Botanical Studies (2008) 49: 25-32.

*

Corresponding author: Email: jyewu@cc.ncu.edu.tw; Tel:

+886-3-422-7971; Fax: +886-3-422-8482.

INTRODUCTION

Pollen tube elongation is a crucial event in the sexual

reproduction of flowering plants. It begins when pollen

germinates on the stigma and then continues to penetrate

the stigmatic tissue, style, and transmitting tract. Finally,

the pollen tube reaches the ovule and delivers the genetic

material for fertilization. Pollen tube elongation involves

many specialized mechanisms that control growth and

navigation in order for the pollen tube to reach the

dedicated destination. These features make the pollen tube

a unique system for the study of not only male fertility in

plants, but also many other distinctive aspects of biology

(Franklin-Tong, 1999; Edlund et al., 2004). One of them

is that, while most plant cells expand through a diffuse

growth mechanism, pollen tube elongation occurs only at

the utmost apex of the tube through a polarized tip growth

mechanism (Hepler et al., 2001).

Because the pollen tube¡¦s growth is restricted to the

tube apex, many molecules must be translocated to the

growing tip. For example, pectic polysaccharides are

assembled in the Golgi apparatus, then transported in

vesicles, and subsequently released at the growing tip

through exocytosis (Sterling et al., 2001). This process

is essential because pectin is a major component of the

cell wall at the growing pollen tube tip (Ferguson et al.,

1998). In addition to the cell wall materials, vesicles

are responsible for carrying nascent membranes with

embedded proteins and secretory molecules to the growing

tube tip. Pollen tube growth also involves the recycling of

excess membranes and uptake of female derived molecules

through endocytosis (Derksen et al., 1995; Parton et al.,

2001). It has been well characterized that an inverted

cone-shaped region adjoining the pollen tube apex is

devoid of organelles and almost exclusively occupied by

vesicles (Franklin-Tong, 1999). These vesicles must retain

their individual specificity so that each of them can find

and fuse with its unique target membrane and deliver its

cargo to its designated compartment during pollen tube tip

growth.

Current models indicate that the recognition between

a vesicle and a target membrane is initially mediated

by tethering factors. Tethering factors can be generally

Arabidopsis HIT1, a putative homolog of yeast tethering

protein Vps53p, is required for pollen tube elongation

Lian-Chin WANG

1

, Ching-Hui YEH

1

, Ronald J. SAYLER

2

, Ya-Yun LEE

1

, Chung-An LU

1

, and

Shaw-Jye WU

1,

*

1

Department of Life Sciences, National Central University, No. 300, Jhong-da Road, Jhong-li City, Taoyuan 320, Taiwan

2

Department of Plant Pathology, 217 Plant Science, University of Arkansas, Fayetteville, AR 72701, USA

(Received March 27, 2007; Accepted September 29, 2007)

ABSTRACT.

The Arabidopsis HIT1 gene encodes a protein that is homologous to the yeast tethering factor

Vps53p, which is involved in retrograde vesicle trafficking from the endosome to the trans-Golgi network.

Although the ethyl methanesulfonate mutagenized hit1-1 allele can be maintained homozygously, T-DNA

insertional hit1-2 and hit1-3 mutants can only be isolated as hemizygous lines. No heterozygous progeny

were produced in outcrosses to wild-type plants using pollen from either hit1-2 or hit1-3 heterozygotes. The

reciprocal cross using pollen from wild-type plants on either hit1-2 or hit1-3 mutants produced heterozygous

and wild-type progeny. In reproductive tissues, HIT1 promoter-driven GUS activity was detected only in

mature pollen and elongated pollen tubes. In vitro pollen germination further showed that only half the pollen

grains from hit1-2 and hit1-3 heterozygote plants produced normal pollen tubes. In contrast, the pollen tube

length of pollen grains from the hit1-1 mutant was reduced compared to that of the wild type. These results

suggest that HIT1 may govern a vesicle trafficking event that is required for pollen tube tip growth during

male gametogenesis and that disruption of HIT1 results in male specific transmission defect. Moreover, while

the hit1-1 mutant is partially functional leading to reduced pollen tube length, hit1-2 and hit1-3 are total-loss-

of-function alleles.

Keywords: Arabidopsis thaliana; Pollen tube; Tip growth; Vesicle tethering factor; Yeast Vps53p protein.

Abbreviations: hit, heat-intolerant; GARP, Golgi-associated retrograde protein; TGN, trans-Golgi network;

VPS, vesicular protein sorting.

PHYSIOLOGY

26

Botanical Studies, Vol. 49, 2008

divided into a group of extended coiled-coil proteins

and a class of oligomeric complexes. These tethers act

to establish physical links, over considerable distances,

between two membranes that are due to fuse (Sztul and

Lupashin, 2006). Despite the fact that many of these

regulators have been identified in plants through genomic

analysis, the question of which of them are essential and

what biological roles they play during pollen tube tip

growth has yet to be resolved. Only POK, a putative

homolog of yeast Vps52p in the Golgi-associated

retrograde protein (GARP) complex, and AtSEC8, a

subunit of the putative exocyst complex, have recently

been shown to regulate pollen tube growth in Arabidopsis

(Lobstein et al., 2004; Cole et al., 2005).

Previously, an ethyl methanesulfonate (EMS)

mutagenized hit1-1 (heat-intolerant) mutant, the growth

of which had been inhibited by high temperatures,

was isolated from Arabidopsis (Wu et al., 2000). The

mutated gene was later identified to encode a homolog of

the yeast tethering factor Vps53p (Lee et al., 2006). In

yeast, Vps51p, Vps52p, Vps53p and Vps54p constitute

a tetrameric GARP tethering complex. This complex

interacts with a SNARE and a Rab GTPase to mediate

retrograde vesicle trafficking from the endosome to the

trans-Golgi network (TGN) (Conibear and Stevens, 2000;

Siniossoglou and Pelham, 2001; Conibear et al., 2003).

Although hit1-1 mutant can be maintained homozygously,

T-DNA insertional hit1-2 and hit1-3 mutants are like the

T-DNA insertional pok mutant that can only be isolated as

a hemizygous line. This finding suggests that HIT1 may

also be involved in pollen tube growth. Here we provide

evidence to support this hypothesis.

MATERIALS AND METHODS

Plant materials and growth conditions

The wild-type Arabidopsis thaliana plants used in

this study were in the Colombia-0 background. Seeds

were obtained from the Lehle Seeds Company (Round

Rock, TX, USA). The hit1-1 mutant line was isolated

from the F

2

progeny of plants mutagenized with EMS, as

described (Wu et al., 2000). The T-DNA insertion alleles

hit1-2 (GABI_100C01) and hit1-3 (SALK_047230)

were obtained from the Max-Planck Institute, Cologne,

Germany (Rosso et al., 2003) and the Arabidopsis

Biological Resource Center at Ohio State University,

Columbus, Ohio, respectively. Plants were either grown in

soil or on Murashige Skoog (MS) salt agar plates at 23

o

C

under a light cycle of 16 h light/8 h dark for both wild type

and hit1-1 mutant. For the selection of T-DNA insertion

lines, seeds were pregerminated on MS-salt agar plates

with sulfadiazine (hit1-2) or kanamycin (hit1-3) according

to the suppliers¡¦ instructions.

Genotyping of T-DNA insertion hit1 mutants

The predicted HIT1 gene has 24 exons with translation

start for the HIT1 transcript located in the second exon,

based on the most current sequence information from the

Arabidopsis Information Resource (TAIR, http://www.

Arabidopsis.org), the mutant supplier¡¦s data, and the open

reading frame derived from cloned cDNA. The hit1-2

allele has a T-DNA inserted in the eighth exon and hit1-3

allele has a T-DNA inserted in the seventh intron (Figure

1a). Genotyping of the hit1-2 lines was performed by

PCR using the HIT1-complemented forward primer (5¡¦-

GCTGGAACGGATTTTTATTTCTGG-3¡¦) and the T-DNA

left border complemented reverse primer (5¡¦-CCCATT

TGGACGTGAATGTAGACAC-3¡¦) to amplify a 601 bp

DNA fragment corresponding to the junction sequence

for hit1-2. Control PCR assays amplified the 909 bp

non-disrupted HIT1 sequence using the same forward

primer with the HIT1-complemented reverse primer (5¡¦-

ATTATTCCCGTGCCAAGTAGG-3¡¦). A similar strategy

was also applied to genotype the hit1-3 lines with HIT1-

complemented forward primer (5¡¦-CCAAACCAGCTCA

TTGTCATTTTG-3¡¦), T-DNA left border complemented

reverse primer (GCGTGGACCGCTTGCTGCAACT-

3¡¦), and HIT1-complemented reverse primer (5¡¦-

GCCTATACGGCACATGCCAAG-3¡¦).

Preparations of the HIT1 promoter construct

The promoter region of HIT1 was amplified with

gene-specific primers 5¡¦-GTGAAGCTTGGCATCAAC

Figure 1. Insertional mutations affecting the hit1 transmission is

male specific. (a) Intron-exon structure of the HIT1 gene. Solid

black lines represent introns, and gray boxes represent exons.

The positions of the point mutation in hit1-1 and the T-DNA

insertion in hit1-2 and hit1-3 are indicated, respectively. The

predicted translation start is specified by an arrow; (b) Progeny

from an outcross using HIT1/hit1-2 heterozygote anthers to

pollinate wild-type plants were genotyped by PCR using a set of

three primers to produce distinct HIT1 and hit1-2 bandings. No

heterozygous progeny were produced; (c) Heterozygous progeny

were produced in an outcross to a HIT1/hit1-2 heterozygote

using pollen from a wild-type homozygote. W, wild type; h,

HIT1/hit1-2 heterozytote; M, molecular marker.

WANG et al. ¡X A tethering protein homolog in male gametophyte

27

ACCATCATCTAAACA-3¡¦ and 5¡¦-GGCCTGCAGTTT

GTATGATAAAACCAAAAATCA-3¡¦ (HindIII and PstI

sites are underlined). The 2.4 kb amplified products were

resolved by electrophoresis, gel purified, and cloned into

pGEMT-Easy vector (Promega, Madison Wisconsin,

USA). After sequence verification, the fragments were

inserted upstream of the £]-Glucuronidase (GUS) reporter

gene followed by the nopaline synthase terminator in

pCAMBIA1300 Ti-derived binary vector (CAMBIA,

Canberra, Australia). Agrobacterium tumefaciens strain

GV3101 was used to deliver this construct into wild-

type plants by vacuum infiltration (Clough and Bent,

1998). Transgenic plants were selected on MS agar

medium containing 25 £gg/mL Hygromycin B for 14 days.

Resistant T1 seedlings were transferred to soil and grown

to maturity. Homozygous T2 lines were selected by

screening their T3 progeny using resistance to Hygromycin

B as the marker. T3 seeds derived from homozygous T2

lines were used for GUS assays.

Histochemical GUS assays

Agar grown whole seedlings were vacuum-infiltrated

with 1 mM 5-bromo-4-chloro-3-indolyl glucuronide

(X-gluc), 29 mM Na

2

HPO

4

, 21 mM NaH

2

PO

4

, and 0.1

% (v/v) Triton X-100 at pH 7.0 and then incubated at 37

o

C to stain for GUS activity. After staining for 4 h, the

samples were washed several times with 70% ethanol to

remove the chlorophyll. For GUS assays on pollen grains,

inflorescences were cut off and prefixed in chloroform:

ethanol:water (3:6:1, v/v/v), 0.1% (v/v) Triton X-100

for 30 min followed by the vacuum-filtration and GUS

staining procedures described above (Procissi et al., 2003).

After staining, anthers at different stages were separated

from flowers, and their contents were released using a

needle under a dissecting microscope.

In vitro pollen germination

The in vitro pollen germination assay was adapted from

previous reports (Li et al., 1999; Fan et al., 2001; Johnson-

Brousseau and McCormick, 2004). The medium used for

pollen germination was prepared using double distilled

water and contained 18% (w/v) sucrose, 0.01% (w/v)

boric acid, 1 mM MgSO

4

, 1 mM CaCl

2

, 1 mM Ca(NO

3

),

and 1% (w/v) agar at pH 7. After heating at 100¢XC for 2

min, the medium was allowed to solidify on glass slides,

forming a 1-mm thick layer. Newly dehisced anthers

were detached from flowers and carefully rubbed onto

the surface of the germinating medium to transfer the

pollen grains. Following the application of pollen, the

slides were immediately put inside a box with water in the

bottom to maintain the relative humidity at 100%. The

entire assembly was then transferred to a growth chamber

at 25¢XC with constant illumination and incubated for 12 h

before examination under a light microscope.

RESULTS

Disruption of the HIT1 gene causing a male-

specific transmission defect

The hit1-2 and hit1-3 alleles can only be isolated as

hemizygous lines, and the siliques produced from the

heterozygous parental plants were fully filled with viable

seeds, suggesting that disruption of HIT1 gene by T-DNA

insertion may result in a male-specific transmission defect

(Lee et al., 2006). To verify that the transmission defect

was indeed male specific, outcrosses were performed

using pollen from hit1-2 heterozygotes to pollinate the

stigma of wild-type plants. PCR-based genotyping

revealed that no heterozygous progeny were produced

(Figure 1b). Outcrosses were also made using pollen from

wild-type plants to pollinate hit1-2 heterozygotes, and the

PCR-based genotyping revealed that both wild-type and

heterozygous progeny were produced (Figure 1c). Similar

results were also found when testing hit1-3 alleles (Table

1), confirming that the transmission defect is specifically

associated with male gametophyte. While T-DNA

insertion in the hit1-2 and hit1-3 alleles showed a strong

male transmission defect, the single base substituted allele

Table 1. Inheritance of mutant HIT1 alleles.

No. of Progeny tested

a

Genotypes of progeny

b

x

2

P

+/+ +/m m/m

Natural self cross of HIT1 heterozygotes

25% 50% 25%

Expected

HIT1/hit1-2

425

54% 46%

0%

251.8 .0.001

HIT1/hit1-3

124

53% 47%

0%

70.8 .0.001

Outcross of HIT1 heterozygotes

50% 50%

--

Expected

HIT1/hit1-2 (¡ñ) ¡Ñ WT (¡ð)

66

100% 0%

--

66.0 .0.001

HIT1/hit1-3 (¡ñ) ¡Ñ WT (¡ð)

58

100% 0%

--

58.0 .0.001

WT (¡ñ) ¡Ñ HIT1/hit1-2 (¡ð)

66

55% 45%

--

0.545

NS

c

WT (¡ñ) ¡Ñ HIT1/hit1-3 (¡ð)

54

54% 46%

--

0.296

NS

c

a

Samples are from three or more individual crosses;

b

+, HIT1; m, mutant hit1 allele;

C

NS, Not significantly different.

28

Botanical Studies, Vol. 49, 2008

hit1-1 was maintained homozygously. These findings

suggest that hit1-2 and hit1-3 are null alleles while hit1-1

is a partially functional allele.

The HIT1

promoter is predominately active in

the male gametophyte

The expression pattern of HIT1 was determined in

plants expressing the £]-glucuronidase reporter gene under

the control of the putative HIT1 promoter, a 2.4 kb DNA

fragment upstream of the HIT1 coding region. In 10-day-

old plantlets, the blue staining was restricted to the root

with the apex region showing enhanced intensity (Figure

2a). In mature plants, GUS staining was observed in

particular reproductive tissues at certain stages of flower

development. More specifically, blue coloration was

detected in the anthers after floral stage 11. At this time,

the petal height has exceeded that of the short stamens,

stigmatic papillae begin to appear, and meiosis of

microspore mother cell is complete, as previously defined

(Figure 2b-c) (Smyth et al., 1990; Sanders et al., 1999).

The blue color appears in the anthers as the filaments

continue to elongate rapidly through floral stage 12. GUS

staining begins to appear in the stigmatic region of the

style at floral stages 13 and 14, as the flower opens and the

long anthers extend to the stigma for pollen release. After

pollination, the style elongates, and the stigma extends

above long anthers, the landmark of floral stage 15. At

this stage, the blue coloration was still apparent in the

stigmatic region but had faded away in the anthers. The

absence of staining was due to the evacuation of pollen

grains, causing the anthers to appear translucent.

To better define the HIT1 promoter activities, GUS

stained tissues were analyzed microscopically. In the

root apex, enhanced GUS activity was observed in the

meristem and elongation zones. In contrast, GUS activity

was not detected in the root cap (Figure 3a). Microscopic

observation also revealed that the blue coloration was

clearly detectable in the pollen grains within the anthers at

stages 12 and 13, but not at stage 11 (Figure 3b-d). Further

examination showed that GUS activity was only expressed

in matured pollen grains, but not in tetrad or unicellular

microspores (Figure 3e-g). Although the blue coloration in

the pollen grains became lighter after pollination, possibly

caused by the exit of cytoplasm, strong GUS staining

occurred in the pollen tubes as they extended along the

papillae cells toward the style (Figure 3h). These results

indicate that the blue coloration observed in the pollinated

stigma came from pollen tubes, not the papillae cells.

Mutations in HIT1

affect pollen tube elongation

To examine if mutation of the HIT1 gene can result

in abnormal pollen tube growth, pollen germination

experiments were performed in vitro. Thirty pollen

grains from each of three flowers per plant were randomly

selected for observation. After 12 h of incubation, the

length of each pollen tube was measured. As shown in

Figure 4, most of the wild-type pollen tubes exceed 300

£gm in length, resulting in a single peak in the distribution

curve. For the hit1-1 mutant, none of the pollen tubes

exceeded 350 £gm. Their lengths were mainly between

50 £gm and 250 £gm, making a central plateau in the

distribution curve. For the HIT1/hit1-2 heterozygous

plants, roughly half of the pollen tubes were shorter

than 50 £gm and half were longer than 300 £gm, creating

two peaks at the opposite sides of the distribution curve.

These peaks may correspond to the hit1-2 and the HIT1

Figure 2. GUS assay of transgenic Arabidopsis plants harboring

HIT1 promoter-driven GUS reporter gene. (a) GUS staining

on a 10-day-old plantlet; (b) GUS staining on an inflorescence.

Blue signals are observed in the anthers of late flowers; (c) A

series of five flower buds on an inflorescence. The detached

flower buds were stained with X-Gluc solution, then the sepals

and petals were removed for photographic documentation.

WANG et al. ¡X A tethering protein homolog in male gametophyte

29

allele, respectively, in the haploid pollen grains (Figure 4).

Similar results were found when the pollen grains from

HIT1/hit1-3 heterozygous plants were examined (data not

shown). Together, these data indicate that HIT1 is crucial

for pollen tube growth. Disruption of HIT1 by T-DNA

insertion restricts pollen tube elongation while hit1-1 is a

partially functional allele resulting in a reduction of pollen

tube length.

DISCUSSION

Vesicle tethering factors are one of the major

components conferring the specificity of membrane

fusion, possibly at the earliest stage, in the course of

vesicle trafficking. Tethering factors can be large multi-

subunit complexes, and at least seven complexes have

been proposed to play roles in vesicle tethering at distinct

trafficking steps (Whyte and Munro, 2002). To date,

yeast and animal systems have provided the basis of our

understanding of vesicle tethering. Many homologs of

these tethering factors were identified in plants through

genomic analysis, but their biological roles remain

unknown (Jurgens and Geldner, 2002; Elias et al., 2003;

Figure 3. Microscopic observation of GUS activity in transgenic Arabidopsis plants harboring HIT1 promoter-driven GUS reporter

gene. (a) The root apex region from a 10-day-old plantlet; (b, c, d) Anthers from flowers at floral stages 11 (b); 12 (c), and 13 (d);

(e) Tetrads; (f) Uninucleate microspore; (g) Mature pollen grains; (h) The stigmatic region of a self-pollinated transgenic plant,

showing blue-colored pollen tubes along papillae cells. The pollen grains may become detached from the pollen tubes during sample

preparation. Some pollen tubes and papillae cells are out of focus. pt, pollen tube. Scare bars: (a, b, c, d) 50 £gm; (e, f, g, h) 25 £gm.

Figure 4. The distribution of pollen tube lengths in vitro. Three

flowers from each plant were picked, and 30 pollen grains from

each flower were randomly chosen for observation for a total

of 90 pollen grains per plant. Three plants from each line were

used to calculate the standard deviations of the mean percent of

pollen tubes at each length for each genotype using SigmaPlot

2000 software (SPSS Inc., Chicago, IL).

30

Botanical Studies, Vol. 49, 2008

Latijnhouwers et al., 2005). Only very recently, functional

characterizations of the plant genes AtSEC8 and POK by

mutant analysis have been reported (Lobstein et al., 2004;

Cole et al., 2005). Each of these genes, however, encodes

a putative subunit of different tethering complexes. HIT1

and POK, on the other hand, encode putative subunits of

the same tethering complex. Therefore, the hit1 mutants

are valuable for both functional studies and for providing

a new approach to dissecting the multifarious vesicle

tethering in plants.

Male gametophyte development occurs through a series

of sequential steps, and disrupting any of these steps can

result in male-specific transmission defects. Whether HIT1

would affect pollen development at earlier developmental

events, such as mitotic cytokinesis, is uncertain. However,

because the HIT1 promoter-GUS fusion activity appeared

in the mature pollen grains and the growing pollen tubes, it

was thought that one function of the HIT1 was to facilitate

the initiation and maintenance of the polarized growth of

pollen tubes.

The fact that HIT1 is a homolog of known

tethering factors provided additional support for this idea.

The reduction in pollen tube length produced by pollen

from hit1-1 plants also lends credence to this hypothesis.

Additionally, male gametophytes with reduced pollen tube

length like those harboring hit1-1 allele can theoretically

cause segregation distortion, due to competition with wild-

type gametophytes. In fact, the analysis of F2 progeny

from a cross between the wild type and a hit1-1 mutant

revealed a 4:1 ratio of wild-type to mutant phenotype,

instead of the standard 3:1 ratio (Wu et al., 2000).

In plants, the elongation of root hair also employs a tip

growth mechanism. Since hit1-2 and hit1-3 can only be

isolated as hemizygous lines, it is not possible to study

the effects of these two T-DNA insertion alleles on root

hair development. However, the hit1-1 mutant did exhibit

reduced root hair length (Wang and Wu, unpublished

data), suggesting that HIT1 may have a general role in

polar growth.

In yeast, Vps51p, Vps52p, Vps53p, and Vps54p

constitute the newly discovered tetrameric GARP tethering

complex (Conibear and Stevens, 2000; Siniossoglou

and Pelham, 2001; Conibear et al., 2003). In addition

to HIT1, which is homologous to Vps53p, Arabidopsis

genes POK and AtVps54 (At1g71270 and At4g19490,

respectively) were recently identified to encode potential

homologs of yeast Vps52p and Vps54p (Lobstein et al.,

2004; Latijnhouwers et al., 2005). Nevertheless, HIT1

and POK promoter-driven GUS staining showed different

expression patterns in the reproductive tissues. While

HIT1 expression was specifically in the haploid male

gametophyte, the expression of POK can be sporophytic

and female oriented, as blue staining was also observed

in ovules (Lobstein et al., 2004). The differences in

GUS expression between the HIT1 and POK constructs

may have due to the fact that the GUS gene in the POK

study was fused with the first ten exons of the POK, and

the GUS gene for HIT1 in the present study was placed

directly downstream of the HIT1 5¡¦ untranslated region.

Alternatively, a gene encoding a homolog of Vps51p has

not been detected in Arabidopsis, and considering the

complexity in cellular organization and secretory proteins

in plants, it has been proposed that the vesicle-trafficking

machinery may have evolved independently in plants

(Jurgens and Geldner, 2002).

The hit1-1 mutant was originally identified by its

susceptibility to heat and osmotic stress (Wu et al.,

2000). This susceptibility made it clear that HIT1 is not

only important in the male gametophyte, but also in

somatic cells. Vesicle trafficking has been proposed to

be essential in plant responses to stress (Levine, 2002).

This process is required for the removal of damaged

membranes and the delivery of the new ones to their target

sites. Indeed, if HIT1 is involved in vesicle tethering

events, the partially functional hit1-1 allele would lead

to inefficient vesicle trafficking, explaining the observed

stress sensitive phenotypes in addition to reduced pollen

tube length. Furthermore, a low level expression of

HIT1 was detected in other vegetative tissues by RNA

gel blot and reverse transcription-PCR (Lobstein et al.,

2004; Lee et al., 2006). Microarray analysis on the other

hand showed that the expression of HIT1 is low and lacks

tissue specificity (Schmid et al., 2005). Although the

data in this report shows that the HIT1 promoter-driven

GUS activity was primarily detected in the root of young

plantlets, a trace amount of GUS activity was detected

in soil-grown rosette leaves (Wang and Wu, unpublished

data). The difference in the expression pattern may be

because of different experimental methods. Taken as a

whole, a single tethering protein homolog may govern

many important cellular events, in both gametophytic and

sporophytic cells, affecting plant growth in different ways

at various developmental stages. Further characterization

and analyses of the hit1 mutants and the function of HIT1

should provide new insights into the importance and the

diverse roles of tethering factors in plants.

Acknowledgement. This work was supported by

National Science Council (Taiwan) through Grant NSC

95-2311-B-008-007-MY2 (S.-J. Wu).

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Botanical Studies, Vol. 49, 2008