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Ann Bot. 2009 August; 104(2): 325–334.
Published online 2009 May 29. doi:  10.1093/aob/mcp127
PMCID: PMC2710899

Pollen source effects on growth of kernel structures and embryo chemical compounds in maize

Abstract

Background and Aims

Previous studies have reported effects of pollen source on the oil concentration of maize (Zea mays) kernels through modifications to both the embryo/kernel ratio and embryo oil concentration. The present study expands upon previous analyses by addressing pollen source effects on the growth of kernel structures (i.e. pericarp, endosperm and embryo), allocation of embryo chemical constituents (i.e. oil, protein, starch and soluble sugars), and the anatomy and histology of the embryos.

Methods

Maize kernels with different oil concentration were obtained from pollinations with two parental genotypes of contrasting oil concentration. The dynamics of the growth of kernel structures and allocation of embryo chemical constituents were analysed during the post-flowering period. Mature kernels were dissected to study the anatomy (embryonic axis and scutellum) and histology [cell number and cell size of the scutellums, presence of sub-cellular structures in scutellum tissue (starch granules, oil and protein bodies)] of the embryos.

Key Results

Plants of all crosses exhibited a similar kernel number and kernel weight. Pollen source modified neither the growth period of kernel structures, nor pericarp growth rate. By contrast, pollen source determined a trade-off between embryo and endosperm growth rates, which impacted on the embryo/kernel ratio of mature kernels. Modifications to the embryo size were mediated by scutellum cell number. Pollen source also affected (P < 0·01) allocation of embryo chemical compounds. Negative correlations among embryo oil concentration and those of starch (r = 0·98, P < 0·01) and soluble sugars (r = 0·95, P < 0·05) were found. Coincidently, embryos with low oil concentration had an increased (P < 0·05–0·10) scutellum cell area occupied by starch granules and fewer oil bodies.

Conclusions

The effects of pollen source on both embryo/kernel ratio and allocation of embryo chemicals seems to be related to the early established sink strength (i.e. sink size and sink activity) of the embryos.

Key words: Zea mays, maize, pollen, kernel, embryo, endosperm, oil, protein, starch, soluble sugars

INTRODUCTION

The oil concentration of mature maize (Zea mays) kernels exhibits a significant degree of genetic variability that enables breeding on this trait. For example, from an open-pollinated maize variety (‘Burr's White’) with an oil concentration of 4·7 %, two populations were divergently selected for their extreme kernel oil concentration values. After 90 generations, kernel oil concentration was 19·3 and 1·1 % for the high and the low oil population, respectively (Dudley and Lambert, 1992). Maize breeding programmes, however, were mainly focused on grain yield increase with little attention to kernel composition (Tollenaar et al., 1992; Edmeades et al., 1993; Echarte et al., 2000; Luque et al., 2006).

Maize kernels with a high oil concentration are preferred in feed rations to livestock and poultry because of their energy value and because they provide a substitute to animal fats (Thomison et al., 2003). The lack of a simultaneous selection programme for both grain yield and kernel oil concentration has promoted a production system for maize of high oil concentration based on pollen source effects on kernel composition (the ‘Xenia effect’ of Bulant and Gallais, 1998): pollen of a high oil concentration pollinator increased kernel oil concentration of the female parental without modifying kernel weight (Letchworth and Lambert, 1998). Increased kernel oil concentration was attributed to both an increased embryo/kernel ratio and an increased embryo oil concentration (Miller and Brimhall, 1954; Dudley et al., 1977; Dudley and Lambert, 1992; Lambert et al., 1998). The above studies were performed on mature kernels and no information on embryo growth and oil accumulation within the embryos was given.

Tanaka and Maddonni (2008) have recently studied pollen source effects on the kernel oil concentration of a hybrid with normal oil content (DK752) self-pollinated and that of the same hybrid but pollinated with a genotype of high oil concentration (5MG). These two genotypes (DK752 and 5MG as female and male genotype, respectively) were included in a commercial high-oil varietals blend of Dekalb-Monsanto Argentina S.A. The different dynamics of embryo/kernel ratio and embryo oil concentration among tested crosses (DK752 × DK752, DK752 × 5MG and 5MG × 5MG) led to a differential kernel oil concentration dynamic during the post-flowering period (Tanaka and Maddonni, 2008). At early stages of kernel growth (<500 °Cd from female flowering, i.e. silking), kernel oil concentration of all crosses was increased as a result of an increase in both embryo/kernel ratio and embryo oil concentration. At latter stages (from 500 °Cd after silking to physiological maturity), kernel oil concentration remained stable (DK752 × DK752 and DK752 × 5MG) or was slightly increased (5MG × 5MG), because the embryo/kernel ratio increased at a lower rate than during the previous period, and embryo oil concentration decreased (DK752 × DK752 and DK752 × 5MG) or remained stable (5MG × 5MG). As pollen source affected embryo growth rate without modifying final kernel weight, we hypothesized that there was differential partitioning of assimilates among kernel structures (i.e. embryo, endosperm and pericarp). Moreover, kernels with contrasting embryo growth promoted by pollen source may exhibit a different anatomical and histological embryo structure (e.g. size of embryonic axis and scultellum, cell number and cell size of the embryo tissues).

To our knowledge, changes to the chemical constituents (soluble sugars, starch, protein and oil) of maize kernel structures (mainly endosperm and embryo) during the post-flowering period have been studied only by Ingle et al. (1965). With regard to embryo compounds, accumulation of proteins and sugars preceded that of oil. Proteins and sugars accumulated at an almost constant rate during the entire period of embryo growth, with a dramatic increase in the accumulation rate of sugars at the end of this period. By contrast, oil accumulated at a low rate at early stages of embryo growth, then accumulated at a high rate during the effective embryo growth period, and accumulated at a decreased rate at later stages. Tanaka and Maddonni (2008) showed that the dynamics of embryo oil accumulation differed among crosses, but data on the deposition of soluble sugars, starch and protein within the embryos were not reported. Interestingly, the effective period of embryo oil accumulation was shorter than that of kernel growth, and the dynamics of embryo oil concentration suggested the existence of compounds deposited late (i.e. proteins, starch and soluble sugars). Hence, a detailed analysis of the oil, protein, starch and soluble sugars deposited within the embryos of the different crosses would aid in our understanding of pollen source effects on embryo oil concentration dynamics.

Reserve lipids in sunflower (Helianthus annuus) fruits and in other oil seeds are laid down in sub-cellular structures known as oil bodies (Huang, 1992; Murphy, 2001). Besides lipids, cells of sunflower embryos also contain protein bodies as reserve compounds (Mantese et al., 2006). Differences among sunflower cultivars in embryo oil concentration have been related to the proportion of protein bodies within cotyledon cells (Mantese et al., 2006). In maize, the link between sub-cellular structures (oil and protein bodies and starch granules) in the scuttellum of embryos and oil concentration has not been explored.

The present study expands upon that of Tanaka and Maddonni (2008) by addressing pollen source effects on (1) the dynamics of pericarp, endosperm and embryo growth; (2) the dynamics of accumulation of oil, protein, starch and soluble sugars in embryos; and (3) the anatomy and histology of mature embryos. Kernels of three crosses were used: (1) single-cross hybrid DK752 self-pollinated (DK752 × DK752), (2) single-cross hybrid DK752 pollinated with a genotype of high oil concentration (5MG; DK752 × 5MG) and (3) the high oil concentration pollinator self-pollinated (5MG × 5MG).

MATERIALS AND METHODS

Two field experiments were conducted at Pergamino (34 °56′S, 60 °34′W), Argentina, during the growing seasons of 2004/05 and 2005/06. The experiments and measurements used in the present study were partially described in Tanaka and Maddonni (2008). Briefly, plots of maize were sown in late October, at a commercial plant population density of 9 plants m−2 and irrigated and fertilized. The effect of two factors on kernel weight and kernel composition of the single-cross maize hybrid DK752 (Dekalb-Monsanto Argentina S.A.) was studied: pollen source and assimilates availability per kernel during the grain filling period (i.e. post-flowering source/sink ratio). Pollen source (maize hybrids DK752 and 5MG, Dekalb-Monsanto Argentina S.A.) was assigned to the main plot and the post-flowering source/sink ratio to the sub-plot. As post-flowering source/sink ratio did not modify embryo/kernel ratio or embryo oil concentration (Tanaka and Maddonni, 2008), the study was restricted to kernels of the pseudo-natural pollination treatment (i.e. plants daily hand-pollinated with fresh pollen to mimic open pollination conditions). During 2005/06, 5MG plants were also self-pollinated. Manual pollinations allowed a similar kernel set among crosses to be reached, preventing any sink effect on post-flowering source activity (Reynolds et al., 2000).

Kernels were sampled from apical ears (three ears per sub-plot) from similar spikelet positions (10–20 from the bottom of the ear) to avoid variations of the traits under study along the ear (Lambert et al., 1967; Tollenaar and Daynard, 1978). Samples were taken weekly from 15 d after silking to physiological maturity. For each sampling date, 60 kernels per sub-plot were collected and separated into three groups to determine: growth of kernel structures (approx. 40 kernels), histological and anatomical structure of the embryos (approx. five kernels), and deposition of oil, protein, starch and soluble sugars in the embryos (approx. 15 kernels). At physiological maturity, five plants per sub-plot were individually sampled and kernel number per plant was counted.

Growth of kernel structures

From the main bulk of kernels (approx. 40), ten were randomly selected and used to measure individual kernel weight and the others were dissected to extract embryos, endosperms and pericarps. Dissections were performed on fresh kernels to facilitate extraction of kernel structures. Entire kernels and kernel structures were dried at 70 °C until a constant weight was reached. The dynamics of kernel growth and those of kernel structures were expressed on a thermal time basis (base temperature 8 °C; Ritchie and NeSmith, 1991) from silking stage onwards.

Accumulation of oil, protein, starch and soluble sugars in embryos

Accumulation of oil, protein, starch and soluble sugars in embryos was determined from early stages of kernel growth to physiological maturity. Measurements were made on the bulk of embryos used to describe embryo growth.

Oil concentration was measured via an exhaustive lipid extraction technique (Rondanini et al., 2003), by using an initial solvent extraction (Hara and Radin, 1978) followed by supercritical CO2 extraction (Am 3-96, AOCS, 1996) of the residual oil in the sample. Protein concentration was calculated as 6·25× the nitrogen concentration measured by the micro Kjeldahl technique (Horwitz et al., 1975) using a Kjeltec 2300 analyser (Foss Tecator AB, Hoeganaes, Sweden). Soluble sugars and starch contents were determined using an anthrone method (Yemm and Willis, 1954). Soluble sugars were extracted from 100 mg of fine material with 80 % ethanol (v/v), using three successive extractions. The supernatant was analysed for the presence of glucose and the residue was boiled for 3 h in 25 mL 3 % HCl (v/v) solution in order to hydrolyse starch (Zhou et al., 2004).

Anatomy and histology of the embryos

Study of embryo anatomy and histology was performed on kernels sampled at physiological maturity. Embryos, dissected from fresh kernels, were immediately embedded in paraffin and serially cut at 10–15 µm with a Minot-type rotary microtome in longitudinal and transversal orientations. Sections stained with Safranin-Fast Green (Johansen, 1940) were observed with a Zeiss fluorescence microscope (Axioplan, Oberkochen, Germany) with a 5× and 40× lens for embryo size and scutellum cell size determinations, respectively. Digital photographs were analysed using Image Tool 3·0 software (Wilcox et al., 2002) to measure the length (longitudinal cut, Fig. 1A) and width (transversal cut, Fig. 1B) of each embryo structure (scutellum and embryonic axis) and scutellum cell size. As the scutellum does not have a uniform width, two measurements of this dimension were made (Fig. 1B). Scutellum cell size was measured from transversal sections of the embryo. Three sub-samples of ten cells were randomly selected and analysed to determine cell size (width and length). The number of cells layers of the scutellum was estimated as the ratio of (1) scutellum width (transversal section) or length (longitudinal section) to (2) cell size.

Fig. 1.
Schematic diagram of a mature maize kernel: (A) longitudinal section and (B) transversal section. Lsc, scutellum length; Lea, embryonic axis length; Wsc and W′sc, scutellum width; Wea and W′ea, embryonic axis width; sc, scutellum; ea, ...

Mature embryos were also used to observe oil and protein bodies and starch granules. For starch granule determinations, small cubical portions (sides 1–2 mm long) of the scutellums were fixed in 3 % glutaraldehyde in phosphate buffer (pH 7·2), dehydrated in an ascending series of ethanol concentrations (from 70 to 95 %) and embedded in resin (Historesin Embbeding Kit, Leica, Wetzlar, Germany). Semi-fine sections (2–3 µm) were cut with an Ultracut III Reichert ultramicrotome (Reichert-Jung, Vienna, Austria) and stained with 0·5 % cresyl violet. Semithin sections (2–3 µm) were also cut and stained with 0·5 % Coomassie Brilliant Blue to observe protein bodies.

For oil body determinations, scutellum samples (sides 0·5–1 mm long) were fixed at 4 °C in a solution 0·1 m glutaraldehyde (1 %) and paraformaldehyde (4 %), in phosphate buffer (pH 7·2), and post-fixed for 4 h at 24 °C in a solution of 1 % osmium tetroxide (O'Brien and McCully, 1981). The tissue was dehydrated in an ascending series of acetone concentrations (from 25 to 100 %) and embedded in resin (Spurr, 1969). Semithin sections (2–3 µm) were cut as described above and stained with 0·5 % Toluidine Blue for light microscopy. Other ultrathin (7–8 nm) sections were also cut and stained with uranyl acetate and lead citrate (Reynolds, 1963) for electron microscopy. Images obtained with the electron microscope were photographed with a Jeol-Jen 1200 EXII TEM at 85 kV.

Statistical analyses

Results were subjected to analysis of variance (ANOVA) to evaluate the effects of treatments and their interaction on measured variables. Correlation analysis was performed to investigate relationships among variables.

Bilinear (eqns 1 and 2) and trilinear models (eqns 1, 3 and 4) with plateau were fitted by using the nonlinear routine of Table Curve v. 3·0 (Jandel TBLCURVE, 1992):

equation image
1

equation image
2

equation image
3

equation image
4

where a is the intercept, b and d the slopes, c and e the breakpoints of the functions and TT thermal time after silking.

A bilinear model with plateau was fitted between kernel structure weight and thermal time, where b and c estimated the growth rate and the thermal time above which kernel structure attained its maximum weight, respectively. Initiation of growth of kernel structures (i.e. lag phase) was calculated as –a/b and the duration of the effective growth period was estimated as c – (a/b). The bilinear model with plateau was also used to describe the dynamics of: (1) entire kernel growth, (2) accumulation of oil, protein, starch and soluble sugars within the embryos, and (3) concentration of protein and soluble sugars in the embryos.

A trilinear model was fitted between embryo oil concentration and thermal time. The parameter b estimated the rate of the first phase, c and e the breakpoint between both phases, and d the rate of the second phase.

Finally, a negative exponential model (eqn 5) was used to describe the dilution of embryo starch concentration from approx. 450 °Cd after silking to physiological maturity:

equation image
5

Treatment effects on model parameters were tested by ANOVA. When model parameters were not affected by treatments, models were re-fitted to the whole data set.

RESULTS

Kernel and kernel structures growth

At physiological maturity, plants of all crosses exhibited a similar kernel number (approx. 536 kernels per plant) and kernel weight (approx. 255 mg per kernel; Table 1). A single pattern described the temporal development of kernel growth (approx. 197 °Cd and 633 °Cd for the lag phase and the effective grain-filling period, respectively, and a growth rate of approx. 0·403 mg °Cd−1).

Table 1.
Growth dynamics (initiation and cessation of growth, growth rate and final weight) of kernel and kernel structures (embryo, endosperm and pericarp) of kernels located at the middle of the ear of DK752 hand-pollinated with two pollinators (DK752 and 5MG) ...

Pericarp growth started approx. 100 °Cd before silking (i.e. the lag phase could not be estimated) and attained maximum value (approx. 15·6 mg) approx. 280 °Cd earlier than maximum kernel weight. Pericarp growth dynamics (approx. 658 °Cd and 0·027 mg °Cd−1 for the effective growth period and growth rate, respectively) were not affected by pollen source (Table 1).

By contrast, both endosperm and embryo attained maximum weights close to physiological maturity, but embryo growth started approx. 80 °Cd later than endosperm growth (Table 1). Pollen source affected endosperm (P < 0·05) and embryo (P < 0·001) growth rates, without affecting the duration of growth of both kernel structures (Table 1). DK752 × 5MG kernels had a lower endosperm growth rate (approx. 0·31 mg °Cd−1) than those of DK752 × DK752 (approx. 0·35 mg °Cd−1), while the embryo growth rate of the former cross was greater (approx. 0·067 mg °Cd−1) than that of the latter (approx. 0·057 mg °Cd−1). 5MG × 5MG kernels exhibited the lowest endosperm growth rate (0·30 mg °Cd−1) and the highest embryo growth rate (0·092 mg °Cd−1), but a similar growth period of both kernel structures to those of the other crosses. Hence, a negative correlation (r = 0·73, P < 0·16) between endosperm and embryo growth rates was found for the whole data set. Consequently, differences among crosses in final embryo/kernel ratio (approx. 12, 15 and 20 % for DK752 × DK752, DK752 × 5MG and 5MG × MG, respectively) were related to pollen source effects on both embryo and endosperm growth rates.

Accumulation of oil, protein, starch and soluble sugars in the embryos

At physiological maturity, embryo oil content of all crosses contributed almost 80 % to total kernel oil. Embryos (Table 2) of DK752 × 5MG exhibited higher final oil content (approx. 15·6 mg) than those of DK752 × DK752 (approx. 10·8 mg). Embryos of 5MG × 5MG yielded the highest oil value (approx. 23·2 mg). Pollen source affected embryo oil accumulation rate without modifying the period of oil accumulation, which ended at close to physiological maturity (Table 2). Consequently, modifications in final embryo oil content were related (r = 0. 93, P < 0·05) to changes in embryo oil accumulation rate. Differences among crosses in embryo oil concentration were detected from 550 °Cd after silking to physiological maturity (Fig. 2A, B and Table 2).

Fig. 2.
Dynamics of embryo oil concentration (A, B), embryo protein concentration (C, D), embryo starch concentration (E, F), and embryo soluble sugars concentration (G, H) during the post-flowering period of DK752 × DK752, DK752 × 5MG and 5MGx5MG ...
Table 2.
Dynamics of oil, protein, starch and soluble sugars accumulation in embryos (initiation and cessation of the accumulation period, accumulation rate, and final content), and final concentrations in the embryos of kernels located at the middle of the ear ...

For all embryos, protein accumulation started approx. 313 °Cd after silking (Table 2) and continued to physiological maturity. Similar to the results described above for embryo oil accumulation, pollen source affected (P < 0·01–0·05) embryo protein accumulation rate, without affecting the period of protein accumulation. Hence, variations in final embryo protein content were related to (r = 0·97, P < 0·01) a pollen source effect on protein accumulation rate. DK752 × 5MG embryos exhibited a higher protein accumulation rate (approx. 0·010 and 0·012 mg °Cd−1 during 2004/05 and 2005/06, respectively) than those of DK752 × DK752 (approx. 0·009 and 0·011 mg °Cd−1 during 2004/05 and 2005/06, respectively). During 2005/06, embryos of 5MG × 5MG yielded the highest protein accumulation rate (approx. 0·015 mg °Cd−1). Hence, at physiological maturity, embryos of 5MG × 5MG and DK752 × 5MG exhibited higher protein content than those of DK752 × DK752, but a lower protein concentration (Table 2), because of the greater embryo weight of the former crosses (Table 1). For each cross, embryo protein concentration increased from 400 °Cd after silking to physiological maturity, when maximum embryo protein concentration was attained (Fig. 2C, D). Mature embryos of DK752 × DK752 exhibited the highest protein concentration.

Starch accumulation in the embryos started earlier (approx. 190 °Cd after silking) than those of the other compounds, and continued at an almost constant rate until physiological maturity (Table 2). Pollen source affected (P < 0·01–0·10) embryo starch accumulation rate, without affecting the period of starch accumulation (Table 2). Therefore, a pollen source effect on embryo starch accumulation rate (r = 0·93, P < 0·05) affected final embryo starch content. DK752 × 5MG embryos exhibited a higher starch accumulation rate (approx. 0·065 mg °Cd−1) than those of DK752 × DK752 (approx. 0·0058 mg °Cd−1). During 2005/06, 5MG × 5MG embryos yielded the highest starch accumulation rate (approx. 0·088 mg °Cd−1). For each cross, embryo starch concentration decreased during the embryo growth period (Fig. 2E, F). Differences among crosses (P < 0·01–0·05) in embryo starch concentration were mainly detected at later stages of embryo growth. At physiological maturity, 5MG × 5MG embryos exhibited the lowest starch concentration.

The effective period of soluble sugar accumulation was shorter than those of the other embryo compounds, mainly due to their longest lag phase (approx. 393 °Cd; Table 2). Embryos of DK752 × DK752 and DK752 × 5MG exhibited a similar pattern (i.e. rates and durations) of soluble sugar accumulation. By contrast, 5MG × 5MG embryos had the highest soluble sugar content, mainly due to their highest accumulation rate (approx. 0·0091 mg °Cd−1). Embryo soluble sugar concentration increased during the post-silking period and reached a maximum at close to physiological maturity (Fig. 2G, H). During 2005/06, 5MG × 5MG embryos attained maximum soluble sugar concentrations approx. 45 °Cd before physiological maturity. Embryos of DK752 × 5MG and 5MG × 5MG exhibited a lower final soluble sugar concentration than those of DK752 × DK752 (Fig. 2G, H and Table 2), as the former crosses had a greater embryo weight than that of the latter (Table 1).

Embryo oil concentration was not related to embryo protein concentration (Fig. 3A). By contrast, embryo oil concentration exhibited a negative correlation with those of the other embryo chemical compounds (Fig. 3B, C). Single linear functions significantly described the relationship between embryo oil concentration and those of starch (r2 = 0·98, P < 0·005) and soluble sugars (r2 = 0·91, P < 0·02) of the whole data set.

Fig. 3.
Embryo oil concentration as a function of embryo protein concentration (A), embryo starch concentration (B) and embryo soluble sugar concentration (C). Each point is the mean of three replicates. Symbols as in Fig. Fig.2.2. Lines indicate models ...

Embryo anatomy and histology

Differences among crosses in embryo weight were related to embryo size (Fig. 4A–C). Histological sections revealed that mature embryos of DK752 × 5MG were longer (P < 0·01–0·05) and wider (P < 0·10–0·01) than those of DK752 × DK752, while kernels of 5MG × 5MG exhibited the longest and widest embryos (Table 3). As the embryonic axis represented a small portion of the embryo, differences among crosses in embryo dimensions were mainly associated with scutellum size (Table 3). Scutellum tissue was characterized by spherical-shaped cells (Table 3), with numerous communications between a cell and its neighbours and the presence of intercellular air spaces (Fig. 4D–F). Scutellum tissue of 5MG × 5MG had the largest cell size and the highest number of cells layers along the transversal section (Fig. 4D–F and Table 3). Differences between scutellum size of DK752 × 5MG and DK752 × DK752 were related only to cell number (Table 3).

Fig. 4.
Photographs of mature maize embryos (A–C) and scutellum cells (D–I) with coalescent oil bodies (o), starch granules (s) and nuclei (n). Embryos were dissected from kernels of DK752 × DK752 (A, D, G), DK752 × 5MG (B, E, ...
Table 3.
Dimensions (length and width) of the embryo, the embryonic axis, scutellum cells and the number of scuttellum cell layers (longitudinal and transversal sections) of kernels located at the middle of the ear of maize hybrid DK752 hand-pollinated with two ...

The presence of oil bodies in scutellum cells was easily distinguishable by light microscopy and most of the bodies coalesced with adjacent ones (Fig. 4D–F). Electron micrographs revealed that oil bodies were tightly packed as a result of compression, but their individuality was still preserved (Fig. 5A, B). These bodies appeared to be more abundant in the scutellum cells of DK752 × 5MG than those of DK752 × DK752, while oil bodies were most plentiful in the scutellum cells of 5MG × 5MG (Figs 4D–F and and5A,5A, B). Starch granules were also distinguishable by light microscopy as large globular structures evenly distributed in the scutellum cells (Fig. 4G–I). The abundance of starch granules of DK752 × 5MG (approx. 10·7 % of the cell area) was lower (P < 0·05–0·10) than that of DK752 × DK752 (approx. 13·8 % of the cell area), while they were rare in scutellum cells of 5MG × 5MG (approx. 7·9 % of the cell area).

Fig. 5.
Photographs of ultrathin sections showing structural details of scutellum cells of DK752 × DK752 (A) and 5MG × 5MG (B). Note the residual cytoplasm (c), proximity between oil bodies (o), presence of starch granules (s) and absence of protein ...

Protein bodies were not detected either by light microscopy (Fig. 4) or by electron microscopy (Fig. 5).

DISCUSSION

The present study broadens previous knowledge of pollen source effects on the oil concentration of mature maize fruits (Miller and Brimhall, 1954; Dudley et al., 1977; Dudley and Lambert, 1992; Lambert et al., 1998), through modifications of both final embryo/kernel ratio and embryo oil concentration. As pollen source did not modify kernel weight (Tanaka and Maddonni, 2008), the present study focused on: (1) growth dynamics of kernel structures (i.e. pericarp, endosperm and embryo); (2) the allocation of principal chemical constituents (i.e. oil, protein, starch and soluble sugars) within the embryos; and (3) the anatomy (i.e. size of the scutellum and embryonic axis) and histology (i.e. scutellum cell number and cell size, presence and abundance of oil and protein bodies and starch granules) of the embryos.

As was previously reported (Watson, 1987), maize kernel structures exhibited different growth dynamics. We have parameterized these dynamics and established an earlier growth period of the pericarps than those of the other kernel structures. Pericarp growth started approx. 100 °Cd before silking, i.e. close to the beginning of ovary growth (Cárcova and Otegui, 2007), and maximum pericarp weight was attained at approx. 550 °Cd from silking, when maize kernels exhibit maximum volume (Gambín et al., 2007). These results suggest a physical constraint of maternal tissue (i.e. the pericarp) to kernel expansion. Pollen source may have affected the duration of growth of kernel structures with paternal origin (Watson, 1987), such as the embryo (1:1 for paternal and maternal origin, respectively) and endosperm (1:2 for paternal and maternal origin, respectively). We could not reject this hypothesis as the kernel structures of parental pollinators (DK752 and 5MG) exhibited a similar duration of growth. By contrast, pollen source revealed a previously undocumented trade-off between embryo and endosperm growth rates, which strongly impacted on final embryo/kernel ratio, but did not affect final kernel weight. As pollen source did not modify either kernel set (i.e. sink size) or post-flowering plant biomass production (data presented in Tanaka and Maddonni, 2008), kernels of all crosses were growing under similar post-flowering source/sink ratios (approx. 137·2 and 239·9 mg per kernel during 2004/05 and 2005/06, respectively). Hence, we speculate that genes from pollen did not affect post-flowering source activity, but could have modulated the partitioning of assimilates between embryo and endosperm tissues, by setting a different sink capacity of the two kernel structures.

The sink capacity of maize kernels has been related to the size (i.e. cell number and cell size) of the main storage tissue, i.e. the endosperm, which is early established during the formative period of the grain (Tollenaar and Daynard, 1978; Reddy and Daynard, 1983; Jones et al., 1996). Under this conceptual framework, differences among maize genotypes in kernel growth rate and kernel weight were related to the early established sink capacity (Borrás et al., 2003; Gambín et al., 2006). In the present study, differences among crosses in endosperm growth rate were detected, but these were not reflected in kernel growth rate. Thus, pollen source effects on both embryo and endosperm growth rates cancelled out the positive relationship between endosperm size and kernel weight observed in maize and other cereals crops (Gleadow et al., 1982; Reddy and Daynard, 1983; Nicolas et al., 1984; Jones et al., 1996). We did not perform a histological study of endospermic tissue, but histological study of the embryos revealed a pollen source effect on scuttelum cell number. As cell division rate commonly precedes cell extension (Kigel and Galili, 1995), and lag phase of the embryos did not differ among crosses, pollen source probably affected cell division rate by establishing at an early stage the sink capacity of this kernel structure. A higher sink activity of the embryos (Doehlert and Lambert, 1991) could also be modulating partitioning of assimilates between endosperm and embryo tissues. These hypotheses need to be tested.

The study also focused on the allocation of different chemical constituents within the embryos to understand the differential embryo oil concentration dynamics among the tested crosses (Tanaka and Maddonni, 2008). Embryo oil concentration dynamics were complementary to those of the other compounds. At early stages of embryo growth, oil concentration increased and starch concentration decreased. By contrast, at later stages of embryo growth, the decrease in embryo oil concentration matched a sharp increase in protein and soluble sugar concentrations and a decelerating drop in starch concentration. These negative trends among embryo compounds were more evident in embryos obtained from a parental of low oil concentration. Thus, pollen source effects on final embryo oil concentration appear to be complex and strongly linked to the concentration of late deposition compounds. A differential profile of active enzymes among the tested crosses probably explains the final destination of imported soluble sugars from vegetative tissues for the synthesis of embryo chemical compounds. Differences among maize populations in kernel oil concentration were related to the early activity of enzymes associated with lipid synthesis in immature embryos (Doehlert and Lambert, 1991).

In contrast to data reported for sunflower cotyledons (Mantese et al., 2006), no relationship between oil and protein concentration of mature embryos was found here. Moreover, the presence of protein bodies in maize embryos was not detected. A different function of maize embryo proteins to those of sunflower embryos may explain these contrasting result. In maize fruits, reserves compounds (i.e. starch and proteins) are mainly located in the endospermic tissue, and embryo proteins (albumin and globulins) are linked to the enzymatic apparatus (Watson, 1987). Interestingly, the present data revealed a previously undocumented negative relationship between embryo oil concentration and both starch and soluble sugar concentration. Histological study of the scuttellum tissues revealed that embryos with the lowest oil concentration exhibited the highest cell area occupied by starch granules and the fewest oil bodies. Previous studies of the carbohydrate metabolism of wheat embryos rejected the hypothesis that embryo starch could be the carbon source for the lipid synthesis at late stages of embryo development (Black et al., 1996). The starch pool, however, could be used as an energy source for germination (Bewley and Black, 1994), and possibly contributes to a higher tolerance to desiccation (Black et al., 1996). Consequently, selection for higher oil content in maize kernels, based on embryo oil content, could adversely affect seed storability (Munamava et al., 2004). In contrast, a higher embryo size has been a decisive trait in breeding programmes of winter cereals for restricted environments, associated with greater seedling vigour (Richards and Lukacs, 2002). Breeding of maize for increasing kernel oil concentration, based on embryo/kernel ratio, probably has no effect on the post-harvest performance of seeds.

In conclusion, physiological, chemical, anatomical and histological studies have clearly shown that pollen source not only affected embryo growth but also embryo chemical compounds. A detailed analysis of the pathways and regulation of the metabolic process involved in the synthesis and deposition of chemical products within the embryos would be necessary to understand the trade-off among oil, starch and soluble sugars.

ACKNOWLEDGEMENTS

We wish to thank to F. Vartorelli, D. Ravetta, M. E. Otegui, G. Zarlavsky, L. Blanco, E. Pagano, A. G. Cirilo and D. Rondanini for their valuable help. This work was supported by the National Council for Research (CONICET. PIP 5540) and Dekalb-Monsanto Argentina.

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