Metabolic engineering of stomatal precursor cells enhances photosynthetic water-use efficiency and vegetative growth under water-deficit conditions inArabidopsis thaliana

Metabolic engineering of stomatal precursor cells enhances photosynthetic water-use efficiency and vegetative growth under water-deficit conditions in Arabidopsis thaliana | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Metabolic engineering of stomatal precursor cells enhances photosynthetic water-use efficiency and vegetative growth under water-deficit conditions in Arabidopsis thaliana View ORCID Profile Jacques W. Bouvier , View ORCID Profile Steven Kelly doi: https://doi.org/10.1101/2024.08.29.610053 Jacques W. Bouvier 1 Department of Biology, University of Oxford , South Parks Road, Oxford, OX1 3RB, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jacques W. Bouvier Steven Kelly 1 Department of Biology, University of Oxford , South Parks Road, Oxford, OX1 3RB, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Steven Kelly For correspondence: steven.kelly{at}biology.ox.ac.uk Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Stomata are epidermal pores that control the exchange of gaseous CO 2 and H 2 O between plants and their environment. Modulating stomatal density can alter this exchange, and thus presents a target for engineering improved crop productivity and climate resilience. Here, we show that stomatal density in Arabidopsis thaliana can be decreased by the expression of a water-forming NAD(P)H oxidase targeted to stomatal precursor cells. We demonstrate that this reduction in stomatal density occurs irrespective of whether the expressed enzyme is targeted to the cytosol, chloroplast stroma, or chloroplast intermembrane space of these cells. We reveal that this decrease in stomatal density occurs in the absence of any measurable impact on stomatal dynamics, or the efficiency or thermal sensitivity of photosynthesis. Consequently, overexpression plants exhibit a higher intrinsic water use efficiency due to an increase in CO 2 fixed per unit water transpired. Finally, we demonstrate that this enhanced water-use efficiency translates to an improvement in vegetative growth and biomass accumulation under water-deficit conditions. Together, these results thus provide a novel approach for enhancing plant productivity through metabolic engineering of stomatal density. Introduction Stomata mediate the exchange of gaseous CO 2 and water vapour between plants and their external environment. Specifically, CO 2 provides the primary substrate of photosynthesis and its uptake is thereby required to fuel plant growth. In contrast, loss of water vapour is integral to the transpiration stream which helps regulate internal water status, temperature, and nutrient uptake. As both CO 2 and H 2 O gases share a single diffusion path but in opposite directions, controlling flux along this path is essential for balancing the physiological demands of the plant. Consequently, optimizing the distribution and behaviour of stomata is a central way in which plants adapt to different terrestrial habitats 1 – 3 . Given their role at the interface between plants and their environment, plants are able to modulate the diffusive conductance of stomata in response to diverse stimuli including light (quality and quantity), humidity, temperature, soil water availability, and atmospheric CO 2 4 . By integrating these signals alongside natural circadian rhythms, stomata open to promote CO 2 fixation during the day and close at night or under adverse conditions to limit excessive water loss. In addition to this complex and dynamic behaviour, stomata also exhibit remarkable developmental plasticity across species and in response to diverse environmental changes. Most notably, stomatal density inversely correlates with soil water availability 5 , temperature 6 , and ambient CO 2 concentration 7 – 13 , and positively correlates with relative air humidity 14 , 15 over both developmental or geological timescales. These environmentally-induced changes in density are generally linked with secondary adjustments in stomatal size (such that higher densities of stomata are generally smaller and vice versa 16 – 19 ), though density always takes precedence over size as the predominant anatomical constraint on leaf gaseous conductance 11 , 20 . Thus, both dynamic and developmental changes in stomata play important roles in the growth and environmental adaptation of plants. The considerable plasticity of stomatal form and function, coupled with the importance of stomata in regulating plant growth and environmental interactions, have together inspired multiple attempts to alter their properties for crop improvement. Pioneering efforts of Farquhar and Richards in the 1980’s used stable carbon isotope ratios to screen for enhanced water use efficiency in wheat 21 , 22 . Subsequently, targeted engineering efforts have included a range of forward genetic approaches which have manipulated the distribution of stomata 23 – 28 , as well as the mechanical 29 , transport 30 , 31 , 40 – 42 , 32 – 39 , and metabolic 34 , 43 , 52 – 56 , 44 – 51 properties of their guard cells. Of all these approaches, altering stomatal density has received the most attention, and a vast array of mutants with both increased 57 , 58 , 67 – 69 , 59 – 66 and decreased 17 , 58 , 72 – 79 , 61 , 64 , 65 , 67 – 71 stomatal densities have been engineered across diverse plant and crop species. A common theme from approaches which have altered stomatal densities has been the genetic manipulation of the expression levels of stomatal development genes. Such targets have included the basic helix-loop-helix transcription factors SPEECHLESS (SPCH) 65 , MUTE 67 , and FAMA 68 which regulate the initiation, proliferation, and differentiation stages of the stomatal lineage, respectively. Engineering success has also been achieved by altering the expression levels of other important components of stomatal development including the EPIDERMAL PATTERNING FACTOR family of signalling peptides 17 , 57 , 76 – 78 , 58 , 63 , 69 – 73 , 75 alongside their receptor components (TOO MANY MOUTHS 66 and the ERECTA protein kinases 62 ), stomatal density and distribution 1 (SDD1) and its respective interactor components 59 , 61 , 74 , and a host of other genes involved in hormone signalling and non-canonical stomatal developmental pathways 60 , 64 , 79 . However, despite these considerable successes, the molecular and biochemical mechanisms that link these gene networks to changes in environmental and physiological cues remain poorly understood. Nicotinamide adenine dinucleotide (NAD + ) presents a potential metabolic nexus that can link physiological changes in the leaf to the genetic regulation of stomatal patterning 80 – 82 . This potential role arises from recent studies which have demonstrated that changes in cellular NAD + status either through exogenous NAD + treatment 81 , knockdown of mitochondrial (NDT1 and NDT2) and peroxisomal (PXN1) NAD + transporters 81 , 82 , or knockdown of poly(ADP-ribose)polymerase 1 gene involved in NAD + recycling 81 all produce plants with decreased stomatal densities in Arabidopsis thaliana . This NAD + -mediated alteration in stomatal patterning is thought to occur via perturbation of abscisic acid (ABA) biosynthesis and signalling, which supresses the activity of SPCH 83 and in turn acts to inhibit cell entry into the stomatal lineage 5 , 84 – 87 . This connection between NAD + and ABA was postulated because changes in NAD + homeostasis were associated with both transcriptional changes in ABA metabolic genes and endogenous levels of this phytohormone, and because alterations in stomatal density in mutants could be rescued by inhibition of ABA biosynthesis 81 . Intriguingly, this also aligns with the known interaction of both NAD + and ABA in regulating stomatal aperture 88 , 89 . Thus, as NAD + is one of the cornerstones of plant metabolism, the recent discovery that it also has a role in regulating stomatal cell differentiation provides a simple and unified route through which changes in physiology or environment directly feedback on stomatal pattering. Water-forming NAD(P)H oxidases (NOX) present a powerful genetically-encoded tool for studying the role of redox status on organismal developmental processes. Specifically, NOX are a class of enzymes which catalyse the four-electron reduction of molecular oxygen in the presence of protons and reducing equivalents NADH and nicotinamide adenine dinucleotide phosphate (NADPH) to produce water and the corresponding NAD + /NADP + electron acceptor in the following stoichiometry [2NAD(P)H + 4H + + O 2 → 2NADP + + 2H 2 O]. In nature, these enzymes are thought to function in both oxidative stress management as well as in balancing the relative levels of oxidised and reduced forms of co-factor pools 90 . Due to this physiological role and the fact that NOX enzymes are highly catalytically efficient, they have been exploited to alter ratios of oxidised and reduced co-factor pools in both fundamental and applied science contexts 91 – 98 . In this study, we aimed to engineer stomatal density through NOX-mediated manipulation of NAD(P) + /NAD(P)H status in stomatal precursor cells. We show that active monomeric NOX can be targeted to different compartments in plant cells. We further show that expression of NOX across these diverse subcellular compartments of stomatal precursor cells leads to a reduction in stomatal density without any adverse effects on stomatal function or leaf-level photosynthesis. Although maximal rates of CO 2 assimilation are not affected in transgenic plants, the intrinsic photosynthetic water-use efficiency is improved such that more CO 2 is fixed per unit water transpired by the leaf. Accordingly, this results in enhanced growth and biomass accumulation under water-deficit conditions, whilst maintaining wild-type levels of growth under well-watered conditions. In summary, this study thus provides a novel and tractable metabolic engineering approach to manipulate stomatal patterning as a potential target to enhance crop productivity and climate resilience. Results NOX can be expressed in plant cells and targeted to multiple subcellular locations Diverse water-forming NAD(P)H oxidases (NOX) enzymes have been characterised across bacteria 94 , 95 , 107 – 116 , 99 , 117 , 100 – 106 , archaea 90 , 118 – 123 , and eukaryotes 124 – 126 . Here, we used an engineered variant of this enzyme from the bacteria Streptococcus mutans ( Sm NOX) for the basis of all experiments. This variant was chosen based on several criteria. First, Sm NOX exhibits NAD(P)H bifunctionality 127 and is thus capable of oxidising co-factor pools in a range of eukaryotic cellular contexts. Second, Sm NOX is functional across a broad pH and temperature range with optimal activities matching physiological conditions in planta 99 , 128 . Third, Sm NOX is monomeric 99 allowing a higher possibility for it to tolerate protein-protein fusions and N-terminal targeting peptides whilst maintaining functional activity. Finally, Sm NOX exhibits negligible secondary H 2 O 2 production that may contribute to undesirable off-target effects 127 , 128 . To assess whether NOX can be produced in plants, we first tested Sm NOX expression using a transient protoplast expression system. For this purpose, mesophyll protoplasts were isolated from mature rosette leaves of A. thaliana and transformed with a genetic construct containing the full-length Sm NOX coding sequence translationally fused to a C-terminal GFP (Supplemental File 1, Figure S1A). Analysis of cell fluorescence using confocal microscopy confirmed that the Sm NOX-GFP fusion was translated in plant cells, and that the enzyme localised to the cytosol when driven in the absence of any additional targeting sequences ( Figure 1A ) in agreement with a predicted bioinformatic localisation (Supplemental File 1, table S1). Download figure Open in new tab Figure 1. Validation of Sm NOX localisation and functional activity in planta . A) The subcellular localisation of Sm NOX in Arabidopsis thaliana protoplasts. Free GFP: protoplasts expressing eGFP (cytosolic control). At RBCS1A::GFP: protoplasts expressing eGFP fused to the C-terminus of the rubisco small subunit (chloroplast stroma control). Cyto- Sm NOX: protoplasts expressing Sm NOX fused at the C-terminus to eGFP (cytosolic localisation). Stromal- SmNOX : protoplasts expressing SmNOX fused at the C-terminus to eGFP and fused at the N-terminus to the chloroplast transit peptide of the photosystem II subunit QA protein (chloroplast stromal localisation). CIMS- Sm NOX: protoplasts expressing SmNOX fused at the C-terminus to eGFP and fused at the N-terminus to the full length outer envelope protein 9 (CIMS; chloroplast intermembrane space localisation). Three separate signals are shown. Green: eGFP fluorescence. Violet: Thylakoid-derived chlorophyll autofluorescence. Grey: bright-field. The merged channel represents all three signals overlaid in a single image. Scale bar = 10 µM. B) Boxplot depicting the rate of NADH oxidation from crude leaf lysate of wild-type (black) and transgenic plants (blue). Abbreviations follow that described in (A). Data represent an average across three independent single copy lines ( n = 4 plants per line). C) As in (B), but for the total NADPH oxidation from crude leaf lysate ( n = 5 plants per line). Differences between transgenic plants and WT are assessed by Fisher LSD post-hoc analysis following a two-way ANOVA, where letters above each box represent statistically significant differences in mean values (p ≤ 0.05). The raw data can be found in Supplemental File 4. Whilst NAD + is produced in the cytosol, the regulatory mechanism that governs cytosolic NAD + levels is dependent on chloroplast NADP + production. Accordingly, we sought to test whether Sm NOX could also be localised closer to the site of NADP + supply in both the chloroplast stroma (the site of NADPH production) and the chloroplast intermembrane space (the site of NADPH/NADH transfer via the malate valve). Sm NOX was successfully localised to the chloroplast stroma (stromal- Sm NOX) when fused to the transit peptide from the photosystem II subunit QA (PSBQA) protein ( Figure 1A ). This stromal localisation was verified by comparison to a stromal control consisting of the full-length rubisco small subunit gene fused to GFP ( Figure 1A ). Moreover, Sm NOX was also successfully targeted to the chloroplast intermembrane space (CIMS- Sm NOX) when fused to the full-length outer envelope protein 9 gene ( Figure 1A ). In summary, Sm NOX can be expressed in plants and can be localised to diverse subcellular compartments including the cytosol (the site of NAD + production), chloroplast stroma (the site of NADP + production), and chloroplast intermembrane space (the site of NAD + /NADP + transfer). Identifying a promoter capable of driving stable and high expression of SmNOX in stomatal precursor cells To investigate whether Sm NOX could affect plant stomatal patterning, it was necessary to next find a promoter which drives high gene expression in undifferentiated stomatal progenitor cells in the leaf epidermis (i.e., during the developmental window prior to onset of stomatal lineage differentiation) (Supplemental File 1, Figure S2A). For this purpose, we analysed a cell-specific transcriptome dataset derived from all major cell types spanning the stomatal developmental trajectory 129 . This revealed that the expression of genes encoding known master-regulators of stomatal development including the transcription factors SPEECHLESS (SPCH), MUTE, and FAMA were expressed at low levels which were considered to be insufficient to achieve Sm NOX-mediated alteration of cellular NAD + status (Supplemental File 1, Figure S2B – S2J). Consequently, we sought to identify an alternative more highly expressed promoter. Interrogation of the above transcriptome data revealed that the gene encoding the chlorophyll a/b binding protein 3 (CAB3) exhibited high mRNA abundance in the target epidermal tissue of interest (Supplemental File 1, Figure S2K). Specifically, CAB3 was ranked as the 36 th (out of ∼28,000) most highly expressed nuclear-encoded gene in this cell type (Supplemental File 1, Figure S2L and S2M), and exhibited transcripts levels that were ∼1,800-fold more abundant than SPCH (0.5 TPM), ∼9,000-fold more abundant than MUTE (0.1 TPM), and ∼425-fold more abundant than FAMA (2.1 TPM), respectively (Supplemental File 1, Figure S2B – S2M). Thus, as CAB3 is one of the most abundant transcripts in the cell type of interest, and as CAB3 is a well-characterised promoter that is extensively used for driving transgene expression in plant synthetic biology 130 , this promoter was selected for use herein. Genetic constructs containing targeted and untargeted variants of the Sm NOX coding sequence were thus cloned under the control of the CAB3 promoter and were transformed into A. thaliana ecotype Col-0 (Supplemental File 1, Figure S1B and Figure S3A and S3B). Homozygous plants from three single-copy insertion events of cyto-SmNOX, stromal-SmNOX and CIMS-SmNOX were used for the basis of all subsequent experiments. Validation of SmNOX expression and function To assess the functional activity of NOX in plants, total rates of NADH and NADPH oxidation were determined using in vitro assays containing crude lysate from mature rosette leaves. As plants possess multiple native enzymes capable of oxidising NADH and NADPH, background oxidation of NADH and NADPH was observed in all cases as expected ( Figure 1B and 1C ). However, the rates of both NADH and NADPH oxidation were significantly increased across all transgenic lines compared to wild-type controls ( Figure 1B and 1C ). Thus, there was successful expression of active Sm NOX in each of the targeted subcellular localisations. It should be noted that the CAB3 promoter also drives high levels of transgene expression in other photosynthetic cells of the mature leaf (Supplemental File 1, Figure S2K – S2M and Figure S4A – S4C). Thus, although our target tissue for expression of Sm NOX is the leaf epidermis, only a portion of the above enzyme activity in the crude leaf lysate corresponds to this cell layer. Moreover, the rates of Sm NOX-mediated NADH/NADPH oxidation measured above (∼ 1.0 μmol m −2 s −1 ) are substantial in the context of an epidermal cell, they are not expected to impact NADH/NADPH pools in photosynthetic mesophyll cells. This is because of both the large pool size of NADH (24 – 55 μM) and NADPH (140 – 180 μM) 131 in the mesophyll tissue, as well as the extraordinary high rates of photosynthetic NADPH production (∼70 μmol g −1 s −1 ) 132 which occurs during the light. Thus, we hypothesized that Sm NOX would be capable of manipulating endogenous levels of NAD + in leaf cells with low photosynthetic activity such as epidermal cells, but would have little or no effect on the NAD + status or photosynthesis of mesophyll cells. SmNOX activity causes reductions in stomatal density Given that Sm NOX was active in the transgenic plants, we next investigated whether this resulted in an alteration in stomatal patterning. For this purpose, the epidermal composition of the abaxial surface of mature rosette leaves from 8-week-old plants was analysed by bright-field microscopy. This analysis revealed that stomatal densities in all Sm NOX-expressing lines were reduced by 20 – 30% compared to wild-type control plants ( Figure 2A ). Thus, changes in cellular NAD(P) + /NAD(P)H ratios, irrespective of the location of perturbation of that ratio, caused a suppression in stomatal development resulting in a reduced density of stomata on leaves. Download figure Open in new tab Figure 2. The effect of Sm NOX expression on stomatal density. A) Boxplot depicting the stomatal density (count mm −2 ) measured on the abaxial surface of mature rosette leaves from wild-type (black) and transgenic plants (blue). WT: wild-type. Cyto- Sm NOX: cytosolic Sm NOX. Stromal- Sm NOX: chloroplast stroma Sm NOX. CIMS- Sm NOX: chloroplast intermembrane space Sm NOX. Data represent an average across three independent single copy lines ( n = 9 – 10 plants per line). B) As in (A), but for the measured leaf mass per area (LMA, g dry mass per m −2 leaf area) of mature rosette leaves ( n = 10 plants per line). Differences between transgenic plants and WT are assessed by Fisher LSD post-hoc analysis following a two-way ANOVA, where letters above each box represent statistically significant differences in mean values (p ≤ 0.05). The raw data can be found in Supplemental File 4. Stomatal density changes have previously been linked with secondary changes in mesophyll development (such that leaves with reduced stomatal numbers are generally thinner and vice versa 133 , 134 ) which can have important consequences for plant physiology 135 – 141 . Thus, we also investigated whether Sm NOX-expression was associated with an alteration in leaf width or cellular density by measuring leaf mass per area (LMA = leaf dry mass / leaf area). This revealed Sm NOX-expressing lines exhibited a small but significant reduction in LMA compared to wild-type ( Figure 2B ). Thus, expression of Sm NOX results in both a decrease in stomatal patterning and anticipated secondary alterations in leaf architecture. SmNOX-induced changes in stomatal density improve plant photosynthetic water-use efficiency As stomatal density is a key determinant of leaf gaseous conductance, we next characterised the effect of Sm NOX expression on plant physiological performance. To test this, we first assessed the photosynthetic parameters of Sm NOX-expressing lines along a light intensity gradient using an infrared open-gas exchange system ( Figure 3A – 3C ). This analysis revealed that there was a small reduction in CO 2 assimilation rate ( A ) between Sm NOX-expressing lines and wild-type plants ( Figure 3A ). However, stomatal conductance ( g s ) was substantially reduced across all Sm NOX-expressing lines ( Figure 3B ), consistent with these mutants exhibiting lower ratios of intercellular CO 2 to ambient CO 2 ( C i / C a ) and reduced rates of transpiratory water loss under high light (Supplemental File 1, Figure S5A and S5B). Sm NOX-expressing lines also exhibited comparable light-saturated rates of CO 2 assimilation ( A sat , Figure 3D , Supplemental File 1, table S2), and substantially lower light-saturated rates of stomatal conductance ( g s sat , Figure 3E , Supplemental File 1, table S2). As a consequence of these combined effects on A and g s , all Sm NOX-expressing lines exhibited a higher intrinsic water use efficiency ( iWUE = A / g s ) across a broad range of light intensities ( Figure 3C and 3F , Supplemental File 1, table S2). Download figure Open in new tab Figure 3. The response of plant gas exchange to light. A) CO 2 assimilation rate ( A , μmol m −2 s −1 ) at different PPFD in wild-type (black) and transgenic plants (blue). WT: wild-type. Cyto- Sm NOX: cytosolic Sm NOX. Stromal- Sm NOX: chloroplast stromal Sm NOX. CIMS- Sm NOX: chloroplast intermembrane space Sm NOX. Data represent mean ± 1 S.E. B) as in (A), but for stomatal conductance ( g s , mol m −2 s −1 ). C) as in (A), but for intrinsic water-use efficiency ( iWUE , μmol mol −1 ). D) Boxplot depicting the light-saturated assimilation rate ( A sat , μmol m −2 s −1 ). The colour scheme and abbreviations follow that described in (A). E) As in (D), but for the light-saturated stomatal conductance ( g s sat , mol m −2 s −1 ). F) As in (D), but for the light-saturated intrinsic water-use efficiency ( iWUE sat , μmol mol −1 ). G) Boxplot depicting the difference in stomatal conductance between the light intensity at which 95% maximum assimilation rate and the light-saturated stomatal conductance are achieved (Δ g s , mol m −2 s −1 ). H) As in (G), but for the difference in light intensity at which 95% maximum assimilation rate and the light-saturated stomatal conductance are achieved (Δ PPFD, μmol m −2 s −1 ). All data represent an average across three independent single copy lines ( n = 7 – 8 plants line). Differences between transgenic plants and WT are assessed by Fisher LSD post-hoc analysis following a two-way ANOVA, where letters above each box represent statistically significant differences in mean values (p ≤ 0.05). The raw data can be found in Supplemental File 4. In addition to an enhanced iWUE , Sm NOX expression also resulted in tighter coordination between rates of A and g s . Specifically, all mutants displayed a reduced “overshooting” of g s at light intensities above those where 95% maximum assimilation was achieved (beyond which neither CO 2 delivery nor light is considered limiting) (Supplemental File 1; Figure 3G and 3H ; Supplemental File 1, Figure S6A – S6F, table S3). This improved iWUE and tighter coupling between A and g s was not associated with a change in any other measured photosynthetic parameter including light compensation point, mitochondrial respiration in the light, apparent quantum yield (i.e., the efficiency by which light can be converted into ATP and NADPH), or the light-saturated rate of electron transport (Supplemental File 1, Figure S5C – S5F, table S2). Moreover, there was no consistent difference in any derived photosynthetic parameter when CO 2 assimilation was measured as a function of sub-stomatal CO 2 concentration (Supplemental File 1, Figure S7, Table S4). This included no change in the maximal CO 2 saturated assimilation rate, CO 2 -compensation point, the transition point between rubisco-limited and RuBP regeneration-limited rates of photosynthesis, the rubisco Michaelis constant for CO 2 in the presence of 21% O 2 air, the carboxylation rate of rubisco, or the maximum electron transport rate through PSII (Supplemental File 1, Figure S6, Table S4). As anticipated, NOX activity was able to alter stomatal density, but was not sufficient to exert any measurable impact on mesophyll cell photosynthesis. Accordingly, Sm NOX-mediated reduction in stomatal density drives both an enhanced photosynthetic water-use efficiency and a tighter coordination between rates of CO 2 assimilation and gas exchange. SmNOX does not affect the kinetics of stomatal movement We next sought to examine whether changes in stomatal density caused by Sm NOX expression were also associated with off-target impacts on stomatal behaviour. Specifically, we chose to assess the speed and magnitude of stomatal responses to light, given that this is one of the most important fluctuating variables that plants face under real-world conditions in the field. Light was also chosen because NADP + is the terminal electron acceptor in the photosynthetic electron transport chain and thus the impact of light on stomatal kinetics had potential to be perturbed by Sm NOX expression. For the purpose of this analysis, the experimental design of 142 was followed. In brief, plants were first acclimated at 100 μmolLJm −2 LJs −1 of light for 60 minutes, followed by a step increase in light intensity to 1000 μmolLJm −2 LJs −1 for 60 minutes, and a final step decrease in light intensity to 100 μmolLJm −2 LJs −1 for 30 minutes, with gas-exchange parameters recorded at one-minute intervals over this entire period. This analysis revealed that all plants experience a normal sigmoidal response of g s in response to step changes in irradiance ( Figure 4A and 4B , Supplemental File 1, Figure S8). Moreover, subsequent model-based interrogation 143 of these data demonstrated that neither the amplitude nor the speed of stomatal responses was different between wild-type and transgenic plants. Specifically, no difference was found in either the modelled initial lag time in g s , the maximum rate of change of g s (calculated from the slope of the exponential phase of the curve), or the time taken for g s to reach new steady-state upon the initial step increase in light intensity from 100 to 1000 μmolLJm −2 LJs −1 ( Figure 4C – 4E , Supplemental File 1, table S5). Similarly, no difference could be detected in the time taken to reach new steady-state g s upon the subsequent step decrease in light intensity from 1000 to 100 μmolLJm −2 LJs −1 ( Figure 4F , Supplemental File 1, table S5). Thus, consistent with the lack of effect of Sm NOX on plant photosynthesis, Sm NOX expression did not impair the dynamic behaviour of stomatal movement. Download figure Open in new tab Figure 4. The temporal response of plant gas exchange to step changes in light intensity. A) The response of CO 2 assimilation rate ( A , μmol m −2 s −1 ) to step changes in light intensity in wild-type (black) and transgenic plants (blue). Shaded and unshaded areas represent periods of light intensity of 100 μmol m −2 s −1 and 1000 μmol m −2 s −1 , respectively. WT: wild-type. Cyto- Sm NOX: cytosolic Sm NOX. Stromal- Sm NOX: chloroplast stromal Sm NOX. CIMS- Sm NOX: chloroplast intermembrane space Sm NOX. Data represent mean ± 1 S.E. B) as in (A), but for stomatal conductance ( g s , mol m −2 s −1 ). C) Boxplot depicting the estimated initial lag in response time of g s (λ, minutes) after the step increase in light intensity from 100 μmol m −2 s −1 to 1000 μmol m −2 s −1 . The colour scheme and abbreviations follow that described in (A). D) As in (C), but for the estimated maximal rate of stomatal opening ( Sl max , mol m −2 s −1 min −1 ) after the step increase in light intensity from 100 μmol m −2 s −1 to 1000 μmol m −2 s −1 . E) As in (C), but for the estimated time taken to achieve new steady state stomatal conductance ( k i , minutes) after the step increase in light intensity from 100 μmol m −2 s −1 to 1000 μmol m −2 s −1 . F) As in (C), but for the estimated time taken to achieve new steady state stomatal conductance ( k d , minutes) after the step decrease in light intensity from 1000 μmol m −2 s −1 to 100 μmol m −2 s −1 . All data represent an average across three independent single copy lines ( n = 3 – 4 plants per line). Differences between transgenic plants and WT are assessed by Fisher LSD post-hoc analysis following a two-way ANOVA, where letters above each box represent statistically significant differences in mean values (p ≤ 0.05). The raw data can be found in Supplemental File 4. SmNOX does not alter plant susceptibility to supra-optimal temperatures One important function of the plant transpiration stream is to mediate the temperature control of plant aerial tissues through evaporative cooling. As such, plants exhibiting reduced stomatal numbers might be more susceptible to thermal damage of photosynthesis under supra-optimal temperatures, owing to impairment of their temperature control mechanism (Supplemental File 1, Figure S5B). To investigate this, we measured plant photosynthesis across step-wise increases in air temperatures from 20 to 45 °C. Under these conditions no difference could be detected in any photosynthesis parameter at any temperature between Sm NOX-expressing lines and wild type plants. In all plants, the rate of A increased with temperature from 20 °C, reaching an optima around 30 °C, and then rapidly declining with increasing temperatures beyond this optima consistent with thermal-induced inactivation of the photosynthetic machinery ( Figure 5A ). Despite an attenuated rate of g s in mutant plants across all conditions, a comparable trend was also observed in this between both mutant and wild-type plants ( Figure 5B ). Moreover, no difference could be detected in the measured leaf temperature of Sm NOX-expressing lines compared to wild-type plants at any given air temperature ( Figure 5C ). Thus, reductions in stomatal density in Sm NOX-expressing lines was not associated with either a change in leaf temperature or any change in the susceptibility to thermal damage of the photosynthetic machinery under supra-optimal temperatures. Download figure Open in new tab Figure 5. The response of plant gas exchange to air temperature (°C). A) CO 2 assimilation rate ( A , μmol m −2 s −1 ) at different air temperatures in wild-type (black) and transgenic plants (blue). WT: wild-type. Cyto- Sm NOX: cytosolic Sm NOX. Stromal- Sm NOX: chloroplast stromal Sm NOX. CIMS- Sm NOX: chloroplast intermembrane space Sm NOX. Data represent mean ± 1 S.E. B) as in (A), but for stomatal conductance ( g s , mol m −2 s −1 ). C) as in (A), but for leaf temperature (°C). All data represent an average across three independent single copy lines ( n = 3 – 5 plants per line). The raw data can be found in Supplemental File 4. SmNOX improves vegetative growth and biomass accumulation under water-deficit conditions Based on the findings that Sm NOX expression caused an enhancement in iWUE without any off-target effects on photosynthetic efficiency, stomatal behaviour, or thermal sensitivity, we next sought to determine whether an impact on plant growth could also be observed. For this purpose, we grew plants in a fully randomised block design under long-day conditions with a 16-hour photoperiods and 250 μmol m −2 s −1 non-limiting light. Under these conditions, no difference in the vegetative growth or performance between transgenic Sm NOX-expressing plants and wild-type plants was detected under well-watered conditions (Supplemental File 1, Figure S9A – S9C). Given the higher iWUE of mutant plants, we next determined whether these improve photosynthesis-water relations resulted in any change in plant growth under water-deficit conditions. For this purpose, plant trays were watered prior to stratification, but water was then withheld for 15-days after the termination of stratification to maintain sub-optimal and deteriorating soil-water saturation levels during vegetative growth (after which watering was resumed when the top layer of soil was visibly dry every ∼7 days). This assay revealed that Sm NOX expressing plants produced more biomass and larger rosettes compared to wild-type plants over equivalent periods of time ( Figure 6A-E ). This result was caused by Sm NOX expressing-lines maintaining higher relative growth rates during water-deficit ( Figure 6F ). Consistent with this enhancement in vegetative growth and tolerance to adverse water conditions, mutants also exhibited earlier times of bolting ( Figure 6G ) and flowering ( Figure 6H ), and produced visibly taller inflorescences relative to wild-type ( Figure 6I ). However, these alterations in vegetative growth and biomass did not translate to improved yields, and all Sm NOX expressing-lines exhibited a reduction in total seed mass ( Figure 6J ). Thus, Sm NOX-expressing plants exhibited enhanced biomass production and tolerance to water-deficit conditions but an overall impairment of seed production. Download figure Open in new tab Figure 6. The vegetative and reproductive performance of plants under water-deficit conditions. (A) The increase in visible rosette area (cm 2 ) over time in wild-type (black) and transgenic plants (blue). WT: wild-type. Cyto- Sm NOX: cytosolic Sm NOX. Stromal- Sm NOX: chloroplast stromal Sm NOX. CIMS- Sm NOX: chloroplast intermembrane space Sm NOX. Data represent mean ± 1 S.E. B) Representative images of wild-type and transgenic plants taken on day 12 post stratification. Scale bar = 2.5 cm. C) As in (B), but for day 15 post stratification. D) As in (B), but for day 18 post stratification. In parts B – D, the same individual plant is shown across the growing period and all images were generated by computationally removing background from the aerial photographs analysed for growth analyses. E) Boxplot depicting the maximum visible rosette area at the end of vegetative growth (day = 20). The colour scheme and abbreviations follow that described in (A). F) As in (E), but for the average percentage growth rate of plants computed over the vegetative growth phase (% increase in rosette area day −1 ). G) As in (E), but for the time taken for bolting to occur in plants (days post stratification). H) As in (E), but for the time taken for flowering to occur in plants (days post stratification). I) As in (E), but for the maximum inflorescence height (cm) measured at day 32 post stratification. J) As in (E), but for the total seed weight harvested from plants after drying (g). All data represent an average across three independent single copy lines ( n = 17 – 20 plants per line for all measurements, except total seed weight where n = 11 – 13 plants per line). Differences between transgenic plants and WT are assessed by Fisher LSD post-hoc analysis following a two-way ANOVA, where letters above each box represent statistically significant differences in mean values (p ≤ 0.05). The raw data can be found in Supplemental File 4. Discussion Manipulation of stomatal density is a key target for enhancing crop productivity and climate resilience under current and future conditions 144 , 145 . In the present study, we demonstrate that driving the expression of a water-forming NAD(P)H oxidase from Streptococcus mutans ( Sm NOX) in stomatal precursor cells causes a reduction in stomatal density in Arabidopsis thaliana . We show that this reduced stomatal density phenotype occurs irrespective of whether Sm NOX is targeted to the cytosol, chloroplast stroma, or chloroplast intermembrane space. We also find that neither Sm NOX expression nor Sm NOX-mediated reductions in stomatal density had any detectable impact on plant CO 2 assimilation rate, the efficiency or thermal sensitivity of photosynthesis, the kinetics of stomatal movement, or plant growth under well-watered conditions. Nonetheless, we discover that all Sm NOX-expressing lines exhibit an enhanced intrinsic water-use efficiency. We also show that this improvement in water-use efficiency translates to an increase in plant vegetative growth and biomass accumulation under water-deficit conditions. Collectively, we thus provide a novel metabolic engineering approach which can be leveraged to alter stomatal patterning thus plant photosynthesis-water relations. Nicotinamide adenine dinucleotide [NAD(H)] and nicotinamide adenine dinucleotide phosphate [NADP(H)] are ubiquitous coenzymes that facilitate cellular energy transfer, redox homeostasis, and signalling in living organisms 146 . Alongside these diverse roles, NAD + has recently been implicated as a negative regulator of stomatal development. Evidence in support of this hypothesis has arisen because perturbations in cellular redox status associated with translational repression of mitochondrial ( At NDT1 and At NDT2) and peroxisomal ( At PXN) NAD + transporter proteins, as well as poly (ADP-ribose) polymerase (PARP) proteins (PARP1, PARP2 and PARP3) involved in NAD + recycling, have all produced A. thaliana mutant plants with reduced stomatal densities 81 , 82 . The mechanism of this developmental effect is proposed to occur via abscisic acid (ABA) which is known to inhibit entry into the stomatal lineage by suppressing the activity of SPEECHLESS (SPCH) 84 , possibly via induction of the cyclin-dependent kinase (CDK) inhibitor gene ICK1 87 , 147 . This interaction between NAD + and ABA was postulated because NAD + transport and metabolism mutants were associated with both transcriptional changes in ABA metabolic genes and reduced SPCH expression 81 . Importantly, exogenous NAD + applications were also sufficient to drive a reduction in stomatal patterning in wild-type Arabidopsis , but not in mutants impaired in ABA biosynthesis or signalling 81 . In the present study, we build on this foundational biology by demonstrating that expression of a water-forming NAD(P)H oxidase in stomatal precursor cells is capable of driving a reduction in stomatal density through perturbation of endogenous cellular redox status. We further show that such a reduction in stomatal density is achieved irrespective of the subcellular compartment in which this enzyme is targeted. Thus, these results collectively demonstrate that NAD + is a viable metabolic handle which can be turned to alter stomatal patterning in synthetic biology applications. The density of stomata imposes an upper anatomical constraint on leaf diffusive conductance and thus has important physiological implications for plant productivity 148 . Despite reduced stomatal density mutants having been generated in a diverse number of disparate species and studies 17 , 58 , 72 – 79 , 81 , 82 , 61 , 64 , 65 , 67 – 71 , the effects of this developmental alteration on plant photosynthesis, water-use efficiency, growth, and yield traits have proven variable across different studies and plant species 149 . In the present study, we characterise the gas exchange properties of our reduced stomatal density mutants across a range of environmental conditions to shed light on their underlying physiology. We demonstrate that these reduced stomatal density plants exhibit an increased intrinsic water-use efficiency ( iWUE ) by maintaining wild-type rates of CO 2 assimilation at lower respective stomatal gaseous conductance. Reductions in stomatal density also caused a tighter co-ordination between the rates of CO 2 assimilation and stomatal conductance, such that a reduced extent of water loss occurred after light-saturated rates of CO 2 assimilation were achieved. However, decreasing stomatal density did not result in any change in the efficiency or thermal sensitivity of photosynthesis and CO 2 assimilation, or the kinetics of stomatal movements. Together, these results align with recent findings from Epidermal Patterning Factor mutants which similarly report reductions in stomatal density without any impact on plant CO 2 assimilation 58 , 71 , 72 , 150 . The biological basis of these collective observations is intriguing. As the rate of photosynthesis at any one time is constrained by the most limiting factor of chloroplast CO 2 delivery (including both stomatal conductance and mesophyll conductance components) and photosynthetic biochemistry 151 , this phenomenon could be explained if stomata are either not limiting to plant photosynthesis under the study conditions or if secondary adjustments in leaf architecture compensate for changes in stomatal density 150 . Future research efforts will be required to further elucidate the complex interplay between stomata, leaf architecture and plant physiology. In addition to the lack of effect of reduced stomatal density on leaf photosynthesis, CO 2 assimilation was also not impacted by the expression of SmNOX under the control of the CAB3 promoter. Thus, whilst Sm NOX was able to perturb NAD + levels to mediate a reduction in stomatal density, it was not sufficient to impair photosynthesis. The reason for this is likely that the large pool sizes of NADH (24 – 55 μM) and NADPH (140 – 180 μM) 131 and the extraordinary high rates of photosynthetic NADPH production (∼70 μmol g −1 s −1 ) 132 would together mask the effect of Sm NOX activity in the mesophyll tissue. By contrast, epidermal cells contribute minimally toward total plant photosynthesis and contain small chloroplasts with high stroma to grana ratio which would produce much lower rates of NADPH 152 , 153 . Thus, although Sm NOX is expressed to a high level throughout diverse photosynthesis tissues using the CAB3 promoter, the impact of this enzyme on cellular NAD + status would be largest in stomatal progenitor cells. Consistent with the lack of impairment of stomatal density or Sm NOX expression on photosynthetic CO 2 assimilation, transgenic plants did not exhibit any difference in plant growth or biomass accumulation under well-watered conditions. However, increased vegetative growth and biomass accumulation was observed under water-deficit conditions. This improved tolerance to adverse water conditions is caused by the more conservative water-input requirements of these plants, driven by both the reduced rates of transpiration and improvement in photosynthetic water-use efficiency and is in agreement with other analyses of reduced stomatal density mutants 71 – 73 , 75 . Moreover, the reduced leaf mass per area measured in Sm NOX-expressing lines would also favour biomass accumulation as this requires a lower resource investment per unit leaf tissue produced. Despite this improved tolerance to adverse water conditions, all Sm NOX-expressing lines exhibited a lower overall seed yield. Similar impairments in plant reproductive traits were also reported in NAD + carrier mutants 82 , 154 and are likely caused by the role of NAD + in seed 155 , 156 and pollen development 157 – 160 . It would be interesting to investigate in future work whether this yield impairment is unavoidable, or whether it could be instead mitigated by use of other promoters with reduced off-target expression effects. Conclusion As global food security is increasingly threatened by climate change, developing more climate-resilient crops presents an important goal for crop breeding and engineering efforts. Altering stomatal density represents a promising avenue to optimise the relationship between plant carbon assimilation, water demand, growth, and yield. Here, we provide a novel metabolic engineering strategy which can be exploited to reduce plant stomatal density by altering the endogenous NAD + status of stomatal precursor cells. We demonstrate that this approach produces plants with improved photosynthesis-water relations and improved productivity under water-deficit conditions without any apparent cost on photosynthetic carbon assimilation or growth under normal conditions. Thus, this approach reveals a potential novel way in which plants can be altered to enhance climate resilience. Materials and Methods Gene accession numbers The following list comprises the accession numbers of the genes used in this study: Sm NOX2 (WP_002268749.1); At RBCS1A (At1g67090); At PSBQA (At4g21280) ; At OEP9 (At1g16000). Gene synthesis A variant of the NAD(P)H oxidase (NOX) enzyme encoded by the Streptococcus mutans nox2 gene but containing amino acid substitutions D192A/V193R/V194H/A199R was chosen for use in this study 127 . This gene (hereinafter, termed Sm NOX) was codon optimised for expression in Arabidopsis thaliana and domesticated to remove the type IIS restriction enzymes BsaI or BpiI. Appropriate flanking regions compatible with Golden Gate Assembly 161 were added to the 5’ and 3’ regions, and the resulting coding sequence was commercially synthesized 161 (TWIST Bioscience, USA). The full-length coding sequence of AtOEP9 and At RBCS1A, as well as a chloroplast stromal transit peptide from AtPSBQA (containing the first 87 amino acids of AtPSBQA including the intact stromal import domain but lacking a complete thylakoid targeting motif 162 ) were subject to these same steps for codon optimisation, domestication, addition of compatible flanking regions for Golden Gate assembly 161 , and gene synthesis. The full-length sequences of each these genes are found in Supplemental File 2. Construct design and assembly Genetic constructs for stable expression in plants were generated by cloning the coding sequence of Sm NOX downstream of the A. thaliana chlorophyll a/b-binding protein 3 ( At CAB3; pTEI071) and upstream of the A. tumefaciens nopaline synthase terminator (nosT; piCH41421) in the Level 1 backbone position forward 2 vector pICH47742. Compositional forms of this Level 1 module were also generated in which Sm NOX was translationally fused at the N-terminus to either the At PSBQA transit peptide or the At OEP9 full-length coding sequence, respectively. Each of these constructs were subsequently assembled into Level 2 Golden Gate modules using the plant transformation vector acceptor backbone pAGM37443 163 with the pFAST-R seed-coat specific fluorescent selection cassette 164 (pICSL70008) and the end Linker position 3 insert (pICH49266). Genetic constructs for transient expression in protoplasts were generated by cloning the coding sequence of Sm NOX translationally fused to a C-terminal enhanced green fluorescent protein (GFP; pJOG176) downstream of the cauliflower mosaic virus 35S promoter (CaMV35S; EC15058) and upstream of the A. tumefaciens nosT (piCH41421) in the Level 1 backbone position forward 2 vector pICH47742. As above, compositional forms of this were also generated in which Sm NOX was translationally fused at the N-terminus to the At PSBQA transit peptide or the At OEP9 full-length coding sequence, respectively. Positive control constructs were also cloned using the same promoter, terminator, and backbone vector but containing only the coding sequence of the GFP (cytosolic fluoresce control) or the coding sequence of GFP translationally fused at the N-terminus to the full-length At RBCS1A gene sequence (chloroplast stroma fluoresce control). All cloning reactions were performed following the Golden Gate one-step one-pot protocol 165 as described in Supplemental File 1. The provenance of commercial vectors used during cloning are also provided in Supplemental File 1. Full-length sequences of each expression cassette generated is provided in Supplemental File 2. The full set of oligonucleotides used for cloning, colony PCR, and Sanger sequencing are found in Supplemental File 3. The thermocycler conditions used for all PCR reactions are also described in Supplemental File 3. Protoplast isolation, transformation, and visualisation Mesophyll protoplasts were isolated from leaves of 4-week old wild-type Arabidopsis thaliana Col-0 plants following the ‘Tape- Arabidopsis Sandwich’ method 167 . Protoplast transformation was performed using the calcium/polyethylene glycol-mediated method 168 , as modified in 167 . Transformed protoplasts were incubated in the dark overnight at 25°C prior to imaging. Cell fluorescence was visualised using a Zeiss LSM 880 Airy Scan confocal microscope. Zeiss ZEN software (Zeiss) was used for confocal laser scanning microscopy and subsequent image processing. Plant material and stable transformation Stable transformation of A. thaliana ecotype Columbia (Col-0) background was performed using the agrobacterium-mediated floral dip method 169 . Positive transformants were initially screened based upon seed fluorescence visualised by epifluorescence microscopy (Leica M165 stereo microscope), and confirmed by genotyping PCR and gel electrophoresis using the OneTaq® 2X Master Mix with Standard Buffer (NEB, Catalog #M0482S) following manufacturer’s instructions for reaction set-up and the oligonucleotides and thermocycler conditions described in Supplemental File 3. Single insertion events were subsequently identified by TaqMan® real-time PCR with fluorescent probes and primers which bind to the TagRFP selectable marker (Idna GENETICS) and homozygous individuals were isolated. For the above purposes of genotyping PCR and TaqMan® real-time PCR, genomic DNA was isolated from mature leaves of T1 plants using the cetyl trimethylammonium bromide (CTAB) method 170 . In total, three single-copy homozygous lines were taken forward as the basis of the analysis herein for each Sm NOX-expressing transgenic plant (cyto- Sm NOX, stromal- Sm NOX and CIMS- Sm NOX). All experiments described are performed on T3 and T4 generation plants. Stomatal density measurements Epidermal impressions from the abaxial surface of healthy and fully-expanded excised leaves of 8-week old plants were obtained following the clear nail polish method 171 . All leaves used for this analysis were of similar size. After mounting on microscope slides, stomatal density was measured using a Leica DMR8 light microscope at ×20 magnification under brightfield view. For each sample, stomatal density counts were calculated across five independent fields of view of the same leaf and across 9 – 10 biological replicates per line. Leaf mass area measurements A leaf puncher was used to harvest a fixed area of tissue from healthy and fully-expanded leaves of 8-week-old plants. Leaf dry mass was subsequently determined after 48 hours incubation at 65 °C. All leaves were of similar size and were harvested in the middle of the photoperiod. Leaf mass area (LMA, gLJm −2 ) was subsequently determined as the fixed leaf area divided by leaf dry mass. In vitro enzyme assays A consistent area of leaf tissue was harvested from healthy and fully-expanded leaves of 6-week-old plants using a leaf puncher during the middle of the photoperiod. Leaf material was flash frozen in liquid nitrogen, ground using metal beads and a tissue lyser (Thermo Scientific, TissueLyser II), and resuspended in 500 μL ice cold extraction buffer (50 mM potassium phosphate, 5 mM MgCl2, 1 mM EDTA, 3% Triton X-100) with 1x protease inhibitor cocktail (Sigma, Catalog #P9599-5ML). Homogenate was centrifuged for 1 minute at 12,000 rpm at 4°C, and total crude protein extract was isolated from supernatant. Enzyme assays were performed following the method described in 127 with some alterations. In brief, all reactions were performed using a total volume of 200 μL in unsealed 96 well plate at 25°C. Typical reaction mixtures contained 50 mM potassium phosphate buffer (pH 7.5) and 250 μM of either NADH or NADPH, were vortexed for saturation with oxygen in air (250 μM), and were initiated by addition of 30 μL undiluted crude leaf lysate. Negative controls containing ddH 2 O in the place of leaf lysate were included in every assay. Consumption of NADH or NADPH was monitored spectrophotometrically at 340 nm using a plate reader (BMG Labtech FLUOstar Omega). Each biological replicate was repeated over two technical replicates wells and rates of enzyme reaction were calculated over the linear part of the curve over a duration of at least 5 minutes. Reaction rates were normalized per unit NADH/NADPH oxidation by standard curve. Leaf gas exchange Gas exchange analyses were performed using a LI-6800 open-path gas exchange system equipped with a 6800-01A fluorometer head (LI-COR, USA) using the 2 cm 2 leaf chamber and 90% red and 10% blue actinic light under normal 21% O 2 air. The youngest fully-expanded leaf was used for each plant and all measurements were taken prior to onset of flowering. Downstream statistical analysis, model fitting, and parameter estimation from leaf gas exchange data was performed as described in Supplemental File 1. Response of plant gas exchange to light followed the method of 172 with some alterations. In brief, plants were first adapted at 2,200 μmol m −2 s −1 for 20 minutes, and photosynthesis was then measured at step-wise intervals of 2200, 2000, 1650, 1300, 1000, 750, 500, 400, 300, 200, 150, 100, 50, 0 and 2200 μmol m −2 s −1 light. The environment was held constant at 400 μmol mol −1 CO 2 , 30°C leaf temperature, 55% relative air humidity, 500 µmol s −1 flow rate, and 10,000 rpm fan speed. No matching was performed during the analysis and a 4-minute wait time was used between each light condition. For the response of plant gas exchange to intercellular CO 2 , plants were first adapted at 400 μmol mol −1 CO 2 for 20 minutes and photosynthesis was then measured at step-wise intervals of 400, 0, 50, 75, 100, 200, 400, 800, 1200, 1600, 400 μmol mol −1 reference atmospheric CO 2 concentrations. The environment was held constant at 1500 µmol m −2 s −1 light, 30°C leaf temperature, 55% relative air humidity, 500 µmol s −1 flow rate, and 10,000 rpm fan speed. Matching of CO 2 and H 2 O was performed before each measurement with a minimum and maximum wait time of 3 and 4 minutes, respectively. Investigation of stomatal kinetics in response to step changes in light followed the method of 142 . In brief, plants were first adapted to 100 μmol m −2 s −1 light for 60 minutes and were then subject to a step increase to 1000 μmol m −2 s −1 for 60 minutes followed by a step decrease back to 100 μmol m −2 s −1 for 30 minutes. The environment was held constant at 400 μmol mol −1 CO 2 , 30°C leaf temperature, 1.8 kPa vapour pressure deficit, 500 µmol s −1 flow rate, and 10,000 rpm fan speed. No matching was performed during the analysis and measurements were taken every minute. For the response of plant gas exchange to temperature, plants were first adapted at 20°C for 20 minutes and photosynthesis was then measured at step-wise intervals of 20, 25, 30, 35, 40 and 45°C reference air temperatures. The environment was held constant at 1500 µmol m −2 s −1 light, 400 μmol mol −1 CO 2 , 500 µmol s −1 flow rate, and 10,000 rpm fan speed. As previously described in the thermal analysis of 173 , it is difficult to maintain VPD at temperatures above 30°C. Thus, leaf vapour pressure deficit (VPD) was maintained at 1.5 kPa for measurements at 20, 25 and 30°C, 2.5 kPa for the measurement at 35°C, 3.5 kPa for the measurement at 40°C and 4.5 kPa for the measurement at 45 °C. Matching of CO 2 and H 2 O was performed before each measurement with a minimum and maximum wait time of 10 and 14 minutes, respectively. Plant growth conditions Seeds were grown in individual pots directly on soil (Levington Seed modular compost) and were stratified for 2 days at 4°C to overcome dormancy. Unless explicitly stated, plants were grown in climate-controlled environment conditions in floor-standing growth chambers (Aralab FitoclimaD1200 PLH) under short-day conditions (8-hour light/16-hour dark cycle) at 21°C, ambient 400 ppm CO 2 , 50% relative humidity, and 125 μmol m −2 s −1 light and were maintained under well-watered conditions. Plant growth assays For analysis of plant growth, after stratification plants were transferred to controlled environment chambers (CERs) at The University of Oxford and grown under a 16-hour/8-hour day/night period, 21°C, and 250 μmol m −2 s −1 light. To minimize confounding and positional effects, plant genotypes were grown in a fully randomised block design and the position of trays were rotated every 2 days. For well-watered experiments, plants were watered on a weekly basis as usual from the base of the tray. For water-deficit growth experiment, plant trays were watered prior to stratification as above, but water was then withheld completely for 15 days after germination to maintain sub-optimal and deteriorating soil-water saturation levels during vegetative growth. After this period, watering was resumed following the method described in the well-watered growth experiments. Aerial photographs were taken daily during vegetative growth. Image processing was performed to correct for orientation and perspective using Yet Another Scanning Wizard (YASW) ( https://github.com/ImageProcessing-ElectronicPublications/yasw ). Computational analysis was performed to derive growth parameters. Green pixels were isolated from images using an in-built script, and visible rosette area was computed with ImageJ software 174 using the in-built ‘Analyze Particles’ function. Time of bolting was measured as the number of days after stratification when the inflorescence was greater than 1 cm in height above the rosette. Time of flowering time was measured as the number of days after stratification at which the first flower was open and petals were visible. Plant height was measured as the maximum height of the primary inflorescence from the base of the rosette at 32 days post stratification. Total seed weight was measured after allowing plants to completely dry for a month. Funding This work was funded by the Royal Society and the European Union’s Horizon 2020 research and innovation program under grant agreement number 637765. JWB was funded by the BBSRC through BB/J014427/1. This research was funded in whole, or in part, by the BBSRC number BB/J014427/1. For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Data Availability All data used in this study is provided in the supplemental material. Author Contributions JWB and SK conceived the study, designed the experiments, interpreted and analyzed the data, and wrote the manuscript. JWB conducted the experiments. Conflicts of interest SK is co-founder of Wild Bioscience Ltd. SK and JWB have applied for a patent on the work presented in this study. References 1. ↵ Edwards , D. , Kerp , H. & Hass , H. Stomata in early land plants: An anatomical and ecophysiological approach . J. Exp. Bot . 49 , 255 – 278 ( 1998 ). OpenUrl CrossRef Web of Science 2. Clark , J. W. et al. The origin and evolution of stomata . Curr. Biol . 32 , R539 – R553 ( 2022 ). OpenUrl 3. ↵ Raven , J. A. Selection pressures on stomatal evolution . New Phytol . 153 , 371 – 386 ( 2002 ). OpenUrl CrossRef Web of Science 4. ↵ Möldenhawer , J. Beyträge zur Anatomie der Pflanzen . in 95 – 107 ( Königl. Schulbuchdruckerei , 1812 ). 5. ↵ Quarrie , S. A. & Jones , H. G. Effects of abscisic acid and water stress on development and morphology of wheat . J. Exp. Bot . 28 , 192 – 203 ( 1977 ). OpenUrl CrossRef Web of Science 6. ↵ Lau , O. S. et al. Direct Control of SPEECHLESS by PIF4 in the High-Temperature Response of Stomatal Development . Curr. Biol . 28 , 1273 – 1280 .e3 ( 2018 ). OpenUrl CrossRef 7. ↵ Woodward , F. I. Stomatal numbers are sensitive to increases in CO2 from pre-industrial levels . Nature 327 , 617 – 618 ( 1987 ). OpenUrl CrossRef Web of Science 8. Beerling , D. J. , McElwain , J. C. & Osborne , C. P. Stomatal responses of the ‘living fossil’ Ginkgo biloba L. to changes in atmospheric CO2 concentrations . J. Exp. Bot . 49 , 1603 – 1607 ( 1998 ). OpenUrl CrossRef Web of Science 9. Woodward , F. I. , Lake , J. A. & Quick , W. P. Stomatal development and CO2: Ecological consequences . New Phytol . 153 , 477 – 484 ( 2002 ). OpenUrl CrossRef Web of Science 10. Woodward , F. I. & Bazzaz , F. A. The responses of stomatal density to CO2 partial pressure . J. Exp. Bot . 39 , 1771 – 1781 ( 1988 ). OpenUrl CrossRef Web of Science 11. ↵ McElwain , J. C. & Chaloner , W. G. Stomatal density and index of fossil plants track atmospheric carbon dioxide in the palaeozoic . Ann. Bot . 76 , 389 – 395 ( 1995 ). OpenUrl CrossRef 12. Franks , P. J. & Beerling , D. J. Maximum leaf conductance driven by CO2 effects on stomatal size and density over geologic time . Proc. Natl. Acad. Sci. U. S. A . 106 , 10343 – 10347 ( 2009 ). OpenUrl Abstract / FREE Full Text 13. ↵ Beerling , D. J. , Osborne , C. P. & Chaloner , W. G. Evolution of leaf-form in land plants linked to atmospheric CO2 decline in the Late Palaeozoic era . Nature 410 , 352 – 354 ( 2001 ). OpenUrl CrossRef GeoRef PubMed Web of Science 14. ↵ Fanourakis , D. , Heuvelink , E. & Carvalho , S. M. P. A comprehensive analysis of the physiological and anatomical components involved in higher water loss rates after leaf development at high humidity . J. Plant Physiol . 170 , 890 – 898 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 15. ↵ Bakker , J. C. Effects of humidity on stomatal density and its relation to leaf conductance . Sci. Hortic. (Amsterdam) . 48 , 205 – 212 ( 1991 ). OpenUrl CrossRef 16. ↵ de Boer , H. J. et al. Optimal allocation of leaf epidermal area for gas exchange . New Phytol . 210 , 1219 – 1228 ( 2016 ). OpenUrl CrossRef PubMed 17. ↵ Doheny-Adams , T. , Hunt , L. , Franks , P. J. , Beerling , D. J. & Gray , J. E. Genetic manipulation of stomatal density influences stomatal size, plant growth and tolerance to restricted water supply across a growth carbon dioxide gradient . Philos. Trans. R. Soc. B Biol. Sci . 367 , 547 – 555 ( 2012 ). OpenUrl CrossRef PubMed 18. Dittberner , H. et al. Natural variation in stomata size contributes to the local adaptation of water-use efficiency in Arabidopsis thaliana . Mol. Ecol . 27 , 4052 – 4065 ( 2018 ). OpenUrl 19. ↵ Buckley , C. R. , Caine , R. S. & Gray , J. E. Pores for Thought: Can Genetic Manipulation of Stomatal Density Protect Future Rice Yields? Front. Plant Sci . 10 , ( 2020 ). 20. ↵ Mcelwain , J. C. , Yiotis , C. & Lawson , T. Using modern plant trait relationships between observed and theoretical maximum stomatal conductance and vein density to examine patterns of plant macroevolution . New Phytol . 209 , 94 – 103 ( 2016 ). OpenUrl CrossRef PubMed 21. ↵ Farquhar , G. D. & Richards , R. A. Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes . Aust. J. Plant Physiol . 11 , 539 – 552 ( 1984 ). OpenUrl CrossRef Web of Science 22. ↵ Condon , A. G. , Richards , R. A. , Rebetzke , G. J. & Farquhar , G. D. Improving Intrinsic Water-Use Efficiency and Crop Yield . Crop Sci . 42 , 122 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 23. ↵ Dow , G. J. , Berry , J. A. & Bergmann , D. C. The physiological importance of developmental mechanisms that enforce proper stomatal spacing in Arabidopsis thaliana . New Phytol . 201 , 1205 – 1217 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 24. Papanatsiou , M. , Amtmann , A. & Blatt , M. R. Stomatal spacing safeguards stomatal dynamics by facilitating guard cell ion transport independent of the epidermal solute reservoir . Plant Physiol . 172 , 254 – 263 ( 2016 ). OpenUrl Abstract / FREE Full Text 25. Yu , Q. et al. RSD1 Is Essential for Stomatal Patterning and Files in Rice . Front. Plant Sci . 11 , ( 2020 ). 26. Wang , H. , Ngwenyama , N. , Liu , Y. , Walker , J. C. & Zhang , S. Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis . Plant Cell 19 , 63 – 73 ( 2007 ). OpenUrl Abstract / FREE Full Text 27. Shpak , E. D. , McAbee , J. M. , Pillitteri , L. J. & Torii , K. U. Stomatal patterning and differentiation by synergistic interactions of receptor kinases . Science (80-.) . 309 , 290 – 293 ( 2005 ). OpenUrl Abstract / FREE Full Text 28. ↵ Nadeau , J. A. & Sack , F. D. Control of stomatal distribution on the Arabidopsis leaf surface . Science (80-.) . 296 , 1697 – 1700 ( 2002 ). OpenUrl Abstract / FREE Full Text 29. ↵ Carroll , S. et al. Altering arabinans increases Arabidopsis guard cell flexibility and stomatal opening . Curr. Biol . 32 , 3170 – 3179 .e4 ( 2022 ). OpenUrl 30. ↵ Wang , Y. et al. Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth . Proc. Natl. Acad. Sci. U. S. A . 111 , 533 – 538 ( 2014 ). OpenUrl Abstract / FREE Full Text 31. ↵ Toh , S. et al. Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen . Front. Plant Sci . 12 , ( 2021 ). 32. ↵ Medeiros , D. B. et al. Enhanced photosynthesis and growth in atquac1 knockout mutants are due to altered organic acid accumulation and an increase in both stomatal and mesophyll conductance . Plant Physiol . 170 , 86 – 101 ( 2016 ). OpenUrl Abstract / FREE Full Text 33. Medeiros , D. B. et al. Impaired malate and fumarate accumulation due to the mutation of the tonoplast dicarboxylate transporter has little effects on stomatal behavior . Plant Physiol . 175 , 1068 – 1081 ( 2017 ). OpenUrl Abstract / FREE Full Text 34. ↵ Antunes , W. C. , de Menezes Daloso , D. , Pinheiro , D. P. , Williams , T. C. R. & Loureiro , M. E. Guard cell-specific down-regulation of the sucrose transporter SUT1 leads to improved water use efficiency and reveals the interplay between carbohydrate metabolism and K+ accumulation in the regulation of stomatal opening . Environ. Exp. Bot . 135 , 73 – 85 ( 2017 ). OpenUrl 35. Flütsch , S. et al. Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth . EMBO Rep . 21 , ( 2020 ). 36. Wang , H. et al. A subsidiary cell-localized glucose transporter promotes stomatal conductance and photosynthesis . Plant Cell 31 , 1328 – 1343 ( 2019 ). OpenUrl Abstract / FREE Full Text 37. Kusumi , K. , Hirotsuka , S. , Kumamaru , T. & Iba , K. Increased leaf photosynthesis caused by elevated stomatal conductance in a rice mutant deficient in SLAC1, a guard cell anion channel protein . J. Exp. Bot . 63 , 5635 – 5644 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 38. Horaruang , W. et al. Engineering a K+ channel ‘sensory antenna’ enhances stomatal kinetics, water use efficiency and photosynthesis . Nat. Plants 8 , 1262 – 1274 ( 2022 ). OpenUrl 39. ↵ Papanatsiou , M. et al. Optogenetic manipulation of stomatal kinetics improves carbon assimilation,water use, and growth . Science (80-.) . 363 , 1456 – 1459 ( 2019 ). OpenUrl Abstract / FREE Full Text 40. ↵ Reyer , A. et al. Channelrhodopsin-mediated optogenetics highlights a central role of depolarization-dependent plant proton pumps . Proc. Natl. Acad. Sci. U. S. A . 117 , 20920 – 20925 ( 2020 ). OpenUrl Abstract / FREE Full Text 41. Huang , S. et al. Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel GtACR1 . Sci. Adv . 7 , ( 2021 ). 42. ↵ Zhou , Y. et al. Optogenetic control of plant growth by a microbial rhodopsin . Nat. Plants 7 , 144 – 151 ( 2021 ). OpenUrl 43. ↵ Daloso , D. M. et al. Guard cell-specific upregulation of sucrose synthase 3 reveals that the role of sucrose in stomatal function is primarily energetic . New Phytol . 209 , 1470 – 1483 ( 2016 ). OpenUrl CrossRef PubMed 44. ↵ Antunes , W. C. , Provart , N. J. , Williams , T. C. R. & Loureiro , M. E. Changes in stomatal function and water use efficiency in potato plants with altered sucrolytic activity . Plant, Cell Environ . 35 , 747 – 759 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 45. Acevedo-Siaca , L. G. et al. Guard-cell-targeted overexpression of Arabidopsis Hexokinase 1 can improve water use efficiency in field-grown tobacco plants . J. Exp. Bot . 73 , 5745 – 5757 ( 2022 ). OpenUrl 46. Lugassi , N. , et al. Expression of Arabidopsis hexokinase in tobacco guard cells increases water-use efficiency and confers tolerance to drought and salt stress . Plants 8 , ( 2019 ). 47. Kelly , G. et al. Guard-Cell Hexokinase Increases Water-Use Efficiency Under Normal and Drought Conditions . Front. Plant Sci . 10 , ( 2019 ). 48. Horrer , D. et al. Blue light induces a distinct starch degradation pathway in guard cells for stomatal opening . Curr. Biol . 26 , 362 – 370 ( 2016 ). OpenUrl CrossRef PubMed 49. Laporte , M. M. , Shen , B. & Tarczynski , M. C. Engineering for drought avoidance: Expression of maize NADP-malic enzyme in tobacco results in altered stomatal function . J. Exp. Bot . 53 , 699 – 705 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 50. Penfield , S. et al. Expression and manipulation of PHOSPHOENOLPYRUVATE CARBOXYKINASE 1 identifies a role for malate metabolism in stomatal closure . Plant J . 69 , 679 – 688 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 51. ↵ Müller , G. L. et al. Improved water use efficiency and shorter life cycle of Nicotiana tabacum due to modification of guard and vascular companion cells . Sci. Rep . 8 , ( 2018 ). 52. ↵ Boxall , S. F. et al. Kalanchoë PPC1 is essential for crassulacean acid metabolism and the regulation of core circadian clock and guard cell signaling genes[CC-BY] . Plant Cell 32 , 1136 – 1160 ( 2020 ). OpenUrl Abstract / FREE Full Text 53. Flütsch , S. et al. Guard cell starch degradation yields glucose for rapid stomatal opening in Arabidopsis . Plant Cell 32 , 2325 – 2344 ( 2020 ). OpenUrl Abstract / FREE Full Text 54. Prasch , C. M. et al. β-amylase1 mutant Arabidopsis plants show improved drought tolerance due to reduced starch breakdown in guard cells . J. Exp. Bot . 66 , 6059 – 6067 ( 2015 ). OpenUrl CrossRef PubMed 55. Kelly , G. et al. The pitfalls of transgenic selection and new roles of AtHXK1: A high level of AtHXK1 expression uncouples hexokinase1 dependent sugar signaling from exogenous sugar . Plant Physiol . 159 , 47 – 51 ( 2012 ). OpenUrl FREE Full Text 56. ↵ Lugassi , N. et al. Expression of Arabidopsis hexokinase in citrus guard cells controls stomatal aperture and reduces transpiration . Front. Plant Sci . 6 , ( 2015 ). 57. ↵ Tanaka , Y. , Sugano , S. S. , Shimada , T. & Hara-Nishimura , I. Enhancement of leaf photosynthetic capacity through increased stomatal density in Arabidopsis . New Phytol . 198 , 757 – 764 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 58. ↵ Franks , P. J. , W. Doheny-Adams , T. , Britton-Harper , Z. J. & Gray , J. E. Increasing water-use efficiency directly through genetic manipulation of stomatal density . New Phytol . 207 , 188 – 195 ( 2015 ). OpenUrl CrossRef PubMed 59. ↵ Schlüter , U. , Muschak , M. , Berger , D. & Altmann , T. Photosynthetic performance of an Arabidopsis mutant with elevated stomatal density (sdd1-1) under different light regimes . J. Exp. Bot . 54 , 867 – 874 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 60. ↵ Schuler , M. L. , Sedelnikova , O. V. , Walker , B. J. , Westhoff , P. & Langdalea , J. A. SHORTROOT-mediated increase in stomatal density has no impact on photosynthetic efficiency . Plant Physiol . 176 , 757 – 772 ( 2018 ). OpenUrl Abstract / FREE Full Text 61. ↵ Bussis , D. , Von Groll , U. , Fisahn , J. & Altmann , T. Stomatal aperture can compensate altered stomatal density in Arabidopsis thaliana at growth light conditions . Funct. Plant Biol . 33 , 1037 – 1043 ( 2006 ). OpenUrl CrossRef Web of Science 62. ↵ Masle , J. , Gilmore , S. R. & Farquhar , G. D. The ERECTA gene regulates plant transpiration efficiency in Arabidopsis . Nature 436 , 866 – 870 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 63. ↵ Sakoda , K. et al. Higher Stomatal Density Improves Photosynthetic Induction and Biomass Production in Arabidopsis Under Fluctuating Light . Front. Plant Sci . 11 , ( 2020 ). 64. ↵ Jalakas , P. , Merilo , E. , Kollist , H. & Brosché , M. ABA-mediated regulation of stomatal density is OST1-independent . Plant Direct 2 , ( 2018 ). 65. ↵ Gudesblat , G. E. et al. SPEECHLESS integrates brassinosteroid and stomata signalling pathways . Nat. Cell Biol . 14 , 548 – 554 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 66. ↵ Yang , M. & Sack , F. D. The too many mouths and four lips mutations affect stomatal production in arabidopsis . Plant Cell 7 , 2227 – 2239 ( 1995 ). OpenUrl Abstract / FREE Full Text 67. ↵ Pillitteri , L. J. , Sloan , D. B. , Bogenschutz , N. L. & Torii , K. U. Termination of asymmetric cell division and differentiation of stomata . Nature 445 , 501 – 505 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 68. ↵ Bergmann , D. C. , Lukowitz , W. & Somerville , C. R. Stomatal development and pattern controlled by a MAPKK kinase . Science (80-.) . 304 , 1494 – 1497 ( 2004 ). OpenUrl Abstract / FREE Full Text 69. ↵ Hepworth , C. , Doheny-Adams , T. , Hunt , L. , Cameron , D. D. & Gray , J. E. Manipulating stomatal density enhances drought tolerance without deleterious effect on nutrient uptake . New Phytol . 208 , 336 – 341 ( 2015 ). OpenUrl CrossRef 70. Dunn , J. et al. Reduced stomatal density in bread wheat leads to increased water-use efficiency . J. Exp. Bot . 70 , 4737 – 4747 ( 2019 ). OpenUrl CrossRef 71. ↵ Caine , R. S. et al. Rice with reduced stomatal density conserves water and has improved drought tolerance under future climate conditions . New Phytol . 221 , 371 – 384 ( 2019 ). OpenUrl CrossRef 72. ↵ Hughes , J. et al. Reducing stomatal density in barley improves drought tolerance without impacting on yield . Plant Physiol . 174 , 776 – 787 ( 2017 ). OpenUrl Abstract / FREE Full Text 73. ↵ Mohammed , U. et al. Rice plants overexpressing OsEPF1 show reduced stomatal density and increased root cortical aerenchyma formation . Sci. Rep . 9 , ( 2019 ). 74. ↵ Yoo , C. Y. et al. The Arabidopsis GTL1 transcription factor regulates water use efficiency and drought tolerance by modulating stomatal density via transrepression of SDD1 . Plant Cell 22 , 4128 – 4141 ( 2010 ). OpenUrl Abstract / FREE Full Text 75. ↵ Wang , C. et al. PdEPF1 regulates water-use efficiency and drought tolerance by modulating stomatal density in poplar . Plant Biotechnol. J . 14 , 849 – 860 ( 2016 ). OpenUrl 76. ↵ Yin , X. et al. CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice . Plant Cell Rep . 36 , 745 – 757 ( 2017 ). OpenUrl CrossRef 77. Hara , K. et al. Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in arabidopsis leaves . Plant Cell Physiol . 50 , 1019 – 1031 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 78. ↵ Hara , K. , Kajita , R. , Torii , K. U. , Bergmann , D. C. & Kakimoto , T. The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule . Genes Dev . 21 , 1720 – 1725 ( 2007 ). OpenUrl Abstract / FREE Full Text 79. ↵ Zhao , J. , Li , A. , Xu , M. , Dai , G. & Chen , J. Genome-wide analysis of the TIFY family in Lycium and the negative regulation of stomatal development by LrJAZ2 gene . Plant Physiol. Biochem . 206 , ( 2024 ). 80. ↵ Falquetto-Gomes , P. , Silva , W. J. , Siqueira , J. A. , Araújo , W. L. & Nunes-Nesi , A. From epidermal cells to functional pores: Understanding stomatal development . J. Plant Physiol . 292 , ( 2024 ). 81. ↵ Feitosa-Araujo , E. et al. Changes in intracellular NAD status affect stomatal development in an abscisic acid-dependent manner . Plant J . 104 , 1149 – 1168 ( 2020 ). OpenUrl 82. ↵ de Souza Chaves , I. , et al. The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in Arabidopsis thaliana . Plant J . 100 , 487 – 504 ( 2019 ). OpenUrl 83. ↵ Yang , X. , Lalitha Gavya , S. , Zhou , Z. , Urano , D. & Lau , O. S. Abscisic acid regulates stomatal production by imprinting a SnRK2 kinase–mediated phosphocode on the master regulator SPEECHLESS . Sci. Adv . 8 , ( 2022 ). 84. ↵ Tanaka , Y. , Nose , T. , Jikumaru , Y. & Kamiya , Y. ABA inhibits entry into stomatal-lineage development in Arabidopsis leaves . Plant J . 74 , 448 – 457 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 85. Lake , J. A. & Woodward , F. I. Response of stomatal numbers to CO2 and humidity: Control by transpiration rate and abscisic acid . New Phytol . 179 , 397 – 404 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 86. Chater , C. et al. Elevated CO2-Induced Responses in Stomata Require ABA and ABA Signaling . Curr. Biol . 25 , 2709 – 2716 ( 2015 ). OpenUrl CrossRef PubMed 87. ↵ Chater , C. C. C. , Oliver , J. , Casson , S. & Gray , J. E. Putting the brakes on: Abscisic acid as a central environmental regulator of stomatal development . New Phytol . 202 , 376 – 391 ( 2014 ). OpenUrl CrossRef PubMed 88. ↵ Hashida , S. N. et al. Nicotinate/nicotinamide mononucleotide adenyltransferase-mediated regulation of NAD biosynthesis protects guard cells from reactive oxygen species in ABA-mediated stomatal movement in Arabidopsis . J. Exp. Bot . 61 , 3813 – 3825 ( 2010 ). OpenUrl CrossRef PubMed 89. ↵ Sun , L. et al. NADK2 positively modulates abscisic acid-induced stomatal closure by affecting accumulation of H2O2, Ca2+ and nitric oxide in Arabidopsis guard cells . Plant Sci . 262 , 81 – 90 ( 2017 ). OpenUrl 90. ↵ Yan , M. , Yin , W. , Fang , X. , Guo , J. & Shi , H. Characteristics of a water-forming NADH oxidase from Methanobrevibacter smithii, an archaeon in the human gut . Biosci. Rep . 36 , ( 2016 ). 91. ↵ Kroutil , W. , Mang , H. , Edegger , K. & Faber , K. Biocatalytic Oxidation of Primary and Secondary Alcohols . ChemInform 35 , ( 2004 ). 92. Turner , N. J. Enantioselective Oxidation of C-O and C-N bonds using oxidases . Chem. Rev . 111 , 4073 – 4087 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 93. de Gonzalo , G. , A. Orden , A. & R. Bisogno , F. New Trends in Organic Synthesis with Oxidative Enzymes . Curr. Org. Chem . 16 , 2598 – 2612 ( 2012 ). OpenUrl 94. ↵ Titov , D. V. et al. Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio . Science (80-.) . 352 , 231 – 235 ( 2016 ). OpenUrl Abstract / FREE Full Text 95. ↵ Cracan , V. , Titov , D. V. , Shen , H. , Grabarek , Z. & Mootha , V. K. A genetically encoded tool for manipulation of NADP+/NADPH in living cells . Nat. Chem. Biol . 13 , 1088 – 1095 ( 2017 ). OpenUrl CrossRef PubMed 96. Vemuri , G. N. , Eiteman , M. A. , McEwen , J. E. , Olsson , L. & Nielsen , J. Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae . Proc. Natl. Acad. Sci. U. S. A . 104 , 2402 – 2407 ( 2007 ). OpenUrl Abstract / FREE Full Text 97. Eiteman , M. A. , Vemuri , G. N. , Altman , E. , Sangurdekar , D. P. & Khodursky , A. B. Overflow Metabolism in Escherichia coli during Steady-State GrowthLJ: Transcriptional Regulation and Effect of the Redox Ratio . Appl. Environ. Microbiol . 72 , 3653 – 3661 ( 2006 ). OpenUrl Abstract / FREE Full Text 98. ↵ Heux , S. , Cachon , R. & Dequin , S. Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism . Metab. Eng . 8 , 303 – 314 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 99. ↵ Higuchi , M. et al. Identification of two distinct NADH oxidases corresponding to H2O2-forming oxidase and H2O-forming oxidase induced in Streptococcus mutans . J. Gen. Microbiol . 139 , 2343 – 2351 ( 1993 ). OpenUrl CrossRef PubMed Web of Science 100. ↵ Schmidt , H.-L , Stöcklein , W. , Danzer , J. , Kirch , P. & Limbach , B. Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis . Eur. J. Biochem . 156 , 149 – 155 ( 1986 ). OpenUrl PubMed 101. Matsumoto , J. , Higuchi , M. , Shimada , M. , Yamamoto , Y. & Kamio , Y. Molecular cloning and sequence analysis of the gene encoding the H2O-forming NADH Oxidase from streptococcus mutans . Biosci. Biotechnol. Biochem . 60 , 39 – 43 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 102. Kawasaki , S. , Ishikura , J. , Chiba , D. , Nishino , T. & Niimura , Y. Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum: Existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria . Arch. Microbiol . 181 , 324 – 330 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 103. Zhang , Y. W. et al. Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus . Enzyme Microb. Technol . 50 , 255 – 262 ( 2012 ). OpenUrl PubMed 104. Geueke , B. , Riebel , B. & Hummel , W. NADH oxidase from Lactobacillus brevis: A new catalyst for the regeneration of NAD . Enzyme Microb. Technol . 32 , 205 – 211 ( 2003 ). OpenUrl CrossRef 105. Hummel , W. & Riebel , B. Isolation and biochemical characterization of a new NADH oxidase from Lactobacillus brevis . Biotechnol. Lett . 25 , 51 – 54 ( 2003 ). OpenUrl CrossRef PubMed 106. ↵ Dishisha , T. , Sabet-Azad , R. , Arieta , V. & Hatti-Kaul , R. Lactobacillus reuteri NAD(P)H oxidase: Properties and coexpression with propanediol-utilization enzymes for enhancing 3-hydroxypropionic acid production from 3-hydroxypropionaldehyde . J. Biotechnol . 289 , 135 – 143 ( 2019 ). OpenUrl 107. ↵ Gao , H. et al. NADH oxidase from Lactobacillus reuteri: A versatile enzyme for oxidized cofactor regeneration . Int. J. Biol. Macromol . 123 , 629 – 636 ( 2019 ). OpenUrl 108. Li , F. L. , Zhou , Q. , Wei , W. , Gao , J. & Zhang , Y. W. Switching the substrate specificity from NADH to NADPH by a single mutation of NADH oxidase from Lactobacillus rhamnosus . Int. J. Biol. Macromol . 135 , 328 – 336 ( 2019 ). OpenUrl 109. Lountos , G. T. et al. The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: Insights into the conversion of O2 into two water molecules by the flavoenzyme . Biochemistry 45 , 9648 – 9659 ( 2006 ). OpenUrl CrossRef PubMed 110. Riebel , B. R. , Gibbs , P. R. , Wellborn , W. B. & Bommarius , A. S. Cofactor Regeneration of NAD+ from NADH: Novel Water-Forming NADH Oxidases . Advanced Synthesis and Catalysis vol. 344 1156 – 1168 https://onlinelibrary.wiley.com/doi/epdf/10.1002/1615-4169%28200212%29344%3A10%3C1156%3A%3AAID-ADSC1156%3E3.0.CO%3B2-%23 ( 2002 ). OpenUrl 111. Park , J. T. , Hirano , J. I. , Thangavel , V. , Riebel , B. R. & Bommarius , A. S. NAD(P)H oxidase v from Lactobacillus plantarum (NoxV) displays enhanced operational stability even in absence of reducing agents . J. Mol. Catal. B Enzym . 71 , 159 – 165 ( 2011 ). OpenUrl 112. Nowak , C. et al. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors . Front. Microbiol . 6 , ( 2015 ). 113. Lopez de Felipe , F. & Hugenholtz , J. Purification and characterisation of the water forming NADH-oxidase from Lactococcus lactis . Int. Dairy J . 11 , 37 – 44 ( 2001 ). OpenUrl CrossRef 114. Sakamoto , M. , Uchimura , T. & Komagata , K. Comparison of H2O-Forming NADH Oxidase from Leuconostoc mesenteroides subsp. mesenteroides NRIC 1541T and H2O2-Forming NADH Oxidase from Sporolactobacillus inulinus NRIC 1133T . J. Ferment. Bioeng . 82 , 531 – 537 ( 1996 ). OpenUrl 115. Gao , H. , Tiwari , M. K. , Kang , Y. C. & Lee , J. K. Characterization of H2O-forming NADH oxidase from Streptococcus pyogenes and its application in l-rare sugar production . Bioorganic Med. Chem. Lett . 22 , 1931 – 1935 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 116. ↵ Stanton , T. B. & Jensen , N. S. Purification and characterization of NADH oxidase from Serpulina (Treponema) hyodysenteriae . J. Bacteriol . 175 , 2980 – 2987 ( 1993 ). OpenUrl Abstract / FREE Full Text 117. ↵ Wang , L. , Chong , H. & Jiang , R. Comparison of alkyl hydroperoxide reductase and two water-forming NADH oxidases from Bacillus cereus ATCC 14579 . Appl. Microbiol. Biotechnol . 96 , 1265 – 1273 ( 2012 ). OpenUrl PubMed 118. ↵ Jia , B. et al. Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2O . FEBS J . 275 , 5355 – 5366 ( 2008 ). OpenUrl CrossRef PubMed 119. Jia , B. et al. An archaeal NADH oxidase causes damage to both proteins and nucleic acids under oxidative stress . Mol. Cells 29 , 363 – 371 ( 2010 ). OpenUrl CrossRef PubMed 120. Nisar , M. A. et al. TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH . J. Biosci. Bioeng . 116 , 39 – 44 ( 2013 ). OpenUrl 121. Rashid , N. , Hameed , S. , Siddiqui , M. A. & Ikram-ul-Haq . Gene cloning and characterization of NADH oxidase from thermococcus kodakarensis . African J. Biotechnol . 10 , 17916 – 17924 ( 2011 ). OpenUrl 122. Wu , X. et al. Application of a novel thermostable NAD(P)H oxidase from hyperthermophilic archaeon for the regeneration of both NAD + and NADP + . Biotechnol. Bioeng . 109 , 53 – 62 ( 2012 ). OpenUrl CrossRef PubMed 123. ↵ Ward , D. E. et al. The NADH oxidase from Pyrococcus furiosus: Implications for the protection of anaerobic hyperthermophiles against oxidative stress . Eur. J. Biochem . 268 , 5816 – 5823 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 124. ↵ Brown , D. M. , Upcroft , J. A. & Upcroft , P. A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis . Eur. J. Biochem . 241 , 155 – 161 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 125. Li , L. & Wang , C. C. A likely molecular basis of the susceptibility of Giardia lamblia towards oxygen . Mol. Microbiol . 59 , 202 – 211 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 126. ↵ Castillo-Villanueva , A. , Méndez , S. T. , Torres-Arroyo , A. , Reyes-Vivas , H. & Oria-Hernández , J. Cloning, Expression and Characterization of Recombinant, NADH Oxidase from Giardia lamblia . Protein J . 35 , 24 – 33 ( 2016 ). OpenUrl PubMed 127. ↵ Petschacher , B. et al. Cofactor specificity engineering of streptococcus mutans NADH oxidase 2 for nad(p)+ regeneration in biocatalytic oxidations . Comput. Struct. Biotechnol. J . 9 , e201402005 ( 2014 ). OpenUrl 128. ↵ Li , F. L. , Shi , Y. , Zhang , J. X. , Gao , J. & Zhang , Y. W. Cloning, expression, characterization and homology modeling of a novel water-forming NADH oxidase from Streptococcus mutans ATCC 25175 . Int. J. Biol. Macromol . 113 , 1073 – 1079 ( 2018 ). OpenUrl 129. ↵ Adrian , J. et al. Transcriptome dynamics of the stomatal lineage: Birth, amplification, and termination of a self-renewing population . Dev. Cell 33 , 107 – 118 ( 2015 ). OpenUrl CrossRef PubMed 130. ↵ Mitra , A. , Choi , H. K. & An , G. Structural and functional analyses of Arabidopsis thaliana chlorophyll a/b-binding protein (cab) promoters . Plant Mol. Biol . 12 , 169 – 179 ( 1989 ). OpenUrl CrossRef PubMed Web of Science 131. ↵ Igamberdiev , A. U. & Gardeström , P. Regulation of NAD- and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves . Biochim. Biophys. Acta - Bioenerg . 1606 , 117 – 125 ( 2003 ). OpenUrl 132. ↵ Wieloch , T. & Sharkey , T. D. Compartment-specific energy requirements of photosynthetic carbon metabolism in Camelina sativa leaves . Planta 255 , ( 2022 ). 133. ↵ Pérez-Bueno , M. L. et al. An extremely low stomatal density mutant overcomes cooling limitations at supra-optimal temperature by adjusting stomatal size and leaf thickness . Front. Plant Sci . 13 , ( 2022 ). 134. ↵ Dow , G. J. , Berry , J. A. & Bergmann , D. C. Disruption of stomatal lineage signaling or transcriptional regulators has differential effects on mesophyll development, but maintains coordination of gas exchange . New Phytol . 216 , 69 – 75 ( 2017 ). OpenUrl CrossRef 135. ↵ Savaldi-Goldstein , S. , Peto , C. & Chory , J. The epidermis both drives and restricts plant shoot growth . Nature 446 , 199 – 202 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 136. Baillie , A. L. & Fleming , A. J. The developmental relationship between stomata and mesophyll airspace . New Phytol . 225 , 1120 – 1126 ( 2020 ). OpenUrl CrossRef 137. Kondo , T. et al. Stomatal density is controlled by a mesophyll-derived signaling molecule . Plant Cell Physiol . 51 , 1 – 8 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 138. Serna , L. & Fenoll , C. Stomatal development in Arabidopsis: How to make a functional pattern . Trends Plant Sci . 5 , 458 – 460 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 139. Bergmann , D. C. Integrating signals in stomatal development . Curr. Opin. Plant Biol . 7 , 26 – 32 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 140. Lundgren , M. R. et al. Mesophyll porosity is modulated by the presence of functional stomata . Nat. Commun . 10 , ( 2019 ). 141. ↵ Vile , D. et al. Arabidopsis growth under prolonged high temperature and water deficit: Independent or interactive effects? Plant, Cell Environ . 35 , 702 – 718 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 142. ↵ McAusland , L. et al. Effects of kinetics of light-induced stomatal responses on photosynthesis and water-use efficiency . New Phytol . 211 , 1209 – 1220 ( 2016 ). OpenUrl CrossRef PubMed 143. ↵ Vialet-Chabrand , S. , Dreyer , E. & Brendel , O. Performance of a new dynamic model for predicting diurnal time courses of stomatal conductance at the leaf level . Plant, Cell Environ . 36 , 1529 – 1546 ( 2013 ). OpenUrl CrossRef Web of Science 144. ↵ Bertolino , L. T. , Caine , R. S. & Gray , J. E. Impact of stomatal density and morphology on water-use efficiency in a changing world . Front. Plant Sci . 10 , ( 2019 ). 145. ↵ Leakey , A. D. B. et al. Water Use Efficiency as a Constraint and Target for Improving the Resilience and Productivity of C 3 and C 4 Crops . Annu. Rev. Plant Biol . 70 , 781 – 808 ( 2019 ). OpenUrl CrossRef 146. ↵ Smith , E. N. , Schwarzländer , M. , Ratcliffe , R. G. & Kruger , N. J. Shining a light on NAD- and NADP-based metabolism in plants . Trends Plant Sci . ( 2021 ) doi: 10.1016/j.tplants.2021.06.010 . OpenUrl CrossRef 147. ↵ Wang , H. et al. ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid . Plant J . 15 , 501 – 510 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 148. ↵ Haworth , M. , Elliott-Kingston , C. & McElwain , J. C. Stomatal control as a driver of plant evolution . J. Exp. Bot . 62 , 2419 – 2423 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 149. ↵ Lawson , T. & Blatt , M. R. Stomatal size, speed, and responsiveness impact on photosynthesis and water use efficiency . Plant Physiol . 164 , 1556 – 1570 ( 2014 ). OpenUrl Abstract / FREE Full Text 150. ↵ Harrison , E. L. , Arce Cubas , L. , Gray , J. E. & Hepworth , C. The influence of stomatal morphology and distribution on photosynthetic gas exchange . Plant J . 101 , 768 – 779 ( 2020 ). OpenUrl 151. ↵ Gago , J. et al. Photosynthesis Optimized across Land Plant Phylogeny . Trends in Plant Science vol. 24 947 – 958 at doi: 10.1016/j.tplants.2019.07.002 ( 2019 ). OpenUrl CrossRef 152. ↵ Barton , K. A. , Wozny , M. R. , Mathur , N. , Jaipargas , E. A. & Mathur , J. Chloroplast behaviour and interactions with other organelles in Arabidopsis thaliana pavement cells . J. Cell Sci . 131 , ( 2018 ). 153. ↵ Barton , K. A. et al. Epidermal pavement cells of arabidopsis have chloroplasts . Plant Physiol . 171 , 723 – 726 ( 2016 ). OpenUrl FREE Full Text 154. ↵ Feitosa-Araujo , E. et al. Downregulation of a Mitochondrial NAD+Transporter (NDT2) Alters Seed Production and Germination in Arabidopsis . Plant Cell Physiol . 61 , 897 – 908 ( 2020 ). OpenUrl 155. ↵ Katoh , A. , Uenohara , K. , Akita , M. & Hashimoto , T. Early steps in the biosynthesis of NAD in arabidopsis start with aspartate and occur in the plastid . Plant Physiol . 141 , 851 – 857 ( 2006 ). OpenUrl Abstract / FREE Full Text 156. ↵ Chai , M. F. et al. NADK2, an Arabidopsis chloroplastic NAD kinase, plays a vital role in both chlorophyll synthesis and chloroplast protection . Plant Mol. Biol . 59 , 553 – 564 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 157. ↵ Cárdenas , L. , McKenna , S. T. , Kunkel , J. G. & Hepler , P. K. NAD(P)H oscillates in pollen tubes and is correlated with tip growth . Plant Physiol . 142 , 1460 – 1468 ( 2006 ). OpenUrl Abstract / FREE Full Text 158. Hashida , S. N. , Takahashi , H. , Kawai-Yamada , M. & Uchimiya , H. Arabidopsis thaliana nicotinate/nicotinamide mononucleotide adenyltransferase (AtNMNAT) is required for pollen tube growth . Plant J . 49 , 694 – 703 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 159. Hashida , S. N. , Kawai-Yamada , M. & Uchimiya , H. NAD+ accumulation as a metabolic off switch for orthodox pollen . Plant Signal. Behav . 8 , ( 2013 ). 160. ↵ Hashida , S. N. et al. NAD+ accumulation during pollen maturation in arabidopsis regulating onset of germination . Mol. Plant 6 , 216 – 225 ( 2013 ). OpenUrl CrossRef PubMed 161. ↵ Marillonnet , S. & Engler , C. Golden Gate Cloning . Methods Mol. Biol . 1116 , 119 – 131 ( 2014 ). OpenUrl CrossRef PubMed 162. ↵ Ma , S. H. et al. The ten amino acids of the oxygen-evolving enhancer of tobacco is sufficient as the peptide residues for protein transport to the chloroplast thylakoid . Plant Mol. Biol . 105 , 513 – 523 ( 2021 ). OpenUrl 163. ↵ Grützner , R. et al. High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns . Plant Commun . 2 , ( 2021 ). 164. ↵ Engler , C. et al. A Golden Gate modular cloning toolbox for plants . ACS Synth. Biol . 3 , 839 – 843 ( 2014 ). OpenUrl CrossRef PubMed 165. ↵ Engler , C. , Gruetzner , R. , Kandzia , R. & Marillonnet , S. Golden gate shuffling: A one-pot DNA shuffling method based on type ils restriction enzymes . PLoS One 4 , ( 2009 ). 166. Woo , H. R. et al. Programming of plant leaf senescence with temporal and inter-organellar coordination of transcriptome in arabidopsis . Plant Physiol . 171 , 452 – 467 ( 2016 ). OpenUrl Abstract / FREE Full Text 167. ↵ Wu , F. H. et al. Tape-arabidopsis sandwich - A simpler arabidopsis protoplast isolation method . Plant Methods 5 , ( 2009 ). 168. ↵ Yoo , S. D. , Cho , Y. H. & Sheen , J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis . Nat. Protoc . 2 , 1565 – 1572 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 169. ↵ Clough , S. J. & Bent , A. F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . Plant J . 16 , 735 – 743 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 170. ↵ Murray , M. G. & Thompson , W. F. Rapid isolation of high molecular weight plant DNA . Nucleic Acids Res . 8 , 4321 – 4326 ( 1980 ). OpenUrl CrossRef PubMed Web of Science 171. ↵ Wu , S. & Zhao , B. Using clear nail polish to make Arabidopsis epidermal impressions for measuring the change of stomatal aperture size in immune response . in Methods in Molecular Biology vol. 1578 243 – 248 ( 2017 ). OpenUrl 172. ↵ López-Calcagno , P. E. et al. Stimulating photosynthetic processes increases productivity and water-use efficiency in the field . Nat. Plants 6 , 1054 – 1063 ( 2020 ). OpenUrl CrossRef 173. ↵ Busch , F. A. & Sage , R. F. The sensitivity of photosynthesis to O2 and CO2 concentration identifies strong Rubisco control above the thermal optimum . New Phytol . 213 , 1036 – 1051 ( 2017 ). OpenUrl CrossRef 174. ↵ Schneider , C. A. , Rasband , W. S. & Eliceiri , K. W. NIH Image to ImageJ: 25 years of image analysis . Nat. Methods 9 , 671 – 675 ( 2012 ). OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted August 30, 2024. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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