Functional characterization of drought-responsive.pdf

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University of Toronto Scarborough
Biological Sciences
Steve Joordens

Physiologia Plantarum 140: 321–333. 2010 Copyright © Physiologia Plantarum 2010, ISSN 0031-9317 Functional characterization of drought-responsive aquaporins in Populus balsamifera and Populus simonii × balsamifera clones with different drought resistance strategies Adriana M. Almeida-Rodriguez , Janice E.K. Cooke ,FrancisYeh a and Janusz J. Zwiazek a∗ aDepartment of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada T6E 2E3 bDepartment of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 Correspondence We have characterized poplar aquaporins (AQPs) to investigate their possible *Corresponding author, functionsin differentialdroughtresponses of Populusbalsamifera and Populus e-mail: [email protected] simonii × balsamifera leaves. Plants were exposed to mild and severe levels Received 23 April 2010; of drought stress and to drought stress recovery treatment, and their responses revised 20 July 2010 were compared with well-watered controls. Compared with P. balsamifera, P. simonii × balsamifera used drought avoidance as the main drought doi:10.1111/j.1399-3054.2010.01405.x resistance strategy, and rapidly reduced stomatal conductance in response to stress. This strategy is correlated with growth rate reductions. ElevenQPs were transcriptionally profiled in leaves from these experiments and five were functionally characterized for water channel activity.IP1;3 and PIP2;5 were among the most highly expressed leaf AQPs that were responsive to drought. Expression of PIP1;3 and five other AQPs increased in response to drought in the leaves of P. simonii × balsamifera but not in P. balsamifera, suggesting a possible role of these AQPs in water redistribution in the leaf tissues.PIP2;5 was upregulated in P. balsamifera, but not in P. simonii × balsamifera, suggesting that this AQP supports the transpiration-driven water flow. Functional characterization of five drought-responsive plasma membrane intrinsicproteins(PIPs) demonstratedthat threePIP2 AQPs (PIP2;2, PIP2;5, PIP2;7) functioned as water transporters in Xenopus laevis oocytes, while the two PIP1 AQPs (PIP1;2 and PIP1;3) did not, consistent with the notion that they may be functional only as heterotetramers. Introduction (drought-tolerant) plants that rapidly reduce leaf water Water availability is among the most limiting factors for potential (ψ leafin response to drought, isohydric (drought-avoidant) plants exercise tight control over plant growth (Bogeat-Triboulot et al. 2007, Touchette water loss mainly through stomatal movements (Tardieu et al. 2007). Therefore, considerable research efforts and Simonneau 1998, McDowell et al. 2008). Since have focused on understanding drought resistance isohydric plants rely on stomatal conductance as the mechanisms in plants. As opposed to anisohydric main drought resistance mechanism, the water content Abbreviations – AQP, aquaporin; CNI, close neighbor interchange; LPI, leaf plastochron index; MIPs, major intrinsic proteins; MP, maximum parsimony; NJ, neighbor joining; NIP, nodulin-like intrinsic protein; PIP, plasma membrane intrinsic protein; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RH, relative humidity; SIP, small basic intrinsic protein; SWC, soil water content; TDR, time domain reflectometry; TIP, tonoplast intrinsic protein; XIP, X-intrinsic protein. Physiol. Plant. 140, 2010 321 of these plants fluctuates less and they tend to be withholdingwater,although AtPIP1;4 and AtPIP2;5 were more susceptible to xylem cavitation compared with upregulated (Alexandersson et al. 2005). Expression of anisohydric plants (McDowell et al. 2008). Although AQPs in roots and aerial parts of A. thaliana plants hybrid poplars are generally considered to be relatively responded differently to mannitol-inducedosmotic stress isohydric (Tardieu and Simonneau 1998), they widely (Jang et al. 2004) or gradual drought stress (Alexanders- vary in their stomatal sensitivity and susceptibility to son et al. 2005). These differential responses ofPsto xylem cavitation (Awad et al. 2010). drought stress are likely because of distinct functions that Hydraulic conductivity of plant tissues plays an AQPs may have in different tissues and the functional important role in drought resistance (Ogasa et al. 2010). differences between various tissues in plants exposed Watertransportinvolvesacombinationofapoplasticand to stress. In some cases, there can be discrepancies in cell-to-cell (symplastic and transmembrane) pathways. AQP expression data reported in different studies (e.g. Transmembrane movement of water is facilitated by Alexandersson et al. 2005, Jang et al. 2004). Variation aquaporins (AQPs) (Maurel et al. 2008), a functional in simulating drought stress under laboratory conditions classofchannelproteinsthatbelongtotheancientfamily and the fact that plants may have diverse resistance strategies under different drought levels (Siemens and of the major intrinsic proteins (MIPs) (Chaumont et al. 2001, Johanson et al. 2001, Maurel et al. 2008, Siefritz Zwiazek 2003) can confound the interpretation of AQP et al. 2002). This gene family has been highly conserved expression patterns reported by different studies. Inthepresentstudy,wecompareda Populus simonii × duringevolution,probablybecauseofthestrictstructural requirements of water channels (Shapiguzov 2004). Five Populus balsamifera hybrid poplar clone, which, similar subfamilies of MIPs have been identified in angiosperms to otherhybridpoplars,has beencharacterizedas having high stomatal sensitivity (Stettler et al. 1996, Tardieu and [plasma membrane intrinsic protein (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-like intrinsic proteins Simonneau 1998), and a P. balsamifera clone with stom- (NIPs), small basic intrinsic proteins (SIPs) and X-intrinsicta that exhibits less sensitivity to drought (Pearce et al. 2006). We hypothesized that the expression of leaf AQPs proteins (XIPs); Johanson et al. 2001, Chaumont et al. 2001, Sakurai et al. 2005, Danielson and Johanson would respond differently in these two poplar clones 2008]. The latter subfamily has been reported recently when the plants are exposed to drought conditions in accordance with their stomatal responses. Therefore, in in the moss Physcomitrella patens, Populus trichocarpa and in a wide variety of dicots, but not in monocots the present study, we subjected the plants of these two or Arabidopsis thaliana (Danielson and Johanson clones to mild drought, severe drought and drought recovery treatments and we compared their physiolog- 2008, Gupta and Sankararamakrishnan 2009). AQPs participate in water dynamics as facilitators of water and ical responses and AQP expression with well-watered small solutes movement across cell membranes (Maurel plants. et al. 2009). Their differential expression between plant tissues and in response to abiotic stresses suggests that Materials and methods they play roles in maintaining water balance in plants Plant material (Luu and Maurel 2005, Parent et al. 2009, Suga et al. 2001, Tyerman et al. 2002). However, the role of AQPs P. balsamifera (L.) (clone AP1004, gender unknown) and in leaf water transport under drought conditions remains P. simonii (Carr.) × P. balsamifera (L.) (clone P38P38, unclear (Heinen et al. 2009). As the expression of plant female) shoot cuttings were provided from single trees AQPs is affected by drought (Aharon et al. 2003), it by Alberta-Pacific Forest Industries (Boyle AB, Canada). could be expected that leaf AQPs that are involved in Ten-centimeter stem segments from 1-year-old trees transpiration-driven water transport are strongly linked were re-hydrated for two days before planting in to stomatal movements. 45 × 340 cm 3 styroblocks (Stuewe and Sons, Tangent, AQP expression in plants subjected to water deficit OR) containing peat moss, vermiculite and Turface cal- stress varies depending on the species, developmen- cined clay (Profile Products, Buffalo Grove, IL) (2:1:1/2) tal stage, growth conditions and severity of drought with 2.75 g l−1 of Nutricote Total controlled-release (Alexandersson et al. 2005, Cocozza et al. 2010, Oono fertilizer (13–13–13; American Horticultural Supplies, et al. 2003, Seki et al. 2001, 2002). For example, in San Marcos, CA). Rooted cuttings were replanted in Nicotiana tabacum, drought stress reduced expression 4-l pots with the same soil composition and grown of NtPIP1;1 and NtPIP2;1 while increasing expres- in the◦greenhouse under semi-controlled conditions sion of NtAQP1 (Mahdieh et al. 2008). In A. thaliana [22/20 C day/night, 18/6 h light/dark, 60% relative leaves, expression of several PIP genes was downregu- humidity (RH)], with watering six times per week until −1 lated in response to gradual drought stress induced by runoff and fertilization (2 g l 15–30–15) once per 322 Physiol. Plant. 140, 2010 week. Plants were 130–160-cm tall at the outset of the (PMS Instruments, Corvallis, OR) was used to measure drought experiments. leaf water potential (ψleaf for LPI 7 as described by A preliminary experiment was used to empirically Wan and Zwiazek (1999). For both the gas exchange define mild drought stress, severe drought stress, and and pressure chamber measurements, one block of stress recovery treatments. Soil apparent permittivity four replicates per treatment was analyzed per day, (Ka) was measured using a time domain reflectome- for a total of n = 8. Within each block, the order ter (TDR; Tektronix 1502B Cable TDR Cable Tester; in which the plants were measured was randomized. Tektronix, Scarborough, ON, Canada), and volumet- For the molecular experiment, complete leaves (with ric water content (θ) or soil water content (SWC) was petioles removed) from LPI 7 to LPI 10 were harvested determined based on the equation for organic soils from each of the six plants and immediately frozen in ◦ (Robinson et al. 2003, Toop et al. 1980). Measurements liquid nitrogen. Samples were stored at −80 C until of stomatal conductance (g s) were carried out with an processed. Li-1600 steady-state porometer (LI-COR, Lincoln, NE). SWC of 20–25% was defined as mild stress, based on Sequence analysis the observation of changes in g wsen compared with well-watered plants. SWC lower than 20% was desig- P. trichocarpa (Torr. & Gray) AQP gene models were nated as severe stress because, at this point, plants were identified in version 2.0 of the assembled, annotated wiltedandtheyshowedlow g .Thrsedaysofre-watering genome ( using following the point of severe stress was designated as P. trichocarpa AQP sequences reported by Gupta and recovery, because at this point g s was comparable to Sankararamakrishnan (2009) as BLAST queries (Altschul that in well-watered plants. et al.1997)(seeAppendixS1).AQP-deducedaminoacid Two duplicate drought experiments were conducted sequences from P. trichocarpa, A. thaliana (L.) Heynh. in this study. One was used for physiological measure- (Johanson et al. 2001; and repre- ments (n = 8) (referred to as the physiological experi- sentative XIPs fromPh. patens (Danielson and Johanson ment) and one for the gene expression analyses (n = 6) 2008) were aligned using CLUSTALW in MEGA v4 (Tamura (referred to as the molecular experiment). In each exper- et al. 2007). Phylogenetic trees were constructed using iment, trees for each of the two poplar clones were maximum parsimony (MP) in MEGA v4. A bootstrap randomly assigned to the different drought treatments, consensus tree of 2000 replicates was obtained, using i.e. mild drought, severe drought, recovery from severe close neighbor interchange (CNI) with search level of drought and well-watered plants (controls). Because of 3, a random addition of 10 replicates, and a complete the diurnal cycle of AQP activity, physiologicalmeasure- deletion for gaps and missing data. No out group was ments (physiological experiment) and tissue harvesting included in the analysis. (molecular experiment) were performed between 08:00 and 11:00 h. In the case of the physiological experi- Quantitative reverse transcription polymerase ment, the plants were measured within two days. For chain reaction both experiments, drought treatments were initiated on different days so that mild drought, severe drought, and Total RNA was extracted using the hexadecyltrimethy- drought recovery treatments ended on the same day. lammonium bromide (CTAB) extraction protocol of For logistical reasons, the physiological experiment was Chang et al. (1993). Three micrograms of total RNA were divided into two blocks of four independent replicates treated with DNase I (New England Biolabs, Ipswich, per treatment per block; the treatments for the two MA) and used as template for first strand cDNA synthesis blocks were initiated one day apart so that the plants usingoligo(dT)23VNandMoloneymurineleukemiavirus from block 1 could be analyzed on one day, and the reverse transcriptase, following manufacturer’s instruc- plants from block 2 could be analyzed the following tions (New England Biolabs). day. Gene-specific qRT-PCR primers were designed in the ▯ Leaves for analysis were selected according to the leaf 3untranslated (UTR)-region using Primer Express v3 plastochron index (LPI; Larson and Isebrands 1971). For (Applied Biosystems, Foster City, CA) (Appendix S2). the physiological experiment, single measurements of Populus AQP genes were selected based on phylo- net photosynthesis (Pn)andg swere measured for LPI 7 genetic proximity to genes associated with drought on eight plants per treatment with an LI-6400 portable responses in other species, or by EST abundance pat- infrared gas analyzer (LI-COR) with an incorporated terns, suggesting relatively high expression in leaves or light source. All measurements were taken between in abiotic stress responses (Sterky et al. 2004). Several 08:00 and 11:00 h. A Scholander pressure chamber potential reference genes were tested for stable levels of Physiol. Plant. 140, 2010 323 transcript abundance across samples to be compared. threshold) for analysis, and dissociation curves were A member of the EF1α gene family was chosen as the verified for each of the genes. reference gene, as transcript abundance corresponding to this gene did not show significant differences across Osmotic water permeability assay −▯Ct treatments when expressed as 2 (P = 0.514 for the Full-length PtdPIP1;2, PtdPIP1;3, PtdPIP2;2, PtdPIP2;5 drought experiment, and P = 0.257 for the tissue com- and PtdPIP2;7 cDNAs were amplified using the Expand parison experiment; Appendix S3). High Fidelity PCR System (Roche, Indianapolis, IN), Transcript abundance was quantified using standard using gene-specific primers incorporating BglII sites on curves for both target and reference genes. Standard both ends (Appendix S2). Full-length cDNAs were sub- curves were constructed from amplicons of full-length cloned into the BglII site of the pAWPLA (pXLII) vector or near full-lengthP. trichocarpa × deltoides cDNAs for [dephosphorylated using calf intestinal alkaline phos- conducting a tissue survey analysis (data not shown) phatase (Invitrogen)],carryingthe 5 -and3 -UTR regions and for the drought experiment described in this study. of the beta-globin gene from Xenopus laevis. Plasmids P. trichocarpa × deltoides cDNAs were either obtained containing the full-length poplar AQPs in the correct from Universite ´ Laval ( or, in the orientation were determined by restriction mapping and case of PtdSIP1;2 and PtdEF1α, cloned via RT-PCR sequencing. Plasmids were linearized downstream of with primers described in Appendix S2 (Sambrook and the 3 -UTR beta-globin sequence using FastDigest SacI Russell 2001). Plasmids were purified using GeneJET (Fermentas) for PtdPIP1;2 and PtdPIP1;3, NgoMIV (New Plasmid Miniprep Kit (Fermentas, Burlington, ON, England Biolabs) for PtdPIP2;7 and Bpu10I (New Eng- Canada) according to the manufacturer’s protocol. Each land Biolabs) for PtdPIP2;2 and PtdPIP2;5. Capped RNA plasmid (30 ng μl −1) was used as template in a PCR (cRNA) was synthesized in vitro by T3 RNA polymerase reaction using universal pri◦ers. PCR conditions were (mMESSAGE mMACHINE T3 kit, Ambion, Austin, TX) as follows: 1 cycle at 94 C for 5 min, followed by utilizing the T3 promoter of the vector. DNA was 40 cycles at 94 ◦C for 40 s, 50 C for 40 s, and 72 C ◦ removed using TURBO DNase, and the cRNA was pre- for 1.5 min, and finishing with an extension step at cipitated for 2 h at −20 C using lithium chloride. cRNA 72 ◦C for 5 min. Amplicons were purified using the quality and quantity were determined using the Nan- QIAquick PCR purification kit (Qiagen, Mississauga, odrop and the 2100 Bioanalyser (Agilent, Santa Clara, ON, Canada), and diluted 1:10 with autoclaved CA). cRNA was aliquotted and stored at −80 C until use. RNase-free water. Concentration was determined using X. laevis oocytes were prepared as previously a Nanodrop ND-1000 spectrophotometer (Thermo described (Cao et al. 1992, Daniels et al. 1996). Fifty Scientific, Wilmington, DE). Target and reference genes −1 nanoliters AQP cRNA (1 mg ml ) or nuclease-free were diluted individually to a final concentration of water were injected to the oocytes using a 10-μl microin- 4 × 10 9 molecules of DNA. Afterward, a pooled serial jector (Drummond Scientific, Broomall, PA). Injected dilution series was made that included all target 7 6 4 oocytes were randomly assigned to two separated six- genes plus EF1α, comprising 4 × 10 ,4 × 10 ,4 × 10 , well cell culture plates, and incubated as described by 4 × 10 3 and 4 × 10 copies of DNA molecules for each Cao et al. (1992). One oocyte was added to one well of gene. a two-well thick-slide filled with regular Barth’s solution Real-time PCR was performed on a 7500 Fast Real- (200 mosmol), and an initial image of the cell was taken. Time PCR system (Applied Biosystems). Three biological Then an oocyte was quickly transferred to the other replicates, each with three technical replicates, were well of the slide containing hypotonic Barth’s solution assayed for each sample. The reference gene was (Barth’s solution diluted five times with distilled water included on each plate. cDNA from a single, pooled for a final concentration of 40 mosmol). Changes in cell RT reaction representing one sample from the control volume were tracked via microscopy images taken at 5-s (well-watered) plants from the drought experiment was intervals for 5 min. Images were analyzed using I MAGE J used as a calibrator to enable plate-to-plate comparison. (version 1.38X, Abramoff et al. 2004). The surface area PCR was carried out in a volume of 10 μl including a from the oocyte picture was obtained automatically with final concentration of 50 ng cDNA, 1× master mix con- IMAGEJ after a known scale was set. Oocyte surface area taining 0.2 m M dNTPs, 0.3 U Platinum Taq Polymerase and volume were calculated based on the sphere surface (Invitrogen), 0.25× SYBR Green and 0.1× ROX◦ PCR area and volume geometric formulas correspondingly. conditions were as follows: 1 cycle at 95 Cofr2mi, The relative volume (V/V ),othe initial transmembrane 40 cycles at 95 ◦C for 15 s, 60 C for 1 min, and a disso- volume flux (J ) and the osmotic water permeability ◦ ◦ v ciation stage including 2 cycles of 95 C for 15 s, 60 C coefficient (P f were calculated based on Zhang and for 1 min. Samples were subjected to auto Ct (cycle Verkman (1991). 324 Physiol. Plant. 140, 2010 Statistical analysis Effects of PtdPIPs on the osmotic water permeability coefficient for the oocytes exposed to hypotonic solu- Data were analyzed using SAS version 9.1 (SAS Institute, tion were evaluated by a nested analysis of variance. The Cary, NC). Effects of drought on g ,P s n, height and alternativehypothesisofdeterminingthestatisticaldiffer- ψ leafwere evaluated by a complete randomized block ences among different oocytes exposed to a hypotonic design analysis of variance. For every treatment, at solution that were injected individually with different least six replicates were used for each poplar clone. PtdPIPs. Pairwise comparisons were tested using the The experimental design included two blocks, four least-square means differences statement. P-values were treatments, and four replicates per treatment in each adjusted with Tukey–Kramer to a P-value of 0.05. block. The alternative hypothesis of determining the statistical differences among treatments for each of the physiological measurements in each of the poplars Results was evaluated. Preplanned comparisons were tested Effects of drought treatments on P ,g , and using the least-square means differences statement n s leaf with Tukey–Kramer P-values adjustment, and/or with height growth contrast statement, adjusting the alpha value to 0.03. It took two days for P. balsamifera to reduce SWC Effects of drought on AQP transcript abundance were to 20–25% (mild drought). In contrast, it took six evaluated by a randomized factorial analysis of variance. days for P. simonii × balsamifera to reach a similar The alternative hypothesis of determining the statistical level. InP. simonii × balsamifera, this mild stress treat- differences among treatments for each of the 11 poplar ment resulted in a reduction of P n by about 60% and AQP genes in each of the poplars was evaluated. Pre- g by about 33% compared with well-watered plants s planned comparisons were tested using the least-square (Fig. 1A,B). In contrast, no significant differences were means differences statement. P-values were adjusted observed in P n and g setween control and mild drought with Tukey–Kramer or LSD adjusted to a P-value of 0.03. stress treatment in P. balsamifera. In plants treated with Fig. 1. Drought responses in two contrasting Populus clones, Populus balsamifera and Populus simonii × P. balsamifera. (An Photosynthesis (P ), (B) stomatal conductancs (gssociated with the percentage of SWC, (C) plant height and (D) leaf watleaf Ten-week-old plants were well watered (control) or subjected to mild water stress, severe water stress, or stress recovery following severe water stress. Significant differences among treatments indicated by letters above each bar (P ≤ 0.05, Tukey-Kramer adjustment). Means ±(n = 6 minimum). Physiol. Plant. 140, 2010 325 severe drought (SWC lower than 20%), both P and g n s wilted in response to severe drought while only a few were reduced but still higher in P. balsamifera compared leaves in some P. simonii × balsamifera plants showed with P. simonii × balsamifera (Fig. 1A,B). Following signs of wilting. Similarly, young leaves of P. balsamifera stress recovery, P n and g is both poplars returned displayed a relatively high extent of necrosis and leaf to the levels measured in well-watered control plants abscission which occurred in only a few P. simonii × (Fig. 1A,B). balsamifera plants (data not shown). Drought stress treatments did not affect heights of P. balsamifera plants. However, P. simonii × balsam- ifera plants were shorter following stress recovery Analysis of the P. trichocarpa AQP family treatment compared with the control and mild stress Fifty-five full-length AQP gene models were identified treatments (Fig. 1C). The ▯ leafexhibited trends similar to in version 2.0 of the P. trichocarpa genome (Appendix Pn and g sn both clones (Fig. 1D). Mild drought did not S4), consistent with the findings of Gupta and Sankarara- significantly affect ▯ leafn P. balsamifera, but decreased makrishnan (2009), who reported on an earlier version ▯ in P. simonii × balsamifera (Fig. 1D). Both poplars leaf of the P. trichocarpa genome. In agreement with Gupta showed a significant reduction of ▯ leafwhen exposed and Sankararamakrishnan (2009), phylogenetic analy- to severe drought and returned to near control levels sis (Fig. 2) demonstrated that the P. trichocarpa AQPs in re-watered plants (Fig. 1D). Leaves of P. balsamifera fall into five subfamilies: the PIPs, TIPs, NIPs, SIPs and Fig. 2. Dendrogram obtained using MP illustrating relationships between members of the AQP family in Populus trichocarpa. P. trichocarpa AQPs are denoted with black diamonds. The AQP family from Arabidopsis thaliana (AtAQPs) (Johanson et al. 2001) and two XIPs previously reported in the moss Physcomitrella patens (PpAQPs) (Danielson and Johanson 2008) are included for comparison. Arrows indicate aquaporins selected for gene expression analysis. 326 Physiol. Plant. 140, 2010 the recently described XIPs (Danielson and Johanson Functional analysis of five PIPs 2008, Gupta and Sankararamakrishnan 2009). Further subgroups of proteins could be identified in the TIP Five PIPs cloned from P. trichocarpa × deltoides were tested for their ability to transport water using alaevis (TIP1, TIP2, TIP3, TIP4, TIP5) and PIP (PIP1 and PIP2) oocyte swelling assay (Fig. 4). Relative volumes of subfamilies. For consistency, the same gene names are used in this paper as those proposed by Gupta and oocytes injected with PtdPIP1;2 and PtdPIP1;3 were similar to those injected with water (controls). In con- Sankararamakrishnan (2009). trast, oocytes injected with one of threedPIP2s showed All 55 AQPs were assigned to chromosomes in v2.0 of the P. trichocarpa genome, compared to only 46 in higher relativevolumes comparedto the controloocytes. The greatest changes in oocyte volumes were observed earlier versions (Gupta and Sankararamakrishnan 2009). for PtdPIP2;5 followed by PtdPIP2;2 and PtdPIP2;7. While 15 of 19 linkage groups (chromosomes) exhib- ited 5 AQP genes or less, linkage group 9 contained All oocytes injected with any of the PIP2s ruptured under hypotonic conditions during the 5-min assay. 9 AQPs, including 5 of the 6 PtXIPs. Several closely The osmotic water permeability coefficient (P )w s related gene pairs could be identified based on dendro- f calculated based on its relationship with the initial trans- gram location and locations within homologous genome membrane volume flux (J ).vP vflues for oocytes injected blocks described by Tuskan et al. (2006), which were based on the v1.1 genome release: PtPIP1;4/PtPIP1;5, with cRNAs corresponding to PtdPIP2;5, PtdPIP2;7 and PtdPIP2;2 were respectively 9-, 3.2- and 5.2-fold higher PtPIP1;3/PtPIP1;2, PtPIP2;4/PtPIP2;3, PtPIP2;1/PtPIP2;2, than those of control oocytes. For the oocytes injected PtTIP1;3/PtTIP1;4, PtTIP1;1/PtTIP1;2, PtTIP2;1/PtTIP2;2, PtNIP3;3/PtNIP3;4, PtNIP3;1/PtNIP3;2, PtSIP1;1/ with the cRNA corresponding to PtdPIP1;3 and Ptd- PIP1;2,theP fvalues were the same or lower than for the PtSIP1;2, PtSIP1;3/PtSIP1;4 and PtSIP2;2/PtSIP2;1. control (the ratios between the Pf values of the putative PtdPIP1 and the control were 1 and 0.64, respectively; Expression profiling of Populus AQPs Fig. 4). A subset of AQPs was selected for a qRT-PCR survey of transcript abundance in leaves of P. balsamifera and Discussion P. simonii × balsamifera subjectedto droughtconditions P. balsamifera and P. simonii × balsamifera exhibit identical to the experiments described above. As PIPs and TIPs have been associated with drought responses contrasting strategies for coping with drought in A. thaliana (Alexandersson et al. 2005, Jang et al. P. balsamifera and its descendant P. simonii × bal- 2004), eight PIPs( PIP1;2, PIP1;3, PIP2;1, PIP2;2, PIP2;3, samifera hybrid are phreatophytic poplars (Amlin and PIP2;4, PIP2;5 and PIP2;7)a dwto TIPs( TIP1;6 and Rood 2003). We have shown that these two poplar TIP2;1)were chosen for gene expression analysis (Fig. 3). clones have different strategies to resist drought stress. One SIP (SIP1;2) was also selected for the analysis. The responses of P. simonii × balsamifera to drought PIP1;2, PIP1;3, PIP2;1, PIP2;2, PIP2;7, TIP1;6 and TIP2;1 can be referred to as drought avoidant, characterized showed statistically significant responses to drought by rapid stomatal closure. This type of drought strat- in P. simonii × balsamifera, but not in P. balsa
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