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Published online 21 November 2006
Published in Crop Sci 46:2581-2589 (2006)
© 2006 Crop Science Society of America
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SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY

Zein Transcription and Endoreduplication in Maize Endosperm are Differentially Affected by Heat Stress

Paulo Monjardinoa, Alan G. Smithb,* and Robert J. Jonesa

a Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, St. Paul, MN 55108
b Dep. of Horticultural Science, Univ. of Minnesota, St. Paul, MN 55108. P. Monjardino, present address: Univ. of Azores, Centro de Biotecnologia dos Açores, 9701-851 Angra do Heroísmo, Portugal

* Corresponding author (alan{at}cbs.umn.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High temperature stress imposed during the cell division stage of maize (Zea mays L.) kernel development adversely affects growth and mature mass. However, the processes affected by heat stress are not known. The goal of this study was to determine the mechanisms by which heat stress affects early zein accumulation in maize kernels. Intact ears of growth chamber–grown plants were subjected to heat stress (continuous 35°C) for 2 or 4 d, starting at 5 d after pollination (DAP). Both the 27 kDa and cluster 1 zeins of subfamily 4 (ZSF4C1) zein mRNA steady-state levels were significantly delayed by 4 d of heat stress (DHS), but were not affected by 2 DHS. Similarly, transcription rates of both zeins were reduced in the endosperm of kernels exposed to 4 DHS treatment up to 17 DAP. The 2 DHS treatment significantly delayed endosperm endoreduplication, up to 17 DAP, whereas 4 DHS significantly repressed it. The lack of coordinate changes among mRNA steady-state levels, transcription rates, and endoreduplication during heat stress indicates that the effects of heat stress on zein transcription rates may not be directly related to alterations in endoreduplication. Instead, zein transcription is most likely affected by a delay in endosperm development.

Abbreviations: CDK, cyclin-dependent kinase • DAP, days after pollination • DHS, days of heat stress • MI, mithramycin A • ZSF4C1, cluster 1 zeins of subfamily 4


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE MECHANISMS by which environmental stress affect plant growth and development have become the focus of recent research. High temperature stress during endosperm cell division can significantly disrupt subsequent maize kernel growth and development by reducing cell division and amyloplast biogenesis, resulting in reduced sink capacity (Jones et al., 1984, 1985; Commuri and Jones, 1999; Engelen-Eigles et al., 2000). Sugar metabolism and starch biosynthesis are sensitive to heat stress (Hanft and Jones, 1986), through the reduction of enzyme levels, activities and the transcript steady-state levels of acid invertase, ADP glucose pyrophosphorylase, and soluble and insoluble starch synthase (Cheikh and Jones, 1995; Duke and Doehlert, 1996). Protein accumulation is also affected by heat stress, but the mechanisms involved are not well understood. The zeins, the predominant group of proteins in the maize kernel, are particularly sensitive to heat stress. Their concentration is significantly reduced during early developmental stages by 4 DHS, whereas the concentration of the other protein fractions (i.e., the albumins plus globulins and glutelins) tends to increase (Monjardino et al., 2005).

The zeins constitute approximately 50 to 60% of the total maize kernel protein. Accumulation of zeins begins around 10 to 12 DAP and continues until the grain reaches physiological maturity. Zeins are a composite of several proteins that differ in alcohol solubility depending on the presence of a reducing agent. Separation by SDS-PAGE resolves components with apparent Mr of 50, 27, 22, 19, 18, 16, 15, and 10 kDa (Wilson, 1991; Chui and Falco, 1995; Woo et al., 2001). The 19 and 22 kDa zeins constitute the major class of zeins (70–90% of the zein fraction). They are encoded by a large multigene family of about 75 to 150 genes (Hagen and Rubenstein, 1981; Wilson and Larkins, 1984), which have been divided into four subfamilies, based on DNA and amino acid sequence analyses (Rubenstein and Geraghty, 1986). Some of these genes, like ZSF4C1 are highly expressed, resulting in significant amounts of accumulated protein, whereas others that contain early in-frame stop codons were found to have either low or no expression (Liu and Rubenstein, 1993). On the other hand, depending on the cultivar, each of the 50-, 27-, 18-, 16-, 15-, and 10-kDa zeins are encoded by only one or two genes. Most of these genes, particularly those that encode the 15- and the 27-kDa zeins are highly expressed throughout kernel development (Marks et al., 1985; Woo et al., 2001). Zein accumulation has been correlated with high levels of mRNA (Larkins et al., 1976; Viotti et al., 1979; Park et al., 1980; Burr and Burr, 1981). Zein mRNAs begin to accumulate in the endosperm by 10 to 12 DAP and reach maximum levels between 18 and 22 DAP (Marks et al., 1985). With the exception of the 10-kDa zein (Cruz-Alvarez et al., 1991), zein synthesis is regulated mainly at the transcriptional level (Motto et al., 1989) but can also be regulated at the post-transcriptional level (Plotnikov and Bakaldina, 1996). Opaque-2 is one of the few genes identified whose product interacts with a conserved sequence (designated "endosperm box") 300 bp upstream of the translation start of most of the prolamins (Lohmer et al., 1991; Schmidt et al., 1992). It encodes a leucine-zipper DNA binding protein, a trans-acting factor that regulates the ZSF4C1 zein transcription (Hartings et al., 1989; Schmidt et al., 1990). It has been demonstrated that the opaque-2 mutation also affects the 10- and 15-kDa zeins (Hunter et al., 2002). Other trans-acting factors that regulate zein synthesis have been identified (Pysh et al., 1993; Carlini et al., 1999; Ciceri et al., 2000), which may interact with Opaque-2 to promote ZSF4C1 zein transcription.

Endoreduplication, the increase in DNA content without cytokinesis, is an important process for maize endosperm development. Around 10 to 12 DAP, following the cessation of mitotic activity, the S-phase alternates with distinct gap phases that lack DNA replication resulting in an exponential increase in nuclear DNA content of endosperm cells. Both nuclear size and endoreduplication peak at 16 to 18 DAP (Kowles and Phillips, 1988). Endoreduplication can be estimated in terms of its C value, the number of copies of its basic haploid genome. In maize endosperm, an average C value of 12.7 has been reported for kernels at 15 DAP (Kowles et al., 1990). However, the endoreduplication of cells in the inner portion of the endosperm, which are the most endoreduplicated, can be as high as 155°C, depending on the cultivar (Kowles and Phillips, 1985).

The mechanisms by which endoreduplication occurs are beginning to be understood: it has been proposed that low cyclin-dependent kinase (CDK) activity permit the formation of prereplication complexes at origins of replication during the G1 phase but inhibit M-phase induction, and the subsequent S-phase CDKs disassembly allows subsequent rounds of replication (reviewed in Edgar and Orr-Weaver, 2001; Larkins et al., 2001). Therefore, oscillations in the activity of S-phase CDKs and loss of M-phase CDK activity permit alternate cycles of DNA synthesis without cell division, thus leading to increasing levels of DNA.

The similar timing of endoreduplication and zein protein accumulation suggests that endoreduplication may be important for zein accumulation (Kowles and Phillips, 1988; Artlip et al., 1995; Cavallini et al., 1995). However, there may not be a direct relationship between endoreduplication and gene expression, because the more endoreduplicated cells accumulate less storage proteins and have lower zein mRNA levels than the peripheral endosperm cells (Dolfini et al., 1992; Woo et al., 2001). Moreover Leiva-Neto et al. (2004) have shown that a 50% decrease in mean C-value in maize endosperm nuclei had little or no effect on protein and starch accumulation.

In this study, the effects of heat stress on the steady state levels and transcription rates of the 27 kDa and the ZSF4C1 zein genes are determined. These data are compared to the effects of heat stress on endoreduplication to determine their interaction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material, Growth Conditions, Heat Treatments, and Sampling
Seeds of inbred W64A were planted in 18-L pots, on a Waukegan silt loam (fine-silty over sandy or sandy-skeletal, mixed, mesic Typic Hapludoll), watered daily, and fertilized with Peters fertilizer 20:10:20 on a weekly basis. Plants were kept in growth chambers, with irradiance levels ranging between 600 and 900 µmol m–2 s–1, at a 25/20°C (day/night) temperature regime, and a 14-h photoperiod, as previously described (Monjardino et al., 2005).

The ear shoots were covered with paper bags before silk emergence, and plants were self- or sib-pollinated 3 to 4 d after silk emergence. At 5 DAP, ears were randomly chosen and assigned to the control (25/20°C continuously) or heat treatments (2 or 4 DHS). Heat stress treatments were imposed on the kernels using 24-V AC electronic temperature controllers (Omron model E5CS-X, Omron Corp., Tokyo), connected to 12.7 by 15.2 cm silicon rubber insulated thermo-foil heating mats (Minco Products, Inc., Minneapolis, MN). A bimetal thermocouple temperature sensor (Type K, Omega Corporation, Stanford, 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. Heating mats were wrapped around each of the selected ears, which were heat stressed by keeping them at a constant 35°C (± 0.1°C) for 2 or 4 d. Control ears were also wrapped with a wire mesh sleeve and heating mats, with a sensor inserted between the inner husks, though disconnected from the controllers. Kernels were sampled from the mid-ear position, in alternate rows, from three different ears for each sampling date (11, 14, 17, 20, 23 DAP) as described by Monjardino et al. (2005). The endosperms were isolated free of pericarp, embryo, and nucellar tissue, and weighed. Endosperms used for RNA analysis were frozen in liquid N and stored at –80°C. Endosperms used for the transcription run-on assays and for flow cytometry were immediately handled after sampling, according to the procedures described below.

RNA Extraction
To isolate total RNA, 400 to 600 mg of frozen (–80°C) endosperm tissue (14, 17, 20, and 23 DAP) were hand-ground in liquid N with a mortar and pestle and extracted with phenol–chloroform, as described by Das et al. (1990). For each heat treatment and sampling date, three independent replicate samples were analyzed. All RNA extraction steps were conducted in RNAase free conditions, and the extracted samples were kept at –80°C until their analysis.

Northern Blots
To assess RNA intactness, total RNA (5 µg) was denatured at 65°C for 10 min in 1x F buffer (20 mM 3-[N-morpholino] propanesulfonic acid, pH 7.0, 1 mM ethylenediaminetetraacetic acid pH 7.0, and 5 mM sodium acetate), 50% formamide (v/v) and 6% (v/v) formaldehyde, and fractionated in a 1.2% (w/v) agarose gel with 1x F buffer, 6% formaldehyde (v/v), and 0.05 µL ethidium bromide solution (5 mg of ethidium bromide per milliliter of 0.1 M ammonium acetate) per milliliter of gel. The fractionated RNA was transferred to Genescreen Plus nylon membrane (NEN, Dupont, Boston), according to the manufacturer's specifications, and RNA was fixed to the membrane by air drying for at least 24 h.

To prepare probes, E. coli were lysed by alkali, and plasmid minipreps were conducted according to the procedures described by Sambrook et al. (1989). The probe specific for the A copy of the 27-kDa zein was a 1.2-kb SphI–SalI fragment of a genomic subclone from inbred line W22 (Geraghty, 1985). For the 22-kDa zeins, a probe was used that hybridizes specifically to ZSF4C1 zeins. The ZSF4C1 probe was a 1.0-kb EcoRI fragment of a cDNA subclone from inbred W22 (Liu and Rubenstein, 1993). The 18S rRNA probe was a 1.7-kb EcoRI fragment of a genomic subclone of tomato (Solanum lycopersicum L.) (Perry and Palukaitis, 1990). Each probe was labeled with [{alpha}-32P]dCTP (specific radioactivity 3000 Ci mmol–1, 10 mCi mL–1) by random oligo labeling with Megaprime labeling kit (Amersham, Piscataway, NJ), following the manufacturer's instructions. To assess the level of mRNA on each membrane, polyuridylic acid (poly-U) oligonucleotides were end labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP (specific radioactivity 3000 Ci mmol–1, 10 mCi mL–1).

For random oligo labeling probes, filters were prehybridized and hybridized at 42°C in 50% (v/v) formamide, 5x Denhardt's (0.1%w/v Ficoll, 0.1% w/v polyvinylpyrrolidone, 0.1% w/v bovine serum albumin), 5x SSPE (5 mM Na2EDTA, 50 mM NaH2PO4, pH 7.5, 0.9 M NaCl), 1% (v/v) lauryl sulfate, and 100 µg mL–1 salmon sperm DNA (Sambrook et al., 1989). Prehybridization and hybridization conditions with [{gamma}-32P]ATP labeled poly-U were similar to those used with random oligo labeled probes, except that the solution stocks were all RNAase free, the prehybridization and hybridization solutions contained only 1% (v/v) lauryl sulfate and 1 M sodium chloride, and hybridization was conducted at 50°C. For successive hybridizations, filters were stripped according to the manufacturer's specifications.

RNA Dot Blots
RNA samples were bound to Genescreen Plus nylon membranes by slow filtration with a dot blot apparatus (Bio-Rad Laboratories, Hercules, CA), following the recommendations by the membrane manufacturer. Five hundred nanograms of total RNA (measured by OD 260 nm) were analyzed per dot, for the 27-kDa and the ZSF4C1 zeins. All samples were analyzed in duplicate. A standard dilution series of RNA was included in each hybridization to ensure that the samples fell within the linear range of the technique. As a control for measurement and loading errors among the RNA samples, the 18S rRNA and poly-U probes were hybridized with a fivefold dilution of each RNA sample. This dilution was necessary to bring the concentration of 18S rRNA and poly-A RNA within the linear range of the assay. The probe preparation procedures used for dot blots were the same that were used for Northern blots.

Run-on Transcription Analysis
Nuclei were isolated from 2 to 5 g of control and 4 DHS endosperm from 14, 17, and 20 DAP freshly harvested kernels as described by Das et al. (1990). Nuclei were resuspended in 50% (v/v) glycerol buffer (50% glycerol, 0.5 M sucrose, 50 mM Trizma-hydrochloride pH 7.5, 5 mM magnesium chloride and 10 mM ß-mercaptoethanol) and stored at –80°C. DNA content of aliquots of nuclei samples stained with Hoechst 33258 dye was measured with a fluorometer (DyNA Quant 200, Hoefer, San Francisco, CA), with calf thymus DNA as a standard (Labarca and Paigen, 1980).

The procedures for the transcription run-on reactions were modified from the method of Cruz-Alvarez et al. (1991). Nuclei equivalent to 15 µg of DNA were used for each individual run-on reaction which contained 25 mM Trizma hydrochloride, pH 7.8, 5 mM magnesium chloride, 10% (v/v) glycerol, 5 mM ß-mercaptoethanol, 75 mM ammonium sulfate, 0.5 mM ATP, CTP, and GTP, 75 units of RNasin, and 300 µCi of [{alpha}-32P]UTP (specific radioactivity 3000 Ci mmol–1, 10 mCi mL–1), in a total volume of 100 µL. To adjust osmolality differences among nuclei samples, specific amounts of 50% (v/v) glycerol buffer were added to the assays.

Assays were conducted at room temperature for 20 min in microtubes placed on a slow rotator with the tubes near horizontal. Reactions were stopped on ice by the addition of 200 units of DNAase I and 100 µmol UTP and then incubated at room temperature for 10 min. For RNA extraction, 120 µL of 10x SET (1 mM Trizma hydrochloride, pH 7.5 and 120 µg proteinase K) were added. One extraction with 1 volume (V) of phenol/chloroform/isoamyl alcohol (1:1:0.04) and one extraction with 1 V of chloroform/isoamyl alcohol (99:1) were performed. The solution was adjusted to 0.3 M sodium acetate, RNA was precipitated with 2.5 V ethanol overnight at –20°C, and the pellet resuspended in TE (10 mM Trizma hydrochloride pH 8.0 and 1 mM EDTA pH 8.0). The UTP-labeled transcripts were analyzed by hybridization to membranes containing gene-specific DNA probes. Plasmid DNA for each probe was purified with a QIAGEN Plasmid Midi Kit (QIAGEN Inc., Valencia, CA). Recombinant plasmids were cut with the restriction enzymes described above to liberate a gene-specific insert. Two micrograms of DNA were denatured and separated electrophoretically on a 0.7% agarose gel, as described in Sambrook et al. (1989). DNA was transferred to Genescreen Plus nylon membrane as described by the manufacturer's instructions. Individual strips containing a specific DNA fragment of interest were cut and air dried to immobilize the DNA to the membrane.

The DNA-gel blot membranes were prehybridized for 4 h at 42°C in 50% formamide (v/v), 5x Denhardt's, 5x SSPE, 1% (v/v) lauryl sulfate, and 100 µg mL–1 salmon sperm DNA. Hybridization with labeled transcripts was done in 1 mL of the same solution in microtubes placed in a slow rotator for 24 h at 42°C, and filters were washed according to the manufacturer's instructions. Run-on transcripts were hybridized to an excess of single stranded DNA from clones corresponding to the 27-kDa zein, ZSF4C1 zeins, 18S rRNA, and to the plasmid pUC 119. Ribosomal RNA was used to standardize the zein transcription levels. The pUC 119 was used to determine nonspecific hybridization and the background was subtracted to calculate the hybridization to each probe.

Quantification of mRNA and Transcription
Northern and dot blots were exposed to a phosphor screen and scanned in a Molecular Dynamics Storm 840 system, and the signal was quantified by ImageQuant v1.1 software (Molecular Dynamics, Sunnyvale, CA). For the mRNA (dot-blot) analysis, all repetitions of each individual heat treatment and sampling date were analyzed at the same time. Steady-state levels of zein mRNA were normalized on the basis of the 18S rRNA and poly-U quantities in each sample. For the run-on analysis, newly transcribed zein mRNAs were normalized on the basis of 18S rRNA.

Flow Cytometry
DNA endoreduplication patterns were determined by flow cytometer after the argon laser was aligned at 488 nm with microsphere DNA beads (Coulter, MDADS II, Epics division, Hialeah, FL). Nuclei preparation and mithramycin A (MI) staining were done according to the procedures described by Kowles et al. (1994). The samples were run at 450 nm since MI is excited at this wavelength. Fluorescence of the dye was detected at 530 nm. Chicken red blood cells were used as standards. The samples were gated to remove the cellular debris from the analysis. Fifteen hundred nuclei were counted for each sample.

For nuclei counting, samples were diluted in MI buffer (described by Kowles et al., 1994) and mixed with a constant number of microsphere beads (internal standard for nuclei counting). Samples were continuously shaken to prevent nuclei from settling. The beads were gated out, and 1500 nuclei were counted, so that the volume of the sample analyzed could be determined as well as the number of nuclei per transcription run-on reaction.

Statistical Analysis
All data were analyzed as complete randomized models for each sampling date. For RNA and endoreduplication analysis, there were three or four replicates. Transcription run-on assays were replicated twice, due to a limited number of kernels. After the ANOVA, the means were compared by LSD at 5 and 1% levels of significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RNA Analysis
Heat stress disrupts protein accumulation in maize kernels with the greatest reductions in zein levels at early developmental stages (Monjardino et al., 2005). The relative 18S rRNA content was similar across treatments, except at 17 DAP (Fig. 1A ). However, the relative poly-A RNA content was numerically greater in the 4 DHS treatment from 17 through 23 DAP, and, at 20 DAP, these differences were significant (Fig. 1B). The 2 DHS treatment did not significantly affect relative poly-A RNA or 18S rRNA content. Since measurements of the 18S rRNA levels were consistent with optical density measurement, 18S rRNA levels were used to normalize loading of RNA-gel blots and dot-blots.


Figure 1
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  		Fig. 1. Relative levels of 18S ribosomal RNA and poly-A RNA in endosperm after 0 (ct), 2 (2d), or 4 (4d) days of heat stress (DHS) treatments. (A) 18S rRNA levels expressed per OD 260 of the RNA preparation. (B) Poly-A RNA levels normalized relative to OD 260 of the RNA preparation. Average RNA relative levels of each heat treatment were compared for each sampling date by LSD; a and b indicate statistically different values at 5% (*) and 1% (**) levels of significance.

 
In inbred W64A, the 27-kDa zein is encoded by a single gene (Das and Messing, 1987), which is similar to the A gene of the inbred W22. Strong hybridization of the 27-kDa zein probe to RNA was observed for all heat treatments (data not shown), which indicates an abundance of these transcripts during endosperm development. However, 27-kDa zein transcript levels differed significantly between control and 4 DHS endosperms at 14 and 17 DAP. Four DHS only delayed accumulation of the 27-kDa zein transcript levels since control levels were reached at 20 and 23 DAP (Fig. 2A ).


Figure 2
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  		Fig. 2. The 27-kDa and ZSF4C1 zein mRNA relative steady-state levels in endosperm after 0 (ct), 2 (2d), or 4 (4d) days of heat stress (DHS) treatments. (A) 27-kDa zein levels normalized relative to 18S rRNA. (B) ZSF4C1 zein levels normalized relative to 18S rRNA. Comparison of means was done by LSD; a and b indicate statistically different values at 5% (*) and 1% (**) levels of significance.

 
The ZSF4C1 set of the 19- plus 22-kDa zeins contains approximately 15 to 20 zein genes per haploid genome of maize (Hagen and Rubenstein, 1981). The proteins of this subfamily have an apparent Mr of 22 kDa. The ZSF4C1 zein genes are the only genes known of subfamily 4 that do not contain inframe stop codons, and their estimated number is 3 to 4 genes per haploid genome (Liu and Rubenstein, 1992). Like the 27-kDa zein, the transcript steady state levels of the ZSF4C1 zeins were high in the endosperm tissue of developing kernels and the 4 DHS treatment caused a significant repression of ZSF4C1 mRNA accumulation at 14 DAP (Fig. 2B).

Endoreduplication
Endosperm endoreduplication was analyzed by evaluating the proportion of nuclei in each individual C class (1C being the maize haploid DNA content). The endoreduplication pattern of control endosperms (peaked at 17 DAP) was similar to those determined previously (Kowles and Phillips, 1985, 1988; Kowles et al., 1990; Schweizer et al., 1995; Engelen-Eigles et al., 2000) (Fig. 3 ). The 2 and 4 DHS endosperms had a significantly slower rate of accumulation of DNA than that observed for control endosperms (Fig. 3). Endoreduplication in the 2 DHS recovered to control levels at 17 and 20 DAP; however, endoreduplication in 4 DHS was significantly lower than control levels at all time points.


Figure 3
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  		Fig. 3. Endosperm endoreduplication expressed as the average C values of nuclei sampled after 0 (ct), 2 (2d), or 4 (4d) days of heat stress (DHS) treatments. Comparison of means was done by LSD; a and b indicate statistically different values at 5% (*) and 1% (**) levels of significance.

 
In the case of the control endosperms, the DNA levels per nucleus ranged from 3C (nonendoreduplicated triploid tissue) to 96C (triploid tissue that has gone through five endoreduplication cycles) (Fig. 4 ). The 2 DHS endosperm's delayed pattern of DNA accumulation was due to a significant reduction of the relative proportion of nuclei in higher C classes at 11 DAP (24C and 48C), 14 DAP (96C), and 17 DAP (12C, 24C, and 48C), which was mirrored by a higher proportion of nuclei in the nonendoreduplicated C classes, for example, the 3C class at 11 and 17 DAP and the 6C class at 14 DAP (Fig. 4A, 4C, 4B, respectively). The 4 DHS treatment caused significant reductions in the proportion of nuclei in the 12 to 96C classes, with the exception of the 12 C class at 11 DAP. The reduction of higher C classes in the 4 DHS was also mirrored by a significant increase in the proportion of nuclei in the 3C class, for all sampling dates (Fig. 4A–4D).


Figure 4
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  		Fig. 4. Relative proportion of endosperm nuclei in each C class, sampled after 0 (ct), 2 (2d), or 4 (4d) days of heat stress (DHS) treatments. (A) 11 days after pollination (DAP). (B) 14 DAP. (C) 17 DAP. (D) 20 DAP. Comparison of means was done by LSD; a and b indicate statistically different values at 5% (*) and 1% (**) levels of significance.

 
Run-on Transcription Analysis
Endosperm nuclei were isolated from the same ears used to analyze transcript and endoreduplication levels. Nuclei yields were similar to those reported by Das et al. (1990). Overall, the 17 and 20 DAP control endosperm samples had the lowest nuclei yields, and the 14 DAP 4 DHS endosperm samples had the highest yield (data not shown). Nuclei prepared for transcription run-on assays by the method of Das et al. (1990) had much lower average endoreduplication levels (Fig. 5 ) relative to those prepared for determination of DNA level by the method of Kowles et al. (1994) (Fig. 3), due to preferential exclusion of the higher C class nuclei (Fig. 6 ).


Figure 5
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  		Fig. 5. Average C values of nuclei isolated for transcription run-on assays after 0 (ct) or 4 (4d) days of heat stress (DHS).

 

Figure 6
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  		Fig. 6. Relative proportion of nuclei in each C class, isolated for transcription run-on assays after 0 (ct) or 4 (4d) days of heat stress (DHS). Endosperm samples were taken at (A) 14 days after pollination (DAP); (B) 17 DAP, and (C) 20 DAP. Comparison of means was done by LSD; a and b indicate statistically different values at 5% (*) and 1% (**) levels of significance.

 
The 4 DHS treatment delayed the increase in transcription rate of the 27-kDa zein from 14 to 17 DAP, but at 20 DAP the transcription rates of this gene recovered to equal or higher levels than those of control kernels (Fig. 7A ). Transcription of the ZSF4C1 zein genes was lower at 14 DAP, similar at 17 DAP, and greater than controls at 20 DAP (Fig. 7B).


Figure 7
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  		Fig. 7. Zein transcription levels in endosperm after 0 (ct) or 4 (4d) days of heat stress (DHS) treatments. (A) 27-kDa zein rate of transcription. (B) ZSF4C1 zein rate of transcription. Rates were normalized relative to 18S rRNA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, the mechanisms by which heat stress disrupts zein accumulation in early developing kernels were investigated. To evaluate zein transcript levels, we assessed the levels of 18S rRNA and poly-A RNA relative to optical density. The 4 DHS treatment had a surprising effect on the relative levels of poly-A RNA. After the 4 DHS, endosperms had ratios of poly-A RNA to optical densities that were higher than in control kernels. The reason for this increase may be related to a heat stress–induced increase in the albumin plus globulin and the glutelin proteins (Monjardino et al., 2005). An increase in the accumulation of these very abundant proteins could result from higher steady-state mRNA accumulation, thus increasing poly-A RNA levels. Considering the less stable levels of poly-A RNA for all heat treatments, we concluded that zein transcript levels would more accurately be expressed relative to 18S rRNA levels.

Previous studies showed that the 27-kDa and 19- plus 22-kDa zein protein accumulation of 4 DHS endosperms was significantly lower than those of control and 2 DHS endosperms up to 17 and 14 DAP, respectively (Monjardino et al., 2005). The 19- plus 22-kDa zein levels were significantly higher in control endosperms as compared to 4 DHS endosperms at 20 DAP. Hence, the 4 DHS treatment delays zein protein accumulation in developing kernels, whereas the 2 DHS treatment had no significant effect. The developmental pattern of the 27-kDa zein mRNA steady-state levels mimics its protein levels for all heat treatments. These data suggest that the heat stress delay in the accumulation of the 27-kDa zein may result from altered transcription rates.

The developmental pattern of the ZSF4C1 zein mRNA accumulation (Fig. 2) was different than the 19- plus 22-kDa zein protein levels (Monjardino et al., 2005). The ZSF4C1 is one of the most highly expressed members of the 19- plus 22-kDa zeins. The 19- plus 22-kDa zein levels increased up to 17 DAP for all heat treatments, whereas ZSF4C1 mRNA steady-state levels reached a plateau at 14 DAP. Another dissimilarity is that at 20 DAP the mRNA steady-state levels of all heat treatments did not differ significantly from controls (Fig. 2B), whereas the protein levels of control and 4 DHS were significantly different (Monjardino et al., 2005). However, the ZSF4C1 zein mRNA steady state levels were reduced by approximately 50% by 4 DHS treatment at 14 DAP.

The rate of transcription was tested as a possible mechanism for the reduction of mRNA steady state levels. The 27-kDa and ZSF4C1 zein transcription rates were delayed by the 4 DHS, in a very similar pattern to the effects of heat stress on mRNA steady-state levels (Fig. 2, 7). These data suggest that the impairment of transcription after heat stress (at 14 and 17 DAP for the 27-kDa zein and at 14 DAP for the ZSF4C1 zeins) may have resulted in the delay of protein (Monjardino et al., 2005) and mRNA accumulation. Later in kernel development (20 DAP), the rate of transcription, the mRNA steady state levels, and the protein levels of the ZSF4C1 zeins do not follow a similar pattern (Fig. 2B, 7B; Monjardino et al., 2005). An explanation may be that late in endosperm development, ZSF4C1 zein transcription may compensate for earlier delays in accumulation due to the 4 DHS treatment. Transcription of the 27-kDa and ZSF4C1 zeins is disrupted by heat stress.

Despite the delay in endoreduplication, 2 DHS endosperms had equivalent steady-state levels of the 27-kDa and ZSF4C1 mRNA and of zein proteins compared to control endosperms. Moreover, the 4 DHS treatment repressed endosperm endoreduplication, but it only delayed the 27-kDa and ZSF4C1 transcription rates, mRNA, and protein accumulation (Monjardino et al., 2005). Therefore endoreduplication and zein transcription levels are differentially affected by heat stress.

The methodology used to isolate nuclei for the transcription run-on assays is based on the separation of nuclei from starch granules by density and resulted in the preferential loss of higher C class nuclei (Fig. 6). However, the transcription levels of the 27-kDa and of the ZSF4C1 zein genes followed a pattern similar to their mRNA steady state levels for most of the sampling dates and heat treatments tested. The function of endoreduplication is generally thought to increase cell metabolic output, mainly by affecting gene expression (Edgar and Orr-Weaver, 2001). Zhao and Grafi (2000) demonstrated that endoreduplicated cells of maize endosperm have a decreased ratio of histone H1/DNA and increased hypophosphorylation levels of HMG-I/Y, as compared with mitotic endosperm cells. Considering that (i) histone H1 is widely known to have a repressive effect on transcription of several genes by promoting compaction of DNA; (ii) hypophosphorylation of HMG-I/Y up-regulates transcription of several genes by assembling and stabilizing complexes of transcriptional factors; and (iii) the 27-kDa zein gene promoter region contains short homopolymeric runs of dA·dT base pairs, a preferential binding site for HMG-I/Y proteins; Zhao and Grafi (2000) propose that endoreduplication increase this zein gene expression. Therefore, the alteration of endoreduplication may result in the delay of zein gene transcription.

However, the lack of correlation between endoreduplication and transcription rates leads to the conclusion that the effects of brief periods of heat stress during endosperm cell division on zein synthesis are not directly associated with endoreduplication. This conclusion is supported by the data of Dolfini et al. (1992), which demonstrated that the inner endosperm cells with higher levels of endoreduplication do not significantly transcribe zein genes. The experiments of Leiva-Neto et al. (2004) further support the lack of correlation between endoreduplication reduction and zein gene expression. However, a role for endoreduplication in promoting gene expression, especially of the genes involved with metabolism of starch and storage proteins is not ruled out by these experiments.

Reduced zein transcription rates of 4 DHS kernels could be due to the reduction of transcription factor activity. Indeed, Opaque2 DNA binding activity is regulated by a phosphorylation–dephosphorylation mechanism that appears to be affected by environmental conditions (Ciceri et al., 1997). Our results do not confirm nor refute this hypothesis. However, none of the characterized factors that bind to the "endosperm box" are capable of affecting a large number of zein genes (Müller et al., 1995). Therefore, a reduction in zein accumulation would require reductions in a majority of the transcription factor activities.

Heat stress has pleiotropic effects including the inhibition of cell division, endoreduplication, starch accumulation, and protein accumulation (Jones et al., 1984; Cheikh and Jones, 1994, 1995; Commuri and Jones, 1999; Engelen-Eigles et al., 2000) that delays kernel development. Hence, by 11 to 14 DAP most of the 4 DHS endosperm cells were probably at an earlier developmental stage, which is also supported by the pattern of poly-A RNA accumulation in developing endosperms (Fig. 1B). As a result, they are also delayed in zein gene transcription.

In summary, heat stress imposed early in the cell division stage resulted in significant delays in zein mRNA steady-state levels, which may have been caused in part by reduced rates of zein gene transcription. The repression of endoreduplication caused by heat stress was not directly associated with the reduced transcription of zein genes.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Joachim Messing for providing a genomic subclone of the 27-kD zein and Dr. Irwin Rubenstein for providing a cDNA subclone of the ZSF4C1 zeins. We thank Dr. John Murray, Jeff Roessler, Rod Felsheim, and Fabíola S. Gil for their critical discussion and technical support. This research was supported in part by the Minnesota Agricultural Experiment Station. Paper No. 051210160.

Received for publication March 1, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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