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Bundle-sheath aquaporins play a role in controlling Arabidopsis leaf hydraulic conductivity a

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Nir Sade , Arava Shatil-Cohen & Menachem Moshelion a

Institute of Plant Sciences and Genetics in Agriculture; Robert H. Smith Faculty of Agriculture, Food, and Environment; Hebrew University of Jerusalem; Rehovot, Israel Published online: 03 Jun 2015.

Click for updates To cite this article: Nir Sade, Arava Shatil-Cohen & Menachem Moshelion (2015) Bundle-sheath aquaporins play a role in controlling Arabidopsis leaf hydraulic conductivity, Plant Signaling & Behavior, 10:5, e1017177, DOI: 10.1080/15592324.2015.1017177 To link to this article: http://dx.doi.org/10.1080/15592324.2015.1017177

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SHORT COMMUNICATION Plant Signaling & Behavior 10:5, e1017177; May 2015; © 2015 Taylor & Francis Group, LLC

Bundle-sheath aquaporins play a role in controlling Arabidopsis leaf hydraulic conductivity Nir Sadey, Arava Shatil-Cohen, and Menachem Moshelion* Institute of Plant Sciences and Genetics in Agriculture; Robert H. Smith Faculty of Agriculture, Food, and Environment; Hebrew University of Jerusalem; Rehovot, Israel y

Current address: Department of Plant Sciences; University of California, Davis; Davis, CA, USA.

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Keywords: abscisic acid (ABA), artificial microRNA (amiRNA), aquaporins (AQPs), bundle sheath, leaf hydraulic conductivity (Kleaf), plasma membrane intrinsic proteins (PIPs)

The role of molecular mechanisms in the regulation of leaf hydraulics (Kleaf) is still not well understood. We hypothesized that aquaporins (AQPs) in the bundle sheath may regulate Kleaf. To examine this hypothesis, AQP genes were constitutively silenced using artificial microRNAs and recovery was achieved by targeting the expression of the tobacco AQP (NtAQP1) to bundle-sheath cells in the silenced plants. Constitutively silenced PIP1 plants exhibited decreased PIP1 transcript levels and decreased Kleaf. However, once the plants were recovered with NtAQP1, their Kleaf values were almost the same as those of WT plants. We also demonstrate the important role of ABA, acting via AQP, in that recovery and Kleaf regulation. These results support our previously raised hypothesis concerning the role of bundlesheath AQPs in the regulation of leaf hydraulics.

The idea that aquaporins (AQPs) control the movement of water into the leaf (i.e., radial hydraulic conductance) has been suggested by several research groups.1-4 Antisense silencing of multiple PIP genes and a single PIP knockout2,3 have been used to demonstrate that down-regulation of AQPs negatively affects leaf hydraulic conductance (Kleaf). As the axial vascular-hydraulic structure of the mature leaf is constant, the assumption is that a substantial portion of the dynamic hydraulic regulation in non-embolized or undamaged xylem is controlled via the radial or extravascular movement of water through the parenchymal tissue that surrounds the xylem elements (Sack & Holbrook, 2006). Recent studies have suggested that, in Arabidopsis, the leaf radial inflow rate is controlled by the vascular bundle sheath.5-7 Tightly wrapped around the entire vascular system, the bundle sheath acts as barrier to efflux from the xylem vessels. It has been suggested that bundle-sheath cells sense stress signals within the xylem sap and respond by changing their ‘acrossthe-pipe-wall’ (radial) hydraulic conductivity accordingly. This most likely occurs via downregulation of the AQP activity in bundle-sheath cells, which leads to a decrease in the osmotic water permeability (P-f) of those cells.7,8 This, in turn, reduces the flow of water into the leaf, which decreases the leaf water potential (Cleaf). Indeed, Prado et al. (2013) demonstrated a significant role for vascular parenchyma

PIP2;1 expression in the regulation of whole-rosette hydraulics. Recently, we demonstrated that multiple and specific silencing of PIP1 genes by artificial microRNAs (amiRNAs) in the whole plant (35S promoter) or specifically in bundlesheath cells (SCR promoter8) reduces Kleaf and further confirmed the role of bundle-sheath PIP1 AQPs in the regulation of leaf hydraulics.9 In the current study, we attempted to restore the Kleaf of these plants by expressing AQP specifically in bundle-sheath cells. In order to reduce the possibility of post-transcriptional or translational regulation, we used a heterologous aquaporin gene from tobacco (NtAQP1), which has been found to play a role in the regulation of plant hydraulic conductivity.10,11 Thus, 35S:mir plants were crossed with SCR:NtAQP1 plants expressing the tobacco Aquaporin1 – NtAQP1 gene under a bundle-sheath-specific promoter [SCARECROW (SCR)] to yield double (SCR:NtAQP1 and 35S:mir1) lines. We then followed PIP expression in the enriched bundle sheath tissue.12 In 35S:mir1–8, all of the PIP1 genes except for PIP1:3 were down-regulated in the enriched bundle sheath tissue. The expression of NtAQP1 in the bundle sheath of the control plants (SCR:NtAQP1) was similar to that observed in the WT control plants, except for the down-regulation of PIP2:2. In the 35S:mir1–8 plants expressing SCR:NtAQP1, we observed PIP1 downregulation similar to that observed in

*Correspondence to: Nir Sade; Email: [email protected]; Menachem Moshelion; Email: [email protected] Submitted: 01/20/2015; Revised: 01/30/2015; Accepted: 02/02/2015 http://dx.doi.org/10.1080/15592324.2015.1017177

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development proceeded in the presence of strong down-regulation of multiple AQP expression in the bundle-sheath cells. Some developmental and or physiological mechanisms might be affected by this silencing. Complete recovery of hydraulic flow was observed when only one AQP was knocked-out and recovered in the vascular system, as recently reported by Prado (2013).4 The fact that NtAQP1exFigure 1. Relative expression profile of the PIP genes in the leaf midveins. Columns indicate the mean ( § SE) pressed in the bundle-sheath PIP transcript levels (quantitative RT-PCR) in 35S:mir1–8 plants, plants with bundle sheath-specific expression cells of WT plants (SCR: of NtAQP1 (SCR:NtAQP1), 35S:mir1–8 plants expressing the bundle-sheath NtAQP1 (35S:mir1–8 £ SCR: NtAQP1) and the control (WT). The presented data were normalized to the WT. Asterisks indicate significant NtAQP1) had no impact on differences (*P < 0.05, n D 5 independent biological repetitions) between treatments and the control based Kleaf in well-irrigated plants on the raw transcript levels, as calculated using Dunnett’s method. Data were normalized to Actin2 levels. (Fig. 3) is surprising, as this channel was previously reported to contribute to hydraulic conductivity under stress.10,11,13 A possible its 35S:mirPIP1 parent line, with the exception of the up-reg- explanation for this phenomenon might be related to an inherulation of PIP1:1. Expression of NtAQP1 and mir164 was ently high Pf of the bundle-sheath cells that allows maximal Kleaf observed only in the SCR:NtAQP1 and 35S:mirPIP1XSCR: under favorable growing conditions. NtAQP1 and 35S:mir1–3and 8 lines, respectively (Fig. 1). In This assumption led us to examine plants treated with addition, SCR:NtAQP1 and 35S:mirPIP1XSCR:NtAQP1plants 10 mM ABA, which is known to decrease Kleaf by inhibiting had similar NtAQP1 transcript levels, suggesting that AQPs in the veins.6,7 Under ABA treatment conditions, we NtAQP1 is not downregulated by amiRNApip1 (Fig. 1). observed a significant decrease in Cleaf in the WT; whereas Kleaf was measured using a detached-leaf approach7 based on SCR:NtAQP1 plants showed higher (less negative) Cleaf levels the determination of the transpiration rate (E) and leaf water than the WT (no differences were observed in the transpirapotential (Cleaf), which were then used to calculate Kleaf (ratio of tion rates of the different plants). This suggests that NtAQP1 E to Cleaf; Fig. 2). The Kleaf of the 35S:mir1–8 line was signifi- facilitates the movement of water into the leaf through the cantly reduced as compared to that of the control. In contrast to bundle sheath and is not post-transcriptionally regulated by the 35S:mir1–8 plants, the Kleaf of the 35S:mir1–8XSCR: ABA. However, the Kleaf of the SCR:NtAQP1 plants was not NtAQP1 line was no different from that of the control. This significantly different from that observed in the control intermediate value suggests that the expression of NtAQP1 in the (Fig. 3). bundle-sheath cells partially restored the transport of water in the The concentration of ABA in the xylem increases under 35S:mir1–8 plants (Fig. 2). An additional possibility is that the abiotic stress conditions14 and this leads to reduced Cleaf increased Kleaf observed in the 35S:mirPIP1XSCR:NtAQP1 and Kleaf via a reduction of the P-f of the bundle-sheath plants is due to the upregulation of PIP1;1 in those plants, as cells, possibly due to reduced expression and/or activity of compared to the 35S:mirPIP1–8 plants (Fig. 1). AQP.2,6,7 Under these conditions, an artificial increase in The fact that Cleaf and Kleaf could be recovered by target- AQP expression in the bundle-sheath cells should increase ing NtAQP1 in the bundle-sheath cells of the silenced plants water influx, thus Cleaf, as observed in our ABA-treated (35S:mir1–8XSCR:NtAQP1) lends additional support to the leaves (Fig. 3). Moreover, post-translational mechanisms that theory that bundle-sheath AQPs can regulate the movement might reduce AQP activity under stress conditions seem not of water into bundle-sheath cells, as well as Kleaf (or xylem to act on NtAQP1, as this channel is known as a stressefflux;4,6,7,9). Nevertheless, the fact that these plants showed induced AQP.11,13 High root hydraulic conductivity was only partial recovery (Fig. 2) could be related to the expres- maintained in stressed tomato plants that over-expressed sion level induced by the SCR promoter. An additional NtAQP1, as compared with the control plants whose root explanation could be related to the fact that those plants hydraulic conductivity was sharply reduced in response to expressed the silencing agent constitutively. Thus, their entire the stress.10

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Figure 2. Leaf hydraulic conductivity (Kleaf) of the AQP-modified lines. Leaves were harvested from each of the tested lines and their petioles were immediately immersed in artificial xylem sap (AXS). After 1 h, (A) Kleaf 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, Cleaf. Asterisks indicate significant differences (*P < 0.05) between a genotype and the WT, as calculated using Dunnett’s method. Data are means ( §SE ) from 14–17 replicates from 2 independent experiments.

Our results emphasize the dynamic role of the bundle sheath in controlling leaf hydraulic conductance in response to vascular signals. These results underscore the importance of the bundle sheath’s role as a control center, balancing soil-root long term signals and the behavior of the stomata, to regulate the leaf’s water status. References 1. Cochard H, Venisse J-S, Barigah TS, Brunel N, Herbette S, Guilliot A, Tyree MT, Sakr S. Putative role of aquaporins in variable hydraulic conductance of leaves in response to light. Plant Physiol 2007; 143:122-33; PMID:17114274; http://dx.doi.org/ 10.1104/pp.106.090092 2. Martre P, Morillon R, Barrieu F, North GB, Nobel PS, Chrispeels MJ. Plasma membrane Aquaporins play a significant role during recovery from water deficit. Plant Physiol 2002; 130:2101-10; PMID:12481094; http://dx.doi.org/10.1104/pp.009019 3. Postaire O, Tournaire-Roux C, Grondin A, Boursiac Y, Morillon R, Schaeffner AR, Maurel C. A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis. Plant Physiol 2010; 152:1418-30; PMID:20034965; http://dx.doi.org/10.1104/pp.109.145326

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Figure 3. Treatment with ABA led to higher leaf water potential (Cleaf) in the SCR:NtAQP1 line. Leaves were harvested from SCR:NtAQP1 and control plants and fed AXS without ABA (-ABA; A) or with 10 mM ABA (CABA; B) through their petioles. After 1 h, Kleaf was calculated for each individual leaf by dividing the whole-leaf transpiration rate, E, by the absolute value of the leaf water potential, Cleaf. The asterisk represents a significant difference between SCR:NtAQP1 and the WT (t test, P < 0.05). Data are means ( §SE ) of values from 12–14 replicates from 2 independent experiments.

4. Prado K, Boursiac Y, Tournaire-Roux C, Monneuse J-M, Postaire O, Da Ines O, Da Ines O, Sch€affner AR, Hem S, Santoni V, et al. Regulation of Arabidopsis leaf hydraulics involves light-dependent phosphorylation of aquaporins in veins. Plant Cell 2013; 25:1029-39; PMID:23532070; http://dx.doi. org/10.1105/tpc.112.108456 5. Ache P, Bauer H, Kollist H, Al-Rasheid KAS, Lautner S, Hartung W, Hedrich R. Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non-invasive pressure probe measurements. Plant J 2010; 62:1072-82; PMID:20345603 6. Pantin F, Monnet F, Jannaud D, Costa JM, Renaud J, Muller B, Simonneau T, Genty B. The dual effect of abscisic acid on stomata. New Phytol 2013; 197:65-72; PMID:23106390; http://dx.doi. org/10.1111/nph.12013

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7. Shatil-Cohen A, Attia Z, Moshelion M. Bundle-sheath cell regulation of xylem-mesophyll water transport via aquaporins under drought stress: a target of xylemborne ABA? Plant J 2011; 67:72-80; PMID:21401747; http://dx.doi.org/10.1111/j.1365-313X.2011.04576.x 8. Sade N, Galle A, Flexas J, Lerner S, Peleg G, Yaaran A, Moshelion M. Differential tissue-specific expression of NtAQP1 in Arabidopsis thaliana reveals a role for this protein in stomatal and mesophyll conductance of CO2 under standard and salt-stress conditions. Planta 2014; 239:357-66; PMID:24170337; http://dx.doi. org/10.1007/s00425-013-1988-8 9. Sade N, Shatil-Cohen A, Attia Z, Maurel C, Boursiac Y, Kelly G, Granot D, Yaaran A, Lerner S, Moshelion M. The role of plasma membrane aquaporins in regulating the bundle sheath-mesophyll continuum and leaf hydraulics. Plant Physiol 2014; 166:1609-20; PMID: 25266632; http://dx.doi.org/10.1104/pp.114.248633

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Plant Cell 2002; 14:869-76; PMID:11971141; http:// dx.doi.org/10.1105/tpc.000901 12. Brown NJ, Palmer BG, Stanley S, Hajaji H, Janacek SH, Astley HM, Parsley K, Kajala K, Quick WP, Trenkamp S, et al. C-4 acid decarboxylases required for C-4 photosynthesis are active in the mid-vein of the C-3 species Arabidopsis thaliana, and are important in sugar and amino acid metabolism. Plant J 2010; 61:122-33; PMID:19807880; http://dx.doi.org/10.1111/j.1365313X.2009.04040.x

13. Mahdieh M, Mostajeran A, Horie T, Katsuhara M. Drought stress alters water relations and expression of PIP-type aquaporin genes in Nicotiana tabacum plants. Plant Cell Physiol 2008; 49:801-13; PMID:18385163; http://dx.doi.org/10.1093/pcp/pcn054 14. Tuteja N. Abscisic Acid and abiotic stress signaling. Plant Signal Behav 2007; 2:135-8; PMID:19516981; http://dx.doi.org/10.4161/psb.2.3.4156

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10. Sade N, Gebretsadik M, Seligmann R, Schwartz A, Wallach R, Moshelion M. The role of tobacco aquaporin1 in improving water use efficiency, hydraulic conductivity, and yield production under salt stress. Plant Physiol 2010; 152:245-54; PMID:19939947; http://dx.doi.org/10.1104/ pp.109.145854 11. Siefritz F, Tyree MT, Lovisolo C, Schubert A, Kaldenhoff R. PIP1 plasma membrane aquaporins in tobacco: from cellular effects to function in plants.

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Bundle-sheath aquaporins play a role in controlling Arabidopsis leaf hydraulic conductivity.

The role of molecular mechanisms in the regulation of leaf hydraulics (K(leaf)) is still not well understood. We hypothesized that aquaporins (AQPs) i...
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