photosynthesis. Here, the decrease in the N2-fixing ability by P-deprivation was due to the depression of both total nodule activity and nodule growth in mashbean and mungbean (Table 4). The reduction of nodule growth and function at P-deficient conditions may be closely related to an insufficient supply of photosynthate from leaves and of P from roots (Table 6). The results showed that nodule P concentration decreased more severely by P-deprivation

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than other plant parts under hydroponic conditions (Table 6). These results suggest that mashbean and mungbean nodules may directly absorb P from the solution culture as found in common bean (P. vulgaris) nodules (Al-Niemi et al. 1998) and soybean nodules (Chaudhary et al. 1997). Relatively more biomass was partitioned to leaves and less to the stem plus petioles in soybean, compared to the other species during P-deprivation (Table 2). P partitioning may play an important role in determining P utilization efficiency. Equally, the partitioning of P to leaves may determine the ability to tolerate to P-deficient conditions. After cessation of P absorption, legumes may express the capability of utilizing the Pi pool for new biomass production. The utilization efficiency of mashbean and mungbean may be lower than soybean because of lower rates of, supply of, and use of, P for photosynthesis and N2-fixation. It has reported that phosphorus use efficiency varies genetically in cowpea (Sanginga et al. 2000), common bean (P. vulgaris) (Vadez and Drevon 2001), and pigeon pea (Vesterager et al. 2006). Vesterager et al. (2006) found that dry-matter production correlated positively with the P uptake and P-use efficiency, whereas the P-use efficiency correlated negatively with the P-uptake efficiency. The whole plant 32P uptake was far higher during P-deprivation in soybean than in mashbean (Table 7) suggesting that P absorption activity in roots of soybean was induced in P-deficient conditions but repressed in P deficient mashbean. P deprivation over the long-term initiates adaptive responses that tend to conserve the supply of Pi thereby maintaining high rates of photosynthesis in plants acclimated to P-deficiency (Rao and Terry 1989, 1995; Rao et al. 1989, 1990). The P-deprivation led to substantial reduction of translocation of P from roots to other parts (data not shown) and the reduction was more pronounced in mashbean, suggesting that plant parts that are P-deficient prevent the translocation of P to other plant parts. It has been reported that in P-deficient plants of pigeon pea and soybean the proportion of Pi decreases but that of organic compound-P increases (Adu-Gyamfi et al. 1989). Therefore, under Pdeficiency, Pi may be responsible for the synthesis of new compounds that reduce the Pi pool, and result in the reduction of Pi translocation. Therefore, ability of soybean to translocate P from roots to shoots, particularly to the leaf may be one determinant factor of tolerance to low-P conditions. Low-P tolerance was shown to be determined by species. A strong genetic component could be inferred. The translocation of P from roots to leaf blades during P-deprivation could be used to select for low P-tolerant cultivars within species. Phosphorus deficiency affects various aspects of plant physiological functioning (Fujita et al. 2003, 2004). For instance, in tomato (Lycopersicon esculentum), at first phosphorus deficiency impairs sink

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activity of stem, subsequently suppresses photosynthetic translocation and deposits carbohydrate in the source leaves, which may result in depression of photosynthetic activity (Fujita et al. 2003). Recently, genes expression induced by suboptimal P supply have been reported (Liu et al. 2005; Tian et al. 2007; Sanchez-Calderon et al. 2006). These reports suggest that the genes involved in tolerance to P deficiency may work on different physiological functioning in plants.

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