1 running title: biogenesis of pollen-coat material in maize tapeta

1

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

www.plantphysiol.orgon February 13, 2018 - Published by Downloaded from Copyright © 2012 American Society of Plant Biologists. All rights reserved.




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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



3

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]



4

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.



5

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



6

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



7

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.



8

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.



9

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



10

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



11

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.



12

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



13

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



15

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).



16

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



17

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



18

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,



19

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.



20

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



21

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.



22

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



23

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



24

(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.



25

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



26

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.



27

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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



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.



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),



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.



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.



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



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



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



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



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

1 running title: biogenesis of pollen-coat material in maize tapeta

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