Green cotyledons of two Hakea species control seedling mass and morphology by supplying mineral nutrients rather than organic compounds


  • Byron B. Lamont,

    Corresponding author
    1. Department of Environmental Biology, Curtin University of Technology, PO Box U1987, Perth, WA 6845, Australia;
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  • Philip K. Groom

    1. Department of Environmental Biology, Curtin University of Technology, PO Box U1987, Perth, WA 6845, Australia;
    2. Current address: School of Natural Sciences, Edith Cowan University, Joondalup, Australia
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Author for correspondence: Byron B. Lamont Tel: +61 89266 7368 Fax: +61 89266 2495 Email:


  • • Hakea species dominate shrubby vegetation on seasonally dry, nutrient-impoverished soils in Australia. We hypothesized that the exposed cotyledons control growth of the young seedling by providing it with mineral nutrients rather than C-based compounds.
  • • Two representative species with differing seed masses were tested. Cotyledon removal caused large reductions in plant mass and dimensions, but little effect on specific leaf area. Cotyledons expanded markedly on germination although their chlorophyll content was much lower than the first leaves. Shading the cotyledons or applying a C-storage compound (inositol) to the soil had no consistent effect on seedling properties.
  • • Cotyledon removal greatly reduced seedling P (especially), N, K, Ca, Mg and Cu, but not Fe or Mn, whereas shading the cotyledons had no effect. Transfer of mineral nutrients from the embryos to the seedlings in their natural soils varied from 90 to 2% (for P and Ca, respectively), and accounted for 79% of total content (P) to negligible (K, Ca, Fe) at 3 months. Cotyledons indirectly increased soil nutrient uptake. Addition of P, N or P + N after cotyledon removal had no benefit but addition of P + N + K + Mg + S restored morphology and nutrient content.
  • • In nutrient and water-limited habitats with abundant light, cotyledons in Hakea spp. might serve to maximize supply of mineral nutrients to the shoots, which then maximize C-supply to the rapidly elongating roots.


Seeds contain both carbon (C) and mineral nutrient reserves (Ashcroft & Murray, 1979; Fenner, 1983; Zhang & Maun, 1991) but there is little direct evidence of how much is exported to the developing seedling and little is known about the relative importance of this on seedling morphology. For example, by comparing growth in a nutrient solution lacking N, and in distilled water, Hanley & Fenner (1997) showed that N content of the seed was largely responsible for the root : shoot ratio (R : S) values in seedlings of three species. By increasing leaf area, stem length or root growth these reserves could have indirect effects on further increasing the plant’s C, water and nutrient supply. For species with green cotyledons, many of which expand greatly after germination (Milberg & Lamont, 1997), there is the potential for C fixed by the cotyledons to be transported to the seedling as well (Marshall & Kozlowski, 1974). For species with large, thick cotyledons, their photosynthesis may be negligible or only enough to balance respiration (Lovell & Moore, 1971; Kitajima, 1992). In other species, photosynthetic activity of the cotyledons provides the mechanism by which nutrients are moved between the cotyledons and seedling (Marshall & Kozlowski, 1975).

Stock et al. (1990) showed that seed-stored N and P made a major contribution to their presence in seedlings of five species from nutrient-impoverished soils, but not K, Ca or Mg which they argued were obtained from the soil after fire when germination was most likely. Seed-stored K but not N or P met the growth requirements of three of four species establishing after fire in Greece (Hanley & Fenner, 1997). In sand, variation in seedling mass was most sensitive to the extent of P export from the cotyledons of 21 species, N was intermediate and K was least of the three (Milberg et al., 1998). There is a strong negative interaction on growth between soil levels of the limiting nutrients and seed-stored levels (Zhang et al., 1990; Milberg et al., 1998). Among four species, the impact of cotyledon removal was least in the species with lowest nutrient storage in the more fertile soil, while it was greatest for the species with greatest nutrient storage, which was otherwise unaffected by soil type (Milberg & Lamont, 1997). In the species with smallest cotyledons, nutrient levels in the cotyledons actually increased over time, while for all species there was a tendency for cotyledon nutrient reserves to contribute less to seedling content in the more fertile soil.

We studied the impact of cotyledon sources of C and eight mineral nutrients on seedling morphology and nutrition of two species, with mean seed masses of 20 (H. lasianthoides) and 67 (H. psilorrhyncha) mg, in the genus Hakea (Proteaceae). These seed sizes were considered intermediate in the genus where 50 species have a range of 3–156 mg (Groom & Lamont, 1996). With 100 taxa in the mediterranean south-western corner of Australia, of the total of 160 species, this genus of sclerophyllous shrubs is well-represented on the most nutrient-impoverished sands and laterites typical of this region (Lamont, 1995). H. lasianthoides is a large shrub in eucalypt woodland on lateritized quartzite over clay where its seeds are released after fire in summer. These germinate that winter and seedlings compete for establishment with cogerminating species, with death of most seedlings, especially competitors for recruitment sites, during the ensuing summer droughts (Lamont et al., 1999; Groom et al., 2001). H. psilorrhyncha is an emergent shrub in scrub-heath on acid sand dunes and similarly competes for resources, especially water, during establishment (Lamont et al., 1993). First-year seedlings of large-seeded species show least summer drought stress and survive preferentially in this environment (Lamont et al., 1993, 1999; Richards & Lamont, 1996).

Seed P concentrations are particularly high in hakeas where it is stored as phytin which releases P and inositol (C6H12O6) on germination (Mitchell & Allsopp, 1984) where inositol could act as an energy source (Mayer & Poljakoff-Mayber, 1975). Initially protected by the testa, the cotyledons are carried above the soil (epigeous) where they spread out (phanerocotylous), enlarge and become green (Lamont & Milberg, 1997; Milberg & Lamont, 1997). In this nutrient and water-limited but high light environment it has been argued that such seeds are an essential source of nutrients, especially P and N, rather than of C, for shoot growth and thence root elongation to reach groundwater as a drought-avoiding strategy over the first summer (Milberg & Lamont, 1997; Milberg et al., 1998). For a 15-cm tall seedling, the taproot may reach a depth of 2 m of more in the first growing season (Enright & Lamont, 1992). The best way to ensure initial survival (water-limited) is to maximize photosynthesis of the shoots (nutrient-limited) to support root growth (C-limited). Stored or photosynthetic sources of C-based compounds from the cotyledons would appear to be quite inadequate for this task.

We therefore hypothesized that:

  • Removal of the cotyledons would have a marked effect on seedling growth and morphology (shoot, leaf and root mass, leaf and stem dimensions, S : R, specific leaf area);
  • Stored or photosynthetic sources of C-based compounds from the cotyledons would make a negligible contribution to seedling development;
  • The role of the cotyledons in seedling development could be replaced by the addition of mineral (rather than organic) nutrients to the soil;
  • Seed nutrient reserves would make a variable direct and indirect contribution to nutrition of the seedling, with N and P supplied essentially from the cotyledons and other nutrients essentially from the soil.

Materials and Methods

Experiment 1

Two species were selected from locations where Hakea species are abundant, one from rocky substrates and the other from deep sand. Fruits of H. lasianthoides B.L. Rye were collected from Walyunga National Park (31°45′ S, 116°5′ E), 30 km NE of Perth, Australia (Groom et al., 2001) along with the A1 horizon soil (depth 150 mm) in which the parent plants were growing – hereafter called loam (Table 1). Fruits of H. psilorrhyncha R.M. Barker were collected from Eneabba (29°53′ S, 115°17′ E), 280 km N of Perth, Australia (Enright & Lamont, 1992), as well as adjacent soil, 5 months after a spring fire – hereafter called sand (Table 1). This experiment was designed to compare the role of cotyledons and externally available nutrients on seedling morphology and N content and whether their roles were interchangeable.

Table 1.  Attributes of experimental soils (concentrations in µg ml−1 soil)
AttributeEneabba sandWalyunga loam
  1. Based on methods and results in Lamont (1995).

Total N315742
Total P 36193
Total K 76932
Available N  3.6  4.9
Available P  1.0  0.3
Available K 15 16
Available Ca414 77
Available Mg 44 53
Interaction effectsNPK (positive)NP (positive)

After harvest, the woody fruits were air-dried to release their seeds, and 20 were weighed individually to obtain their air-dry mass. Seeds were germinated on moist paper towelling at 15°C and 1-wk-old germinants were transplanted to course aquarium sand (Lonestar®, Lapis Lustre, CA, USA). The soil was held in 150 × 25 mm plastic tubes (Cone-tainers®, Portland, OR, USA) suspended on racks in an unshaded and nonair-conditioned glasshouse (Stanford University, CA, USA) during September–November. Mean minimum weekly temperature at the soil surface was 12.8°C and maximum was 29.3°C. Mean PAR was 766 µmol m−2 s−1 on a clear day (recorded at 2 h intervals with a Li-Cor quantum sensor, LI-185B, Li-Cor, Lincoln, NE, USA) and 311 µmol m−2 s−1 on an overcast day. Until treatments were begun, seedlings were given distilled water.

Once the emerged cotyledons had completed their lateral spread after a further week, the treatments were begun (earlier removal of cotyledons caused death in H. psilorrhyncha; Milberg & Lamont, 1997). Both cotyledons were sliced off with a razorblade at the nodes from 16 random seedlings (−) and 16 were left intact (+). Treatments were maintained for 12 wk when almost half the cotyledons had dehisced. Half within each batch were flushed daily with a balanced nutrient solution (Peters® 24(% N)−8(% P2O5)−16(% K2O) fertilizer, Grace and Co., Fogelsville, PA, USA), at the 100 ppm N recommended level plus 2 mg l−1 CaCl2·2H2O (solution M) while the other treatments continued to receive distilled water only. Projected areas of 10 embryos (essentially the cotyledons) before emergence from the testas, oven dry mass and projected areas of 10 harvested cotyledons at the start of the experiment (initial) and at the end (final), and total chlorophyll (a + b) content at the start were determined on three cotyledons as described in Milberg & Lamont (1997). At harvest, root systems were washed free of sand and rinsed in distilled water. As the entire root system produced during the experiment was still alive at the time of harvest there was no need to take possible root turnover into account in these figures. Plants were divided into shoots and roots and dried at 70°C for 2 d and weighed separately. Stem length to the cotyledon nodes was measured and the number of mature leaves counted. Ratios were graphed as their two components so that the causes of any differences could be identified. A line corresponding to the ratio was drawn through the control (+) so that other ratios could be compared visually. As we were specifically interested in whether the −M treatment was able to substitute for + compared with the other two combinations, we used the nonparametric Kruskal–Wallis multiple range test on the four treatments at the P < 0.05 level of significance (Zar, 1996).

The 1–2 uppermost mature leaves produced per plant during the experiment had their areas and dry weights taken. The means were used for estimating specific leaf area (SLA = projected area/mass) per plant, taking into account the cylindrical shape of the leaves of H. psilorrhyncha by multiplying this expression by 0.7854 (Witkowski & Lamont, 1991). Five replicates of final shoots and roots per treatment and species were also analysed for total N by standard Kjeldahl digestion and titration.

Experiment 2

In this experiment, the two species were used to determine how the cotyledons control seedling morphology and mineral nutrition. Germinants were placed in 170 × 40 mm tubes as before but this time in their natural soil (see Experiment 1 and Table 1) so that the relative importance of these could be compared with contribution of the cotyledons. Once the first pair of leaves had appeared both cotyledons were sliced off six random batches of 5–6 seedlings. Some were used for surface area (6) and chlorophyll content (3) determination, while the rest were oven-dried and kept for later nutrient analysis. One batch was designated as the minus cotyledon control (−) and received only distilled water, another received 5 mmol l−1 NH4NO3 twice a week and distilled water at other times (−N), another received 2 mmol l−1 NaH2PO4 (−P), another a combination of these (−PN), the next was the same combination plus 6 mmol l−1 KCl and 2 mmol l−1 MgSO4 (−M), and the last, 6 mmol l−1 myo-inositol (C6H12O6, soluble breakdown product of phytin in seeds; Mayer & Poljakoff-Mayber, 1975) (−S). These concentrations were based on the Long Ashton formula for nutrient solutions (Hewitt & Smith, 1975), except P was 20% the recommended level, while inositol was set at a level comparable with the major nutrients. As P toxicity symptoms in the −P, −PN and −M treatments for H. psilorrhyncha were evident by 9 wk, nutrient applications were reduced to once per week throughout. This experiment was undertaken from mid-November to the first week of February with mean temperatures of 9.4°C and 25.6°C and midday light at 150–500 µmol m−2 s−1.

Cotyledons were left on the next five batches of 5–6 seedlings. One batch was designated as the plus cotyledon control (+), another received the five minerals as before (+M), another the five minerals plus inositol as above (+MS), another inositol only (+S), and the last had the cotyledons shaded with an aluminium cap fitted over the mouth of the tube and around the stem of the seedling (+D). A quantum sensor under three caps on tubes in the glasshouse gave readings of 12–17 µmol m−2 s−1 PAR light vs uncovered of 700–810 µmol m−2 s−1. Surface soil temperatures measured with a thermocouple gave 23.1–23.7°C under five caps, and 23.1–23.9°C uncovered (NS by t-test). After 12 wk, the experiment was harvested, when about half the cotyledons had fallen off.

The same attributes were assessed as in the previous experiment. Part of the youngest mature leaf of three replicates of the + and +M treatments was used for chlorophyll analysis and converted to a wholeleaf basis using dry mass equivalents, and the projected surface areas of these leaves were taken as well. Leaf length, central width and thickness (veins did not protrude) of mature leaves initiated since the start of the experiment (3–6) were measured with calipers. Leaf density was calculated from (thickness.LSA)−1 (Witkowski & Lamont, 1991). All results were graphed in pairs of related attributes and ratios compared visually by drawing the ratio line through +0. All 11 treatments, including ratios, were compared by Tukey’s HSD multiple range test at the P < 0.05 level of significance (Zar, 1996).

Five whole plants (excluding cotyledons) of each of treatments +, +D, −, −PN, −M and +M were analysed separately for their nutrient content, as well as bulked seeds (minus testas), and initial and final cotyledons. N was determined colorimetrically with indophenol blue after standard Kjeldahl digestion using a Technicon autoanalyser at the WA Chemical Laboratories, East Perth. P, K, Ca, Mg, Cu, Fe and Mn were determined by inductively coupled plasma atomic emission spectroscopy of the same digest. As results were obtained on a concentration basis they were multiplied by mass per individual to give total nutrient content per plant, seed or cotyledon. All six treatments were compared by Tukey’s HSD multiple range test at the P < 0.05 level of significance.

Amount of nutrient transferred from cotyledons to the seedling was calculated by subtracting final cotyledon content from seed (embryo) content. (Means were used throughout for these exercises.) This amount was subtracted from total nutrient content of the + plant to give amount transferred from the soil. Nutrient content of the − seedling was subtracted from the amount transferred from the soil of the + seedling to obtain the additional amount of nutrient obtained from the soil when cotyledons were present. The amount already transferred from the cotyledons to the seedling at the start of the experiment was estimated from seed content minus initial cotyledon content. This was subtracted from the − plant to give the amount obtained from the soil in the absence of cotyledons. Percentage transfer of nutrient from the embryo to the seedling was given by (embryo – final cotyledon)/embryo. Contribution of the embryo to + seedling content was estimated as (embryo – final cotyledon)/seedling. Indirect contribution of cotyledons to soil uptake was given as [(+ seedling) – (− seedling)]/[(+ seedling) – (embryo – final cotyledon)]. Direct and indirect contribution of the embryo to total seedling content was calculated from {(embryo – final cotyledon) + [(+ seedling) – (− seedling)]}/(+ seedling).


Experiment 1

Table 2 shows that cotyledon area of H. Iasianthoides increased by 6.8 times after discarding the testa, while H. psilorrhyncha increased by 5.5 times. Cotyledon mass of H. Iasianthoides increased by 67% during the experiment, but that of H. psilorrhyncha did not change. While initial cotyledon mass of H. psilorrhyncha was three times that of H. Iasianthoides, its chlorophyll content was double (Table 2).

Table 2.  Seed and cotyledon attributes (mean ± SD) of two Hakea species
Attribute H. lasianthoidesH. psilorrhyncha
  1. n = 10, except for seed mass, n= 20, and chlorophyll, n= 3. Initial, 2 wk after germination; final, at harvest (12 wk after germination).

Air-dry seed mass (mg) 20.1 ± 3.5 67.1 ± 1.9
Oven-dry total cotyledon mass (mg)– initial13.1 ± 1.4 39.6 ± 3.0
 – final21.9 ± 1.9 38.3 ± 6.8
Area per cotyledon (mm2)– at germination11.6 ± 2.7 31.5 ± 6.3
 – initial91.0 ± 15.0172.6 ± 30.0
SLA of cotyledons (m2 kg−1)– initial4.154.36
Cotyledon chlorophyll content (µg) – initial72.0 ± 8.3156.5 ± 15.8

Figure 1a shows for H. Iasianthoides that added nutrients (M) were almost able to replace the removed cotyledons (−) in controlling root and shoot mass and their ratio (+ and −M were NS), with a disproportionate increase in shoot mass for +M only. For H. psilorrhyncha, added nutrients increased values of these three attributes for – but made no difference to +. The – treatment had little N content, while the differences between + and −M treatments were NS (Fig. 1b). N concentrated in the shoots of the +M treatment. Leaf production and stem length showed a similar pattern as N distribution (Fig. 1c). The + and −M treatment differences were NS, with longest internodes for +M and shortest for −. Leaf size more than halved when the cotyledons were removed from both species but there was no effect on SLA (Fig. 1d). Added nutrients increased leaf area more than leaf mass, so that SLA increased. The + and −M treatment differences were NS for H. Iasianthoides.

Figure 1.

Morphological attributes (mean ± SE) for 12-wk-old seedlings of two Hakea species for four treatments in sand culture: open circles, cotyledons removed; no added nutrients (−); open squares, cotyledons intact, no added nutrients (+); closed circles, cotyledons removed, added nutrients (−M), closed squares, cotyledons intact, added nutrients (+M). Lines are the ratios between each pair of attributes for controls (+): any point lying on the line has the same ratio. (a) Shoot vs root mass (line, equal R : S). (b) Shoot vs root N (line, equal R : S on N basis). (c) Number of leaves vs stem length (line, equal internode length). (d) Leaf mass vs area (line, equal SLA).

Experiment 2

Figure 2a shows there were no significant changes in cotyledon area between the start and end of this experiment. Area of representative leaves was five times greater than the cotyledons in control H. lasianthoides but only half that of cotyledons in H. psilorrhyncha. Total chlorophyll content tended to fall with time although less so in the full nutrient-amended treatment. Chlorophyll content was two orders of magnitude higher in the leaves, with the nutrient-treated plants 150% (H. lasianthoides) and over 200% (H. psilorrhyncha) higher than the controls. For H. lasianthoides, cotyledon removal almost halved root and shoot mass but R : S was unaffected (Fig. 2b). Addition of N, P and inositol (S) made no difference to the – values but PN and a combination of major nutrients (M) did. Adding M to the controls (+M) did not change the values, neither did covering the cotyledons to stop photosynthesis (+D). The highest three R : S values, although NS, were produced by the S treatments, due to −MS and +S having the highest root masses (also NS). For H. psilorrhyncha, removal of the cotyledons reduced shoot mass to 40% of the controls which was cancelled by addition of M but not by N, P or PN. Addition of S had no effect. D caused a 28% reduction in shoot mass, while +M caused a 164% increase. The patterns for roots were similar, although the 28% reduction in D was NS, while the +M and +S were NS. R : S for – was 1.4–2.1 times the other treatments.

Figure 2.

Cotyledon and seedling morphology (mean ± SE) for 12-wk-old seedlings of two Hakea species after various treatments in their natural soils: −, cotyledons removed; +, cotyledons intact; L, leaf value; I, initial value; F, final value; D, containers capped so that cotyledons in dark; S, inositol (soluble C source) added to soil; N, NH4NO3 added; P, NaH2PO4 added; PN, P + N added; M, P + N + K + Mg + S added; MS, M + S added. Lines are the ratios between each pair of attributes for controls (+): any point lying on the line has the same ratio. (a) Cotyledon and leaf chlorophyll content vs surface area (line, equal chlorophyll concentration). (b) Shoot vs root mass (line, equal R : S). (c) Number of leaves vs stem length (line, equal internode length). (d) Leaf length vs width (line, equal leaf shape). (e) Leaf mass vs area (line, equal SLA). (f) Leaf density vs thickness (line, equal SLA).

Leaf production and stem length were reduced by 35–40% after cotyledon removal in H. lasianthoides but internode length was unaffected (Fig. 2c). P, PN and M cancelled the negative effect on both. D and S had no effect while +M had the highest values although NS. Leaf production fell 28% and stem length 45% in H. psilorrhyncha with a reduction in internode length. Only the M treatments cancelled or overcompensated for these falls. D and S had no effect. −M and +M had significantly higher values than the other treatments, including longer internodes for +M. Fig. 2d shows a negligible reduction in leaf dimensions and shape with cotyledon removal for H. lasianthoides. All but −N and −S increased leaf size. There was a 60% reduction in leaf width and 40% in length with a shape change following cotyledon removal in H. psilorrhyncha. Addition of S and P restored leaf width but only N and M restored length as well. +D and −M had relatively narrow leaves, like −.

Cotyledon removal caused no significant reduction in leaf area, mass or SLA in H. lasianthoides (Fig. 2e). M treatments had larger leaves than the – treatments while +M had a 30% higher SLA than the others. Cotyledon removal caused a 74% fall in leaf area, a 57% fall in mass and a 40% reduction in SLA in H. psilorrhyncha. Only −M restored these three attributes while S had no effect. +M had the largest leaves without a significant reduction in SLA. Leaves of H. lasianthoides were insignificantly thinner and denser after cotyledon removal (Fig. 2f). Only −P and −S were significantly denser than +M and thinner than +. Leaf thickness fell by 58% and density increased by four times after cotyledon removal in H. psilorrhyncha. All but P + N restored these to + levels. +D had thinner, denser leaves than +.

Table 3 shows a marked reduction in mass and all major nutrients, but not always among the minor nutrients, following cotyledon removal from both species. Covering the cotyledons had no effect on biomass or nutrient content. Addition of P + N and P + N + K + Mg + S (M) restored biomass and nutrient content in H. lasianthoides (except Mg for P + N). N and P made no difference to biomass or content of most nutrients in H. psilorrhyncha but N was raised to + levels, whereas P was five times higher than + and Mn was almost half −. M overcompensated following cotyledon removal for N, P and K content but not biomass, Ca, Fe or Mn in this species, while Cu fell. There was no difference in biomass or nutrient content between −M and +M for H. lasianthoides. Biomass increased by 36% but nutrient content was maintained between −M and +M for H. psilorrhyncha.

Table 3.  Total plant mass and content of eight nutrients for two Hakea species grown in their natural soils
 Mass (mg)N (mg)P (mg)K (mg)Ca (mg)Mg (mg)Cu (µg)Fe (mg)Mn (µg)
  1. Treatments: +, cotyledons retained; −, cotyledons removed; D, cotyledons shielded from light; PN, P and N added to irrigation water; M, all major nutrients, except Ca, added. Results are mean for five replicates with different letters indicating differences at P < 0.05 by Tukey’s HSD multiple comparison test. Concn. = Concentration: total nutrient content/plant mass.

H. lasianthoides
+ 451.0a 5.95b0.19a 2.62bc2.74a0.90a 4.62a1.12a 55.23ab
+D 457.6a 6.14b0.24a 2.75bc2.70a0.94a 4.17a0.90ab 54.32ab
 275.2b 3.55c0.09b 1.48d1.87b0.54b 3.46a0.99ab 47.84b
−PN 425.1a 6.59ab0.25a 2.17cd2.24a0.66b 4.02a1.11a 64.71ab
−M 436.3a 7.43ab0.24a 5.25a2.32a1.14a 4.63a0.91ab 85.36a
+M 452.6a 8.23a0.28a 4.04ab2.62a1.13a 4.81a0.73b 71.26a
P   0.0003 0.00010.0001 0.00010.03980.0001 0.33190.0123  0.0003
Concn. ratio + : − 1.021.29 1.080.891.02 0.810.69  0.70
H. psilorrhyncha
+ 743.1bc 7.40b0.91c 1.63b5.30ab1.85b 8.41a0.28a 96.10ab
+D 537.6c 6.91b0.90c 1.39b4.37bc1.56bc10.63a0.27a 72.30b
 354.3d 3.32c0.29d 1.05c3.18c1.03cd 4.06b0.23ab 58.92bc
−PN 280.2d 9.23b4.74b 1.31bc3.12c0.94d 3.96b0.11b 32.10d
−M 820.1b14.65a7.48a12.14a5.63ab2.86a 5.65b0.35a118.50a
+M1117.7a16.03a7.30a14.44a7.43a3.34a 8.28ab0.28a130.80a
P   0.0001 0.00010.0001 0.00010.00010.0001 0.00020.0002  0.0001
Concn. ratio + : − 1.061.50 0.740.790.86 0.990.58  0.78

Figure 3 illustrates the contribution of cotyledons to nutrient content of 12-wk-old seedlings of the two Hakea species. For all 8 nutrients, cotyledons from the larger-seeded species contributed greater amounts than from the small-seeded species. Even though H. lasianthoides was only 60% as heavy as H. psilorrhyncha, it contained larger amounts of K and Fe, while the reverse was true for the other nutrients, except perhaps for N. Table 4 shows 80–85% of N, about 95% of P to under 30% of Ca were exported from the cotyledons to the seedling in both species. A much greater proportion of K, Ca, Mg, Cu and Fe was exported from the large-seeded species. Contribution of embryo-stored nutrients to the total seedling content varied from 75% for P in both species to negligible levels of K, Ca, Fe and Mn in H. lasianthoides. Proportional uptake of nutrients from the soil due to the presence of the cotyledons was 30–65% for both species, except for lower levels for Cu and Mn in H. lasianthoides and Fe for both species. The direct and indirect contribution of the embryo to total seedling content varied from 11% for Fe in H. lasianthoides to 93% for P in H. psilorrhyncha.

Figure 3.

Nutrient content of 12-wk-old seedlings of Hakea lasianthoides (left) and H. psilorrhyncha (right). The upper two portions together represent original embryo content, while the lower two represent the amount absorbed from the soil in the absence of cotyledons (−) and the additional amount due to the presence of cotyledons (+) (a) N. (b) P. (c) K. (d) Ca. (e) Mg. (f) Cu. (g) Fe. (h) Mn. Cotyledon residue, dark grey portion; uptake from cotyledons, light grey portion; soil uptake (+ cotyledons minus − cotyledons), open portion; soil uptake (− cotyledons), closed portion.

Table 4.  Distribution of eight mineral nutrients within seedlings of Hakea lasianthoides (Hl) and H. psilorrhyncha (Hp) on a percentage basis
Initial embryo content transferred to seedling (%)80.985.492.197.3 6.990.4
Contribution of embryo to total seedling content (%) 0.339.5 0.1 3.2 2.813.011.851.3 0.1 3.5 0.9 6.7
Indirect contribution of cotyledons to uptake from soil (%)31.641.346.766.743.634.231.741.340.845.622.837.211.017.813.339.9
Direct and indirect contribution of embryo to total seedling content (%)44.769.683.192.943.860.231.843.142.552.631.169.411.120.714.243.9


The presence of cotyledons controls the early dimensions and growth attributes of these two Hakea species. Following cotyledon removal, the marked reductions in plant mass, stem length and area per leaf, and increase in R : S, have been noted in other species (Harmer, 1990; Zhang & Maun, 1991; Andersson & Frost, 1996). However, the higher R : S could be an artefact of the seedlings having a substantial root but not shoot system before these experiments began. In addition, leaf production, internode length, leaf mass, and nutrient content and concentration were reduced in our study, pointing to the pivotal role of seed reserves in general development of the seedling.

Addition of a solution of all major nutrients to seedlings + or − cotyledons resulted in an increase in values of 10 morphological attributes and N content, with relative indices such as R : S (on a mass and N basis), SLA, leaf width : length and density tending to remain constant or decrease. This indicates that mineral nutrient supply was limiting absolute growth but not necessarily relative dimensions. It was possible to add balanced nutrient solutions to − cotyledon seedlings to equate most of these attributes to the + cotyledon seedlings (Figsure 1 and 2). This suggests that the cotyledons control seedling mass and morphology via the extent of mineral nutrient reserves. This is supported by the marked reduction in nutrient content of the − cotyledon seedlings despite access to soil nutrients (Table 3).

The larger-seeded species not only contained higher amounts of the eight nutrients in its cotyledons, they were more efficiently exported to the seedlings grown in their natural soils (Table 3; Milberg & Lamont, 1997). This may have been a question of demand as well as supply. The greatest positive difference in nutrient concentration for seedlings + and − cotyledons by far was for P, followed by N (Table 3). This indicates that P is the most important nutrient supplied by the cotyledons. Indeed, over 90% of stored P was exported to the seedlings, with over 80% of N (Table 4). These results are consistent with the conclusion of Fenner & Lee (1989) that larger-seeded species had a relatively reduced demand for external P but not necessarily for other nutrients. Milberg et al. (1998) showed seedling mass for nine species from the same region as these hakeas was most responsive to the amount of P exported from the seed, followed by N then K (no other nutrients were measured). For five species from similar nutrient-impoverished, fire-prone environments, Stock et al. (1990) obtained a similar order of export for P and N, but < 20% transport of K, Ca or Mg, and they argued that the last three were supplied from postfire ash. For H. psilorrhyncha grown in its natural postfire soil which produced no growth response by Avena fatua until N, P and K were added (Lamont, 1995), 29–90% of its K, Ca and Mg (and 50–91% of Cu, Fe and Mn) were obtained from its cotyledons (Table 4). For H. lasianthoides in its more fertile soil (N and P but not K-limited), little K and Ca were remobilized in contrast with 21–78% of Mg, Cu, Fe and Mn. Thus, the cotyledons may also be major sources of K, Ca and Mg, even following fire when recruitment normally occurs (Richards & Lamont, 1996).

We conclude that the cotyledons are a general source of mineral nutrients whose supply is inversely proportional to the most limiting in the species natural environment tempered by their mobility in plants (Fig. 3, Fenner & Lee, 1989). For example, Ca, Fe and Mn are poorly mobile in plants, and soil sources largely accounted for their levels in the seedlings (Table 4). This general nutrient function of the cotyledons is further supported by: the ineffectiveness of P, N or P + N but success with P + N + K + Mg + S (other combinations were not tried) in restoring the morphology of − cotyledon seedlings; and the 11–67% indirect contribution of cotyledons to soil uptake of these eight nutrients via their effect on enlarging the root system. However, P was the only case in which efficient export of nutrients from the cotyledons implied a reduced role for the roots in uptake of that nutrient (Table 4, Fig. 3). Thus, it was not possible to identify if any remobilized nutrients selectively control particular morphological attributes – a nutrient omission experiment would be required (Hanley & Fenner, 1997). If root growth is C-limited and shoot growth nutrient-limited (Milberg & Lamont, 1997), it seems likely that P, and to a lesser extent, N are shunted preferentially into the leaves, enhancing their photosynthetic capacity. Certainly, added N accumulated in the shoots rather than the roots (Fig. 1b). In that case, it is at first surprising that SLA was usually unaffected by cotyledon removal. However, closer examination shows that the presence of the cotyledons not only increased leaf production, the leaves were larger, thicker and less dense (Figs 1 and 2), all features favouring C-fixation (making SLA misleading as an index of C-fixing capacity; Niinemats, 1999).

These two species had cotyledons ≥ 1 mm thick, placing them in category 2 where their photosynthetic rates are just enough to balance respiration (Kitajima, 1992). Certainly, their chlorophyll content was negligible compared with that of the early leaves (Fig. 2a). In fact, covering them had no major effect on seedling morphology or nutrient content (except leaves of H. lasianthoides were narrower and denser), unlike results of Marshall & Kozlowski (1975) for nutrient transport. However, they do not fit into the third category of Lovell & Moore (1971) as the cotyledons expanded markedly and their longevity reached 3 months. Inositol, the main C-storage compound (as phytin) in hakea seeds and mobilized during germination, had no effect on morphology when applied via the soil to − cotyledon seedlings, except that there was greater root mass for all three treatments in which it was used on H. lasianthoides (Fig. 2). This is some support for our proposition that early root growth is C-limited, but, in view of the negligible effect on H. psilorrhyncha, that nutrients were sufficient to restore morphology, and ignorance of the amount of inositol actually stored, the importance of the result is unclear. Certainly, there was no net export of C, as the mass of senescent cotyledons was as high or higher than their initial mass (Table 1).

We conclude that the cotyledons of these two species are a major source of mineral nutrients but not organic compounds for directing growth of the young seedling (contrast Ashcroft & Murray (1979), Mulligan & Patrick (1985) and Zhang & Maun (1991) for other species with green cotyledons). Our results support the hypothesis that, in nutrient and water-limited habitats with abundant light, such cotyledons serve to maximize mineral nutrient supply to the shoots, which can then maximize C supply to the roots (Milberg et al., 1998). This facilitates root growth and enhances nutrient uptake (Fig. 3). More importantly, it promotes rapid elongation of the taproot (up to 2 m or more) which increases the chances of maintaining contact with soil water during the first summer drought (Enright & Lamont, 1992; Richards & Lamont, 1996; Milberg & Lamont, 1997).

This indirect contribution of cotyledons to root growth is the key to successful seedling recruitment following every fire, and cannot be viewed just as an ‘emergency’ mechanism to deal with extreme events or rare growing conditions (Westoby et al., 1996). Small-seeded species have no such capacity and must rely on enhanced lottery processes through greater fecundity and/or drought-tolerance mechanisms (Lamont & Witkowski, 1995; Richards & Lamont, 1996). Having secured contact with soil water over summer, surviving plants produce an extensive system of surface lateral roots with hairy root clusters the following wet season that enhance nutrient uptake, especially P (Lamont, 1973). Cotyledon-stored nutrients have an essential role in seedling establishment in this environment but finally make a negligible contribution to the nutrient budget of these long-lived shrubs. Even after 18 months, cotyledon-supplied nutrients only account for 2–10% of the total in H. psilorrhyncha (syn. H. obliqua) as a result of subsequent soil uptake (Pate & Dell, 1982).


BL undertook these experiments while a Senior Fulbright Fellow at Stanford University, California (hosted by Hal Mooney) and prepared the original manuscript while an Erskine Fellow at the University of Christchurch, New Zealand (hosted by Dave Kelly). The technical support of Heather Lamont, Phil Read and Vicky Whitten was invaluable. Comments on the manuscript from Per Milberg and Mike Fenner and the referees were much appreciated.