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Running title: Biogenesis of pollen-coat material in maize tapeta
Corresponding author:
Dr. Anthony Huang
Center for Plant Cell Biology
Department of Botany and Plant Sciences
University of California
Riverside, CA 92521
email: [email protected]
phone: 1-951-827-4783
fax: 1-951-827-4437
Research Area:
Biochemical Processes and Macromolecular structure
Plant Physiology Preview. Published on January 30, 2012, as DOI:10.1104/pp.111.189241
Copyright 2012 by the American Society of Plant Biologists
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The maize tapetum employs diverse mechanisms to synthesize and store proteins and
flavonoids and transfer them to the pollen surface
Yubing Li1, Der Fen Suen1, Chien-Yu Huang1, Shung-Yee Kung1,2 and Anthony H. C. Huang1,2,*
1 Center for Plant Cell Biology
Department of Botany and Plant Sciences
University of California
Riverside, CA 92521
2 Institute of Plant and Microbial Biology
Academia Sinica, Taipei, Taiwan 11529
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Footnotes:
This work was supported by U.S. Department of Agriculture-National Research Initiative Grant
2005-02429 and a Taiwan National Science Council grant.
Corresponding author:
* Corresponding author; email [email protected]
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ABSTRACT
In anthers, the tapetum synthesizes and stores proteins and flavonoids, which will be transferred
to the surface of adjacent microspores. The mechanism of synthesis, storage and transfer of these
pollen-coat materials in maize (Zea mays, L.) differs completely from that reported in
Arabidopsis, which stores major pollen-coat materials in tapetosomes and elaioplasts. On maize
pollen, 3 proteins--glucanase, xylanase and a novel protease, ZmPCP, -- are predominant. During
anther development, glucanase and xylanase transcripts appeared at a mid-developmental stage,
whereas protease transcript emerged at a late-developmental stage. Protease and xylanase
transcripts were present only in the anther tapetum of the plant, whereas glucanse transcript
distributed ubiquitously. ZmPCP belongs to the cysteine protease family but has no closely
related paralogs. Its nascent polypeptide has a putative N-terminal endoplasmic reticulum (ER)-
targeting peptide and a propeptide. All 3 proteins were synthesized in the tapetum and were
present on mature pollen after tapetum death. Electron microscopy of tapetum cells of mid-late
developmental stages revealed small vacuoles distributed throughout the cytoplasm and
numerous secretory vesicles concentrated near the locular side. Immunofluorescence microscopy
and subcellular fractionation localized glucanase in ER-derived vesicles in the cytoplasm and the
wall facing the locule, xylanase in the cytosol, protease in vacuoles, and flavonoids in
subdomains of ER rather than in vacuoles. The nonoverlapping subcellular locations of the 3
proteins and flavonoids indicate distinct modes of their storage in tapetum cells and transfer to
the pollen surface, which in turn reflect their respective functions in tapetum cells or pollen
surface.
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INTRODUCTION
An anther in a flower usually has 4 layers of cells enclosing a fluidic locule, in which
microspores mature to become pollen (Bewley et al., 2000; Goldberg et al., 1993; Hesse et al.,
1993; McCormick, 2004; Scott et al., 2004). Cells of the outer 3 layers are highly vacuolated and
presumed to be metabolically less active. Cells of the innermost layer, the tapetum, have densely
packed cytoplasm and carry out active metabolism. They control maturation of the microspores.
Despite the importance of tapetum cells in sexual reproduction, we have minimal information
about their mode of functioning.
Tapetum cells in Brassicaceae species have been characterized to some extent, much
more so than those in other species (Hsieh and Huang, 2005 and 2007; Murgia et al., 1991;
Owen and Makaroff, 1995; Platt et al., 1998; Wu et al., 1997). Early in development, the
tapetum cells are involved in active secretion of molecules into the locule for maturation of the
microspores from tetrads to solitary entities, on which the outer exine wall gradually appear. Late
in development when the microspores become binucleate with a single large and then multi-
smaller vacuoles, the tapetum cells become warehouses for temporary storage. In Brassicaceae
species, they are packed with two predominant storage organelles, the elaioplasts and
tapetosomes. Elaioplasts are plastids containing abundant steryl-ester droplets and minimal
thylaloids, and tapetosomes are storage organelles, each having numerous oleosin-coated alkane
droplets linked ionically with flavonoid-containing vesicles. At the conclusion of development,
the tapetum cells undergo programmed cell death (PCD) (Wu and Cheung, 2000; Huang et al.,
2011) and release the storage materials, which become the coat of mature pollen. These materials
include steryl esters from the elaioplasts as well as alkanes, oleosins, and flavonoids from the
tapetosomes. Steryl esters and alkanes are lipids waterproofing the pollen. Oleosins are
amphipathic proteins aiding storage of coat materials in the tapetum cells and emulsification of
these materials on the pollen surface and, subsequently, assisting water uptake from the stigma to
the pollen for germination. Flavonoids are UV-absorbing molecules for protecting the nucleic
acids in haploidic pollen (Winkel-Shirley, 2001; Hsieh and Huang 2007).
Tapetum cells in other plant species have been less investigated (Hesse et al., 1993).
Among these other species, maize has been the most studied. Electron microscopy studies have
shown that tapetum cells at a late developmental stage in maize do not possess elaioplasts and
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tapetosomes (Horner et al., 1993; Skvarla and Larson, 1966; this report), and so the pollen-coat
materials have to be synthesized, stored and delivered via other mechanisms.
Maize pollen surface components external to the plasma membrane can be separated into
2 biochemically distinct morphological parts: the wall in the interior and the non-covalently
linked coat external to it (Suen et al., 2003). The pollen wall consists of largely sporopollenin
and includes transiently associated proteins (Allen and Lonsdale, 1993; Bosch and Helper, 2005;
Chay et al, 1992; Li et al., 2003; Rubinstein et al., 1995; and Suen et al., 2003, Wu et al., 2001),
which are synthesized in the microspore or pollen interior and secreted to the wall and exterior
before, during or immediately after pollen germination. These proteins include polygalacturonase
and several wall-modulating proteins, which consist of expansin, pectin methylesterase, profilin,
cation-binding protein, pollen allergen (trypsin inhibitor), extensin and others. The transcripts of
these proteins in the microspores and mature pollen appear late during anther development, and
their levels persist or increase after germination (Suen et al., 2003). Thus, these wall hydrolases
and modulating proteins likely exert a role after the pollen tube has penetrated the stigma and
may hydrolyze and modulate the wall of the cells along the pollen tube track in the carpel for
advancement of the pollen tube toward the ovary.
Maize pollen coat, which is on the surface of the pollen wall, contains proteins,
flavonoids and lipids. These coat molecules are largely if not exclusively synthesized in the
adjacent tapetum (Hesse et al., 1993). The coat consists of only 3 major proteins: a 70-kDa
1,3:1,4-β-glucanase (Suen et al., 2003, termed glucanase hereafter), a 35-kDa endo-xylanase
(Bih et al., 1999; termed xylanase) and a previously mentioned but unstudied 25-kDa protein
(Suen et al., 2003). In developing anthers, the transcripts of the glucanase and xylanase appear
earlier than those of the microspore-synthesized wall proteins (preceding paragraph). The
glucanase transcript is present in the tapetum and many other maize organs (via genome database
searches [Suen et al., 2003] and experimentation [to be described in this report]), and the
function of the glucanase in the pollen coat is unknown. This glucanase differs from the well-
known tapetum-synthesized and secreted glucanase that hydrolyzes the tetrad microspore wall;
the latter enzyme appears at a very early stage of anther development (Suen et al., 2003). The
xylanase transcript is restricted to the tapetum in anthers and nowhere else in a whole plant. The
nascent xylanase is an inactive precursor of 60 kDa, which is processed at both the N and C
termini to the active 35-kDa enzyme (Wu et al., 2002). Maize and other cereals have Type II cell
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wall, which contains predominantly cellulose and hemicellulose, and xylan is a major component
of hemicellulose (Carpita and Mann, 2000). Use of an anti-sense approach has revealed that the
pollen-coat xylanase acts on the stigma cell wall, thereby creating an opening for entry of the
emerging pollen tube (Suen et al., 2007). Maize pollen coat contains flavonoids, which are
mostly quercetin and isorhamnetin glycosides (Ceska and Styles, 1984). In diverse plant species,
pollen coat flavonoids protect the haploid genome against UV irradiation (Hsieh and Huang,
2007), and in some species such as maize, they are also involved in pollen germination and tube
growth (Mo et al., 1992). Pollen coat flavonoids are derived from the tapetum (Stanley and
Linsken, 1974). The flavonoids in Brassicaceae tapeta are synthesized in ER and then
temporarily stored in the tapetosomes (Hsieh and Huang, 2007). Their synthesis and storage in
tapeta of other species, including maize, is unknown. Minimal lipids are present in the pollen
coat of maize, a wind-pollinating species, and no or few lipid droplets are present in the tapetum
cells immediately before tapetum PCD.
The mechanisms whereby maize tapetum cells synthesize the pollen-coat proteins and
flavonoids, store them in the cells and transfer them to the pollen surface are unknown. In the
current report, we present experimental findings to delineate these mechanisms, which differ
completely from those in the studied Brassicaceae tapetum cells. We also report the identification
and characterization of the previously unstudied maize pollen-coat 25-kD cysteine protease.
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RESULTS
The maize 25-kDa pollen-coat protein is a cysteine protease, whose nascent polypeptide has
an N-terminal ER targeting peptide and a propeptide
Mature maize pollen was washed with diethyl ether to yield a coat fraction. Proteins in
the fraction were resolved by SDS-PAGE (Fig. 1A). Three proteins predominated and could
represent less than 1% of the total pollen proteins via our visual estimation of the protein stains
in the gel. They appeared as sharp bands on a SDS-PAGE gel and thus did not seem to represent
degraded proteins from dead tapetum cells. The 70-kDa protein was a glucanase (Suen et al.,
2003) and the 35-kDa protein a xylanase (Bih et al., 1999; Wu et al., 2002). The 25-kD protein
has been observed but not studied (Suen et al., 2003), and we explored its identity and properties.
A 14-residue sequence near the N-terminus of the 25-kDa protein was obtained by micro-
sequencing (Fig. S1). This sequence matches an assembled but incomplete EST sequence of a
TIGR maize gene (TC342635), and we further obtained the complete sequence (termed ZmPCP
in this report; Genbank Accession NP_001146834). The deduced polypeptide has 35.3 kDa and
352 residues, including, beginning from the N terminus, a 28-residue endoplasmic reticulum
(ER) targeting sequence, a 113-residue N-terminal propeptide (NTPP, which would cover the
active site of a hydrolase for more refined control of the in vivo activity) and a 211-residue
mature protein. The NTPP has a peptidase C1A motif ERFNIN (Fig. S1) that is conserved
among studied plant, mammalian and microbial cysteine proteases (Groves et al., 1998). The
mature protein has the characteristics of a cysteine protease, possessing the catalytic residues
glutamine, cysteine, histidine and asparagine of C1 family peptidases (Fig. S1). The ER targeting
signal was predicted with SignalP 4.0, and the other domains were analyzed with NCBI
Conserved Domain Database. A search of proteins with sequences similar to that of ZmPCP in
Genbank retrieved two closely related homologs. One is from sorghum (Genebank Accession
EER94674; 85% identity with ZmPCP) and the other from rice (Genebank Accession
EEC84502; 58% identity). The sorghum and rice proteins were retrieved from results of large-
scale DNA/RNA sequencing (Paterson et al., 2009; Tanaka et al., 2008), and their properties
have not been studied and reported. A Blast for maize proteins related to ZmPCP retrieved 17
distantly related paralogs (sequence identity ranging from 40-50%), of which 12 are predicted
proteins from large-scale DNA/RNA sequencing without functional analysis (Alexandrov et al.
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2009). The other 5 paralogs have been studied, including AAB70820 (Mir1), AAB88262 (Mir2)
and AAB88263 (Mir3) in calli and leaves (Pechan et al., 1999; Leopez et al., 2007); BAA08244
(CCP1) and BAA08245 (CCP2) in developing and germinated seed (Domoto et al., 1995) and
senescing leaves (Griffiths et al., 1997). An unrooted phylogenetic tree of these 17 maize
cysteine proteases and ZmPCP is shown in Fig. 1B, and an alignment of their sequences is shown
in Fig. S1. ZmPCP falls into the same clade (SEN102) with 3 other predicted maize cysteine
proteases (Genebank Accessions ACG25394, ACG30386 and ACF82315 in Fig. 1B); these 3
other maize cysteine proteases have not been characterized and reported. A member of the
SEN102 clade was first identified in daylily (Hemerocallis) taple, in which the protease gene
transcription is up-regulated during flower senescence, and the protease may be involved in
protein hydrolysis at a late stage of senescence (Valpuesta et al., 1995; Cuerrero et al. 1998).
A rice protease (OsCP1; Genebank Accession BAF16127) has been reported to be
tapetum specific, and its transcript peaks during early anther development (Lee et al., 2004). Its
amino acid sequence is less similar to those of the 18 maize cysteine proteases (complete
sequences shown in Fig. S1). We studied this rice protease gene (OsCP1) in rice anthers by RT-
PCR. Our results confirmed the early appearance of the OsCP1 transcript during anther
development (data not shown). The early appearance (developmental stages of maize are defined
in the following section; developmental stages of rice were defined earlier [Huang et al., 2011])
of this rice tapetum protease transcript (OsCP1) suggests that the protease is not directly
involved in tapetum PCD at a late stage of development.
We synthesized a short peptide of the sequence, QARRYACSRSRAAQ, which is unique
to ZmPCP (Fig. S1). Polyclonal antibodies were raised against this peptide for further studies of
the protease.
Transcripts of the 3 pollen-coat hydrolases were present in the tapetum of anthers but had
different temporal profiles
The 3 hydrolases were the predominant proteins in the pollen-coat fraction (Fig. 1A).
Glucanase transcript in anthers was present in tapetum cells and not in microspores or mature
pollen (Suen et al., 2003). Nevertheless, it also existed in many other maize organs (Fig. 1D).
Xylanase transcript in anthers was also present in tapetum cells and absent in microspores and
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mature pollen (Wu et al., 2002) as well as other maize organs (Fig. 1D). Protease transcript was
similar to xylanase transcript in being restricted to tapetum cells (Fig. 1C) and absent in pollen
and other organs (Fig. 1D).
Developing anthers were divided into 4 stages (see Materials and Methods) for the study
of appearance of the transcripts and proteins of the 3 pollen-coat hydrolases. Briefly, at stage 1,
the microspores were in a tetrad structure. At stage 2, the microspores were solitary and had the
exine wall. At stages 3 and 4, the microspores were binucleate and trinucleate, respectively.
During anther development, glucanase and xylanase transcripts appeared at stages 2 and 3 (Fig.
1E). Protease transcript was absent in stages 2 and 3 and emerged only at stage 4. The
developmental profiles of these 3 transcripts reflected those of their encoded proteins (Fig. 2).
Both glucanase and xylanase appeared in stage-3 anthers and peaked in stage-4 anthers; they
were also present in pollen (Fig. 2) and specifically restricted to the pollen coat (Wu et al., 2002;
Suen et al., 2003). Xylanase appeared initially as a 60-kDa precursor, which was then converted
to the final active 35-kDa xylanase (Wu et al., 2002). Protease appeared only in stage-4 anthers
and then in the pollen coat. The antibody preparations against these 3 hydrolases were highly
specific in immunoblotting, recognizing only antigens of the expected molecular weights in the
tapetum cells; the only exception was that the antibodies against the xylanase recognized an
unknown protein of 55 kDa of the microspore/pollen interior (Fig. 2; Wu et al., 2002). These
antibodies were suitable for immunofluorescence microscopy.
Tapetum cells had small vacuoles distributed throughout the cytoplasm and secretory
vesicles concentrated near the locular side
A maize anther has 4 layers of cells enclosing the locule, in which microspores mature.
Cells of the outer 3 anther layers are highly vacuolated, usually each with a large central vacuole.
Cells of the innermost layer, the tapetum, had dense cytoplasm throughout anther development
(Fig. 3).
Each tapetum cell at stages 1-2 had 1 or 2 nuclei, which began to disintegrate at stage 3.
During development, the cell had abundant rough ER and some Golgi, mitochondria and
proplastids. Numerous vacuoles of 0.5-2 μm in diameter distributed throughout the cytoplasm.
Many secretory vesicles of 0.2-0.5 μm in diameter were present; they, in comparison to the
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vacuoles, were smaller, with a more electron-dense, granular matrix, and concentrated near the
locular side of the cytoplasm (Fig. 3B). At stage 4, the cell became elongated, concomitant with
elongation of the anther; its wall facing the locule had dissolved, and the cell gradually detached
from the 3 outer anther cell layers.
Immuno-CLSM of tapetum cells localized glucanase in ER-vesicles near the locule,
xylanase in the cytosol and protease in vacuoles
Subcellular locations of the 3 hydrolases in anthers of different developmental stages
were examined with immunofluorescence confocal laser scanning microscopy (CLSM). Figure 4
shows results at the cellular level in one anther lobe. Glucanase was barely detected in a stage-2
anther but localized in the tapetum in stage-3 and -4 anthers. In these anthers, most glucanase
was present in the tapetum near the locule and between adjacent tapetum cells connecting to the
locule. Some glucanase was present on the microspore surface in stage 3 and less so in stage 4.
Xylanase exhibited a similar developmental pattern but distributed evenly, rather than near the
locule, in tapetum cells. In addition, the microspores in stage 2-4 anthers showed antigenic signal
(Fig. 4). This signal represented an unknown 55-kDa protein present inside the microspores
throughout development; this protein was recognized by the antibody preparation via
immunoblotting (Fig. 2; Wu et al., 2002). Protease was present only in tapetum cells in stage-4
anthers and was absent on the microspores.
Immunofluorescence CLSM at the subcellular level in tapetum cells was performed with
antibodies against the hydrolases and known subcellular organelle markers (Fig. 5). Glucanase
located near the locule in a tapetum cell in a stage-3 anther, whereas calreticulin, a marker of ER,
distributed in the ER network throughout the cell (Fig. 5A). At the outermost region of the cell
nearest the locule, glucanase (shown in red) existed in the absence of calreticulin (green),
whereas slightly interior to this region, glucanase and calreticulin overlapped (appeared as
yellow). The former region could represent the wall outside the plasma membrane, and the latter
region could correspond to the nearby protoplast where abundant secretory vesicles existed (Fig.
3). Glucanase was not detected in other regions of ER, presumably because the secretory vesicles
were only transiently associated with specific regions of the ER facing the locule. Thus,
glucanase was associated with ER-derived secretory vesicles and was secreted.
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Xylanase in a tapetum cell in a stage-3 anther distributed throughout the cytoplasm and
neither overlapped with glucanase or calreticulin nor exhibited a specific pattern (Fig. 5A). Thus,
xylanase was in the cytosol.
Protease was scarce in stage-3 anthers (Fig. 2), and so we examined its subcellular
location in tapetum cells in stage-4 anthers. At this developmental stage, tapetum cells had lost
most of their cell wall and were partly detached from the outer anther cell layers. Unlike
glucanase and xylanase, protease was associated with small subcellular particles of 1-2 μm in
diameter throughout the cytoplasm (Fig. 5B). The protease-associated particles were also the
locations of γ-TIP (tonoplast intrinsic protein) (Pedrazzini et al., 1997) and V-PPase (vacuolar
pyrophosphatase) (Sarafian et al., 1992), both markers of cell vacuoles. They were not ER-
derived secretory vesicles, because V-PPase (Fig. 5B) and protease (not shown) did not overlap
with glucanase-associated secretory particles near the locule. Antibody preparations against
tobacco γ-TIP and pea V-PPase were specific for the respective vacuolar enzymes in maize
anther extracts, as revealed in immunoblotting analyses (Fig. 5C). The overall results indicate
that protease was present in vacuoles of tapetum cells at a late stage of anther development.
The above subcellular localization results on the 3 hydrolases with CLSM are consistent
with computer software predictions on the basis of amino acid sequences. Two software
programs, PSORT (Nakai and Kanehisa, 1991) and TargetP (Emanuelsson et al., 2000; Nielsen
et al., 1997), suggest that the 3 hydrolases are not in chloroplasts or mitochondria. Glucanase is
predicted to be in the secretory pathway; its nascent polypeptide has a putative 23-residue N-
terminal ER-targeting signal but no ER-retention signal, and therefore glucanase may be
associated with the ER-secretory machinery. Xylanase is predicted not to associate with
subcellular structures. Protease is predicted to be in the ER-secretory pathway; its sequence of
having a putative 28-residue N-terminal ER-targeting signal and an immediate downstream 113-
residue NTPP (identified via a comparison with other proteases; Hatsugai et al., 2006) leads to
the prediction of its location in vacuoles.
Subcellular fractionation of tapetum cells by density gradient centrifugation separated the
3 hydrolases into different fractions
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The subcellular locations of the 3 hydrolases were further explored by biochemical
subcellular fractionation. An extract of stage-3 and -4 anthers, obtained after mild grinding to
yield mostly tapetum subcellular materials and after removing the glucanase-containing tapetum
wall fragments by filtration (see Materials and Methods), was subjected to sucrose density
gradient centrifugation (Fig. 6). Xylanase, protease and glucanase peaked at different gradient
fractions, although their peaks overlapped.
Xylanase was present only in fractions at the top of the gradient. These fractions
contained cellular materials (cytosol) that were not associated with subcellular structures.
Protease migrated into the gradient, peaking at fraction #7 (density 1.07 g.cm-3). It was
also present in the top fractions. The distribution of protease along the gradient followed that of
V-PPase, a vacuole marker. The findings are consistent with protease being in vacuoles in
tapetum cells. The small vacuoles in tapetum cells were heterogeneous in size, as shown by EM
(Fig. 3) and CLSM (Fig. 5B), and perhaps also in contents. Their content is largely water and
other small molecules, and therefore vacuoles had a low buoyant density. This low density, plus
the small and heterogeneous sizes of the vacuoles, resulted in a wide distribution of the
organelles along the gradient.
Glucanase moved into the gradient and peaked at fraction #9 (density 1.09 g.cm-3), and
no soluble glucanase was at the top of the gradient. The observed buoyant density of the
glucanase is that expected of small secretory vesicles containing glucanase after 4-h
centrifugation (Huang et al., 1983). Calreticulin, a marker of ER, had a wider distribution along
the gradient but encompassed the glucanase-peak fractions. This wide distribution is expected,
because calreticulin was associated with all ER present throughout the cell and secretory vesicles
containing glucanase located near the locule side of the cell (Fig. 5A).
Fraction 9 possessing peak glucanase in the gradient contained largely vesicles with
morphology and size similar to those of secretory vesicles in situ observed by EM (Fig. 6C).
These secretory vesicles had an electron opaque matrix and differed from the vacuoles that
possessed a distinctly less opaque matrix. Our attempts to observe isolated vacuoles by EM in
fractions 7 and 8 possessing peak protease were unsuccessful, probably because the fragility of
the organelles and the fractions being highly contaminated with cytosolic materials prevented
proper processing of the fractions for EM.
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Flavonoids in tapetum cells were present in ER-subdomains and not in vacuoles
Pollen-coat flavonoids are derived from the tapetum (Stanley and Linskens, 1974). In the
current study of developing maize anthers, flavonoids were almost absent in stages 1 and 2 but
abundant in stages 3 and 4 and then in the coat of mature pollen, as revealed in a TLC analysis
(Fig. 7A). Maize anther and pollen-coat flavonoids were present as flavonoid glycosides (Ceska
and Styles, 1984), whose flavonoid moieties were mostly quercetin and isorhamnetin (Fig. 7A);
the findings are consistent with those of maize pollen flavonoids reported earlier (Ceska and
Styles, 1984).
We examined the subcellular locations of the flavonoids in stage-3 and -4 tapetum cells.
Fixed anthers were sectioned into small segments and stained with diphenylboric acid 2-
aminoethyl ester (DPBA) and then subjected to immunofluorescence analyses (see Materials and
Methods). Stage-3 and -4 tapetum cells did not have an extensive ER network of a secretory
nature facing the locular side; rather, the ER network, localized with the ER marker calreticulin,
distributed throughout the cytoplasm (Fig. 3B and Fig. 7B). DPBA-flavonoids were colocalized
with portions of the calreticulin, which presumably represented ER subdomains (Fig. 7B); this
colocalization is similar to that in the Brassica tapetum, in which the flavonoids are initially
associated with ER (Hsieh and Huang, 2007). In contrast, DPBA-flavonoids were not
colocalized with V-PPase, a marker of vacuoles, and thus flavonoids were absent in the vacuoles.
The 3 hydrolases and flavonoids were deposited on the pollen surface at a late stage of
anther development
Microspores removed from stage-3 and -4 anthers and mature pollen were subjected to
immuno-treatment for observation of the 3 hydrolases on their surface (Fig. 8). Microspores of
stages 3 and 4 had little hydrolases on the surface. A trace amount of glucanase, but not the other
2 hydrolases, could be detected on the microspore surface at stage 3 (Fig. 8); this observation is
consistent with the secretory nature of glucanase (Fig. 4A and 5). Slightly more glucanase and a
trace amount of protease appeared on the microspore surface at stage 4 (Fig. 8) when the tapetum
began its PCD; these observed hydrolases signals in relatively small amounts might be authentic
or represent technical background noises. Finally, all the 3 hydrolases became abundant on
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mature pollen when the tapetum PCD completed. The findings are consistent with the tapetum
cells maintaining their integrity and possessing the hydrolases (glucanase retained in the wall or
present transiently on its way to the microspore surface, and xylanase and protease in the
protoplasts) until stage 4 when PCD commenced.
Similarly, in stage-3 anthers, DPBA-flavonoids were present mainly in tapetum cells and
almost absent on the microspore surface (Fig. 7C). After tapetum PCD in late stage 4, most of
the flavonoids were present on the microspore surface (Fig. 7C).
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DISCUSSIONS
In anthers, tapetum cells synthesize, store and transfer proteins and non-protein
molecules, especially flavonoids, to the microspore surface, where the molecules carry out
specific functions. The mechanisms of these syntheses, storage and transfers, as well as their
protein constituents, are largely unknown. Recent studies have shown that in Brassicaceae
species, tapetum cells synthesize proteins (oleosins), alkanes and steryl esters, and flavonoids
and store them in tapetosomes and elaioplasts, for bulk and simultaneous discharge to the
microspore surface upon PCD (Hsieh and Huang, 2005; 2007). Maize and rice tapetum cells do
not have storage tapetosomes and elaioplasts. Instead, maize tapetum cells use 3 different
mechanisms for the synthesis, storage, and transfer of the 3 pollen-coat proteins and yet another
mechanism for flavonoids. Glucanase, with its N-terminal ER-targeting signal peptide in the
nascent protein, is synthesized on ER and secreted via the subcellular secretory pathway; it likely
digests the wall of the tapetum cell and perhaps subsequently coordinates with the pollen-coat
xylanase in hydrolyzing the stigma wall for pollen tube penetration. Xylanase is synthesized and
stored in the cytosol as a large inactive protein and upon PCD activated via proteolysis and
released to the microspore surface; it functions after release from the pollen surface onto the
stigma to hydrolyze the stigma wall for pollen tube penetration (Suen and Huang, 2009).
Protease, with its N-terminal ER-targeting signal peptide in the nascent protein, is synthesized on
ER as a large inactive protein and reaches via the subcellular secretory pathway numerous small
vacuoles; likely it is released from the vacuoles upon PCD to proteolyze the cellular contents and
then is retained on the pollen surface, where it possibly exert an additional function on the stigma
similar to those for the xylanase and glucanase. Flavonoids are synthesized on the cytosolic side
of ER (Winkel-Shirley, 2001) and transferred to the ER lumen and then secretory vesicles; it
protects the haploid genome from UV irradiation (Hsieh and Huang, 2007) and plays an
additional role as regulators in pollen germination and tube growth (Mo et al., 1992).
The functions of pollen-coat proteins originated from tapetum cells have been examined
only recently, and minimal information is available. Self-incompatibility signal proteins in the
pollen coat of several plant species, including those of Brassica, come from sporophytic cells,
presumably the tapetum (Kachroo et al., 2004; Kao and Tsukamoto, 2004). In Brassica and
Arabidopsis, the amphipathic oleosins are the predominant proteins in the pollen coat and were
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originated from the storage tapetosomes in tapetum cells (Ting et al., 1998; Wu et al., 1997). A
mutational loss of the major pollen-coat oleosin led to the pollen grain being inefficient in taking
up water from the stigma for germination and pollen tube growth (Mayfield and Pruess, 2000).
This observation may reflect the amphiphatic oleosins (A) serving as an essential ingredient for
water uptake from the stigma to the pollen, (B) acting as an emulsifying agent for the proper
coating of the pollen coat materials that include very hydrophobic lipids (alkanes), fairly
hydrophobic steryl esters and relatively hydrophilic flavonoids (Hsieh and Huang, 2004), (C)
carrying the highly hydrophobic alkanes from the tapetum tapetosomes, in which small alkane
droplets are stabilized with surface oleosins, to the pollen surface, and/or (D) being a non-
functional leftover after their stabilization of alkane droplets in storage tapetosomes in tapetum
cells. Regardless, oleosins are absent in the pollen coat of maize and rice and likely many other
species.
The function of xylanase, one of the 3 pollen-coat proteins in maize, has been
documented. Pollen-coat xylanase acts on the stigma surface, where it hydrolyzes the stigma
xylan wall to generate a hole for entry of the pollen tube. Before xylanase is released from
tapetum cells, it is present as an inactive 60-kDa pre-xylanase (Wu et al., 2002) residing in the
cytosol (this report). The protein in the pollen coat is an active 35-kDa xylanase, with 198 and 48
residues of the N and C termini, respectively, of the pre-xylanase been removed. The proteolytic
processing of the pre-xylanase in vivo and in vitro is precise, and the final active xylanase is
highly resistant to further proteolysis. If xylanase were released from tapetum cells as an active
form early on before PCD, it would encounter its substrate in the tapetum wall facing the locule
before it could reach the microspore surface. Such an undesirable condition is prevented via
storage of the pre-xylanase in the cytosol, where no xylan is available, before its activation and
release upon PCD.
The functions of the other two maize pollen-coat proteins, glucanase and protease, remain
to be elucidated. We can speculate on their functions from available information, especially the
current findings of the mechanisms of storage and transfer of the two proteins. The gene
encoding the glucanase is expressed in not just the anther tapetum but also diverse organs and
tissues. Its transcript in the anther appears first at stage 2 and peaks at stage 3; the developmental
profile of its encoded protein follows slightly after that of the transcript. During stages 2-3, the
microspores have already become solitary; thus, this glucanase is not the glucanase for the
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hydrolysis of the callose wall of the microspore tetrad (Suen and Huang, 2007). This glucanase is
synthesized and secreted in stage-2 and -3 tapetum cells, which still maintain the wall facing the
locule (Fig. 5A), and the majority of the enzyme is not detected on the microspore surface until
PCD (Fig. 8). It is initially concentrated in vesicles near the locular side of the cell and then
secreted. Its detection in the tapetum wall and not in the locule fluid (Figs 4 and 5) could be due
to its high concentration during its action in the tapetum wall and/or its low concentration in the
locule fluid on its way to the microspore surface. This glucanase could function in hydrolyzing
the tapetum cell wall facing the locule and remains with the wall remnant until PCD, after which
it is absorbed onto the microspore surface. Alternatively or in addition, the glucanase in the
pollen coat may work coordinately with xylanase (Suen et al., 2003) in generating a hole on the
stigma wall for entry of the pollen tube. The gene encoding this glucanase expresses in diverse
tissues and organs (Fig. 1D) and thus may carry out a role of modulating the wall linkage and
thus properties for general cell growth and development.
The pollen-coat protease, ZmPCP, is synthesized at a very late stage, stage 4, of anther
development. This is the only known tapetum protease that appears at such a late stage of anther
development in any species. The protease may be directly related to PCD of the tapetum, either
alone or together with other proteases. The protease is synthesized, presumably in the RER, and
processed during or after its transfer to the vacuole. According to the current concept of plant
PCD (Hatsugai et al., 2006; Trobacher et al., 2006), PCD-related proteases, whether trigger or
downstream proteases, are present in vacuoles and are self-activated upon lysis of the tonoplast
and plasma membrane. PCD proteases would be stable after self-activation, resisting further
proteolysis by other identical protease molecules or different proteases. The maize tapetum
protease could be the tapetum-specific trigger protease, because its gene is expressed only in the
tapetum at a very late stage of development in the whole plant. If so, the enzyme could be
present in the pollen coat due to a fortuitous absorption of the very stable protease leftover after
PCD and serves no future function. However, we cannot rule out that the protease plays an
additional role on the stigma surface.
Pollen coat flavonoids are derived from the tapetum (Stanley and Linsken, 1974).
Locations of flavonoids at the subcellular level or even tissue level in different plant cells are
largely unknown due to a major technical difficulty. Most plant flavonoids, with a few
exceptions such as the colorful flavonoids in large central cell vacuoles, are colorless glycosides,
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which could not be detected in situ with known flavonoid dyes for CLSM. Recently, our lab
developed an in situ glycoside hydrolysis procedure that could remove the sugar moieties from
flavonoid glycosides mildly such that subcellular structures and protein antigenicity can be
maintained for immunofluorescence colocalization studies. With this method, we demonstrated
that in Brassicaceae tapeta, flavonoids are present first in ER and then packaged in tapetosomes
(Hsieh and Huang, 2007). In the current study with maize tapetum cells, the flavonoids after
synthesis in ER remain largely in ER subdomains rather than move to the vacuoles, as could
have been speculated. It is possible that this flavonoid location in ER subdomains is common in
tapeta of plant species with no tapetosomes and may even be so in many non-tapetum cells in
plants.
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MATERIALS AND METHODS
Plant materials
Maize (Zea mays L.; inbred B73) plants were grown in a greenhouse. Anthers were
classified into four developmental stages (Bih et al., 1999). At stage 1, the anthers filled about
one-third of the floret. Each microspore mother cell had become a tetrad of microspores. At stage
2, the anthers filled up about one-half of the floret. Microspores had been released from the tetrad
and were covered with the exine. At stage 3, the anthers filled up about two-thirds of the floret.
Microspores had become larger and binucleate, and the single large vacuole developed into
multiple small vacuoles. At stage 4, the anthers filled up the floret completely. Microspores were
trinucleate. Starch accumulates in the microspore, which was dark as observed by bright-field
microscopy. Fresh pollens were collected as described (Wu et al., 2002).
Total anther extraction
Anthers in different stages were homogenized in 1× SDS-PAGE loading buffer (Suen and
Huang, 2007) with a mortar and pestle. The homogenate was boiled and then centrifuged at
16,000 g for 10 min. The supernatant was used as the total anther extract.
Preparation of pollen total and coat extracts
Fresh pollen was homogenized in chloroform/methanol (2:1, v/v) with a mortar and
pestle, and the extract was dried under a stream of nitrogen (Suen et al., 2003). This was the
pollen total extract. Fresh pollen was washed with diethyl ether (1 g pollen per 10 ml ether) in a
capped tube, and the tube was subjected to repeated inversions. The tube was centrifuged at 800
g for 10 min. The supernatant was retained and dried under a stream of nitrogen. This was the
pollen coat fraction.
Subcellular fractionation
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All solutions contained 0.05 M HEPES-NaOH, pH 7.5. Stage-3 and -4 anthers in a 0.6 M
sucrose solution at 4 ˚C were chopped with a razor blade and then homogenized gently with a
mortar and pestle. The homogenate was filtered through a Nitex cloth (20 × 20 μm pore size).
The filter retained most microspores (approximately 70 μm in diameter) and unlyzed outer
anther cells that were more difficult to be broken than wall-depleted tapetum cells. The filtrate
was placed on a linear gradient (0.8-M to 2.3-M sucrose solution) in a 5-ml tube. The gradient
was centrifuged at 28,000 rpm for 3 h at 4 ˚C in a Beckman SW55 rotor (Palo Alto, CA, USA).
Fractions of 0.2 ml each were collected from the bottom of the gradient. Densities of fractions
were checked with a refractometer (Milton Roy, PA, USA), analyzed for proteins by SDS-PAGE
and immuno-blotting, and for organelles by EM.
SDS-PAGE and immunoblot analyses
SDS-PAGE of 12.5% (w/v) polyacrylamide and immunoblotting were performed as
described (Wu et al., 1997). Preparations of anti-35 kDa xylanase (Bih et al., 1999), anti-
glucanase (Suen et al., 2003), anti-protease (current preparation), anti-calreticulin (Coughlan et
al., 1997), anti-V-PPase (Sarafian et al., 1992) and anti-γTIP (Pedrazzine et al., 1997), and HRP-
conjugated goat anti-rabbit-IgG were used. A synthetic polypeptide of the sequence,
QARRYACSRSRAAQ, unique to the maize pollen-coat protease (Fig. S1), was used to prepare
rabbit antibodies via a commercial source (Kim et al., 2002).
Flavonoid extraction and analyses
Preliminary tests showed that most flavonoids in maize anther extracts were in
glycosylated forms. Total extracts of anthers of different developmental stages were acidified in
2 N HCl at 80 ˚C for 1 h. Deglycosylated flavonoids in samples were extracted twice with an
equal volume of isoamyl alcohol at 80 ˚C. The samples were dried under a stream of nitrogen
gas, and the residues were dissolved in methanol for TLC. TLC plates were developed in
toluene:ethylacetate:formic acid:water (50:40:10:5, v/v/v/v) and sprayed with 0.5% (w/v) DPBA
(Sigma D9754-1G) in methanol for flavonoid staining (van der Meer et al., 1992). The plates
were observed and photographed on top of a UV light source.
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Fluorescence confocal microscopy
The procedures followed those described (Li et al., 2002). Anthers were fixed in 4%
paraformaldehyde, 0.05 M K-phosphate buffer, pH 7.4, for 12 h at 4 ˚C. Fixed anthers were
dehydrated in serial ethanol solutions and then embedded in paraffin. Paraffin sections 6-mm
thick were placed on a glass slide. After paraffin removal and rehydration, sections on slides
were incubated in PBS (10 mM Na phosphate, pH 7.4; 138 mM NaCl; 2.7 mM KCl) containing
5% defatted milk power for 1 h at 20 ˚C, and then PBST (PBS containing 0.05% Tween-20)
twice each for 5 min.
For single immunolabeling, sections were incubated with primary antibodies for 12 h at 4
˚C, washed with PBST 3 times each for 5 min, and incubated with Cy5-conjugated goat anti-
rabbit-IgG antibodies (1:200) (Jackson Product 211-005-109) for 1 h at 20 ˚C. Slides were
washed with PBST 3 times for 10 min each, mounted with antifade solution (Molecular Probes,
S-2828; Eugene, OR) and observed by confocal microscopy. For double immunolabeling, Cy3-
conjuated Fab fragment goat anti-rabbit IgG (H+L) and Cy5-conjugated goat anti-rabbit IgG
(H+L) were used. Antifade solution (Molecular Probes, S-2828; Eugene, OR, USA) was used to
protect labeled samples from laser bleaching. Slides were then mounted with a cover slip and
sealed with nail polish. Primary antibodies were anti-calreticulin, anti-xylanase, anti-glucanase,
anti-protease-1 and anti-VPPase. Confocal images were collected with a Leica TCS SP2
confocal microscopy system (Heidelberg, Germany) with a 63 × 1.2 water immersion objective
(HC ×PL APO). Confocal images of sequential scans and 3-D images reconstruction of
sequential confocal images were taken with Leica confocal software (LCS Lite). Images were
processed with Adobe Photoshop software (San Jose, CA, USA).
For double flavonoid-labeling with DPBA (Sigma-Aldrich) and immunolabeling (Hsieh
and Huang, 2007), anther sections were fixed in 4% paraformaldehyde, 1 x PBS (10 mM K-
phosphate, pH 7.4, 138 mM NaCl, and 2.7 mM KCl), and 0.15 M sucrose at 4 ˚C for 16 h. After
fixation, sections were washed with PBST (1 x PBS and 0.1% Tween 20) for 10 min twice and
treated with 0.4 M HCl at 56 ˚C for 30 min, and then washed with PBST for 10 min twice.
Sections after acid treatment were blocked with a blocking solution (3% milk, 1 x PBS) at 25 ˚C
for 1 h and then treated with 1:50 dilution of primary antibodies in 1% milk and 1 x PBS at 25 ˚C
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for 1 h. After washed with PBST for 10 min twice, sections were treated with DPBA and
secondary antibody (0.5% DPBA, 0.01% Triton X-100, 10% glycerol, 1X PBS, and 1:100
cyanine 5-conjugated goat antibodies against rabbit IgG [Jackson Immuno Research
Laboratories, West Grove, PA] for 2 h at 25 ˚C, and then washed twice. Finally, sections were
mounted with antifade solution and observed with Leica SP2 confocal microscopy. DPBA and
cyanine 5 were excited with 488- and 633-nm lines, respectively; the emissions were detected at
490–550 nm and 650-750 nm, respectively.
Electron microscopy
Anthers were fixed with paraformaldehyde and glutaraldehyde; postfixed with OsO4;
dehydrated; embedded in Spurr; sectioned; post-stained with uranyl acetate and lead citrate; and
observed with a Philips EM400 electron microscope (FEI), all as described (Platt et al., 1998).
Fraction #9 (in ~0.75M sucrose) representing the glucanase peak in the sucrose gradient
(Fig. 6) was mixed with an equal volume of 2.5% glutaraldehyde and 0.75 M sucrose for 5 h at 4
˚C. The mixture was diluted to 0.6 M sucrose with 0.4 M sucrose solution in 0.05 M HEPES-
NaOH, pH 7.5. The diluted mixture was placed on top of a membrane (Americon cellulose
membrane YM-10) in a 0.8-ml centrifuge tube (Beckman-Coulter, Fullerton, CA) and
centrifuged at 28,000 rpm for 3 h at 4 ˚C. Materials retained on the membrane were covered with
a drop of 5% agar, and after agar solidification, washed twice with 0.1 M K-phosphate, pH 7.2.
The sample was fixed with 1% OsO4 in 0.1 M K-phosphate, pH 7.2, for 4 h at 4 ˚C and subjected
to dehydration, embedding, sectioning and post-staining as described for anther sections.
RNA Extraction and RT-PCR
Total RNA was extracted (Kim et al., 2002) from anthers of different developmental
stages, mature pollen and pollen germinated for 20 min in a liquid medium (Suen and Huang,
2007). The cDNA template for PCR was synthesized from total RNA with an oligo(dT)15 primer
(Sambrook et al., 1989). For the gene encoding the pollen-coat 70-kDa β-glucanase, the 5' primer
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(5'-CAGATCGAGAGGGCCAACGC-3') and 3' primer (5'-CTTGACGAGGCGGGGGTCC-3')
were designed from the coding region of GLA (ZmGLA3, Genbank AY344632). For the gene
encoding the pollen-coat xylanase, the 5' primer (5'-GGGAGGCATGACCGCTTACT-3') and 3'
primer (5'-CTTGGTGACCGCGTTGCCG-3') were designed from the coding region of XYN
(ZmXYN1) (AF149016). For the gene encoding the pollen-coat protease, the 5' primer (5'-
ATGGCTCTCTTCCGTGCCGC-3') and 3' primer (5'- TTATTTGTATAGTTCATCCAT-3')
were designed from the coding region of the maize EST (TC342635) in TIGR database
(http://www.tigr.org); the complete cDNA sequence was obtained via DNA sequencing and
registered as Genbank EU117211. For the gene encoding the pollen-wall 10-kDa extensin
protein, a 5’ primer (5’-CACCAACAATGGCCTCCAGG-3’) and a 3’ primer (5’-
GCTGAGTGTGTCTATAGCGTG-3’) were designed from the coding region of a maize EST
(AY111779). For a maize actin gene, the 5' primer (5'-GGTTACTCCTTCACCACGAC-3') and
3' primer (5'-CAGACACTGTACTTCCTCTCAG-3') were designed from the coding region of
Maz56 (Genbank U60514). The purified PCR product was 32P-labeled with Prime-A-Gene
labeling kit (Promega, Madison, WI) and employed as a probe for RNA blot hybridization.
For RNA blot hybridization, each sample of 30 µg total RNA was fractionated by
electrophoresis with a 1.2% formaldehyde gel and then blotted onto a Hybond-N membrane
(Amersham Bioscience, Piscataway, NJ). The RNA-blotted membrane was prehybridized at 65
˚C in K phosphate, pH 7.2, 7% SDS, 1% bovine serum albumin, and 0.01 M EDTA, pH 8.0 for 4
h; hybridized with 32P-labeled probes (preceding paragraph) for 12 h; then washed with 2 x SSC,
0.1% SDS for 20 min; 1 x SSC, 0.1% SDS for 20 min; and 0.1 x SSC, 0.1% SDS for 20 min, all
at 65 ˚C.
In Situ Hybridization
Riboprobe was synthesized with DIG Nonradioactive RNA Labeling Kit (Boehringer,
Mannheim, Germany). A PCR fragment of ZmPCP (preceding section) was cloned into pGEM-T
vector (Promega). The plasmid was digested with NotI or SpeI for the synthesis of sense or
antisense riboprobes. The product was alkali-hydrolyzed with Na-carbonate buffer (120 mM
Na2CO3, 80 mM NaHCO3, pH 10.3) at 65 ˚C for 70 min. The riboprobe product was precipitated
and then dissolved in water pretreated with DEPC.
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Procedures for in situ hybridization followed those described (Qu et al., 2003). Anthers
of stage 2.5 were fixed with 4% paraformaldehyde for 4 h and embedded in paraffin. Ten-μm
thick sections on poly-L-Lysine-coated slides were treated with 0.2 M HCl for 15 min, washed
with PBS for 5 min twice and then treated with protease K in prehybridization buffer (100 mM
Tris-HCl and 50 mM EDTA, pH 8.0) at 37 ˚C for 30 min. After being rinsed with TBS (50 mM
Tris-HCl, pH 7.4; 150 mM NaCl) twice, the sections were incubated in TBS containing 2 mg/ml
glycine at 20 ˚C for 2 min and then postfixed with 3.7% formaldehyde in PBS at 20 ˚C for 20
min. Sections were washed with TBS for 5 min twice, acetylated with acetic anhydride (0.25%
acetic acid in 0.1 M triethanolamine-HCl, pH 8.0) at 20 ˚C for 10 min and dehydrated with serial
ethanol solutions. A hybridization solution (10 mM Tris-HCl, pH 6.8; 10 mM Na-phosphate
buffer, pH 6.8; 5 mM EDTA. pH 8.0), 40% [v/v] deionized formamide, 10% [v/v] dextran
sulfate, 300 mM NaCl, 1 mg/ml yeast tRNA, RNase inhibitor [Promega] and 400 ng/ml DIG-
labeled riboprobe) was added onto the sections. After being treated with 50% formamide at 50
˚C for 12 h, sections were washed twice with 2 x SSC at 37 ˚C and incubated with 20 μg mL
RNase A (Promega) at 37 ˚C for 30 min, followed by washes each for 1 h with 2 x SSC, 1 x
SSC, 0.1 x SSC at 65 ˚C. Immunological detection was performed with the Nucleic Acid
Detection Kit (Boehringer Mannheim) according to the supplier's protocol. Samples were
observed under bright field with a Leica DMLB microscope.
Phylogenetic analysis
Full-length amino acid sequences of 17 maize cysteine proteases closest to that of
ZmPCP were obtained from Blast of Genebank using ZmPCP sequence as the query. An
unrooted phylogenetic tree was constructed with use of the neighbor-joining method based on
the matrix of multiple sequence alignments of the 18 cysteine proteases created through
ClustalW. CLC Sequence Viewer (6.5.2) (www.clcbio.com) was used for phylogenetic tree
construction, and bootstrapping analysis was performed (replicates=100).
ACKNOWLEDGEMENTS
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This work was supported by the U.S. Department of Agriculture (NRI grant 2005-
02429). We thank Drs. Sean Coughlan and Tony Kinny of DuPont (Wilmington, DE) for
antibodies against castor bean calreticulin, Dr. Alessandro Vitale of Consiglio Nazionale Delle
Ricerche (Milano, Italy) for antibodies against tobacco γ-TIP, and Dr. Philip Rea of University of
Pennsylvania for antibodies against pea V-PPase.
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33
LEGENDS
Figure 1. Properties of a maize cysteine protease, ZmPCP.
(A) SDS-PAGE of proteins of maize pollen total extract and coat. The gel was stained with
Coomassie blue. The coat sample (20x) and the total extract (1x) were obtained from a fixed
amount of pollen. Molecular weights are shown on the right. The 3 coat proteins are β-glucanase
(70 kDa), xylanase (35 kDa) and cysteine protease (25 kDa).
(B) An unrooted phylogenetic tree of ZmPCP and the most similar 17 maize cysteine proteases.
Members of the SEN102 homolog clade are shown in bold. Bootstrap values are labeled.
(C) In situ hybridization of ZmPCP transcript in anthers. Sections of anthers of stage 3 were
hybridized with an anti-sense or sense RNA probe for ZmPCP. Definition of developmental
stage 3 is presented in Fig. 2 legend. Two lobes of an anther are shown. epi, epidermis; en,
endothecium; ml, middle layer; arrow, microspore; arrowhead, tapetum.
(D) RT-PCR of RNA extracted from different organs with gene-specific primers for GLA
(ZmGLA), XYN (ZmXYN), PCP (ZmPCP) and a maize actin gene.
(E) RNA blot hybridization of RNA from anthers of progressive developmental stages (1 to 4),
mature pollen (MP) and germinated pollen (GP). The extensin gene encodes a pollen extensin,
which is synthesized in the microspores at a late stage of anther development and pollen during
germination (Suen et. al., 2003). The lower panel shows a similar gel that had been stained with
ethidium bromide to reveal the 25S and 16S RNA.
Figure 2. SDS-PAGE and immunoblot analyses of extracts of maize anthers of progressive
developmental stages and mature pollen.
The various developmental stages are defined mainly by the morphology of the microspores. At
stage 1, the microspores were in a tetrad structure. At stage 2, the microspores were solitary and
had the exine wall. At stages 3 and 4, the microspores were binucleate and trinucleate,
respectively. The various extracts applied to the gel represent those from an equal number of
anthers. Gels were stained for proteins or subjected to immunoblot analyses with antibodies
against glucanase (70 kDa), xylanase (60 and 35 kDa) and protease (25 kDa). In the immunoblot
for xylanase, the antibodies recognized a constitutive, unknown protein of 55 kDa inside the
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34
microspores or pollen (P), an inactive 60-kDa pre-xylanase and an active mature 35-kDa
xylanase (Wu et al., 2002). Molecular weights are shown on the right.
Figure 3. Electron microscopy images of maize tapetum cells of progressive developmental
stages of 1-4.
Definition of the various developmental stages is presented in Fig. 2 legend.
At stage 1, the tapetum cell had 1 nucleus, and its cytoplasm was densely packed with RER and
secretary vesciles, as well as some Golgi, mitochondria and proplastids. At stage 2, the tapetum
cell had 1 or 2 nuclei, and the cytoplasm had lesser organelles. At stage 3, the tapetum cell had
lost its nuclei, and small vacuoles appeared. At stage 4, the tapetum cell became elongated with
its wall facing the locule dissolved; it had larger vacuoles and less-dense cytoplasmic contents.
Panel B is an enlarged picture of a portion of the stage-3 tapetum cell in panel A. Many secretory
vesicles of 0.2-0.5 μm in diameter were present; they, in comparison to the vacuoles, were
smaller, with a more electron-dense, granular matrix, and concentrated near the locular side of
the cytoplasm (Fig. 3B).
The locule (L), microspore (M), tapetum cell (T), outer anther cell (A), nucleus (n), vacuole (va),
vesicle (v), mitochondrion (m) and lipid body (l) are labeled.
Figure 4. Bright-field Differential Interference Contrast and immunofluorescence microscopy
images of maize anther lobes.
One lobe of an anther of each progressive developmental stage is shown. Immunofluorescence
images were obtained after treating samples with antibodies against the indicated proteins. Cy5-
conjugated secondary antibodies were used. Excitation wavelength of 648 nm and emission
wavelength of 645-700 nm were instituted, under which autofluorescence of the pollen wall was
not detected. Identical CLSM settings (laser power and detection gain) were used for all images
of each hydrolase. In the anther lobe, several layers of outer anther wall cells (A) and the
tapetum (T) together enclosed the locule (L) in which microspores (M) matured.
Figure 5. Immunofluorescence microscopy images of single tapetum cells of maize anthers for
subcellular localization of glucanase, xylanase and protease.
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35
Panels A and B show microscopy images of stage-3 and -4 tapetum cells, respectively, and the
cell circumference is marked with a dotted white line. Samples were double-labeled with
antibodies against two proteins, which could include calreticulin (ER marker), γ-TIP and V-
PPase (both vacuole markers). The nucleus (n) and locule (L) are labeled. Tapetum cells in panel
B had lost most of their cell wall and did not maintain a rigid shape. Panel C shows the
specificity of the antibodies against V-PPase and γ-TIP in total extracts of stage-3 anthers. The
extracts were subjected to SDS-PAGE, and the gel was stained for proteins or treated for
immunoblotting. Lanes 1 and 3 show Coomassie blue stained gel, and lanes 2 and 4 reveal
immunoblotting (IB) results. Molecular weights are shown on the right, and the recognized
antigens are indicated with arrows.
Figure 6. Sucrose density gradient centrifugation of maize tapetum extract for subcellular
localization of glucanase, xylanase and protease.
Extract of stage-3 and -4 anthers, representing materials mostly from tapetum cells (see
Experimental Procedures), was subjected to gradient centrifugation, and the gradient was
fractionated.
Panel A shows a plot of sucrose densities against fraction numbers of the gradient. Locations of
peak xylanase, protease and glucanase are indicated.
Panel B shows SDS-PAGE gels of the fractions followed by protein staining or immunoblot
analyses with antibodies against xylanase, protease, V-PPase (vacuole marker), glucanase (Glu)
and calreticulin (ER marker). Molecular weights are shown on the right, and the recognized
antigens are indicated with arrows.
Panel C shows EM pictures of a portion of a tapetum cell in situ and isolated vesicles in the
fraction containing peak glucanase (fraction #9). The cell in situ contained vesicles (v) with a
relatively electron-dense matrix, which can be distinguished from the vacuoles (va) with a less
electron-dense matrix.
Figure 7. Flavonoids in maize anthers analyzed with TLC and CLSM.
Panel A. TLC analysis of flavonoids in extracts of anthers of different developmental stages and
in mature pollen coat. The flavonoid glycosides in the samples were first acid-hydrolyzed to free
flavonoids for TLC analysis and DPBA staining. Location of the markers, quercetin (Q),
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36
kaempferol (K) and isorhamnetin (I) on the TLC plate are indicated; some faster moving
molecules stained as red or blue are unidentified.
Panel B. Sections of stage-3 anthers were double-labeled with DPBA for flavonoids (shown in
green) and antibodies against organelle markers (calreticulin for ER and V-PPase for vacuoles)
(shown in red). The tapetum cell circumference is marked with white dotted lines.
Panel C. Sections of stage-3 and -4 anthers labeled with DPBA for flavonoids (shown in green).
Before lysis of the tapetum cell (circumference of the cell marked with a white dotted line),
flavonoid signals were confined largely in the tapetum cell, and the adjacent microspore had
minimal signals, which, upon high magnification, appeared to be inside near the outermost
boundary of the pollen protoplast. After tapetum cell lysis (circumference of the cell ghost
marked with a white dotted line), flavonoid signals appeared mostly on the microspore surface.
Figure 8. Bright-field, auto- and immuno-fluorescence microscopy images of stage-3 and -4
maize microspores and mature pollen.
Microspores removed from anthers and mature pollen were subjected to immuno treatment. Cy5-
conjugated secondary antibodies were used. Excitation wavelength of 648 nm was used, and
emission wavelength of 645-700 nm were used for instituted. For visualization of
autofluorescence of microspores/pollen, excitation wavelength of 488 nm and emission
wavelength of 530-580 nm were used. Identical CLSM settings (laser power and detection gain)
were used for all images.
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37
Supplemental information
Figure S1. Sequence alignments of 17 maize cysteine proteases and 1 rice cysteine protease
most similar to ZmPCP. Full-length sequences of ZmPCP, 17 maize cysteine proteases and 1 rice
cysteine protease (OsCP1, BAF16172) most similar to that of ZmPCP were made into multiple
sequence alignments using ClutalW. Amino acid residues are shown in a color gradient (dark
pink - dark blue) to represent residue conservation from 0 to 100%. Black triangles locate the
conserved peptidase catalytic residues: Gln, Cys, His and Asn. Along or immediately above or
below the ZmPCP sequence, the red box marks the 14-residue sequence obtained from
microsequencing; the black box marks the sequence of the synthetic peptide used for polyclonal
antibodies generation; the black broken line marks the predicted signal peptide; the black line
marks the NTPP domain sequence; and # marks the ERFNIN motif residues.
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B C
D
ZmGLA
ZmXYN
ZmPCP
Actin
Anti- sense
Sense
20 µm
GP MP 4 3 2 1
ZmXYN ZmPCP
EtBr stained
ZmGLA
Extensin
A
113 kDa 1X
20X
tota
l
coat
92
52
35
21 ACF84854 ACG38044 AAB70820 ACF87096 AAB88262
ACG38442 ACF82315 ACG30386 ZmPCP ACG25394
BAA08244 BAA08245
ACG36860 ACG37911 ACF87422 ACF78748 ACG42266
AAB88263
100
100 100
100
100 96
65
67
64
71 100
100 100 98
99
46
E
epi en ml
epi en ml
Li et al. Figure 1
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1 2 3 4 P kDa 113 92
52
35
21
60
35
70
25
Xyl
anas
e G
luca
nase
Pr
otea
se
Imm
nobl
ots
Stai
ned
gel
Li et al. Figure 2
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va
3 4
L n
1
1 µm
1 µm 1 µm
A
B
1 µm
va
l
m
A
T
M
v
n
2
1 µm L
L M
Li et al. Figure 3
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Xyl
anas
e G
luca
nase
Pr
otea
se
2 3 4
Stage
T
A
M
80µm 80µm 80µm
L
Brightfield IF Brightfield IF Brightfield IF
Li et al. Figure 4
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Glucanase Merged
n
L 4 µm
Xylanase Merged
Calreticulin
Calreticulin
Xylanase Glucanase Merged
4 µm
L
n
n L
4 µm
Merged
Merged
γ-TIP
V-PPase
V-PPase
Protease
Protease
Glucanase Merged
A
C
B
4 µm
4 µm
L
4 µm
66
27
kDa kDa
92
52
35
21
92
52
35
21
Gel IB IB Gel V-PPase γ-TIP
1 2 3 4
Stage 3 Stage 4
Li et al. Figure 5
1.05
1.1
1.15
1.2
1.25
2 4 6 8 10 12 14 16 18 20 22 24 C
ell i
n si
tu
Peak
glu
cana
se f
ract
ion
(#9)
Fraction number
Den
sit (
g.cm
-3)
kDa 109 55
34 30
20
2 4 6 7 8 9 10 11 13 15 17 19
60
35
25
66
70
55
Zm
XY
L Z
mPC
P V-
PPas
e Z
mG
LA
C
al
Imm
unob
lots
St
aine
d ge
l
200 nm
200 nm
va
m
v
A
B C
Li et al. Figure 6
1 2 3 4 Pollen Stage
DPBA Calreticulin Merged
DPBA V-PPase Merged
K I Q
Origin
5 µm
A
B
C
20 µm
DPBA W/ DIC
Stag
e 3
Stag
e 4
(late
)
20 µm
5 µm
Li et al. Figure 7
Lit et al. Figure 8
Brightfield Auto IF Merged M
atur
e
Glu
cana
se
Xyl
anas
e Pr
otea
se
Glu
cana
se
Xyl
anas
e Pr
otea
se
Glu
cana
se
Xyl
anas
e Pr
otea
se
Stag
e 3
Stag
e 4
20 µm
20 µm
20 µm