The role of plasma membrane aquaporins in regulating the bundle sheath-mesophyll continuum and leaf hydraulics.

Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and extravascular, radial hydraulic conductance in leaves (K(leaf)), in particular, is still fairly limited. We hypothesized that the AQPs of the vascular bundle sheath (BS) cells regulate K(leaf). To examine this hypothesis, AQP genes were silenced using artificial microRNAs that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to target all five AQP genes from the PLASMA MEMBRANE-INTRINSIC PROTEIN1 (PIP1) subfamily. Our results show that the constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and BS osmotic water permeability (P(f)), mesophyll conductance of CO2, photosynthesis, K(leaf), transpiration, and shoot biomass. Plants in which the PIP1 subfamily was silenced only in the BS (SCARECROW:microRNA plants) exhibited decreased mesophyll and BS Pf and decreased K(leaf) but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We excluded the possibility of SCARECROW promoter activity in the mesophyll. Hence, the fact that SCARECROW:microRNA mesophyll exhibited reduced P(f), but not reduced mesophyll conductance of CO2, suggests that the BS-mesophyll hydraulic continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling leaf water status under normal and limited-water conditions is discussed.

1 0 0 cellular water transport (Maurel, 1997;Kaldenhoffet al., 2007). Arabidopsis thaliana has  It has been suggested that AQPs play a role in regulating K leaf (reviewed by Sack AQPs on leaf hydraulics is not fully understood. Many of the studies that have investigated the role of AQPs in regulating plant 1 1 7 hydraulic conductance were performed using classical reverse-genetic approaches. For  In an attempt to cope with these challenges, we employed methodology involving 1 2 7 artificial microRNA (amiRNA), which exploits endogenous miRNA precursors to 1 2 8 generate small RNA sequences (sRNA) that direct gene-silencing in the cell. AmiRNA-1 2 9 mediated gene-silencing is a useful method for inducible and partial gene inactivation, as in the regulation of hydraulic conductivity (Kaldenhoff et al., 1998;Martre et al., 2002;1 4 4 Siefritz et al., 2002;Postaire et al., 2010;Prado et al., 2013). Therefore, we chose to 1 4 5 silence the PIP1 subfamily, using artificial microRNA. To that end, we designed a single 1 4 6 sequence of synthetic miRNA containing a consensus sequence of 21 nucleotides 1 4 1 2 targeted by the amiRNA. This might be related to the fact that these putative targets have 2 9 0 somewhat higher rates of sequence mismatch (4-5 mismatches; Figure S5). From the 2 9 1 physiological point of view, the fact that PIP2 genes were down-regulated does not 2 9 2 interfere with the notion that AQPs (either PIP1s or others) can determine the membrane 2 9 3 osmotic water permeability and K leaf . The reduction in P f observed in the constitutively silenced plants (Figure 3) was expected, 2 9 7 as decreased mesophyll P f has been observed previously in AQP knock-out and antisense lines (Martre et al., 2002;Postaire et al., 2010). The role of PIPs in controlling K leaf and 2 9 9 radial influx, in particular, is an interesting question. Research in this area has shown that  Our data confirm these findings, with a significant reduction in P f , as well as K ros (2013). Thus, our reverse-genetic approach effectively reduced PIP expression and 3 1 2 cellular activity and, as result, leaf tissue hydraulic conductivity decreased. Moreover, 3 1 3 using the detached-leaf method, we were able to measure leaf water efflux (E) and, 3 1 4 separately, estimate the ratio between influx and out-flux through Ψ leaf . These was not due to decrease in E (i.e., a decrease in water influx, but not stomatal 3 1 7 conductance; Figure 6B, C). A Ψ leaf reduction with no significant change in transpiration was also observed in intact leaves (from plants grown in soil, as opposed to excised 3 1 9 leaves with submerged petioles and an unlimited water supply; Figure S6). These results support the hypothesized role of BS AQPs in regulating the 3 2 1 movement of water into BS cells and K leaf (or xylem efflux), as suggested by the 3 2 2 hydraulic g s feedback theory (i.e., down-regulation of AQPs reduces K leaf , which reduces  The role of PIP1 AQPs in controlling gas-exchange parameters 3 2 7 Recent studies have pointed to the ability of PIP1s to conduct CO 2 , as well as water 3 2 8 (Uehlein et al., 2003(Uehlein et al., , 2008Sade et al., 2014). It has been suggested that AtPIP1;2 may Siefritz et al., 2002;Flexas et al., 2006;Secchi et al., 2013Secchi et al., , 2014. Indeed, gas-exchange  (Table I). Recently, Atpip1;2 mutants were reported to have lower  However, in both studies (Postaire et al., 2010;Heckwolf et al., 2011) (Table II), yet did exhibit reduced mesophyll P f , suggest the possibility that a BS-3 4 5 mesophyll hydraulic signal controls K leaf , yet, further research is needed to elucidate this  Down-regulation of BS AQPs (SCR:mir1 plants) was found to be effective at the the silenced BS cells of those plants ( Figure 8A). Unexpectedly, the mesophyll P f of those 3 5 2 plants was reduced as well ( Figure 8B), which raises questions about how it is controlled.

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We excluded the possibility of SCR promoter activity in the mesophyll, as this 3 5 4 promoter is known to be expressed specifically in the BS and not at all in the mesophyll 3 5 5 (Wysocka-Diller et al., 2000;Shatil-Cohen et al., 2011;Sade et al., 2014). In addition, it 3 5 6 is not likely that the silencing agent moves to the mesophyll because: 1) microRNA is 3 5 7 known to be cell autonomous (Alvarez et al., 2006); 2) the relative expression of the leaf margin area (i.e., enriched mesophyll) was similar to that of the control ( Figure S8).

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Since the silencing of the microRNA agent in a cell should not vary between the different PIPs, we ruled out the possibility of its presence in the mesophyll cells. In fact, SCR:mir plants showed mild stress symptoms, in the form of reductions in 3 6 5 their K leaf , Ψ leaf and BS and mesophyll P f , but did not exhibit other stress symptoms of 3 6 6 reduced transpiration, g m or reduced photosynthesis [nor did they exhibit increased ABA  This physiological phenotype suggests that the P f and K leaf are very sensitive and 3 7 1 the first to change once conditions become less than optimal (before any reduction in 3 7 2 stomatal conductance or increase in ABA levels). This raises a question regarding the  Previous studies addressing this question suggested the existence of a generalized 3 7 6 sensitivity cascade of plants' physiological responses to water stress. Accordingly, the 3 7 7 first symptoms to appear at the onset of stress are turgor loss and the inhibition of cell 3 7 8 growth, followed by gradual increases in ABA levels, the initiation of stomatal closure  Our results in SCR:mir plants suggest that the imposed reduction in K leaf might 3 8 2 serve as hydraulic feed-forward signal that regulates the mesophyll P f prior to the 3 8 3 appearance of secondary and more severe signals, such as increased ABA levels and 3 8 4 reduced g s . Two more pieces of information support this hydraulic-stress signal cascade  Second, mesophyll g m , which was reduced under the constitutive 35s:mir promoter  These results as well as the literature concerning the generalized sensitivity studies have suggested that radial unloading of water from the xylem to the leaf is tightly needed to elucidate the interaction between the BS and the mesophyll, with respect to 4 3 0 their hydraulic functions and the specific roles of stress and AQP in this interaction. In 4 3 1 addition, it would be interesting to use the microRNA approach to target the PIP2 4 3 2 subfamily and the entire PIP family. All plants were grown in potting mix containing (w/w) 30% vermiculite, 30% peat, 20%   LhG4 and ER-GFP was subcloned behind an operator array in the BJ36 vector (Moore et  Those lines were crossed to generate plants in which GFP was expressed specifically in (stated in the text as 'control', a similar cross was made with WT Columbia plants). All   bath solution to 500 mOsm hypotonic solution). P f was determined using a numerical 4 9 0 approach, an offline curve-fitting procedure using the PFFIT program, as described in Whole-rosette hydraulic conductance (K ros ) 4 9 6 Hydrostatic K ros was measured on 21 days old plants, as described in Postaire et al.,  The transpiration rate (E) was measured using a Li-Cor 6400 gas-exchange system was followed immediately by the determination of leaf water potential (Ψ leaf ) using a 10 µM ABA for 1 h before measurement. Gas-exchange measurements 5 3 0 Gas-exchange measurements were assayed using a LI-6400 portable gas-exchange  were measured on WT plants ( Figure S10) The amount of blue light was set to 10% of 5 3 7 the photosynthetically active photon flux density, to optimize stomatal aperture. VPD using an open gas-exchange system with an integrated fluorescence chamber head (LI-   Mesophyll conductance of CO 2 (g m ) was calculated according to (Harley et al., 1992), as immediately frozen in liquid nitrogen.

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Total RNA was extracted using Tri-Reagent (Molecular Research Center; Cincinnati, 5 5 7 OH, USA) and treated with RNase-free DNase (Fermentas; Vilnius, Lithuania). cDNA samples and data analysis was performed using Rotor-Gene 6000 series software 1.7.  sharp peak in the melting curve indicates that a single, specific DNA species was  Total RNA was extracted from Arabidopsis leaves using Tri-Reagent (Molecular Research Center). Ten μ g of total RNA were separated according to size on a 1%   Leaves were harvested, immersed in 96% ethanol and incubated at 50°C for 3 h. The for an overnight incubation. Leaves were cut and immediately immersed (petiole-deep) in concentrated (1g*l-1) CW,  Following the clearing procedure, leaves were washed with water and put on microscope 6 2 2 slides. Images were taken using the NIS-Elements software (Japan) and the Leica 6 2 3 MZFLIII stereomicroscope (Germany) on which a Nikon DS-Fi1 digital camera was 6 2 4 mounted. Images were later analyzed to determine leaf vein area using the ImageJ was used in all images and the area of the veins was determined and calculated relative to 6 2 7 the total (ellipse) leaf area.    intact leaves from soil-grown, BS-silenced AtPIP1 plants.       rosette from a ~20 day-old 35S:mir1-3 plant exposed to prolonged night (11-21 h of 6 5 2 darkness).                    Nucleic Acids Research 31: 3406-3415.  deliberate mismatches between the amiRNApip1 and the AtPIP1 gene subfamily. WT, as calculated using Dunnett's method. were normalized to Actin2. between a genotype and the WT, as calculated using Dunnett's method. μ mol m −2 s −1 , ~25°C, and 400 μ mol mol −1 CO 2, as described by Flexas et al., 2007).

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Data are given as means ± SE (n, number of independent biological repetitions). A N : 9 4 8 photosynthesis; C i : sub-stomatal CO 2 concentration; g s : stomatal conductance; g m : 9 4 9 mesophyll CO 2 concentration; E: transpiration.    Presented data were normalized to the WT. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, N = 3-7 independent biological repetitions) between treatments and the control, as calculated based on the raw transcript levels using Dunnett's method. Data were normalized to Actin2.  . Constitutive silencing of the AtPIP1 AQP subfamily decreased the P f of bundle sheath (BS) and mesophyll cells. Columns indicate the mean P f (±SE) of control (black bars), 35S:mir1-8 (gray bars) and 35S:mir1-3 (white bars) of (A) BS protoplasts (n = 62, n = 15 and n = 24, respectively) and of (B) mesophyll protoplasts (n = 17, n = 12 and n = 14, respectively). An asterisk indicates a significant differences (*P < 0.05, **P < 0.001) between a genotype and the WT, as calculated using Dunnett's method.   . Relative expression profile of the PIP genes in the leaf midveins. Columns indicate the mean (± SE) PIP transcript levels (quantitative RT-PCR) in 35S:mir1-3, 35S:mir1-8 and the control (WT). The presented data were normalized to the WT. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, n = 5-6 independent biological repetitions) between treatments and the control based on the raw transcript levels, as calculated using Dunnett's method. Data were normalized to Actin2 levels.  . Relative expression profile of the PIP genes in the midveins of SCR:mir1 leaves. Columns indicate the mean (± SE) expression levels (q-RT-PCR analysis) of all PIP transcript levels in SCR:mir1 and control leaves. Presented data were normalized to the control. An asterisk indicates a significant difference between a treatment and the control based on the raw transcript levels (t test, p < 0.05, n = 7-10 independent biological repetitions). Data were normalized to Actin2 levels.  mesophyll protoplasts from SCR:mir1 (gray bar; n = 32 and n = 24 respectively) and control protoplasts (black bar; n = 25 and n = 25, respectively). An asterisk indicates a significant difference between a treatment mean and that of the control (t test, P < 0.05).  . Leaf hydraulic conductivity (K leaf ) was lower in the BS-silenced AtPIP1 leaves. Leaves from SCR:mir1 and control lines were harvested and immersed in artificial xylem sap (AXS). After 1 h, (A) K leaf was calculated for each individual leaf by dividing (B) the whole-leaf transpiration rate, E, by (C) the absolute value of the leaf water potential, Ψ leaf . An asterisk indicates a significant difference between lines (t test, P < 0.001). Data are means (±SEs) of values from 18-31 replicates from three independent experiments.