Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts

Abstract

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.

Fig. 1

Water permeability coefficient (P_os) measured in lily (Lilium longiflorum) control pollen grain protoplasts (only gold particles, not plasmid coated) or protoplasts obtained from pollen grains bombarded with gold beads coated with AtSUC3::GFP and LeLAT52::GFP plasmids. Small data points indicate individual experiments. Large data points are mean ± SD. The mean P_os value of controls is indicated by the solid line; the grey box indicates the SD of control values.

Fig. 2

Typical volume transients (swelling curves) of protoplasts obtained from lily (Lilium longiflorum) pollen grains transformed with Arabidopsis thaliana plasma membrane intrinsic protein 1;1 (AtPIP1;1)/cyan fluorescent protein (CFP) plasmids (a), AtPIP1;2/CFP plasmids (b), AtPIP2;1/yellow fluorescent protein (YFP) plasmids (c), or AtPIP2;2/YFP plasmids (d). The calculated water permeability coefficient (P_os) and the respective parameters α and β (see Materials and Methods) are given for each individual protoplast. The images show the protoplast at t = 0 min as bright field images (upper left quarter) and fluorescence images (lower left quarter). The right half of the image shows the protoplast at the end of the swelling curve (t_final). Bars, 20 μm.

Fig. 3

Summary of water permeability coefficient (P_os) values measured in control and transformed lily (Lilium longiflorum) pollen grain protoplasts as well as in control and transformed pollen tube protoplasts. Individual P_os values and mean ± SD are presented. Solid lines and grey boxes indicate the mean and the corresponding SD of the respective control P_os values. The respective mean ± SD of P_os values for each treatment is given in μm s⁻¹ and the number in brackets indicates the number of individual protoplasts measured. P_os values significantly different from control data are indicated, with significance values (P) calculated using Student’s t-test (ST) and the Mann–Whitney test (MW).

Fig. 4

Single-cell RT-PCR of Arabidopsis thaliana plasma membrane intrinsic protein 1;1 (AtPIP1;1)-transformed and control pollen grain protoplasts from lily (Lilium longiflorum). Fluorescent as well as control pollen grain protoplasts were collected and immediately frozen in liquid nitrogen. cDNA was generated by reverse transcription with AtPIP1;1-specific reverse primer, followed by PCR with an AtPIP1;1-specific primer pair. Amplified product was separated by agarose gel electrophoresis, blotted onto a membrane and detected using a digoxigenin-labelled specific AtPIP1;1 probe. Lane 1, water control; lanes 2–5, individual, transformed protoplasts; lanes 6–8, nontransformed protoplasts.

Fig. 5

Swelling curves of protoplasts obtained from lily (Lilium longiflorum) pollen grains co-transformed with Arabidopsis thaliana plasma membrane intrinsic protein 1;1 (AtPIP1;1)/AtPIP2;2 (a) and AtPIP1;2/AtPIP2;1 (b). The images show the protoplast at t = 0 min as bright field images (upper left quarter) and as fluorescence images (lower left quarter). The right half of the image shows the protoplast at the end of the swelling curve (t_final). Bars, 20 μm.

Fig. 6

Typical swelling curve of a pollen tube protoplast transformed with Arabidopsis thaliana plasma membrane intrinsic protein 2;1 (AtPIP2;1) (a) and pollen tube growth rates measured in pollen tubes of control and transformed pollen grains (b). The images show the protoplast at t = 0 min as bright field images (upper left quarter) and as fluorescence images (lower left quarter). The right half of the image shows the protoplast at the end of the swelling curve (t_final). Numbers in brackets give the number of individual experiments whereas the numbers in the boxes give the mean growth rate ± SD in μm min⁻¹. Bar, 20 μm.