Plant Physiol. (1998) 116: 485-494
Developmental and Hormonal Regulation of Genes Coding for
Proline-Rich Proteins in Female Inflorescences and Kernels of
Maize1
Matilde Josè-Estanyol and
Pere Puigdomènech*
Departament de Genètica Molecular, Centre
d'Investigació i Desenvolupament, Consejo Superior de
Investigaciones Científicas, Jordi Girona Salgado 18-26, 08034 Barcelona, Spain
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ABSTRACT |
The pattern of expression of two
genes coding for proteins rich in proline, HyPRP
(hybrid proline-rich protein) and HRGP
(hydroxyproline-rich glycoprotein), has been studied in maize
(Zea mays) embryos by RNA analysis and in situ
hybridization. mRNA accumulation is high during the first 20 d
after pollination, and disappears in the maturation stages of
embryogenesis. The two genes are also expressed during the development
of the pistillate spikelet and during the first stages of embryo
development in adjacent but different tissues. HyPRP
mRNA accumulates mainly in the scutellum and HRGP mRNA
mainly in the embryo axis and the suspensor. The two genes appear to be
under the control of different regulatory pathways during
embryogenesis. We show that HyPRP is repressed by
abscisic acid and stress treatments, with the exception of cold
treatment. In contrast, HRGP is affected positively by
specific stress treatments.
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INTRODUCTION |
The use of recombinant DNA techniques has led to the
identification and sequence determination of a number of structural
protein components of the plant cell wall (Cassab and Varner, 1988;
Showalter, 1993
). These include different types of HRGPs and Pro-rich
proteins, Gly-rich proteins, arabinogalactan proteins, and lectins. In
most of these the protein sequence is highly repetitive. A good example in maize (Zea mays) is HRGP, one of the main proteins
extractable from the cell wall (Kieliszewski and Lamport, 1987; Stiefel
et al., 1988). Its mRNA is abundant in dividing maize tissues and highly accumulated in provascular cells (Stiefel et al., 1990). The
HRGP gene responds to mechanical stress and to ethylene
treatment by increasing its mRNA levels (Tagu et al., 1992). The
protein is rich in Pro, Lys, and Thr, and these amino acids form the
repetitive motifs that constitute most of its primary structure, which
is polymorphic between different maize varieties (Raz et el., 1991). Homologous proteins have been described in related species such as
teosinte, sorghum, and rice (Raz et al., 1991, 1992; Caelles et al.,
1992).
Recently, a number of proteins other than HRGPs that are also rich in
Pro or Gly have been described in several plant species. These proteins
have characteristic signal peptides (probably necessary for the
translocation of the protein to the cell wall), repetitive Pro- or
Gly-rich domains, and a Cys-rich C-terminal domain that sometimes shows
similarity to proteins with a defensive or a storage function. Because
of the presence of two domains with such distinct features, these
proteins have been called hybrid Pro-rich proteins (Josè and
Puigdomènech, 1993, 1994). In at least two cases (Cheung et al.,
1993; Domingo et al., 1994) proteins belonging to this class have been
located in the cell wall using immunologic techniques.
A gene encoding a protein with a Pro-rich domain and
a hydrophobic, Cys-rich C-terminal domain has been described in maize and named HyPRP (zmHyPRP) (Josè-Estanyol et al.,
1992). Partially homologous sequences have been described in other
plant species (Salts et al., 1992). The mRNA corresponding to this gene
in maize accumulates mainly during embryogenesis. A low level of mRNA
accumulation was also found in the pistillate spikelet before anthesis
(Josè-Estanyol et al., 1992). Maize embryogenesis follows a
characteristic pattern of development (Abbé and Stein, 1954).
After anthesis, a single-celled zygote develops by division into the
proembryo, which is radially symmetric. At the transition stage
asymmetry is introduced by the formation of an internal, wedge-shaped
meristematic region in the upper part of the embryo. At the coleoptilar
stage this region gives rise to the shoot apex, the surrounding
coleoptilar ring, and the root apex. After leaf primordium
differentiation, subsequent stages follow. After the embryogenic
period, ABA is induced and embryo maturity (during stage 6) is finally
attained (Quatrano, 1987; Sheridan and Clark, 1987).
In situ hybridization studies indicated that at stage 2 of maize
embryogenesis, the HyPRP gene is expressed in parenchymal cells surrounding the developing vascular system in the embryo axis, as
well as in subepidermal cells, showing a high level of mRNA
accumulation in the scutel-lum (Josè-Estanyol et al., 1992). In contrast, the accumulation of HRGP mRNA shows a pattern
complementary to that observed for HyPRP in the sense that
in the immature embryo one gene is expressed in tissues in which the
other is not expressed. In particular, HRGP mRNA is found
abundantly in axis provascular cells, where HyPRP is not
found (Josè-Estanyol et al., 1992; Ruiz-Avila et al., 1992). The
mRNA of HyPRP disappears when ABA accumulation begins in the
developing embryo with the initiation of the maturation and dessication
phases (Jones and Brenner, 1987). We have proposed (Josè-Estanyol
et al., 1992) that ABA could repress HyPRP expression, as
its mRNA level is maintained in ABA-deficient maize mutants
(vp2). In this paper we describe the cell types in which
HyPRP and HRGP are expressed in the pistillate
spikelet at different stages of ear development and during the first
stages of embryo development before leaf primordia appearance. The
effects of ABA and different physiological situations have also been
studied.
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MATERIALS AND METHODS |
Biological Materials
Unless otherwise stated, the plant material used was derived from
seeds of a maize (Zea mays cv W64A) pure inbred line grown in a greenhouse in Barcelona, Spain. Spikelets, kernels, and excised treated and untreated immature embryos were collected at different developmental stages and stored at 
80°C. For stress treatments immature embryos excised at stage 2 of development were treated with a
hormone solution (10 µm ABA), as described by Vilardell et al. (1990), a high-osmoticum solution (0.25 m NaCl), or
cold treatment (4°C) for different times. As a control, excised
embryos were placed in a humid chamber. Heterozygous caryopses of the viviparous-2 (vp2) mutant of maize were obtained
from Dr. R.J. Lambert (Maize Genetic Stock Center, University of
Illinois, Urbana). Homozygous vp2 kernels at stage 2 were
selected on the basis of white, colorless endosperm.
RNA Preparation and Gel-Blot Analysis
RNA was extracted by the guanidinium-HCl procedure (Logemann et
al., 1987). All RNAs were checked for RNA quality by nondenaturing electrophoresis on 1.5% agarose gels and by EtBr staining, and the
concentrations were adjusted by reading the
A260. Total RNAs were separated on gels
containing 1.5% agarose-formaldehyde (Lehrach et al., 1977). The gels
were blotted onto nylon membranes (Hybond-N, Amersham), treated as
described by the manufacturer, and hybridized at 65°C for at least
12 h in a phosphate solution (Church and Gilbert, 1984) with
specific probes. Fragments from the HyPRP (Josè-Estanyol et al., 1992) and HRGP (Raz et al.,
1992) coding regions and from the RAB28 (Pla et al., 1991)
and H4 (Philipps et al., 1986) coding and 3
-nontranslated
regions were labeled by random priming to a specific activity of
108 cpm/µg. After hybridization, filters were
washed to 20 mm phosphate stringency at 65°C and
autoradiographed. Experiments were repeated at least three times.
In Situ Hybridization
Ears or their excised spikelets, kernels, and treated and
untreated immature embryos were collected at different developmental stages and submerged in 84:11:5 ethanol:formaldehyde:glacial acetic acid fixing solution. The whole procedure of in situ hybridization, including paraffin inclusion and tissue dissection in a microtome (Reichert, Germany), was performed essentially according to the method
of Langdale et al. (1988). A 670-bp DdeI and a 660-bp
FockI-SnabI fragment of the 5-coding region of HyPRP and
HRGP genes, respectively, were cloned in a pBluescript SK+
vector (Stratagene) and used as a template for the synthesis of sense
and antisense riboprobes. Transcripts from T3 and T7 promoters were
produced following the instructions of the manufacturers, using
35S-CTP (37 TBq mmol 
1;
Amersham). The final concentration of the probes was 0.1 µg/mL, and
each slide was hybridized with 0.5 kBq of labeled probe. Hybridization was then performed as described previously (Langdale et al., 1988). The
slides were exposed using Kodak NTB-2 emulsion, and stained after
developing with 0.5% (w/v) fast green in 95% ethanol or 1% toluidine
blue in 1% sodium tetraborate. The photographs were taken using an
automated camera on a light microscope (Axiophot, Zeiss).
| |
RESULTS |
Developmental Expression of HyPRP and
HRGP Genes in Maize Ears
The gene coding for the maize HyPRP is mainly expressed in young
embryos and has a low level of expression in developing female inflorescences (Josè-Estanyol et al., 1992). HRGP mRNA
accumulates throughout embryo development, mainly where the level of
mRNA corresponding to genes expressed in proliferating tissues, such as
histone genes, is very high (Ruiz-Avila et al., 1992). In this study
the accumulation of HyPRP and HRGP mRNA was
measured using RNA analysis and in situ hybridization techniques during
the development of the pistillate spikelet, the immature female flower
(Kiesselbach, 1980; Schmidt et al., 1993). Several stages of
development were studied, from undifferentiated ear spikelet primordia
to just before anthesis. At this stage differentiation is completed and silks are just longer than the ear (2.5-mm pistillate spikelet diameter). Nonpollinated pistillate spikelets up to 4.0 mm in size were
also studied.
HyPRP mRNA accumulated in developing pistillate spikelets
until anthesis (Fig. 1A). At this point,
HyPRP expression decreased and disappeared when pollination
did not occur (Fig. 1A). Following anthesis a low level of mRNA was
observed in young kernels, which reached a maximum around 6 to 10 d after pollination, when the maize embryo begins to develop
(Josè-Estanyol et al., 1992). HyPRP mRNA was not
observed in the silk (Fig. 1A). Conversely, when the HRGP
probe was used, a high level of mRNA accumulation was observed in the
silk and, to a lesser extent, in the spikelet, where expression
increased when anthesis did not occur (Fig. 1A). After pollination
HRGP expression is maintained in the kernel (Ruiz-Avila et
al., 1991).
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| Figure 1.
Accumulation of HyPRP and
HRGP mRNA in different tissues. A, Accumulation of
HyPRP and HRGP mRNA in maize female
inflorescences. Total mRNA (30 µg per lane) from maize ears, from
their isolated pistillated spikelets, or from their respective silks at
different stages of ear development were subjected to electrophoresis
on agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with HyPRP and HRGP probes.
Exposure time was 10 d for HyPRP and 1 d for
the HRGP probe, with the exception of silk samples,
which were exposed for only 2 h. 1, Ear length of 0.6 cm; 2, excised ear spikelets sharing 0.5-cm silk length; 3 through 5, excised
ear spikelets without silks (at left) or their corresponding silks (at
right). The stage of development is indicated by the length of the
respective silks: 3, 4.5-cm silk length; 4, 10.5-cm silk length; 5, silks just extending above the ear; 6 and 7, excised ear spikelets
without silks (at left) or their corresponding silks (at right),
excised from an unpollinated ear after anthesis time. The stage of
development is indicated by the number of days after anthesis time: 6, 5 d; 7, 12 d. The arrow indicates anthesis (An) time in ear
development. B, RNA gel blot of mRNA extracted from excised mature,
dehydrated, and germinated maize embryos. E30, E40, E50, and E60, immature embryos
30, 40, 50, and 60 d after pollination, respectively;
Eax60 and Esc60, embryo axis and scutellum, respectively, from dry kernels 60 d after pollination; 1d to 4d, excised coleoptile-nodular region from embryos 1 to 4 d after germination. Exposure time was 10 d for the HyPRP
probe and 1 d for HRGP.
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Accumulation of HyPRP mRNA in maize embryo begins to
decrease progressively after 18 d of anthesis (Josè-Estanyol
et al., 1992), and accumulation of HRGP begins to decrease
after 30 d (Ruiz-Avila et al., 1991). Neither HyPRP nor
HRGP mRNA was detectable during maturation of the embryo
(between 40 and 60 d after pollination) or in the dry embryo (Fig.
1B). Both genes began to be re-expressed in the embryo shortly after
germination, when the growth of the plant began. HyPRP mRNA
was transiently observed at a low level, but was no longer detectable
during vegetative growth, whereas HRGP mRNA accumulated at
high levels in the plantlet (Fig. 1B).
In situ hybridization was carried out to define the pistillate spikelet
cell types responsible for HyPRP and HRGP mRNA
accumulation. Figure 2 shows the results
of such a study on a 2-mm longitudinal section placed in the middle of
a 6-mm ear. In this zone it is possible to observe immature female
flowers at different stages of development (Kiesselbach, 1980; Schmidt
et al., 1993). HyPRP mRNA was accumulated at a very low
level in parenchymal cells present at the border of each
differentiating spikelet below the differentiating floral primordia
(Fig. 2a). This is the region of the ear where HRGP
expression is localized in the floret primordia below the epidermis and
between growing spikelets (Fig. 2b).
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| Figure 2.
Cellular localization of HyPRP and
HRGP mRNA in longitudinal sections from maize female
inflorescences. a and b, Longitudinal section from maize ear at a very
early stage of spikelet organ differentiation. In situ hybridization of
an HyPRP (a) and an HRGP (b) antisense
RNA probe with a longitudinal section from an ear 0.6 cm in length.
HyPRP slides were exposed for 22 d, whereas HRGP was exposed for 7 d. c through g, Longitudinal
sections from mature pistillate spikelets of maize just before anthesis
time. c, In situ hybridization of an HyPRP sense RNA
probe with a mature pistillate spikelet external longitudinal section.
d through e, In situ hybridization of an HyPRP antisense
RNA probe with a mature pistillate spikelet central and external
longitudinal section, respectively. f through g, In situ hybridization
of an HRGP antisense RNA probe with a mature pistillate
spikelet and central and external longitudinal sections, respectively.
Bars = 250 µm (a and b) and 200 µm (c-g).
HyPRP slides were exposed for 18 d, whereas
HRGP was exposed for 7 d. es, Embryo sac; fp,
floral primordia; i, integuments; n, nucellus; ow, ovary wall; p.ch,
placento-chalazal region; pv, provascular tissues; r, rachis; s, silk;
sp, spikelet primordia; and vt, vascular tissue. All of the sections
were examined under dark-field microscopy. Hybridization is visible as
blue areas.
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Figure 2, c through g, shows the analysis of two different longitudinal
sections of a young maize pistillate spikelet just before anthesis,
when the different cells of the vascular tissue, placento-chalazal
region, ovary, and embryo sac are well differentiated and the silks
nearly extend above the ear (10.5 cm). In an internal longitudinal
section containing the embryo sac, HyPRP mRNA (Fig. 2d)
appeared to be nearly absent from the flower, where only a low level of
expression remains around the placento-chalazal region, but not around
the differentiated vascular tissue. In the accompanying abortive
flower, which is in an earlier developmental stage similar to the one
observed for 0.5-cm, silk-length spikelets (not shown), HyPRP mRNA was abundant in the cells surrounding the
provascular emerging cells. When adjacent sections were hybridized with
the HRGP probe (Fig. 2f), mRNA was observed around the
embryo sac in the nucellus, in the ovary wall epidermis, and in the
stylar canal. In the differentiated vascular tissue, expression was
localized in cells accompanying fibers and xylem-differentiated
structures.
When an external longitudinal section from the same stage of
development was studied, HyPRP mRNA was only accumulated in
the area surrounding the placento-chalazal region (Fig. 2e). In a similar section, HRGP mRNA (Fig. 2g) was accumulated in
cells placed at the internal border of the placental side of the
placento-chalazal region, as well as in the nucellus, in the epidermis
of ovary wall and in the silk. The same results were observed when
similar longitudinal sections of spikelets in an intermediate stage of development (silk lengths of 4.5 cm) were studied. The only difference was that at this stage starch grains in the cortex were present (data
not shown).
mRNA Accumulation of HyPRP and HRGP
in Maize Kernels
In situ hybridization (Fig. 3) of
kernels between anthesis and 9 d after pollination showed
that at the proembryo and transition phase of embryo development (Fig.
3, a and d), HyPRP mRNA was faintly detected in the
embryo, whereas HRGP mRNA was already detectable in a polar
manner in cells in which the suspensor was beginning to be developed
(Fig. 3, b and e). HRGP mRNA continued to be highly
abundant in the suspensor until the coleoptilar stage was
reached (Fig. 3, h and k). Dark (Fig. 3, b, e, h, and k)- and bright
(Fig. 3, c, f, i, and l)-field exposures corresponding to the same
sections for HRGP are presented for a better comparison of
the anatomy of the region where the gene is expressed. In contrast, HyPRP mRNA was not clearly observed until the embryo reached
the coleoptilar stage (Fig. 3, g and j). At this time HyPRP
was accumulated in the regions differentiating to scutellum.
HyPRP mRNA was also observed at this stage in the first
layer of endocarp cells of developing pericarp (Fig. 3j). At this time
the HRGP mRNA was accumulated in the suspensor and in cells
surrounding the emerging shoot meristem (Fig. 3k), as well as in the
developing exocarp (not shown).
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| Figure 3.
Cellular localization of HyPRP and
HRGP mRNA in longitudinal sections from maize kernel. a
through c, In situ hybridization with HyPRP (a) and
HRGP (b-c) antisense probes with longitudinal sections
of maize kernels at the proembryo stage of development. d through f, In
situ hybridization with HyPRP (d) and
HRGP (e-f) antisense probes with longitudinal sections
of maize kernels at embryo transition phase of development. g through
i, In situ hybridization with HyPRP (g) and
HRGP (h-i) antisense probes with longitudinal sections
of maize kernel at embryo initial coleoptilar stage of development. j
through l, In situ hybridization with HyPRP (j) and
HRGP (k and l) antisense probes with maize kernels at
embryo coleoptilar stage of development. Bars = 50 µm (a, b, d,
and e); 25 µm (c and f); 100 µm (g-i); and 250 µm (j-l).
Sections were examined under light- or dark-field microscopy.
Hybridization is visible as blue areas in a, b, d, e, g, h, and k, and
as green areas in j.
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Accumulation of HyPRP and HRGP mRNA
in Maize Embryos under Different Physiological Conditions: Effect
of ABA
HyPRP is expressed preferentially in immature embryos
during morphogenesis, and its mRNA level disappears when the maturation stage starts after ABA begins to accumulate in the embryo (Quatrano, 1987; Josè-Estanyol et al., 1992). It has been proposed that ABA
could repress HyPRP expression, as its mRNA levels are
maintained until the onset of germination in maize vp2
mutant plants that are deficient in ABA (Josè-Estanyol et al.,
1992). An ABA-responsive element (Guiltinan et al., 1990) is present in
its promoter (Josè-Estanyol et al., 1992). RNA blots were used to
analyze the effect of exogenous ABA on HyPRP expression in
freshly excised, immature maize embryos at stage 2 of development from
wild type (Fig. 4) and in vp2
mutant maize plants (Robertson, 1955) (Fig.
5).
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| Figure 4.
Accumulation of HyPRP and
HRGP mRNA in young embryos after different stresses.
Total RNAs (10 µg) from young, freshly excised embryos 16 d
after pollination (at stage 2 of embryo development) subjected to the
treatments indicated or not treated (control) were subjected to
electrophoresis and hybridized with HyPRP,
HRGP, RAB28, and H4
probes. Hybridization and exposure times were similar for all blots.
Numbers indicate hours of treatment before freezing for RNA
extraction.
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| Figure 5.
RNA gel-blot analysis of mRNA extracted from
vp2 embryos after ABA treatment. Total RNAs (10 µg)
from vp2 embryos at stage 2 of development treated for
different times with ABA were subjected to electrophoresis and
hybridized with HyPRP, HRGP, and
RAB28 probes. Numbers indicate hours of treatment.
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HyPRP mRNA decreased dramatically in excised, immature
wild-type embryos after 6 h of incubation with exogenous ABA;
after 24 h it was undetectable (Fig. 4). In contrast, the mRNA
levels of RAB28, induced by ABA in late embryogenesis (Pla
et al., 1991), increased. When the effect of exogenous ABA on embryos
from vp2 mutant maize plants was studied a similar effect
was found (Fig. 5). This indicates that the applied hormone
simultaneously represses HyPRP and activates
RAB28. A control experiment using the same conditions but in
the absence of ABA is not possible because after 24 h in buffer
conditions, the embryos germinate and the expression of
HyPRP stops, as occurs during seed germination (see Fig.
1B). For this reason, we used embryos excised from the ear and
maintained in a humid chamber without being subjected to any stress or
damage as controls (Fig. 4). Under these conditions expression of
HyPRP and HRGP was maintained and expression of
RAB 28 was increased. HyPRP mRNA accumulation at
first decreased but in the absence of ABA the gene was not repressed
(Fig. 4).
Figure 4 also shows that HRGP expression can be arrested in
excised, immature wild-type embryos after 24 h of exogenous ABA treatment, although expression increased from 2 to 6 h. Similar results were observed for HRGP when the same experiment was
performed with immature vp2 mutant embryos (Fig. 5). In both
cases the mRNA levels increased slightly at the beginning of the ABA
treatment and then disappeared. When H4, a marker of cell division
(Philipps et al., 1986), was used cessation of H4 gene expression was
observed (Fig. 4) as a result of the arrest of cell division in the
induction of dormancy by ABA (Quatrano, 1987).
Aside from the regulation of HyPRP and HRGP mRNA
levels related to embryo development, specific physiological conditions
such as NaCl or cold treatment (4°C) may have an effect on mRNA
accumulation. Results shown in Figure 4 indicate that NaCl treatment of
excised embryos slightly increased HyPRP mRNA levels after
2 h of treatment, but that after 1 d these were reduced to
less than zero and were also lower than the control. ABA plus NaCl
treatments also increased HyPRP mRNA accumulation within
2 h of application, but after 6 h this was reduced to a low
level, as was also observed with ABA treatment. Cold treatment
gradually increased HyPRP mRNA (Fig. 4). HyPRP
was never induced by cold, wounding, or fungal attack in germinated or
young plants (results not shown). Salinity and cold treatment induced
an increased level of HRGP mRNA to a maximum around 6 h
of NaCl and 48 h of cold treatment. A simultaneous treatment with
ABA plus NaCl led to a maximum expression of HRGP after
2 h (Fig. 4).
The results described were correlated to those obtained from two other
genes expressed in the axis and scutellum of immature maize embryos,
RAB28, an ABA-responsive gene, and histone H4, a
marker of cell division. RAB28 gene expression was induced
after NaCl treatment. ABA plus NaCl showed a cooperative effect, which reached a maximum only 2 h after treatment, but after cold
treatment only low levels of mRNA accumulation were observed (Fig. 4).
In contrast, H4 expression was always arrested after cold or
NaCl plus ABA treatment and markedly diminished after NaCl treatment, which confirmed that after stress treatment the embryo cell division program was arrested, as it was after ABA treatment (Fig. 4).
Previous in situ hybridization studies (Josè-Estanyol et al.,
1992) indicated that HyPRP mRNA in immature embryos at stage 2 of development is observed mainly in parenchymal cells surrounding the developing vascular system in the axis and in cells with a high
level of mRNA transcription in the scutellum. In contrast, the
HRGP gene shows a pattern of expression mainly associated with tissues involved in vascular development on the axis of immature embryos but not in the scutellum. These two genes thus appeared to be
expressed in adjacent tissues. We wondered whether the changes of
expression that are shown by nothern analysis after stress treatment
have an effect on the tissue distribution revealed using in situ
hybridization. For cellular localization analysis, only results with
the HRGP probe are presented, as HyPRP mRNA
usually disappeared after stress treatment and no changes were observed after cold treatment (not shown).
In situ hybridization studies showed that when we compare the
hybridization pattern of HRGP on sections of embryos at
stage 2 of development without exogenous ABA treatment (Fig.
6a) with the one observed after 24 h
of exogenous ABA treatment (not shown) or after the appearance of high
ABA levels in the embryo (at stage 3) (Jones and Brenner, 1987), the
HRGP mRNA accumulation disappeared from the developing
vascular bundles of the coleoptile and foliar primordia (Fig. 6b),
although it was still observed in the parenchymal cells surrounding
them. No change was observed in a similar transverse section
corresponding to the radicular region. After cold treatment of immature
maize embryos at stage 2 of development, a modified pattern of
HRGP expression was also observed in a transverse section at
the coleoptilar level. HRGP mRNA levels increased in
parenchymal cells surrounding the vascular coleoptile bundle and below
the foliar primordium epidermis (see Fig. 6c and bright-field
microscope plates for a more detailed analysis).
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| Figure 6.
In situ hybridization of three transverse sections
of maize embryo coleoptiles. a, Location of HRGP mRNA in maize embryos 16 d after pollination (at stage 2 of development) examined under dark-field microscopy. b, Location of HRGP mRNA in maize
embryos 25 d after pollination (at stage 3 of embryo development)
examined under dark-field microscopy. c, Location of
HRGP mRNA in embryos 16 d after pollination (at
stage 2 of development) after 24 h of cold treatment at 4°C
examined under dark-field microscopy. d and e, Details of the same
section shown in c are examined under bright-field microscopy. d,
Foliar primordium detail. e, Vascular bundle detail of the coleoptile.
c, Coleoptile; e, epidermis; fp, foliar
primordium; sc, scutellum; sp, scutellar procambium; and vb,
vascular bundle. Bar = 250 µm for a through c, and 25 µm for d
and e. Hybridization is visible as blue-green areas in a through c, and
as dots in d through e.
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DISCUSSION |
HRGP and HyPRP belong to an expanding family
of plant genes encoding proteins with a Pro-rich domain (Josè and
Puigdomènech, 1993, 1994). In immature maize embryos,
HyPRP mRNA has been shown by in situ hybrization to
accumulate in cells having the features of parenchyma around the
emerging provascular cells of the embryo axis. In this organ
HyPRP mRNA is accumulated in tissues adjacent to but not
coincident with tissues accumulating HRGP mRNA, which is
expressed in axis provascular cells. In this organ, expression of these
genes appears to be mutually exclusive. Although the immature embryo is
the location of maximum levels of HyPRP mRNA, it has been
shown that HyPRP also appears to be expressed at low levels
in nonpollinated pistillate spikelets prior to anthesis as well as in
kernels 6 to 10 d after pollination. Another feature of this gene
is that its mRNA disappears (Josè-Estanyol et al., 1992) from the
embryo when the maturation stage is attained and the ABA content
increases (Jones and Brenner, 1987). The two main aims of the present
report were: (a) to analyze the cell types accumulating
HyPRP and HRGP mRNA in developing female
inflorescences and kernels of maize at the first stages of development
to understand how the expression of these genes is regulated as a
function of the development of specific cell types; and (b) to analyze
the response of these genes under hormonal and stress treatments in the
embryo.
The HyPRP mRNA during the development of the pistillate
spikelet (Kiesselbach, 1980; Schmidt et al., 1993) is mainly associated with parenchymal cells surrounding initial floral meristems, initial developing vascular tissue, or the placento-chalazal region; when anthesis is achieved it disappears. The HRGP mRNA is very
abundant in the floral meristem during spikelet differentiation and
then decreases until pollination takes place. In the silk HRGP mRNA levels are always very high (Fig. 1). During development of the pistillate spikelet, HRGP mRNA is observed in the
provascular cells, where it has been observed in other organs (Stiefel
et al., 1990). After anthesis, HRGP mRNA is observed in the
exocarp cell layer of kernel pericarp in differentiation, whereas in
the embryo its mRNA is especially abundant in the suspensor.
During the first stages of kernel development (Kiesselbach, 1980;
Sheridan and Clark, 1987; van Lammeren, 1987), HyPRP mRNA is
observed in the first cell layer of ectodermic tissue, which originates
from maternal ovary wall that differentiates into the pericarp after anthesis and in the embryo proper after the first scutellar cells are
differentiated. The study of HyPRP and HRGP cell
expression during female inflorescence and kernel development indicates
that these genes always show a different pattern of expression.
The mRNA levels corresponding to HyPRP and HRGP
decrease after stages 3 and 4 of development, respectively, and no mRNA
can be detected in the dry embryo. Both genes are activated during germination, but in the case of HyPRP this is a short,
transient effect followed by a definitive ending of HyPRP
gene expression, whereas HRGP is actively expressed during
the growth of the seedling. This transient expression of
HyPRP is consistent with the hypothesis that embryo
maturation and dessication are in fact a temporal interruption of
embryogenesis, which is restarted in early germination. Immediately
after germination, normal vegetative growth appears and
HyPRP is no longer detected in the adult plant.
The expression of HyPRP is interrupted when the
concentration of ABA in the embryo increases at stage 3 of development,
and expression of HRGP is interrupted at stage 4 (Jones and
Brenner, 1987; Quatrano, 1987). Although both genes are repressed
during embryo development, they are probably under the control of
different regulatory signals. It is shown here that HyPRP is
under the negative control of ABA, as observed after exogenous ABA
treatment of excised, wild-type maize embryos. Moreover, in excised
vp2 maize embryos deficient in ABA and supplied with
exogenous ABA, the HyPRP mRNA level decreased in parallel to
the increase in a typical ABA-induced gene, RAB28. It may
then be proposed that the same mechanisms or signals that act upon
RAB28 to increase its mRNA level also act on HyPRP, but in
the opposite direction. In addition, in the 5-flanking region of the
HyPRP gene a consensus ABA-responsive element is
present, although it is not known whether it regulates this effect
(Guiltinan et al., 1990). In contrast, the HRGP mRNA level
in the wild type and in vp2 mutants is first transiently increased and then repressed in response to ABA application. In maize
HRGP arrest might be associated with the arrest of cell division in the induction of dormancy occurring as a result of ABA
action (Quatrano, 1987). This behavior is specific for maize HRGP. In rice plantelets the homologous gene (Caelles et
al., 1992) responds positively to ABA stress but not to wounding (Guo et al., 1994), indicating that maize and rice HRGP genes are
differentially regulated and respond to different stresses. The rice
gene promoter includes a sequence in agreement with the ABA-responsive
element described by Guiltinian et al. (1990), whereas this is lacking in maize.
NaCl treatment reduces HyPRP mRNA
accumulation to a low level following a pattern that is opposite to the
induction of the RAB28 gene. In this situation the
HRGP mRNA level is increased, in contrast to what was
observed after ABA treatment. This result may indicate that
HRGP is sensitive to salt stress in an ABA-independent manner. The correlation of the expression of the HRGP and
H4 genes in the seedling indicated the need for
HRGP in the new cell walls after division (Ludevid et al.,
1990; Ruiz-Avila et al., 1991). HRGP expression after NaCl
treatment is increased, although cell division has been arrested, as
judged by the low histone H4 mRNA level. We conclude that
after NaCl treatment, as was also observed after ethylene treatment
(Tagu et al., 1992), HRGP expression is not correlated with
the accumulation of histone H4 mRNA, and therefore the response is
independent of cell division.
Cold treatment increases HyPRP expression. Cold responses
are mediated in some cases by ABA (Klee and Estelle, 1991), but this is
apparently not the case for HyPRP expression, in which the
cold response is clearly ABA independent, as indicated by the lack of
RAB28 induction. This finding is consistent with the hypothesis that
after cold treatment the signal necessary for RAB28
induction and HyPRP repression is absent, and for this
reason, HyPRP expression is not reduced and RAB28 is not
induced. Other genes responding to dehydration or cold treatment in an
ABA-independent manner have been described (Guerrero et al., 1990;
Yamaguchi-Shinozaki and Shinozaki, 1994). Different results are
obtained with the HRGP gene. After cold treatment or during
ABA induction or treatment, cells accumulating HRGP mRNA are
different from those accumulating the same mRNA at normal temperatures,
or in the absence of ABA, as seen by in situ hybridization (Fig. 6).
HRGP behavior indicates that after the process of cold or
initial ABA treatment the maize embryo is not maintained in the
previous state but, rather, is now in a new state, which has an effect
on either the transcriptional status of the HRGP gene or its
mRNA stability.
In conclusion, the two genes coding for Pro-rich proteins in maize
studied here allow the study of specific processes occurring during
female inflorescence and at the first stages of maize kernel development. HRGP marks the regions where meristematic and
cell division activities are high. Its mRNA accumulates particularly in
the provascular cells and the silks in female inflorescences, and in
suspensor cells of immature embryos. HyPRP follows the formation of parenchymal cells surrounding the provascular system of
pistillate spikelets and scutellum formation in maize embryos. The
expression of both genes is reduced by ABA treatment. However, there is
fast repression by ABA in the case of HyPRP, whereas there
may be an indirect effect for HRGP due to the cessation of
meristematic activity, which appears first to be transiently induced by
the hormone.
| |
FOOTNOTES |
1
This study was supported by grants from the Plan
Nacional de Investigación Científica y Técnica (no.
BIO94-0734) and from the European Communities (no. BIO4-CT96-0210), and
was carried out within the framework of the Centre de Referència
de Biotecnologia de la Generalitat de Catalunya.
*
Corresponding author; e-mail pprgmp{at}cid.csic.es; fax
34-3-204-5904.
Received May 27, 1997;
accepted October 21, 1997.
| |
ABBREVIATIONS |
Abbreviations:
HRGP, hydroxyproline-rich glycoprotein.
HyPRP, hybrid Pro-rich protein.
| |
ACKNOWLEDGMENT |
We are thankful to Dr. Montserrat Pagés for furnishing the
RAB28 probe.
| |
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