Planta (2014) 240:553–564 DOI 10.1007/s00425-014-2106-2
Original Article
Hydraulic adjustments in aspen (Populus tremuloides) seedlings following defoliation involve root and leaf aquaporins Juan Liu · María A. Equiza · Alfonso Navarro‑Rodenas · Seong H. Lee · Janusz J. Zwiazek
Received: 7 May 2014 / Accepted: 5 June 2014 / Published online: 24 June 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Main conclusion Changes in root and leaf hydraulic properties and stimulation of transpiration rates that were initially triggered by defoliation were accompa‑ nied by corresponding changes in leaf and root aqua‑ porin expression. Aspen (Populus tremuloides) seedlings were subjected to defoliation treatments by removing 50, 75 % or all of the leaves. Root hydraulic conductivity (Lpr) was sharply reduced in plants defoliated for 1 day and 1 week. The decrease in Lpr could not be prevented by stem girdling and it was accompanied in one-day-defoliated plants by a large decrease in the root expression of PIP1,2 aquaporin and an over twofold decrease in hydraulic conductivity of root cortical cells (Lpc). Contrary to Lpr and Lpc, 50 and 75 % defoliation treatments profoundly increased leaf lamina conductance (Klam) after 1 day and this increase was similar in magnitude for both defoliation treatments. Transpiration rates (E) rapidly declined after the removal of 75 % of leaves. However, E increased by over twofold in defoliated plants after 1 day and the increases in E and Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2106-2) contains supplementary material, which is available to authorized users. J. Liu · M. A. Equiza · A. Navarro‑Rodenas · S. H. Lee · J. J. Zwiazek (*) Department of Renewable Resources, University of Alberta, 442 Earth Sciences Bldg, Edmonton, AB T6G 2E3, Canada e-mail: [email protected] Present Address: J. Liu Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming 650224, Yunnan, China
Klam were accompanied by five- and tenfold increases in the leaf expression of PIP2;4 in 50 and 75 % defoliation treatments, respectively. Defoliation treatments also stimulated net photosynthesis after 1 day and 3 weeks, although the increase was not as high as E. Leaf water potentials remained relatively stable following defoliation with the exception of a small decrease 1 day after defoliation which suggests that root water transport did not initially keep pace with the increased transpirational water loss. The results demonstrate the importance of root and leaf hydraulic properties in plant responses to defoliation and point to the involvement of PIP aquaporins in the early events following the loss of leaves. Keywords Gas exchange · Hydraulic conductivity · Stem girdling · Water potential · Water transport Abbreviations E Transpiration Klam Leaf lamina hydraulic conductance KLEAF Absolute leaf hydraulic conductance Kr Root hydraulic conductance Lpc Cell hydraulic conductivity Lpr Root hydraulic conductivity PIP Plasma membrane intrinsic protein Pn Net photosynthesis Introduction Defoliation is caused by a variety of biotic and abiotic factors and is a major traumatic event which threatens plant’s survival. Large-scale defoliation events in trees are common, especially during periodical outbreaks of defoliating insects such as tent caterpillars (Cooke et al. 2011). In
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trembling aspen (Populus tremuloides), epidemic outbreaks of forest tent caterpillar (Malacosoma disstria Hbn.) occur on an average at about 10-year intervals (Churchill et al. 1964; Cooke et al. 2011) and can lead to complete defoliation of entire tree stands. Plants which are commonly exposed to defoliation have developed various adaptations to prevent the loss of leaves and to cope with its consequences once defoliation takes place. Fully defoliated aspen trees produce new leaves by precocious break of the lateral buds; however, if defoliation is only partial, lateral buds may remain inhibited until the following growth season (Donaldson and Lindroth 2008). The loss of photosynthetic area and the resulting depletion of energy reserves are among the main concerns in defoliated trees (Churchill et al. 1964; Landhäusser and Lieffers 2012). In trembling aspen trees, the recovery of root energy reserves may take up to 2 years following partial defoliation (Landhäusser and Lieffers 2012) and affect long-term hydraulic performance of trees (Anderegg and Callaway 2012). On the other hand, compensatory responses which may offset some of the loss of foliage have been reported for plants in which partial defoliation stimulated photosynthetic rates in the remaining leaves (Thomson et al. 2003; Turnbull et al. 2007; Quentin et al. 2012). The transient increase in photosynthesis may be accompanied by an increase in transpiration rates or stomatal conductance (Gálvez and Tyreee 2009; Quentin et al. 2012). However, the signaling pathways leading to these responses remain unclear. The loss of foliage alters the water and hormonal balance in trees. The water demand of a partly defoliated tree will depend on the degree of the photosynthetic compensation response and the involvement of stomatal conductance in this response. In fully defoliated trees, the demand for water will be greatly reduced until the expansion of leaf primordia commences in lateral buds and new leaves emerge. Partial defoliation lowers the tree’s transpirational area and causes a rapid increase in the root-to-shoot mass ratio. Therefore, adjustments in the energy-costly maintenance processes of root hydraulic conductance could be desirable to reflect changes in the transpirational demand. Plant water transport is driven by fast, transpirationdriven and slow, osmotically driven forces (Steudle and Peterson 1998). In both cases, water moves along the leastresistance pathways and there is extensive evidence which points to aquaporins as the principal structures responsible for regulating water flow resistance in roots and leaves (Javot and Maurel 2002; Heinen et al. 2009; Shatil-Cohen et al. 2011). The synthesis and opening of aquaporins are energy-demanding (Kamaluddin and Zwiazek 2001). Therefore, adjustments of root hydraulic conductivity to reflect changes in the total transpirational demand may help conserve limited energy resources which are needed
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in defoliated plants for defense mechanisms, wound repair, and re-growth of new leaves. The role of leaf aquaporins in regulating water transport processes is still the subject of an intensive debate (Heinen et al. 2009; Shatil-Cohen et al. 2011). If leaf aquaporins play a significant role in regulating leaf water transport in aspen, the effect of defoliation on leaf water flow properties offers an excellent opportunity to study the role of leaf aquaporins since the removal of leaves changes an overall hydraulic balance of the plant. It has been estimated that the resistance of leaves to water transport accounts for as much as 80 % of the total plant hydraulic resistance (Sack and Holbrook 2006). Leaf hydraulic conductance has been often correlated with photosynthetic capacity, maximum stomatal conductance and stomatal pore area per leaf area (Aasamaa and Sõber 2001; Sack et al. 2003; Brodribb et al. 2005). If defoliation triggers an increase in photosynthesis and transpiration of the remaining leaves, it is likely that the increased water transport to the leaves is facilitated by the increased hydraulic conductance, possibly mediated by aquaporins. Increased root (Lee et al. 2008; Laur and Hacke 2013) and leaf (Lee et al. 2009; Lopez et al. 2013) hydraulic conductance has been often reported to accompany increased transpirational demand and may be accompanied by the increased expression of aquaporins (Laur and Hacke 2013; Lopez et al. 2013). In the present study, we examined the effects of partial and complete defoliation on gas exchange, water transport and aquaporin expression in trembling aspen (Populus tremuloides) seedlings. The study was carried out to examine the adjustments in water relations of trembling aspen seedlings following defoliation and the processes involved in these responses. We examined the hypothesis that defoliation would (1) decrease root hydraulic conductivity to reflect reduced transpirational demand of the partly defoliated plants and (2) increase leaf hydraulic conductance to keep up with the increased water demand of the remaining leaves (compensation response). We also hypothesized that these changes would be accompanied by changes in the aquaporin expression in leaves and roots, which would impact cell-to-cell water transport. To study a possible role of chemical signals originating in shoots on plant hydraulic responses to defoliation, we also girdled stems of plants prior to defoliation to block the basipetal transport through the phloem and vascular cambium.
Materials and methods Experiment 1: defoliation For the defoliation experiment, trembling aspen (Populus tremuloides Michx.) seeds, locally collected in Edmonton,
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AB, Canada, were germinated and seedlings grown in 2.8-l pots filled with the Metromix (Sun Gro Horticulture Canada, Vancouver, BC, Canada) sand mixture (2:1, v/v) and grown in the greenhouse for 5 months before the start of defoliation treatments under the following conditions: 22/18 °C (day/night) temperature, relative humidity of 55–75 % relative humidity. Supplemental lighting in the greenhouse provided a photosynthetic flux density of 400 μmol m−2 s−1 at the top of the plants and extended the photoperiod to 16 h. Seedlings were watered daily and fertilized weekly with a 1 % (w/v) solution of commercial 20:20:20 (N:P:K) fertilizer. Defoliation treatments were carried out by removing every second leaf (50 % defoliation), or every three out of four leaves (75 % defoliation) from the entire length of the stem. Intact seedlings with no removed leaves served as defoliation control (0 % defoliation). The measurements were carried out 1 day, 1 and 3 weeks following defoliation in leaves and roots of six plants per treatment. Transpiration rates (E) and root hydraulic conductance (Kr) were also measured immediately (within 10 min) following defoliation. Experiment 2: defoliation and girdling For the defoliation and girdling experiment, one-year-old container-grown trembling aspen seedlings were obtained from Coast to Coast Reforestation Inc., Edmonton, AB, Canada, and grown in the greenhouse under the conditions described for Experiment 1. The seedlings were potted in 2.8-l pots filled with a commercial potting mix composed of peat moss (55–65 %), perlite, dolomitic limestone and gypsum. (Sunshine LA4 mix, Sun Gro Horticulture Canada Ltd) and fertilized with a controlled-release fertilizer (Osmocote, 13:13:13, N:P:K) applied at 20 g per pot. The plants started to flush 1 week after being transferred to the greenhouse and they were grown for 2 months before the commencement of defoliation and girdling treatments. The seedlings were subjected to the following treatments: no defoliation + no girdling (control), no defoliation + girdling, 100 % defoliation + no girdling, and 100 % defoliation + girdling. The girdling treatment involved the removal of a 1-cm ring of bark (periderm and phloem) at 2 cm from the base of the main stem about 1 h before defoliation. Bark was carefully removed with a razor blade while keeping the stem wet using a water sprayer. The girdle section was immediately wrapped with ParafilmTM and aluminum foil to prevent desiccation. For all measurements, six plants per treatment were taken for measurements after 1 day (approximately 24 h), 10 days (when buds started to flush in completely defoliated plants), and 3 weeks following defoliation. To minimize possible diurnal effects, defoliation treatments for Experiment 1 and 2 were carried out 2–6 h after
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the onset of photoperiod and the plant measurements were carried out interchangeably for the different treatments. Root hydraulic conductance (Kr) and conductivity (Lpr) measurements Root hydraulic conductance (Kr) was measured for excised root systems with the high-pressure flow meter (HPFM; Dynamax Inc., Houston, House, TX, USA) as previously described (Voicu et al. 2009; Calvo-Polanco et al. 2012). Each root system was subjected to the increasing pressure from 0 to 0.4 MPa and Kr was calculated from the slope of the linear portion of the graph produced by the high-pressure flow meter software and expressed in kg MPa−1s−1. Roots were immersed in water in a graduated cylinder and the volume of each root system was determined from the volume of displaced water. To obtain Lpr, Kr value was divided by root volume and expressed in kg MPa−1s−1cm−3 (Kamaluddin and Zwiazek 2002). There were six root systems per treatment taken for Kr measurements (n = 6) in both experiments. Cell hydraulic conductivity (Lpc) measurements Cell pressure probe measurements were carried out as previously described (Lee et al. 2012) in roots from seven control (non-defoliated) aspen plants and seven plants subjected to 75 % defoliation treatment for 1 day (approximately 24 h). A single cortical cell was punctured at a distance of about 20 mm from the root tip with a silicon oil-filled microcapillary (5- to 6-μm tip diameter). The position of cortical cell was estimated from the depth of the insertion of the microcapillary tip inside the root. The measurements of half-times of water exchange (T1/2) were carried out for up to 20 min. The hydraulic conductivity of root cortical cells (Lpc) was calculated using the following equation (Steudle 1993):
Lpc = V × ln (2)/{A × T1/2 × (ε + π i )},
(1)
where ε is cell elastic modulus, V is the cell volume, A is the cell surface area πi is osmotic cell pressure. Cell dimensions for V and A were examined microscopically in the root cortical cells and πi was estimated from the steady-state P. Cell elastic modulus (ε) was calculated from changes in cell volumes (ΔV) produced by the cell pressure probe and corresponding changes in cell turgor (ΔP) (Steudle 1993), i.e.: (2)
ε = V × ∆P/∆V Leaf water potentials and gas exchange measurements
A Scholander pressure chamber was used to measure water potentials of fully expanded leaves (Wan and Zwiazek 1999) 1 day, 1 and 3 weeks following defoliation
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(Experiment 1). There were six leaves from six plants per treatment (non-defoliated control, 50 and 75 % defoliation) (n = 6). Net photosynthesis and transpiration rates were measured for the defoliation treatments (Experiment 1) with a portable open-flow photosynthesis system (LI-6400, LI-COR Inc, Lincoln, NE, USA) equipped with a LI6400B red/blue LED light source. Light intensity was set at 400 µmol m−2 s−1 and reference CO2 was set at 400 µmol mol−1 using the 6400-01 CO2 mixer. Air temperature was 21 ± 0.15 °C and vapor pressure deficit (VPD) 1.3 ± 0.3 kPa. When readings were stable, data were logged every 30 s during a 5-min period. Fully elongated upper leaves were taken for the measurements in six plants per treatment (n = 6) immediately following defoliation as well as 1 day, 1 and 3 weeks following defoliation. Leaf lamina hydraulic conductance (Klam) measurements For absolute leaf hydraulic conductance (KLEAF) measurements, a fully elongated leaf in Experiment 1 was excised with a razor blade near the stem, at approximately the midpoint of the shoot height, and immediately immersed in a glass container filled with distilled water. The leaf was attached to the HPFM and water was forced into the leaf at constant pressure between 0.3–0.45 MPa depending on the leaf size (Voicu et al. 2009). A computer recorded the water flow rate (Q, kg s−1) and the applied pressure (P, MPa). The values were determined every 2 s and saved as means every 8 s (Voicu et al. 2009) to compute KLEAF, (kg s−1MPa−1) as Q/P after Q stabilized (1–2 min). After the measurement of KLEAF, the leaf petiole was excised with a razor blade from the leaf lamina and the hydraulic conductance of the petiole (KPET, kg s−1MPa−1) was measured in the same manner as for the whole leaf. Petiole hydraulic conductivity (Kpet) was calculated from KPET values expressed on the petiole length basis (Sack et al. 2002; Voicu et al. 2009). The absolute hydraulic conductance of the leaf lamina (KLAM, kg s−1MPa−1) was calculated as KLAM = 1/[(1/KLEAF)−(1/KPET)] (Sack et al. 2002). KLAM was used to calculate lamina hydraulic conductance (Klam, kg s−1MPa−1cm−2) after determining leaf area with a leaf area meter (Li-3100, LI-COR, Lincoln NE) (Sack et al. 2002). Aquaporin expression in roots and leaves RNA was isolated from roots and leaves of P. tremuloides, using spin columns (RNeasy Plant Mini Kit, Qiagen, Venlo, Netherlands) according to the manufacturer’s instruction. The concentration of RNA was quantified by measuring the absorbance at 260 nm. For cDNA synthesis
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with integrated removal of genomic DNA contamination, the QuantiTect Reverse Transcription kit (Qiagen) was used according to the manufacturer’s instruction with 1 µg of total RNA, using RT primer mix (Qiagen). Gene expression of PtPIP1;1 (AJ849323.1), PtPIP1;2 (AJ849322.1), PtPIP2;1 (AJ849324.1), PtPIP2;2 (AJ849325.1), PtPIP2;3 (AJ849326.1), PtPIP2;4 (AJ849327.1), PtPIP2;5 (AJ849328.1) was studied by quantitative real-time PCR (qRT-PCR) using 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). These PIPs were selected since they were reported to be among the major stress responsive functional aquaporins in poplars (Marjanovic´ et al. 2005; Almeida-Rodriguez et al. 2010) and their sequences were available in the database. The primer sets used to amplify the genes in the synthesized cDNA are shown in Supplemental Table S1. The primers were designed in the 3′ UTR with the online software Primer 3 v 0.4.0 according to specific criteria, which included a predicted melting temperature of 60 ± 2 °C, a primer length of 18–24 nucleotides, a product size of 80– 150 bp, and a GC content of 45–60 %. Each 10-μl reaction contained 1 μl of a 1:10 dilution of the cDNA, 50 nM of each primer and 5 μl of 2X QPCR Mastermix (Molecular Biology Service Unit, Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada). The 2X QPCR Mastermix is a proprietary mix containing Tris (pH 8.3), KCl, MgCl2, glycerol, Tween 20, DMSO, dNTPs, ROX as a normalizing dye, SYBR Green (Molecular Probes) as the detection dye, and an antibody-inhibited Taq polymerase. The PCR program consisted of a 2-min incubation at 95 °C to activate the antibody-inhibited Taq polymerase, followed by 35 cycles of 45 s at 95 °C and 60 s at 62 °C, at which temperature the fluorescence signal was measured. The specificity of the PCR amplification procedure was checked with a heat dissociation protocol (from 70 to 100 °C), after the final cycle of the PCR. The efficiency of the primer set was evaluated by performing real-time PCR on several dilutions of cDNA. The results obtained for the different treatments were normalized to the PtJip1 (AJ407583) levels (Grunze et al. 2004), which were amplified with the primers shown in Supplemental Table S1. Real-time PCR experiments were carried out in three plants, with the Ct determined in triplicate. The relative transcription levels were calculated using the 2–ΔCt method (Livak and Schmittgen 2001). Negative controls without cDNA were used in all PCR reactions. Statistical analyses In both experiments and for each measurement date, oneway ANOVA was used to analyze treatment effects on gas exchange, water potential, Lpr, and Klam (SigmaPlot version
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12.0, Systat Software, San José, CA, USA). Separation of means was performed by Tukey’s HSD test at α = 0.05. Data were transformed to meet the normality and homoscedastic postulates. Normality was tested by the Kolmogorov–Smirnov’s test and homogeneity of variance by Levene Median’s test. Untransformed values are presented for easier interpretation. For the gene expression analysis, measurements were transformed to the natural logarithm (Rieu and Powers 2009). The expression data were subjected to one-way ANOVA and Tukey’s test at α = 0.05 using the SPSS (version 15) statistical package. For the cell hydraulic conductivity, the data were analyzed using unpaired t test (α = 0.05).
Results Leaf water potentials and gas exchange Leaf water potential decreased in plants subjected to 75 % defoliation for 1 day (Fig. 1a). There were no differences in leaf water potential between control and defoliated plants when measured 1 and 3 weeks following defoliation (Fig. 1a). Transpiration rates (E) increased by more than twofold in one-day-defoliated plants compared with non-defoliated plants and the magnitude of the increase was not affected by the degree of defoliation (Fig. 1b). 1 week after defoliation, E values were similar in defoliated and non-defoliated plants and there was another small increase in defoliated plants after 3 weeks (Fig. 1b). To determine the immediate response, E was also measured continuously during the first 10 min following 75 % defoliation and in non-defoliated plants. The E values measured 2.68 ± 0.1 mmol H2O m−2 s−1 at 10 min following defoliation and 2.71 mmol H2O m−2 s−1 in the non-defoliated group (mean, n = 6 ± SE). At 10 min following 75 % defoliation, E values measured 2.03 ± 0.2 mmol H2O m−2 s−1 compared with 2.58 mmol H2O m−2 s−1 in non-defoliated plants (mean, n = 6 ± SE, P = 0.032). Similarly to E, net photosynthesis (Pn) increased in defoliated plants after 1 day and 3 weeks and there was no effect when measured in plants defoliated for 1 week (Fig. 1c). However, in contrast to E, the increases in Pn in defoliated plants were relatively small (Fig. 1c). Root hydraulic conductivity (Lpr), leaf lamina hydraulic conductance (Klam) and cell hydraulic conductivity (Lpc) One day following defoliation, root hydraulic conductivity (Lpr) was reduced by 26 and 51 in 50- and 75 %-defoliated plants, respectively, compared with control (Fig. 2a). The decreases in both groups of defoliated plants were
Fig. 1 Leaf water potentials (Ψw) (a), transpiration rates (E) (b) and net photosynthetic rates (Pn) (c) in aspen seedlings after 1 day, 1 and 3 weeks following 50 and 75 % defoliation. Means (n = 6) ± SE are shown. Different letters above the bars indicate statistically significant differences at α = 0.05 determined by Tukey’s HSD test
smaller and of similar magnitude compared with control 1 week after defoliation (Fig. 2a). There was no effect of 50 % defoliation on Lpr after 3 weeks, but some reduction of Lpr was still noticeable in 75 % defoliation treatment (Fig. 2a). Another group of seedlings was taken for the measurements of short-term responses of Lpr to 75 % defoliation. Ten minutes following defoliation, Lpr measured
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Effects of girdling on Lpr of defoliated plants To determine whether the signal inhibiting Lpr is transferred from leaves to roots in the living tissues outside the secondary xylem, we girdled some of the seedlings prior to 100 % defoliation treatment. Approximately 10 days after defoliation, some of the lateral buds started flushing and producing lateral shoots. Three weeks following defoliation, the lateral shoots and leaves were still growing. Girdling had no effect on Lpr (Fig. 4) and did not affect the decrease in Lpr triggered by defoliation which was measured in the roots of plants defoliated for one and 10 days (Fig. 4). Three weeks following leaf removal, defoliated and control seedlings had similar Lpr (Fig. 4). Aquaporin expression in roots and leaves
Fig. 2 Root hydraulic conductivity (Lpr) (a) and leaf lamina hydraulic conductance (Klam) (b) in aspen seedlings after 1 day, 1 and 3 weeks following 50 and 75 % defoliation. Means (n = 6) ± SE are shown. Different letters above the bars indicate statistically significant differences at α = 0.05 determined by Tukey’s HSD test
5.0 ± 0.42 (mean ± SE, n = 6) MPa−1 s−1 cm−3 × 10−7 compared with 5.4 ± 0.43 (mean ± SE, n = 6) MPa−1 s−1 cm−3 × 10−7 in non-defoliated plants (P = 0.506). In plants of the same experimental group that were subjected to 75 % defoliation for 1 day (approximately 24 h), Lpr was 3.7 ± 0.44 (mean ± SE, n = 6) MPa−1 s−1 cm−3 × 10−7 compared with 5.6 ± 0.29 (mean ± SE, n = 6) MPa−1 s−1 cm−3 × 10−7 measured in non-defoliated plants (P = 0.004). Leaf lamina hydraulic conductance (Klam) increased by about fourfold in plants following 1 day of 50 and 75 % defoliation (Fig. 2b). Defoliation had no effect on Klam after 1 week (Fig. 2b), however, Klam increased in 50 %-defoliated plants after 3 weeks (Fig. 2b). One day following 75 % defoliation, half-times of water exchange (T1/2) of root cortical cells increased by approximately twofold compared with non-defoliated controls (Fig. 3a). This increase corresponded to a similar magnitude decrease in Lpc (Fig. 3b).
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Several plasma membrane intrinsic proteins (PIPs) were selected for a qRT-PCR survey of transcript abundance in roots and leaves of control plants and plants defoliated by 50 and 75 %. PIP1;1, PIP1;2, and PIP 2;4 were the most highly expressed in the roots (Fig. 5) and PIP1;2 and PIP 2;4 were the most highly expressed in the leaves (Fig. 6). One day following defoliation treatment, the most striking change of the measured aquaporins in roots was a sharp (more than 50-fold) decrease in PIP1;2 in the roots of 50- and 75 %-defoliated plants (Fig. 5a). Other changes included a decrease in PIP2;2 in the 50 % defoliation treatment and increases in PIP2;5 in the 50 % and 75 % defoliation treatments (Fig. 5a). Root levels of PIP2;4 were reduced by over twofold in the 50 % defoliation treatment after 1 week and by 27 % after 3 weeks (Fig. 5b, c). There were also relatively minor increases of root PIP 1;1, PIP1;2, and PIP2;1 levels in the 50 % defoliation treatment after 3 weeks (Fig. 5c). Defoliation resulted after 1 day in a drastic change in the PIP expression profile in the leaves with a sharp increase (five- to tenfold) in the PIP2;4 transcript levels in 50 and 75 % defoliation treatments (Fig. 6a). There were also some increases in expression of PIP1;1 and PIP2;5 and decreases in expression PIP1;2 in defoliated plants (Fig. 6). Similarly to roots, the changes observed after one and 3 weeks of defoliation were relatively minor compared with one-day-defoliated plants with increases in PIP1;1 and PIP2;1 in 75 % defoliation treatment after 1 week (Fig. 6a) and 3 weeks (Fig. 6b), respectively.
Discussion Defoliation is a common traumatic event with a major impact on the hydraulic architecture of the affected plant. In our study, aspen plants subjected to partial
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Fig. 3 Cell-pressure probe traces showing turgor pressure (P) and half-times of water exchange (T1/2) (a) and cell hydraulic conductivity (Lpc) (b) of root cortical cells in control (non-defoliated) aspen seedlings and in seedlings subjected to 75 % defoliation for 1 day. On the right side in a, the cell pressure probe trace shows quick changes in pressure imposed for the determination of cell volumetric elastic modulus (ε). Means (n = 7) ± SE are shown. Different letters above the bars indicate statistically significant differences (P ≤ 0.05) determined by the t test
Fig. 4 Effects of girdling and removal of all leaves (100 % defoliation) on root hydraulic conductivity (Lpr) in aspen seedlings after 1 day, 10 days, and 3 weeks following defoliation. Stems were girdled prior to leaf removal. Means (n = 6) ± SE are shown. Different letters above the bars indicate statistically significant differences at α = 0.05 determined by Tukey’s HSD test
defoliation decreased E within several minutes following defoliation. However, this initial response was followed by an over twofold increase in E compared with
control plants when measured 1 day following defoliation. Changes in E were accompanied by changes in Pn, but of a lower magnitude suggesting that the stomatal conductance was not likely the limiting factor to photosynthesis in defoliated plants. Defoliated and non-defoliated plants had similar intercellular CO2 concentrations (data not shown). On the other hand, since some of the PIP2 aquaporins were recently demonstrated to be involved in facilitating CO2 transport (Mori et al. 2014), we cannot exclude the possibility that the increased PIP2;4 expression in the leaves of partly defoliated plants could facilitate CO2 transport and stimulate Pn. However, similarly to the barley HvPIP2;4 aquaporin, PIP2;4 from aspen has methionine residue present at position 254 suggesting that it may not have CO2 transporting properties (Mori et al. 2014). The photosynthetic compensation response has been frequently observed following defoliation (Tschaplinski and Blake 1995; Quentin et al. 2012; Eyles et al. 2013). The response varies in magnitude between studied plants and may or may not be accompanied by increases in E and (or) stomatal conductance (Gálvez and Tyreee 2009; Quentin et al. 2012). The explanations for the photosynthetic compensation response include changes in source–sink relationship (Tschaplinski and Blake 1995; Pinkard et al. 2011;
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Fig. 5 Relative expression of the PtPIP1;1, PtPIP1;2, PtPIP2;1, PtPIP2;2, PtPIP2;3, PtPIP2;4, PtPIP2;5 genes determined by quantitative real-time PCR in roots of non-defoliated (Control), 50 %-defoliated (50 %) and 75 %-defoliated (75 %) aspen plants following 1 day (a), 1 week (b) and 3 weeks (c) following defoliation. The bars show the means (n = 3) ± SE. Significant differences are indicated by different letters as determined by Tukey’s test (α = 0.05)
Eyles et al. 2013) and the increase in maximum carboxylation rate and RuBP regeneration (Turnbull et al. 2007). Root metabolites (Meinzer and Grantz 1990) and
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carbohydrates (Eyles et al. 2013) have been implicated as possible signaling molecules for up-regulation of gas exchange in defoliated plants.
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Fig. 6 Relative expression of the PtPIP1;1, PtPIP1;2, PtPIP2;1, PtPIP2;2, PtPIP2;3, PtPIP2;4, PtPIP2;5 genes determined by quantitative real-time PCR in leaves of non-defoliated (Control), 50 %-defoliated (50 %) and 75 %-defoliated (75 %) aspen plants after 1 day (a), 1 week (b) and 3 weeks (c) following defoliation. The bars show the means (n = 3) ± SE. Significant differences are indicated by different letters were determined by Tukey’s test (α = 0.05)
Increases in E measured 1 day after partial defoliation in aspen seedlings were accompanied by decreases in leaf water potentials suggesting that the rates of water loss due to increased E likely exceeded the rates of water uptake.
A similar transient decrease in stem water potential which accompanied an increase in stomatal conductance was reported in hybrid poplar (Populus trichocarpa × deltoides) plants exposed to increased light level (Laur and Hacke
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2013). In our study, plant hydraulic adjustments did not take place immediately following defoliation as evidenced by the absence of Lpr responses when measured 10 min following defoliation. However, within 1 day following 50 and 75 % defoliation, Lpr decreased to about 75 and 50 %, respectively, compared with the control (non-defoliated) plants. Lpr slowly recovered in defoliated plants but the reduction was still present after 3 weeks in plants subjected to 75 % defoliation. In Citrus plants, partial defoliation resulted in higher water potentials and root conductivities, but did not affect stomatal conductance which was probably limited by other factors (Syvertsen 1994). Higher whole plant hydraulic conductance was also reported for defoliated Eucalyptus globulus seedlings (Quentin et al. 2012). Since the resistance of leaves to water transport accounts for 30–80 % of the total plant hydraulic resistance (Sack and Holbrook 2006), it is likely that the overall effect of defoliation on plant hydraulic conductance may be influenced by the decreased resistance caused by leaf removal and possible changes in leaf hydraulic conductance triggered by defoliation. In our study, Klam increased by about six times in one-day defoliation treatments and returned to the control level 1 week following defoliation. However, 3 weeks after defoliation, 50 %-defoliated plants had again higher Klam and E compared with non-defoliated plants, likely reflecting renewed growth of newly flushed shoots. Diurnal changes in leaf hydraulic conductance largely reflected those in stomatal conductance in Populus trichocarpa and P. nigra leaves suggesting that a regulation of both processes in leaves may be linked (Lopez et al. 2013). The decrease in Lpr is a logical rapid response of plants to the large loss of transpiration area and the resulting decrease in water demand in plants which lost 50 or 75 % of their foliage. However, the large increases of E in oneday-defoliated plants at least partly compensated for the lost transpiration area. A similar phenomenon was reported for partly defoliated sugarcane (Meinzer and Grantz 1990). The decrease of Lpr at the time the transpiration rates increased is in contrast to the commonly observed responses to other environmental stresses where E and stomatal conductance were usually synchronized with Lpr (Wan et al. 2001; Kamaluddin and Zwiazek 2001, 2002). The decrease in leaf water potentials of one-day-defoliated plants suggests that the reduction of Lpr was among the factors which limited water supply to the leaves. To examine whether the decrease in Lpr involved changes in cell-to-cell water flow in roots, we compared Lpc in root cortical cells of non-defoliated (control) plants and in plants subjected to 75 % defoliation treatment for 1 day. Since the decrease in Lpc was of a similar magnitude to the decrease in Lpr, we concluded that the inhibition of cell-to-cell transport in roots was the principal factor responsible for the decrease in Lpr.
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The increase in Klam in defoliated plants which accompanied the increase in transpiration rates demonstrates the importance of a dynamic regulation of leaf water transport to a plant’s water balance. The pivotal role of aquaporins in root water transport regulation has been well documented (Maurel et al. 2010; Lee et al. 2012). More recently, the importance of leaf aquaporins in hydraulic responses to environmental cues received considerable attention (Cochard et al. 2007; Voicu et al. 2009; Lopez et al. 2013; Pou et al. 2013). Changes in hydraulic properties of roots and leaves of aspen seedlings that were triggered by defoliation were accompanied by profound changes in the expression of both root and leaf aquaporins. In the roots of one-day-defoliated plants, the decrease in Lpr was accompanied by a large decrease in PIP1;2 expression while in the leaves, the most striking change was the large increase in the expression of PIP2;4. Both PIP1;2 and PIP2;4 were among the highly expressed PIPs in roots and leaves of aspen seedlings, respectively, and following defoliation, PIP2;4 showed higher expression in the leaves than any other PIP. These PIPs were reported to be expressed in all studied organs of P. tremula × tremuloides rooted cuttings, however, the isoform PIP1;2 showed an increase in mycorrhizal roots where Lpr was also increased (Marjanovic´ et al. 2005). Although PIP1 isoforms show very low water permeability in heterologous expression, their water permeability increases when they interact with PIP2 isoforms in a heterotetrameric complex (Fetter et al. 2004; Temmei et al. 2005; Zelazny et al. 2007). In fact, PtPIP1;3 from Populus trichocarpa, which shares 99 % identity with PIP1;2 from P. tremuloides, was shown to play a major role in refilling of embolized vessels (Secchi and Zwieniecki 2010) and was found to be responsive to drought in P. simonii × balsamifera (Almeida-Rodriguez et al. 2010). PIP2 aquaporins have been shown to be the major water channels in plants (Chrispeels et al. 2001). PIP2;4 shows 99 % similarity with P. trichocarpa PIP2;4 which, when expressed in oocytes, increased their permeability by fivefold (Secchi et al. 2009). Putative orthologs of PIP2;4 include JrPIP2;1 and JrPIP2;2 from Junglas regia and AtPIP2;5 from A. thaliana. There is growing evidence of the importance of leaf and root aquaporins in adjusting plant hydraulic properties to transpirational demand. In J. regia, the increase in PIP transcript levels in response to light was reported to be functionally linked to the increase in leaf hydraulic conductance (Cochard et al. 2007). The increased gene expression of several root aquaporins including PIP1;1, PIP1;2, PIP1;3, PIP2;3; PIP2;4, and PIP2;5 was reported in P. trichocarpa × deltoids exposed to increased humidity and light levels (Laur and Hacke 2013). In both cases, the changes in root aquaporin expression were accompanied by changes in root water flow (Laur and Hacke 2013). Exposure to light
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also induced gene expression of several leaf aquaporins and triggered an accumulation of PIP2 proteins in leaves of P. trichocarpa and P. nigra which was accompanied by increases in leaf hydraulic conductance (Lopez et al. 2013). In our study, the reduction in root PIP1;2 levels and the increase in leaf PIP2;4 did not occur in plants after 1 and 3 weeks of defoliation suggesting that the longer term regulation of hydraulic properties also involved other mechanisms. Both root (Laur and Hacke 2013) and leaf (Lopez et al. 2013) PIP aquaporin expression followed a complex pattern in response to light in the studied poplars and it was suggested that they are regulated through adjustments in the levels of transcripts and proteins in response to environmental factors (Lopez et al. 2013). To investigate possible signals triggering changes in Lpr and root PIP expression, we girdled the stems of seedlings above the root collar prior to complete defoliation. Similarly to partial defoliation, complete defoliation reduced Lpr by about 50 % and was carried out to remove a possible source of chemical signals (Schachtman and Goodger 2008; Eyles et al. 2013) transported in the phloem from the leaves and which could affect Lpr. Girdling treatment did not affect Lpr in non-defoliated plants and did not prevent the reduction of Lpr in plants defoliated for 1 and 10 days. Therefore, it is unlikely that the carbohydrates (Eyles et al. 2013) or other phloem-transported signaling molecules (Schachtman and Goodger 2008) could be responsible for the inhibition of Lpr in defoliated aspen seedlings. It is also unlikely that sparse wood axial parenchyma (Ache et al. 2010) could provide a significant alternative pathway for basipetal transport to roots in girdled seedlings (Wan et al. 2006). In conclusion, partial and complete defoliation of aspen seedlings triggered a decrease in Lpr. One day after the defoliation treatment, the decrease in Lpr was proportional to the degree of defoliation and was accompanied by the decrease in Lpc of root cortical cells as well as a profound reduction in the root expression levels of PIP1;2. Partial (50 and 75 %) defoliation also increased Klam in one-daydefoliated seedlings and the magnitude of this increase was similar in both defoliation treatments. The increase in Klam was accompanied by an over 100 % increase in E and a five- to tenfold increase in the leaf expression of PIP2;4. The decrease of Lpr in defoliated plants could not be prevented by girdling suggesting that the response was not likely due to chemical signals which originated in the shoots. The results point to the importance of root and leaf aquaporins in the coordination of plant hydraulic responses to defoliation and in adjustments of plant hydraulic properties to changes in transpirational demand. Author contribution JL carried out two defoliation experiments and conducted physiological measurements; MAE carried out girdling and short-term experiments and
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conducted physiological measurements; AN-R carried out PCR analyses; SHL carried out cell pressure probe measurements, JJZ designed the study, wrote and revised the manuscript. All authors participated in data analyses and manuscript preparation. Acknowledgments This work was supported through the Discovery Research Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to JJZ.
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Planta (2014) 240:553–564 Sack L, Cowan PD, Jaikumar N, Holbrook NM (2003) The ‘hydrology’ of leaves: co-ordination of structure and function in temperate woody species. Plant Cell Environ 26:1343–1356 Schachtman DP, Goodger JQD (2008) Chemical root to shoot signaling under drought. Trends Plant Sci 13:281–287 Secchi F, Zwieniecki M (2010) Patterns of PIP gene expression in Populus trichocarpa during recovery from xylem embolism suggest a major role for the PIP1 aquaporin subfamily as moderators of refilling process. Plant Cell Environ 33:1285–1297 Secchi F, Maciver B, Zeidel ML, Zwieniecki MA (2009) Functional analysis of putative genes encoding the PIP2 water channel subfamily in Populus trichocarpa. Tree Physiol 29:1467–1477 Shatil-Cohen A, Attia Z, Moshelion M (2011) Bundle-sheath cell regulation of xylem-mesophyll water transport via aquaporins under drought stress: a target of xylem-borne ABA? Plant J 67:72–80 Steudle E (1993) Pressure probe techniques: basic principles and application to studies of water and solute relations at the cell, tissue and organ level. In: Smith JAC, Griffith H (eds) Water deficits: plant responses from cell to community. Bios Scientific Publishers, Oxford, pp 5–36 Steudle E, Peterson CA (1998) How does water get through roots? J Exp Bot 49:775–788 Syvertsen JP (1994) Partial shoot removal increases net CO2 assimilation and alters water relations of Citrus seedlings. Tree Physiol 14:497–508 Temmei Y, Uchida S, Hoshino D, Kanzawa N, Kuwahara M, Sasaki S, Tsuchiya T (2005) Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation. FEBS Lett 579:4417–4422 Thomson VP, Cunningham SA, Ball MC, Nicotra AB (2003) Compensation for herbivory by Cucumis sativus through increased photosynthetic capacity and efficiency. Oecologia 134:167–175 Tschaplinski T, Blake TJ (1995) Growth and carbohydrate status of coppice shoots of hybrid poplar following shoot pruning. Tree Physiol 15:333–338 Turnbull TL, Adams MA, Warren CR (2007) Increased photosynthesis following partial defoliation of field-grown E. globulus seedlings is not caused by increased leaf N. Tree Physiol 27:1481–1492 Voicu MC, Cooke JK, Zwiazek JJ (2009) Aquaporin gene expression and apoplastic water flow in bur oak (Quercus macrocarpa) leaves in relation to the light response of leaf hydraulic conductance. J Exp Bot 60:4063–4075 Wan X, Zwiazek JJ (1999) Mercuric chloride effects on root water transport in aspen (Populus tremuloides) seedlings. Plant Physiol 121:939–946 Wan X, Zwiazek JJ, Lieffers VJ, Landhäusser S (2001) Effect of low temperature on root hydraulic conductance in aspen (Populus tremuloides) seedlings. Tree Physiol 21:691–696 Wan X, Landhäusser SM, Lieffers VJ, Zwiazek JJ (2006) Signals controlling root suckering and adventitious shoot formation in aspen (Populus tremuloides). Tree Physiol 26:681–687 Zelazny E, Borst JW, Muylaert M, Batoko H, Hemminga MA, Chaumont F (2007) FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization. Proc Natl Acad Sci USA 104:12359–12364
Original Article
Hydraulic adjustments in aspen (Populus tremuloides) seedlings following defoliation involve root and leaf aquaporins Juan Liu · María A. Equiza · Alfonso Navarro‑Rodenas · Seong H. Lee · Janusz J. Zwiazek
Received: 7 May 2014 / Accepted: 5 June 2014 / Published online: 24 June 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Main conclusion Changes in root and leaf hydraulic properties and stimulation of transpiration rates that were initially triggered by defoliation were accompa‑ nied by corresponding changes in leaf and root aqua‑ porin expression. Aspen (Populus tremuloides) seedlings were subjected to defoliation treatments by removing 50, 75 % or all of the leaves. Root hydraulic conductivity (Lpr) was sharply reduced in plants defoliated for 1 day and 1 week. The decrease in Lpr could not be prevented by stem girdling and it was accompanied in one-day-defoliated plants by a large decrease in the root expression of PIP1,2 aquaporin and an over twofold decrease in hydraulic conductivity of root cortical cells (Lpc). Contrary to Lpr and Lpc, 50 and 75 % defoliation treatments profoundly increased leaf lamina conductance (Klam) after 1 day and this increase was similar in magnitude for both defoliation treatments. Transpiration rates (E) rapidly declined after the removal of 75 % of leaves. However, E increased by over twofold in defoliated plants after 1 day and the increases in E and Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2106-2) contains supplementary material, which is available to authorized users. J. Liu · M. A. Equiza · A. Navarro‑Rodenas · S. H. Lee · J. J. Zwiazek (*) Department of Renewable Resources, University of Alberta, 442 Earth Sciences Bldg, Edmonton, AB T6G 2E3, Canada e-mail: [email protected] Present Address: J. Liu Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming 650224, Yunnan, China
Klam were accompanied by five- and tenfold increases in the leaf expression of PIP2;4 in 50 and 75 % defoliation treatments, respectively. Defoliation treatments also stimulated net photosynthesis after 1 day and 3 weeks, although the increase was not as high as E. Leaf water potentials remained relatively stable following defoliation with the exception of a small decrease 1 day after defoliation which suggests that root water transport did not initially keep pace with the increased transpirational water loss. The results demonstrate the importance of root and leaf hydraulic properties in plant responses to defoliation and point to the involvement of PIP aquaporins in the early events following the loss of leaves. Keywords Gas exchange · Hydraulic conductivity · Stem girdling · Water potential · Water transport Abbreviations E Transpiration Klam Leaf lamina hydraulic conductance KLEAF Absolute leaf hydraulic conductance Kr Root hydraulic conductance Lpc Cell hydraulic conductivity Lpr Root hydraulic conductivity PIP Plasma membrane intrinsic protein Pn Net photosynthesis Introduction Defoliation is caused by a variety of biotic and abiotic factors and is a major traumatic event which threatens plant’s survival. Large-scale defoliation events in trees are common, especially during periodical outbreaks of defoliating insects such as tent caterpillars (Cooke et al. 2011). In
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trembling aspen (Populus tremuloides), epidemic outbreaks of forest tent caterpillar (Malacosoma disstria Hbn.) occur on an average at about 10-year intervals (Churchill et al. 1964; Cooke et al. 2011) and can lead to complete defoliation of entire tree stands. Plants which are commonly exposed to defoliation have developed various adaptations to prevent the loss of leaves and to cope with its consequences once defoliation takes place. Fully defoliated aspen trees produce new leaves by precocious break of the lateral buds; however, if defoliation is only partial, lateral buds may remain inhibited until the following growth season (Donaldson and Lindroth 2008). The loss of photosynthetic area and the resulting depletion of energy reserves are among the main concerns in defoliated trees (Churchill et al. 1964; Landhäusser and Lieffers 2012). In trembling aspen trees, the recovery of root energy reserves may take up to 2 years following partial defoliation (Landhäusser and Lieffers 2012) and affect long-term hydraulic performance of trees (Anderegg and Callaway 2012). On the other hand, compensatory responses which may offset some of the loss of foliage have been reported for plants in which partial defoliation stimulated photosynthetic rates in the remaining leaves (Thomson et al. 2003; Turnbull et al. 2007; Quentin et al. 2012). The transient increase in photosynthesis may be accompanied by an increase in transpiration rates or stomatal conductance (Gálvez and Tyreee 2009; Quentin et al. 2012). However, the signaling pathways leading to these responses remain unclear. The loss of foliage alters the water and hormonal balance in trees. The water demand of a partly defoliated tree will depend on the degree of the photosynthetic compensation response and the involvement of stomatal conductance in this response. In fully defoliated trees, the demand for water will be greatly reduced until the expansion of leaf primordia commences in lateral buds and new leaves emerge. Partial defoliation lowers the tree’s transpirational area and causes a rapid increase in the root-to-shoot mass ratio. Therefore, adjustments in the energy-costly maintenance processes of root hydraulic conductance could be desirable to reflect changes in the transpirational demand. Plant water transport is driven by fast, transpirationdriven and slow, osmotically driven forces (Steudle and Peterson 1998). In both cases, water moves along the leastresistance pathways and there is extensive evidence which points to aquaporins as the principal structures responsible for regulating water flow resistance in roots and leaves (Javot and Maurel 2002; Heinen et al. 2009; Shatil-Cohen et al. 2011). The synthesis and opening of aquaporins are energy-demanding (Kamaluddin and Zwiazek 2001). Therefore, adjustments of root hydraulic conductivity to reflect changes in the total transpirational demand may help conserve limited energy resources which are needed
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in defoliated plants for defense mechanisms, wound repair, and re-growth of new leaves. The role of leaf aquaporins in regulating water transport processes is still the subject of an intensive debate (Heinen et al. 2009; Shatil-Cohen et al. 2011). If leaf aquaporins play a significant role in regulating leaf water transport in aspen, the effect of defoliation on leaf water flow properties offers an excellent opportunity to study the role of leaf aquaporins since the removal of leaves changes an overall hydraulic balance of the plant. It has been estimated that the resistance of leaves to water transport accounts for as much as 80 % of the total plant hydraulic resistance (Sack and Holbrook 2006). Leaf hydraulic conductance has been often correlated with photosynthetic capacity, maximum stomatal conductance and stomatal pore area per leaf area (Aasamaa and Sõber 2001; Sack et al. 2003; Brodribb et al. 2005). If defoliation triggers an increase in photosynthesis and transpiration of the remaining leaves, it is likely that the increased water transport to the leaves is facilitated by the increased hydraulic conductance, possibly mediated by aquaporins. Increased root (Lee et al. 2008; Laur and Hacke 2013) and leaf (Lee et al. 2009; Lopez et al. 2013) hydraulic conductance has been often reported to accompany increased transpirational demand and may be accompanied by the increased expression of aquaporins (Laur and Hacke 2013; Lopez et al. 2013). In the present study, we examined the effects of partial and complete defoliation on gas exchange, water transport and aquaporin expression in trembling aspen (Populus tremuloides) seedlings. The study was carried out to examine the adjustments in water relations of trembling aspen seedlings following defoliation and the processes involved in these responses. We examined the hypothesis that defoliation would (1) decrease root hydraulic conductivity to reflect reduced transpirational demand of the partly defoliated plants and (2) increase leaf hydraulic conductance to keep up with the increased water demand of the remaining leaves (compensation response). We also hypothesized that these changes would be accompanied by changes in the aquaporin expression in leaves and roots, which would impact cell-to-cell water transport. To study a possible role of chemical signals originating in shoots on plant hydraulic responses to defoliation, we also girdled stems of plants prior to defoliation to block the basipetal transport through the phloem and vascular cambium.
Materials and methods Experiment 1: defoliation For the defoliation experiment, trembling aspen (Populus tremuloides Michx.) seeds, locally collected in Edmonton,
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AB, Canada, were germinated and seedlings grown in 2.8-l pots filled with the Metromix (Sun Gro Horticulture Canada, Vancouver, BC, Canada) sand mixture (2:1, v/v) and grown in the greenhouse for 5 months before the start of defoliation treatments under the following conditions: 22/18 °C (day/night) temperature, relative humidity of 55–75 % relative humidity. Supplemental lighting in the greenhouse provided a photosynthetic flux density of 400 μmol m−2 s−1 at the top of the plants and extended the photoperiod to 16 h. Seedlings were watered daily and fertilized weekly with a 1 % (w/v) solution of commercial 20:20:20 (N:P:K) fertilizer. Defoliation treatments were carried out by removing every second leaf (50 % defoliation), or every three out of four leaves (75 % defoliation) from the entire length of the stem. Intact seedlings with no removed leaves served as defoliation control (0 % defoliation). The measurements were carried out 1 day, 1 and 3 weeks following defoliation in leaves and roots of six plants per treatment. Transpiration rates (E) and root hydraulic conductance (Kr) were also measured immediately (within 10 min) following defoliation. Experiment 2: defoliation and girdling For the defoliation and girdling experiment, one-year-old container-grown trembling aspen seedlings were obtained from Coast to Coast Reforestation Inc., Edmonton, AB, Canada, and grown in the greenhouse under the conditions described for Experiment 1. The seedlings were potted in 2.8-l pots filled with a commercial potting mix composed of peat moss (55–65 %), perlite, dolomitic limestone and gypsum. (Sunshine LA4 mix, Sun Gro Horticulture Canada Ltd) and fertilized with a controlled-release fertilizer (Osmocote, 13:13:13, N:P:K) applied at 20 g per pot. The plants started to flush 1 week after being transferred to the greenhouse and they were grown for 2 months before the commencement of defoliation and girdling treatments. The seedlings were subjected to the following treatments: no defoliation + no girdling (control), no defoliation + girdling, 100 % defoliation + no girdling, and 100 % defoliation + girdling. The girdling treatment involved the removal of a 1-cm ring of bark (periderm and phloem) at 2 cm from the base of the main stem about 1 h before defoliation. Bark was carefully removed with a razor blade while keeping the stem wet using a water sprayer. The girdle section was immediately wrapped with ParafilmTM and aluminum foil to prevent desiccation. For all measurements, six plants per treatment were taken for measurements after 1 day (approximately 24 h), 10 days (when buds started to flush in completely defoliated plants), and 3 weeks following defoliation. To minimize possible diurnal effects, defoliation treatments for Experiment 1 and 2 were carried out 2–6 h after
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the onset of photoperiod and the plant measurements were carried out interchangeably for the different treatments. Root hydraulic conductance (Kr) and conductivity (Lpr) measurements Root hydraulic conductance (Kr) was measured for excised root systems with the high-pressure flow meter (HPFM; Dynamax Inc., Houston, House, TX, USA) as previously described (Voicu et al. 2009; Calvo-Polanco et al. 2012). Each root system was subjected to the increasing pressure from 0 to 0.4 MPa and Kr was calculated from the slope of the linear portion of the graph produced by the high-pressure flow meter software and expressed in kg MPa−1s−1. Roots were immersed in water in a graduated cylinder and the volume of each root system was determined from the volume of displaced water. To obtain Lpr, Kr value was divided by root volume and expressed in kg MPa−1s−1cm−3 (Kamaluddin and Zwiazek 2002). There were six root systems per treatment taken for Kr measurements (n = 6) in both experiments. Cell hydraulic conductivity (Lpc) measurements Cell pressure probe measurements were carried out as previously described (Lee et al. 2012) in roots from seven control (non-defoliated) aspen plants and seven plants subjected to 75 % defoliation treatment for 1 day (approximately 24 h). A single cortical cell was punctured at a distance of about 20 mm from the root tip with a silicon oil-filled microcapillary (5- to 6-μm tip diameter). The position of cortical cell was estimated from the depth of the insertion of the microcapillary tip inside the root. The measurements of half-times of water exchange (T1/2) were carried out for up to 20 min. The hydraulic conductivity of root cortical cells (Lpc) was calculated using the following equation (Steudle 1993):
Lpc = V × ln (2)/{A × T1/2 × (ε + π i )},
(1)
where ε is cell elastic modulus, V is the cell volume, A is the cell surface area πi is osmotic cell pressure. Cell dimensions for V and A were examined microscopically in the root cortical cells and πi was estimated from the steady-state P. Cell elastic modulus (ε) was calculated from changes in cell volumes (ΔV) produced by the cell pressure probe and corresponding changes in cell turgor (ΔP) (Steudle 1993), i.e.: (2)
ε = V × ∆P/∆V Leaf water potentials and gas exchange measurements
A Scholander pressure chamber was used to measure water potentials of fully expanded leaves (Wan and Zwiazek 1999) 1 day, 1 and 3 weeks following defoliation
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(Experiment 1). There were six leaves from six plants per treatment (non-defoliated control, 50 and 75 % defoliation) (n = 6). Net photosynthesis and transpiration rates were measured for the defoliation treatments (Experiment 1) with a portable open-flow photosynthesis system (LI-6400, LI-COR Inc, Lincoln, NE, USA) equipped with a LI6400B red/blue LED light source. Light intensity was set at 400 µmol m−2 s−1 and reference CO2 was set at 400 µmol mol−1 using the 6400-01 CO2 mixer. Air temperature was 21 ± 0.15 °C and vapor pressure deficit (VPD) 1.3 ± 0.3 kPa. When readings were stable, data were logged every 30 s during a 5-min period. Fully elongated upper leaves were taken for the measurements in six plants per treatment (n = 6) immediately following defoliation as well as 1 day, 1 and 3 weeks following defoliation. Leaf lamina hydraulic conductance (Klam) measurements For absolute leaf hydraulic conductance (KLEAF) measurements, a fully elongated leaf in Experiment 1 was excised with a razor blade near the stem, at approximately the midpoint of the shoot height, and immediately immersed in a glass container filled with distilled water. The leaf was attached to the HPFM and water was forced into the leaf at constant pressure between 0.3–0.45 MPa depending on the leaf size (Voicu et al. 2009). A computer recorded the water flow rate (Q, kg s−1) and the applied pressure (P, MPa). The values were determined every 2 s and saved as means every 8 s (Voicu et al. 2009) to compute KLEAF, (kg s−1MPa−1) as Q/P after Q stabilized (1–2 min). After the measurement of KLEAF, the leaf petiole was excised with a razor blade from the leaf lamina and the hydraulic conductance of the petiole (KPET, kg s−1MPa−1) was measured in the same manner as for the whole leaf. Petiole hydraulic conductivity (Kpet) was calculated from KPET values expressed on the petiole length basis (Sack et al. 2002; Voicu et al. 2009). The absolute hydraulic conductance of the leaf lamina (KLAM, kg s−1MPa−1) was calculated as KLAM = 1/[(1/KLEAF)−(1/KPET)] (Sack et al. 2002). KLAM was used to calculate lamina hydraulic conductance (Klam, kg s−1MPa−1cm−2) after determining leaf area with a leaf area meter (Li-3100, LI-COR, Lincoln NE) (Sack et al. 2002). Aquaporin expression in roots and leaves RNA was isolated from roots and leaves of P. tremuloides, using spin columns (RNeasy Plant Mini Kit, Qiagen, Venlo, Netherlands) according to the manufacturer’s instruction. The concentration of RNA was quantified by measuring the absorbance at 260 nm. For cDNA synthesis
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with integrated removal of genomic DNA contamination, the QuantiTect Reverse Transcription kit (Qiagen) was used according to the manufacturer’s instruction with 1 µg of total RNA, using RT primer mix (Qiagen). Gene expression of PtPIP1;1 (AJ849323.1), PtPIP1;2 (AJ849322.1), PtPIP2;1 (AJ849324.1), PtPIP2;2 (AJ849325.1), PtPIP2;3 (AJ849326.1), PtPIP2;4 (AJ849327.1), PtPIP2;5 (AJ849328.1) was studied by quantitative real-time PCR (qRT-PCR) using 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). These PIPs were selected since they were reported to be among the major stress responsive functional aquaporins in poplars (Marjanovic´ et al. 2005; Almeida-Rodriguez et al. 2010) and their sequences were available in the database. The primer sets used to amplify the genes in the synthesized cDNA are shown in Supplemental Table S1. The primers were designed in the 3′ UTR with the online software Primer 3 v 0.4.0 according to specific criteria, which included a predicted melting temperature of 60 ± 2 °C, a primer length of 18–24 nucleotides, a product size of 80– 150 bp, and a GC content of 45–60 %. Each 10-μl reaction contained 1 μl of a 1:10 dilution of the cDNA, 50 nM of each primer and 5 μl of 2X QPCR Mastermix (Molecular Biology Service Unit, Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada). The 2X QPCR Mastermix is a proprietary mix containing Tris (pH 8.3), KCl, MgCl2, glycerol, Tween 20, DMSO, dNTPs, ROX as a normalizing dye, SYBR Green (Molecular Probes) as the detection dye, and an antibody-inhibited Taq polymerase. The PCR program consisted of a 2-min incubation at 95 °C to activate the antibody-inhibited Taq polymerase, followed by 35 cycles of 45 s at 95 °C and 60 s at 62 °C, at which temperature the fluorescence signal was measured. The specificity of the PCR amplification procedure was checked with a heat dissociation protocol (from 70 to 100 °C), after the final cycle of the PCR. The efficiency of the primer set was evaluated by performing real-time PCR on several dilutions of cDNA. The results obtained for the different treatments were normalized to the PtJip1 (AJ407583) levels (Grunze et al. 2004), which were amplified with the primers shown in Supplemental Table S1. Real-time PCR experiments were carried out in three plants, with the Ct determined in triplicate. The relative transcription levels were calculated using the 2–ΔCt method (Livak and Schmittgen 2001). Negative controls without cDNA were used in all PCR reactions. Statistical analyses In both experiments and for each measurement date, oneway ANOVA was used to analyze treatment effects on gas exchange, water potential, Lpr, and Klam (SigmaPlot version
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12.0, Systat Software, San José, CA, USA). Separation of means was performed by Tukey’s HSD test at α = 0.05. Data were transformed to meet the normality and homoscedastic postulates. Normality was tested by the Kolmogorov–Smirnov’s test and homogeneity of variance by Levene Median’s test. Untransformed values are presented for easier interpretation. For the gene expression analysis, measurements were transformed to the natural logarithm (Rieu and Powers 2009). The expression data were subjected to one-way ANOVA and Tukey’s test at α = 0.05 using the SPSS (version 15) statistical package. For the cell hydraulic conductivity, the data were analyzed using unpaired t test (α = 0.05).
Results Leaf water potentials and gas exchange Leaf water potential decreased in plants subjected to 75 % defoliation for 1 day (Fig. 1a). There were no differences in leaf water potential between control and defoliated plants when measured 1 and 3 weeks following defoliation (Fig. 1a). Transpiration rates (E) increased by more than twofold in one-day-defoliated plants compared with non-defoliated plants and the magnitude of the increase was not affected by the degree of defoliation (Fig. 1b). 1 week after defoliation, E values were similar in defoliated and non-defoliated plants and there was another small increase in defoliated plants after 3 weeks (Fig. 1b). To determine the immediate response, E was also measured continuously during the first 10 min following 75 % defoliation and in non-defoliated plants. The E values measured 2.68 ± 0.1 mmol H2O m−2 s−1 at 10 min following defoliation and 2.71 mmol H2O m−2 s−1 in the non-defoliated group (mean, n = 6 ± SE). At 10 min following 75 % defoliation, E values measured 2.03 ± 0.2 mmol H2O m−2 s−1 compared with 2.58 mmol H2O m−2 s−1 in non-defoliated plants (mean, n = 6 ± SE, P = 0.032). Similarly to E, net photosynthesis (Pn) increased in defoliated plants after 1 day and 3 weeks and there was no effect when measured in plants defoliated for 1 week (Fig. 1c). However, in contrast to E, the increases in Pn in defoliated plants were relatively small (Fig. 1c). Root hydraulic conductivity (Lpr), leaf lamina hydraulic conductance (Klam) and cell hydraulic conductivity (Lpc) One day following defoliation, root hydraulic conductivity (Lpr) was reduced by 26 and 51 in 50- and 75 %-defoliated plants, respectively, compared with control (Fig. 2a). The decreases in both groups of defoliated plants were
Fig. 1 Leaf water potentials (Ψw) (a), transpiration rates (E) (b) and net photosynthetic rates (Pn) (c) in aspen seedlings after 1 day, 1 and 3 weeks following 50 and 75 % defoliation. Means (n = 6) ± SE are shown. Different letters above the bars indicate statistically significant differences at α = 0.05 determined by Tukey’s HSD test
smaller and of similar magnitude compared with control 1 week after defoliation (Fig. 2a). There was no effect of 50 % defoliation on Lpr after 3 weeks, but some reduction of Lpr was still noticeable in 75 % defoliation treatment (Fig. 2a). Another group of seedlings was taken for the measurements of short-term responses of Lpr to 75 % defoliation. Ten minutes following defoliation, Lpr measured
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Effects of girdling on Lpr of defoliated plants To determine whether the signal inhibiting Lpr is transferred from leaves to roots in the living tissues outside the secondary xylem, we girdled some of the seedlings prior to 100 % defoliation treatment. Approximately 10 days after defoliation, some of the lateral buds started flushing and producing lateral shoots. Three weeks following defoliation, the lateral shoots and leaves were still growing. Girdling had no effect on Lpr (Fig. 4) and did not affect the decrease in Lpr triggered by defoliation which was measured in the roots of plants defoliated for one and 10 days (Fig. 4). Three weeks following leaf removal, defoliated and control seedlings had similar Lpr (Fig. 4). Aquaporin expression in roots and leaves
Fig. 2 Root hydraulic conductivity (Lpr) (a) and leaf lamina hydraulic conductance (Klam) (b) in aspen seedlings after 1 day, 1 and 3 weeks following 50 and 75 % defoliation. Means (n = 6) ± SE are shown. Different letters above the bars indicate statistically significant differences at α = 0.05 determined by Tukey’s HSD test
5.0 ± 0.42 (mean ± SE, n = 6) MPa−1 s−1 cm−3 × 10−7 compared with 5.4 ± 0.43 (mean ± SE, n = 6) MPa−1 s−1 cm−3 × 10−7 in non-defoliated plants (P = 0.506). In plants of the same experimental group that were subjected to 75 % defoliation for 1 day (approximately 24 h), Lpr was 3.7 ± 0.44 (mean ± SE, n = 6) MPa−1 s−1 cm−3 × 10−7 compared with 5.6 ± 0.29 (mean ± SE, n = 6) MPa−1 s−1 cm−3 × 10−7 measured in non-defoliated plants (P = 0.004). Leaf lamina hydraulic conductance (Klam) increased by about fourfold in plants following 1 day of 50 and 75 % defoliation (Fig. 2b). Defoliation had no effect on Klam after 1 week (Fig. 2b), however, Klam increased in 50 %-defoliated plants after 3 weeks (Fig. 2b). One day following 75 % defoliation, half-times of water exchange (T1/2) of root cortical cells increased by approximately twofold compared with non-defoliated controls (Fig. 3a). This increase corresponded to a similar magnitude decrease in Lpc (Fig. 3b).
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Several plasma membrane intrinsic proteins (PIPs) were selected for a qRT-PCR survey of transcript abundance in roots and leaves of control plants and plants defoliated by 50 and 75 %. PIP1;1, PIP1;2, and PIP 2;4 were the most highly expressed in the roots (Fig. 5) and PIP1;2 and PIP 2;4 were the most highly expressed in the leaves (Fig. 6). One day following defoliation treatment, the most striking change of the measured aquaporins in roots was a sharp (more than 50-fold) decrease in PIP1;2 in the roots of 50- and 75 %-defoliated plants (Fig. 5a). Other changes included a decrease in PIP2;2 in the 50 % defoliation treatment and increases in PIP2;5 in the 50 % and 75 % defoliation treatments (Fig. 5a). Root levels of PIP2;4 were reduced by over twofold in the 50 % defoliation treatment after 1 week and by 27 % after 3 weeks (Fig. 5b, c). There were also relatively minor increases of root PIP 1;1, PIP1;2, and PIP2;1 levels in the 50 % defoliation treatment after 3 weeks (Fig. 5c). Defoliation resulted after 1 day in a drastic change in the PIP expression profile in the leaves with a sharp increase (five- to tenfold) in the PIP2;4 transcript levels in 50 and 75 % defoliation treatments (Fig. 6a). There were also some increases in expression of PIP1;1 and PIP2;5 and decreases in expression PIP1;2 in defoliated plants (Fig. 6). Similarly to roots, the changes observed after one and 3 weeks of defoliation were relatively minor compared with one-day-defoliated plants with increases in PIP1;1 and PIP2;1 in 75 % defoliation treatment after 1 week (Fig. 6a) and 3 weeks (Fig. 6b), respectively.
Discussion Defoliation is a common traumatic event with a major impact on the hydraulic architecture of the affected plant. In our study, aspen plants subjected to partial
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Fig. 3 Cell-pressure probe traces showing turgor pressure (P) and half-times of water exchange (T1/2) (a) and cell hydraulic conductivity (Lpc) (b) of root cortical cells in control (non-defoliated) aspen seedlings and in seedlings subjected to 75 % defoliation for 1 day. On the right side in a, the cell pressure probe trace shows quick changes in pressure imposed for the determination of cell volumetric elastic modulus (ε). Means (n = 7) ± SE are shown. Different letters above the bars indicate statistically significant differences (P ≤ 0.05) determined by the t test
Fig. 4 Effects of girdling and removal of all leaves (100 % defoliation) on root hydraulic conductivity (Lpr) in aspen seedlings after 1 day, 10 days, and 3 weeks following defoliation. Stems were girdled prior to leaf removal. Means (n = 6) ± SE are shown. Different letters above the bars indicate statistically significant differences at α = 0.05 determined by Tukey’s HSD test
defoliation decreased E within several minutes following defoliation. However, this initial response was followed by an over twofold increase in E compared with
control plants when measured 1 day following defoliation. Changes in E were accompanied by changes in Pn, but of a lower magnitude suggesting that the stomatal conductance was not likely the limiting factor to photosynthesis in defoliated plants. Defoliated and non-defoliated plants had similar intercellular CO2 concentrations (data not shown). On the other hand, since some of the PIP2 aquaporins were recently demonstrated to be involved in facilitating CO2 transport (Mori et al. 2014), we cannot exclude the possibility that the increased PIP2;4 expression in the leaves of partly defoliated plants could facilitate CO2 transport and stimulate Pn. However, similarly to the barley HvPIP2;4 aquaporin, PIP2;4 from aspen has methionine residue present at position 254 suggesting that it may not have CO2 transporting properties (Mori et al. 2014). The photosynthetic compensation response has been frequently observed following defoliation (Tschaplinski and Blake 1995; Quentin et al. 2012; Eyles et al. 2013). The response varies in magnitude between studied plants and may or may not be accompanied by increases in E and (or) stomatal conductance (Gálvez and Tyreee 2009; Quentin et al. 2012). The explanations for the photosynthetic compensation response include changes in source–sink relationship (Tschaplinski and Blake 1995; Pinkard et al. 2011;
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Fig. 5 Relative expression of the PtPIP1;1, PtPIP1;2, PtPIP2;1, PtPIP2;2, PtPIP2;3, PtPIP2;4, PtPIP2;5 genes determined by quantitative real-time PCR in roots of non-defoliated (Control), 50 %-defoliated (50 %) and 75 %-defoliated (75 %) aspen plants following 1 day (a), 1 week (b) and 3 weeks (c) following defoliation. The bars show the means (n = 3) ± SE. Significant differences are indicated by different letters as determined by Tukey’s test (α = 0.05)
Eyles et al. 2013) and the increase in maximum carboxylation rate and RuBP regeneration (Turnbull et al. 2007). Root metabolites (Meinzer and Grantz 1990) and
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carbohydrates (Eyles et al. 2013) have been implicated as possible signaling molecules for up-regulation of gas exchange in defoliated plants.
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Fig. 6 Relative expression of the PtPIP1;1, PtPIP1;2, PtPIP2;1, PtPIP2;2, PtPIP2;3, PtPIP2;4, PtPIP2;5 genes determined by quantitative real-time PCR in leaves of non-defoliated (Control), 50 %-defoliated (50 %) and 75 %-defoliated (75 %) aspen plants after 1 day (a), 1 week (b) and 3 weeks (c) following defoliation. The bars show the means (n = 3) ± SE. Significant differences are indicated by different letters were determined by Tukey’s test (α = 0.05)
Increases in E measured 1 day after partial defoliation in aspen seedlings were accompanied by decreases in leaf water potentials suggesting that the rates of water loss due to increased E likely exceeded the rates of water uptake.
A similar transient decrease in stem water potential which accompanied an increase in stomatal conductance was reported in hybrid poplar (Populus trichocarpa × deltoides) plants exposed to increased light level (Laur and Hacke
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2013). In our study, plant hydraulic adjustments did not take place immediately following defoliation as evidenced by the absence of Lpr responses when measured 10 min following defoliation. However, within 1 day following 50 and 75 % defoliation, Lpr decreased to about 75 and 50 %, respectively, compared with the control (non-defoliated) plants. Lpr slowly recovered in defoliated plants but the reduction was still present after 3 weeks in plants subjected to 75 % defoliation. In Citrus plants, partial defoliation resulted in higher water potentials and root conductivities, but did not affect stomatal conductance which was probably limited by other factors (Syvertsen 1994). Higher whole plant hydraulic conductance was also reported for defoliated Eucalyptus globulus seedlings (Quentin et al. 2012). Since the resistance of leaves to water transport accounts for 30–80 % of the total plant hydraulic resistance (Sack and Holbrook 2006), it is likely that the overall effect of defoliation on plant hydraulic conductance may be influenced by the decreased resistance caused by leaf removal and possible changes in leaf hydraulic conductance triggered by defoliation. In our study, Klam increased by about six times in one-day defoliation treatments and returned to the control level 1 week following defoliation. However, 3 weeks after defoliation, 50 %-defoliated plants had again higher Klam and E compared with non-defoliated plants, likely reflecting renewed growth of newly flushed shoots. Diurnal changes in leaf hydraulic conductance largely reflected those in stomatal conductance in Populus trichocarpa and P. nigra leaves suggesting that a regulation of both processes in leaves may be linked (Lopez et al. 2013). The decrease in Lpr is a logical rapid response of plants to the large loss of transpiration area and the resulting decrease in water demand in plants which lost 50 or 75 % of their foliage. However, the large increases of E in oneday-defoliated plants at least partly compensated for the lost transpiration area. A similar phenomenon was reported for partly defoliated sugarcane (Meinzer and Grantz 1990). The decrease of Lpr at the time the transpiration rates increased is in contrast to the commonly observed responses to other environmental stresses where E and stomatal conductance were usually synchronized with Lpr (Wan et al. 2001; Kamaluddin and Zwiazek 2001, 2002). The decrease in leaf water potentials of one-day-defoliated plants suggests that the reduction of Lpr was among the factors which limited water supply to the leaves. To examine whether the decrease in Lpr involved changes in cell-to-cell water flow in roots, we compared Lpc in root cortical cells of non-defoliated (control) plants and in plants subjected to 75 % defoliation treatment for 1 day. Since the decrease in Lpc was of a similar magnitude to the decrease in Lpr, we concluded that the inhibition of cell-to-cell transport in roots was the principal factor responsible for the decrease in Lpr.
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The increase in Klam in defoliated plants which accompanied the increase in transpiration rates demonstrates the importance of a dynamic regulation of leaf water transport to a plant’s water balance. The pivotal role of aquaporins in root water transport regulation has been well documented (Maurel et al. 2010; Lee et al. 2012). More recently, the importance of leaf aquaporins in hydraulic responses to environmental cues received considerable attention (Cochard et al. 2007; Voicu et al. 2009; Lopez et al. 2013; Pou et al. 2013). Changes in hydraulic properties of roots and leaves of aspen seedlings that were triggered by defoliation were accompanied by profound changes in the expression of both root and leaf aquaporins. In the roots of one-day-defoliated plants, the decrease in Lpr was accompanied by a large decrease in PIP1;2 expression while in the leaves, the most striking change was the large increase in the expression of PIP2;4. Both PIP1;2 and PIP2;4 were among the highly expressed PIPs in roots and leaves of aspen seedlings, respectively, and following defoliation, PIP2;4 showed higher expression in the leaves than any other PIP. These PIPs were reported to be expressed in all studied organs of P. tremula × tremuloides rooted cuttings, however, the isoform PIP1;2 showed an increase in mycorrhizal roots where Lpr was also increased (Marjanovic´ et al. 2005). Although PIP1 isoforms show very low water permeability in heterologous expression, their water permeability increases when they interact with PIP2 isoforms in a heterotetrameric complex (Fetter et al. 2004; Temmei et al. 2005; Zelazny et al. 2007). In fact, PtPIP1;3 from Populus trichocarpa, which shares 99 % identity with PIP1;2 from P. tremuloides, was shown to play a major role in refilling of embolized vessels (Secchi and Zwieniecki 2010) and was found to be responsive to drought in P. simonii × balsamifera (Almeida-Rodriguez et al. 2010). PIP2 aquaporins have been shown to be the major water channels in plants (Chrispeels et al. 2001). PIP2;4 shows 99 % similarity with P. trichocarpa PIP2;4 which, when expressed in oocytes, increased their permeability by fivefold (Secchi et al. 2009). Putative orthologs of PIP2;4 include JrPIP2;1 and JrPIP2;2 from Junglas regia and AtPIP2;5 from A. thaliana. There is growing evidence of the importance of leaf and root aquaporins in adjusting plant hydraulic properties to transpirational demand. In J. regia, the increase in PIP transcript levels in response to light was reported to be functionally linked to the increase in leaf hydraulic conductance (Cochard et al. 2007). The increased gene expression of several root aquaporins including PIP1;1, PIP1;2, PIP1;3, PIP2;3; PIP2;4, and PIP2;5 was reported in P. trichocarpa × deltoids exposed to increased humidity and light levels (Laur and Hacke 2013). In both cases, the changes in root aquaporin expression were accompanied by changes in root water flow (Laur and Hacke 2013). Exposure to light
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also induced gene expression of several leaf aquaporins and triggered an accumulation of PIP2 proteins in leaves of P. trichocarpa and P. nigra which was accompanied by increases in leaf hydraulic conductance (Lopez et al. 2013). In our study, the reduction in root PIP1;2 levels and the increase in leaf PIP2;4 did not occur in plants after 1 and 3 weeks of defoliation suggesting that the longer term regulation of hydraulic properties also involved other mechanisms. Both root (Laur and Hacke 2013) and leaf (Lopez et al. 2013) PIP aquaporin expression followed a complex pattern in response to light in the studied poplars and it was suggested that they are regulated through adjustments in the levels of transcripts and proteins in response to environmental factors (Lopez et al. 2013). To investigate possible signals triggering changes in Lpr and root PIP expression, we girdled the stems of seedlings above the root collar prior to complete defoliation. Similarly to partial defoliation, complete defoliation reduced Lpr by about 50 % and was carried out to remove a possible source of chemical signals (Schachtman and Goodger 2008; Eyles et al. 2013) transported in the phloem from the leaves and which could affect Lpr. Girdling treatment did not affect Lpr in non-defoliated plants and did not prevent the reduction of Lpr in plants defoliated for 1 and 10 days. Therefore, it is unlikely that the carbohydrates (Eyles et al. 2013) or other phloem-transported signaling molecules (Schachtman and Goodger 2008) could be responsible for the inhibition of Lpr in defoliated aspen seedlings. It is also unlikely that sparse wood axial parenchyma (Ache et al. 2010) could provide a significant alternative pathway for basipetal transport to roots in girdled seedlings (Wan et al. 2006). In conclusion, partial and complete defoliation of aspen seedlings triggered a decrease in Lpr. One day after the defoliation treatment, the decrease in Lpr was proportional to the degree of defoliation and was accompanied by the decrease in Lpc of root cortical cells as well as a profound reduction in the root expression levels of PIP1;2. Partial (50 and 75 %) defoliation also increased Klam in one-daydefoliated seedlings and the magnitude of this increase was similar in both defoliation treatments. The increase in Klam was accompanied by an over 100 % increase in E and a five- to tenfold increase in the leaf expression of PIP2;4. The decrease of Lpr in defoliated plants could not be prevented by girdling suggesting that the response was not likely due to chemical signals which originated in the shoots. The results point to the importance of root and leaf aquaporins in the coordination of plant hydraulic responses to defoliation and in adjustments of plant hydraulic properties to changes in transpirational demand. Author contribution JL carried out two defoliation experiments and conducted physiological measurements; MAE carried out girdling and short-term experiments and
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conducted physiological measurements; AN-R carried out PCR analyses; SHL carried out cell pressure probe measurements, JJZ designed the study, wrote and revised the manuscript. All authors participated in data analyses and manuscript preparation. Acknowledgments This work was supported through the Discovery Research Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to JJZ.
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