Relationships between shoot to root ratio ... - Wiley Online Library

Plant, Cell and Environment (1999) 22, 949–958

ORIGINAL ARTICLE

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Relationships between shoot to root ratio, growth and leaf soluble protein concentration of Pisum sativum, Phaseolus vulgaris and Triticum aestivum under different nutrient deficiencies M. ANDREWS 1, J. I. SPRENT 2, J. A. RAVEN 2 & P. E. EADY 1 Ecology Centre, University of Sunderland, Sunderland SR1 3SD, UK and 2Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK 1

ABSTRACT Relations between shoot to root dry weight ratio (S : R), total plant dry weight (DW), shoot and plant N concentration and leaf soluble protein concentration were examined for pea (Pisum sativum L.), common bean (Phaseolus vulgaris L.) and wheat (Triticum aestivum L.) under different nutrient deficiencies. A regression model incorporating leaf soluble protein concentration and plant DW could explain greater than 80% of the variation in S : R within and between treatments for pea supplied different concentrations of NO3– or NH4+ in solid substrate; pea and bean supplied different concentrations of N, P, K and Mg in liquid culture; and wheat supplied different concentrations of N, P, K, Mg, Ca and S in liquid culture. Addition of shoot or plant N concentration to the model explained little more of the variation in S : R. It is concluded that results are consistent with the proposal that macronutrient effects on S : R are primarily mediated through their effects on protein synthesis and growth. Key-words: Phaseolus vulgaris; Pisum sativum; Triticum aestivum; dry matter partitioning; macronutrients; nitrogen; pea; pinto bean; protein; wheat.

INTRODUCTION Supply of all macronutrients (N, P, S, K, Mg and Ca) can affect the partitioning of dry matter between shoot and root of higher plants. There is general agreement that shoot to root dry weight ratio (S : R) decreases when growth is limited by N supply (Andrews 1993), S supply (Clarkson, Saker & Purves 1989; Zsoldos et al. 1990; Ingestad & Ägren 1991) or P supply (Adalsteinsson & Jensen 1988; Fredeen, Rao & Terry 1989; Rufty, MacKown & Israel 1990; Ingestad & Ägren 1991; Cakmak, Hengeler & Marschner 1994). In contrast, S : R has been reported to decrease or increase with decreased growth associated with K deficiency (Asher & Ozanne 1967; Drew 1975; Steen 1984; Cakmak et al. 1994; Ericsson 1995), Mg deficiency (Will 1961; Cakmak et al. 1994; Ericsson & Kähr 1995) or Ca deficiency (Joham 1957; Ericsson 1995). There are reports for several higher plant species that S : R Correspondence: Dr Mitchell Andrews. © 1999 Blackwell Science Ltd

changes with growth/development independently of nutrient supply (Bastow-Wilson 1988; Gedroc, McConnaughay & Coleman 1996). Therefore, in some cases, the apparent response of S : R to nutrient supply may have been a growth/ontogenetic effect. Nevertheless, there are several studies which unequivocally show nutrient effects on S : R that are independent of growth/development (Andrews 1993; Cakmak et al. 1994). There is no general agreement as to the mechanism(s) of macronutrient effects on S : R. Bastow-Wilson (1988) reviewed models for the control of dry matter partitioning between shoot and root of higher plants and concluded that changes in S : R in response to a wide range of environmental factors, including macronutrient supply, conform to the Thornley (1972) model. In this model the factors which determine S : R are the supply of C and N substrates by shoot and root, respectively, transport of these substrates between shoot and root and incorporation of these substrates into plant structure. It was argued that structural growth of shoot and root is colimited by the local C and N substrate concentrations and that this growth acts as a sink for substrates to which further substrates diffuse from the points of supply. It was further argued that the rate of transport of C and N substrate from shoot to root and root to shoot, respectively, is proportional to the concentration gradient divided by a resistance. Hence, a decrease in C substrate acquisition would result in an increase in S : R while a decrease in N substrate acquisition would cause S : R to decrease. Dewar (1993) developed a model in which C, N and water interact to determine S : R. In this model it was assumed that a fraction of the N taken up by the root is translocated via the xylem to the shoot where it is transferred to the phloem. The remaining fraction is transferred directly to the root phloem. Phloem translocation of C and N downwards from shoot to root is driven by a shoot to root gradient of labile C in accordance with the Münch pressure flow mechanism. An essential feature of the Thornley (1972) and Dewar (1993) models is that gradients of C and N substrate coexist between shoot and root with C substrate concentration greater in shoot than root and N substrate concentration greater in root than shoot. This is unlikely to be always the case. For example, NO3– is likely to be the main form of N utilized by plants in disturbed soils but N compounds required for structural 949

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growth (primarily proteins) are produced from amino acids. Therefore, although the root is the main site of NO3– uptake, the site of NO3– assimilation (production of amino acids) is the source of N which can be used for growth. Considerable data indicate that for many higher plants, the shoot is the main site of NO3– assimilation at low and high external NO3– concentrations (Andrews 1986). The Thornley (1972) and Dewar (1993) models can not explain a decrease in S : R with decreased NO3– supply for species such as common bean (Phaseolus vulgaris L., hereafter referred to as bean) which have the shoot as their main site of NO3– assimilation regardless of NO3– supply (Andrews 1986; Andrews, Zerihun & Watson 1995). Also, in relation to the Thornley (1972) and Dewar (1993) models, carbohydrate accumulation in shoots has been associated with increased S : R of bean on low K or Mg supply (Marschner Kirkby & Cakmak 1996). Marschner et al. (1996) proposed that S : R increases when K or Mg availability limits growth as both play a distinct role in the export of photosynthate from shoot to root via the phloem and hence, a deficiency of either results in impaired transport of photosynthate from shoot to root, accumulation of carbohydrates in the shoot and increased S : R. However, this proposal does not explain the decrease in S : R with decreased K or Mg supply which has been reported to occur (Will 1961; Asher & Ozanne 1967; Drew 1975; Steen 1984). Most studies which examined the N effect on S : R of higher plants used NO3– as the N source (Andrews 1993). For several herbaceous species grown in solid substrate, plant growth rate increases from a very low value at 0·1 mol m–3 applied NO3– to a maximum in the range 1–5 mol m–3 NO3– then changes little or decreases with increased NO3– supply to 20 mol m–3 but tissue N concentration and S : R increase with increased NO3– throughout (Andrews 1993; Andrews et al. 1995). For several species, a positive linear relationship was found between S : R and whole plant or shoot N concentration (%N; Hirose 1986; Ingestad & Ägren 1991; Boot, Schildwacht & Lambers 1992; Lieffering, Andrews & McKenzie 1993; Andrews et al. 1995). Andrews (1993) proposed that the increase in S : R with increased NO3– supply regardless of its effect on growth is due to an increase in reduced N relative to C substrate for shoot growth in conjunction with the proximity of the shoot to the C supply. Specifically, increased NO3– supply results in increases in NO3– uptake, NO3– assimilation and tissue organic N concentration. The increase in organic N concentration is likely to be due to increases in a range of N-containing molecules but mainly amino acids and proteins. Soluble protein is likely to be of particular importance as it usually constitutes around 50% of total N in leaves although insoluble protein is likely to increase in parallel with soluble protein and also constitute a substantial fraction of organic N (Evans 1989; Anderson et al. 1997; Zerihun, McKenzie & Morton 1998). Nitrate uptake, NO3– assimilation and protein synthesis are energy-requiring processes hence the increase in organic N concentration reflects an increased proportion of energy/C derived from photosynthesis being utilized in processing NO3–.

However, N is a component of chlorophyll and photosynthetic enzymes and hence can influence photosynthesis greatly. If increased processing of NO3– results in increased photosynthate available for structural growth, shoot dry weight (DW) will increase relative to root DW due to proximity of the shoot to the C source and increased organic N available for structural growth. Also, if growth increases, part of the NO3– effect on S : R may be a growth/ontogenetic effect. Nitrogen use efficiency (C gain per unit N per unit time) decreases with increased organic N concentration. If organic N concentration increases but the photosynthate available for structural growth changes little or decreases, S : R will still increase as again the shoot will realize a greater proportion of its growth potential due to its proximity to the source of C and the availability of reduced N for growth. This proposal is similar to the Thornley (1972) model in that structural growth is colimited by local C and N substrate concentrations but it does not rely on a gradient of N between root and shoot. There are several reports that at similar total plant DW, S : R is greater with NH4+ than with NO3– as N source (Cox & Reisenhauer 1973; Timpo & Neyra 1983; Bowman & Paul 1988; Troelstra, Wagenaar & Smant 1992). However, where tested in these studies, the tissue N concentration for plants of similar DW was also greater with NH4+ than with NO3–. Andrews (1993) proposed that the greater S : R with NH4+ than with NO3– at similar plant DW was due to a greater reduced N relative to C availability with NH4+. Subsequent work on barley (Hordeum vulgare L.) and bean found that the relationship between S : R and tissue N concentration was similar with the two N forms (Lieffering et al. 1993; Andrews et al. 1995). For bean, the relationship between S : R and tissue N concentration was similar with NO3–, NH4+ or glutamine as the N source. This is further evidence that N effects on S : R are independent of site of N assimilation and that tissue N concentration is an important factor determining S : R. There are reports that P, K, S and Mg deficiency effects on S : R are dependent on N supply (Davidson 1969; Boote 1977; Fisher & Benson 1983; Zsoldos et al. 1990; Ericsson 1995). There are also reports that uptake of NO3– decreases with decreased P, K and S supply, assimilation of NO3– decreases with decreased P and K supply and protein levels decrease under S and P limitation (Blevins et al. 1978; Schjørring 1986; Clarkson et al. 1989; Lauer et al. 1989; Rufty et al. 1990). In the present study, relations between S : R and total plant DW, root and shoot N concentration and leaf soluble protein concentration were examined for pea (Pisum sativum L.) that was supplied different concentrations of NO3– or NH4+ and pea, bean and wheat (Triticum aestivum L.) that were supplied different concentrations of macronutrients under otherwise similar conditions. Our hypothesis is that N concentration, N-form and macronutrient effects on S : R are mediated through effects on N utilization (Andrews, Raven & Sprent 1996) and growth. It is proposed that protein level is of particular importance as this © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 949–958

Macronutrient effects on shoot : root

reflects the availability of N substrate for growth. If our hypothesis is correct, then across treatments, there should be a positive correlation between S : R and tissue N concentration, tissue protein concentration and growth. MATERIALS AND METHODS Plant material Untreated seeds of pea cv. Pilot, bean cv. Othello and wheat cv. Minx were obtained, respectively, from Wearside Seeds, Sunderland, UK, Dr B. A. McKenzie, Lincoln University, Canterbury, New Zealand and Plant Breeding International, Cambridge, UK. Experimental procedures All experiments were carried out in a glasshouse under natural daylight in which the temperature was maintained above 15 °C. The initial and repeat experiment 1 on pea were carried out in the summer (13 July – 27/28 August) and spring (26 February – 4 April), respectively. Plants were harvested at the onset of flowering in the initial experiment 1. In experiment 1, seeds of pea were germinated in moist (distilled water) perlite in the glasshouse. Seedlings with a shoot length of approximately 20 mm were transferred to 800 cm3 volume pots (one plant per pot) containing equal volumes of vermiculite and perlite which was flushed every 2 d with basal nutrient solution (Andrews, Love & Sprent 1989) containing the appropriate N treatment. The N treatments were 0·5, 1, 2, 3, 4, 5, 6 and 10 mol m–3 N supplied as KNO3 or (NH4)2SO4. Potassium concentration was made equal in all treatments by the addition of K2SO4 as required but SO4 – concentration was not balanced. The pH of all solutions was in the range 5·7–5·9. At harvest, the leaf from the fourth node behind the main stem apex of each plant was detached for determination of soluble protein concentration (Read & Northcote 1981). Plants were then divided into root and shoot, dried at 70 °C for 5 d and weighed. Dried material was ground and total N concentration of 0·5–1 mg samples was determined using a Carlo Erba 1106 CHN Elemental Analyser (Strumentazione, Milan, Italy). The initial and repeat experiments 2, 3 and 4 on pea, bean and wheat, respectively, were carried out over 24 to 37 d periods during late spring to mid-summer (1 May – 26 July). The initial and repeat experiments did not overlap in time. The experiments were carried out in liquid culture. The basal nutrient solution used was that of Cakmak et al. (1994) which contained K2SO4 (0·88 mol m–3), Ca(NO3)2 (2·0 mol m–3), KH2PO4 (0·25 mol m–3), MgSO4 (1·0 mol m–3), KCl (10 mmol m–3), H3BO3 (10 mmol m–3), Fe EDTA (20 mmol m–3), MnSO4 (1 mmol m–3), ZnSO4 (1 mmol m–3), CuSO4 (0·1 mmol m–3) and (NH4)6 Mo7O24 (0·01 mmol m–3). The low P (10 mmol m–3), K (50 mmol m–3) and Mg (20 mmol m–3) treatments used in experiments 2–4 were as described by Cakmak et al. (1994). These treatments were used as they were reported to result in substantially different S : R but similar growth rates (Cakmak et al. 1994). © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 949–958

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A low N treatment (0·1 mol m–3 Ca (NO3)2 plus 1·9 mol m–3 CaCl2) was also included. The pH of all nutrient solutions was in the range 5·25–5·35. The seeds of each species were germinated in moist perlite in the glasshouse. Seedlings with a shoot length of ≈ 30 mm were transferred to 2 dm3 pots (one plant per pot) which contained the appropriate nutrient solution. All nutrient solutions were renewed once, 14 to 17 d after seedling transfer. At harvest, leaf soluble protein concentration and shoot and root DW and N concentration were determined. For pea and bean, the soluble protein concentration was determined in the leaf from the fourth node behind the main stem apex of each plant. For wheat, the soluble protein concentration was determined in the most recently fully expanded main stem leaf (i.e. ligule had formed; Andrews, McKenzie & Jones 1991). The initial and repeat experiment 5 on wheat were carried out side by side in late summer/early autumn (18/19 September – 20/21 October). The basal and low N, P and Mg nutrient solutions used in experiments 2–4 were included in experiment 5. The low K treatment was changed from 50 to 25 mmol m–3 and low S (5 mmol m–3) and Ca (50 mmol m–3) treatments were added. In all treatments, concentrations of all macronutrients except the deficient nutrient were made equal to those in the basal nutrient solution by the addition of the appropriate Na or Cl salt as required. This resulted in treatment differences in Na and Cl concentrations. However, concentrations of Na and Cl were less than 4 mol m–3 in all treatments and unlikely to affect growth or S : R (Greenway & Munns 1980; Hawkins & Lewis 1993). The pH of all solutions was in the range 5·25–5·35. At harvest, shoot and root DW and N concentration and leaf soluble protein concentration were determined for all plants. Treatment replication and data analysis All experiments were of completely randomized design. There were three replicate plants for all treatments in experiments 1 and 5 and five replicate plants for all treatments in experiments 2–4. An analysis of variance was carried out on data for total plant DW, S : R, total plant and shoot N concentration and leaf soluble protein concentration from all experiments. All effects described as significant have a probability P < 0·01. In experiment 1, an analysis of covariance was carried out to test whether the relationships between S : R and total plant DW, total plant N concentration, shoot N concentration and leaf soluble protein concentration were affected by the form of N supplied. This analysis of covariance and linear and multiple regression analyses were used to determine if there were significant (P < 0·05) relationships between S : R and total plant DW, total plant N concentration, shoot N concentration and leaf soluble protein concentration in experiment 1. Means stated as significantly different in experiments 2–5 are on the basis of an LSD (P < 0·05) value. In experiments 2–5, treatment effects and ranking of treatments with respect to S : R, plant DW, plant and shoot N concentration and leaf soluble protein concentration were similar with the initial and repeat experiments and with pooled data from the initial and repeat experiments;

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the results from pooled data are presented for ease of description. Linear and multiple regression analyses were used to determine if S : R and total plant DW, total plant N concentration, shoot N concentration and leaf soluble protein concentration were significantly correlated in experiments 2–5. Replicate values as opposed to treatment means were used in all regression analyses. In all experiments, shoot N concentration was highly correlated with total plant N concentration (straight line regression gave an R2 > 98·6 in all cases) and substitution of plant N concentration with shoot N concentration in the regression models did not explain a greater proportion of the variation in S : R. Because of this, details of total plant N only are given in the results. All data were analysed using the Minitab Version 12 (Minitab Inc., Pennsylvania, USA) package.

RESULTS N form effects on growth, S : R and N and soluble protein concentration of pea Analysis of variance of data obtained in the initial and repeat experiment 1 on pea showed that plant DW, S : R,

plant N concentration and leaf soluble protein concentration were dependent on N concentration and the form applied. In the initial experiment 1, plant DW increased with increased applied NH4+ concentration to 6 mol m–3 then decreased with increased applied NH4+ to 10 mol m–3 (Fig. 1a). Plant DW increased with increased applied NO3– to 10 mol m–3. At all applied N concentrations, plant DW was greater with NO3– than with NH4+. The S : R, plant N concentration and leaf soluble protein concentration increased with increased applied NO3– and NH4+ concentration over the entire range used (results not shown). At all applied N concentrations, the plant N concentration was either greater with NH4+ than with NO3– or similar with the two N forms but S : R and leaf soluble protein concentration were greater with NO3–. Analysis of covariance in the initial experiment 1 revealed significant positive relationships between S : R and plant DW, S : R and plant N concentration, and S : R and leaf soluble protein concentration. The relationship between S : R and plant DW was not affected by the form of N supplied (F1,45 = 0·59, P = 0·447); S : R increased steadily with increased plant DW (Fig. 1b). The relationships between S : R and tissue N concentration and S : R

Figure 1. Effect of different concentrations of NO3– (● ●, ●) and NH4+ (■ ■, ■) on plant dry weight and the relationships between shoot to root dry weight ratio (S : R) and plant dry weight, plant N concentration and leaf soluble protein concentration. Open and closed symbols represent the initial and repeat experiments, respectively. Each point is the mean value of three replicates. Details of regression analysis carried out on the replicate values are given in the text. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 949–958

Macronutrient effects on shoot : root

and leaf soluble protein concentration were dependent on N form (F1,45 = 97·0, P < 0·0001 and F1,45 = 17·1, P < 0·0001, respectively; Fig. 1c & d). The S : R increased with tissue N concentration with NO3– or NH4+ as N source but at a similar N concentration, S : R was substantially greater with NO3–. Similarly, S : R increased with leaf soluble protein concentration with NO3– or NH4+ as N source and at a similar leaf soluble protein concentration was greater with NO3– although in this case, the magnitude of the difference between N forms was small. Linear regression analysis indicated significant positive correlations between S : R and plant DW (R2 = 81·6%; F1,46 = 204, P < 0·0001), S : R and plant N concentration (R2 = 37·7%; F1,46 = 27·8, P < 0·0001) and S : R and leaf soluble protein concentration (R2 = 90·6%; F1,46 = 433, P < 0·0001). A multiple regression model of S : R incorporating leaf soluble protein concentration, plant DW and plant N concentration gave an R2 value of 91·8%. This value was only slightly greater than that with leaf soluble protein concentration alone although partial regression coefficients indicated that S : R was significantly positively correlated with leaf soluble protein concentration (t = 5·66, P < 0·0001) and plant DW (t = 2·18, P = 0·035): S : R and plant N concentration were not significantly related in this analysis (t = 0·29, P = 0·77). For all treatments, plant DW was substantially less in the repeat experiment 1 than in the initial experiment 1 (Fig. 1a). In the repeat experiment 1, plant DW increased with increased applied NH4+ concentration to 4–5 mol m–3 then decreased with increased NH4+ supply thereafter; the plants supplied 10 mol m–3 NH4+ died before completion of the experiment. Plant DW increased with applied NO3– concentration to 4–5 mol m–3 also but changed little with increased NO3– thereafter. As in the initial experiment 1, S : R, plant N concentration and leaf soluble protein concentration increased with increased applied NO3– and NH4+ concentration over the entire range used (results not shown). Linear regression again indicated significant positive correlations between S : R and plant DW (R2 = 52·1%; F1,43 = 45·0, P < 0·0001), S : R and plant N concentration (R2 = 58·6%; F1,43 = 60·7, P < 0·0001) and S : R and leaf soluble protein concentration (R2 = 84·8%; F1,43 = 239, P < 0·0001). Partial regression coefficients obtained from multiple regression indicated a significant positive correlation between S : R and leaf soluble protein concentration (t = 6·13, P < 0·0001) but not S : R and plant DW (t = 0·98,

Pea

Low Mg Basal Low K Low P Low N LSD

P = 0·33) or S : R and plant N concentration (t = 1·58, P = 0·12). When data for the initial and repeat experiment 1 were pooled, linear regression indicated a much stronger positive correlation between S : R and plant DW (R2 = 86·6%; F1,91 = 590, P < 0·0001) than with S : R and leaf soluble protein concentration (R2 = 29·4%; F1,91 = 37·4, P < 0·0001) or S : R and plant N concentration (R2 = 11·2%; F1,91 = 11·5, P < 0·001). With pooled data, a regression model of S : R incorporating plant DW and leaf soluble protein concentration gave an R2 value of 88·6% (F1,90 = 16·6, P < 0·001); the addition of plant N concentration to the model did not increase the R2 value significantly. N, P, K and Mg effects on growth, S : R and N and soluble protein concentration of pea and bean For pea and bean in experiments 2 and 3, respectively, S : R, plant DW, plant N concentration and leaf soluble protein concentration were dependent on nutrient treatment (Table 1). For pea, S : R decreased with nutrient treatment in the order low Mg > basal = low K > low P > low N. In contrast, the plant DW decreased with treatment in the order basal = low K > low P = low Mg = low N whereas plant N concentration was less with low N than with all other nutrient solutions but otherwise, did not differ between treatments. Ranking of leaf soluble protein concentration across treatments was similar to that for S : R. For pea, linear regression indicated a significant strong positive correlation between S : R and leaf soluble protein concentration (R2 = 76·0%; F1,48 = 152, P < 0·0001). Linear regression also indicated a significant but weak positive correlation between S : R and plant N concentration (R2 = 19·3%; F1,48 = 11·5, P < 0·001) but S : R and plant DW were not significantly correlated (R2 = 4·3; F1,48 = 2·16, P = 0·148). Partial regression coefficients obtained from multiple regression indicated a significant strong positive correlation between S : R and leaf soluble protein content (t = 148, P < 0·0001) and weaker negative correlations between S : R and plant DW (t = –4·73, P < 0·0001) and S : R and plant N concentration (t = –2·73, P = 0·009). A regression model of S : R using leaf soluble protein concentration and plant DW had an R2 value of 83·6% (F1,46 = 21·3, P < 0·001). Addition of plant N concentration to the model increased the R2 value to 85·9% (F1,45 = 7·35, P < 0·01). For bean in experiment 3, as for pea in experiment 2, S : R decreased with treatment in the order low Mg > basal

Bean

S:R

DW (g)

N (%)

Prot (%)

S:R

DW (g) N (%)

Prot (%)

7·03 4·84 4·44 3·12 2·03 0·792

0·96 2·20 1·98 1·17 0·79 0·396

3·95 4·04 4·13 4·33 1·86 0·569

15·9 14·0 14·1 10·1 4·2 1·79

6·17 4·66 4·29 2·65 1·87 0·543

1·70 2·87 1·40 1·47 1·33 0·291

15·2 13·7 13·1 10·7 5·3 0·92

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 949–958

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3·62 2·81 3·11 3·65 1·50 0·151

Table 1. Shoot to root dry weight ratio (S : R), total plant dry weight (DW) and N concentration and leaf soluble protein (prot) concentration of pea and bean supplied basal nutrient solution or basal nutrient solution deficient in N, P, K or Mg. Within columns, calculated F-values from an analysis of variance across treatments exceeded the critical value for P < 0·0001

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= low K > low P > low N. For bean as for pea, linear regression indicated that there was a significant, strong, positive correlation between S : R and leaf soluble protein concentration (R2 = 80·5%; F1,48 = 199, P < 0·0001). Linear regression also indicated significant but weaker positive correlations between S : R and plant DW (R2 = 17·6%; F1,48 = 10·2, P = 0·003) and S : R and plant N concentration (R2 = 30·7%; F1,48 = 21·3, P < 0·0001). A regression model of S : R using leaf soluble protein content, plant DW and plant N content had an R2 value of 82·7 (F2,45 = 5·8, P < 0·01) which was only slightly greater than with leaf soluble protein alone. N, P, K, Mg, Ca and S effects on growth, S : R and N and soluble protein concentration of wheat For wheat in experiment 4, S : R decreased with treatment in the order basal > low K = low Mg > low P = low N (Table 2). In experiment 4, as in experiments 1–3, linear regression indicated a significant, strong, positive correlation between S : R and leaf soluble protein concentration (R2 = 79%; F1,48 = 180, P < 0·0001). There were also significant strong positive correlations between S : R and plant DW (R2 = 51·5%; F1,48 = 50·9, P < 0·0001) and S : R and plant N concentration (R2 = 70·5%; F1,48 = 115, P < 0·0001). A regression model of S : R incorporating plant DW, plant N concentration and leaf soluble protein concentration gave an R2 value of 84·9% which was greater than that with leaf soluble protein alone. Partial regression coefficients indicated significant correlations between S : R and plant DW (t = 3·54, P < 0·001) and S : R and plant N content (t = 3·85, P < 0·0001) but S : R and leaf soluble protein content were not significantly correlated (t = 1·11, P = 0·272). In experiment 5 on wheat, S : R decreased with treatment in the order basal > low Mg > low S > low Ca > low P > low K > low N (Table 3). As in experiment 4, linear regression indicated significant positive correlations between S : R and leaf soluble protein concentration (R2 = 56·9%; F1,40 = 52·9, P < 0·0001), S : R and plant DW (71·5%; F1,40 = 100, P < 0·0001) and S : R and plant N concentration (R2 = 70·0%; F1,40 = 93·4, P < 0·0001). A regression model of S : R

Table 2. Shoot to root dry weight ratio (S : R), total plant dry weight (DW) and N concentration and leaf soluble protein (prot) concentration of wheat supplied basal nutrient solution or basal nutrient solution deficient in N, P, K or Mg. Within columns, calculated F-values from an analysis of variance across treatments exceeded the critical value for P < 0·0001

Basal Low K Low Mg Low P Low N LSD

S:R

DW (g)

N (%)

Prot (%)

4·49 4·09 3·96 1·90 1·63 0·360

1·28 1·22 0·75 0·65 0·60 0·225

4·79 3·86 4·82 3·20 1·16 0·161

18·5 16·7 16·2 12·2 6·6 1·49

Table 3. Shoot to root dry weight ratio (S : R), total plant dry weight (DW) and N concentration and leaf soluble protein (prot) concentration of wheat supplied basal nutrient solution or basal nutrient solution deficient in N, P, K, Mg, S and Ca. Within columns, calculated F-values from an analysis of variance across treatments exceeded the critical value for P < 0·0001

Basal Low Mg Low S Low Ca Low P Low K Low N LSD

S:R

DW (g)

N (%)

Prot (%)

6·12 5·57 3·52 2·52 2·03 1·28 1·05 0·174

1·03 0·69 0·83 0·38 0·49 0·19 0·39 0·080

5·72 5·44 5·06 4·74 4·12 3·08 1·34 0·219

16·3 16·1 11·7 15·3 13·1 11·2 7·1 0·59

incorporating plant DW, plant N concentration and leaf soluble protein concentration gave an R2 value of 87·2%. Partial regression coefficients indicated significant positive correlations between S : R and plant DW (t = 7·08, P < 0·0001) and S : R and leaf soluble protein content (t = 3·22, P = 0·003) but S : R and plant N content were not significantly correlated (t = –0·17, P = 0·891). DISCUSSION Macronutrient effects on S : R There are reports for many higher plant species of increased S : R associated with increased growth due to increased N supply (Andrews 1993). This was found to be the case for pea, bean and wheat in all experiments carried out (Fig. 1, Tables 1, 2 & 3). In addition, in experiment 1, S : R of pea increased with increased N (NO3– or NH4+) supply above that which gave maximum growth. Again, this response was reported previously for other species and unequivocally shows that there is a N effect on S : R outside a growth effect (Andrews 1993; Andrews et al. 1995). The supply of macronutrients other than N can affect S : R. Reports are consistent that S : R decreases when growth is limited by P or S supply (Adalsteinsson & Jensen 1988; Clarkson et al. 1989; Fredeen et al. 1989; Rufty et al. 1990; Zsoldos et al. 1990; Ingestad & Ägren 1991; Cakmak et al. 1994). Similarly, for pea and bean under P deficiency and wheat under P and S deficiency, the S : R decreased (Tables 1, 2 & 3). However, there is inconsistency in the literature with respect to the effects of K, Mg and Ca deficiency on S : R (Asher & Ozanne 1967; Drew 1975; Steen 1984; Ingestad & Ägren 1991; Cakmak et al. 1994; Ericsson & Kähr 1995). Recent work by Cakmak et al. (1994) found the S : R of bean to increase under K and Mg deficiency. For bean in experiment 3, K deficiency did not affect S : R despite causing a substantial decrease in plant DW whereas for wheat in experiments 4 and 5, K deficiency resulted in a decrease in S : R (Tables 1, 2 & 3). The basal and low K nutrient solutions used in experiments 3 and 4 were those of Cakmak et al. (1994) and therefore © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 949–958

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these results, in conjunction with those in the literature, show that the direction of response of S : R to K deficiency is not consistent. Similarly, Mg deficiency resulted in increases in S : R of pea and bean but caused a decrease in the S : R of wheat (Tables 1, 2 & 3). For wheat, Ca deficiency, as with deficiency of all other macronutrients, resulted in a decrease in S : R (Tables 2 & 3). This finding contrasts with the report of an increase in S : R of Ca-deficient cotton (Gossypium hirsutum L.) plants during vegetative growth (Joham 1957). Therefore, a comparison of results obtained here with those in the literature indicates that with Ca deficiency, as with Mg and K deficiency, the direction of response in S : R is not consistent. The mechanism(s) of N concentration and form effects on S : R For pea in the initial and repeat experiment 1, there was a strong positive correlation between S : R and tissue N concentration with NO3– as N source (Fig. 1c): this response was reported previously for other species (Hirose 1986; Ingestad & Ägren 1991; Boot et al. 1992; Lieffering et al. 1993; Andrews et al. 1995). There was also a strong positive correlation between S : R and tissue N concentration with NH4+ as N source but in contrast with previous findings for barley and bean (Lieffering et al. 1993; Andrews et al. 1995), the relationship between S : R and tissue N concentration was substantially different with NO3– or NH4+ as N source (Fig. 1c). Usually there is a close positive correlation between leaf soluble protein concentration and plant N concentration but feeding NO3– or NH4+ can alter the relative proportions of the different N fractions in a plant (Goyal & Huffaker 1984; Chaillou et al. 1991) and the N use efficiency (C gain per unit N per unit time) (Raven, Wollenweber & Handley 1992). If it is assumed that leaf soluble protein concentration reflects plant soluble protein concentration then this was the case for pea which had a larger proportion of the total N concentration as soluble protein in NO3–-fed plants and N use efficiency was greater with NO3– than with NH4+. The extent of these differences with N form were such that in the initial and repeat experiment 1, the relationships between S : R and leaf soluble protein concentration and S : R and plant DW were similar with the two N forms (Fig. 1b & d). Our hypothesis in experiment 1 was that N concentration and form effects on S : R are primarily mediated through effects on N utilization, in particular protein level, and growth. Results obtained in experimentt 1 are consistent with this proposal as regression models of S : R incorporating plant DW, plant N concentration and leaf soluble protein concentration could explain 85% or more of the variation in S : R within and across the initial and repeat experiment. In the initial and repeat experiment 1, the correlation between S : R and leaf soluble protein concentration was much stronger than that between S : R and plant N concentration and inclusion of plant N concentration in the regression model explained little more of the variation in S : R. This indicates that leaf soluble protein concentration © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 949–958

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is more important than overall plant N status in determining S : R. It is proposed that this is the case primarily because proteins are the main N-containing molecules required for structural growth (Cassab & Varner 1988; Carpita 1996) and although insoluble as well as soluble protein is required for growth, it is likely that both increase in parallel and so leaf soluble protein concentration reflects N substrate availability for growth. The results in experimentt 1 also indicate that leaf soluble protein concentration or plant DW can be the major factor affecting S : R. Specifically, in the initial and repeat experiment 1, differences in leaf soluble protein concentration could explain 85–90% of the variation in S : R within and across treatments and addition of DW to the regression model explained little more of the variation in S : R. However, when data for the initial and repeat experiment 1 were pooled, differences in plant DW could explain more than 85% of the variation in S : R and addition of leaf soluble protein concentration to the regression model explained little more of the variation in S : R. For plants on similar N supply, DW was substantially greater in the initial than in the repeat experiment 1 (note that experiments were carried out at different times of year) and as a consequence, the range in plant DW was extended when data for the two experiments were pooled: also, values were fairly evenly spread across this range (Fig. 1a). In contrast, leaf soluble protein concentration for plants on similar N supply was in most cases lower in the initial than in the repeat experiment 1. If S : R of pea increases with DW independently of leaf soluble protein concentration, as appears to be the case, then the closer correlation between S : R and plant DW with the increased range in plant DW is as expected. Serial harvests of plants on different N supply under controlled environment conditions are required to fully determine the extent of the growth/development effect on S : R of pea. In the initial and repeat experiment 1 as was reported previously, S : R increased with increased N supply above that which gave maximum growth (Fig. 1). This increase in S : R was associated with increased leaf soluble protein concentration. Previously, it was proposed that at NO3– concentrations above that which give maximum growth, energy/C utilized in processing N could result in less C available for structural growth and hence could be a factor affecting S : R (Andrews 1993; Andrews et al. 1996). For this to be the case, C utilization in processing N must be of a magnitude which could affect dry matter partitioning between shoot and root. There is general agreement that for higher plants usually 25–65% of C fixed in photosynthesis is lost via respiration (Byrd, Sage & Brown 1992; Amthor 1994; Zerihun et al. 1998). Zerihun et al. (1998) calculated the photosynthetic costs associated with the utilization of NO3–, NH4+ or glutamine as N source and concluded that costs were similar with the different N forms particularly in the longer term when costs due to turnover of protein (primarily soluble protein) dominate the total cost of N utilization. It has been estimated that 25–60% of total respiratory loss is due to protein turnover (Penning de Vries 1975; Barneix et al. 1988; de Visser, Spitter & Bouma 1992).

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However, Zerihun et al. (1998) argue on the basis of their calculations and recent reports in the literature (e.g. Bouma et al. 1994) that it is more likely that 5–25% of total respiratory loss is due to N utilization with the proportion increasing with increased protein concentration. On the basis of these values, it was concluded that photosynthate consumption due to N utilization is not substantial relative to the overall C balance of plants and hence could have little effect on S : R. This conclusion is not correct. For example, consider a plant of a particular protein concentration and photosynthetic rate per unit DW where for every 1 C fixed, 0·4 C is lost via respiration with 0·06 C lost (15% of total respiratory C loss) due to protein turnover. If of the 0·6 C remaining, 0·45 C is partitioned to the shoot and 0·15 C is partitioned to the root then if C distribution reflects dry matter distribution, as is usually the case (Andrews, MacFarlane & Sprent 1985; Sutherland et al. 1985; Andrews et al. 1989) this would result in a S : R of 3. If protein concentration doubles and hence the amount of C respired in processing protein doubles but photosynthetic rate per unit DW does not change then in this case, for every 1 C fixed, 0·46 C is utilized in overall respiration (NB only 26% of total respiratory C loss is due to protein turnover) and 0·54 C is left for structural growth. If of this 0·54 C, 0·45 C is again partitioned to the shoot this leaves only 0·09 C for transport to the root and as argued above would result in a S : R of 5·1. It is stressed that because the root is almost always smaller than the shoot (N.B. usually substantially smaller than the shoot), then small changes in total plant C balance could greatly affect S : R. The mechanism(s) of N, P, K, Mg, S and Ca effects on S : R Our hypothesis in experiments 2–5 was that the different macronutrient effects on S : R are primarily mediated through their effects on N utilization, in particular protein level, and growth. Results obtained provide strong evidence that this is the case as in all experiments, a regression model of S : R incorporating plant DW, plant N concentration and leaf soluble protein concentration could explain more than 80% of the variation in S : R within and across treatments. In experiments 4 and 5 on wheat, correlations between S : R and plant N concentration and S : R and leaf soluble protein concentration were equally strong and regression models of S : R incorporating plant DW and plant N concentration or plant DW and leaf soluble protein concentration could explain more than 80% of the variation in S : R. However, in experiments 2 and 3 on pea and bean, respectively, as in experiment 1 on pea, S : R was more closely correlated with leaf soluble protein concentration than with plant N concentration. This is further evidence that leaf soluble protein concentration is more important than overall plant N status in determining S : R. In experiments 2 and 3, multiple regression analysis indicated a weak negative correlation between S : R and tissue N concentration as well as a positive correlation between S : R and leaf soluble protein concentration. This at first

seems contradictory to the linear regression analysis which in both experiments gave a positive correlation between S : R and tissue N concentration. However, this is interpreted as indicating that for a given N concentration, S : R increases with the protein fraction (Table 1). Results obtained in experiment 1 on pea, indicate that changes in leaf soluble protein concentration or plant DW can be the major factor affecting S : R. Similarly, in experiments 2–4, differences in leaf soluble protein concentration could explain 76–81% of the variation in S : R within and across the macronutrient treatments but in experiment 5 which had the greatest number of treatments and the greatest relative differences in plant DW, differences in plant DW accounted for more than 70% of the variation in S : R. In addition, multiple regression analysis indicated that leaf soluble protein concentration and plant DW can interact to determine S : R. For wheat in experiment 5, multiple regression indicated significant positive correlations between S : R and plant DW and S : R and leaf soluble protein concentration and inclusion of both in a regression model explained almost 90% of the variation in S : R within and across treatments. Furthermore, in experiment 1 on pea and experiment 4 on wheat, multiple regression indicated a weak positive correlation between S : R and plant DW in addition to a strong positive correlation between S : R and leaf soluble protein concentration. However, in experiment 2 on pea, multiple regression indicated a weak negative correlation between S : R and plant DW in addition to a strong positive correlation between S : R and leaf soluble protein concentration. In experiment 2, values for the Mg deficiency treatment strongly influenced the data analysis. When this treatment was omitted, linear regression indicated that differences in leaf soluble protein concentration could explain 86·8% of the variation in S : R within and between treatments and addition of plant DW to the regression model did not explain significantly more of the variation in S : R. In comparison with the other treatments, the Mg deficiency treatment had the greatest leaf soluble protein concentration, the lowest equal DW and the greatest S : R (Table 1). It is possible that high leaf soluble protein concentration associated with low growth rate results in exceptionally high S : R due to energy/C utilized in protein turnover being of increased relative importance. Alternatively, there may be a Mg-specific effect on S : R; exceptionally high values of S : R have been reported for bean under Mg deficiency (Cakmak et al. 1994). Nevertheless, in experiment 2, differences in leaf soluble protein concentration could explain 76% of the variation in S : R within and across treatments thus indicating that any Mg-specific effect on S : R is minor relative to its effect on leaf soluble protein concentration. Also with bean in experiment 2 and wheat in experiments 4 and 5 there was no obvious Mg-specific effect on S : R (Tables 1, 2 & 3). Thus, it is concluded that results obtained in experiments 2–5 provide strong evidence in support of the proposal that macronutrient effects on S : R are primarily mediated through their effects on leaf protein concentration and growth. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 949–958

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