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Effects of Nutrient Supply on Pre-emergence Growth and Nutrient Absorption in Wheat (Triticum aestivumL.) and Sugarbeet (Beta vulgarisL.)
Annals of Botany 81 : 665–672, 1998
Effects of Nutrient Supply on Pre-emergence Growth and Nutrient Absorption in Wheat (Triticum aestivum L.) and Sugarbeet (Beta vulgaris L.) C A R O L Y N E D U$ R R and B. M A R Y INRA, UniteU d’Agronomie, rue Fernand Christ, 02007 Laon Cedex, France Received : 19 November 1997
Returned for revision : 16 January 1998
Accepted : 10 February 1988
Nutrient absorption in wheat and sugarbeet was studied during pre-emergence growth by adding 0, 7, 10±5 or 14 mol m−$ nitrogen (N) to the growth medium. Seedling growth and carbon, N and "&N contents of the seedling parts were measured. Differences between the natural abundance of "&N in seeds and in nutrient solution were used to determine the proportion of N in the organs originating from seed reserves and from absorption. Absorption began later for wheat than for sugarbeet and had less effect on seedling growth. The absorbed N was found mainly in roots. Compared to wheat, sugarbeet seedling N content was greatly altered and the hypocotyl showed increased elongation when nutrients were added. Most of the absorbed N was found in the radicle and hypocotyl with less in the cotyledons. Sugarbeet seedling emergence and early growth could be decreased by adverse conditions occurring after sowing by affecting mineral availability in the soil or through altered root absorption. # 1998 Annals of Botany Company Key words : Triticum aestium L., wheat, Beta ulgaris L., emergence, natural isotopic composition, seedling, seed reserves.
INTRODUCTION Seed reserve depletion and the transition to autotrophic growth have been studied for many species (Black, 1956 ; Cooper and MacDonald, 1970 ; Bouaziz and Hicks, 1990 ; Shanmuganathan and Benjamin, 1992 ; Maillard et al., 1994 ; Tamet, Durr and Boiffin, 1994 ; Durr and Boiffin, 1995). The relative carbon contributions of seed reserves and photosynthesis during early seedling growth have been studied using radiocarbon (Pinto Contreras and Gaudille' re, 1987) and changes in the natural abundance of the stable isotope "$C (Dele! ens, Gre! gory and Bourdu, 1984). The relative contributions of mineral seed reserves and nutrient uptake from the substrate by seedlings have been less studied. Various experiments have reported contradictory effects on seed emergence of fertilizer placement in the soil (MacWilliam, Clements and Dowling, 1970 ; Hegarty, 1976 ; Durrant and Mash, 1989 ; Andrews, Scott and MacKenzie, 1991). Thus, there is a need to separate the effects of added minerals on germination sensu stricto, defined as protrusion of the radicle from the seed, and pre-emergence growth, which is the stage after germination until the seedling reaches the soil surface, in order to better understand these contradictory effects. High concentrations of minerals in water have an adverse effect on germination rates during seed inhibition prior to radicle protrusion (MacWilliam et al., 1970 ; Durrant and Mash, 1989). This is due to the decrease in water potential around the seed when minerals are added (Hegarty, 1976). Less is known about the period after radicle protrusion when mineral absorption begins, and about the seedlings’ mineral requirements. Several studies have shown that absorption may begin early (Krigel, 1967, in subterranean 0305-7364}98}05066508 $25.00}0
clover ; MacWilliam et al., 1970, in pasture plants ; Maillard et al., 1995, in walnut seedlings) and that there is more hypocotyl growth in sugarbeet when potassium nitrate is added (Durrant and Mash, 1989). The present study was undertaken to determine the effects of nutrient supply on seedling growth during the pre-emergence growth of two species : sugarbeet (Beta ulgaris L.), a dicotyledonous, epigeal species with perispermic reserves which are completely transferred to the embryo before emergence (Du$ rr and Boiffin, 1995) and wheat (Triticum aestium L.), a monocotyledon with endosperm reserves that are used even after emergence (Pinto Contreras and Gaudille' re, 1987). Seedlings were grown with or without added nutrients. Natural nitrogen isotope composition was used to determine the relative contributions of the two nitrogen sources in the organs of the seedling.
MATERIALS AND METHODS Experiments were performed in a growth chamber in the dark to simulate pre-emergence growth. Pots sown with wheat were first kept at 5 °C for 3 d to break seed dormancy. They were then incubated at 20 °C with the pots sown with sugarbeet. The seeds were taken from a single seedlot (Beta ulgaris L. ‘ Vega ’, monogerm variety for sugarbeet and Triticum aestium L. ‘ Ventura ’ for wheat). One seed weight range, close to the mean seed weight of the seedlot, was selected for each species : 7±0–8±9 mg for sugar beet (mean seed weight without pericarp, i.e. embryoperisperm ¯ 2±4 mg) and 38±0–42±9 mg for wheat seeds. The initial fresh weight of each seed was recorded so that the initial seed weight of each harvested seedling was known. The seed
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DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings
water content (SWC, relative to the seed dry weight) was measured on samples of 100 seeds, making it possible to estimate the seed dry weight of each seed as follows : SDW ¯ SFW}(1SWC) with SWC ¯ 0±112 for sugarbeet and 0±114 for wheat. Seeds were sown at a depth of 2 cm in pots (7¬11 cm) filled with white sand (150–210 µm grain diameter ; bulk density 1±47 kg dm−$). This was done to anchor seeds and roots, but the pots were kept in the dark even after the seedlings were visible at the soil surface. This method was chosen to allow seedlings to grow as they would under natural conditions. Each treatment involved 40 pots with five seeds per pot. The gravimetric water content was increased to 0±2 kg kg−" before sowing by watering the sand with either deionized water or with nutrient solution (Saglio and Pradet, 1980). Before dilution, this solution contained 700 mol m−$ KNO , 750 mol m−$ NH NO , 650 mol m−$ $ % $ Ca(NO ) , 275 mol m−$ KH PO , 50 mol m−$ NaCl, 157 mol # % $ # m−$ MgSO , plus micronutrient supplements. The solution % was diluted in deionized water at a rate of 2, 3 or 4 ml l−", to obtain a N concentration of 7, 10±5 or 14 mol m−$ N—a concentration of 14 mol m−$ N being commonly used to grow adult plants. Treatments were designated as D0 for pure water, D2, D3 and D4 for the three dilutions. D4 was not used for sugarbeet. The soil water content used was determined by preliminary germination (radicle protrusion) tests with deionized water, using the same sowing technique and with maximum germination rate at a water content of 0±15–0±25 kg kg−". Germination rates were also measured for the various mineral supplies using 0±20 kg kg−" water content. Water content was kept constant throughout the experiments by adding deionized water. Atmospheric relative humidity was 80 %. Seedlings were harvested every day or half day for growth measurements. The first samples were taken 2 d after sowing for sugarbeet and 4 d after sowing for wheat. Samples of ten to 15 seeds per treatment (two pots for wheat and three for sugarbeet) were taken and measurements were made on germinated seeds only. The radicle, coleoptile containing folded leaves and seed residue were separated for wheat. The radicle, hypocotyl, cotyledons, seed residue and pericarp were separated for sugarbeet seedlings. The length of each organ was measured. Each part of the seedling was dried for 48 h at 70 °C and weighed separately. The carbon and total nitrogen contents of each part were measured, along with the "&N isotopic composition of their total N, using a mass spectrometer (VG SIRA 9) linked to an automatic combustion analyser (Carlo Erba NA 1500). A single sample consisted of the five seedlings from a sampled pot, and two or three replicates were analysed for each sampling date. Seed residues were also analysed for each sampling date and treatment for sugarbeet and for the D0 and D2 treatments for wheat. Carbon, total nitrogen and isotopic composition were determined on three replicates of five seeds of the chosen seed weight of each species. The "&N abundance in a material is defined as the number of "&N atoms over the number of ("&N"%N) atoms. Results of analyses are expressed as the "&N atom % excess, i.e. "&N abundance of the sample minus air "&N abundance, which is used as reference (abundance : 0±3663 %). The nutrient solution was not labelled and its "&N atom % excess was
0±0004 (mean of five replicates, s.d. ¯ 0±0003 %), whereas the atom % excess of the seed was about 0±0020 %. We attempted to exploit natural differences in the excess of seeds and the nutrient solution. Experiments ended when elongation of the emerging organ ceased (8 d for wheat coleoptiles and 12–16 d for sugarbeet hypocotyls). The cumulated thermal time from sowing (Td) was calculated as follows : i=d
Td ¯ 3 (θi®θb) i=" where Td is the time at sampling date d in degree-days (°Cd), θi the daily mean temperature in the growth chamber and θb the threshold value of 0 °C for wheat (Gate, 1996) and 3±5 °C for sugarbeet (Du$ rr and Boiffin, 1995).
RESULTS Germination and seedling growth There was no difference in germination (sensu stricto) rates in response to any treatment for either species. The 50 and 95 % germination rates were obtained at 35 and 45 °Cd for sugarbeet and 20 and 30 °Cd for wheat, respectively. Changes in dry mass of the wheat seedling and seed residue after germination were similar for all treatments until 110–120 °Cd (Fig. 1 A). Seedlings grew more slowly thereafter in D0 (P ! 0±05), with a smaller decrease in seed residue (P ! 0±01). This difference was due to a smaller increase in coleoptile and leaf dry mass in the D0 treatment (P ! 0±01) ; the root dry mass was not affected (Fig. 1 B and C). Leaves broke through the coleoptile at 110–120 °Cd in all treatments and there was less coleoptile elongation after this point for treatment D0 only (P ! 0±05, Fig. 1 D). There were no differences in seed residue dry matter between any of the sugarbeet treatments (Fig. 1 A). Seedling dry mass was similar for treatments D0, D2 and D3 until 75 °Cd and then markedly smaller in D0 than in D2 and D3 (P ! 0±05 after 100 °Cd). Radicle dry mass was slightly higher in D0 than in other treatments after about 60 °Cd (P ! 0±05 after 100 °Cd, Fig. 1 B). Hypocotyl mass and length varied with treatment becoming smaller in D0 after 75 °Cd and after 100 °Cd in D2 (P ! 0±05, Fig. 1 C and D). Cotyledon masses also differed after 100 °Cd, but this was not related to treatment (not shown).
Carbon partitioning and net balance Changes in the amount of carbon in the seed residue and seedling were similar in wheat for all treatments until 110 °Cd, with a constant pattern of C partitioning between shoot and roots (Fig. 2). Thereafter, there was a smaller increase in seedling carbon mass in D0 than in other treatments with a smaller decrease in the amount of carbon in the seed residue and less C in the shoot. Amounts of carbon in roots did not differ between treatments. Changes in the amounts of carbon in the seed residue and seedling were similar for all sugarbeet treatments (Fig. 2 A).
DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings Wheat
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F. 1. Changes in dry mass and length of wheat and sugarbeet seedlings with thermal time in the dark. Seeds}seedlings were watered with nutrient solutions containing 0 (D0 ; D), 7 (D2 ; E), 10±5 (D3 ; +) or 14 mol m−$ N (D4 ; _). Vertical bars indicate s.e. on the last sampling date (n ¯ 2 for wheat, n ¯ 3 for sugarbeet). °Cd, Degree days ; b, threshold value for thermal time calculation.
The C partitioning pattern between organs was similar for all treatments, except that there was slightly more carbon in the radicle in D0 (Fig. 2 B–D). The pattern of net carbon balance (i.e. the amount of carbon in the seedling and seed residue compared to the amount of carbon in the initial seed) was similar between treatments for both species (Fig. 3). Twenty per cent of the
total C was lost at the end of the experiment in the case of wheat, 50 % in the case of sugarbeet. Nitrogen net balance and absorption The net nitrogen balance was about 100 % in D0 throughout the experiment for wheat and sugarbeet (Fig. 3).
DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings
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F. 2. Amount of carbon in wheat and sugarbeet during D0 (D), D2 (E), D3 (+) and D4 (_) treatments (see Fig. 1 for details). Vertical bars indicate s.e. on the last sampling date (n ¯ 2 for wheat, n ¯ 3 for sugarbeet). °Cd, Degree days ; b, threshold value for thermal time calculation.
The nitrogen balance of D0 did not differ from that of D2 until about 100 °Cd for wheat. Thereafter, the net balance increased and reached a maximum of 125 % at 120 °Cd. Differences began at about 60 °Cd for sugarbeet : the net balance increased and reached a maximum of 150 % at 100–110 °Cd for both the D2 and D3 treatments.
The amounts of N in the coleoptile and radicle of wheat were markedly smaller after 100 °Cd in D0 compared to the other treatments (Fig. 4). For sugarbeet, differences in the amounts of N between D0 and the other treatments developed very rapidly in the hypocotyl and radicle. The difference was generally smaller in the cotyledons (Fig. 4).
DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings
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F. 3. Carbon and nitrogen net balances for wheat and sugarbeet during D0 (C D, N *), D2 (C E, N +) and D3 (C y, N _) treatments (see Fig. 1 for details). °Cd, Degree days ; b, threshold value for thermal time calculation.
Initially, the "&N atom % excess in the various parts of the seedling was similar (Fig. 5 A–D). When no nutrient solution was added (D0), the "&N atom % excess of the seedlings remained constant at about 0±0020 % for both species. The "&N composition of wheat seedling in D2, D3 and D4 was similar to D0 until 110–120 °Cd (Fig. 5). Thereafter, "&N atom % excess in D2, D3 and D4 decreased compared to D0 due to changes in the "&N atom % excess in roots, particularly in D3 and D4, indicating N absorption, while concentrations did not differ in shoots (Fig. 5 B and C). There were differences in the isotopic composition of sugarbeet seedlings from the first sampling date (Fig. 5 A). "&N atom % excess was similar in D2 and D3 and was lower than that in D0 after 30 °Cd (P ! 0±05). It reached the same minimum value, just over 0 % excess, at about 100 °Cd. Changes in composition differed with seedling part (Fig. 5 B–D). Zero values were reached at 60 °Cd for the radicle. Negative values, very close to that of the nutrient solution, were reached at 75 °Cd for the hypocotyl. "&N atom % excess remained higher in the cotyledons. Values were similar to those of D0 until 60–75 °Cd and then decreased, but remained significantly higher than those in the radicle and hypocotyl. DISCUSSION The concentration of minerals used had no effect on germination (radicle protrusion) rates for either species. Seed reserve depletion was unchanged for a long time with differences occurring only for wheat once the leaves had broken through the coleoptile ; carbon net balances were unaffected. Differences in C partitioning were very small with slightly more C in the root when no nutrient was added. Other studies have similarly shown that seed reserve depletion is unaffected by various growth conditions (light or dark, Cooper and MacDonald, 1970 ; water potential, Bouaziz and Hicks, 1990 ; or low temperature, Tamet et al., 1994). Total seedling dry mass, the dry mass of each part and the elongation of the coleoptile were unchanged for wheat until the leaves broke through the coleoptile at about 110– 120 °Cd. Thereafter, the coleoptile and leaves grew more when nutrient solution was added, with more C being
depleted from the seed reserves and going to the shoot. The net nitrogen balance exceeded 100 % thereafter and the "&N atom % excess of roots decreased. Absorption began at this time and the N absorbed mostly accumulated in the roots. Andrews et al. (1991) also reported that addition of NO − $ did not affect root and shoot dry weights in wheat, but that fresh weights and nitrate reductase activity were increased. This effect was particularly evident at the time of first leaf expansion. The final lengths of coleoptiles were rather short compared to those of sugarbeet hypocotyls (Fig. 1 D). One reason may be that the mesocotyl never elongated in wheat seedlings in these experiments. Wheat is generally sown at about 2–3 cm under field conditions. The coleoptile reaches this length at about 100–110 °Cd (Fig. 1 D). Emergence should already have taken place under common field conditions when absorption begins. The coleoptile was broken through by leaves at about 100–110 °Cd in all treatments. Branching roots and new roots appeared at this time and absorption started. If emergence is delayed by deep sowing or soil crusting, the coleoptile will be longer when absorbed minerals are supporting growth and this could help the seedling to reach the soil surface. However, the coleoptile loses much of its rigidity and strength when broken by leaves. Thus, the final emergence rate would not be much improved. Changes in seedling growth and absorption started earlier in sugarbeet seedlings. There was more hypocotyl growth and less radicle growth when nutrients were supplied. The net nitrogen balance exceeded 100 % after 50 °Cd, but the "&N composition of the radicle was affected as soon as it protruded at 35–40 °Cd, showing that absorption had begun. "&N atom % excess in the radicle decreased to a level very close to that of the added solution showing that almost all of the N in the radicle came from the added solution. Similar results were obtained in the hypocotyl. The decrease in "&N atom % excess occurred later in cotyledons and their "&N composition was consistently higher. Less added nitrogen was translocated to these organs, but about 50 % of the N was taken up at the end of the experiment from the added N. The radicle and the hypocotyl elongated most rapidly. The cotyledons remained small until emergence. Carbon from the seed reserves and cotyledons (Du$ rr and
DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings
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F. 4. Amount of nitrogen in wheat and sugarbeet during D0 (D), D2 (E), D3 (+) and D4 (_) treatments (see Fig. 1 for details). Vertical bars indicate s.e. on the last sampling date (n ¯ 2 for wheat, n ¯ 3 for sugarbeet). °Cd, Degree days ; b, threshold value for thermal time calculation.
Boiffin, 1995) and absorbed N were mostly translocated to the hypocotyl maximizing the elongation rate. Seedling emergence forces are also higher when seedlings are grown with nutrients (Souty and Rode, 1993 ; unpub. res.). The N content in the cotyledon at the start of photosynthesis (the cotyledons alone photosynthesize until the first leaves appear) will be higher if N is present in the nutrient solution. Crop emergence and early growth could both be altered if
the mineral supply is reduced, due to lower soil availability or local concentration, or to poor root absorption. Wheat and sugarbeet seedlings both absorb minerals from the growth medium during their C heterotrophic growth but absorption begins later for wheat, the timing being related to emergence time in common field conditions. These two species differ in the size of their seed reserves and depletion patterns. This may be related to the time at which
DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings Wheat
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F. 5. Wheat and sugarbeet "&N atom % excess in the whole seedling and in the seedling parts for D0 (D), D2 (E), D3 (+) and D4 (_) treatments (see Fig. 1 for details). Vertical bars indicate the mean s.e. per sampling date (n ¯ 2 for wheat, n ¯ 3 for sugarbeet). °Cd, Degree days ; b, threshold value for thermal time calculation.
absorption begins. Absorption begins earlier in dicotyledonous pasture seedlings, even in legume seedlings, than in grasses (MacWilliam et al., 1970). These results help us to better understand the effects of certain seed treatments or fertilizer placement. Fertilizer placement may increase mineral concentrations in soil surrounding seedling roots and improve early absorption. However, more detailed
observations on such treatments are required, particularly in field conditions. In conclusion, the time at which absorption began was identified by comparing seedlings grown with and without added nitrogen. Differences in the "&N isotopic composition of the nutrient solution and seeds made it possible to determine the time at which absorption began and where the
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absorbed nitrogen went. Therefore this simple method not only allowed us to quantify the N absorption rate but also to chart the fate of added N in the plant. A C K N O W L E D G E M E N TS We thank O. Delfosse, C. Dominiarzick and C. Leforestier for technical assistance and F. Devienne and G. Richard for their helpful advice. The English text was checked by Dr O. Parkes. LITERATURE CITED Andrews M, Scott WR, MacKenzie BA. 1991. Nitrate effects on preemergence growth and emergence percentage of wheat (Triticum aestium L.) from different sowing depths. Journal of Experimental Botany 42 : 1449–1454. Black JN. 1956. The influence of seed size and depth of sowing on preemergence and early vegetative growth of subterranean clover (Trifolium subterraneum L.). Australian Journal of Agricultural Research 7 : 98–109. Bouaziz A, Hicks DR. 1990. Consumption of wheat seed reserves during germination and early growth as affected by soil water potential. Plant and Soil 128 : 161–165. Cooper CS, MacDonald PW. 1970. Energetics of early seedling growth in corn (Zea mays L.). Crop Science 10 : 136–139. Dele! ens E, Gregory N, Bourdu R. 1984. Transition between seed reserve use and photosynthetic supply during development of maize seedlings. Plant Science Letters 37 : 35–39. Du$ rr C, Boiffin J. 1995. Sugarbeet (Beta ulgaris L.) seedling growth from germination to first leaf stage. Journal of Agricultural Science, Cambridge 124 : 427–435.
Durrant MJ, Mash SJ. 1989. Stimulation of sugarbeet hypocotyl extension with potassium nitrate. Annals of Applied Biology 115 : 367–374. Gate P. 1996. Ecophysiologie du bleU . Paris : ITCF Tecdoc Lavoisier. Hegarty TW. 1976. Effects of fertilizer on the seedling emergence of vegetable crops. Journal of Science of Food and Agriculture 27 : 962–968. Krigel I. 1967. The early requirement for plant nutrients by subterranean clover seedlings (Trifolium subterraneum). Australian Journal of Agricultural Research 18 : 879–886. MacWilliam JR, Clements RJ, Dowling PM. 1970. Some factors influencing the germination and early seedling development of pasture plants. Australian Journal of Agricultural Research 21 : 19–32. Maillard P, Dele! ens E, Daudet FA, Lacointe A, Frossard JS. 1994. Carbon and nitrogen partitioning in walnut seedlings during the acquisition of autotrophy through simultaneous "$CO and "&NO # $ long-term labelling. Journal of Experimental Botany 45 : 203–210. Pinto Contreras M, Gaudille' re JP. 1987. Efficacite! de la croissance du ble! lors du passage a' l’autotrophie. Plant Physiology and Biochemistry 25 : 35–42. Saglio P, Pradet A. 1980. Soluble sugars, respiration and energy charge during aging of excised maize root tips. Plant Physiology 66 : 516–519. Shanmuganathan V, Benjamin LR. 1992. The influence of sowing depth and seed size on seedling emergence time and relative growth rate in spring cabbage (Brassica oleracea var. capitata L.). Annals of Botany 69 : 273–276. Souty N, Rode C. 1993. Emergence of sugar beet seedlings from under different obstacles. European Journal of Agronomy 2 : 213–221. Tamet V, Du$ rr C, Boiffin J. 1994. Croissance des plantules de carotte de la germination jusqu’a' l’apparition des premie' res feuilles. Acta Horticulturae 354 : 17–25.
Effects of Nutrient Supply on Pre-emergence Growth and Nutrient Absorption in Wheat (Triticum aestivum L.) and Sugarbeet (Beta vulgaris L.) C A R O L Y N E D U$ R R and B. M A R Y INRA, UniteU d’Agronomie, rue Fernand Christ, 02007 Laon Cedex, France Received : 19 November 1997
Returned for revision : 16 January 1998
Accepted : 10 February 1988
Nutrient absorption in wheat and sugarbeet was studied during pre-emergence growth by adding 0, 7, 10±5 or 14 mol m−$ nitrogen (N) to the growth medium. Seedling growth and carbon, N and "&N contents of the seedling parts were measured. Differences between the natural abundance of "&N in seeds and in nutrient solution were used to determine the proportion of N in the organs originating from seed reserves and from absorption. Absorption began later for wheat than for sugarbeet and had less effect on seedling growth. The absorbed N was found mainly in roots. Compared to wheat, sugarbeet seedling N content was greatly altered and the hypocotyl showed increased elongation when nutrients were added. Most of the absorbed N was found in the radicle and hypocotyl with less in the cotyledons. Sugarbeet seedling emergence and early growth could be decreased by adverse conditions occurring after sowing by affecting mineral availability in the soil or through altered root absorption. # 1998 Annals of Botany Company Key words : Triticum aestium L., wheat, Beta ulgaris L., emergence, natural isotopic composition, seedling, seed reserves.
INTRODUCTION Seed reserve depletion and the transition to autotrophic growth have been studied for many species (Black, 1956 ; Cooper and MacDonald, 1970 ; Bouaziz and Hicks, 1990 ; Shanmuganathan and Benjamin, 1992 ; Maillard et al., 1994 ; Tamet, Durr and Boiffin, 1994 ; Durr and Boiffin, 1995). The relative carbon contributions of seed reserves and photosynthesis during early seedling growth have been studied using radiocarbon (Pinto Contreras and Gaudille' re, 1987) and changes in the natural abundance of the stable isotope "$C (Dele! ens, Gre! gory and Bourdu, 1984). The relative contributions of mineral seed reserves and nutrient uptake from the substrate by seedlings have been less studied. Various experiments have reported contradictory effects on seed emergence of fertilizer placement in the soil (MacWilliam, Clements and Dowling, 1970 ; Hegarty, 1976 ; Durrant and Mash, 1989 ; Andrews, Scott and MacKenzie, 1991). Thus, there is a need to separate the effects of added minerals on germination sensu stricto, defined as protrusion of the radicle from the seed, and pre-emergence growth, which is the stage after germination until the seedling reaches the soil surface, in order to better understand these contradictory effects. High concentrations of minerals in water have an adverse effect on germination rates during seed inhibition prior to radicle protrusion (MacWilliam et al., 1970 ; Durrant and Mash, 1989). This is due to the decrease in water potential around the seed when minerals are added (Hegarty, 1976). Less is known about the period after radicle protrusion when mineral absorption begins, and about the seedlings’ mineral requirements. Several studies have shown that absorption may begin early (Krigel, 1967, in subterranean 0305-7364}98}05066508 $25.00}0
clover ; MacWilliam et al., 1970, in pasture plants ; Maillard et al., 1995, in walnut seedlings) and that there is more hypocotyl growth in sugarbeet when potassium nitrate is added (Durrant and Mash, 1989). The present study was undertaken to determine the effects of nutrient supply on seedling growth during the pre-emergence growth of two species : sugarbeet (Beta ulgaris L.), a dicotyledonous, epigeal species with perispermic reserves which are completely transferred to the embryo before emergence (Du$ rr and Boiffin, 1995) and wheat (Triticum aestium L.), a monocotyledon with endosperm reserves that are used even after emergence (Pinto Contreras and Gaudille' re, 1987). Seedlings were grown with or without added nutrients. Natural nitrogen isotope composition was used to determine the relative contributions of the two nitrogen sources in the organs of the seedling.
MATERIALS AND METHODS Experiments were performed in a growth chamber in the dark to simulate pre-emergence growth. Pots sown with wheat were first kept at 5 °C for 3 d to break seed dormancy. They were then incubated at 20 °C with the pots sown with sugarbeet. The seeds were taken from a single seedlot (Beta ulgaris L. ‘ Vega ’, monogerm variety for sugarbeet and Triticum aestium L. ‘ Ventura ’ for wheat). One seed weight range, close to the mean seed weight of the seedlot, was selected for each species : 7±0–8±9 mg for sugar beet (mean seed weight without pericarp, i.e. embryoperisperm ¯ 2±4 mg) and 38±0–42±9 mg for wheat seeds. The initial fresh weight of each seed was recorded so that the initial seed weight of each harvested seedling was known. The seed
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DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings
water content (SWC, relative to the seed dry weight) was measured on samples of 100 seeds, making it possible to estimate the seed dry weight of each seed as follows : SDW ¯ SFW}(1SWC) with SWC ¯ 0±112 for sugarbeet and 0±114 for wheat. Seeds were sown at a depth of 2 cm in pots (7¬11 cm) filled with white sand (150–210 µm grain diameter ; bulk density 1±47 kg dm−$). This was done to anchor seeds and roots, but the pots were kept in the dark even after the seedlings were visible at the soil surface. This method was chosen to allow seedlings to grow as they would under natural conditions. Each treatment involved 40 pots with five seeds per pot. The gravimetric water content was increased to 0±2 kg kg−" before sowing by watering the sand with either deionized water or with nutrient solution (Saglio and Pradet, 1980). Before dilution, this solution contained 700 mol m−$ KNO , 750 mol m−$ NH NO , 650 mol m−$ $ % $ Ca(NO ) , 275 mol m−$ KH PO , 50 mol m−$ NaCl, 157 mol # % $ # m−$ MgSO , plus micronutrient supplements. The solution % was diluted in deionized water at a rate of 2, 3 or 4 ml l−", to obtain a N concentration of 7, 10±5 or 14 mol m−$ N—a concentration of 14 mol m−$ N being commonly used to grow adult plants. Treatments were designated as D0 for pure water, D2, D3 and D4 for the three dilutions. D4 was not used for sugarbeet. The soil water content used was determined by preliminary germination (radicle protrusion) tests with deionized water, using the same sowing technique and with maximum germination rate at a water content of 0±15–0±25 kg kg−". Germination rates were also measured for the various mineral supplies using 0±20 kg kg−" water content. Water content was kept constant throughout the experiments by adding deionized water. Atmospheric relative humidity was 80 %. Seedlings were harvested every day or half day for growth measurements. The first samples were taken 2 d after sowing for sugarbeet and 4 d after sowing for wheat. Samples of ten to 15 seeds per treatment (two pots for wheat and three for sugarbeet) were taken and measurements were made on germinated seeds only. The radicle, coleoptile containing folded leaves and seed residue were separated for wheat. The radicle, hypocotyl, cotyledons, seed residue and pericarp were separated for sugarbeet seedlings. The length of each organ was measured. Each part of the seedling was dried for 48 h at 70 °C and weighed separately. The carbon and total nitrogen contents of each part were measured, along with the "&N isotopic composition of their total N, using a mass spectrometer (VG SIRA 9) linked to an automatic combustion analyser (Carlo Erba NA 1500). A single sample consisted of the five seedlings from a sampled pot, and two or three replicates were analysed for each sampling date. Seed residues were also analysed for each sampling date and treatment for sugarbeet and for the D0 and D2 treatments for wheat. Carbon, total nitrogen and isotopic composition were determined on three replicates of five seeds of the chosen seed weight of each species. The "&N abundance in a material is defined as the number of "&N atoms over the number of ("&N"%N) atoms. Results of analyses are expressed as the "&N atom % excess, i.e. "&N abundance of the sample minus air "&N abundance, which is used as reference (abundance : 0±3663 %). The nutrient solution was not labelled and its "&N atom % excess was
0±0004 (mean of five replicates, s.d. ¯ 0±0003 %), whereas the atom % excess of the seed was about 0±0020 %. We attempted to exploit natural differences in the excess of seeds and the nutrient solution. Experiments ended when elongation of the emerging organ ceased (8 d for wheat coleoptiles and 12–16 d for sugarbeet hypocotyls). The cumulated thermal time from sowing (Td) was calculated as follows : i=d
Td ¯ 3 (θi®θb) i=" where Td is the time at sampling date d in degree-days (°Cd), θi the daily mean temperature in the growth chamber and θb the threshold value of 0 °C for wheat (Gate, 1996) and 3±5 °C for sugarbeet (Du$ rr and Boiffin, 1995).
RESULTS Germination and seedling growth There was no difference in germination (sensu stricto) rates in response to any treatment for either species. The 50 and 95 % germination rates were obtained at 35 and 45 °Cd for sugarbeet and 20 and 30 °Cd for wheat, respectively. Changes in dry mass of the wheat seedling and seed residue after germination were similar for all treatments until 110–120 °Cd (Fig. 1 A). Seedlings grew more slowly thereafter in D0 (P ! 0±05), with a smaller decrease in seed residue (P ! 0±01). This difference was due to a smaller increase in coleoptile and leaf dry mass in the D0 treatment (P ! 0±01) ; the root dry mass was not affected (Fig. 1 B and C). Leaves broke through the coleoptile at 110–120 °Cd in all treatments and there was less coleoptile elongation after this point for treatment D0 only (P ! 0±05, Fig. 1 D). There were no differences in seed residue dry matter between any of the sugarbeet treatments (Fig. 1 A). Seedling dry mass was similar for treatments D0, D2 and D3 until 75 °Cd and then markedly smaller in D0 than in D2 and D3 (P ! 0±05 after 100 °Cd). Radicle dry mass was slightly higher in D0 than in other treatments after about 60 °Cd (P ! 0±05 after 100 °Cd, Fig. 1 B). Hypocotyl mass and length varied with treatment becoming smaller in D0 after 75 °Cd and after 100 °Cd in D2 (P ! 0±05, Fig. 1 C and D). Cotyledon masses also differed after 100 °Cd, but this was not related to treatment (not shown).
Carbon partitioning and net balance Changes in the amount of carbon in the seed residue and seedling were similar in wheat for all treatments until 110 °Cd, with a constant pattern of C partitioning between shoot and roots (Fig. 2). Thereafter, there was a smaller increase in seedling carbon mass in D0 than in other treatments with a smaller decrease in the amount of carbon in the seed residue and less C in the shoot. Amounts of carbon in roots did not differ between treatments. Changes in the amounts of carbon in the seed residue and seedling were similar for all sugarbeet treatments (Fig. 2 A).
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F. 1. Changes in dry mass and length of wheat and sugarbeet seedlings with thermal time in the dark. Seeds}seedlings were watered with nutrient solutions containing 0 (D0 ; D), 7 (D2 ; E), 10±5 (D3 ; +) or 14 mol m−$ N (D4 ; _). Vertical bars indicate s.e. on the last sampling date (n ¯ 2 for wheat, n ¯ 3 for sugarbeet). °Cd, Degree days ; b, threshold value for thermal time calculation.
The C partitioning pattern between organs was similar for all treatments, except that there was slightly more carbon in the radicle in D0 (Fig. 2 B–D). The pattern of net carbon balance (i.e. the amount of carbon in the seedling and seed residue compared to the amount of carbon in the initial seed) was similar between treatments for both species (Fig. 3). Twenty per cent of the
total C was lost at the end of the experiment in the case of wheat, 50 % in the case of sugarbeet. Nitrogen net balance and absorption The net nitrogen balance was about 100 % in D0 throughout the experiment for wheat and sugarbeet (Fig. 3).
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F. 2. Amount of carbon in wheat and sugarbeet during D0 (D), D2 (E), D3 (+) and D4 (_) treatments (see Fig. 1 for details). Vertical bars indicate s.e. on the last sampling date (n ¯ 2 for wheat, n ¯ 3 for sugarbeet). °Cd, Degree days ; b, threshold value for thermal time calculation.
The nitrogen balance of D0 did not differ from that of D2 until about 100 °Cd for wheat. Thereafter, the net balance increased and reached a maximum of 125 % at 120 °Cd. Differences began at about 60 °Cd for sugarbeet : the net balance increased and reached a maximum of 150 % at 100–110 °Cd for both the D2 and D3 treatments.
The amounts of N in the coleoptile and radicle of wheat were markedly smaller after 100 °Cd in D0 compared to the other treatments (Fig. 4). For sugarbeet, differences in the amounts of N between D0 and the other treatments developed very rapidly in the hypocotyl and radicle. The difference was generally smaller in the cotyledons (Fig. 4).
DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings
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F. 3. Carbon and nitrogen net balances for wheat and sugarbeet during D0 (C D, N *), D2 (C E, N +) and D3 (C y, N _) treatments (see Fig. 1 for details). °Cd, Degree days ; b, threshold value for thermal time calculation.
Initially, the "&N atom % excess in the various parts of the seedling was similar (Fig. 5 A–D). When no nutrient solution was added (D0), the "&N atom % excess of the seedlings remained constant at about 0±0020 % for both species. The "&N composition of wheat seedling in D2, D3 and D4 was similar to D0 until 110–120 °Cd (Fig. 5). Thereafter, "&N atom % excess in D2, D3 and D4 decreased compared to D0 due to changes in the "&N atom % excess in roots, particularly in D3 and D4, indicating N absorption, while concentrations did not differ in shoots (Fig. 5 B and C). There were differences in the isotopic composition of sugarbeet seedlings from the first sampling date (Fig. 5 A). "&N atom % excess was similar in D2 and D3 and was lower than that in D0 after 30 °Cd (P ! 0±05). It reached the same minimum value, just over 0 % excess, at about 100 °Cd. Changes in composition differed with seedling part (Fig. 5 B–D). Zero values were reached at 60 °Cd for the radicle. Negative values, very close to that of the nutrient solution, were reached at 75 °Cd for the hypocotyl. "&N atom % excess remained higher in the cotyledons. Values were similar to those of D0 until 60–75 °Cd and then decreased, but remained significantly higher than those in the radicle and hypocotyl. DISCUSSION The concentration of minerals used had no effect on germination (radicle protrusion) rates for either species. Seed reserve depletion was unchanged for a long time with differences occurring only for wheat once the leaves had broken through the coleoptile ; carbon net balances were unaffected. Differences in C partitioning were very small with slightly more C in the root when no nutrient was added. Other studies have similarly shown that seed reserve depletion is unaffected by various growth conditions (light or dark, Cooper and MacDonald, 1970 ; water potential, Bouaziz and Hicks, 1990 ; or low temperature, Tamet et al., 1994). Total seedling dry mass, the dry mass of each part and the elongation of the coleoptile were unchanged for wheat until the leaves broke through the coleoptile at about 110– 120 °Cd. Thereafter, the coleoptile and leaves grew more when nutrient solution was added, with more C being
depleted from the seed reserves and going to the shoot. The net nitrogen balance exceeded 100 % thereafter and the "&N atom % excess of roots decreased. Absorption began at this time and the N absorbed mostly accumulated in the roots. Andrews et al. (1991) also reported that addition of NO − $ did not affect root and shoot dry weights in wheat, but that fresh weights and nitrate reductase activity were increased. This effect was particularly evident at the time of first leaf expansion. The final lengths of coleoptiles were rather short compared to those of sugarbeet hypocotyls (Fig. 1 D). One reason may be that the mesocotyl never elongated in wheat seedlings in these experiments. Wheat is generally sown at about 2–3 cm under field conditions. The coleoptile reaches this length at about 100–110 °Cd (Fig. 1 D). Emergence should already have taken place under common field conditions when absorption begins. The coleoptile was broken through by leaves at about 100–110 °Cd in all treatments. Branching roots and new roots appeared at this time and absorption started. If emergence is delayed by deep sowing or soil crusting, the coleoptile will be longer when absorbed minerals are supporting growth and this could help the seedling to reach the soil surface. However, the coleoptile loses much of its rigidity and strength when broken by leaves. Thus, the final emergence rate would not be much improved. Changes in seedling growth and absorption started earlier in sugarbeet seedlings. There was more hypocotyl growth and less radicle growth when nutrients were supplied. The net nitrogen balance exceeded 100 % after 50 °Cd, but the "&N composition of the radicle was affected as soon as it protruded at 35–40 °Cd, showing that absorption had begun. "&N atom % excess in the radicle decreased to a level very close to that of the added solution showing that almost all of the N in the radicle came from the added solution. Similar results were obtained in the hypocotyl. The decrease in "&N atom % excess occurred later in cotyledons and their "&N composition was consistently higher. Less added nitrogen was translocated to these organs, but about 50 % of the N was taken up at the end of the experiment from the added N. The radicle and the hypocotyl elongated most rapidly. The cotyledons remained small until emergence. Carbon from the seed reserves and cotyledons (Du$ rr and
DuX rr and Mary—Nutrient Absorption by Sugarbeet and Wheat Seedlings
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F. 4. Amount of nitrogen in wheat and sugarbeet during D0 (D), D2 (E), D3 (+) and D4 (_) treatments (see Fig. 1 for details). Vertical bars indicate s.e. on the last sampling date (n ¯ 2 for wheat, n ¯ 3 for sugarbeet). °Cd, Degree days ; b, threshold value for thermal time calculation.
Boiffin, 1995) and absorbed N were mostly translocated to the hypocotyl maximizing the elongation rate. Seedling emergence forces are also higher when seedlings are grown with nutrients (Souty and Rode, 1993 ; unpub. res.). The N content in the cotyledon at the start of photosynthesis (the cotyledons alone photosynthesize until the first leaves appear) will be higher if N is present in the nutrient solution. Crop emergence and early growth could both be altered if
the mineral supply is reduced, due to lower soil availability or local concentration, or to poor root absorption. Wheat and sugarbeet seedlings both absorb minerals from the growth medium during their C heterotrophic growth but absorption begins later for wheat, the timing being related to emergence time in common field conditions. These two species differ in the size of their seed reserves and depletion patterns. This may be related to the time at which
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F. 5. Wheat and sugarbeet "&N atom % excess in the whole seedling and in the seedling parts for D0 (D), D2 (E), D3 (+) and D4 (_) treatments (see Fig. 1 for details). Vertical bars indicate the mean s.e. per sampling date (n ¯ 2 for wheat, n ¯ 3 for sugarbeet). °Cd, Degree days ; b, threshold value for thermal time calculation.
absorption begins. Absorption begins earlier in dicotyledonous pasture seedlings, even in legume seedlings, than in grasses (MacWilliam et al., 1970). These results help us to better understand the effects of certain seed treatments or fertilizer placement. Fertilizer placement may increase mineral concentrations in soil surrounding seedling roots and improve early absorption. However, more detailed
observations on such treatments are required, particularly in field conditions. In conclusion, the time at which absorption began was identified by comparing seedlings grown with and without added nitrogen. Differences in the "&N isotopic composition of the nutrient solution and seeds made it possible to determine the time at which absorption began and where the
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absorbed nitrogen went. Therefore this simple method not only allowed us to quantify the N absorption rate but also to chart the fate of added N in the plant. A C K N O W L E D G E M E N TS We thank O. Delfosse, C. Dominiarzick and C. Leforestier for technical assistance and F. Devienne and G. Richard for their helpful advice. The English text was checked by Dr O. Parkes. LITERATURE CITED Andrews M, Scott WR, MacKenzie BA. 1991. Nitrate effects on preemergence growth and emergence percentage of wheat (Triticum aestium L.) from different sowing depths. Journal of Experimental Botany 42 : 1449–1454. Black JN. 1956. The influence of seed size and depth of sowing on preemergence and early vegetative growth of subterranean clover (Trifolium subterraneum L.). Australian Journal of Agricultural Research 7 : 98–109. Bouaziz A, Hicks DR. 1990. Consumption of wheat seed reserves during germination and early growth as affected by soil water potential. Plant and Soil 128 : 161–165. Cooper CS, MacDonald PW. 1970. Energetics of early seedling growth in corn (Zea mays L.). Crop Science 10 : 136–139. Dele! ens E, Gregory N, Bourdu R. 1984. Transition between seed reserve use and photosynthetic supply during development of maize seedlings. Plant Science Letters 37 : 35–39. Du$ rr C, Boiffin J. 1995. Sugarbeet (Beta ulgaris L.) seedling growth from germination to first leaf stage. Journal of Agricultural Science, Cambridge 124 : 427–435.
Durrant MJ, Mash SJ. 1989. Stimulation of sugarbeet hypocotyl extension with potassium nitrate. Annals of Applied Biology 115 : 367–374. Gate P. 1996. Ecophysiologie du bleU . Paris : ITCF Tecdoc Lavoisier. Hegarty TW. 1976. Effects of fertilizer on the seedling emergence of vegetable crops. Journal of Science of Food and Agriculture 27 : 962–968. Krigel I. 1967. The early requirement for plant nutrients by subterranean clover seedlings (Trifolium subterraneum). Australian Journal of Agricultural Research 18 : 879–886. MacWilliam JR, Clements RJ, Dowling PM. 1970. Some factors influencing the germination and early seedling development of pasture plants. Australian Journal of Agricultural Research 21 : 19–32. Maillard P, Dele! ens E, Daudet FA, Lacointe A, Frossard JS. 1994. Carbon and nitrogen partitioning in walnut seedlings during the acquisition of autotrophy through simultaneous "$CO and "&NO # $ long-term labelling. Journal of Experimental Botany 45 : 203–210. Pinto Contreras M, Gaudille' re JP. 1987. Efficacite! de la croissance du ble! lors du passage a' l’autotrophie. Plant Physiology and Biochemistry 25 : 35–42. Saglio P, Pradet A. 1980. Soluble sugars, respiration and energy charge during aging of excised maize root tips. Plant Physiology 66 : 516–519. Shanmuganathan V, Benjamin LR. 1992. The influence of sowing depth and seed size on seedling emergence time and relative growth rate in spring cabbage (Brassica oleracea var. capitata L.). Annals of Botany 69 : 273–276. Souty N, Rode C. 1993. Emergence of sugar beet seedlings from under different obstacles. European Journal of Agronomy 2 : 213–221. Tamet V, Du$ rr C, Boiffin J. 1994. Croissance des plantules de carotte de la germination jusqu’a' l’apparition des premie' res feuilles. Acta Horticulturae 354 : 17–25.