Published online November 21, 2006
Zein Transcription and Endoreduplication in Maize Endosperm are Differentially
Affected by Heat Stress
Paulo Monjardino, Alan G. Smith,* and Robert J. Jones
ABSTRACT
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
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
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, tran-
scription 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 alter-
ations in endoreduplication. Instead, zein transcription is most likely
affected by a delay in endosperm development.
T HE 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 sub-
sequent maize kernel growth and development by
reducing cell division and amyloplast biogenesis, result-
ing 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 re-
duction of enzyme levels, activities and the transcript
steady-state levels of acid invertase, ADP glucose pyro-
phosphorylase, 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 con-
centration is significantly reduced during early develop-
mental 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
P. Monjardino and R.J. Jones, Dep. of Agronomy and Plant Genetics;
A.G. Smith, 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 Heroı́smo, Portugal.
Received 1 Mar. 2006. *Corresponding author (alan@cbs.umn.edu).
Published in Crop Sci. 46:2581–2589 (2006).
Seed Physiology, Production & Technology
doi:10.2135/cropsci2006.03.0136
ª Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
2581
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 depend-
ing 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 di-
vided 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 ker-
nel development (Marks et al., 1985; Woo et al., 2001).
Zein accumulation has been correlated with high lev-
els 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 regu-
lated mainly at the transcriptional level (Motto et al.,
1989) but can also be regulated at the post-transcrip-
tional level (Plotnikov and Bakaldina, 1996). Opaque-2
is one of the few genes identified whose product inter-
acts 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 tran-
scription (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 with-
out cytokinesis, is an important process for maize endo-
sperm development. Around 10 to 12 DAP, following the
cessation of mitotic activity, the S-phase alternates with
Abbreviations: CDK, cyclin-dependent kinase; DAP, days after pol-
lination; DHS, days of heat stress; MI, mithramycin A; ZSF4C1, clus-
ter 1 zeins of subfamily 4.
 2582 CROP SCIENCE, VOL. 46, NOVEMBER–DECEMBER 2006
distinct gap phases that lack DNA replication resulting
in an exponential increase in nuclear DNA content of
endosperm cells. Both nuclear size and endoreduplica-
tion 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 por-
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
tion of the endosperm, which are the most endoredupli-
cated, can be as high as 1558C, 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 in-
duction, and the subsequent S-phase CDKs disassembly
allows subsequent rounds of replication (reviewed in
Edgar and Orr-Weaver, 2001; Larkins et al., 2001). There-
fore, 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 pro-
tein 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 endore-
duplication and gene expression, because the more endo-
reduplicated cells accumulate less storage proteins and
have lower zein mRNA levels than the peripheral endo-
sperm cells (Dolfini et al., 1992; Woo et al., 2001). More-
over 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 endoredupli-
cation to determine their interaction.
MATERIALS AND METHODS
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 be-
tween 600 and 900 mmol m22 s21, at a 25/208C (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 (25/208C continuously) or heat treat-
ments (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 Corpora-
tion, 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 358C (6 0.18C) 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 de-
scribed 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 2808C. Endosperms used for the
transcription run-on assays and for flow cytometry were im-
mediately handled after sampling, according to the procedures
described below.
RNA Extraction
To isolate total RNA, 400 to 600 mg of frozen (2808C)
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 con-
ducted in RNAase free conditions, and the extracted samples
were kept at 2808C until their analysis.
Northern Blots
To assess RNA intactness, total RNA (5 mg) was denatured
at 658C for 10 min in 13 F buffer (20 mM 3-[N-morpholino]
propanesulfonic acid, pH 7.0, 1 mM ethylenediaminetetra-
acetic 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 13 F buffer, 6% formaldehyde (v/v), and
0.05 mL 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 ny-
lon membrane (NEN, Dupont, Boston), according to the manu-
facturer’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 de-
scribed 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 spe-
cifically 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 ly-
copersicum L.) (Perry and Palukaitis, 1990). Each probe was la-
beled with [a-32P]dCTP (specific radioactivity 3000 Ci mmol21,
10 mCi mL21) by random oligo labeling with Megaprime
labeling kit (Amersham, Piscataway, NJ), following the manu-
facturer’s instructions. To assess the level of mRNA on each
membrane, polyuridylic acid (poly-U) oligonucleotides were
end labeled with T4 polynucleotide kinase and [g-32P]ATP
(specific radioactivity 3000 Ci mmol21, 10 mCi mL21).
 MONJARDINO ET AL.: HEAT STRESS AND ZEIN ACCUMULATION
For random oligo labeling probes, filters were prehybridized
and hybridized at 428C in 50% (v/v) formamide, 53 Denhardt’s
(0.1%w/v Ficoll, 0.1% w/v polyvinylpyrrolidone, 0.1% w/v
bovine serum albumin), 53 SSPE (5 mM Na2EDTA, 50 mM
NaH2PO4, pH 7.5, 0.9 M NaCl), 1% (v/v) lauryl sulfate, and
100 mg mL21 salmon sperm DNA (Sambrook et al., 1989).
Prehybridization and hybridization conditions with [g-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 solu-
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
tions contained only 1% (v/v) lauryl sulfate and 1 M sodium
chloride, and hybridization was conducted at 508C. For suc-
cessive hybridizations, filters were stripped according to the
manufacturer’s specifications.
RNA Dot Blots
RNA samples were bound to Genescreen Plus nylon mem-
branes 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 five-
fold 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 b-mercaptoethanol) and stored at 2808C. DNA con-
tent 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 stan-
dard (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 mg of DNA were used for each in-
dividual run-on reaction which contained 25 mM Trizma hy-
drochloride, pH 7.8, 5 mM magnesium chloride, 10% (v/v)
glycerol, 5 mM b-mercaptoethanol, 75 mM ammonium sul-
fate, 0.5 mM ATP, CTP, and GTP, 75 units of RNasin, and
300 mCi of [a-32P]UTP (specific radioactivity 3000 Ci mmol21,
10 mCi mL21), in a total volume of 100 mL. To adjust osmo-
lality 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 hori-
zontal. Reactions were stopped on ice by the addition of
200 units of DNAase I and 100 mmol UTP and then incubated
at room temperature for 10 min. For RNA extraction, 120 mL
of 103 SET (1 mM Trizma hydrochloride, pH 7.5 and 120 mg
proteinase K) were added. One extraction with 1 volume (V)
of phenol/chloroform/isoamyl alcohol (1:1:0.04) and one ex-
traction with 1 V of chloroform/isoamyl alcohol (99:1) were
performed. The solution was adjusted to 0.3 M sodium ace-
2583
tate, RNA was precipitated with 2.5 V ethanol overnight at
2208C, 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 mem-
branes 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 manu-
facturer’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 428C in 50% formamide (v/v), 53 Denhardt’s, 53
SSPE, 1% (v/v) lauryl sulfate, and 100 mg mL21 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 428C, and filters were washed according to the manu-
facturer’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 deter-
mine nonspecific hybridization and the background was sub-
tracted 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 (Molec-
ular 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 de-
bris from the analysis. Fifteen hundred nuclei were counted for
each sample.
For nuclei counting, samples were diluted in MI buffer (de-
scribed 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 nu-
clei 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,
 2584 CROP SCIENCE, VOL. 46, NOVEMBER–DECEMBER 2006
there were three or four replicates. Transcription run-on as-
says were replicated twice, due to a limited number of ker-
nels. After the ANOVA, the means were compared by LSD
at 5 and 1% levels of significance.
RESULTS
RNA Analysis
Heat stress disrupts protein accumulation in maize
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
kernels with the greatest reductions in zein levels at
early developmental stages (Monjardino et al., 2005).
The relative 18S rRNA content was similar across treat-
ments, except at 17 DAP (Fig. 1A). However, the rela-
tive 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 rela-
tive poly-A RNA or 18S rRNA content. Since mea-
surements 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.
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 abun-
dance of these transcripts during endosperm develop-
ment. 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).
The ZSF4C1 set of the 19- plus 22-kDa zeins con-
tains 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).
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. Com-
parison of means was done by LSD; a and b indicate statistically
different values at 5% (*) and 1% (**) levels of significance.
 MONJARDINO ET AL.: HEAT STRESS AND ZEIN ACCUMULATION 2585

Endoreduplication
Endosperm endoreduplication was analyzed by eval-
uating the proportion of nuclei in each individual C class
(1C being the maize haploid DNA content). The endo-
reduplication 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
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

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.
In the case of the control endosperms, the DNA levels
per nucleus ranged from 3C (nonendoreduplicated trip-
loid tissue) to 96C (triploid tissue that has gone through
five endoreduplication cycles) (Fig. 4). The 2 DHS en-
dosperm’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 non-
endoreduplicated 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).

Run-on Transcription Analysis

Endosperm nuclei were isolated from the same ears
used to analyze transcript and endoreduplication levels.

Fig. 4. Relative proportion of endosperm nuclei in each C class, sam-

pled after 0 (ct), 2 (2d), or 4 (4d) days of heat stress (DHS) treat-
ments. (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.

Nuclei yields were similar to those reported by Das et al.

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.
(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).
The 4 DHS treatment delayed the increase in tran-
scription rate of the 27-kDa zein from 14 to 17 DAP,
 2586 CROP SCIENCE, VOL. 46, NOVEMBER–DECEMBER 2006
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
Fig. 5. Average C values of nuclei isolated for transcription run-on
assays after 0 (ct) or 4 (4d) days of heat stress (DHS).
but at 20 DAP the transcription rates of this gene re-
covered to equal or higher levels than those of control
kernels (Fig. 7A). Transcription of the ZSF4C1 zein
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.
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 transcrip-
tion. (B) ZSF4C1 zein rate of transcription. Rates were normalized
relative to 18S rRNA.
genes was lower at 14 DAP, similar at 17 DAP, and
greater than controls at 20 DAP (Fig. 7B).
DISCUSSION
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 rela-
tive 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 glob-
ulin 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. Con-
sidering the less stable levels of poly-A RNA for all
heat treatments, we concluded that zein transcript lev-
els 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 endo-
sperms 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
 MONJARDINO ET AL.: HEAT STRESS AND ZEIN ACCUMULATION
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 pro-
tein 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.
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
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 devel-
opment (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 transcrip-
tion 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 endo-
sperms 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 transcrip-
tion levels are differentially affected by heat stress.
The methodology used to isolate nuclei for the tran-
scription 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 sam-
pling dates and heat treatments tested. The function of
endoreduplication is generally thought to increase cell
metabolic output, mainly by affecting gene expression
2587
(Edgar and Orr-Weaver, 2001). Zhao and Grafi (2000)
demonstrated that endoreduplicated cells of maize en-
dosperm 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 repres-
sive effect on transcription of several genes by promot-
ing compaction of DNA; (ii) hypophosphorylation of
HMG-I/Y up-regulates transcription of several genes
by assembling and stabilizing complexes of transcrip-
tional 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 pro-
teins; Zhao and Grafi (2000) propose that endoredupli-
cation 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 endoredu-
plication 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 di-
rectly associated with endoreduplication. This conclu-
sion 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 be-
tween endoreduplication reduction and zein gene expres-
sion. 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 regu-
lated by a phosphorylation–dephosphorylation mecha-
nism 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 char-
acterized 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 transcrip-
tion factor activities.
Heat stress has pleiotropic effects including the inhibi-
tion of cell division, endoreduplication, starch accumula-
tion, 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 di-
vision 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 re-
pression of endoreduplication caused by heat stress was
not directly associated with the reduced transcription of
zein genes.
 2588 CROP SCIENCE, VOL. 46, NOVEMBER–DECEMBER 2006
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 Fabı́ola S.
Gil for their critical discussion and technical support. This
research was supported in part by the Minnesota Agricultural
Experiment Station. Paper No. 051210160.
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
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