Mutation of Aquaporin Gene PIP 2 ; 5 Postpones Pollen Hydration in Arabidopsis

In flowering plants with dry stigmas, pollen hydration involves water movement, which may be facilitated by aquaporins. To explore the possibility underlying this biological process, we identified and characterized a mutant with a T-DNA insertion in PIP2;5, which encodes an aquaporin with water channel activity in the PIP2 subfamily. We monitored the pollination process (pollen hydration, germination, and pollen tube growth) of wild type pollen on stigmas of the mutant and wild type. Pollen hydration was postponed on the stigmas of the mutant, compared with that on wild type stigmas. However, pollen tube germination and growth was unaffected in the mutant. The PIP2;5 protein was located in the cell plasma membrane and was preferentially expressed in the stigma. Based on our results, we concluded that PIP2;5 might play an important role in water movement during pollen hydration.


Introduction
During sexual reproduction in flowering plants, water movement between cells and tissues is essential for anther dehiscence and pollen tube development.Pollination is initiated when desiccated pollen grains hydrate on the stigma [1].The regulated passage of water from the stigma to pollen is important for successful pollen tube development in flowering plants with dry stigmas.
The pollen grains that land on dry stigmas mobilize their lipid-rich pollen coat to form an interface between the two cell surfaces.This interface converts into a histochemically distinguishable structure that is thought to promote water flow [2,3].Water, nutrients, and other small molecules must be transported through two membranes from the stigma papillae to the pollen grain for successful pollen hydration and germination.
Aquaporins are membrane proteins that permit fast transport of water through cellular membranes.These proteins contain six membrane-spanning helices and five loops (loops A to E) with N-and C-termini residing in the cytosol [4,5].Plant aquaporins can be subdivided into four major groups: plasma membrane-intrinsic proteins (PIPs), tonoplast-intrinsic proteins (TIPs), nodulin26-like-intrinsic proteins (NIPs), and small and basic membrane-intrinsic proteins (SIPs) [6,7].The abundance of aquaporins in reproductive organs suggests that these proteins function in plant reproduction.In tobacco, PIP1 and PIP2 show distinct expression patterns during stigma and anther development, and may contribute to the development of these organs in different ways [8,9].In potato, PIP2a was shown to be highly expressed in the pistil and anther tissues [10].PIP1 aquaporins are expressed in the anther and stigma epidermis in Brassica sp.[11][12] and OsPIP1;1 and OsPIP4;1 are highly expressed in rice anthers [13].Six out of 35 aquaporin genes in the Arabidopsis genome are known to be expressed in pollen [11].Among them, TIP1;3 is expressed in vesicles and vacuoles of vegetative cells, and TIP5;1 is expressed in vacuoles of sperm cells [11,[14][15][16].TIP5;1 and TIP1;3 are involved in the nitrogen metabolic pathway during pollen tube growth [17,18].NIP4;1 and NIP4;2 are two pollen-specific aquaporins; NIP4;1 functions during pollen development and germination, while NIP4;2 functions exclusively during pollen tube growth [19].As well as being expressed in pollen, AtNIP7;1, AtSIP1;1, and AtSIP2;1 are expressed in other tissues.AtNIP7;1 is expressed in microspores [20], while AtSIP1;1 and AtSIP2;1 are expressed in the vascular tissues of the flower petal, stigma, stamens, and pollen [21].
As pollen hydration involves the movement of water, it is possible that aquaporins function in the stigma to facilitate this process.Previously, PIPs were found to be expressed in the stigma of Arabidopsis [12,13].To explore the role of PIP aquaporins expressed in the stigma in pollen hydration, we screened a number of PIP mutants obtained from Arabidopsis Biological Resource Center (ABRC).We found a mutant with a T-DNA insertion in PIP2;5 that disrupted expression of the gene and resulted in slower pollen hydration on the stigma during pollination, comparing with that in wild type.Thus, we conclude that PIP2;5 played an important role in water movement during pollen hydration.

Characterization of pip2;5 mutant
To understand the role of aquaporins in pollen hydration, we tried to isolate Arabidopsis T-DNA insertion mutants showing delayed pollen hydration from the T-DNA insertion collection of ABRC.
One mutant (SALK_072405) with delayed hydration was identified.In this mutant, the T-DNA was inserted in the second intron of the genomic sequence of AT3G54820 (1,215 bp downstream from the initiation codon ATG) (Figure 1A).AT3G54820 encodes the protein PIP2;5, which belongs to the PIP2 subfamily.The genotype of the heterozygous plants (pip2;5/+) among the progenies was identified by a PCR-based method using gene-specific primers and the T-DNA primer.The genotype ratio of the wild type, heterozygous, and homozygous plants among the offspring of heterozygous plants was about 1:2:1 (59:121:62), indicating the presence of a single T-DNA insertion.We investigated the effect of the T-DNA insertion on the transcript level of PIP2;5 by RT-PCR.For pip2;5 homozygous mutant plants, no product was obtained, indicating full knock-out of PIP2;5 (Figure 1B).The pip2;5 homozygous plants were used for further analysis.

Postponed hydration of pollen grains on pip2;5 stigma
We examined the viability of pip2;5 pollen grains by Alexander staining.No difference in viability was identified between the mature pollen grains of the pip2;5 mutant and wild type (supplemental Figure 1A, B).Next, we examined the germination and tube growth of pollen in vitro.The pollen germination ratio and pollen tube morphology of the pip2;5 mutant was comparable with those of wild type in vitro (Supplemental Figure 1C, D).
Pollen grains were considered to be fully hydrated when the width remained constant for 10 min.We determined the hydration completion time for pollen from wild type and the pip2;5 mutant on stigmas of the pip2;5 mutant and wild type.First, when wild type and pip2;5 pollen grains were placed on a wild type stigma, the hydration completion time show no difference between the wild type and pip2;5 pollen grains (data not showed).Next, the pollen grains were placed on wild type and pip2;5 stigmas.Wild type pollen initiated hydration rapidly following it contact with on the wild type stigma (hydration completed by 9.5 ± 2.7 min after pollination.n = 61, Figure 2), however the pollen showed a severe delay in the completion of hydration on the pip2;5 stigma (12.8 ± 5.2 min to complete hydration, n = 61, P<<0.01; Figure 2).By 12 min after pollination, 92% of wild type pollen grains had completed hydration on the wild type stigma, whereas only 36% had completed hydration on the stigma of the pip2;5 mutant.The results suggest that pollen hydration is postponed on the pip2;5 stigma.
After hydration, the pollen grain germinates and produces a pollen tube that will penetrate the stigma and grow through the style and along the transmitting tract to enter the ovule.To detect whether pollen tube growth was affected in the pip2;5 mutant, we examined pollen tube growth in the pistil by histochemical analysis of emasculated wild type and pip2;5 pistils hand-pollinated with pollen of PLAT52::GUS expressing plants at 0.5, 1.5, 2.5, 4.5, 6, or 9 hours after pollination (HAP).The pollen tube growth in the style and transmitting tract was normal in pip2;5 plants (Supplemental Figure 2).Seed set was also normal in pip2;5 self-pollinated plants (data not shown).These results imply that the PIP2;5 mutation affected pollen hydration on the stigma.

Complementation of pip2;5 mutant
To confirm that the pip2;5 delayed-pollen-hydration phenotype was indeed due to a mutation in PIP2;5, we amplified the predicted promoter (3,800 bp) and coding sequence (CDS) of PIP2;5, subcloned them into PMDC107 vector (conferring hygromycin resistance to transgenic plants), and introduced them into the pip2;5 homozygote via infiltration with A. tumefaciens.Seven lines harboring PPIP2;5::PIP2;5-GFP were obtained by resistance screening and fluorescence detection.
Five transformants can rescue the hydration defect of the mutants (average pollen hydration completion completion time, 9.4 ± 2.6 min; n = 56, combined data; P<<0.01, Figure 2, 3).Therefore, the pip2;5 mutant phenotype could be converted to a normal phenotype by constitutively expressing the wild type PIP2;5 CDS.
The function of aquaporins is closely linked to their subcellular localization; therefore, we constructed P35S::GFP-PIP2;5 to investigate the subcellular localization of PIP2;5.The T-DNA insertion caused a truncated protein, designated as PIP2;5m, which contains the first and second exons of PIP2;5.The subcellular localization of PIP2;5 was in the plasma membrane (PM).In contrast, GFP-PIP2;5m showed a clear cytoplasmic and PM localization (Figure 3).This result indicates that the N terminus is important for determining the PM localization of the PIP2;5 protein.

Preferential expression of PIP2;5 in the stigma
The papilla cells of the stigma control whether a pollen grain hydrates or not, so, if PIP2;5 does play a role in regulating water transport at the stigma surface, it might be expressed in the papilla cells of the stigma.Previous Affymetrix analysis showed PIP2;5 had a particularly strong transcript expression level in the stigma [22].To determine whether this was the case, we detected the expression level of PIP2;5 protein in the developing flowers of transgenic Arabidopsis harboring PPIP2;5::PIP2;5-GUS.The results showed that PIP2;5 was expressed in the style and the papilla cells of stages 9-14 flowers (Figure 4 A-H).In the pistil, GUS activity was detected in the papilla cells and the top part of the style close to the stigma.Strong GUS activity was detected in the transmitting tract.In stage 15 flowers, the weak GUS signals were detected in the papilla and style.In stage 16 flowers, GUS activity was observed at low levels in the anthers but not in pollen grains (data not shown).

Upregulation of PIP2;5 by cold treatment
Abiotic stresses have adverse effects on plant growth and productivity.PIP2;5 is thought to be involved in water transport in plants and, as a consequence, it may play a role in water deficit tolerance.The overexpression PIP2;5 in transgenic Arabidopsis plants showed no advantage under normal conditions but presented a rapid water loss under drought stress conditions [23].In our study, pip2;5 mutants did not show any growth alteration in standard medium and on plates with 50 mM NaCl, 100 mM NaCl.pip2;5 mutants only showed reduced slightly roots length on plates with 200 mM sorbitol (Supplemental Figure 3).It indicated that pip2;5 mutants might be sensitive to and Osmotic stress, but not to salt stress.
Similar to water and salt stresses, cold stress is an important abiotic stress factor that significantly limits plant growth and development.To understand the effect of cold stress on PIP2;5 expression, transgenic Arabidopsis seedlings (20 days after germination and flowers at stage 14) harboring PPIP2;5::PIP2;5-GUS were subjected to a cold treatment at 4°C for 6 days.In response to the cold treatment, GUS signals were enhanced in the root, leaves, and papilla cells (Figure 4I-L).Real-time PCR analysis confirmed that the PIP2;5 transcript levels were upregulated in the inflorescences and seedlings after the cold treatment (Figure 4M).

Discussion
The pollen hydration process is typically completed quickly.The water used for pollen hydration is derived from the stigma in flowering plants with dry stigmas.It is impossible that the free diffusion of water molecules provides sufficient water for timely pollen hydration.The plasma membrane is the first barrier limiting water exchange in plant cells, and the rate and capacity of its water transport is mainly determined by aquaporins.Therefore, there must be different aquaporins that are expressed and functioning in the stigma.The PIP aquaporins appear to play a major role in controlling membrane water permeability.This class of aquaporins can be grouped into two subfamilies: PIP1 and PIP2.We found that PIP2;5 was expressed in papilla cells and the transmitting tract with increasing strength at stages 9-14 before pollination.The pollen grains on the homozygous pip2;5 mutant stigma showed delayed hydration, longer average hydration time, and lower hydration frequency at every time point (Figure 2).However, subsequent pollen tube growth in the style and transmitting tract was normal in pip2;5 plants (Supplemental Figure 2).Based on our results, we speculate that the PIP2;5 mutation affects the transport activity or density of aquaporins in the stigma papillae, thereby affecting the pollen hydration rate.The molecular mechanisms of this process require further study.
Cold stress, including freezing and chilling, is one of the major stresses affecting the growth and development of plants, and it can result in huge crop losses.In plants, aquaporins are believed to play an important role in maintaining water homeostasis not only under normal growth conditions but also under various stress conditions.For example, the cotton aquaporin GhTIP1 ecotypes have a defined a narrow temperature window for pollen germination and pollen tube growth.Another study reported that pollen germination rates dropped to <50% at temperatures above and below 22°C [27].In our study, we found that PIP2;5 was preferentially expressed in the stigma and that the transcript level of PIP2;5 increased after a cold treatment (Figure 4I-M).
Based on these results, we speculate that at optimum temperature, the PIP2;5 protein may be involved in the pollen-stigma interaction, and under cold conditions, increased expression of PIP2;5 in the stigma would create more favorable moisture conditions for pollen germination.
Next, our research will focus on this this problem.

Pollen assays
Pollen hydration assays on the stigma were performed by hand-pollinating emasculated wild type and mutant stigmas and observing hydration of pollen grains under a Zeiss Axioscope microscope (Carl Zeiss MicroImaging Inc. Thornwood, NY, USA) until no further change in pollen diameter was observable.During this process, images were captured every 30 s. Pollen diameter was measured using ImageJ software and data were analyzed using SigmaPlot 11.0.

Confocal microscopy
The P35S::GFP-PIP2;5 and P35S::GFP-PIP2;5m constructs were introduced into tobacco leaves via infiltration with A. tumefaciens for transient expression.The excitation and emission wave lengths for GFP fluorescence were 488 nm and 505-530 nm, respectively.Images were captured with a TCS SP5II confocal laser scanning microscope (Leica, Wetzlar, Germany).

GUS assays
Staining to detect GUS activity was performed according to Sieburth and Meyerowitz [29].
The seedlings used in these experiments were 20 days old and had stage 14 flowers.The expression of the construct was determined by GUS staining, as described above, and by RT-PCR.

; 1 was
shown to play a role in the response to cold stress [24] and PIPs in rice have also been shown to play important roles in re-establishing the water balance after chilling [25].Overexpression of PIP2;5 was shown to alleviate inhibition of plant growth under low temperature and to facilitate Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 25 July 2018 doi:10.20944/preprints201807.0471.v1seed germination of transgenic Arabidopsis under cold stress [23, 26].The Columbia and Ler

Figure 1 .
Figure 1.(A) Schematic representation of PIP2;5 genomic organization and T-DNA position in SALK-072405.Exons of PIP2;5 are represented by black boxes and introns by lines.Triangle

Figure 2 .
Figure 2. Hydration of wild type pollen on wild type and pip2;5 homozygous stigma.(A-D) Wild type pollen status at 1 minute hydration of pollen (MHP), 9 MHP, 16 MHP, and 20 MHP on wild type stigma; (E-H) Wild type pollen status at 1 MHP, 9 MHP, 16 MHP, and 20 MHP on pip2;5 homozygous stigma; (I-L) Wild type pollen status at 1 MHP, 9 MHP, 16 MHP, and 20 MHP on pip2;5+PPIP2;5:: PIP2;5 transgenic stigma.Bar =50 µm in (A)-(L).(M) Statistical analysis of pollen hydration time; (N) Box plot showing completion of hydration.In (M) and (N), Completion is defined as the time elapsed from the first change in diameter to the time at which final diameter is achieved.Box plot depicts median, 25 th and 75 th percentiles, and full range of values.Single spots represent outlying points, defined as being further from 75 th percentile than spread between 25 th and 75 th percentiles.