Rh-PIP2;1, a Rose Aquaporin Gene, Is Involved intially decreased the petal fresh weight, while 1-MCP...

Rh-PIP2;1, a Rose Aquaporin Gene, Is Involved inEthylene-Regulated Petal Expansion1[C][W][OA]

Nan Ma, Jingqi Xue, Yunhui Li, Xiaojing Liu, Fanwei Dai, Wensuo Jia, Yunbo Luo, and Junping Gao*

Department of Ornamental Horticulture (N.M., J.X.,Y. Li, F.D., J.G.), Department of Pomology (W.J.), andLaboratory of Fruit Biology, College of Food Science and Nutritional Engineering (Y. Luo), ChinaAgricultural University, Beijing 100193, China

Aquaporins are water channel proteins that facilitate the passage of water through biological membranes and play a crucialrole in plant growth. We show that ethylene treatment significantly reduced petal size, inhibited expansion of petal abaxialsubepidermal cells, and decreased petal water content in rose (Rosa hybrida ‘Samantha’). Here, we report the isolation of aplasma membrane aquaporin (PIP) gene, Rh-PIP2;1, and characterized its potential role in ethylene-inhibited petal expansion.Rh-PIP2;1 is mainly localized on the plasma membrane and belongs to the class 2 subfamily of PIP proteins. We show that Rh-PIP2;1 is an active water channel. The transcripts of Rh-PIP2;1 are highly abundant in petal epidermal cells, especially in theabaxial subepidermal cells. The expression of Rh-PIP2;1 is highly correlated with petal expansion and tightly down-regulatedby ethylene. Furthermore, we demonstrate that in Rh-PIP2;1-silenced flowers, petal expansion was greatly inhibited andanatomical features of the petals were similar to those of ethylene-treated flowers. We argue that Rh-PIP2;1 plays an importantrole in petal cell expansion and that ethylene inhibits petal expansion of roses at least partially by suppressing Rh-PIP2;1expression.

The gaseous phytohormone ethylene modulatesvarious plant developmental processes, including or-gan growth, flowering, fruit ripening, leaf senescence,and abscission (Abeles et al., 1992; Wang et al., 2002).Particularly, the inhibitory role of ethylene in organgrowth has been well documented (Pierik et al., 2006).In Arabidopsis (Arabidopsis thaliana), reduction of hy-pocotyl elongation in dark-grown seedlings occurredmerely 15 min after the plant was subjected to ethyl-ene treatment (Binder et al., 2004). Ethylene-inhibitedgrowth was also found in stems and petioles ofArabidopsis (Smalle and Van der Straeten, 1997), rootsof Rumex and cucumber (Cucumis sativus; Visser et al.,1997; Pierik et al., 1999), and leaves of Poa species(Fiorani et al., 2002). In flower development, ethylenehas been reported to be mainly involved in sexualdetermination (Yamasaki et al., 2003) and petal senes-cence (Woltering and van Doorn, 1988; van Doorn andvan Meeteren, 2003; van Doorn and Woltering, 2008).

Especially, several reports proved that ethylene alsoplayed an important role in floral organ abscission(Patterson and Bleecker, 2004; Butenko et al., 2006). Inroses (Rosa spp.), Reid et al. (1989a, 1989b) showed thatethylene could promote or inhibit petal growth in acultivar-dependent manner. However, little is knownconcerning the regulatory mechanisms of ethylenein petal development during the process of floweropening.

It has been reported that ethylene inhibits plantorgan growth by regulating cell elongation and ex-pansion processes. Ethylene impedes cell expansion inroots along the direction of the long axis but enhancescell radial expansion (Chadwick and Burg, 1967).Ethylene induces the formation of apical hypocotylhooks by regulating asymmetrical cell elongation onthe adaxial and abaxial sides of the apical hook regionin Arabidopsis and pea (Pisum sativum; Lehman et al.,1996). Moreover, Arabidopsis ethylene constitutiveresponse mutant plants (ctr1) are tiny and their leafcell sizes are also much smaller (Kieber et al., 1993),while the ethylene-insensitive mutants (e.g. ers1 andetr1-1) are larger and their larger leaf area is thought tobe caused by increased cell size (Bleecker et al., 1988;Hua et al., 1995). Cell expansion requires the syner-gistic action of cell wall loosening, deposition of cellwall materials, and water absorption. In Rumex, eth-ylene can influence petiole elongation by regulatingexpression of the genes related to cell expansion(Vreeburg et al., 2005). However, little is known aboutthe mechanism of ethylene-regulated cell expansion.

Aquaporins (AQPs) are the primary channels ofwater transport across biological membranes. AQPs

1 This work was supported by the National Natural ScienceFoundation of China (grant no. 30671480) and the National HighTechnology Research and Development Program (863 Program) ofChina (grant no. 2006AA100109).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Junping Gao ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.120154

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are a class of small (24–34 kD) transmembrane proteinsand are localized on various cellularmembranes (Maureland Chrispeels, 2001). Plasmamembrane intrinsic pro-teins (PIPs), which are plasma membrane-associatedAQPs, can be classified into two major subgroups, PIP1and PIP2. PIP2 has a shorter N terminus and a longerC terminus than PIP1 (Johansson et al., 2000). PIP2usually has a higher channel activity than PIP1 (Danielset al., 1994; Yamada et al., 1995; Moshelion et al., 2002;Fetter et al., 2004).AQPs have been implicated in many developmental

processes in plants, including cell expansion, organmovement, andelongation (for review, seeTyermanet al.,2002). In Arabidopsis, reduction of AtPIP1;2 (previ-ously namedAtPIP1b) mRNA levels decreased cellularpermeability and increased rootmass (Kaldenhoff et al.,1998). Overexpression ofAtPIP1;2 in tobacco (Nicotianatabacum) significantly increased plant growth rate andresulted in larger plants (Aharon et al., 2003). Thetranscriptional activity of a tobacco AQP,NtAQP1, washighly associatedwith cell expansion regions (e.g. youngpetioles and corolla; Siefritz et al., 2004). In tobacco,PIP1 andPIP2 showeddistinct expressionpatternsdur-ing stigma and anther development, and they mightcontribute to the development of these organs in dif-ferent ways (Bots et al., 2005a, 2005b). In Arabidopsis,eight PIP genes were detectable in flower buds, sug-gesting the involvement of PIPs in flower development(Quigley et al., 2002). In tulip (Tulipa spp.), temperature-dependent changes of a putative plasma membrane-associated AQP activity were associated with petalopening and closing (Azad et al., 2004). However, verylimited direct evidence is available to support the roleof PIPs in petal expansion.Roses have been one of the most important ornamen-

tal crops in the floriculture industry for centuries, andflowering is one of the most important traits. Generally,flower opening requires programmatic expansion ofpetals. Considering that postmitotic development ofpetals mainly depends on water uptake-driven cellexpansion, it is highly possible that AQPs play animportant role in petal expansion.Our previous studies showed that ethylene could

accelerate the flower-opening process in roses andcould result in irregular petal shape (Ma et al., 2006;Xue et al., 2008). In this study, we found that ethylenesignificantly reduced petal size and water content inroses and inhibited petal expansion by inhibiting cellexpansion. Furthermore, we identified a rose AQP genefrom the public EST collection and demonstrated thatthis gene was tightly down-regulated by ethylene andwas involved in the regulation of rose petal expansion.

RESULTS

Ethylene Inhibits Petal Expansion

We have reported that ethylene plays a crucial rolein the flower-opening process of roses (Ma et al., 2006;Xue et al., 2008). Here, we further investigated the

effects of ethylene on petal expansion during floweropening. Our results showed that ethylene treatmentresulted in vertically compressed flowers with de-creased flower height-to-flower diameter ratio in rose(Rosa hybrida ‘Samantha’). An ethylene action inhibitor,1-MCP (for 1-methylcyclopropene), was used to in-hibit the ethylene effects because of its high specificityof action and lack of deleterious side effects (Sereket al., 1995; Sisler et al., 1996; Sisler and Serek, 1997;Cho and Cosgrove, 2002; Tanaka et al., 2005; Iwai et al.,2006). Expectably, 1-MCP increased the ratio signifi-cantly and resulted in vertically stretched flowers (Fig.1, A and B). In addition, ethylene significantly de-creased both petal length and width, while 1-MCPincreased petal length significantly (Fig. 1C).

During normal flower opening, although the petalsexpanded significantly, their thickness significantlydecreased, especially in the upper and middle regions(25% and 50% of the petal length from the top, respec-tively). Ethylene treatment significantly increasedpetal thickness in the upper and middle regions, while1-MCP significantly decreased petal thickness in theupper region (Table I).

Ethylene Inhibits Cell Expansion of Petals

In general, petal cells cease division right beforeanthesis (Drews et al., 1992; Martin and Gerats, 1993;Guterman et al., 2002; Dafny-Yelin et al., 2005). Con-sequently, during flower opening, petal growth isdriven mainly by cell expansion. Here, we determinedthe effects of ethylene on petal cell expansion.

Scanning electron microscopy showed that the petalcould be roughly divided into five sections anatomi-cally: adaxial epidermis, parenchyma cell, vascularbundle, abaxial subepidermis (AbsE), and abaxialepidermis (Fig. 2A). Among the five sections, AbsEcells appeared to have a regular shape and to form aflat layer. The expansion of AbsE cells was tightlyrelated to petal expansion (Supplemental Figs. S1 andS2). Therefore, we examined the size of AbsE cells bycounting the number of cells in a 1,360- 3 1,024-mm2

microscope visual area. We found that ethylene treat-ment significantly increased cell numbers per visualarea, indicating a significant decrease in cell size, while1-MCP significantly reduced cell numbers, especiallyin the upper region of petals, indicating a significantincrease in cell size (Fig. 2B). Interestingly, ethylenetreatment also resulted in interlocking AbsE cells ofirregular shape, suggesting that normal cell polarexpansion might be impeded by ethylene (Fig. 2B).These results indicated that ethylene inhibited petalexpansion, at least partially, through the inhibition ofAbsE cell expansion.

Ethylene Reduces Water Content of Petals

As indicated above, petal growth during floweropening is driven mainly by cell expansion, a processof rapid water uptake. In this study, we investigated

Rh-PIP2;1 in Ethylene-Regulated Rose Petal Expansion

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petal water content during flower opening and uponethylene or 1-MCP treatment. The fresh weight ofpetals in control flowers increased rapidly during thefirst 4 d of flower opening and then started to decrease,while the dry weight stayed at a steady level duringthe first 7 d of the flower-opening process (Fig. 1D).During flower opening, ethylene treatment substan-tially decreased the petal fresh weight, while 1-MCPsignificantly increased the fresh weight. However, nosignificant differences in dry weight were observedbetween the control flowers and those treated withethylene or 1-MCP (Fig. 1D). This indicated that eth-ylene caused significant petal water loss while 1-MCPincreased petal water content during flower opening.

Cloning and Characterization of Rh-PIP2;1

Since AQPs play important roles in regulating thewater intake of plant cells and ethylene reduces thewater content of rose petals, it is highly possible thatethylene impedes petal expansion of roses by regulat-ing certain AQPs. To identify potential AQPs involvedin petal expansion, we first searched the public roseEST database for sequences with similarity to knownAQPs. Among the identified rose AQP-like ESTs, one(GenBank accession no. BQ104371) showed approxi-mately 20-fold increase in expression in rose petalsduring the early period of flower opening, accordingto a microarray analysis (Guterman et al., 2002).Therefore, we characterized this EST for its functionalrole in rose petal expansion.

The full-length cDNA of the EST was isolated viaRACE. The full transcript is 1,145 bp in length andcontains an 846-bp open reading frame, a 91-bp5#-untranslated region (UTR), and a 208-bp 3#-UTR.The deduced amino acid sequence contains the sig-nature motifs of plasma membrane-associated AQPs(GGGANXXXXGYand TNPARSL/FGAAI/VI/VF/YN)and shares significant homology with plasma mem-brane AQPs from Arabidopsis, grape (Vitis vinifera),Brassica spp., and poplar (Populus spp.; Fig. 3). Basedon the length of its C and N termini, this gene wasclassified as a class 2 subfamily PIP protein. Therefore,we named it Rh-PIP2;1.

We then determined the localization of Rh-PIP2;1 byexpressing a 35S::GFP-Rh-PIP2;1 fusion gene in Arabi-

Figure 1. Morphological changes of cut rose flowers in response toexogenous ethylene. A, Vertical (left) and side (right) views of flowersafter ethylene and 1-MCP treatments. Flowers were exposed to air(control), 10 mL L21 ethylene, or 2 mL L21 1-MCP for 24 to 72 h, and thephotographs were taken at the end of individual treatment times.Twenty-five flowers were used in each treatment, and representativeresults are shown. B, The ratio of flower height to flower diameter. Each

data point represents the mean 6 SE (n = 15). Asterisks indicatesignificant differences (Student’s t test; P , 0.05) between untreatedcontrol and ethylene- or 1-MCP-treated flowers. C, The size of petalsafter ethylene and 1-MCP treatments. The length and width of petalswere measured on the 2nd d after 24-h treatments. One petal waschosen randomly from the second layer of each flower. Doubleasterisks indicate significant differences in length or width betweenuntreated control and ethylene- or 1-MCP-treated flower petals (n = 15,P , 0.01). D, Changes of petal fresh weight (top blue lines) and dryweight (bottom red lines) after ethylene and 1-MCP treatments. Eachdata point represents the mean 6 SE (n = 15). Asterisks indicatesignificant differences (Student’s t test; P , 0.05) between untreatedcontrol and ethylene- or 1-MCP-treated flower petals.

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896 Plant Physiol. Vol. 148, 2008



dopsis. To determine a more detailed localization, weisolated protoplasts from the transgenic Arabidopsisharboring the 35S::GFP-Rh-PIP2;1 cassette. We foundthat the green fluorescence was primarily distributed onthe plasma membrane while also distributed on someinternal membranes (Fig. 4A). Thus, we conductedcolocalization of GFP-Rh-PIP2;1 with a mCherry (a redfluorescent protein mutant derived from mRFP1 that isa monomeric mutant of DsRed [Shaner et al., 2004])-labeled plasma membrane marker (PM-rk; The Arabi-dopsis Information Resource stock no. CD3-1007) and

an endoplasmic reticulum marker (ER-rk; The Arabi-dopsis Information Resource stock no. CD3-959; Nelsonet al., 2007), respectively. The results indicated that Rh-PIP2;1 was mainly distributed on the plasmamembraneand much less on the endoplasmic reticulum (Fig. 4B).

We also examined the spatial expression pattern ofRh-PIP2;1 in petals through in situ hybridization. A216-bp fragment in the 3# end of Rh-PIP2;1was used togenerate digoxigenin (DIG)-labeled RNA antisenseand sense probes. In situ hybridization of Rh-PIP2;1transcripts in petals at the early stages of flower

Table I. Effect of ethylene on petal thickness (mm)

Petal thickness was measured at the upper, middle, and bottom regions (25%, 50%, and 75% of thepetal length from the top, respectively). Flowers were treated with ethylene or 1-MCP on the first day ofopening duration for 24 h. Day 0, The first day of flower opening. Day 3, The third day of flower opening.Data represents means from 15 replicates 6SE. Different letters in the same column indicate significantdifferences according to Duncan’s multiple range test (P , 0.05).

Treatments Upper (25%) Middle (50%) Bottom (75%)

Control (day 0) 459.2 6 10.3 a 574.6 6 21.4 a 820.2 6 56.2 aControl (day 3) 183.7 6 26.5 b 446.8 6 15.1 b 844.1 6 65.8 aEthylene (day3) 358.2 6 38.2 a 567.2 6 14.3 a 886.4 6 35.0 a1-MCP (day 3) 113.9 6 34.3 c 461.4 6 10.8 b 854.1 6 37.6 a

Figure 2. Inhibition of the cell elongation of AbsE inpetals by ethylene. A, The typical anatomic structureof a petal at flower full opening stage. Left, A scan-ning electron microscope image. Bar = 50 mm. Right,A light microscope image with semithin section.Bar = 200 mm. AbE, Abaxial epidermis; AdE, adaxialepidermis; PC, parenchyma cells; VB, vascular bun-dle. B, Traces of outlines of AbsE cells (top) and cellnumbers (bottom) in different regions of petals. Bars =200 mm. AbsE cells of petals were photographedusing a Nikon IX-71 microscope. Petal samples weretaken as a 0.5-cm 3 0.4-cm slice at 25%, 50%, and75% of the petal length from petal top on the 2nd dafter 24-h treatments. The slices were fixed in form-aldehyde and then cleared in ethanol. The traceswere drawn using Photoshop 7.0 software. Fifteenflowers were used in each treatment, and represen-tative results are shown. Cell numbers were countedusing ImageJ software in a visual field of 1,360 31,024 mm2. Each data point represents the mean6 SE

(n = 15). Different letters in the same row indicatesignificant differences between different treatmentsaccording to Duncan’s multiple range test (P, 0.05).





opening showed that the expression of Rh-PIP2;1 wasmuch stronger in AbsE than in adaxial epidermis, andthe expression was also detected in parenchyma cellsbut at a relatively lower level (Fig. 4C).

Water Channel Activity of Rh-PIP2;1

Plant PIP2 proteins usually have high water channelactivities, whereas PIP1 proteins are often inactive orhave low activities (Kaldenhoff and Fischer, 2006).Here, we tested the water channel activity of Rh-PIP2;1by comparing osmotic water permeability (Pf) of leafprotoplasts between Arabidopsis wild-type (ecotypeColumbia [Col]) plants and transgenic plants harbor-ing the 35S::Rh-PIP2;1 cassette. The number of leafprotoplasts with high Pf was significantly higher in

transgenic plants (Fig. 5A). As shown in Figure 5B, onaverage, the Pf value of leaf protoplasts in transgenicplants was significantly higher than that in the wildtype (14.4 and 6.6, respectively; P , 0.01). This indi-cates that Rh-PIP2;1 is an active water channel.

Regulation of Rh-PIP2;1 Expression by Ethylene

We determined the tissue-specific expression pat-tern of Rh-PIP2;1 by RNA gel-blot analysis. As shownin Figure 6A, Rh-PIP2;1 was mainly expressed inpetals. We then determined its expression levels inpetals over the course of flower opening. Rh-PIP2;1was highly expressed within the first 2 d of floweropening, when petal expansion was the most active.The expression of Rh-PIP2;1 decreased dramatically

Figure 3. Alignment of deduced amino acid sequence of Rh-PIP2;1with seven PIPs from other plants. The accession numbers ofthe used amino acid sequences are as follows: VvPIP2;2 (grape), AAF71820; SoPIP2;1 (spinach), AAA99274; AtPIP1;1(Arabidopsis), NP_191702; AtPIP1;2 (Arabidopsis), CAB37860; AtPIP2;1 (Arabidopsis), NP_001030851; AtPIP2;2 (Arabidopsis),NP_181254; and AtPIP2;7 (Arabidopsis), P93004. Red lines under the putative amino acid sequence indicate signaturesequences of plasma membrane-associated AQPs. [See online article for color version of this figure.]

Ma et al.




after 3 d of flower opening, when the petal expansionprocess was almost completed (Fig. 6B). These resultsindicated that Rh-PIP2;1 expression was highly corre-lated with the petal expansion process.We further investigated the effects of ethylene and

its action inhibitor, 1-MCP, on Rh-PIP2;1 expression inpetals during the first 72 h of flower opening. Ethylenetreatment significantly inhibited the expression of Rh-PIP2;1, while 1-MCP maintained its expression at ahigher level for a longer time compared with thecontrols (Fig. 6C).We then examined Rh-PIP2;1 expression in petals on

a narrower time scale during the first 24 h of ethyleneor 1-MCP treatment in order to understand howquickly Rh-PIP2;1 responds to ethylene. The resultsshowed that the expression of Rh-PIP2;1 changedwithin 0.5 to 1 h upon ethylene treatment (Fig. 6D),suggesting a quick response of Rh-PIP2;1 expression toethylene.

Characterization of Rh-PIP2;1-Silenced Flowers

We functionally characterized the role of Rh-PIP2;1in petal expansion by silencing Rh-PIP2;1 in roseflowers using a virus-induced gene silencing (VIGS)approach. The 290-bp fragment of Rh-PIP2;1 3# end

was used to construct the TRV-Rh-PIP2;1 silencingvector. We successfully silenced Rh-PIP2;1 in rosepetals (Fig. 7A), although the efficiency of silencing(about 9:300) was much lower than that reported intobacco and tomato (Solanum lycopersicum; Liu et al.,2002; Fu et al., 2005). Flowers with silenced Rh-PIP2;1had petals with significantly decreased length andwidth as well as reduced water content (Fig. 7B), aphenotype similar to that of ethylene-treated flowers.Rh-PIP2;1 silencing also resulted in significantly in-creased cell numbers of AbsE per microscope visualarea in the upper region of petals, indicating that thesilencing reduced the cell volume; however, the cellshapes of AbsE were more regular than those ofethylene-treated petals (Fig. 8).

In addition, we also obtained 12 partially Rh-PIP2;1-silenced flowers, and their phenotypes were moresimilar to those of the wild-type flowers (Figs. 7 and 8).

DISCUSSION

Ethylene Influences Petal Expansion by ModulatingAbsE Cells in Roses

In plant development, proliferative growth andpostmitotic expansion are coordinated and modulated

Figure 4. The intracellular localization of Rh-PIP2;1and in situ hybridization of Rh-PIP2;1. A, The intra-cellular localization of Rh-PIP2;1 in Arabidopsis leafmesophyll cell protoplast. The GFP-Rh-PIP2;1 fusiongene was inserted in pRTL2 and transformed intoArabidopsis leaf protoplast for transient expression.Imaging of the protoplast-expressed 35S::GFP-Rh-PIP2;1 fusion was conducted with a laser scanningconfocal microscope (Nikon C1). Arrows indicateinternal membrane localization of Rh-PIP2;1. Bar =20 mm. B, GFP-Rh-PIP2;1 localization is comparedwith a mCherry-labeled endoplasmic reticulum (ER)marker (ER-rk; CD3-959) and a mCherry-labeledplasma membrane (PM) marker (PM-rk; CD3-1007).At least 20 protoplasts were examined in expressingeach marker. Bars = 10 mm. C, Pattern of Rh-PIP2;1mRNA accumulation examined by in situ hybridiza-tion. Rh-PIP2;1was detected by DIG-labeled 3#-UTRRNA antisense (left) and sense (right) probes in trans-verse sections of cut rose petals. Bar = 200 mm.





by phytohormones like auxin, brassinosteroids, GA,and ethylene (Mizukami, 2001; Ingram and Waites,2006). In Arabidopsis, auxin, abscisic acid, GA, andbrassinosteroids can enhance longitudinal expansionof many organs, such as leaves and roots, whileethylene increases lateral expansion of hypocotylsand roots (Saab et al., 1990, 1992; Spollen et al., 2000;

for review, see Mizukami, 2001). Compared with wild-type plants, ethylene overexpression (eto) and consti-tutive response (ctr1) mutants have smaller organs,while ethylene-insensitive (etr) mutant plants appearto have larger leaves and floral organs (Ecker, 1995).Ethylene causes asymmetric cell elongation and re-sults in formation of the apical hooks of pea (Pecket al., 1998). Treatment with ethylene or its precursor,1-aminocyclopropane-1-carboxylic acid, greatly in-hibits cell elongation in Arabidopsis roots (Le et al.,2001; De Cnodder et al., 2005). Thus, ethylene can playan important role in suppressing organ expansion inplants. Ethylene promotes flower opening in carnation(Dianthus caryophyllus; Jones and Woodson, 1997), or-chid (Phalaenopsis spp.; Bui and O’Neill, 1998), andPetunia (Tang and Woodson, 1996). In rose, ethylenemay promote or inhibit flower opening in a cultivar-dependent manner (Reid et al., 1989a, 1989b). In ourprevious work, we observed that ethylene promotedflower opening in Samantha rose (Cai et al., 2002; Maet al., 2005, 2006; Xue et al., 2008). Despite these reportsdescribing the effects of ethylene on ornamental plantflower opening, little is known about the effect ofethylene on petal expansion.

In this study, we show that ethylene treatmentsignificantly reduced petal size (Fig. 1C) and resultedin vertically compressed flowers (Fig. 1, A and B). Inaddition, ethylene suppressed the expansion of AbsEcells and resulted in cells with irregular shapes (Fig.2B). These findings strongly suggest that inhibitedexpansion of AbsE cells may contribute to the inhi-bition of petal expansion by ethylene.

Rh-PIP2;1 Is Involved in Petal Expansion of Rose

PIPs belong to a multigene family in higher plants,and 13 PIPs have been identified in both Arabidopsis(Johanson et al., 2001) and maize (Zea mays; Chaumontet al., 2001). It has been reported that high levels of PIPexpression are usually associated with developmental

Figure 5. Histogram of Pf of leaf protoplasts (A) and statistical analysis(B) from Arabidopsis wild-type (Col) and transgenic (35S::Rh-PIP2;1)plants. Leaves of 20- to 22-d-old plants were harvested, and threedifferent preparations of protoplasts were used. For each line, the Pf of75 protoplasts was measured, and the initial diameter of protoplastswas about 60 mm. Error bars represent SE (n = 75). The double asteriskindicates a significant difference in Pf between wild-type (Col) andtransgenic (35S::Rh-PIP2;1) plants (n = 75; t test; P , 0.01).

Figure 6. The expression of Rh-PIP2;1 in cutrose petals. A, Rh-PIP2;1 expression in differ-ent organs. P, Petal; Se, sepal; St, stamen; G,gynoecium; Re, receptacle; L, leaf. B, Rh-PIP2;1 expression in petals during normalflower opening. Lanes 0 to 6 indicate vasedays. C and D, Rh-PIP2;1 expression in re-sponse to ethylene in petals. Flowers wereexposed to air (control), 10 mL L21 ethylene,or 2 mL L21 1-MCP. Lanes 0 to 72 indicatetreatment hours. Each lane contains 10 mg oftotal RNA. Ethidium bromide-stained rRNAwas used as an internal control to normalizethe amount of total RNA. Total RNA wasisolated from three individual samples ateach time point, and all of the hybridizationswere repeated at least three times. Represen-tative results are shown.

Ma et al.




processes requiring substantial water intake, includ-ing root elongation and expansion (Javot et al., 2003;Ranathunge et al., 2004), anther dehiscence (Bots et al.,2005a, 2005b), and petiole movement (Moshelion et al.,2002; Siefritz et al., 2004). During flower opening, petalexpansion is mainly driven by cell expansion, a pro-

cess of rapid water uptake (Drews et al., 1992; Martinand Gerats, 1993; Guterman et al., 2002). In this study,we show that petal fresh weight increased quickly anddry weight stayed at a steady level during petalexpansion in rose, indicating that petal expansionshould be primarily caused by water uptake-driven

Figure 7. Silencing of the Rh-PIP2;1 gene in rose petals by VIGS. Rose branches were infiltrated with Agrobacterium containingTRV alone (TRV, pTRV1 + pTRV2) or TRV carrying a fragment of Rh-PIP2;1 (TRV-Rh-PIP2;1, pTRV1 + pTRV2-Rh-PIP2;1). A, RT-PCR analysis of Rh-PIP2;1 in the Rh-PIP2;1-silencing flower petals. The first-strand cDNAwas generated from 1 mg of total RNAand was used to amplify Rh-PIP2;1 and the 18S rRNA gene using gene-specific primers. The PCR cycles was 27 and 18 for Rh-PIP2;1 and 18S rRNA, respectively. Completely, Completely silenced flowers; partially, partially silenced flowers; non, nosilenced flowers. B, The phenotype of Rh-PIP2;1-silencing flowers (top) and petal sizes and water content (bottom). Thephotographs were taken on the 7th d after infiltration. The petal size and water content were determined on the 7th d afterinfiltration as described in Figure 1. Each data point represents the mean6 SE (n = 15 for control, TRV-Rh-PIP2;1 [non], and TRV;n = 9 for TRV-Rh-PIP2;1 [completely]; n = 12 for TRV-Rh-PIP2;1 [partially]).

Figure 8. Silencing of Rh-PIP2;1 inhibits the cell elongation of the AbsE in petals. Petal samples were taken as a 0.5-cm3 0.4-cmslice at 25% of the length from petal top on the 7th d after infiltration and treated as described in Figure 2B. Bars = 200 mm. Cellnumbers were counted as described in Figure 2C. Completely, Completely silenced flowers; partially, partially silenced flowers;non, no silenced flowers. Each data point represents the mean6 SE (n = 15 for control, TRV-Rh-PIP2;1 [non], TRV, and ethylenetreatment; n = 9 for TRV-Rh-PIP2;1 [completely]; n = 12 for TRV-Rh-PIP2;1 [partially]). Different letters indicate significantdifferences between different treatments according to Duncan’s multiple range test (P , 0.05).





cell expansion in rose (Fig. 1D). In addition, we alsoshowed that ethylene significantly inhibited petal ex-pansion and reduced petal water content. Thus, wespeculated that PIPs could be involved in the processof ethylene-inhibited petal expansion. Guterman et al.(2002) reported an AQP-like EST (BQ104371) thatexhibited a 20-fold increase of mRNA concentrationin petals of opened flowers compared with unopenedbuds, suggesting that it has a potential role in rosepetal expansion. In this study, we further functionallycharacterized this EST, which we named Rh-PIP2; 1, byinvestigating its role in ethylene-inhibited petal ex-pansion.

The deduced amino acid sequence of Rh-PIP2;1shares high homology with members of the PIP2subfamily from other plant species (Fig. 3). Rh-PIP2;1was expressed predominantly in petals, and its tran-script abundance was tightly correlated with theflower-opening process (Fig. 6, A and B). In addition,among petal tissues, Rh-PIP2;1 expression was stron-ger in AbsE, whose expansion is tightly related to theincrease of petal size during flower opening (Fig. 4C).These results strongly suggest that Rh-PIP2;1 is in-volved in petal expansion.

In general, PIPs are localized on the plasma mem-brane. However, Siefritz et al. (2001) reported that atobacco PIP2 protein, NtAQP1, was also localized onsome endomembranes in protoplasts. We also foundthat Rh-PIP2;1 was mainly plasma membrane local-ized, but its presence was also observed in someinternal membrane compartments. Furthermore, us-ing a set of subcellular fluorescence-labeled markers(Nelson et al., 2007), we proved that the internalmembrane where Rh-PIP2;1 was localized was theendoplasmic reticulum (Fig. 4, A and B). MammalianAQPs and plant TIPs (for tonoplast intrinsic proteins)can shuttle between subcellular compartments in re-sponse to various stimuli (reviewed by Luu andMaurel, 2005). However, little is known concerningthe dynamic localizations of plant PIPs. Recently,Zelazny et al. (2007) reported that PIP interactioncould regulate their subcellular localization. Thus,further analysis would be necessary to understandthe mechanisms of Rh-PIP2;1 dynamic localization.

Ethylene Regulates Rh-PIP2;1 Expression in Rose Petals

PIPs are likely to play divergent and unique roles inplants with fewer functional redundancies (Luu andMaurel, 2005). The expression of PIP genes can beregulated by developmental and environmental cuesat both the transcriptional and posttranscriptionallevels (Kjellbom et al., 1999; Hachez et al., 2006). Atthe transcriptional level, PIP gene expression is regu-lated by various environmental factors, including light(Cochard et al., 2007), drought (Aroca et al., 2006;Galmes et al., 2007), cold (Jang et al., 2004), salt (Janget al., 2004; Boursiac et al., 2005), and phytohormones,including gibberellic acid (Phillips and Huttly, 1994;Suga et al., 2002), abscisic acid (Kaldenhoff et al., 1993;

Zhu et al., 2005; Aroca et al., 2006), and brassinolide(Morillon et al., 2001).

At the posttranscriptional level, AQP channel activ-ities are modulated by several mechanisms, includ-ing protein phosphorylation, pH, protein trafficking,and protein oligomerization (Luu and Maurel, 2005;Hachez et al., 2006). Phosphorylation at certain resi-dues of some AQPs is thought to be an importantmodification mechanism for channel activity. For in-stance, the channel activities of an Arabidopsis a-TIPgene, AtTIP3;1, the spinach (Spinacia oleracea) SoPIP2;1(PM28A), and the soybean (Glycine max) nodulin 26are all regulated through protein phosphorylation(Maurel et al., 1995; Johansson et al., 1996; Guentheret al., 2003). Recently, Tornroth-Horsefield et al. (2006)reported that phosphorylation-induced structural re-modeling led to channel gating of SoPIP2;1.

The involvement of ethylene in the modification ofAQP channel activity has been suggested in a fewreports. A general inhibitor of AQP activities, HgCl2,decreased ethylene-enhanced water flow in aspen(Populus spp.) roots (Kamaluddin and Zwiazek,2002). A protein kinase inhibitor, okadaic acid, couldinhibit the ethylene-induced phosphorylation in mungbean (Vigna radiata) hypocotyls (Kim et al., 1997),orchid petals (Wang et al., 2001), and pea (Berryet al., 1996; Kwak and Lee, 1997), and the inhibitoralso inhibited the phosphorylation of AQP PM28A inspinach leaves (Johansson et al., 1996). Since the phos-phorylation of AQPs is considered to be an importantregulatory mechanism controlling channel activity(Tornroth-Horsefield et al., 2006), it is reasonablyspeculated that ethylene may be involved in the reg-ulation of water channel activities via the proteinphosphorylation/dephosphorylation pathway.

However, little direct evidence has been obtained ateither the transcriptional or posttranscriptional level tosupport the role of AQPs in ethylene-regulated watertransport.

In this work, we found that ethylene substantiallydecreased the transcript levels of Rh-PIP2;1 (Fig. 6, Cand D). The suppressing effect of ethylene on Rh-PIP2;1 expression was observed within only 0.5 to 1 hof the treatment (Fig. 6D), suggesting that Rh-PIP2;1expression is likely regulated directly by ethylene.Additionally, the Rh-PIP2;1-silenced flowers exhibitedsimilar morphological and anatomical phenotypes toflowers treated by ethylene (Figs. 7 and 8), furthersupporting the notion that ethylene inhibits petalexpansion of rose by suppressing Rh-PIP2;1 expres-sion. Taken together, we argue that Rh-PIP2;1 is able torespond to ethylene promptly at the transcriptionallevel and is involved in ethylene-regulated petal ex-pansion. However, we cannot rule out the possibilitythat ethylene also contributes to regulating petal wateruptake and petal expansion by influencing the preex-isting Rh-PIP2;1 protein activity. However, furtherwork is required to clarify this possibility.

In this study, we also found that although the Rh-PIP2;1-silenced and ethylene-treated flowers exhibited

Ma et al.




similar morphological and anatomical phenotypes,overall the ethylene-treated flowers showed a moresevere phenotype regarding the inhibited petal expan-sion. This indicates that some other ethylene-regulatedgenes might also be involved in ethylene-inhibitedpetal expansion.On the other hand, it is possible that the cross talk

between different hormones is involved in the regula-tion of plant organ expansion. In maintaining leafexpansion of Arabidopsis and tomato, and primaryroot elongation of Arabidopsis, endogenous abscisicacid was thought to play an important role by restrict-ing ethylene production (Sharp et al., 2000; Spollenet al., 2000; LeNoble et al., 2004).In Arabidopsis, the inhibitory effect of ethylene on

GA-induced root elongationwas accomplished throughenhancing the stability of the conserved repressors ofGA signaling, GAI (GA insensitive) andRGA (repressorof ga1-3; Achard et al., 2003), suggesting that there maybe cross talk in influencing root elongation betweenethylene and GA.Taken together, ethylene, abscisic acid, and GA may

synergistically influence petal expansion by regulatingrelevant genes, like AQP, at the transcriptional and/orthe posttranscriptional level. However, further studiesare required to understand the possible mechanisms.

MATERIALS AND METHODS

Plant Materials

Cut roses (Rosa hybrida ‘Samantha’) were harvested at stage 2 of flower

opening from a local commercial grower and placed in water immediately.

Flower-opening stages were as defined by Wang et al. (2004) and Ma et al.

(2005): stage 0, unopened bud; stage 1, partially opened bud; stage 2, com-

pletely opened bud; stages 3 and 4, partially opened flower; stage 5, fully

opened flower with anther appearance (yellow); stage 6, fully opened flower

with anther appearance (black). The flowers were delivered to our laboratory

within 1 h of harvest. Their stemswere then cut to a length of 25 cmunderwater

and placed in deionized water, which was refreshed daily. For all analyses in

this study, samples were collected from outermost ray petals of the flowers.

Treatment of Flowers with Ethylene and 1-MCP

Based on our previous work (Ma et al., 2006), we used 10 mL L21 ethylene

or 2 mL L21 1-MCP for treatments. The flowers were sealed in a 64-L chamber

with ethylene, 1-MCP, or regular air as the control at 25�C for different periods

of time. NaOH (1 mol L21) was put into the chamber to prevent the

accumulation of CO2. After treatment, flowers were placed in a vase with

deionized water under the following controlled conditions: approximately

23�C to 25�C room temperature, approximately 30% to 40% relative humidity,

and 12/12-h light/dark photoperiodwith an illumination of approximately 40

mmol m22 s21.

Microscopic Examination and Cell Counting

Petal samples were taken as 0.5-cm 3 0.4-cm slices from regions at 25%,

50%, and 75% of the petal length from the top. For scanning electron

microscopy, slices of the petal middle region (50% of the length) of fully

opened flowers were selected and then fixed and processed according to a

standard protocol (Bowman et al., 1989). The slices were flat mounted on

either their adaxial or abaxial surface, or fractured to reveal internal anatomy

transverse to the petal longitudinal axis. Scanning electron microscopy was

performed using a Philip S3400N apparatus.

AbsE cell photography and cell counting were performed as described by

Dewitte et al. (2007). In brief, rose flowers were treated at the 1st d (day 0) of

opening for 24 h, and petal samples were taken on the 2nd d after the

respective treatments. Petal samples were taken as 0.5-cm 3 0.4-cm slices at

25%, 50%, and 75% of the petal length from the petal top. The slices were fixed

in formaldehyde and then cleared in ethanol. AbsE cells of the slices were

photographed using a Nikon IX-71 camera. The traces were drawn using

Photoshop 7.0 software. Fifteen flowers were used in each treatment.

Numbers of AbsE cells were counted using ImageJ software in a visual

field of 1,360 3 1,024 mm2.

Rh-PIP2;1 Subcellular Localization

Rh-PIP2;1 cDNA was amplified with primers Rh-PIP2;1_up (5#-GGACG-

AGCTGTACAAGATGACGAAGGAAGTG-3#) and Rh-PIP2;1_down (5#-GCG-

TGGATCCTTAGTTGGAGGGGTTGCTCCG-3#). gfp cDNAwas amplified with

primers gfp_up (5#-ATCCCATGGATGGTGAGCAAGGGCGAGGAGCT-3#)and gfp_down (5#-CACTTCCTTCGTCATCTTGTACAGCTCGTCC-3#). The re-

sulting fragments were used to generate the GFP-Rh-PIP2;1 fusion gene by

overlapping PCR. The fused fragment was inserted into pGEM T-Easy vector

(Promega) and subjected to sequencing for verification. After NcoI and BamHI

digestion, the cassette was inserted into pRTL2 to construct the pRTL-GFP-Rh-

PIP2;1 transient expression vector. Transient expression was conducted accord-

ing to Kovtun et al. (2000). Arabidopsis (Arabidopsis thaliana) leaf mesophyll

protoplasts were isolated from well expanded leaves of 3- to 4-week-old plants.

Leaf sections of roughly 1 g were cut into 0.5- to 1-mm strips with a razor blade.

The strips were placed into 15 mL of preheated enzyme solution (1% cellulase

R10, 0.25% macerozyme R10, 0.4 M mannitol, 20 mM KCl, and 20 mM MES, pH

5.7) and gently shaken at 23�C in darkness for 3 h. Then, the protoplasts were

filtered and spun at 1,000 rpm for 1min. The resulting pelletswerewashed once

by cold W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM Glc, and

2 mM MES, pH 5.7), suspended in the same solution, kept on ice for 30 min,

centrifuged at 1,000 rpm for 1 min, and then resuspended in MMg solution

(0.4 M mannitol, 15 mM MgCl2, and 4 mM MES, pH 5.7). Around 15 mg of

plasmid DNAwas mixed with 5 3 104 protoplasts in 0.1 mL of MMg solution.

An equal volume of 40% (w/v) PEG4000 (Fluka) in 0.1 M Ca(NO3)2 and 0.4 M

mannitol solution of pH 10 was added and gently mixed, and then the mixture

was incubated at 23�C for 25min. After incubation, a 4-fold volumeW5 solution

was added and mixed gently, and the mixture was centrifuged at 1,000 rpm for

2 min. The pellets were resuspended gently in 1 mL of W5 solution and were

incubated at 23�C for 20 to 22 h in darkness. All transformation was performed

three times, and assays were repeated on at least three separate occasions.

pRTL-GFP-Rh-PIP2;1 was digested by HindIII, and the 35S::GFP-Rh-

PIP2;1-Nos cassette was inserted into the binary vector pGreen 0229 and

transformed into Agrobacterium tumefaciens GV3101. Arabidopsis plants were

infiltrated with Agrobacterium using the floral dip method (Clough and Bent,

1998). The GFP florescence was visualized using a confocal microscopy

(Nikon).

Colocalization experiments were performed using the mCherry-labeled

plasma membrane marker (PM-rk; CD3-1007) and the endoplasmic reticulum

marker (ER-rk; CD3-959) purchased from the Arabidopsis Biological Resource

Center (Nelson et al., 2007). The plasmids of CD3-1007 and CD3-959 were

separately transformed into protoplasts of Arabidopsis transgenic plants

overexpressing GFP-PIP2;1. Imaging of protoplasts was conducted with a

laser scanning confocal microscope (Nikon C1). The excitation wavelengths

for GFP and mCherry were 488 and 543 nm, respectively, and the emission

filter wavelengths were 505 to 530 nm for GFP and 560 to 615 nm for mCherry.

In Situ Hybridization

In situ hybridization was performed as described by Xu et al. (2005) with

minor modifications. A 216-bp fragment at the 3# end of Rh-PIP2;1 was

amplified using the following primer pairs: 5#-CCTCCAACTAAATGAGAA-

GAAGC-3# and 5#-GCTAATACGACTCACTATAGGGCAAAACACCACTA-

CTCATTGAC-3# for the antisense probe and 5#-GCTAATACGACTCACTA-

TAGGGCCTCCAACTAAATGAGAAGAAGC-3# and 5#-CAAAACACCACT-

ACTCATTGAC-3# for the sense probe. The PCR products were purified

(Axyprep DNA gel extraction kit; Axygen) and used as the template to

generate the antisense or sense probe via in vitro transcription using the DIG

RNA labeling mix (Roche).

Petal samples were fixed in cold phosphate-buffered saline (pH 7.0)

containing 4% (w/v) paraformaldehyde overnight at 4�C. After being washed

twice with cold phosphate-buffered saline, the samples were dehydrated with

an ethanol series and then embedded in Paraplast plus (Sigma). Subsequently,





the samples were cut into 7-mm sections and mounted on slides pretreated

with 0.01% poly-L-Lys (Sigma). The hybridization was performed at 43�Covernight with a 0.1 to 0.2 ng mL21 RNA probe concentration. After washing

and digestion by RNase A, the slides were incubated with Anti-Digoxigenin

AP-Conjugate (Roche). Hybridizations were visualized using nitroblue tetra-

zolium/5-bromo-4-chloro-3-indolyl phosphate stock solution (Roche) and

photographed with an Olympus BX51 microscope.

Measurement of Pf on Protoplasts

The Pf of Rh-PIP2;1 in leaf protoplasts of Arabidopsis wild-type and

transgenic plants was measured according to Ramahaleo et al. (1999),

Moshelion et al. (2004), and Cabanero et al. (2006).

In brief, leaves of 20- to 22-d-old plants were harvested, and three different

preparations of protoplasts were generated. Fifteen-microliter protoplast

suspensions were dispersed on a microscope slide, and the same amount of

water was added to decrease the osmotic potential of the medium to half. Cell

volumes were captured with an inverted Nikon microscope (IX-71), and

images were acquired at 3-s intervals for a total of 90 s using a CCD camera

(DP 70; Nikon). Protoplast swelling was analyzed using the ImageJ program

(http://rsb.info.nih.gov/ij/). The Pf values were calculated according to the

equation from Zhang and Verkman (1991): Pf = (V0/S0)[k/Vw(Osmolin 2Osmolout)], where V0 is the initial cell volume, S0 is the initial cell surface area,

Vw is the molar volume of water (18 cm3 mol21), k is the fitted exponential rate

constant of the initial phase of the swelling, and osmolin and osmolout are the

internal and external osmolarities, respectively. Measurements of osmolarity

(mosmol kg21) were carried out using an osmometer (Fiske ONE-TENTM

Osmometer).

For each line, the Pf of 75 protoplasts was measured, and the initial

diameter of the protoplasts was about 60 mm.

RNA Extraction, Northern-Blot Analysis, and

Reverse Transcription-PCR

The full-length cDNA of Rh-PIP2;1was obtained by RACE (SMART RACE

cDNA Amplification; Clontech) and deposited into GenBank under accession

number EU572717. Procedures for total RNA extraction and RNA gel-blot

analysis were reported by Ma et al. (2006). Petals were ground with liquid

nitrogen and homogenized with the preheated extraction buffer (200 mM

sodium tetraborate decahydrate, 30 mM EGTA, 1% deoxycholic acid sodium

salt, 10 mM dithiothreitol, 2% polyvinylpyrrolidone 40, and 1% Nonidet P-40).

RNAwas first precipitated overnight with 2 M LiCl, then washed by 2 M LiCl

and dissolved in 1 M Tris-Cl (pH 7.5). Subsequently, the RNAwas precipitated

with 100% ethanol at 280�C for 2 h. For RNA gel-blot analysis, 10 mg of total

RNAwas fractionated on 1.2% agarose gels containing 2.5% (v/v) formalde-

hyde. The 3#-UTR region of Rh-PIP2;1 was used as the probe. DIG-labeled

probes were generated by PCR using a PCR DIG probe synthesis kit

(Boehringer Mannheim). Hybridization was carried out overnight at 46�C.The membranes were washed twice in 23 SSC at 37�C and twice in 0.13 SSC

at 59�C, with 30 min for each wash, and then were incubated with Anti-

Digoxigenin AP-Conjugate (Roche). The membranes were then subjected to a

chemiluminescent reaction with CDP-Star according to the manufacturer’s

protocol (Roche) and exposed to Fuji Medical X-Ray Film (Fuji Photo Film).

All of the hybridizations were performed with at least three biological

replicates.

For reverse transcription (RT)-PCR, first-strand cDNA was synthesized

from 1 mg of total RNA using SuperScript Moloney murine leukemia virus

reverse transcriptase (Promega). Semiquantitative RT-PCR was performed as

described by Fu et al. (2005). The primers used for RT-PCR were 5#-TTG-

AAAATGACGAAGGAAGTGAGCGA-3# and 5#-CCGGATCAGAGAGAC-

CTTACGAG-3#, which were outside the region used for VIGS. This ensured

that only the endogenous Rh-PIP2;1 transcripts were examined in VIGS

plants. The 18S rRNA gene was used as an internal control for RNA quantity.

The PCR product amplified from a reaction without reverse transcriptase was

used as the negative control. The PCR products were analyzed on 1.0%

agarose gels and imaged by AlphaImager 2200 (Alpha Innotech).

Silencing of Rh-PIP2;1 in Rose Flowers by VIGS

The pTRV1 and pTRV2 VIGS vectors (described by Liu et al., 2002) were

kindly provided by Dr. S.P. Dinesh-Kumar at Yale University. The Rh-PIP2;1

silencing was performed as described by Fu et al. (2005). A 302-bp fragment at

the 3# end of Rh-PIP2;1 was amplified using primers 5#-CGTTCTAGAGTG-

CAGCCGCGTACCACCAG-3# with an XbaI restriction site and 5#-CATCTC-GAGGATTGGCAAAACACCACTA-3# with an XhoI restriction site. The

resulting product was cloned into pTRV2 to form the pTRV2-Rh-PIP2;1

construct. pTRV2-Rh-PIP2;1, as well as the pTRV1 and pTRV2 vectors, were

transformed into Agrobacterium GV3101 by electroporation. The Agrobacterium

strain GV3101 containing pTRV1, pTRV2, or pTRV2-Rh-PIP2;1 was grown at

28�C in Luria-Bertani medium supplemented with 10 mM MES, 20 mM

acetosyringone, and 50 mg L21 kanamycin for about 24 h. Agrobacterium cells

were harvested and suspended in the infiltration buffer (10 mMMgCl2, 150 mM

acetosyringone, and 10 mM MES, pH 5.6) to a final optical density at 600 nm of

around 1.0. A mixture of Agrobacterium cultures containing pTRV1 and

pTRV2-Rh-PIP2;1 in a ratio of 1:1 (v/v), as well as a mixture containing

pTRV1 and pTRV2 in a 1:1 ratio (which served as the negative control), were

shaken at room temperature for 4 h before vacuum infiltration.

For vacuum infiltration, rose flower branches at flower stage 1 were placed

into the bacterial suspension solution and infiltrated by vacuum at 30 mmHg

for 30 s. After release of the vacuum, the flower branch base was washed by

deionized water and kept in deionized water for about 10 d at 18�C. The petalswere collected when flowers fully opened, approximately at the 7th d after the

infiltration.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number EU572717.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Anatomic structure of a petal by scanning elec-

tron microscopy.

Supplemental Figure S2. Petal AbsE cell expansion during flower opening.

Supplemental Figure S3. The quantification of the in situ signals of Fig-

ure 4C.

Supplemental Figure S4. The quantification of the northern hybridization

signals in Figure 6, C (left) and D (right).

ACKNOWLEDGMENTS

We are grateful to the laboratory of Dr. Shu-Nong Bai for their kind help

with in situ hybridization. We also thank Dr. Zhangjun Fei for critical review

of the manuscript and Ms. Sara Zimmer and Ryan McQuinn for proof-

reading.

Received April 1, 2008; accepted August 10, 2008; published August 20, 2008.

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