Nitrogen Deficiency Slows Leaf Development and Delays Flowering in Narrow-leafed Lupin

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1 Annals of Botany 79: 43 49, 1997 Nitrogen Deficiency Slows Leaf Development and Delays Flowering in Narrow-leafed Lupin QIFU MA*, NANCY LONGNECKER* and MILES DRACUP* * Cooperatie Research Centre for Legumes in Mediterranean Agriculture, Soil Science, Uniersity of Western Australia, Nedlands, Western Australia 697, Agriculture Western Australia, Locked Bag 4, Bentley, Western Australia 6983 Received: 16 May 1996 Accepted: 1 November 1996 Effects of nitrogen (N) supply on leaf and flower development in Lupinus angustifolius L. cv Merrit were examined in a temperature-controlled glasshouse. Low N supply ( or 4 mmn) had little effect on leaf initiation but slowed leaf emergence on the main stem compared with plants receiving high N supply (6 or64mmn), or with symbiotic N -fixation. Plants experiencing transient N deficiency had slower leaf emergence than plants with a continuous supply of 64 mmn. Nitrogen supply did not affect the time of floral initiation, which occurred within 4 weeks of sowing, by which time nine to ten leaves had emerged. However, the flowering of low-n plants was delayed by 68 to 2 C d (i.e d) even though they had fewer leaves. The effect of N deficiency on flowering time was largely a result of slower leaf emergence Annals of Botany Company Key words: Lupinus angustifolius L., nitrogen, leaf, flower initiation, thermal time, plastochron, phyllochron. INTRODUCTION Several studies have shown that nutrient deficiencies can affect the rate of leaf emergence, although usually only when the nutrients concerned [e.g. nitrogen (N), copper (Cu)] are severely deficient (Dale and Wilson, 1978; Longnecker, Kirby and Robson, 1993a; Longnecker, Slater and Robson, 1993b; Longnecker and Robson, 1994). Delayed maturity is also commonly observed in nutrient-deficient plants, e.g. N, phosphorus (P) (Peaslee, 1977), Cu (Loneragan, Snowball and Robson, 198) and manganese (Mn) (Longnecker, Graham and Card, 1991). Lupinus angustifolius is a comparatively new, but important, seed legume crop in southern Australia, where about 14 million hectares are grown each year (Australian Grain, 1996). Although L. angustifolius is a legume, even a properly-inoculated crop can become N-deficient at various stages of growth. For instance, since lupin crops are generally grown on N-infertile soils, N deficiency can develop before appreciable amounts of N have been fixed (about to 6 weeks, Farrington et al., 1977). Also, low seed P content impairs root nodulation in L. angustifolius (Thomson, Bell and Bolland, 1992), and waterlogging, which is common in the mediterranean environment of southern Australia, depresses N -fixation (Farrington et al., 1977), leading to pale, N-deficient crops (Setter and Belford, 199). Furthermore, symbiotic N -fixation stops when terminal drought begins, usually during rapid grain filling (Farrington et al., 1977). Thus N deficiency has the potential to affect lupin growth, development and yield. Since N deficiency during the early stages of growth could interfere with initiation and development of leaves and flowers, this study examines the effects of continuous or transient N deficiency on main stem leaf and inflorescence development. Plants of L. angustifolius were grown with or without inoculation, and the supply of mineral N was manipulated at various stages of development. Measurements were then made of rates of leaf initiation and emergence, leaf growth, floral initiation and flowering time. MATERIALS AND METHODS Seeds of L. angustifolius L. cv. Merrit were sown in white sand at a rate of four per pot and later thinned to one plant per pot (size 27 mm or PVC tube of diameter 1 mm and depth 4 mm, with holes at the bottom). Five separate experiments were conducted in a temperaturecontrolled glasshouse, each with different N treatments arranged in a completely randomized design (see Table 1 for details). In expts 3 and, when applying N fluctuations, the sand was thoroughly flushed with deionized water in order to achieve a complete N change. For the N -fixation treatment, seeds were inoculated with 1 ml rhizobia suspension per seed (Group WU4) at sowing. The temperature regimes of day (6 18 h)night (18 6 h) were 1813 C, C or 2313 C. Daylength also differed between experiments due to variations in date of sowing (Table 1). A combination of ammonium nitrate, calcium nitrate and potassium nitrate was used, giving a 1: 9 ratio of NH :NO except for expt 4 where the ratio was 1:1. In addition to the N treatments, the plants received the following basal nutrients (µm): 6, K SO ; 3, KH PO ;, MgSO 7H O; 128, Ca+;, H BO ; 38, ZnSO 7H O; 2, CuSO H O;, MnSO H O; 1, Na MoO 2H O; 4, FeNaEDTA. The concentration of $. bo Annals of Botany Company

2 44 Ma et al. Nitrogen Deficiency Affects Lupin Deelopment TABLE 1. Treatments of each experiment Avearge day- Temp (C) Nitrogen Sowing date length (h) (daynight) treatment (mm) Expt 1, 28 Jul , 4, 16, 64 Expt 2, 7 Mar , 64 Expt 3, 1 Jun , 6, *1. 6. N -fixing Expt 4, 3 Jun , 64, N -fixing Expt, 1 Mar , 64, N -fixing, 4 for 23 d 64, 64 for 23 d 4 for 2 weeks 64 Average daylength between sowing and flowering was taken as the number of hours from civil twilight to civil twilight, when the centre of sun disc is 6 below the horizon (Kiesling, 1982). * Plants were supplied with 1mM N for the first 3 weeks and 6 mmn thereafter. Plants were supplied with 4 mmn until floral initiation (23 d after sowing) and 64 mmn thereafter. Plants were supplied with 64 mmn until floral initiation (23 d after sowing), followed by 4 mmn for 2 weeks and 64 mmn thereafter. Ca+ was based on the amount of Ca(NO ) 4H O used in the 64 mmn treatment, and CaSO was added as necessary to maintain a constant Ca+ supply between low and high N treatments. During the growing season, each pot or tube was watered three times a week with ml nutrient solution after the soil had been flushed with deionized water to prevent nutrient accumulation. Soon after seedling emergence, shoots were dissected every 2 3 d and examined, using a binocular microscope, to count emerged and initiated leaves and determine the timing of floral initiation. Emerged leaves were leaves which had begun to unfold from the apical bud, as defined by Dracup and Kirby (1993). Any uninoculated plants which had developed nodules were discarded. The N content of the shoot material used in dissections was analysed using a CHN- elemental analyser (LECO Corporation, 1991). The final area of individual main stem leaves was measured 4 weeks after flowering using an electronic planimeter. For a given treatment, the time of flowering was taken to be the date at which half of the plants had flowered, i.e. when the standard petal of the lowest flower on the main stem raceme was erect (Dracup and Kirby, 1996). Air temperatures inside the glasshouse during the experiments were monitored electronically and recorded hourly by a datalogger (Unidata Australia). Accumulated thermal time (C d after sowing) was calculated from the average of daily maximum and minimum temperatures above a base temperature of C (Dracup, Davies and Tapscott, 1993; Dracup and Kirby, 1993). Regression analysis was done using Minitab (Minitab Release 8, Minitab Inc., State College, PA, USA) and a general linear model was used for the regression of numbers of emerged leaves or primordia against accumulated thermal time. The linear model explained over 97% of the variation (except in expt 1 where r was 94 98%) in leaf emergence rate, and improvements by fitting a quadratic or cubic model were marginal. A Student s t-test was used to compare the slopes of linear regressions, and the inverse of the slope is reported as the phyllochron (C d per emerged leaf) or plastochron (C d per leaf primordium). Analysis of variance was conducted on the other variables measured. RESULTS Leaf initiation There was no significant effect of N deficiency (4 mm)on rates of leaf initiation (Table 2). Inoculated plants had a similar plastochron to that of the plants supplied with sufficient mineral N. Leaf emergence N deficiency caused a decrease in the rate of main stem leaf emergence (Table 3). Plants supplied with between and 16 mm N had a higher phyllochron (i.e. slower leaf emergence) than plants supplied with 6 or 64mM N. Leaves emerged at similar rates in inoculated plants or those supplied with high mineral N. Fluctuation in N supply during the vegetative phase also affected the rate of main stem leaf emergence. Plants supplied with 1 mmn for the first 3 weeks (expt 3), or with 4 mm N until completion of leaf initiation (23 d after sowing, expt ), and 6 or64mm N thereafter, showed slower leaf emergence than plants supplied continuously with 6 or64mm N (Table 3). Transient treatment of TABLE 2. Plastochrons for leaes on the main stem of nodulated plants or plants treated with different rates of added N Plastochron (C dprimordium) Sowing date d.f. 4 mmn 64mMN N -fixing Expt 2, 7 Mar (1) (13) Expt 4, 3 Jun () (224) (177) Expt, 1 Mar (77) (8) (71) The plastochron was taken from the reciprocal of the slope of the number of initiated primodia plus emerged leaves s. accumulated thermal time. Values in parentheses are s.d. of the mean. Within each row, plastochrons were not significantly different (LSD ).

3 Ma et al. Nitrogen Deficiency Affects Lupin Deelopment 4 TABLE 3. Phyllochrons for leaes on the main stem of nodulated plants or plants treated with different rates of added N Nitrogen treatment mm Sowing date d.f. Phyllochron ( Cdemerged leaf) Expt Jul a 442ab 44b 328c 127 (4) (173) (144) (118) Expt Mar a 348b 6 (122) (64) Expt 3 1 * Jun N -fixing 431a 43ab 376c 394b 132 (11) (14) (87) (94) Expt Jun N -fixing 488a 387b 377b 114 (9) (8) (4) Expt Mar. 199 N -fixing 397a 347b 3c 299d 34d 631 (44) (3) (36) (3) (31) The phyllochron was taken from the reciprocal of the slope of the number of emerged leaves s. accumulated thermal time. Values in parentheses are s.d. of the mean. Within each row, phyllochrons with different superscripts are significantly different (LSD. ). *,, See Table 1 caption for a description of N fluctuation treatments. TABLE 4. Effects of N treatment on leaf number and thermal time to flowering (%) of the main stems Nitrogen Sowing treatment Main stem % plants date (mm) leaf number flowering C d) Expt (16)a 124 (1)a 28 Jul (.39)a 124 (1)a (48)b 11 (1)b 64 (71)c (7)c d.f. 8 Expt (47)a 1173 (18)a 7 Mar (18)b 1 ()b d.f. Expt (92)a 1364 (13)a 1 Jun *1 6 (69)a 1232 (13)b 6 266(18)b 12 (13)bc N -fixing 4 (1)a 1188 (13)c d.f. 8 Expt 4 4 3(2)a 1224 ()a 3 Jun (79)a 93 (6)b N -fixing 21 (83)a 61 (6)c d.f. 1 6 Expt 4 223(7)a 7 (7)a 1 Mar (8)a 22 (16)b (7)b (13)b (62)b 988 ()c N -fixing 231 (72)a 923 (7)d d.f Within one experiment, means with different superscripts are significantly different (LSD ). Values in the brackets are s.d. of the mean. *,, See Table 1 for a description of N fluctuation treatments. 64 mmn plants with 4 mmn for 2 weeks after completion of leaf initiation slowed leaf emergence compared with the continuous 64 mmn treatment (Table 3). Low N supply caused a decrease in the number of main stem leaves. In four of the five experiments, the number of main stem leaves was reduced by 2 3 for low N

4 46 Ma et al. Nitrogen Deficiency Affects Lupin Deelopment A B mm N (LN) 6.4 mm N (HN) Leaf position on main shoot C 1 LN HN D 1 LN HN HN LN Individual leaf area (cm 2 ) 1 E N 2 -fixing Individual leaf area (cm 2 ) FIG. 1. Effects of different rates of added N or N -fixation on final area of individual main stem leaves. Fluctuations in N supply were started at the time of floral initiation. Horizontal bars represent s.e. of the mean. Arrows indicate the emerging leaf at which the change in N supply took place (expt ) plants (4, 1 mm) compared with high N plants (6, 64 mm). The number of main stem leaves of the inoculated plants was lower than for plants supplied with high N in two out of three experiments (Table 4). Low N supply before completion of leaf initiation also resulted in fewer leaves on the main stem (expts 3 and ). Leaf size Final size of individual leaves on the main stem was also affected by N treatment. Leaves of plants grown with 4 mm N became progressively smaller from the base upwards, whereas they became progressively larger on plants grown with 64 mmn (Fig. 1A, B). Plants grown with 4 mm N until completion of leaf initiation and then transferred to 64 mm N (when nine leaves had emerged) had larger leaves from leaf seven onwards than plants grown with continuous 4 mmn (Fig. 1A, C). When grown continuously at 64 mm N, however, all leaves above leaf four were larger than on plants grown at 64 mm N only from completion of leaf initiation. Transient N deficiency of 64 mm N plants (which received 4 mm N for 2 weeks) caused a marked decrease in the final size of the leaves emerging during those 2 weeks and also of several leaves emerging after the plants were resupplied with 64 mm N (Fig. 1B, D). The areas of leaves three to of inoculated plants were similar to those of the high N plants but the first two and the uppermost five (21 to ) leaves were generally smaller (Fig. 1B, E). Flowering time Although the number of leaves initiated was affected by N supply, there was no discernible effect on the time of floral initiation (flower primordium was observed on the same day for all the N treatments, within 4 weeks of sowing). However, low N supply (, 4, 1 mm) delayed flowering in each experiment, even though these plants had fewer leaves than high N plants (6, 64 mm) (Table 4). The difference in flowering time between low and high N plants

5 Ma et al. Nitrogen Deficiency Affects Lupin Deelopment 47 Shoot N concentration (%) A plants on day 16. After day 18, both high N and inoculated plants showed little change in shoot N concentration, though the former had consistently higher shoot N than the latter. The shoot N concentration of the 4 mmn plants continued to decline. The apparent effect of N supply on shoot growth occurred later than on shoot N concentrations. Shoot dry weight was similar on day 21 for all the N treatments (Fig. 2B) but, thereafter, plants supplied with 64 mmn, and the inoculated plants, maintained higher rates of growth (with more than 4% N in shoots) than plants supplied with 4 mmn (less than 3% N in shoots). The onset of rapid growth in the N - fixing plants appeared to be slightly delayed compared with plants supplied with high N. 3. B 1 Days after sowing DISCUSSION Leaf initiation and emergence Shoot dry wt (g/plant) Days after sowing FIG. 2. A, Shoot N concentration and B, dry weight of the inoculated plants, or plants supplied with 64or4mMN from 7 28 d after sowing (expt ). Each data point is the mean of seven plants with the vertical bars representing s.e. The arrow shows the time at which root nodules were first observed on the inoculated plants., 64mMN;,4mM N;,N -fixing. varied between 68 and 2 C d (i.e d). The smallest differences occurred in the experiments sown in Mar. with long, but declining, daylengths, whereas the biggest differences occurred in the experiments sown in Jun. and Jul. with short, but increasing, daylengths. For plants receiving a fluctuating N supply (4 to64mm,or64to4 then to 64 mm), flowering time was intermediate between those of plants receiving the high and low N treatments. Inoculated plants flowered a little earlier (22 6 C d, i.e. 2 3 d) than plants supplied with 64 mmn. Inoculated plants generally had one less main stem leaf, but a similar rate of leaf emergence, compared with plants supplied continuously with 64 mmn (Tables 3, 4). Shoot nitrogen concentration and growth Shoot N concentration declined during the early growth of inoculated plants and plants receiving mineral N, but was about 1% higher in plants supplied with 64 mmn than in inoculated plants or plants supplied with 4 mm Nupto day 18 (Fig. 2A). Nodules were first observed on inoculated 3 Seeds of L. angustifolius contain five to six leaf primordia (Dracup and Kirby, 1993). Counting of leaf primordia started 1 week after sowing, by which time two leaves had emerged and eight to nine primordia were already present on each main stem apex. Thereafter, leaf primordia were initiated with a plastochron of to 3 C d per primordium, the value depending largely on the daylength of individual experiments but not on the levels of mineral N supply. This is consistent with the value of 24 to 26 C d per primordium observed in field-grown plants in Western Australia by Dracup and Kirby (1993, 1996). Leaf initiation on the main stem was complete within 4 weeks of sowing, by which time eight to nine leaves had emerged on the main stem and cotyledons were beginning to yellow (on 4 mmn and inoculated plants). Although low N supply did not cause a decrease in the rate of leaf initiation, it resulted in a decrease in the rate of leaf emergence. The difference in phyllochron between the 4 and 64 mmn plants was about 7 to C d per emerged leaf, but leaf emergence was not affected by low N supply until after the completion of leaf initiation (e.g. day 23, Fig. 2). At that stage, the shoot N concentration of 4 mm N plants had fallen below 3% and shoot growth had decreased by 22%. Slower leaf emergence at low levels of N supply (, 2 and 3 mm N) was also observed in Triticum aestium L. by Longnecker et al. (1993a). Longnecker and Robson (1994) found that N deficiency caused a decrease in the rate of leaf emergence in T. aestium when shoot dry matter production was approx. 4% or less of maximum. These findings indicate that the rate of leaf emergence is less sensitive to N deficiency than is plant growth. In the present study, plants transferred from 4to64mM N after completion of leaf initiation had lower rates of leaf emergence than plants supplied continuously with 64 mm N. Plants supplied with 64 mmn throughout, except for 2 weeks of 4 mm N immediately after completion of leaf initiation, also had slower leaf emergence. Similarly, low N supply before the double ridge stage (four to five leaves) in wheat had greater effects on the rate of leaf emergence than the same treatment imposed later (Longnecker and Robson, 1994). In the field, transient N deficiency caused by delayed

6 48 Ma et al. Nitrogen Deficiency Affects Lupin Deelopment or poor nodulation, or waterlogging during the early growth of lupin crops, could lengthen the period of leaf emergence and delay flowering time. Leaf size The area of the leaves of field-grown L. angustifolius plants increases progressively up the main stem (Dracup and Kirby, 1993), as also seen in the N -fixing treatment of this study. However, in the present study, this trend was reversed when plants were grown with 4 mmn, and only the lowest four leaves were comparable in size to those on plants grown with 64 mmn, or using fixed N. Presumably N from seed reserves was adequate for the growth of the first few leaves but insufficient for the subsequent leaves if other sources of N were limited. The lowest two leaves and the uppermost leaves of N -fixing plants were generally smaller than those of the plants supplied with 64 mm N. This could be due to limited N supply before the onset of N -fixing andor competition between leaves and nodules for carbohydrate. In later growth, the supply of N from N - fixation to the uppermost leaves may be insufficient for maximum growth because of the demands of concurrent lateral branch growth. When plants were transferred from 4 to64mm Nat completion of leaf initiation (when nine leaves had emerged), leaves from leaf seven onwards were larger than corresponding leaves on the 4 mm N plants, but always (except for leaves one to four) smaller than those on the 64 mmn plants. The failure of higher leaves on the plants transferred from low to high N supply to reach the same size as those on the 64 mm N plants shows the irreversible consequences of early low N supply. Similarly, when plants with ten emerged leaves were transferred from 64to4mM N for 2 weeks and then back to 64 mmn, leaves from leaf nine upwards were consistently smaller than on plants supplied continuously with 64 mm N. This indicates that transient N deficiency can affect not only the final size of leaves growing during the period of low N supply, but also several leaves emerging after that period. The reduced size of leaves emerging after the period of low N supply might be due to reduced cell division during the period of low N supply and to lowered availability of photoassimilates later because of lower total leaf area (compared with high N plants). The sensitivity of leaf growth to early N deficiency could, therefore, result in slower canopy development and affect yield potential. Studies with split applications of N fertilizer to cereal crops show that early application stimulates leaf growth and tiller formation, whereas late application promotes leaf area duration and grain filling (Spiertz and Vos, 1983; Pearson and Jacobs, 1987). Raising N supply at the double ridge stage in wheat can have significant effects on spikelet number, grain number and weight per ear (Langer and Liew, 1973). Time to floral initiation and flowering In this study, plants receiving mineral N (4 or64mm) and N -fixing plants initiated reproductive development at the same time, but low N supply delayed flowering by 68 to 2 C d (i.e. up to 14 d). The delay in flowering was due mainly to slower rates of leaf emergence. There was an interaction between N nutrition and daylength in the determination of flowering time: the smallest differences in flowering time between low and high N plants occurred in the experiments sown in Mar. under long, but declining, daylengths, whereas the largest differences occurred in the experiments sown in Jun. and Jul. under short, but increasing, daylengths. Long days in March may have caused an acceleration in apical development in the early growth stages and reduced the effects of N deficiency. In summary, lupin leaf development was sensitive to low N supply or temporary N deficiency during early growth and, as a consequence, flowering was delayed. This may indicate that the imposition of stress, even for a short period, causing delay or suppression in nodulation or N - fixation, could have adverse effects on canopy development and yield potential of a lupin crop. In southwest Australia, available soil N is low (1 3%) and early N deficiency in lupin plants may develop before appreciable amounts of N have been fixed. Starter-N fertilizer (e.g. 1 kg N ha at sowing), which could be a useful practice for rapid canopy development, leading to higher grain yield, is currently being investigated in the field. Alternatively, genotypic variation in the onset of N -fixation in lupins warrants investigation. ACKNOWLEDGEMENTS We thank Grains Research and Development Corporation, Australia for funding this research. Statistical assistance of Dr Nick Galwey is gratefully acknowledged. LITERATURE CITED Australian Grain Grain Yearbook 1996, Toowoomba. Dale JE, Wilson RG A comparison of leaf and ear development in barley cultivars as affected by nitrogen supply. Journal of Agricultural Science 9: 3 8. Dracup M, Davies C, Tapscott H Temperature and water requirements for germination and emergence of lupin. Australian Journal of Experimental Agriculture 33: Dracup M, Kirby EJM Patterns of growth and development of leaves and internodes of narrow-leafed lupin. Field Crops Research 34: 9 2. Dracup M, Kirby EJM Lupin deelopment guide. Perth, Western Australia: University of WA Press. Farrington P, Greenwood EAN, Titmanis ZV, Trinick MJ, Smith DW Fixation, accumulation, and distribution of nitrogen in a crop of Lupinus angustifolius cv. Unicrop. Australian Journal of Agricultural Research 28: Kiesling TC Calculation of the length of day. Agronomy Journal 74: Langer RHM, Liew FKY Effects of varying nitrogen supply at different stages of the reproductive phase on spikelet and grain production and on grain nitrogen in wheat. Australian Journal of Agricultural Research 24: Loneragan J, Snowball K, Robson A Copper supply in relation to content and redistribution of copper among organs of the wheat plant. Annals of Botany 4: Longnecker N, Graham RD, Card G Effects of manganese deficiency on the pattern of tillering and development of barley (Hordeum ulgare cv. Galleon). Field Crops Research 28: 8 2.

7 Ma et al. Nitrogen Deficiency Affects Lupin Deelopment 49 Longnecker N, Kirby EJM, Robson A. 1993a. Leaf emergence, tiller growth, and apical development of nitrogen-deficient spring wheat. Crop Science 33: Longnecker N, Slater J, Robson A. 1993b. Copper supply and the leaf emergence rate of spring wheat. Plant and Soil 1/16: Longnecker N, Robson A Leaf emergence of spring wheat receiving varying nitrogen supply at different stages of development. Annals of Botany 74: 1 7. Pearson CJ, Jacobs BC Yield components and nitrogen partitioning of maize in response to nitrogen before and after anthesis. Australian Journal of Agricultural Research 38: 1 9. Peaslee DE Effects of nitrogen, phosphorus, and potassium nutrition on yield, rates of kernel growth and grain filling periods of two corn hybrids. Communications in Soil Science and Plant Analysis 8: Setter T, Belford RK Waterlogging: how it reduces plant growth and how plants can overcome its effects. Western Australian Journal of Agriculture 31: 1. Spiertz JHJ, Vos NMD Agronomic and physiological aspects of the role of nitrogen in yield formation of cereals. Plant and Soil 7: Thomson BD, Bell RW, Bolland MDA Low seed phosphorus concentration depresses early growth and nodulation of narrowleafed lupin (Lupinus angustifolius cv. Gungurru). Journal of Plant Nutrition 1: