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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.
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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).