PMC

Alignment of deduced amino acid sequence of Rh-PIP2;1 with seven PIPs from other plants. The accession numbers of the 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 signature sequences of plasma membrane-associated AQPs. [See online article for color version of this figure.]

Histogram of P_f 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 three different preparations of protoplasts were used. For each line, the P_f of 75 protoplasts was measured, and the initial diameter of protoplasts was about 60 μm. Error bars represent se (n = 75). The double asterisk indicates a significant difference in P_f between wild-type (Col) and transgenic (35S∷Rh-PIP2;1) plants (n = 75; t test; P < 0.01).

Silencing of Rh-PIP2;1 inhibits the cell elongation of the AbsE in petals. Petal samples were taken as a 0.5-cm × 0.4-cm slice at 25% of the length from petal top on the 7th d after infiltration and treated as described in . Bars = 200 μm. Cell numbers were counted as described in . Completely, Completely silenced flowers; partially, partially silenced flowers; non, no silenced flowers. Each data point represents the mean ± se (n = 15 for control, TRV-Rh-PIP2;1 [non], TRV, and ethylene treatment; n = 9 for TRV-Rh-PIP2;1 [completely]; n = 12 for TRV-Rh-PIP2;1 [partially]). Different letters indicate significant differences between different treatments according to Duncan's multiple range test (P < 0.05).

The expression of Rh-PIP2;1 in cut rose petals. A, Rh-PIP2;1 expression in different organs. P, Petal; Se, sepal; St, stamen; G, gynoecium; Re, receptacle; L, leaf. B, Rh-PIP2;1 expression in petals during normal flower opening. Lanes 0 to 6 indicate vase days. C and D, Rh-PIP2;1 expression in response to ethylene in petals. Flowers were exposed to air (control), 10 μL L⁻¹ ethylene, or 2 μL L⁻¹ 1-MCP. Lanes 0 to 72 indicate treatment hours. Each lane contains 10 μg of total RNA. Ethidium bromide-stained rRNA was used as an internal control to normalize the amount of total RNA. Total RNA was isolated from three individual samples at each time point, and all of the hybridizations were repeated at least three times. Representative results are shown.

Silencing of the Rh-PIP2;1 gene in rose petals by VIGS. Rose branches were infiltrated with Agrobacterium containing TRV 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 cDNA was generated from 1 μg of total RNA and 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, no silenced flowers. B, The phenotype of Rh-PIP2;1-silencing flowers (top) and petal sizes and water content (bottom). The photographs were taken on the 7th d after infiltration. The petal size and water content were determined on the 7th d after infiltration as described in . Each data point represents the mean ± 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]).

The intracellular localization of Rh-PIP2;1 and in situ hybridization of Rh-PIP2;1. A, The intracellular localization of Rh-PIP2;1 in Arabidopsis leaf mesophyll cell protoplast. The GFP-Rh-PIP2;1 fusion gene was inserted in pRTL2 and transformed into Arabidopsis leaf protoplast for transient expression. Imaging of the protoplast-expressed 35S∷GFP-Rh-PIP2;1 fusion was conducted with a laser scanning confocal microscope (Nikon C1). Arrows indicate internal membrane localization of Rh-PIP2;1. Bar = 20 μm. B, GFP-Rh-PIP2;1 localization is compared with a mCherry-labeled endoplasmic reticulum (ER) marker (ER-rk; CD3-959) and a mCherry-labeled plasma membrane (PM) marker (PM-rk; CD3-1007). At least 20 protoplasts were examined in expressing each marker. Bars = 10 μm. C, Pattern of Rh-PIP2;1 mRNA accumulation examined by in situ hybridization. Rh-PIP2;1 was detected by DIG-labeled 3′-UTR RNA antisense (left) and sense (right) probes in transverse sections of cut rose petals. Bar = 200 μm.

Morphological changes of cut rose flowers in response to exogenous ethylene. A, Vertical (left) and side (right) views of flowers after ethylene and 1-MCP treatments. Flowers were exposed to air (control), 10 μL L⁻¹ ethylene, or 2 μL L⁻¹ 1-MCP for 24 to 72 h, and the photographs were taken at the end of individual treatment times. Twenty-five flowers were used in each treatment, and representative results are shown. B, The ratio of flower height to flower diameter. Each data point represents the mean ± se (n = 15). Asterisks indicate significant differences (Student's t test; P < 0.05) between untreated control and ethylene- or 1-MCP-treated flowers. C, The size of petals after ethylene and 1-MCP treatments. The length and width of petals were measured on the 2nd d after 24-h treatments. One petal was chosen randomly from the second layer of each flower. Double asterisks indicate significant differences in length or width between untreated control and ethylene- or 1-MCP-treated flower petals (n = 15, P < 0.01). D, Changes of petal fresh weight (top blue lines) and dry weight (bottom red lines) after ethylene and 1-MCP treatments. Each data point represents the mean ± se (n = 15). Asterisks indicate significant differences (Student's t test; P < 0.05) between untreated control and ethylene- or 1-MCP-treated flower petals.

Inhibition of the cell elongation of AbsE in petals by ethylene. A, The typical anatomic structure of a petal at flower full opening stage. Left, A scanning electron microscope image. Bar = 50 μm. Right, A light microscope image with semithin section. Bar = 200 μm. AbE, Abaxial epidermis; AdE, adaxial epidermis; PC, parenchyma cells; VB, vascular bundle. B, Traces of outlines of AbsE cells (top) and cell numbers (bottom) in different regions of petals. Bars = 200 μm. AbsE cells of petals were photographed using a Nikon IX-71 microscope. Petal samples were taken as a 0.5-cm × 0.4-cm slice at 25%, 50%, and 75% of the petal length from petal top on the 2nd d after 24-h treatments. The slices were fixed in formaldehyde and then cleared in ethanol. The traces were drawn using Photoshop 7.0 software. Fifteen flowers were used in each treatment, and representative results are shown. Cell numbers were counted using ImageJ software in a visual field of 1,360 × 1,024 μm². Each data point represents the mean ± se (n = 15). Different letters in the same row indicate significant differences between different treatments according to Duncan's multiple range test (P < 0.05).