Hydrogen peroxide permeability of plasma membrane aquaporins of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Although aquaporins have been known to transport hydrogen peroxide (H₂O₂) across cell membranes, the H₂O₂-regulated expression patterns and the permeability of every family member of the plasma membrane intrinsic protein (PIP) toward H₂O₂ have not been determined. This study investigates the H₂O₂-regulated expression levels of all plasma membrane aquaporins of Arabidopsis thaliana (AtPIPs), and determines the permeability of every AtPIP for H₂O₂ in yeast. Hydrogen peroxide treatment of Arabidopsis down-regulated the expression of AtPIP2 subfamily in roots but not in leaves, whereas the expression of AtPIP1 subfamily was not affected by H₂O₂ treatment. The growth and survival of yeast cells that expressed AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 was reduced in the presence of H₂O₂, while the growth of yeast cells expressing any other AtPIP family member was not affected by H₂O₂. These results show that only certain isoforms of AtPIPs whose expression is regulated by H₂O₂ treatment are permeable for H₂O₂ in yeast cells, and suggest that the integrated regulation of aquaporin expression by H₂O₂ and the capacity of individual aquaporin to transport H₂O₂ are important for plant response to H₂O₂.     

Mechanisms and effects of retention of over-expressed aquaporin AtPIP2;1 in the endoplasmic reticulum

Plasma membrane intrinsic proteins (PIPs) are aquaporins that mediate water transport across the plant plasma membrane (PM). The present work addresses, using Arabidopsis AtPIP2;1 as a model, the mechanisms and significance of trafficking of newly synthesized PIPs from the endoplasmic reticulum (ER) to the Golgi apparatus. A functional diacidic export motif (Asp4-Val5-Glu6) was identified in the N-terminal tail of AtPIP2;1, using expression in transgenic Arabidopsis of site-directed mutants tagged with the green fluorescent protein (GFP). Confocal fluorescence imaging and a novel fluorescence recovery after photobleaching application based on the distinct diffusion of PM and intracellular AtPIP2;1-GFP forms revealed a retention in the ER of diacidic mutated forms, but with quantitative differences. Thus, the individual role of the two acidic Asp4 and Glu6 residues was established. In addition, expression in transgenic Arabidopsis of ER-retained AtPIP2;1-GFP constructs reduced the root hydraulic conductivity. Co-expression of AtPIP2;1-GFP and AtPIP1;4-mCherry constructs suggested that ER-retained AtPIP2;1-GFP may interact with other PIPs to hamper their trafficking to the PM, thereby contributing to inhibition of root cell hydraulic conductivity.     

Relationship between chloroplastic H2O2 and the salicylic acid response

Reactive oxygen species (ROS) act as signaling molecules for regulating plant responses to abiotic and biotic stress and there exist source- and kind-specific pathways for ROS signaling. Recently, we created a novel system for producing H₂O₂ in Arabidopsis chloroplasts by chemical-dependent thylakoid membrane-bound ascorbate peroxidase (tAPX) silencing using an estrogen-inducible RNAi method. Microarray analysis revealed that the expression of a large set of genes was altered in response to tAPX silencing, some of which are known to be involved in pathogen response/resistance. Furthermore, we found that tAPX silencing enhances the levels of salicylic acid (SA) and the response to SA, a central regulator for biotic stress response. In this addendum, we describe the relationship between chloroplastic H₂O₂ and SA in stress response, and discuss the function of the kind- and source-specific ROS signaling in SA-mediated stress response.     

Phosphorylation of the zinc finger transcriptional regulator ZAT6 by MPK6 regulates Arabidopsis seed germination under salt and osmotic stress

C₂H₂-type zinc finger proteins (ZFPs) play diverse roles in plant response to abiotic stresses. ZAT6, an Arabidopsis C₂H₂-type ZFP, has been reported to regulate root development and nutrient stress responses. However, its roles in regulation of abiotic stress response are incompletely known. Here, we demonstrate that salt or osmotic stress triggers a strong increase in ZAT6 expression in leaves. Transgenic plants overexpressing ZAT6 showed improved seed germination under salt and osmotic stress. Intriguingly, ZAT6 interacts with a stress-responsive mitogen-activated protein kinase MPK6 in vitro and in planta. ZAT6 is phosphorylated by both recombinant and plant endogenous MPK6. Serine 8 and serine 223 in ZAT6 were identified as the sites phosphorylated by MPK6. In contrast to wild-type form of ZAT6, overexpression of phosphorylation mutant form did not display significantly enhanced salt and osmotic stress tolerance. Altogether, our results suggest that phosphorylation by MPK6 is required for the functional role of ZAT6 in seed germination under salt and osmotic stress.     

The response of Arabidopsis root water transport to a challenging environment implicates reactive oxygen species- and phosphorylation-dependent internalization of aquaporins

Aquaporins, which facilitate the diffusion of water across biological membranes, are key molecules for the regulation of water transport at the cell and organ levels. We recently reported that hydrogen peroxide (H₂O₂) acts as an intermediate in the regulation of Arabidopsis root water transport and aquaporins in response to NaCl and salicylic acid (SA).¹ Its action involves signaling pathways and an internalization of aquaporins from the cell surface. The present addendum connects these findings to another recent work which describes multiple phosphorylations in the C-terminus of aquaporins expressed in the Arabidopsis root plasma membrane.² A novel role for phosphorylation in the process of salt-induced relocalization of AtPIP2;1, one of the most abundant root aquaporins, was unraveled. Altogether, the data delineate reactive oxygen species (ROS)-dependent signaling mechanisms which, in response to a variety of abiotic and biotic stresses, can trigger phosphorylation-dependent PIP aquaporin intracellular trafficking and root water transport downregulation.Key words: reactive oxygen species, aquaporin, phosphorylation, cell signaling, stress, protein relocalization, root water transportPlants can regulate their water uptake capacity i.e. their root hydraulic conductivity (Lp_r) on a short term (minutes to hour) basis through regulation of plasma membrane (PM) aquaporins of the Plasma membrane Intrinsic Protein (PIP) subfamily.³ It has been known for a long time that salt stress (NaCl), as many other abiotic stresses such as cold, anoxia or nutrient deprivation, induces an inhibition of Lp_r in many plant species.³ In the recent study by Boursiac et al. (2008),¹ we identified SA as a new inhibitory increased the accumulation of ROS in roots, it was hypothesized that H₂O₂ or other ROS may have a central role in the regulation of root water transport in response to various biotic or abiotic stimuli. When Arabidopsis roots were treated with mM concentrations of exogenous H₂O₂, Lp_r was inhibited within minutes by up to 90%. These findings are consistent with previous reports showing that ROS can downregulate water transport in cucumber and maize roots or in the algae Chara corallina.^4–7 H₂O₂ and possibly other derived ROS may modulate the Lp_r through signaling mechanisms or by a direct oxidative gating of aquaporins. The latter hypothesis, which has been favored in previous studies by Steudle and colleagues,^6,7 was investigated by Boursiac et al., by functionally expressing aquaporins in Xenopus oocytes and by testing their sensitivity to external H₂O₂. The results show that Arabidopsis aquaporins are insensitive to direct oxidation by H₂O₂ or hydroxyl radicals. Thus, these and complementary pharmacological analyses on excised roots rather support a role for H₂O₂ as a second messenger that connects environmental stimulus perception to water transport regulation in plant roots. The additional finding that H₂O₂ can be transported by aquaporins^8,9 opens the possibility of intricate loop mechanisms whereby these proteins may interfere with their own regulation. For example, active PIP aquaporins could facilitate the diffusion within the cell of NADPH-oxidase derived apoplastic H₂O₂, which in turn would activate signaling pathways acting on PIP activity and/or subcellular localization.In a previous study, we monitored the subcellular localization of AtPIP1;2 and AtPIP2;1, two of the most abundant PIPs in roots, by expression in transgenic Arabidopsis of fusions with the green fluorescent protein (GFP).¹⁰ We observed that a 100 mM NaCl treatment induced in 2–4 hours an increased intracellular labeling which was interpreted as an intracellular relocalization of the two aquaporins.¹⁰ In our more recent study, both a 150 mM NaCl and a 0.5 mM SA treatments induced an intracellular labeling by GFP-PIP1;2 and PIP2;1-GFP fusions, with a “fuzzy” pattern or at the level of spherical bodies. Preventing the NaCl- or SA-dependent accumulation of ROS with exogenous catalase was able to almost completely counteract the effects of the two stimuli on the localization pattern of the PIP2;1-GFP fusion. In addition, the inhibition of Lp_r by SA was also counteracted at 33% by the catalase treatment. Altogether, the data stress the importance of an ROS-induced relocalization of aquaporins in the regulation of root water transport. Yet, we still miss quantitative data and complementary pharmacological evidence to determine the exact contribution of aquaporin relocalization with respect to other aquaporin regulatory mechanisms.Another recent work by our group has, however, provided deeper insights into the mechanisms of stress-induced relocalization of aquaporins in plants.² Our group identified by mass spectrometry multiple adjacent phosphorylation sites (up to 4 in the case of AtPIP2;4) in the C-terminus of aquaporins expressed at the root plasma membrane.² Phosphorylation of AtPIP2;1, which shows a simpler profile with only two sites at Ser280 and Ser283, was studied in closer detail by site-directed mutagenesis and expression in transgenic Arabidopsis of GFP-PIP2;1 fusions. A Ser283Ala mutation, which mimics a constitutively dephosphorylated Ser283, induced a marked intracellular accumulation of GFP-PIP2;1 in resting conditions. Because no phenotype was observed after a Ser280Ala mutation, the data suggest a specific role for Ser283 phosphorylation in the proper targeting of the protein. When plants were treated by 100 mM NaCl for 2 to 4 hours, the wild type (WT) and Ser280Ala mutant forms of GFP-PIP2;1 showed similar intracellular staining, in both “fuzzy” structures or spherical bodies. On the contrary, the Ser283Ala mutant did not label any spherical body. Interestingly, a Ser283Asp mutation that mimics a constitutively phosphorylated Ser283 resulted in a salt-induced labeling of spherical bodies similar to the one observed with WT GFP-PIP2;1 whereas no “fuzzy” staining was observed. Therefore, the phosphorylation status of Ser283 seems to determine the redistribution of AtPIP2;1 towards fuzzy structures (non-phosphorylated Ser283) or spherical bodies (phosphorylated Ser283). Although the nature of these intracellular structures remains to be identified, we now consider the possibility that the spherical bodies correspond to the late endosome/prevacuolar compartment that orientates aquaporins towards a degradation pathway whereas the fuzzy structures may act as a storage compartment for subsequent relocalization of PIP aquaporins to the PM, and rapid recovery of the PM water permeability. Although we favor the idea that the intracellular labeling shown by GFP-PIP2;1 in response to salt originates from aquaporins relocalized from the PM, newly synthesized proteins may also contribute to this pattern.Prak et al., also developed an absolute quantification method to show that the phosphorylation profile of AtPIP2;1 at the root plasma membrane was altered upon 100 mM NaCl and 2 mM H₂O₂ treatments. Whereas NaCl decreased the abundance of phosphorylated Ser283, H₂O₂ enhanced the overall phosphorylation of the AtPIP2;1 C-terminus. These observations add another level of complexity to the mechanisms of stimulus-induced and phosphorylation- dependent relocalisation of plant aquaporins uncovered in our group. Although one of the primary effects of NaCl is undoubtedly an accumulation of ROS, the difference in phosphorylation patterns observed in response to H₂O₂ and NaCl treatments may come from quantitative and kinetic differences in ROS patterns between the two treatments or from additional regulations activated by salt.We note that phosphorylation of PIP aquaporins had already been investigated in detail.^11–13 In particular, studies with spinach SoPIP2;1 has pointed to two phosphorylation sites, Ser115 in the first cytoplasmic loop (loop B) and Ser274 at the C-terminus, as important for modulating the water transport activity of this aquaporin after expression in Xenopus oocytes. A role for these two sites in aquaporin gating was also deduced from the atomic structure of SoPIP2;1.¹⁴ Whereas Ser280 in AtPIP2;1 corresponds to Ser274 in SoPIP2;1, the functional role of sites equivalent to Ser283 in AtPIP2;1 had not been considered previously in any other PIP. To our knowledge, the study by Prak et al., provides the first evidence in plants for a role of phosphorylation on the relocalization of aquaporins and highlights the importance of multiple phosphorylations sites in the C-terminus of aquaporins, as has been recently shown in human Aquaporin-2.^15,16Overall, the advance provided by our two recent studies delineates a working model (Fig. 1), whereby multiple abiotic and biotic stresses, which all induce an accumulation of ROS, activate common signaling pathways to downregulate root water transport. We have provided evidence that some of these pathways are calcium- and/ or protein kinase-dependent. One regulatory mechanism triggered by these pathways is the relocalization of aquaporins into intracellular “fuzzy” structures or bigger spherical bodies. For AtPIP2;1, the sorting between these structures is determined in part by the phosphorylation status of Ser283, which ultimately may control the cellular fate of the protein for degradation or remobilization to the PM. A coming challenge will be to determine how this and other cellular mechanisms quantitatively contribute to the integrated regulation of water transport at the cell and tissue (whole root) levels. Another avenue for future research will be to identify the molecular components involved in upstream ROS-dependent cell signaling and aquaporin phosphorylation. These studies will tell us how the regulation of root water uptake in parallel to the regulation of transpiration allows the plant to preserve its water status when it is continuously challenged by multiple stresses.Open in a separate windowFigure 1Tentative model of regulation of root hydraulic conductivity (Lp_r) through reactive oxygen species (ROS) signaling. Multiple biotic and abiotic stimuli such as NaCl or salicylic acid can induce an intra- and/or extracellular accumulation of ROS by acting on their production, degradation or transport. The stimulus-induced ROS in turn activate signaling pathways involving protein kinases and cytosolic calcium. These events result in changes in the phosphorylation and subcellular localization patterns of plasma membrane (PM) aquaporins (PIPs). In particular, endocytosis can direct PIPs towards various intracellular compartments for subsequent recycling at the PM or degradation. Phosphorylation can interfere with this routing process, but also determines the intrinsic water transport activity (gating) of PM localized PIPs. The possibility exists that signaling components directly act on PIP gating, recycling or degradation through phosphorylation- and endocytosis-independent pathways (not shown). In addition, transport of H₂O₂ by PIP aquaporins may provide retroactive effects of aquaporins on upstream signaling events. Aquaporin activity at the PM determines root cell water permeability, which contributes to most of Lp_r in Arabidopsis. The overall scheme shows how stress-induced ROS signaling results in an inhibition of PIP aquaporin activity and, as a consequence, in an overall downregulation of Lp_r.     

Unravelling mitochondrial retrograde regulation in the abiotic stress induction of rice ALTERNATIVE OXIDASE 1 genes

Mitochondrial retrograde regulation (MRR) is the transduction of mitochondrial signals to mediate nuclear gene expression. It is not clear whether MRR is a common regulation mechanism in plant abiotic stress response. In this study, we analysed the early abiotic stress response of the rice OsAOX1 genes, and the induction of OsAOX1a and OsAOX1b (OsAOX1a/b) was selected as a working model for the stress‐induced MRR studies. We found that the induction mediated by the superoxide ion (O_2·^‐)‐generating chemical methyl viologen was stronger than that of hydrogen peroxide (H₂O₂). The addition of reactive oxygen species (ROS) scavengers demonstrated that the stress induction was reduced by eliminating O_2·^‐. Furthermore, the stress induction did not rely on chloroplast‐ or cytosol‐derived O_2·^‐. Next, we generated transgenic plants overexpressing the superoxide dismutase (SOD) gene at different subcellular locations. The results suggest that only the mitochondrial SOD, OsMSD, attenuated the stress induction of OsAOX1a/b specifically. Therefore, our findings demonstrate that abiotic stress initiates the MRR on OsAOX1a/b and that mitochondrial O_2·^– is involved in the process.     

A Medicago truncatula EF-Hand Family Gene,MtCaMP1, Is Involved in Drought and Salt Stress Tolerance

Background

Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize gens of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis.

Methodology/Principal Findings

Transgenic Arabidopsis seedlings expressing MtCaMP1exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na⁺ in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H₂O₂ and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress.

Conclusions/Significance

The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na⁺ accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na⁺ under drought and salt stress would protect plants from water default and Na⁺ toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.     

Glutathione reductase gene expression depends on chloroplast signals in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Glutathione reductase (EC 1.6.4.2) is one of the main antioxidant enzymes of the plant cell. In Arabidopsis thaliana, glutathione reductase is encoded by two genes: the gr1 gene encodes the cytosolic-peroxisomal form, and the gr2 gene encodes the chloroplast-mitochondrial form. Little is known about the regulation of expression of plant glutathione reductase genes. In the present work, we have demonstrated that gr2 (but not gr1) gene expression in Arabidopsis leaves changes depending on changes in redox state of the photosynthetic electron transport chain. Expression of both the gr1 and gr2 genes was induced by reactive oxygen species. In heterotrophic suspension cell culture of Arabidopsis, expression of both studied genes did not depend on H₂O₂ level or on changes in the redox state of the mitochondrial electron transport chain. Our data indicate that chloroplasts are involved in the regulation of the glutathione reductase gene expression in Arabidopsis.     

The Functional and Regulatory Mechanisms of the Thellungiella salsuginea Ascorbate Peroxidase 6 (TsAPX6) in Response to Salinity and Water Deficit Stresses

Soil salinization is a resource and ecological problem in the world. Thellungiella salsuginea is becoming a new model plant because it resembles its relative species, Arabidopsis thaliana, in small genome and short life cycle. It is highly tolerant to salinity and drought stresses. Ascorbate peroxidase (APX) is an enzyme that clears H₂O₂ in plants. The function and molecular and regulation mechanisms of APX in T. salsuginea have rarely been reported. In this study, an APX gene, TsApx6, was cloned from T. salsuginea and its responses to abiotic stresses in transgenic Arabidopsis were studied. Under high salinity treatment, the expression of TsApx6 was significantly induced. Under drought treatment, overexpression of TsApx6 increased the survival rate and reduced leaf water loss rate in Arabidopsis. Compared to the wild type plants, high salinity treatment reduced the concentrations of MDA, H₂O₂ and proline but elevated the activities of APX, GPX, CAT and SOD in the TsApx6-overexpressing plants. Meanwhile, germination rate, cotyledon greening, and root length were improved in the transgenic plants compared to the wild type plants under salt and water deficit conditions. Based on these findings, TsApx6 has an important function in the resistance of plants to certain abiotic stresses. The TsApx6 promoter sequence was obtained using Genome Walking technology. Bioinformatics analysis indicated that it contains some cis-acting elements related to stress response. The treatments of salt, dehydration, and ABA induced the expression of Gus gene under the regulation of the TsApx6 promoter. Mutation analysis showed that the MBS motif present in the TsApx6 promoter might be a key negative regulatory element which has an important effect on the growth and developmental process of plants.     

Apple RING finger E3 ubiquitin ligase MdMIEL1 negatively regulates salt and oxidative stresses tolerance

RING-finger-containing E3 ubiquitin ligases play important roles in plant response to biotic and abiotoc stresses. In this study, through homology analysis, a Malus× domestica MYB30-Interacting E3 Ligase 1 gene, MdMIEL1, was identified and subsequently cloned from apple ‘Gala’ (Malus×domestica). MdMIEL1 contained a zinc finger domain close to N-terminus and a RING finger domain close to Cterminus. Expression of MdMIEL1 was significantly induced by NaCl and H₂O₂ treatments. Further study demonstrated that the MdMIEL1-overexpressing Arabidopsis and apple calli were less tolerance to salt stress than wild-type control. In addition, transgenic plants had higher levels of reactive oxygen species (ROS) (H₂O₂ and O₂ ^–). And transgenic Arabidopsis and apple calli exhibited more sensitive phenotype to H₂O₂ treatment, which was associated with increased levels of ROS. These findings indicate MdMIEL1 is an important regulator involved in plant response to salt and oxidative stresses tolerance.     

Exogenous ethanol treatment alleviates oxidative damage of Arabidopsis thaliana under conditions of high-light stress

Abiotic stresses, such as high light and salinity, are major factors that limit crop productivity and sustainability worldwide. Chemical priming is a promising strategy for improving the abiotic stress tolerance of plants. Recently, we discovered that ethanol enhances high-salinity stress tolerance in Arabidopsis thaliana and rice by detoxifying reactive oxygen species (ROS). However, the effect of ethanol on other abiotic stress responses is unclear. Therefore, we investigated the effect of ethanol on the high-light stress response. Measurement of chlorophyll fluorescence showed that ethanol mitigates photoinhibition under high-light stress. Staining with 3,3′-diaminobenzidine (DAB) showed that the accumulation of hydrogen peroxide (H₂O₂) was inhibited by ethanol under high-light stress conditions in A. thaliana. We found that ethanol increased the gene expressions and enzymatic activities of antioxidative enzymes, including ASCORBATE PEROXIDASE1 (AtAPX1), Catalase (AtCAT1 and AtCAT2). Moreover, the expression of flavonoid biosynthetic genes and anthocyanin contents were upregulated by ethanol treatment during exposure to high-light stress. These results imply that ethanol alleviates oxidative damage from high-light stress in A. thaliana by suppressing ROS accumulation. Our findings support the hypothesis that ethanol improves tolerance to multiple stresses in field-grown crops.     

Volatile organic compounds of some Trichoderma spp. increase growth and induce salt tolerance in Arabidopsis thaliana

Many beneficial effects of Trichoderma spp. on plant growth and/or resistance to biotic/abiotic stresses can result from the production of bioactive compounds including volatile organic compounds (VOCs). We evaluated the effects of the volatile mixtures from 13 strains of different Trichoderma species on induction of tolerance to salt stress (100 mM NaCl) as well as growth promotion of Arabidopsis thaliana. Plants responded differently due to the presence of VOCs from various Trichoderma species ranging from both growth promotion and induction of salt tolerance to no significant changes under any of the conditions tested. In plants exposed for 2 weeks to VOCs of the selected strain, i.e. Trichoderma koningii, there was less H₂O₂ accumulation under salt stress compared to that in control plants. This result may reflect the possible role of VOCs of this strain in plant protection against oxidative damage under salt stress. Together, induction of salt tolerance using VOCs should be added to the known mechanisms of plant vigor enhancement by Trichoderma spp.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> regulates root system architecture by modulating the polar transport and redistribution of auxin

The accumulation and redistribution of the plant hormone auxin plays a crucial role in root development and patterning. Plants can alter their root system architecture (RSA) to adapt to different biotic and abiotic stresses. In addition, reactive oxygen species (ROS), such as H₂O₂, are known to increase in plants undergoing stress. Here, we present evidence that H₂O₂ can regulate auxin accumulation and redistribution through modulating polar auxin transport, leading to changes in RSA. Plants exposed to different concentrations of H₂O₂ formed a highly branched root system with abundant lateral roots and a shorter primary root. Monitoring of the auxin responsive DR5::GUS indicated that auxin accumulation decreased in lateral root primordia (LRP) and emerging lateral root tips. In addition, polar auxin transport, including both basipetal and acropetal transport modulated by AUX1 and PIN protein carriers, was involved in the process. Taken together, our results suggest that H₂O₂ could regulate plastic RSA by perturbing polar auxin transport as a means of modulating the accumulation and distribution of auxin.     

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

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.