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a Dep. of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA
b Dep. of Horticultural Science, University of Minnesota, St. Paul, MN 55108, USA
* Corresponding author (jones012{at}tc.umn.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: DAP, days after pollination • DHS, days of heat stress • Mr, apparent molecular weight
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maize protein accumulation seems less susceptible to heat stress (Bhullar and Jenner, 1985; Wilhelm et al., 1999) and water stress (Ober et al., 1991) than does starch metabolism. In some cases, it has been reported that protein concentration is positively correlated with high temperature during cereal grain growth (Campbell and Davidson, 1979; Campbell et al., 1981; Kolderup, 1975; Stone and Nicolas, 1998a, 1998b). However, considering that starch constitutes approximately 80% of the endosperm mass and that its accumulation is severely repressed by heat stress, the percentage protein increase on a dry weight basis may be simply a reflection of the proportionately lower starch weight (Bhullar and Jenner, 1985; Stone and Nicolas, 1998a; Wilhelm et al., 1999). Therefore, protein content (mass per kernel) rather than protein concentration may provide a more biologically relevant indication of how protein accumulation is affected by heat stress.
Maize endosperm proteins can be divided into albumins, globulins, prolamins (zeins), and glutelins (Osborne, 1908). During early stages of kernel development (10–12 DAP), albumins and globulins represent the predominant form of endosperm protein. At later stages (20 DAP through physiological maturity), zeins and glutelins constitute the majority of endosperm protein content. Zeins are the most abundant proteins; they account for 50 to 60% of the total maize endosperm protein at physiological maturity. Zeins accumulate exclusively in the endosperm in an insoluble form in protein bodies and can withstand desiccation for long periods. Separation by SDS-PAGE resolves mainly zein components with apparent molecular weight (Mr) of 27 or 28, 22, 19, 16 or 17, 14 or 15, and 10 kDa (Wilson, 1991). Recently, two other categories have been identified, the Mr 50- and 18-kDa zeins (Chui and Falco, 1995; Woo et al., 2001).
The glutelin fraction is particularly difficult to characterize because its solubility is restricted to acids and bases. These proteins accumulate during endosperm development, have amino acid composition typical of the storage proteins, and are metabolized during seed germination. Glutelin comprises 30% or more of the endosperm protein, yet its origin and complexity are not well understood. Because they are extracted after all the other proteins have been removed, this fraction can contain residual amounts of the other proteins, mainly the zeins (Bietz and Wall, 1973).
Albumins and globulins are diverse proteins, playing various roles in kernel growth and development. At physiological maturity, most of the albumins and globulins are located in the embryo. The role of these proteins during germination is not clear. However, some proteins, such as those located in the aleurone layer and the scutellum, could act as metabolic enzymes, whereas others may be used as a nitrogen source.
The goal of this investigation was to increase our understanding of the mechanism by which heat stress disrupts kernel development in maize by studying the effects of short-term heat stress treatments on endosperm protein accumulation during the cell division phase of maize kernel development. Amino acid incorporation into endosperm protein fractions and developmental pattern of the 27-kDa zein accumulation were assessed to determine whether heat stress effect on zein accumulation was repressed at the level of synthesis or degradation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Field Study
The field experiment was a repeat of the growth chamber-study, and was established in field plots at St. Paul, MN, USA (45° N, 93°10' W). Seeds were planted on 21 May 1996, in field plots with distance between rows of 75 cm and established on the same soil type used in the growth chamber study and fertilized to soil test recommendations (112 kg ha–1 of ammonium nitrate, 34% N). Plants were thinned to a density of approximately 50 000 plants ha–1 at the three- to four-leaf stage.
In both experiments, ear shoots were covered with paper bags before silk emergence, and plants were self- or sib-pollinated at 3 to 4 d after silk emergence. At 5 d after pollination (DAP), 30 ears were randomly chosen and assigned to the control (25/20°C day/night temperature, for the growth chamber experiment; and ambient temperature, which ranged from 12°C to 30°C, for the field experiment) or heat treatments [2 or 4 d of heat stress (DHS) at 35°C]. The continuous 35°C treatments were chosen to simulate a global climate change environment in which there would be no significant difference between day and night time temperatures. Heat stress treatments were imposed (as previously described by Commuri and Jones, 2001) on the intact ears of both growth chamber and field-grown plants using 24V AC electronic temperature controllers (Omron model E5CS-X, Omron Corporation, Japan), connected to 12.7 x 15.2 cm silicon rubber insulated thermo-foil heating mats (Minco Products, Inc., Minneapolis, MN). A bimetal thermocouple temperature sensor (Type K, Omega Corporation, Stamford, CT) was used to monitor the temperature of the ear. To place the temperature sensor at the mid-ear position, an incision in the outer husks was made with a scalpel, while leaving the inner husks intact. A wire mesh sleeve was placed over the ear to facilitate heat conduction over the entire ear, but still allowed air to circulate. A heating mat was wrapped around each of the 20 ears selected (10 for each heat treatment) and kept at constant 35°C (±0.1°C) for 2 or 4 d. Ten randomly chosen ears were used as controls, in which an incision on the outer husks was also made with a scalpel. The control ears were wrapped with a wire mesh sleeve and heating mats, though disconnected from the controllers.
Kernels were sampled from the mid-ear position, in alternate rows, from three or four different ears for each sampling date (11, 14, 17, 20, 23, 27, 32 DAP), and subsequently were separated into the four major components of endosperm, embryo, pedicel, and pericarp and then weighed. Sampling from each ear occurred two or three times.
All tissue was freeze-dried and weighed again, except for the endosperms of field-grown plants used in the L-[35S]-methionine incorporation studies, which were stored at –80°C until analyzed.
Protein Extraction
Freeze-dried endosperms were pulverized in a Wig-L-Bug Dental Amalgamator (Crescent Dental Mfg. Co., Lyons, IL, USA) in preparation for protein extraction and analysis. Forty to 50 mg of endosperm dry meal was defatted with 1 mL acetone (with agitation, for 30 min) and then air-dried. Protein fractions were then extracted from the defatted meal according to Culley et al. (1984), but modified as follows. Each extraction step was performed twice with 1 mL of solvent by mixing vigorously and placing the microfuge tubes on a rotator. The albumin plus globulin fraction was extracted with 0.5 M NaCl solution, each time for 30 min., and then the meal was washed once with water and the supernatant discarded. Zeins were then extracted twice with 70% (v/v) ethanol and 2% (v/v) ß-mercaptoethanol, once overnight and once for 1 h. The glutelin fraction remaining in the meal was then extracted with 0.05 M NaOH for 30 min. Centrifugations were all performed at 12000 g for 1 min in a microcentrifuge and samples then stored at –20°C.
For the L-[35S]-methionine incorporation study, protein fractions were extracted following the same procedures described above with a few differences. Four hundred to 500 mg of frozen endosperms of field-grown ears were homogenized in 5 mL acetone with a Polytron homogenizer (Brinkman Instruments, Inc., Westbury, NY). Each extraction step was performed twice with 2.5 mL of solvent. After glutelin extraction, residual proteins were extracted once with 1% (w/v) SDS for 3 h. Centrifugations were all performed at 1500 g for 10 min at 4°C.
Protein Analysis
Fractions containing 5 to 120 µg protein were assayed twice according to the method of Lowry modified by Hartree (1972) with bovine serum albumin (BSA) as a standard. Glutelins were assayed directly. The albumin plus globulin fraction was concentrated by trichloroacetic acid precipitation (10:1 v/w), and resuspended in 1 volume of 0.1 M NaOH before assay. Zein samples were lyophilized and dissolved in 70% ethanol before assaying.
Zein composition was analyzed twice by reverse phase-HPLC (Waters, Milford, MA), following the procedures proposed by Paulis and Bietz (1986) and Wilson (1991), with a few modifications. Vydak (Hesperia, CA, USA) C18 (5 µm, 250 x 4.6 mm) columns were used for both the growth chamber and the field experiments. Columns were equilibrated in 38% acetonitrile and 0.1% trifluoroacetic acid, and were held at 50°C. The columns were eluted starting at 38% acetonitrile, increasing at 0.971% min–1 for 7 min, at 0.419% min–1 for 12.4 min, at 0.154% min–1 for 13 min, at 0.714% min–1 for 5.6 min, at 0.568% min–1 for 16.2 min, ending at 65.2%. Trifluoroacetic acid content was maintained at 0.1%. The eluate was monitored at 220 nm. Zeins were separated into five categories: the 27-kDa, the 19- plus 22-kDa, the 16-kDa, 14-kDa, and the 10-kDa zeins. The 50- and 18-kDa zeins were not detectable in SDS-PAGE and therefore were not considered in this analysis. The 19- and 22-kDa zeins were not separated by HPLC. Therefore, the effects of heat stress on these proteins were analyzed in aggregate, rather than by measuring individual subfamilies (Rubenstein and Geragthy, 1986). Their concentration in the endosperm dry meal was estimated by each zein fraction's relative content multiplied by the total zein levels, for each replicate. Protein concentration was expressed on dry meal basis, whereas protein content was expressed per endosperm.
L-[35S]-methionine Incorporation
For L-[35S]-methionine incorporation studies, 400 to 500 mg of freshly isolated endosperms from field-grown ears were equilibrated for 30 min in 3 mL of aerated buffer osmoticum. The osmoticum contained 10 mM 2-[N-morpholino]ethanesulfonic acid (adjusted to pH 5.5 with NaOH), 150 mM of sucrose (adapted from Griffith et al., 1987), and 50 mM of amino acids. The amino acid mixture contained on an equivalent proportion bases all of the L-amino acids found by Lyznik et al. (1985) in placento-chalazal region of 20 DAP maize kernels. The millimolar concentrations were Asn, 4.00; Asp, 1.00; Thr, 1.50; Ser, 3.75; Gln, 22.00; Glu, 1.50; Pro, 0.25; Gly, 1.25; Ala, 5.00; Val, 1.50; Ile, 0.75; Leu, 1.00; Tyr, 0.75; Phe, 0.25; His, 1.75; Lys, 2.25; Arg, 1.25. Following the equilibration step, kernels were incubated for 3 h in L-[35S]-methionine (specific radioactivity 600 Ci mmol–1, 10 mCi mL–1), 4 µCi mL–1 buffer osmoticum. Incorporation of radiolabeled methionine was terminated by three washes of 2, 3, and 5 min duration with solution similar in composition to the equilibration buffer to remove free space radiolabeled methionine (adapted from Gifford and Thorne, 1985).
One milliliter of each fraction of the protein extraction sequence was mixed with 10 mL of Ecosint A scintillation fluid (National Diagnostics, Atlanta, GA, USA), and radioactivity was counted in a Beckman LS6800 (Beckman Instruments, Inc., Irvine, CA, USA). This procedure was repeated twice.
Nonincorporated L-[35S]-methionine was calculated by the sum of radioactivity in the acetone and the trichloroacetic acid soluble fraction. Incorporation of L-[35S]-methionine in solubility class protein fractions was calculated by the relative proportion of radioactivity in each fraction.
Statistical Analysis
In both experiments, data were analyzed as a completely randomized model, for each sampling date. In the growth chamber study, there were three replicates whereas in the field study there were four replicates. Treatment effects were analyzed by analysis of variance (ANOVA) procedure and means were compared by LSD at 5 and 1% levels of probability.
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RESULTS |
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The effects of heat stress on individual zeins were also significant (Fig. 5 A–J). At early developmental stages, the 27-kDa, 19 plus 22-, 16-, 15-, and 10-kDa zeins, even though encoded by one or several genes (see review by Feix and Quayle, 1993), were similarly affected by heat stress. Their concentrations were significantly lower in 4 DHS than in control kernels, at 14, 17, or 20 DAP, depending on the protein and the experimental conditions. Of all the zeins, the 10-kDa zein was the least abundant and least affected by heat stress (Fig. 5 E and J). At later developmental stages, the 27-kDa zein concentration developmental pattern differed from the other zeins (Fig. 5A and F). For both control and heat stressed kernels, the 27-kDa zein steady state levels dropped after 23 DAP, mainly in kernels from field-grown plants (Fig. 5 F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The embryo and endosperm were particularly sensitive to heat stress (Fig. 2). The 2-DHS kernels were either smaller than control or had a normal phenotype, whereas the 4-DHS kernels had ovary cavities that were only partially filled with endosperm and the embryos were of a reduced size (Fig. 1). These results support previous findings reported by Cheikh and Jones (1995), Commuri and Jones (1999), and Engelen-Eigles et al. (2000). However, the pericarp and the pedicel (i.e., maternal tissue) were less affected by heat stress (Fig. 3). An increase in kernel pericarp thickness has been previously reported (Cheikh and Jones, 1995; Commuri and Jones, 1999), which, when combined with the smaller size kernels, resulted in pericarp weights similar to control values at 32 DAP.
Previous studies have reported protein accumulation in cereals to be slightly affected by heat stress (Bhullar and Jenner, 1985; Stone and Nicolas, 1998a, 1998b; Wilhelm et al., 1999) and water stress (Ober et al., 1991). However, this study shows significant effects when heat stress is imposed during the early phase of kernel development. Maize endosperm protein composition was significantly affected by heat stress and protein concentration was also mildly affected by heat stress (Fig. 4). Among the protein fractions, zeins were the most affected by heat stress (Fig. 4 and 5). Within the zein fraction, accumulation was repressed but paralleled control accumulation, except for the 27-kDa zein in the growth-chamber experiment (Fig. 5 A), in which it remained at lower levels for most of the period of kernel development. In the field experiment, the concentration of the 27-kDa zein of 4-DHS endosperms was lower only up to 20 DAP, and its concentration decreased at later stages for all heat treatments (Fig. 5 F). The dramatic reduction of zein content after the 4 DHS may be due to the significant reduction of zein concentration during the 14- to 20-DAP period of growth.
It is important to determine whether the rate of zein synthesis or degradation is the mechanism through which heat stress affects zein accumulation and L-[35S]-methionine incorporation. Storage proteins appear to be stable during kernel development. The strong parallelism between protein and transcript localization (Woo et al., 2001) and the existence of proteinase inhibitors in endosperm tissue throughout cereal kernel development (Kirsi and Mikola, 1971; Mahoney et al., 1984) support the protein degradation hypothesis. Synthesis, rather than degradation may largely determine the accumulation of storage proteins. However, under stress conditions the activity of proteolytic enzymes may increase. Messenger RNAs encoding cysteine proteinases accumulate in drought and salt stressed Arabidopsis plants (Koizumi et al., 1993). Heat shock also increases proteolytic activity in cells. Ubiquitin transcripts have been shown to increase in maize seedlings after heat shock treatment (Christensen et al., 1992), but it remains to be determined whether this also occurs in a developing kernel.
The L-[35S]-methionine incorporation studies alone cannot determine how the 4-DHS treatment repressed zein accumulation because, like the protein steady state levels, the proportion of nonincorporated to protein incorporated L-[35S]-methionine is affected by synthesis and degradation rates of endosperm proteins. However, the steady state levels of the 27-kDa zein may give an indication of the extent of storage protein degradation. Previous work using germinating seeds showed that the 27-kDa zein tends to be the first storage protein to be degraded (de Barros and Larkins, 1990; Mitsuhashi and Oaks, 1994). These data are supported by the location of the 27-kDa zein in the outer-most portion of the protein body (Lending et al., 1988; Lending and Larkins, 1989; Woo et al., 2001), which predisposes it to degradation. Hence, when the 27-kDa zein steady state levels are combined with L-[35S]-methionine incorporation data they indicate the magnitude by which the ratio of synthesis to degradation was affected by heat stress.
Considering that (i) the proportion of nonincorporated L-[35S]-methionine decreased between 11 and 14 DAP at a time that its incorporation into the zein fraction was repressed by 4 DHS and (ii) the 27-kDa zein steady state levels did increase up to 20 DAP, it may be postulated that zein synthesis, rather than its degradation, was repressed by 4 DHS in early developmental stages.
In field-grown kernels, the 27-kDa zein concentration decreased between 23 and 27 DAP, but this trend was not significantly affected by heat stress treatments. However, in the 4-DHS kernels, the proportion of L-[35S]-methionine incorporated into the zeins and the albumins plus globulins decreased. These data indicated that the degradation rate of the 27-kDa zein surpassed its synthesis, which in field-grown kernels is concomitant with the approach of physiological maturity. Therefore, during the latter stage of kernel development, protein degradation rather than protein synthesis, may have contributed to decreased L-[35S]-methionine incorporation into the zeins. However, these data do not suggest that the apparent protein degradation is exacerbated by heat stress since in field-grown kernels, the decline in the steady state levels of the 27-kDa zein between 27 and 32 DAP was not significantly affected by the treatments. These data may suggest, however, that the effect could be a part of the natural progression of kernel development in that as the grain approaches physiological maturity, a small fraction of its reserves are degraded. Clearly, the difference between 27-kDa zein levels of kernels from growth chamber-and field-grown plants suggest that further studies are required to assess whether zein degradation occurs throughout kernel development and to determine whether zein degradation is affected by heat stress.
In conclusion, kernel growth was severely repressed by 2 and 4 DHS imposed early in the endosperm cell division phase. Protein concentration was affected by 4 DHS, but to a lesser extent than kernel mass, which still resulted in a significant disruption of endosperm protein content. The zeins were the protein fraction most affected by heat stress. Specific zein fractions responded similarly to heat stress and were significantly lower in 4-DHS endosperms during the early developmental stages, after which they recovered. As indicated by L-[35S]-methionine incorporation between the protein fractions, and by the developmental accumulation pattern of the 27-kDa zein, zein synthesis, rather than degradation, was most affected by heat stress applied early in development. At later developmental stages, degradation occurred, as a part of the kernel's natural progression toward physiological maturity but does not appear to be strongly affected by heat stress.
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ACKNOWLEDGMENTS |
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Received for publication April 1, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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