Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol154.Issue1.2000:

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Comparison of drought tolerance in nitrogen-fixing and inorganic

nitrogen-grown common beans

Anı´bal R. Lodeiro *, Paola Gonza´lez, Andrea Herna´ndez, Laura J. Balague´,

Gabriel Favelukes

Facultad de Ciencias Exactas,Instituto de Bioquı´mica y Biologı´a Molecular,Uni6ersidad Nacional de La Plata(UNLP),Calles47y115,

1900La Plata,Argentina

Received 6 September 1999; received in revised form 15 November 1999; accepted 16 November 1999

Abstract

In this work, we evaluated how the use of alternative N sources affects drought-stress tolerance in common beans. To this end, plants were cultivated employing either N2fixation or two levels of inorganic nitrogen: 1 mM NH4NO3 (limiting) or 10 mM

NH4NO3 (sufficient). Drought was imposed by withholding watering at 30 days after planting (DAP) — coinciding with

flowering. At 20 DAP, growth and N content were significantly higher in NH4NO3-sufficient plants than in N2-fixing and

NH4NO3-limited beans. At later times, only N2-fixing and NH4NO3-sufficient plants continued assimilating N and growing, with

the NH4NO3-sufficient plants being consistently bigger. After 10 days of stress (40 DAP), desiccation was evident, but only

NH4NO3-sufficient plants suffered drought-induced senescence. After 20 days of stress (50 DAP), N content increased in

NH4NO3-sufficient but not in N2-fixing beans, despite the latter’s lesser state of wilt. Pod dry weight dropped 43% in

NH4NO3-sufficient beans with respect to well-watered plants, while remaining constant in N2-fixing beans. Under drought

conditions, the number of pods limited pod yield regardless of the nitrogen source used; nevertheless, the translocation of soluble matter to pods continued in both NH4NO3-sufficient and N2-fixing beans. We conclude that common beans grown under

conditions of N2 fixation were more drought tolerant than those provided with sufficient levels of NH4NO3. The most

Keywords:Bean; Drought; N2fixation; Nitrogen nutrition

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1. Introduction

The extent of damage caused by drought stress in plants depends on, among other factors, nitro-gen nutrition [1,2]. In crop legumes, atmospheric nitrogen may be fixed via the nitrogenase pathway in the root nodules, and/or inorganic nitrogen in solution may be assimilated from the soil princi-pally through the nitrate-reductase reaction se-quence in leaves. Preferential nitrogen assimilation by either one of these routes might lead to modifi-cations in plant architecture, or have diverse

influ-ences on drought tolerance because of the differences in the utilisation of energy and reduc-ing power characteristic of those pathways [3,4]. There are thus important practical consequences in ascertaining whether or not drought stress has different impacts on N2-fixing and inorganic-N-fed

crop legumes.

The response to drought conditions shares many properties with ageing [5]. Thus, after pro-longed dry periods, an acceleration in senescence-related processes, along with a premature cleavage of proteins and a translocation of the resultant peptides and amino acids, might be expected. In-deed, both in ageing and in drought stress, oxida-tive damage seems to increase and to be, at least in part, responsible for triggering senescence, in * Corresponding author. Tel.: +54-221-4250497; fax: +

54-221-4256809.

E-mail address:[email protected] (A.R. Lodeiro)

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leaves as well as nodules [6,7]. The plant defence against oxidative damage involves changes in cata-lase activity and the operation of the glutathione-ascorbate cycle, both of which functions contribute to the detoxification of cells through the scavenging of active oxygen species [6 – 8].

Depending on the degree of senescence and the tissues affected, the translocation of soluble car-bon and nitrogen compounds to developing pods might be limited. Moreover, in N2-fixing plants,

the impact of transient-drought damage on grain yield depends on the developmental stage at which the stress takes place, with the effect being more severe if the adverse conditions arise during the reproductive period when the N2-fixation rate is

maximal [9].

Since plant growth, N assimilation and the men-tioned translocations are affected differently by the means of N nutrition, the influence of drought on these three processes might differ, thus giving rise to dissimilar tolerance mechanisms. This pos-sibility, however, does not apply to all the legumes tested, in that some species adopted different drought-tolerance strategies under conditions of nitrate feeding and N2fixation, whereas others did

not; and this adaptation, in turn, altered the resis-tance level achieved with each N-assimilation pathway [10 – 12]. In response to increased salinity, nitrate-fed chick-pea and faba bean plants were more drought tolerant than their corresponding N2 fixers [13,14]. Extrapolations of osmotic-duress

responses to drought stress, however, should be made with care, since osmotic and drought-in-duced effects on nodule O2 permeability and

en-ergy metabolism have not proved to be as similar as originally thought (see Ref. [15]).

Drought conditions limit the yield of the com-mon bean (Phaseolus 6ulgaris) in northwest

Ar-gentina owing to the combined effects of high temperature and periods of scant rainfall during the growing season. These crops are grown under conditions of either N2 fixation or inorganic-N

fertilisation without regard to the possible differ-ential impact of these modes of cultivation on drought tolerance. Moreover, plant breeding, in-cluding selection for drought tolerance, is per-formed mainly with nitrate-fertilised plants as opposed to N2-fixing ones, under the assumption

that the same drought-tolerance phenotypes will be expressed. Since, as already described, the use of different N-assimilation pathways might lead to

dissimilar levels of drought tolerance, we sought to evaluate whether or not the manipulation of the N source during plant growth might have a favorable influence on drought resistance. We also wished to examine whether drought limits plant yield by affecting the same or different parameters with each N source. To this end, in the work reported here, we have compared the sensitivity of the common bean to severe drought stress during its reproductive stage under conditions requiring the utilisation of different N-assimilation pathways for growth.

2. Materials and methods

2.1. Plant growth and conditions for imposing drought stress

We cultivated Dor 500 black beans (provided by EERA-INTA, Cerrillos, Argentina) at 28/20°C day/night temperature in a greenhouse having un-controlled relative humidity that varied by around 60/90% day/night. Seeds were surface sterilised with 20% (v/v) commercial bleach followed by six sterile-distilled water washes before placing them for germination on 1.5% (w/v) aqueous agar [16]. We planted 3-day-old uncontaminated seedlings (length around 3 – 4 cm) at two seedlings per 2.5-l pot filled with sterile vermiculite, and immediately watered the pots with a sterile N-free plant-nutri-ent solution that contained, in g/l distilled water: CaCl2·2H2O, 0.10; MgSO4·7H2O, 0.12; KH2

-PO4·3H2O, 0.17; K2HPO4, 0.20; NaCl, 0.06; ferric

citrate, 0.005; and micronutrients from Fa˚hraeus solution [17] (pH 7.0). NH4NO3 was included as

indicated later.

The nitrogen source provided throughout culti-vation varied as follows. Of the 108 plants watered with the plant-nutrient solution at the beginning of the experiment, we inoculated one-third with

Rhizobium etli CE3 (provided by C. Quinto, UNAM, Me´xico, and grown to late exponential phase in yeast-extract mannitol broth [18]), while we fertilised the other two-thirds by adding 1.0 mM NH4NO3to one-third and 10.0 mM NH4NO3

to the other via the plant-nutrient solution at planting. We irrigated with sterile distilled water every 2 days and provided one watering with 1:5-diluted nitrogen-free plant-nutrient solution at 15 days after planting (DAP). With one-half of the

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plants provided with each nitrogen source, we then stopped the watering at 30 DAP. Finally, we sampled four plants per time interval (for each nitrogen source, either with or without continued watering) at 20, 30, 35, 40, and 50 DAP.

Relative water content (RWC) was determined in leaves as previously described [19]. For each time interval, at 3 h after the onset of the pho-tophase, we excised 2.5 cm diameter discs from the middle of leaflets from the uppermost fully ex-panded leaves and recorded the fresh, turgid (discs soaked in double-distilled water up to constancy of weight), and dry (discs heated at 80°C down to constancy of weight) weights of each disc. We then calculated the RWC as: 100×[(fresh weight−dry weight)(turgid weight−dry weight)−1_]. _Disc

weight divided by the disc area gave the leaf weight per unit area.

2.2. Biochemical and analytical determinations

Total organic-N content of oven-dried shoots and pods were measured by the Kjeldahl method [20].

Soluble proteins from leaves and nodules frozen at −135°C were extracted by first grinding the samples with a pestle in a precooled mortar after the addition of insoluble polyvinylpolypyrollidone powder, and then placing the ground tissue for thawing in 25 mM phosphate buffer (pH 7.0), containing 1 mM CaCl2·2H2O. After clarification

of the resulting suspension by centrifuging at 12 000×g for 10 min at 4°C, followed by cloth filtration of the supernatant, we dispensed 1 ml aliquots for storage at −80°C until further pro-cessing. We estimated the concentration of total proteins in the extracts by the method of Bradford [21].

We determined tissue-protease content using 12.5 mg ml−1 _{azocasein as the substrate after}

Malik et al. [22], except that one unit of protease activity was defined as the amount of enzyme producing an increase in absorbance at 440 nm of 0.1 h−1 _{at 37°C.}

Catalase activity was measured by following the decomposition of 20 mM H2O2 at 240 nm [23].

One unit of catalase activity was defined as the amount of enzyme that degraded 1 mmol H2O2

min−1 _{at 30°C.}

We assayed ascorbate peroxidase with 0.1 mM ascorbate plus 1 mM H2O2 by following the

de-composition of ascorbate at 265 nm and 30°C, and correcting those values for the autooxidation of ascorbate in the absence of H2O2 [8]. One unit of

ascorbate-peroxidase activity was defined as the amount of enzyme that degraded one nanomole of ascorbate per minute.

Finally, tissue-polypeptide composition was analysed by discontinuous sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [24] followed by silver staining [25]. Carbonic an-hydrase from bovine erythrocytes (29 kDa), egg albumin (45 kDa) and bovine serum albumin (66 kDa) were used as molecular-weight standards. 2.3. Statistics

We carried out two independent experiments that yielded essentially the same results. We thus present here the mean values9the standard devia-tions (S.D.) from one of these, as being representa-tive of both. In addition, an analysis of variance (ANOVA) and the Tukey test [26] were performed to determine the statistical significance of differ-ences among mean values.

3. Results and discussion

3.1. Growth determined by nitrogen a6ailability

Dor 500 black bean is a cultivar of indetermi-nate growth which, under our present greenhouse conditions, completed the full expansion of the first trifoliate leaf at 20 DAP, started flowering at 30 DAP, and was in pod development and grain filling at between 40 and 50 DAP. These physio-logical states were reached at the same times by all the plants, independent of the N treatment re-ceived (except for pod development in 1 mM NH4NO3-fed plants; see later). Plant size and

growth rate, however, varied according to the nitrogen source. At 20 DAP, the appearance as well as the fresh and dry weights of the N2-fixing

and the 1 mM NH4NO3-fed plants were similar,

whereas the 10 mM NH4NO3-fed plants were

larger (Table 1). The greater green mass of the 10 mM NH4NO3-fed plants resulted, at least in part,

from increased leaf area rather than from thicker leaves, since the leaf fresh and dry weights per unit area did not vary significantly throughout the experiment (not shown). This smaller size of both

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the N2-fixing and the 1 mM NH4NO3-fed plants

indicated that they were N-limited at this growth stage in comparison with the 10 mM NH4NO3-fed

beans. The subsequent rate of increase in shoot fresh weight at 30, 40, and 50 DAP was highest for the 10 mM NH4NO3-fed plants, intermediate for

the N2-fixing beans (Table 1; see also our

prelimi-nary communication [27]), and lowest at virtually 0 mg day−1_{for the 1 mM NH}

4NO3-grown plants.

Likewise, the increase in shoot dry weight also ceased in these latter plants in contrast to the progressively greater values seen with those of the other two experimental groups.

As these results suggested, after 30 DAP, the nitrate available to plants fed NH4NO3 at 1 mM

had apparently dropped to levels sufficient to pro-duce nitrogen starvation, thus precluding further assimilation of nitrogen; whereas the plants of the other two groups were able to continue this pro-cess, although at a lower rate in beans fixing

atmospheric N2 than in the plants fed 10 mM

NH4NO3 (Fig. 1, shaded bars). This latter

differ-ence is in agreement with the low efficiency of the nitrogenase pathway in the common bean [28,29]. To underscore these contrasting features, we will hereafter refer to plants fed 1 and 10 mM NH4NO3 as NH4NO3-limited and NH4NO3

-suffi-cient beans, respectively.

3.2. Effects of nitrogen content and growth on desiccation

We evaluated the progress of drought stress through calculation of the RWC of leaves at 20, 30, 35, 40, and 50 DAP (Fig. 2). By the first 5 days after withdrawal of watering (30 – 35 DAP), the RWC values remained essentially unchanged, sug-gesting that the plants had not yet suffered dehy-dration stress. By 40 DAP, however, the RWC of both the unwatered NH4NO3-sufficient and N2

-Table 1

Fresh and dry weights of different plant parts from common beans cultivated on different nitrogen sources, either with or without drought stressa

DAP Plant nitrogen source

N2Fixation 1 mM NH4NO3 10 mM NH4NO3

Drought

Drought Watering

Watering

Watering Drought

Shoot fresh weight per plant(g)

20 2.95 A – 3.16 A – 7.24 B –

5.86 A 4.22 A 3.70 A 11.34 B

30 5.92 A 11.38 B

40 8.96 A 6.90 A 4.51 B 4.08 B 17.97 C 6.46 A

22.40 C 4.21 B

4.38 B 4.32 B

5.90 B 13.40 A

50 (T)b

50 (L+S)c _{8.56 A} _{3.44 B} _{4.38 B} _{4.21 B} _{16.06 C} _{2.73 B}

4.84 A 2.46 B 0 0 6.34 A 1.59 B

50 (P)d

Shoot dry weight per plant(g)

20 0.39 A – 0.45 A – 0.74 B –

0.74 A 0.70 A 0.66 A

30 0.60 A 1.39 B 1.45 B

1.45 A

40 1.54 A 0.71 B 0.56 B 3.01 C 1.70 A

2.52 A 1.76 A 0.69 B

50 (T) 0.70 B 4.74 C 2.26 A

1.96 A 1.14 B 0.69 B 0.70 B 3.74 C 1.69 AB

50 (L+S)

0.56 A 0.62 A 0 0 1.00 B 0.57 A

50 (P)

Root dry weight per plant(g)

0.37 A –

0.21 B –

0.44 A –

0.38 A 0.34 A 0.22 B

30 0.26 AB 0.59 C 0.61 C

0.45 A 0.72 C

40 0.57 A 0.18 B 0.29 B 0.67 C

0.23 B 0.22 B

0.24 B 0.89 C

0.41 A

50 1.03 C

a_{Plants were either normally watered, or else watering was withheld at 30 DAP. Values followed by different letters (within}

each row) differ significantly atPB0.05 according to the ANOVA and Tukey test.

b_{(T), Total shoot weight (including leaves, stems and pods) was considered.} c_(L₊_{S), Leaves}₊_{stems weight was considered.}

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Fig. 1. Shoot nitrogen content per plant at 20, 30, 40, and 50 DAP. Shaded bars, well-watered plants; open bars, drought-stressed plants. N1=1 mM NH4NO3-fed beans; N10=10

mM NH4NO3-fed plants; F=N2-fixing plants. The striped

portion of the bars represents the nitrogen content of pods. The full height of the bars represents the total N content in shoots (leaves+stems+pods). Error bars indicate the S.D.

fixing beans dropped, with the values for NH4NO3-sufficient plants suggesting the onset of

senescence. This pattern continued to 50 DAP, with the NH4NO3-sufficient beans being now at

the point of death. By contrast, the RWC of NH4NO3-limited plants stayed constant

through-out this same period.

In drought-stressed beans, shoot fresh weight remained constant in the N2-fixing and the

NH4NO3-limited beans, but diminished in the

NH4NO3-sufficient plants (Table 1). In the N2

-fixing beans, the rate of increase in shoot dry matter was slightly slower than with well-watered plants, whereas in the NH4NO3-sufficient group,

this parameter was markedly decreased by drought conditions relative to the corresponding value for the watered beans (Table 1; see also Ref. [27]). The root mass of this latter group increased between 40 and 50 DAP in response to drought, while that of N2-fixing plants actually diminished (Table 1).

The shoot:root ratio diminished significantly at 50 DAP only in the unwatered NH4NO3-sufficient

beans, being about one-half of the value for their well-watered counterparts. By contrast, the limit-ing factor in the growth of NH4NO3-limited plants

would appear to be the NH4NO3 supply rather

than the availability of water (Table 1).

N content increased under drought stress in the NH4NO3-sufficient but not in the N2-fixing beans

(Fig. 1, open bars), although the latter did not become as withered as the former (Fig. 2). This difference in response to water withdrawal agrees with the high sensitivity of nodule energy metabolism [30] and the nitrogenase system [31] to hydration state, both functions being abruptly in-hibited at the onset of drought.

The greater availability of N to the NH4NO3

-sufficient beans from the time of their early devel-opment led to larger plants with more extensive leaf expansion; this difference made them more sensitive to desiccation when confronted with an abrupt reduction in the water supply. The NH4NO3-limited beans, however, grew less rapidly

and were also less sensitive to drought conditions, while N2-fixing plants manifested an intermediate

response. With these latter plants, in contrast to those fertilised with nitrate, atmospheric N2 (their

sole nitrogen source) did not become available immediately after planting because the N2

-assimi-lating system requires the inoculation, root inva-sion, and formation of root nodules by the

Fig. 2. Relative water content as a function of time (DAP). Filled symbols, well-watered plants, open symbols, drought-stressed plants from the time of 30 DAP. Triangles, 10 mM NH4NO3-fed beans; circles, N2-fixing plants; squares, 1 mM

NH4NO3-fed plants. Error bars indicate the S.D. Where not

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Rhizobium bacteria followed by the differentiation of the nodule rhizobia to bacteroids, at which stage the nitrogenase pathway becomes functional [32]. This entire process takes at least 10 days from the time of inoculation. This lag period was reflected in the fact that at 20 DAP, the N2-fixing

plants were still rather similar to the NH4NO3

-lim-ited beans with respect to all the parameters tested, although these same N2-fixing plants later

became much less nitrogen limited at subsequent stages in their growth.

Apart from their greater size, the continuous N assimilation by the NH4NO3-sufficient beans even

under drought stress might have contributed to their lower desiccation tolerance. Low levels of external nitrate reduce stomatal conductance and transpiration within 2 – 5 days after a reduction in N level [33]. Plants with low levels of N supply, moreover, have a smaller leaf area [34 – 36] and retain more water under drought stress, since the sensitivity of the stomatal response to water deficit is increased by N deficiency [1]. This enhancement could result in part from an increased release of abscisic acid from the symplast into the apoplast in response to a drop in N availability [2]. In soybean, shoot:root ratios and root-growth re-sponses under drought conditions were similar to those reported here: in inorganic-N-fed soybeans, but not in N2-fixing plants, root mass increased

during drought stress [10]. Moreover, the threshold value of leaf-water potential for stom-atal closure was higher for N2-fixing than for

inorganic-N-fed plants [10]. This difference could imply that in the N2-fixing plants, photosynthetic

capability could be diminished earlier on after the cessation of watering. Drought stress inhibits ni-trogenase activity in soybean nodules through a decrease in respiratory capacity [37]; which reduc-tion could, in turn, be caused by a prompt and pronounced downregulation of sucrose synthase, thereby preventing sucrose cleavage within the nodule [30].

Alternatively, nascent oxygen along with the oxygen-active species that arises during drought stress can cause irreversible damage to the nitroge-nase protein.

3.3. Senescence

To evaluate the importance of oxidative damage and other senescence-related effects in the

com-mon bean as a consequence of drought stress, we measured catalase, ascorbate peroxidase, and protease activities, enzymes which are indicators of these processes [8,38,39]. Since NH4NO3-limited

plants were quite insensitive to water withdrawal and produced no pods, thus becoming irrelevant to the issue of grain yield (see below); we restricted our analyses to the NH4NO3-sufficient plants as

compared with the N2-fixing beans.

In the nodules of the N2-fixing plants, fresh

weight decreased sharply both with age and as a result of drought, although the total dry mass remained constant throughout (not shown). The total soluble protein content also remained con-stant as well as the protease, catalase, and ascor-bate-peroxidase specific activities (expressed per milligram of tissue protein; cf. Table 2). Qualita-tive analysis of the polypeptide profile by SDS-PAGE (Fig. 3) corroborated that general polypeptide integrity was maintained. This obser-vation, in agreement with a previous study in nodules from soybean plants under moderate drought stress [30], indicates that nodule proteins did not undergo massive degradation and translo-cation under conditions of either ageing or drought stress. In addition, the lack of detectable changes in catalase and ascorbate peroxidase could indicate that oxygen-active species have not risen to dangerous levels. Our results are not consistent with those obtained in nodules from pea plants subjected to a similar drought-stress treat-ment [7], where total soluble-protein content along with the levels of catalase, ascorbate peroxidase, and other antioxidant enzymes decreased under drought stress. Therefore, the determinate nodules in common bean and soybean would appear to be more protected against drought-induced senes-cence and oxidative damage than the indetermi-nate nodules in pea.

In leaves, the total protein decreased, while the specific protease activity increased with ageing and with water deprivation as well, except for the nitrogen-fixing plants at 40 DAP. As already men-tioned, NH4NO3-sufficient drought-stressed beans

were completely dried up by 50 DAP, with only few necrosed leaves remaining on the plants. We therefore did not carry out any measurements on these samples. Specific protease activity varied similarly with age for both the N2-fixing and the

NH4NO3-sufficient plants, ranging from 4.63 and

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Table 2

Nodule and leaf protease, catalase, and ascorbate-peroxidase specific activities in N2-fixing or 10 mM NH4NO3-fed beans at

progressive ages and in different hydration statesa

Age (DAP)b _Protein

Drought

Organ Protease activityc _{Catalase activity}d _{Ascorbate peroxidase}

activitye

(mg plant−1₎ _{(U mg protein}−1₎ _{(U mg protein}−1₎

(U mg protein−1₎

Nodules No 40 12.46 A 2.00 A 7.20 A 67.2 A

Yes 40 11.82 A 2.02 A 7.86 A 85.2 A

50 10.00 A 2.78 A

No 8.37 A 69.6 A

50 9.89 A 2.72 A 6.90 A 51.3 A

Yes

No 40 54.31 A

Leaves, N2- 4.63 A 2.85 A 43.8 A

fixing _Yes ₄₀ _{51.68 A} _{3.83 A} _{3.06 A} _{68.4 A}

50 28.02 B 9.38 B

No 2.10 A 56.7 A

50 5.11 C 15.40 C 3.15 A 62.7 A

Yes

75.6 A

No 40

Leaves, 64.18 A 3.74 A 4.20 A

NH4NO3- Yes 40 25.32 B 6.34 AB 4.29 A 47.1 A

fed No 50 32.43 B 9.21 B 4.53 A 135.9 B

50 NDf _ND

Yes ND ND

a_{Values followed by different letters (within each plant organ) differ significantly at}_P_B_{0.01 according to the ANOVA and}

Tukey test.

b_{DAP, days after planting.}

c_{One unit of protease activity is expressed as the amount of enzyme that produces a}_D_A

440value of 0.1 h−1at 37°C. d_{One unit of catalase activity is expressed as the amount of enzyme that decomposes 1}_m_{mol H}

2O2min−1at 30°C.

e_{One unit of ascorbate peroxidase activity is expressed as the amount of enzyme that oxidizes 1 nmol ascorbate min}−1_{at 30°C.} f_{ND, Not determined.}

Fig. 3. SDS-PAGE polypeptide profiles from plant tissues. Approximately 20 mg protein were loaded per lane. Panel a, leaves (lanes 1 – 4) or nodules (lanes 5 and 6) at 40 DAP (lanes 1, 3 and 5) or 50 DAP (lanes 2, 4 and 6) from well-watered plants grown on the indicated nitrogen source. Panel b, leaves (lanes 1 – 4) or nodules (lanes 5 and 6) of well-watered plants (lanes 1, 3 and 5) or drought-stressed plants (lanes 2, 4 and 6), all at 40 DAP, grown on the indicated nitrogen source. Migration of molecular weight standards is denoted by lines at the right, while the positions of the polypeptides of molecular weight 30, 55, 66 and 78 kDa referred to in the text are marked by arrows at the left.

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at 50 DAP, respectively, representing correspond-ing 2.0- and 2.5-fold increases (Table 2). These increases correlated with a visible degradation of tissue proteins at both of these time intervals, especially in PAGE bands of approximate molecu-lar weight 66 and 55 kDa, the last presumably being the large subunit of Rubisco [40] (Fig. 3a; compare the relative intensities of these bands with that of the 78 kDa species for each lane).

A comparison of the activities and polypeptide profiles at 40 DAP for the stressed and non-stressed plants (i.e. at the middle of the drought period) reveals a small increase in protease specific activity in the NH4NO3-sufficient beans (Table 2),

whereas degradation of the 66 and 55 kDa polypeptides is not yet detectable by SDS-PAGE (Fig. 3b). Nevertheless, an increase in the intensity of the ca. 30 kDa band could be observed for the drought-stressed inorganic-nitrogen-fed plants (Fig. 3b; compare the relative intensity of this band with that of 55 kDa for each lane), indicat-ing a stress-induced change in protein degradation in these plants. No change in the specific activity of either catalase or ascorbate peroxidase was observed as a result of drought conditions; only an increase in the specific activity of ascorbate perox-idase was found to occur with age, at between 40 and 50 DAP, in the NH4NO3-sufficient beans

(Table 2). Common bean leaves thus appeared to be sensitive to drought stress-induced senescence with respect to protease activity and total protein maintenance, but not as regards antioxidant-en-zyme activities; an overall response similar in cer-tain respects to N2-fixing pea plants, the leaves of

which showed significant decreases in both total soluble protein and antioxidant activities, under a similar drought stress [6].

According to these criteria, NH4NO3-sufficient

were more sensitive to drought-induced senescence than N2-fixing plants, in agreement with the

afore-mentioned RWC values (Fig. 2).

3.4. Pod de6elopment and nitrogen translocation

Pod fresh and dry weights were the highest for the NH4NO3-sufficient well-watered plants, but

similar for the NH4NO3-sufficient and the N2

-fixing beans under drought stress (Table 1 and Fig. 4), whereas in NH4NO3-limited plants, no

pods developed either with or without watering. Therefore, while pod yield dropped to 25%

mea-sured as fresh weight or to 57% according to dry weight in the NH4NO3-sufficient beans, this

parameter declined only to 51% by fresh weight and even remained constant by dry weight in the N2-fixing plants (Table 1). The distribution of pod

dry weights shows that pod number was equally restricted by drought with both sources of N (Fig. 4). This observation suggests that some part of the process between flowering and pod establishment had been interrupted early in the drought period in both the NH4NO3-fed plants and the N2-fixing

beans, although grain filling in already established pods continued despite the drought stress in both types of plants. This effect actually compensated for the drop in pod number in the N2-fixing plants

(Fig. 4). We conclude that the effect of drought stress on pod number is probably unrelated to N nutrition; rather, it appears to be a very early effect of drought, perhaps involving an imbalance between ABA and gibberellic acid [1,2,41].

N translocation to pods between 40 and 50 DAP was quite efficient even in the drought-stressed NH4NO3-sufficient plants; however, less

N, was accumulated in pods of unwatered N2

-fixing beans (Fig. 1, open bars, striped portion).

4. Conclusions

N2-Fixing common beans survived better and

sustained a higher proportion of pod yield under conditions of drought stress than did the NH4NO3-sufficient plants. The pods of the former,

however, contained less N than did those of the NH4NO3-grown beans, perhaps because of a

higher drought sensitivity on the part of their nitrogenase system. Drought tolerance in N2-fixing

plants depended at least in part on the lack of external N during their early development, the absence of which led to a smaller leaf-transpira-tion area.

In both N2-fixing and NH4NO3-sufficient plants,

pod number, but not grain filling, was limiting for yield under drought stress, despite the aforemen-tioned differences in the plant’s state of desiccation.

These results would point to the convenience in the use of N2 fixation to increase the survival of

common bean crops during water deficits, and would furthermore suggest the focusing of plant breeding for drought tolerance on the following traits: (i) an enhanced tolerance to water limitation

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Fig. 4. Distribution of individual pod dry weights from four plants at 50 DAP. The pod weights indicated on the abscissa are represented vertically along the ordinate in the same order as they were present at different heights on the plant stem. Each pod is denoted by an arbitrary number shown on the ordinate. Panel a, well watered N2-fixing beans; panel b, drought-stressed

N2-fixing beans; panel c, well-watered 10 mM NH4NO3-fed plants; panel d, drought-stressed 10 mM NH4NO3-fed beans.

in nitrogenase activity; and (ii) a diminished sensi-tivity in flowering and pod establishment at early stages of desiccation. In such plants, enhanced yields could be obtained since in our experiments, nodule senescence and grain filling in established pods appeared to be less sensitive to stress.

Acknowledgements

This work was supported by the Universidad Nacional de La Plata and by the Comisio´n de Investigaciones Cientı´ficas de la Provincia de Buenos Aires, both in Argentina. The authors thank Eng. Alejandro Perticari for performing part of the Kjeldahl determinations and Dr Don-ald F. Haggerty for a critical reading and editing of the manuscript.

References

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[6] I. Iturbe-Ormaetxe, P.R. Escuredo, C. Arrese-Igor, M. Becana, Oxidative damage in pea plants exposed to water deficit or paraquat, Plant Physiol. 116 (1998) 173 – 181.

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[14] M.P. Cordovilla, F. Ligero, C. Lluch, Growth and nitro-gen assimilation in nodules in response to nitrate levels in

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plants were still rather similar to the NH4NO3

-lim-ited beans with respect to all the parameters tested, although these same N2-fixing plants later

became much less nitrogen limited at subsequent stages in their growth.

Apart from their greater size, the continuous N assimilation by the NH4NO3-sufficient beans even

during drought stress [10]. Moreover, the threshold value of leaf-water potential for stom-atal closure was higher for N2-fixing than for

inorganic-N-fed plants [10]. This difference could imply that in the N2-fixing plants, photosynthetic

Alternatively, nascent oxygen along with the oxygen-active species that arises during drought stress can cause irreversible damage to the nitroge-nase protein.

3.3. Senescence

To evaluate the importance of oxidative damage and other senescence-related effects in the

com-mon bean as a consequence of drought stress, we measured catalase, ascorbate peroxidase, and protease activities, enzymes which are indicators of these processes [8,38,39]. Since NH4NO3-limited

plants were quite insensitive to water withdrawal and produced no pods, thus becoming irrelevant to the issue of grain yield (see below); we restricted our analyses to the NH4NO3-sufficient plants as

compared with the N2-fixing beans.

In the nodules of the N2-fixing plants, fresh

NH4NO3-sufficient plants, ranging from 4.63 and

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Table 2

Nodule and leaf protease, catalase, and ascorbate-peroxidase specific activities in N2-fixing or 10 mM NH4NO3-fed beans at

progressive ages and in different hydration statesa

Age (DAP)b _Protein

Drought

Organ Protease activityc _{Catalase activity}d _{Ascorbate peroxidase}

activitye

(mg plant−1₎ _{(U mg protein}−1₎ _{(U mg protein}−1₎

(U mg protein−1₎

Nodules No 40 12.46 A 2.00 A 7.20 A 67.2 A

Yes 40 11.82 A 2.02 A 7.86 A 85.2 A

50 10.00 A 2.78 A

No 8.37 A 69.6 A

50 9.89 A 2.72 A 6.90 A 51.3 A

Yes

No 40 54.31 A

Leaves, N2- 4.63 A 2.85 A 43.8 A

fixing _Yes ₄₀ _{51.68 A} _{3.83 A} _{3.06 A} _{68.4 A}

50 28.02 B 9.38 B

No 2.10 A 56.7 A

50 5.11 C 15.40 C 3.15 A 62.7 A

Yes

75.6 A

No 40

Leaves, 64.18 A 3.74 A 4.20 A

NH4NO3- Yes 40 25.32 B 6.34 AB 4.29 A 47.1 A

fed No 50 32.43 B 9.21 B 4.53 A 135.9 B

50 NDf _ND

Yes ND ND

a_{Values followed by different letters (within each plant organ) differ significantly at}_P_B_{0.01 according to the ANOVA and}

Tukey test.

b_{DAP, days after planting.}

c_{One unit of protease activity is expressed as the amount of enzyme that produces a}_D_A

440value of 0.1 h−1at 37°C. d_{One unit of catalase activity is expressed as the amount of enzyme that decomposes 1}_m_{mol H}

2O2min−1at 30°C.

e_{One unit of ascorbate peroxidase activity is expressed as the amount of enzyme that oxidizes 1 nmol ascorbate min}−1_{at 30°C.} f_{ND, Not determined.}

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which showed significant decreases in both total soluble protein and antioxidant activities, under a similar drought stress [6].

According to these criteria, NH4NO3-sufficient

were more sensitive to drought-induced senescence than N2-fixing plants, in agreement with the

afore-mentioned RWC values (Fig. 2).

3.4. Pod de6elopment and nitrogen translocation

Pod fresh and dry weights were the highest for the NH4NO3-sufficient well-watered plants, but

similar for the NH4NO3-sufficient and the N2

-fixing beans under drought stress (Table 1 and Fig. 4), whereas in NH4NO3-limited plants, no

pods developed either with or without watering. Therefore, while pod yield dropped to 25%

mea-sured as fresh weight or to 57% according to dry weight in the NH4NO3-sufficient beans, this

parameter declined only to 51% by fresh weight and even remained constant by dry weight in the N2-fixing plants (Table 1). The distribution of pod

N translocation to pods between 40 and 50 DAP was quite efficient even in the drought-stressed NH4NO3-sufficient plants; however, less

N, was accumulated in pods of unwatered N2

-fixing beans (Fig. 1, open bars, striped portion).

4. Conclusions

N2-Fixing common beans survived better and

sustained a higher proportion of pod yield under conditions of drought stress than did the NH4NO3-sufficient plants. The pods of the former,

however, contained less N than did those of the NH4NO3-grown beans, perhaps because of a

higher drought sensitivity on the part of their nitrogenase system. Drought tolerance in N2-fixing

plants depended at least in part on the lack of external N during their early development, the absence of which led to a smaller leaf-transpira-tion area.

In both N2-fixing and NH4NO3-sufficient plants,

pod number, but not grain filling, was limiting for yield under drought stress, despite the aforemen-tioned differences in the plant’s state of desiccation.

These results would point to the convenience in the use of N2 fixation to increase the survival of

common bean crops during water deficits, and would furthermore suggest the focusing of plant breeding for drought tolerance on the following traits: (i) an enhanced tolerance to water limitation

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N2-fixing beans; panel c, well-watered 10 mM NH4NO3-fed plants; panel d, drought-stressed 10 mM NH4NO3-fed beans.

Acknowledgements

References

[2] J.W. Radin, D.L. Hendrix, The apoplastic pool of ab-scisic acid in cotton leaves in relation to stomatal closure, Planta 174 (1988) 180 – 186.

[3] C.P. Vance, G.H. Heichel, Carbon in N2fixation:

limita-tion or exquisite adaptalimita-tion, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42 (1991) 373 – 392.

[4] S. Schubert, Nitrogen assimilation by legumes — pro-cesses and ecological limitations, Fert. Res. 42 (1995) 99 – 107.

[5] L. Stoddart, H. Thomas, Leaf senescence, in: F.A. Loewus, W. Tanner (Eds.), Encyclopedia of Plant Physi-ology. New Series, vol. 14A, Springer-Verlag, Berlin, 1982, pp. 592 – 636.

(5)

[6] I. Iturbe-Ormaetxe, P.R. Escuredo, C. Arrese-Igor, M. Becana, Oxidative damage in pea plants exposed to water deficit or paraquat, Plant Physiol. 116 (1998) 173 – 181.

[7] Y. Gorgocena, I. Iturbe-Ormaetxe, P.R. Escuredo, M. Becana, Antioxidant defenses against activated oxygen in pea nodules subjected to water stress, Plant Physiol. 108 (1995) 753 – 759.

[8] S. Sheokand, K. Swaraj, Natural and dark-induced nod-ule senescence in chickpea: nodnod-ule functioning and H2O2

scavenging enzymes, Biol. Plant. 38 (1996) 545 – 554. [9] J.J. Pen˜a-Cabriales, J.Z. Castellanos, Effects of water

stress on N2fixation and grain yield ofPhaseolus6ulgaris

L, Plant Soil 152 (1993) 151 – 155.

[10] M.I. Mı´nguez, F. Sau, Responses of nitrate-fed and nitrogen-fixing soybeans to progressive water stress, J. Exp. Bot. 40 (1989) 497 – 502.

[11] F. Sau, M.I. Mı´nguez, Response to water stress and recovery of nitrate-fed and nitrogen-fixing faba bean, J. Exp. Bot. 41 (1990) 1207 – 1211.

[12] M.C. Antolıń, J. Yoller, M. Sańchez-Dıáz, Effects of temporary drought on nitrate-fed and nitrogen- fixing alfalfa plants, Plant Sci. 107 (1995) 159 – 165.

[13] D.L.N. Rao, P.C. Sharma, Alleviation of salinity stress in chickpea byRhizobium inoculation or nitrate supply, Biol. Plant. 37 (1995) 405 – 410.

[14] M.P. Cordovilla, F. Ligero, C. Lluch, Growth and nitro-gen assimilation in nodules in response to nitrate levels in

Vicia faba under salt stress, J. Exp. Bot. 47 (1996)

203 – 210.