Stomatal Function Requires Pectin De-methyl-esterification of the Guard Cell Wall

1Probing Arabidopsis thaliana leaf sections with a panel of 36 monoclonal antibodies by fluorescence microscopy revealed a range of antibody-binding patterns, including clear differences in the composition of guard cell walls compared to epidermal or mesophyll cells (Figure 1; Figure S1; Table S1). Homogalacturonan (HGA) is a polysaccharide of α-1,4-linked galacturonic acid (GalA) residues and is the predominant form of pectin in A. thaliana. It is synthesized at the Golgi apparatus and secreted from cells in a highly methyl-esterified form. These methyl ester groups can subsequently be removed by enzymatic activity in the cell wall, allowing for a range of methyl-esterification states. A broad range of HGA methyl-esterification patterns are recognized by the JIM7 antibody, and Figure 1A shows uniform JIM7 binding within the walls of guard cells, epidermal pavement, and mesophyll cells, indicating a wide distribution of HGA. HGA can form pectate calcium cross-links when continuous stretches of GalA residues are blockwise de-esterified, and this can be detected using the 2F4 antibody. De-esterified, calcium cross-linked HGA was not detected in the guard cell walls or in neighboring cell walls but, instead, was limited to the junction regions between cells of the epidermis (Figure 1B). In contrast, relatively unesterified HGA (detected by antibody LM19) showed strong labeling in guard cell and epidermal cell walls (Figure 1C). Highly methyl-esterified HGA, as indicated by antibody LM20, was excluded from guard cells but was abundant in the junctions between guard cells and neighboring epidermal cells (Figure 1D). The LM20 signal was observed in the cell walls of epidermal pavement and mesophyll cells, as well as the cuticular ledges of guard cells. This specific pattern of pectins contrasted with the more uniform signal observed following immunolocalization with antibodies against other cell wall components, such as xyloglucan (LM25) (Figure 1E), while signal was absent when the primary antibody was not included (Figure 1F). Out of 36 cell wall antibodies tested, LM20 (against high methyl-esterified HGA) revealed a clear guard-cell-specific pattern (Figure S1; Table S1) being absent from guard cell walls but detected strongly in neighboring cell walls.

2These results can be compared with previous investigations of guard cell wall composition. For example, analysis of Commelina communis has shown that guard cells are rich in pectin but did not report on the differential methyl-esterification patterns described here. Our data show that both highly methyl-esterified and calcium cross-linked blockwise de-esterified HGA are excluded from the guard cells and that un-esterified HGA is the predominant form of pectin in the guard cell wall. Pectic arabinans have previously been implicated as being important for guard cell movements via ectopic application of carbohydrate-modifying enzymes. Thus, our data support the idea that the structural properties of the pectin network are important for guard cell function. We took a molecular genetic approach to test this hypothesis.

3The enzymes that modify plant cell wall pectins are typically encoded by large gene families. For example 66, 35, and 89 pectin methylesterase genes have been annotated in A. thaliana, Oryza sativa, and Populus trichocarpa, respectively. The encoded proteins contain pectin methylesterase (PME), or both PME and pectin methylesterase inhibitor domains (proPME proteins). The PME and proPME enzymes control the methyl-esterification status of HGA by removing methyl ester groups from HGA. This large number of genes has made it difficult to attribute specific pectin modifications of cell walls to particular physiological properties. As our experiments identified guard cells as having a distinct pectin methyl-esterification status, we sought to identify genes encoding pectin-modifying enzymes with a guard-cell-specific expression pattern. We focused on a proPME gene, PME6 (TAIR: AT1G23200), which is expressed at >36-fold higher levels in guard cell protoplasts relative to mesophyll cell protoplasts and is also expressed during seed coat development. PME6 expression has previously been shown to be repressed in scap1, a mutant with altered expression of cell wall modification genes and a resultant change in methyl-esterification state. Negi et al. proposed that PME6 might act downstream of SCAP1 to elicit at least part of the stomatal phenotype observed.

4PME6 encodes a single PMEI domain and a PME domain that contains the two conserved active-site aspartic acid residues necessary for PME activity, as well as an N-terminal signal peptide, suggesting that it is a secreted protein. A PME6 promoter β-glucuronidase (GUS) fusion construct containing approximately 1,400 bp upstream of the start codon (proPME6::GUS) was stably introduced into A. thaliana, and GUS histochemical localization indicated that this DNA region directs expression predominantly in mature guard cells (Figure S2A). Analysis of transcriptome data indicated that PME6 mRNA accumulates to a high level in the scrm-D mutant, which has an excess of mature guard cells and to a lower level in mutants in which epidermal cell differentiation is blocked at the pavement cell stage (spch) or at the stage of meristemoid formation (scrm-D mute) (Figure S1B). These data suggest that PME6 is expressed in guard cells at a relatively late stage of differentiation. Transcriptome data indicated that PME6 is also expressed in siliques, and analysis of the proPME6::GUS lines confirmed this. Analysis of a pme6 mutant (described later) did not reveal any change in seed germination or seed weight, so our further investigation focused on stomatal function. To investigate the function of PME6, we obtained an A. thaliana line with a Ds transposon inserted within the PME6 gene (hereinafter referred to as pme6-1) from the Nottingham Arabidopsis Stock Centre. PCR and RT-PCR analyses showed that pme6-1 homozygous plants harbor an insertion in the single intron of the PME6 gene (Figure S2C) and have no detectable expression of the PME6 mRNA transcript (Figure S2D). Complementation of the pme6-1 line by introducing a native PME6 gene construct under the control of the proPME6 promoter restored the levels of PME6 mRNA in two independent lines (proPME6::pme6) (Figure S2D).

5Immunolocalization analyses of pme6-1 revealed a major change in the methyl-esterification status of guard cell wall pectins. We first confirmed that the guard cells of L. erecta wild-type (WT) plants (the pme6-1 background) showed strong binding of LM19 (Figures 2A and 2B), indicating an abundance of relatively unesterified pectin. There was an absence of LM20 binding (Figures 2E and 2F), indicating that highly methyl-esterified pectin is absent from the guard cells. In contrast, pme6-1 plants had a reduction in the levels of de-esterified pectin in the guard cell, as indicated by the weaker binding of LM19 (Figures 2C and 2D) and abundant highly esterified pectin, as indicated by LM20 binding (Figures 2G and 2H). These data indicate that the structure of the HGA component of the pectin network has been altered in the pme6-1 knockout line and, in particular, that the pectin of guard cells is more highly methyl-esterified in plants lacking PME6. JIM7-binding patterns remained consistent between the WT and pme6-1 (Figures 2I–2L), indicating that the differences observed in Figures 2A–2H were due to an alteration in the methyl-esterification status of the guard cells rather than a change in the overall distribution of HGA in the cell wall. The pattern of methyl-esterification in guard cells was highly reproducible, as shown in Figures 2Q–2S. pme6-1 lines complemented with a proPME6::PME6 construct showed a restoration of the WT methyl-esterification pattern (Figure S3). Analysis of controls (Figures 2M–2P) indicated that the patterns observed in Figures 2A–2L did not simply reflect patterns of cell wall thickness or overall distribution of cellulose. These data indicate that PME6 is crucial for the de-methyl-esterification of guard cell wall HGA.

6To study the functional significance of the changing pectin methyl-esterification status of the guard cells, we investigated the stomatal opening and closure responses of pme6-1. Figure 3A shows the stomatal aperture response in isolated epidermal strips exposed to buffers supplied with elevated (1,000 ppm) or decreased (0 ppm) levels of CO2. Exposure to elevated CO2 caused WT stomatal apertures to decrease, and CO2-free air caused WT apertures to increase, as previously reported. In contrast, pme6-1 stomatal apertures were relatively insensitive to CO2, with the responses to both elevated and decreased CO2 being lost. The stomatal aperture response to CO2 was restored in the complemented lines. A restricted ability of pme6-1 stomata to respond to abscisic acid, a classical regulator of stomatal function, was also observed (Figure S4E), suggesting that the altered pectin methyl-esterification status of the guard cells was affecting a fundamental property of the stomata.

7Since stomata play a major role in controlling the water relations of the plant, thermal imaging was used to investigate the effects of the pme6-1-altered guard cell wall properties at the whole-plant level by gauging leaf temperature as a measure of evaporative cooling (which is tightly linked to stomatal function). Under well-watered growth conditions, there were minimal differences in temperature between the pme6-1, the WT, and complemented mutant lines (Figure 3B). However, under drought conditions, pme6-1 plants were significantly cooler than the WT and complemented lines (Figures 3B and 3C), probably due to a higher rate of transpiration through their more open stomata. These results are consistent with the data in Figure 3A, indicating that the pme6-1 mutant stomata have a more restricted range of stomatal opening/closure as a result of altered guard cell wall properties and a more restricted response to ABA (Figure S4). To further investigate the physiological outcome of altered stomatal performance in the pme6-1 mutant, we conducted infrared gas exchange analysis to assess stomatal conductance (gs), in response to shifts in CO2 conditions. Under ambient CO2 conditions, the pme6-1 leaves had a higher gs compared to the WT (Figure 4A). When exposed to elevated CO2, the pme6-1 gs value decreased slightly but remained higher than the WT value. However, when the CO2 level was decreased to sub-ambient, both pme6-1 and WT gs increased but the maximal level achieved by the pme6-1 leaves plateaued at a lower level than that of the WT; thus, the overall dynamic range of gs shown by the pme6-1 leaves was lower than for the WT. Unlike stomatal aperture measurements in epidermal peels, gs is influenced not only by the guard cells but also by a variety of physiological processes in the whole leaf and plant, which may counteract the actions of individual stomata, leading to an amelioration of the response seen in isolated peels. The data in Figure 4A indicate that pme6-1 stomata have a more restricted dynamic range of opening/closure that cannot be completely compensated for at the whole plant level, thus leading to altered water relationships.

8An altered dynamic range of guard cell swelling and deflation was also indicated by direct observation of stomatal pore area after immersion in an osmoticum expected to decrease turgor pressure. Despite being subjected to a similar decrease in osmotic potential (1.23 MPa), the pme6-1 stomatal pores remained significantly larger than those of the WT (Figure 4B). Analysis of guard cells by electron microscopy did not reveal any overt difference in surface shape or size (Figures S2E and S2F), and overall cell ultrastructure appeared similar in the two genotypes (Figures S2G and S2H), suggesting that the different behavior of the stomata was not due to large-scale change in cell structure but rather to some alteration in the mechanical properties of the cell wall. The differential response to a similarly imposed biophysical challenge via mannitol treatment supports the idea that the pectin structure in the guard cells sets the mechanics of the cellular complex, thus limiting the range of size change possible. It has been postulated that the methyl-esterification status of pectin influences the ability of HGA domains to associate via Ca2+ cross-links and that the degree of Ca2+ cross-linking has a major effect on the mechanical stiffness of the cell wall matrix. Indeed, previous work on guard cell walls suggested that arabinan side chains of the rhamnogalacturonan-I pectic domain associated with HGA might play a role in physically separating HGA domains, thus influencing Ca2+ cross-linking. Since the formation of pectic network structures will be strongly dependent on the methyl-esterification status of HGA, PMEs can be predicted to play a major role in determining the overall mechanical properties of the cell wall. However, although recent evidence strongly supports the role of pectin methyl-esterification status in cell wall mechanics, simple inference of mechanics based on pectin methyl-esterification has proven problematic, since it appears to be highly dependent on cellular context. Our data suggest a situation in which a reduction in pectin de-esterification leads to guard cells that are relatively stiffer, limiting potential changes in cell size.

9From our gas exchange analysis, we derived A/Ci (photosynthesis rate/internal CO2 concentration) curves relating instantaneous carbon assimilation rate to CO2 level (Figure 4D). At ambient CO2, both pme6-1 and WT leaves showed similar assimilation rates; however, at elevated CO2, a much higher rate was measured in pme6-1. As CO2 level rises relative to O2, it is expected that photorespiration in C3 plants (such as Arabidopsis) will decrease, thus leading to a higher net assimilation rate. Leaves with stomata that show a decreased closure response to CO2, as observed here for pme6-1, might be expected to have higher internal CO2 levels and, thus, a greater increase in assimilation rate than the WT, as indicated in Figure 4D. To investigate the outcome of this at a whole-plant level, we compared the growth of WT and pme6-1 mutant plants under ambient and elevated CO2. pme6-1 plants were smaller than WTs when grown under ambient CO2 (Figures 4E and 4F). When grown under elevated CO2, the pme6-1 and WT plants were larger than the equivalent plants grown under ambient CO2 (as expected), but there was no difference in size between mutants and WTs (Figures 4E and 4G). Gas exchange analysis confirmed that the more limited dynamic range in gs observed in the pme6-1 leaves under ambient CO2 conditions was maintained when the plants were grown under elevated CO2 (Figure 4C), indicating that the underlying, more limited, dynamic range of stomatal function was also present in the mutant under these growth conditions, as expected for a genetically determined change in cell wall structure.

10Stomatal size and density are well-characterized parameters known to influence leaf performance under various environments. In the wake of rising atmospheric CO2 levels, there has been much interest in understanding how stomatal parameters provide an insight into past evolutionary events linked to earlier environments and into the potential modification of stomata to create crop plants better attuned to present and predicted climates. The potential role of the guard cell wall in setting and modulating the response dynamics of stomata has been underexplored. Since our analysis did not indicate any differences in stomatal density or stomatal index between pme6-1 and WT leaves (Figures S4A–S4D), the most plausible interpretation of our data is that that modulation of the pectin matrix of guard cells leads to altered wall properties so that the stomata are mechanically limited in their responses to exogenous cues, thus altering plant-water relations.

11Despite a firm theoretical basis for the importance of differential guard cell wall stiffness in the mechanism of stomata opening and closure in response to altered turgor pressure, experimental evidence has often been correlative, e.g., measurements of cell wall thickening, observations of cellulose microfibril orientation. Indeed, despite a wealth of physiological data on turgor pressure and ion fluxes, and intricate details of molecular signaling in guard cells, the causal relationship between guard cell wall structure/composition and stomatal function has been surprisingly underexplored. The most insightful data have come from experiments in which the exogenous supply of cell-wall-modifying enzymes suggested an important role for pectic arabinans in stomatal function. However, the nature of the genes involved and, indeed, formal genetic evidence to support this hypothesis, are lacking. In this paper, we provide molecular data to show that not only do Arabidopsis guard cells have a specific HGA methyl-esterification status (unesterified), but also that this status is required for normal stomatal function. The simplest interpretation of our data is that abnormal pectin methyl-esterification alters mechanical properties of the guard cells, leading to an inability to show appropriate opening and closure in response to environmental signals. Due to the importance of the mechanical properties of guard cells in setting the dynamics of stomatal opening and, thus, whole-plant/water relations, this alteration in guard-cell-specific cell wall gene expression leads to a poorer ability of the leaf to control water loss under drought conditions and poorer growth of the plants under ambient CO2 levels. Interestingly, this growth defect was overcome when the mutant plants were grown at elevated CO2, indicating that, under certain conditions, altered guard cell wall mechanics are not detrimental. In summary, our data indicate that, in addition to the well-explored regulation of stomatal dynamics via signal transduction signals acting on ion transport to vary turgor pressure, cell wall modification plays an important role in setting the overall limits of the system. Although this study focused on the role of pectins, it is clear that cell wall matrix components function together to influence mechanical properties, and exploring the roles of both structural carbohydrates, such as xyloglucans and cellulose, and modulating protein factors, such as expansins, will allow a deeper understanding of the system. Allied to this, targeted modulation of guard cell wall structure provides a novel avenue for the future manipulation of stomatal function.

12Details of the experimental procedures are available in the Supplemental Information.

13Conceptualization, A.J.F. and J.E.G.; Investigation, S.A., L.H., N.E., A.B., M.L., Y.V, and A.J.F.; Writing – Original Draft, A.J.F., J.E.G., and S.A.; Writing – Review & Editing, A.J.F., J.E.G., S.A., L.H., J.P.K., Y.V., and H.V.S.; Resources, J.P.K.; Supervision, A.J.F. and J.E.G.; Funding Acquisition, A.J.F., J.E.G., and H.V.S.