Synthesis and import of GDP-L -fucose into the Golgi affect plant – water relations

(cid:1) Land plants evolved multiple adaptations to restrict transpiration. However, the underlying molecular mechanisms are not sufﬁciently understood. (cid:1) We used an ozone-sensitivity forward genetics approach to identify Arabidopsis thaliana mutants impaired in gas exchange regulation. (cid:1) High water loss from detached leaves and impaired decrease of leaf conductance in response to multiple stomata-closing stimuli were identiﬁed in a mutant of MURUS1 (MUR1), an enzyme required for GDP-L -fucose biosynthesis. High water loss observed in mur1 was independent from stomatal movements and instead could be linked to metabolic defects. Plants defective in import of GDP-L -Fuc into the Golgi apparatus phenocopied the high water loss of mur1 mutants, linking this phenotype to Golgi-localized fucosylation events. However, impaired fucosylation of xyloglucan, N-linked glycans, and arabinogalactan proteins did not explain the aberrant water loss of mur1 mutants. (cid:1) Partial reversion of mur1 water loss phenotype by borate supplementation and high water loss observed in boron uptake mutants link mur1 gas exchange phenotypes to pleiotropic consequences of L -fucose and boron deﬁciency, which in turn affect mechanical and morphological properties of stomatal complexes and whole-plant physiology. Our work emphasizes the impact of fucose metabolism and boron uptake on plant – water relations.


Introduction
The leaves and stems of terrestrial plants are covered with a hydrophobic layer called the cuticle, which directs the majority of plant gas exchange toward stomata.Stomata are epidermal pores surrounded by pairs of guard cells.Guard cells respond to multiple environmental factors, for example light, leaf internal CO 2 concentration, drought, air humidity, pathogens and air pollutants such as ozone (O 3 ), to optimize CO 2 uptake and control water loss, or prevent the entry of pathogens into the leaf tissue.Perception of stomata-closing stimuli initiates a complex series of guard cell signaling events ultimately leading to guard cell plasma membrane depolarization and activation of K + out channels (Merlot et al., 2007;Hedrich, 2012;Sierla et al., 2016;Yamauchi et al., 2016;Pei et al., 2022).The release of ions into the apoplast leads to a decrease in osmotic pressure inside the guard cells, which provokes an efflux of H 2 O from the guard cell cytoplasm and vacuole.The consequent drop in guard cell turgor pressure results in closure of stomatal pores (Franks et al., 1998).
To allow the volume and pressure changes, guard cell walls exhibit a high degree of elasticity, which is determined by specialized wall composition (Amsbury et al., 2016;Merced & Renzaglia, 2018;Carroll et al., 2022).Plants deficient in pectin modifications exhibit defects in stomatal movements, which suggests that the status of pectin crosslinking determines the mechanical properties of guard cell walls (Amsbury et al., 2016;Chen et al., 2021).Furthermore, the cellulose microfibrils that fan out radially from the pore (see Shtein et al., 2017 for a recent visualization) provide a hoop reinforcement that limits the increase in guard cell radius and promotes guard cell elongation during the stomatal opening (Woolfenden et al., 2017).Moreover, the stiffness of guard cell walls is not uniform; the most rigid areas are localized at the guard cell poles and ventral walls directly surrounding the pore (Carter et al., 2017).Taken together, next to guard cell signaling, the mechanics of the guard cell walls has a profound role in the execution of stomatal movements (Woolfenden et al., 2018;Yi et al., 2019).Importantly, the expansion and flexing of guard cells need to overcome the turgor pressure of the neighboring epidermal cells.Recently, Nieves-Cordones et al. (2022) found that decreased pavement cell turgor, observed in mutants of K + RECTIFYING CHAN-NEL 1 (KC1) channel subunit due to decreased K + accumulation, results in wider stomatal apertures, higher stomatal conductance, and elevated loss of water from detached leaves.These results demonstrate that the counter pressure exerted onto the guard cells by the pavement cells is required for stomatal closure.
The synthesis of cell wall glycan polymers relies on the availability of nucleotide sugars that constitute the activated precursor forms serving as a donor of sugar moieties (Bar-Peled & O'Neill, 2011).The importance of nucleotide sugar synthesis and transport can be exemplified by the requirement of GDP-L-fucose for proper growth and development.The synthesis of GDP-L-Fuc is initiated by GDP-D-mannose 4,6dehydratases (GMD1) and MURUS1 (MUR1/GMD2) that catalyze the conversion of GDP-D-mannose to GDP-4-keto-6deoxy-D-mannose (Supporting Information Fig. S1; Bonin et al., 1997Bonin et al., , 2003)).
To understand the processes controlling plant gas exchange, ozone (O 3 ) can be used as an apoplastic ROS donor to stimulate stomatal closure (Overmyer et al., 2000;Kollist et al., 2007;Vahisalu et al., 2010).Plants deficient in O 3 -induced stomatal closure, or those in which epidermal integrity is affected, receive high doses of O 3 that triggers the formation of visible hypersensitive response-like lesions.Here, we describe the identification of the MUR1 mutant from an O 3 -sensitivity screen and show that synthesis and import of GDP-L-fucose into the Golgi play an important role in regulating plant gas exchange.Our results are consistent with the hypothesis that the gas exchange phenotypes observed in mur1 mutants are independent from stomatal movements and we propose that changes in stomatal morphology, and likely also elevated water vapor permeability of mesophyll/epidermal barriers, contribute to the high rates of water loss observed in mur1 mutants.

Materials and Methods
See Methods S1 for detailed description of methods used in this study.

Plant material and growth conditions
Mutant T7-9/mur1-10 was identified from the genetic screen involving the use of environmental simulation chambers (ExpoSCREEN, Helmholtz Zentrum M€ unchen) as described earlier (Sierla et al., 2018).Aside from mutant T7-9/mur1-10 that was in pGC1:YC3.6genetic background and qua1-1 (Ws-4 background), all lines used in this study were in Col-0 background.See Dataset S1 for description of all Arabidopsis thaliana (L.) Heynh.lines used in this study.E.coli and Agrobacterium tumefaciens strains carrying the hpGFT1 construct (Rautengarten et al., 2016) were obtained from Joshua L. Heazlewood.T1 hpGFT1 plants were generated as described before (Rautengarten et al., 2016).Two-wk-old T1 plants were transplanted to soil and grown for additional 3 wk as described below.The same selection procedure has been applied to select for empty vector lines.Other control lines were grown in parallel, except the selection antibiotic was not included in the growth medium.
Unless specified otherwise, seeds were suspended in 0.1% agarose solution, vernalized in the dark for 2 d at 4°C and sown on a 1 : 1 mixture of peat and vermiculite.Plants were grown in controlled growth rooms under 12 h : 12 h, light (200 lmol m À2 s À1 ) : dark cycle, 22°C : 18°C (day : night), 60% : 70% relative humidity.If not otherwise specified, experiments were performed on 3.5-wk-old soil-grown plants.

Water loss assay
Two middle-aged leaves of 3.5-wk-old soil-grown plants were cut and dried abaxial side up at room temperature for 2 h, unless specified otherwise.Mass of leaves was determined before and after drying, and water loss was calculated as percentage of initial fresh weight loss.

Ion leakage assay
The ion leakage was performed by collecting whole rosettes of 3.5-wk-old soil-grown plants into 15 ml of MilliQ water and measuring the conductance of the solution after 6 and 18 h using conductivity meter (FiveEasy FE30; Mettler-Toledo, Columbus, OH, USA).Later, samples were frozen (À20°C) and thawed at room temperature, and the final conductance measurement was performed.Ion leakage was calculated as (%) of conductance recorded after thawing.

Toluidine blue dye-exclusion assay
Toluidine blue dye-exclusion assays (Tanaka et al., 2004) were performed on middle-aged leaves of 3.5-wk-old soil-grown plants.Two 4 ll drops of staining solution (0.05% w/v toluidine blue, 0.01% v/v Tween 20) were applied to adaxial side of the leaves, one on each side of the central vein.Subsequently, plants were covered with transparent lids to maintain high humidity.After 2 h, leaves were abundantly sprayed with water to remove the staining solution.After water has evaporated, leaves were detached and photographed with Nikon D5100 camera equipped with AF-S Micro Nikkor 40 mm 1 : 2.8G objective (Nikon, Tokyo, Japan).

Infrared imaging
Three middle-aged leaves were detached and imaged with Optris PI 450 infrared camera (Optris GmbH, Berlin, Germany) equipped with 10 mm lens.Images were captured at 2-s interval, and leaf temperatures were extracted with the use of OPTRIS PI CONNECT (v.2.9.2147.0)software.
Waxes were extracted by individually dipping 5-6 middleaged leaves of 5-wk-old soil-grown plants into chloroform for 30 s.The samples were dried under a stream of nitrogen, and analysis of wax monomers was performed as described previously (Fahlberg et al., 2019).
Approximately 0.5 g of leaf material collected from 4.5-wk-old soil-grown plants was processed as described earlier (Jenkin & Molina, 2015).Levels of individual cutin components were analyzed by GC-MS.

Gas exchange analysis
The gas exchange experiments were performed with multicuvette gas exchange system (PlantInvent Ltd, Tartu, Estonia) as described previously (Sierla et al., 2018).Leaf conductance was calculated as transpiration divided by the difference in molar concentrations of water vapor inside the leaf and in the ambient air in the measuring chamber (Kollist et al., 2007).To characterize minimal leaf conductance, plants were incubated for at least 1 h in darkness at 800 ll l À1 CO 2 to induce maximal stomatal closure.After stabilization of leaf conductance, rosettes were detached from the roots with a razor blade and leaf conductance measurements were continued for 64 min.The stabilized new levels of leaf conductance were defined as minimal leaf conductance.

Mapping by next-generation sequencing
Rosettes were harvested in bulk and used for preparation of nuclear DNA.The nuclear DNA-enriched sample was sequenced (2 9 150 bp) using NextSeq 500 sequencer (Illumina, San Diego, CA, USA) to an c. 50-fold genome coverage at DNA Sequencing and Genomics Laboratory, Institute of Biotechnology, University of Helsinki.Data were analyzed according to the procedure described earlier (Sun & Schneeberger, 2015).

Expression analysis by qPCR
For gene expression analysis of T-DNA insertion mutants, plants were grown vertically on half-strength Murashige & Skoog medium (Duchefa Biochemie, Haarlem, the Netherlands) in controlled growth chambers (model MLR-350; Sanyo, Osaka, Japan) under 12 h : 12 h, light (130-160 lmol m À2 s À1 ) : dark cycle, 22°C : 18°C (day : night).For every biological replicate (three in total), c. 10 whole 2-wk-old plants were pooled, frozen, and ground in liquid nitrogen.For analysis of GFT1 transcript level, whole rosettes of T1 hpGFT1 plants (minus two middleaged leaves that were used for water loss assay) were frozen in liquid nitrogen and ground with a mortar and pestle.RNA isolation, cDNA synthesis, qPCR, and data analysis were performed as described previously (Xu et al., 2015).

Stomatal morphology
Cotyledons (one cotyledon per plant, four-six plants per line per biological replicate, three biological replicates) were processed (see Methods S1), and serial images (18009 magnification, 10% overlap) were taken with Quanta FEG 250 (Thermo Fisher Scientific, Waltham, MA, USA) scanning electron microscope at the Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki.Images were stitched in IMAGEJ (Schindelin et al., 2012) implementing MIST plugin (Chalfoun et al., 2017).For each cotyledon, from 1 to 2.5 mm 2 area was analyzed, containing 100-350 stomatal complexes.Stomatal morphology was assessed visually and assigned into one of the four categories: normal appearance, not determined, covered, and abnormally large/obstructed.The size of stomatal pores was determined with the use of the IMAGEJ.
For observation of stomata on abaxial side of true leaves c. 4 9 4 mm fragments of middle-aged leaves were excised, mounted on aluminum stubs, and plunged into slush nitrogen.Samples were coated with 5 nm platinum in Quorum PP3010T Cryo-SEM preparation system, and SEM images were acquired with JEOL JIB-4700F Multi Beam System at the OtaNano-Nanomicroscopy Center, Aalto University.Five to six 0.2 mm 2 images per each leaf fragment were captured, and the stomatal density and morphology were assessed with the help of IMAGEJ.

Projected rosette area
Rosettes were photographed with Nikon D5100 camera equipped with AF-S Micro Nikkor 40 mm 1 : 2.8G objective (Nikon).The projected rosette area was determined with IMAGEJ.

Atomic force microscopy
For AFM experiments, seeds were stratified for 7 d at 4°C, then grown in a 3 : 1 compost : perlite mix.Growth conditions were as follows: light intensity 170 lmol m À2 s À1 , 12 h : 12 h, day : night, (21°C) : (17°C), 60% humidity.Leaves from c. 21-d-old plants were analyzed as described previously (Carter et al., 2017).Dissected and plasmolyzed (0.55 M mannitol; minimum 45 min) leaf blocks (c. 5 9 5 mm square) were indented using a Nano Wizard 3 AFM (JPK Instruments, Berlin, Germany) mounted with a nominal 5 nm diameter pyramidal indenter (Windsor Scientific, Slough, UK) on a cantilever of nominal 45 N m À1 stiffness.Cantilever stiffness was calibrated using the thermal tuning method available in the JPK controller software.Cantilever sensitivity was calibrated by performing indentation measurements on a glass slide and was repeated for each experiment.
For each leaf block, an area of 100 9 100 lm was indented by splitting the area into a grid of 128 9 128 indentations.Indentations were stopped when an indentation force of 1000 nN was reached, which corresponds to an indentation depth of between 100 and 1000 nm.Force indentation curves were analyzed with the JPKSPM Data Processing software (JPK Instruments; v.spm 5.0.69) using the following steps: Raw voltage values converted to force values using the calibrated cantilever sensitivity and stiffness, baseline corrected for offset and tilt, displacement offset corrected, indentation calculation, and material property calculation using a Hertzian indentation model to the approach curve.Use of the Hertzian model assumes an infinite homogenous elastic half space, which is clearly not the case for the leaf surface.For this reason, we report results as an apparent modulus (E a ).Retraction curves were discarded due to numerous complications with adhesion between the tip and sample surface.

Observation of dark-elongated hypocotyls by confocal microscopy
Seeds were stratified, surface-sterilized, and sown on agarsolidified (0.8% w/v) 1/2 MS (Duchefa Biochemie) supplemented with 0.5 g l À1 MES pH 5.8 and 1% sucrose.Following 7 h exposure to light, plates were incubated vertically for 4 d in controlled growth chambers (MLR-350; Sanyo) at 23°C in darkness.Seedlings were stained with 0.2 mg ml À1 propidium iodide (PI, Sigma-Aldrich) and imaged using Leica Stellaris 8 confocal microscope equipped with the Leica Application Suite X package.Propidium iodide (PI) excitation was performed using a 552 nm solid-state laser, and fluorescence was detected at 600-650 nm.Stacks of 1024 9 1024 pixels optical section were generated with a Z interval of 1 lm.3D images were reconstructed using the Leica Application Suite LAS X 3D v.4.5.0.In every biological replicate (3 in total), 5-10 plants per line were imaged.

Measurement of stomatal apertures
Plants were sprayed with a solution containing 5 lM ABA (Duchefa), 0.012% (v/v) Silwet, and 0.05% (v/v) ethanol or mock solution.After 45 min, two middle-aged leaves per plant were detached and the abaxial side was immediately coated with Xantopren M Mucosa (Kulzer GmbH, Hanau, Germany).After 24 h, the molds were peeled off and coated with transparent nail polish.The nail polish imprints were imaged with the use of Leica DMLB microscope equipped with Leica 209 Fluotar objective.Two images per leaf were captured, one per each side of the central vein.Images were analyzed with IMAGEJ, and stomatal apertures were calculated as stomatal length : width ratio.

Lack of MUR1 results in high rates of water loss
To identify novel regulators of plant gas exchange, we performed a multistep forward genetic screen based on O 3 -sensitivity (Sierla et al., 2018;Takahashi et al., 2022).The screen was performed on EMS-mutagenized pGC1:Yellow Cameleon 3.6 (YC3.6,Yang et al., 2008) line.Aside from O 3 -sensitivity, the water loss assay, also known as 'mass loss of detached leaves, MLD' (Duursma et al., 2019), was used in this screen as a simple indicator of minimal leaf conductance (g min ).The g min is a sum of cuticular conductance (g cuti ) and residual stomatal conductance observed after the detachment of leaves (minimal stomatal conductance, g s-min ).Elevated water loss can be observed in mutants impaired in stomatal closure (Vahisalu et al., 2008;Hõrak et al., 2016;Sierla et al., 2018) or cuticular/epidermal integrity, for example those deficient in cutin biosynthesis (Bessire et al., 2007;Jakobson et al., 2016) or epidermal cell adhesion (Bouton et al., 2002;Mouille et al., 2007).
From this screen, we isolated mutant T7-9 that exhibited high water loss from detached leaves (Fig. 1a) and altered gas exchange dynamics in response to O 3 , high CO 2 concentration, ABA, and darkness (Fig. S2).To identify the T7-9 causative mutation, we applied the SHOREmap backcross pipeline (Hartwig et al., 2012).Approximately 21% of the BC1 F2 plants (123 out of 593) exhibited increased water loss, indicating that the trait was determined by a single recessive mutation.
Screening of the mutant lines for the five candidate genes (Table S1) revealed that three independent mutant lines: mur1-1, mur1-2 (Reiter et al., 1993;Bonin et al., 1997) and mur1-9 (SALK_057153, Fig. S3), carrying mutations within AT3G51160 (Fig. 1b), exhibited highly elevated water loss (Fig. 1c), while mutant lines for the remaining candidate genes did not (Fig. S4).Monitoring of leaf temperature during the water loss experiment revealed that, in Col-0, detachment of leaves significantly increased leaf temperature while the temperature of mur1 leaves closely resembled that of ghr1-3 indicating comparable rates of g min (Fig. S5).

Research
New Phytologist Therefore, we investigated the level of GDP-L-Fuc in the T7-9 mutant.Like in other mur1 mutants, we were not able to detect this metabolite in T7-9, suggesting complete loss of MUR1 enzymatic activity (Fig. 1d).Finally, an allelism test between T7-9 and mur1-2 mutant revealed lack of complementation, confirming that the T7-9 MUR1 E175K mutation (hereafter referred to as mur1-10) conferred its high water loss (Fig. 1e).

Impaired import of GDP-L-fucose into the Golgi apparatus results in high rates of water loss
Following the identification of mur1 mutant, we set out to investigate the water loss of mutants impaired in the GDP-L-Fuc salvage pathway and import into the Golgi.The water loss of fkgp mutants (fkgp-1, fkgp-2, Kotake et al., 2008) was similar to that of Col-0 (Fig. S6), indicating that the L-fucose salvage pathway has little impact on plant gas exchange.Further, we focused on characterization of the GDP-L-Fuc transporter GFT1.Loss-of-function mutants of GFT1 are not viable; therefore, we utilized the hairpin RNAi strategy used earlier by Rautengarten et al. (2016) to generate GFT1 knockdown plants (hpGFT1).A total of 66 independent hpGFT1 T1 plants were selected and transplanted to soil.As observed before (Rautengarten et al., 2016), the hpGFT1 plants exhibited varying growth phenotypes, that is reduced projected rosette area, short petioles and wavy leaves (Fig. S7a).
For each of the T1 hpGFT1 plant that survived in soil (64 plants), we measured the projected rosette area, loss of water from detached leaves, and the GFT1 transcript level (58 plants).The hpGFT1 T1 plants exhibited varying water loss (Fig. 2a) and residual GFT1 transcript level (Fig. 2b) and displayed a clear negative correlation between these two traits (Figs 2c, S7b).Similarly, the projected rosette area was also negatively correlated with the water loss (Fig. S7c).The majority of hpGFT1 plants with GFT1 transcript level lower than 10% of that observed in the empty vector control displayed water loss comparable to that of mur1 mutants (Fig. 2c).Thus, we conclude that not just the synthesis but also the import of GDP-L-Fuc into the Golgi lumen is required for normal plant gas exchange.

MUR1 is required for regulation of plant gas exchange
To further characterize the gas exchange dynamics in mur1 mutants, we subjected them to a variety of treatments provoking stomatal movements and followed time-resolved whole-rosette transpiration and leaf conductance (Kollist et al., 2007;Jakobson et al., 2016).Gas exchange measurements were performed for mur1-1 and mur1-2 mutants (Reiter et al., 1993;Bonin et al., 1997) using ghr1-3 (Sierla et al., 2018) and ht1-2 (Hashimoto et al., 2006) as nonresponsive controls in closure and opening assays, respectively.
Similarly, a decreased response upon treatment with elevated CO 2 concentration (800 ll l À1 ), 5 lM ABA spray or application of darkness during the light period was observed in mur1 mutants (Figs 3c-h The stimuli provoking stomatal opening, such as exposure to low CO 2 concentration (400 ? 100 ll l À1 ) or increase in light intensity (150 ?500 lmol m À2 s À1 ), did not show any differences between mur1 mutants and the wild-type (WT; Fig. S10).
To estimate minimal leaf conductance, we treated mur1 and Col-0 plants with a combination of stomata-closing stimuli (elevated CO 2 concentration and darkness) for 1 h, after which the rosettes were separated from roots to ensure maximal stomatal closure.Such treatment triggered reduction of leaf conductance in all tested lines; however, in mur1 mutants the leaf conductance stabilized at values two times higher than in Col-0 (Fig. S11).Taken together, our data indicate that stomata of mur1 plants responded to O 3 , high CO 2 concentration, ABA, and darkness; however, with no regard to the stimulus, the post-treatment leaf conductance always remained higher than that of the WT plants indicating general impairment in gas exchange regulation.

MUR1 is involved in stomatal development
To assess stomatal development, we performed scanning electron microscopy-based examination of abaxial epidermis of mur1-1 and mur1-2 cotyledons.No consistent phenotype related to stomatal density was observed in mur1 mutants.The stomatal density in mur1-2 mutant was higher than in mur1-1, which had a similar stomatal density to Col-0 (Fig. 4a).The average size of stomatal pores was moderately increased in the mur1-1 mutant, while in mur1-2 there were no significant differences compared with Col-0 (Fig. 4b).Notably, in cotyledons of both mutants we sporadically observed abnormally big stomatal complexes (4-5% of total stomata, Fig. S12a) that were irregular in shape and often visibly obstructed (Fig. S12b).Moreover, a fraction of mur1 stomata exhibited aberrant structure of the outer cuticular ledge (OCL).In the most severe cases (5-10% of stomata, Fig. S12a), stomatal pores appeared covered with cuticle (Fig. 4c).
Examination of middle-aged rosette leaves, that is leaves which were used for water loss assay, by means of cryo-SEM revealed similar phenotypes (Dataset S3a), except no abnormally big/ obstructed stomata were detected.The stomatal density was elevated in mur1-2 (Fig. 4d), and in both mutants, c. 10% of stomata were covered with OCLs (Fig. 4e).Overall, despite the differences in stomata size and morphology, in the majority of experiments, the daytime whole-rosette leaf conductance of mur1 mutants did not differ significantly from that of the WT plants (Figs S8E, S13).We however observed that the leaf conductance of mur1-2 tended to be smaller than that of Col-0 (Fig. S13).This difference is probably associated with the higher frequency of 'covered' stomata observed in this mutant (Fig. 4e).
High rate of water loss is independent of stomatal movements in mur1 mutants A series of experiments was performed to elucidate whether altered plant gas exchange dynamics observed in mur1 mutants is related to impaired guard cell signaling or stomatal movements.We crossed mur1-1 and mur1-2 to slac1-4 (Vahisalu et al., 2008), aba2-11 (Gonz alez-Guzm an et al., 2002), ost1-3 (Yoshida et al., 2002), and ghr1-3 (Sierla et al., 2018) and assayed the stomatal function of the double mutants via water loss assay.In every double mutant, an additive effect of combining two mutations could be observed (Fig. 5a), indicating that the phenotypes observed in mur1 plants were independent from the canonical guard cell signaling pathways.
Because of the additive effects and the previously documented role of MUR1 in cell wall development, we investigated the mechanical properties of mur1 guard cell walls with atomic force microscopy (Carter et al., 2017).The patterning of the apparent modulus (E a ) in the stomatal complexes of mur1 mutants was comparable to that of the control lines.However, comparison of the absolute E a values derived from the AFM scans indicated that mur1 mutants had significantly stiffer pavement cells (approximately twofold increase on average) and the same difference was observed when values obtained for guard cell walls were compared (Fig. 5b).We therefore conclude that lack of MUR1 affected mechanical properties of the leaf epidermis.
To investigate whether these properties affect stomatal movements, we assessed the closure of mur1 stomata in response to ABA spray by means of microscopic imaging of stomatal width : length ratio in epidermal imprints.For this, whole rosettes of Col-0, mur1-1, mur1-2, and the ABA-unresponsive ghr1-3 mutant (Sierla et al., 2018) were treated with ABA and the stomatal apertures were recorded after 45 min.This treatment led to a pronounced decrease of stomatal aperture in Col-0 while the ghr1-3 mutant was largely unresponsive (Fig. 5c).In contrast to ghr1-3, the stomatal apertures of both mur1 mutants decreased nearly to the same extent as those of Col-0 plants, indicating capability for the execution of stomatal movements in response to ABA (Fig. 5c).Analogical treatment with ABA performed 1 h before leaf excision was not sufficient to suppress the elevated water loss of mur1 mutants (Fig. S14) indicating little correlation between the outcome of water loss assay and initial aperture of stomatal pores.
Furthermore, by microscopic real-time imaging of stomatal movements, we investigated whether mur1 stomata close during the water loss assay.In these experiments, ost1-3 mutant was used as a control.Before leaf excision, stomatal apertures were significantly larger in ost1-3 while no differences between Col-0 and mur1 mutants were observed (Fig. 5d,e).Leaf excision induced a decline, and stomata of mur1 mutants reacted to this treatment similarly as those of Col-0 (Fig. 5d,e; Video S1).The stomatal apertures of ost1-3 remained significantly higher than those of Col-0 and mur1 throughout the experiment.Moreover, a large number of ost1-3 stomata were open at the final time point of the experiment (45 min).We therefore concluded that high water loss observed in mur1 mutants was uncoupled from stomatal movements.
Additionally, we investigated whether reduction of leaf conductance observed in mur1 mutants during the day/night transition (Fig. S8e) can affect the outcome of water loss assay.For this, we performed water loss experiments 2 h following the onset of illumination and the night period, respectively.In these experiments, ost1-3 and ghr1-3 mutants were used as controls because ost1-3 exhibits high g s during the day and a clear decrease during the night while ghr1-3 maintains high g s irrespectively of the diurnal rhythm (Sierla et al., 2018).In ost1-3 mutant, values recorded at night decreased by half compared with daytime water loss, suggesting that the decrease in g s observed during day/night transition was sufficient to limit the nocturnal water loss in this mutant.In contrast, the night-time water loss of ghr1-3 and both mur1 mutants was nearly as high as that recorded during the day, albeit a statistically significant difference was observed in mur1-1 (Fig. 5f).We therefore conclude that high nocturnal water loss observed in mur1 mutants was independent of the decrease in leaf conductance observed during the day/night transition.

Epidermal permeability, cuticle composition, and cell adhesion in mur1 mutants
The apparent lack of correlation between the whole-rosette leaf conductance and stomatal movements as well as high nocturnal water loss suggested that mur1 mutants lose water through nonstomatal paths.Therefore, we set out to assess their cuticular and epidermal integrity.No phenotypes related to cuticle

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New Phytologist permeability were detected in mur1 mutants by means of toluidine blue dye-exclusion assay (Fig. S15a); however, a moderate but consistent (albeit not always statistically significant) increase in whole-rosette ion leakage could be observed (Figs 6a, S16).
The total content of cutin monomers in mur1 mutants was comparable to Col-0; however, differences in relative abundance of monomers could be observed (Table S2).The analysis of cuticular wax composition revealed much more pronounced differences (Fig. 6b; Table S3), with total wax levels nearly doubled in mur1 mutants compared with Col-0.In both mutants, increased abundance of fatty alcohols, fatty acids, and sterols was observed, while the abundance of long-chain aldehydes was reduced in mur1-2 (Fig. 6b; Table S3).Taken together, we conclude that impaired function of MUR1 had little effect on total cutin content but led to changes in cutin composition and overproduction of cuticular wax.
Additionally, we tested whether lack of GDP-L-Fuc leads to loss of epidermal cell adhesion as the water loss of the cell adhesion mutant quasimodo2 (qua2-1; Mouille et al., 2007) was comparable to that of mur1 (Fig. 6c).Indeed, during observation of dark-elongated hypocotyls of mur1 mutants we sporadically observed symptoms of loss of cell adhesion, visible as peeling of epidermal cells; however, this phenotype was not as severe as that of qua2-1 (Fig. S17).Moreover, we found that cell adhesion mutants qua2-1 and qua1-1 (Bouton et al., 2002) stained strongly with toluidine blue (Fig. S15b) and exhibited ion leakage much higher than that of mur1 mutants (Fig. 6a).In qua2, the loss of cell adhesion requires ESMERALDA1 (ESMD1) and can be suppressed by introduction of the esmd1-1 mutation (Verger et al., 2016).We found that the water loss and ion leakage were nearly fully suppressed in qua2-1 esmd1-1 double mutant but not in mur1 esmd1-1 double mutants (Figs 6c, S16).Finally, during the examination of mur1 epidermis by means of cryo-SEM we did not detect any morphological signs indicative of loss of cell adhesion that are otherwise readily visible in cell adhesion mutants (Verger et al., 2018;Lorrai et al., 2021;Dataset S3b,c).We therefore conclude that the lack of GDP-L-fucose had a minor effect on the adhesion of epidermal cells, and loss of epidermal cell adhesion was not the cause of elevated water loss observed in mur1 mutants.

High water loss in boron uptake mutant
To investigate whether high water loss of mur1 was related to the dimerization of RG-II, we grew mur1 mutants in soil supplemented with 1 mM borate.Such treatments were previously shown to compensate for the deficiency in RG-II crosslinking (O'Neill et al., 2001).We observed a significant decrease in water loss of mur1 mutants grown in the presence of 1 mM borate compared with the control conditions (Fig. 7a).A similar trend was observed in Col-0, slac1-4, and ghr1-3 mutants, albeit to a much lower extent, possibly indicating general reduction in transpiration to prevent excess B accumulation (Macho-Rivero et al., 2018).To validate this finding, we utilized plants lacking the REQUIRES HIGH BORON1 (BOR1) boron transporter required for boron xylem loading (Noguchi et al., 1997;Takano et al., 2002) as the impaired uptake of boron was previously demonstrated to affect not only the dimerization of RG-II (Miwa et al., 2013;Panter et al., 2019) but also membrane integrity (Cakmak et al., 1995;Han et al., 2008).We found that a soilgrown bor1-3 mutant (Kasai et al., 2011) exhibited high water loss which could be fully reverted by supplementing the soil with 50 lM borate, while lower concentrations (10 and 20 lM) had less effect (Fig. 7b).The water loss of mutants impaired in fucosylation of xyloglucan (mur2-1; Vanzin et al., 2002); arabinogalactan proteins (fut4 fut6; Tryfona et al., 2014) and N-glycans: fucta fuctb (Strasser et al., 2004), fuctc-1 (Rips et al., 2017) was comparable to that of Col-0 (Fig. 7c), suggesting that high water loss observed in mur1 was not related to impaired fucosylation of these cell wall components.Collectively, our data supported the hypothesis that high water loss observed in mur1 plants is related to a deficiency in RG-II crosslinking and/or membrane integrity.

Discussion
Synthesis of GDP-L-fucose and boron uptake are required for normal plant gas exchange Here, we report the identification of impaired regulation of gas exchange in plants lacking MUR1an enzyme catalyzing the first step in de novo GDP-L-Fuc synthesis pathway (Bonin et al., 1997).Plants lacking GDP-L-Fuc exhibited high loss of water from detached leaves (Fig. 1) and impaired decrease in leaf conductance in response to O 3 , ABA, darkness, and high CO 2 concentration (Figs 3, S8, S9; Dataset S2).High water loss observed in mur1 appears independent from canonical guard cell signaling pathways (Fig. 5a) and was also detected in plants impaired in import of GDP-L-Fuc into the Golgi apparatus (Fig. 2).Therefore, we conclude that not only synthesis but also the import of GDP-L-Fuc into the Golgi lumen is required for normal gas exchange.
The fucosylation of cell wall components is thought to occur in the Golgi apparatus (Fig. S1; Chou et al., 2015;Strasser, 2016), and the majority of fucose is incorporated into the cell wall via the Golgi-derived vesicles (Anderson et al., 2012).We excluded the possibility that phenotypes observed in mur1 might be linked to impaired fucosylation of AGPs, N-linked glycans, and xyloglucan (Fig. 7c).The reversion of the mur1 water loss phenotype by borate supplementation (Fig. 7a), and high water loss observed in bor1-3 mutant (Fig. 7b), suggests that the phenotype was linked either directly or indirectly, to the structure and dimerization of RG-II.Recently, Panter et al. (2023) arrived at the same conclusions.
Depletion of GDP-L-Fuc also likely impairs protein o-fucosylation.The only characterized Arabidopsis protein o-fucosyltransferase SPINDLY (Zentella et al., 2017) was shown to be required for the great majority of o-fucosylation events (Bi et al., 2023).However, we did not detect high water loss in spy mutants (data not shown).Moreover, SPY is a nucleocytoplasmic protein while our data suggest that import of GDP-L-fucose into Golgi is required to restrict water loss (Fig. 2).Taken together,

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we conclude that high water loss observed in mur1 mutants was related to impaired RG-II crosslinking.However, based on data generated in this work, we cannot exclude additional functions of GDP-L-Fuc and boron, that could also contribute to gas exchange regulation (discussed further below).

Stomatal movements and cuticular barriers in mur1 mutants
Our AFM data indicated that the cell walls of mur1 guard cells and epidermal cells were significantly stiffer than that of WT plants (Fig. 5b).While mechanical properties of guard cell walls are expected to change the dynamics of stomatal movements (Jones et al., 2003;Amsbury et al., 2016;Rui et al., 2017), our data clearly indicate that mur1 stomata were capable of closure (Fig. 5c-e; Video S1).Similar conclusions can be drawn from studies by Zhang et al. (2019) and Panter et al. (2023) who demonstrated nearly complete closure of mur1 stomata in response to ABA treatment.Moreover, we found that mur1 stomatal responses to opening stimuli were not impaired (Fig. S10), suggesting that altered mur1 cell wall mechanics does not have a profound effect on stomatal movements.Finally, we found that pretreatment with ABA or night stimulus which clearly decreased leaf conductance of mur1 mutants (Fig. S8e) had little effect on mur1 water loss (Figs S14,5f).Thus, we conclude that high water loss observed in mur1 mutants cannot be readily compared to that observed in classical stomatal mutants such as ghr1 or ost1 and likely has a different mechanistic origin.Despite moderate increase in ion leakage (Fig. 6a) and changes in cutin composition (Fig. 6b; Table S2), mur1 mutants did not exhibit elevated leaf conductance (Fig. S13) or staining with toluidine blue (Fig. S15a; Lorrai et al., 2021;Panter et al., 2023) that is otherwise typical for cuticular and cell adhesion mutants (Jakobson et al., 2016;Fig. S15).In agreement with these data, we did not detect morphological symptoms of loss of epidermal cell adhesion in mur1 mutants (Dataset S3) and found that the introduction of esmd1-1 mutation into the mur1 background did not reduce its water loss (Fig. 6c).
Loss of epidermal/cuticular barriers caused by impaired cutin deposition (Bessire et al., 2007;Voisin et al., 2009) or loss of epidermal cell adhesion (Lorrai et al., 2021) was found to cause strong resistance to Botrytis cinerea.However, in the same study Lorrai et al. (2021) observed no increased resistance to this pathogen in mur1 mutant, suggesting no radical changes in the permeability of epidermal/cuticular barriers.Taken together, our data suggest that gas exchange phenotypes observed in mur1 did not stem from general defects in cutin synthesis or epidermal cell adhesion, that is elevated g cuti .

How do mur1 mutants lose water?
Due to the limited phenotypical similarities between mur1 and classical mutants impaired in stomatal movements, cutin synthesis or epidermal cell adhesion, mur1 mutants cannot be clearly placed into any of these categories.The apparent lack of correlation between measured stomatal apertures and leaf conductance and transpiration (Figs 3e, 5c), leaf conductance and water loss (Figs S8e,5f) as well as normal stomatal movements following leaf detachment (Fig. 5d,e; Video S1) suggest that mur1 stomata are permeable to water vapor even in the closed state.This phenomenon, that is high minimal stomatal conductance, is thought to significantly contribute to g min (Kerstiens, 1996;Duursma et al., 2019;Machado et al., 2021).High g s-min can be associated with higher stomatal density, imperfect closure of stomata and possibly other structural features of guard cells or mesophyll.Our data (Fig. 4a,d), combined with earlier reports (Zeng et al., 2011;Panter et al., 2023) indicate that the stomatal density in mur1 mutants is not elevated.The size of stomatal complexes was somewhat elevated in mur1 mutants (Fig. 4b; Zeng et al., 2011;Panter et al., 2023); however, we found that this did not significantly increase the steady-state leaf conductance or transpiration (Figs S8-S10, S13).Thus, the gas exchange profiles of mur1 mutants might be explained by the imperfect closure of stomata that likely leads to an increase in minimal stomatal conductance.
An explanation for the elevated g s-min might be offered by the observation of stomatal morphology in mur1 mutants.In agreement with earlier reports (Zeng et al., 2011;Panter et al., 2023), we found that mur1 stomata exhibit a range of structural defects, most notably, altered structure of outer cuticular ledges (Figs 4, S12; Dataset S3).It is tempting to speculate that changes in  S2, S3) might be responsible for the OCL-related phenotypes.Importantly, the same structural defects were recently identified also in boron uptake mutant (Panter et al., 2023) which implies a link between uptake of boron and morphology of OCLs.However, it cannot be excluded that the cuticle composition changes are a secondary effect of stress signaling (Voxeur et al., 2017;Chen et al., 2022).The precise function of OCLs is still poorly characterized (Hunt & Gray, 2020); however, changes in OCL structure provide little explanation for additive water loss observed in double mutants of mur1 and guard cell signaling components (Fig. 5a).
The excision of leaves leads to a rapid decrease in leaf water potential, leading to transient increase in g s (usually described as 'wrong way response') and initial drop in leaf temperature, which is ultimately followed by maximal stomatal closure with g s values reaching g s-min (Powles et al., 2006) and increase in leaf temperature (Fig. S5).Under conditions of no water supply observed after leaf excision, the relative humidity in the substomatal cavities and mesophyll air spaces is expected to drop below saturation, ultimately triggering, and linking the evaporation rate to the active control of plasma membrane hydraulic conductivity (Wong et al., 2022), while primary cell walls were proposed to influence plant-water relations over 40 yr ago (Jarvis & Slatyer, 1970) and emerge as important regulators of mesophyll conductance to CO 2 (Evans, 2021), within the mesophyll the major resistance to water vapor is attributed mostly to plasma membrane aquaporins (Wong et al., 2022).Aside from controlling cell wall porosity (Fleischer et al., 1999), RG-II was demonstrated to form borate-dependent linkages with plasma membrane sphingolipids in cultured Rosa cells (Voxeur & Fry, 2014) and boron deficiency affects membrane integrity (Cakmak et al., 1995;Han et al., 2008).Increased ion (Figs 6a, S16) and chlorophyll leakage (Lorrai et al., 2021) observed in mur1 mutants likely stem from the same causes.It is thus possible that elevated membrane permeability, which otherwise does not affect the steady-state leaf conductance, gains significance under conditions of severe water deficit, contributing to high water loss observed in mur1 mutants.However, the relative contribution of stomatal morphology and membrane integrity to the observed gas exchange phenotypes awaits further investigation.

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We conclude that the abnormal gas exchange regulation observed in mur1 is linked to altered mechanical and morphological properties of stomatal complexes likely accompanied by additional defects, for example impaired membrane integrity.In summary, our study highlights the key role of fucose metabolism and boron uptake in determining plant-water relations.Table S1 High-frequency single nucleotide polymorphisms identified in BC1F2 mapping population of Arabidopsis thaliana T7-9 mutant.
Table S2 GC-MS analysis of absolute and relative abundance of cutin monomers in Arabidopsis thaliana mur1 mutants.
Table S3 GC-MS analysis of cuticular wax composition in mur1 mutants.
Video S1 Stomatal movements of Arabidopsis thaliana mur1 mutants and control lines (Col-0 and ost1-3) observed after leaf detachment.Leaves were detached from the rosette at t = 5 min ('Excision'), and the imaging was continued for c. 40 min.
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, S8b-d, S9b-d; Dataset S2).During diurnal light/dark cycles, the transition to darkness induced a rapid drop in transpiration and leaf conductance of Col-0 plants while mur1 mutants exhibited a much less pronounced response (Figs S8e, S9e).

Fig. 2
Fig. 2 Effect of impaired import of GDP-L-Fuc into Golgi on the water loss.(a) Leaf fresh weight loss of 64 independent Arabidopsis thaliana hpGFT1 T1 plants (hp, hairpin), and control lines (Col-0, EVCempty vector control, mur1-1, mur1-2, slac1-4, and ghr1-3), recorded after 2 h.Data points represented values obtained for separate plants.Data bars represent means AE SD (for control lines n = 11-12 plants).(b) Variability in GFT1 transcript level observed in 58 independent A. thaliana hpGFT1 T1 plants.Data points represent values obtained for separate plants.Data bars represent means AE SD; Col-0 n = 3 plants, EVC n = 10 plants.(c) Residual GFT1 transcript level and fresh weight loss observed in 58 independent A. thaliana hpGFT1 T1 plants.Each dot represents values obtained for an independent hpGFT1 T1 plant.

Fig. 5
Fig. 5 Functional analysis of mur1 stomata.(a) Leaf fresh weight loss of Arabidopsis thaliana double mutants obtained after crossing slac1-4, ghr1-3, aba2-11, and ost1-3 with mur1-1 and mur1-2 recorded after 1 h.Data bars represent means AE SD (n = 13-16 plants).Asterisks denote statistical differences (***, P < 0.001) to respective single mutant lines (slac1-4, ghr1-3, aba2-11, and ost1-3) according to one-way ANOVA followed by Sid ak's post hoc test.Experiment was repeated three times with similar results.Results of the representative experiment are shown.(b) Average apparent Young's modulus (E a ) values derived from atomic force microscopy (AFM) scans of pavement cells (PC) and guard cells (GC) of control (Col-0, YC3.6) and mur1 A. thaliana plants.Bars represent means AE SD (n = 2-3 plants, 2 stomata per plant).Asterisks denote statistical differences according to twoway ANOVA, followed by Tukey's post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001) to respective control lines (Col-0, YC3.6) observed within a cell type.(c) Normalized stomatal width : length ratio observed on epidermal imprints of abaxial side of A. thaliana middle-aged leaves 45 min after spray with 5 lM abscisic acid (ABA) or mock solution.Data bars represent means AE SD, n = 8 plants analyzed in four biological replicates (two plants per replicate, two leaves per plant, at least 30 stomata per plant).Within each replicate, values were normalized to mock-treated Col-0.Asterisks denote statistical differences (*, P < 0.05; ***, P < 0.001) to Col-0 within each treatment according to two-way ANOVA followed by Sid ak's post hoc test.(d) Time course of stomatal pore width : length ratios observed on abaxial side of middle-aged leaves of Arabidopsis thaliana mur1 mutants and control lines (Col-0 and ost1-3) after leaf detachment.Leaves were detached at t = 0. Data points represent means AE SE n = 24-28 stomata per genotype (six plants per genotype, typically 3-5 stomata per plant).(e) Statistical analysis of data presented in panel (d).Data points represent values recorded for individual stomata, red horizontal bars represent means AE SE.Red asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001) indicate significant differences to Col-0 within each time point according to two-way ANOVA followed by Dunnett's post hoc test.(f) Leaf fresh weight loss of A. thaliana mur1 mutants and control lines (Col-0, ost1-3 and ghr1-3) recorded after 2 h.Experiments were performed 2 h after the beginning (Day), or end (Night), of the light period.Data bars represent means AE SD (n = 6-8 plants).Asterisks (*, P < 0.05; ***, P < 0.001) denote statistical differences (Day vs Night) within each genotype according to two-way ANOVA followed by Sid ak's post hoc test.Experiment was repeated four times with similar results.Results of the representative experiment are shown.

Fig. 6
Fig. 6 Epidermal integrity in mur1 mutants.(a) Ion leakage in 6 and 18 h expressed as % of total ion content.Data bars represent means AE SD (n = 5-6 plants).Asterisks denote statistical differences according to twoway ANOVA, followed by Tukey's post hoc test (***, P < 0.001) to Col-0 observed within each time point.Experiment was repeated seven times with similar results.Results of the representative experiment are shown.(b) Analysis of wax content by GC-MS.Data bars represent means AE SD (n = 8 plants) calculated based on data presented in Supporting Information Table S3.Asterisks denote statistical differences (**, P < 0.01; ***, P < 0.001) to Col-0 within each compound group, according to two-way ANOVA followed by Dunnett's post hoc test.(c) Leaf fresh weight loss of double mutants obtained after crossing mur1-1, mur1-2 and qua2-1 with esmd1-1, recorded after 2 h.Data bars represent means AE SD (n = 7-8 plants).Asterisks denote statistical differences (***, P < 0.001; ns, not significant) to Col-0 (black) or respective single mutant lines (red), according to one-way ANOVA followed by Sid ak's post hoc test.Experiment was repeated four times with similar results.Results of the representative experiment are shown.(a-c) Experiments performed on whole rosettes (a) or middle-aged leaves (b, c) of 3.5-(a, c) or 5-wk-old (b) soil-grown Arabidopsis thaliana plants.

Fig. 7
Fig. 7 Effect of borate supplementation on water loss.(a) The effect of borate supplementation on the leaf fresh weight loss of Arabidopsis thaliana mur1 mutants and control lines (Col-0, ghr1-3, and slac1-4).Bars represent means AE SD (n = 14-16 plants).Data analyzed with two-way ANOVA followed by Sid ak's post hoc test.Asterisks denote statistical significances (**, P < 0.01; ***, P < 0.001) of treatment effect within each genotype.(b) The influence of borate supplementation on leaf fresh weight loss of Arabidopsis thaliana bor1-3 mutant and control lines.Data bars represent means AE SD (n = 10-16 plants per genotype per concentration).Asterisks denote statistical differences within each genotype, according to two-way ANOVA, followed by Tukey's post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001), to plants watered with tap water.(c) Leaf fresh weight loss of A. thaliana fucosyltransferase mutants and control lines recorded after 2 h.Data bars represent means AE SD (n = 10-12 plants).Asterisks denote statistical differences (***, P < 0.001) to Col-0 according to one-way ANOVA followed by Dunnett's post hoc test.(a-c) Experiments were repeated five (a), three (b, c) times with similar results.Results of the representative experiments are shown.

Fig. S6
Fig. S6 Water loss of mutants impaired in L-Fuc salvage pathway.

Fig. S12
Fig. S12 Frequency and phenotypes of atypical stomata observed in cotyledons of Arabidopsis thaliana mur1 mutants.

Fig. S14
Fig. S14 Effect of ABA pretreatment on water loss of mur1 mutants.

Fig. S17
Fig. S17 Phenotyping of cell adhesion in elongated hypocotyls of qua2-1 and mur1 mutants.Methods S1 Detailed description of methods used in this study.