Final Report Summary - KINPLANTS (Regulation of inward K+ channel activity in Arabidopsis by the Shaker subunit AtKC1: molecular mechanisms and role in control of stomatal opening and plant adaptation to water stress)
Plant Shaker channels form the major K+ conductance of the plasma membrane, thereby mediating large K+ fluxes required for plant growth and development. Also, owing to the role of K+ ion in control of the cell osmotic potential, they are involved in the modulation of stomatal movements, which is necessary for allowing CO2 uptake while limiting transpirational water loss. Thus, they contribute to plant adaptation to changes in water availability and air humidity. Shaker channels are homo- or heterotetramers associating 4 Shaker subunits. In the model plant Arabidopsis, the Shaker subunit AtKC1 is not able to form a functional homotetrameric channel at the plasma membrane. Instead, it is retained in the endoplasmic reticulum (ER). However, when co-expressed with other Shaker subunits, it forms heteromeric channels targeted to the plasma membrane and endowed with new functional properties. Thus, AtKC1 behaves as a silent regulatory subunit. The atkc1 mutant displays a remarkable and pleiotropic phenotype, including reduced growth and impaired stomatal control leading to increased transpirational water loss. The project proposes an integrated approach associating molecular, biochemical and biophysical approaches, along with cell biology and whole plant physiology, in order to unravel the role that AtKC1 plays in stomatal regulation and adaptation to water stress. Different aspects will be investigated: the molecular determinants of AtKC1 activity (for instance, regulatory partner proteins), and physiological impacts of AtKC1 activity on whole plant and guard cell physiology under different environmental and hormonal conditions. Concerning the latter aspect, three working hypotheses have been considered: (i) a direct role of AtKC1 in guard cell turgor and stomatal functioning (via the regulation of inward K+ currents), (ii) a role in the control of the turgor of adjacent epidermal cells, leading to a modulation of stomatal aperture by compression, and (iii) a more indirect effect, via a role in the regulation of the membrane conductance to K+ and of the membrane potential, and thereby in the control of auxin transport (possibly from the hydathodes) that would affect both growth and stomatal opening. These three hypotheses are not mutually exclusive. To gain knowledge on this issue, physiological aspects have been studied in wild type and atkc1 KO mutant plants, using the Phenopsis phenotyping platform for whole plant transpiration measurements and electrophysiology techniques for measuring K+ channel activity and membrane potential changes in epidermal and guard cells. The atkc1 mutant plants have been complemented using constructs targeting expression of AtKC1 in individual leaf cell types where the endogenous AtKC1 gene has been reported to be expressed, e.g. in guard cells, epidermal cells, hydathodes and trichomes. Cell turgor has been assessed using concentrated osmolyte solutions. The whole set of results indicates that AtKC1 plays a major role in the control of stomatal aperture by a mechanism that was never reported so far at the molecular level, involving control of the counter-pressure that epidermal cells exert on guard cells.
Main S&T results/foregrounds
The two main objectives proposed in the project, i.e. the identification of molecular determinants of AtKC1 activity and the physiological impacts of AtKC1 activity on whole plant transpiration, were addressed in parallel. Regarding the molecular determinants of AtKC1 activity, during the previous stage of the scientist in the host lab, molecular determinants involved in the ER retention of AtKC1 were identified. Briefly, the strategy relied on exchanging sequences between AtKC1 (retained in the ER) and KAT2 (Shaker subunit that is targeted to the plasma membrane (PM)). Chimeric channels were transiently expressed in tobacco protoplasts to assess their subcellular localization. As a result, precise sequences regulating the subcellular localisation of AtKC1 and KAT2 were characterised. These results have led to a manuscript (in press) and two contributions to international conferences (see dissemination of foreground section below). Such work was finished during the first months of the fellowship. On the other hand, search for interacting partners of AtKC1 by the split-ubiquitin system (SUS) yielded less promising results during the initial stage of the project. Interaction of putative candidate proteins identified with AtKC1 as a bait could not be further confirmed (putative false positives?). However, interaction of AtKC1 with other Shaker subunits, with which AtKC1 was previously shown to interact and also regulated (Pilot et al. 2003, Jeanguenin et al. 2011), seemed consistent. Thus, we decided to explore whether these Shaker partners participated together with AtKC1 in the control of plant transpiration. For that purpose, establishment of double mutant lines, exhibiting loss of function of AtKC1 and another Shaker subunit, was carried out. We succeeded to produce two different types of double KO mutants (atkc1 KO plus other Shaker subunit KO). For both of them, no interaction was observed between the phenotype of atkc1 mutant plants and that of the other Shaker subunit in comparison to the double mutant phenotype. Both types of double mutant plants behaved as atkc1 KO mutant plants, which points to a dominant/specific effect of AtKC1 in the regulation of stomatal opening.
With respect to the second objective, physiological impacts of AtKC1 activity on whole plant and guard cell physiology, preliminary experiments were performed with the aim to determine whether AtKC1 activity in guard cells regulated stomatal opening. They relied on characterisation of transgenic atkc1 KO mutant plants expressing AtKC1 only in guard cells (cell-specific complementation). The latter was achieved by fusing a guard-cell specific promoter to the AtKC1 gene in the transgene. Interestingly, these plants behaved similarly to atkc1 KO mutant plants and thus suggested that AtKC1 activity is required in other leaf cell types, probably epidermal cells, to display proper stomatal opening. Then cell-specific expression of AtKC1 in different leaf cell types (other than guard cells) in which native AtKC1 is expressed (namely trichomes, hydathodes and epidermal cells; Pilot et al. 2003), was started. Promoter sequences expected to drive cell-specific expression were fused to the AtKC1 cDNA and introduced in atkc1 mutant plants. In parallel, in other set of experiments, plants expressing cell-specific promoters fused to the reporter gene GUS (β-glucuronidase) were obtained to confirm the cell-specific expression of these promoters. While these plants were amplified in the greenhouse, experiments on atkc1 mutant plants to further characterize the mutant phenotypes were carried out. Whole plant transpiration experiments on adult plants were performed on sealed pots under a constant water regime. In agreement with the previous results obtained in the host lab with epidermis peels, mutant plants displayed larger transpiration rates during light and dark periods. These experiments provided us with quantitative data about the contribution of AtKC1 activity to total biomass and water use efficiency. Additionally, putative effect of auxins on the stomatal movements of atkc1 mutant plants (hypothesis (iii)) was tested on atkc1 mutant epidermal peels and was ruled out. Taking into consideration all these results, hypothesis (i) and (iii) seemed less certain and we focused further research on testing hypothesis (ii), especially by the characterization of plants expressing AtKC1 in a cell-specific manner.
Cell-specific expression of AtKC1 in leaf cells in which AtKC1 is expressed (namely trichomes, hydathodes and epidermal cells, besides guard cells; Pilot et al. 2003), was assessed. First, site-directed expression by these promoters in the corresponding cell types was confirmed in transgenic plants expressing a cell-specific promoter::GUS fusion in which GUS staining was observed in the expected cell types. Second, with respect atkc1 KO plants expressing cell-specific promoter::AtKC1 fusions, results in independent experiments showed that expression of AtKC1 in epidermal cells (achieved by two different promoters) allowed the corresponding atkc1 KO complemented plants to behave like wild type plants with respect stomatal aperture values. These results gave strong support to the hypothesis based on the role of the backpressure of epidermal cells on guard cells. To gain insights in the contribution of epidermal cells to stomatal aperture, the pressure probe technique was initially employed. However, epidermal cells have appeared to be not thick enough for the pressure probe methodology and values obtained could not be unequivocally attributable to epidermal cells. Then, alternative tests on epidermal peels were performed. Such tests relied on the use of different external concentration of osmolytes to induce changes in stomatal aperture values. As a result, behavior of atkc1 KO epidermal peels was markedly sensitive to external osmolytes in comparison to that of wild-type epidermal peels and evidenced a partial loss of mechanical resistance (provided by epidermal cells) of atkc1 KO guard cells to open and close. Finally, electophysiological recordings with single-barreled microelectrodes were carried out on epidermal cells from whole wild-type and atkc1 KO leaves. Results showed significant differences in membrane polarization of epidermal cells from each genotype. Thus, we hypothesize that a change in membrane polarization leads to altered K+ accumulation on epidermal cells and thereby giving rise to a loss of backpressure on guard cells. It is worth to note that such backpressure depends on the activity of the Shaker regulatory subunit AtKC1 in epidermal cells.
Potential impact
Understanding of the mechanisms involved in the control of plant transpiration is basic to develop new strategies aiming at improving plant water use efficiency. In the present project, the role of a Shaker regulatory subunit, AtKC1, in the regulation of plant transpiration by aiding epidermal cells to maintain backpressure on guard cells has been assessed. The latter mechanism has been described during the past decades in several plant species (Roelfsema and Hedrich, 2005) but the acting molecular entities have remained elusive. Such a discovery allows access to biotechnological approaches based on transgenic plants with improved performance. With this regard, since epidermal cell backpressure tunes stomatal aperture during both night and day periods, according to the transpiration defects of atkc1 mutant plants, it is thus a very attractive target mechanism to improve water use efficiency during the whole plant life. Moreover, since epidermal cell backpressure is present in several plant species, it is likely that the information gained on the AtKC1 contribution to stomatal opening by maintaining backpressure can be translatable to crops.
As dissemination of foreground activities, two articles have been accepted for publication and a third one based on the contribution of AtKC1 in plant transpiration is in preparation. Moreover, two contributions to international conferences have been made (see section A).
References
1. Jeanguenin L, Alcon C, Duby G, Boeglin M, Chérel I, Gaillard I, Zimmermann S, Sentenac H, Véry AA. (2011) Plant J 67:570-582.
2. Pilot G, Gaymard F, Mouline K, Cherel I, and Sentenac H (2003) Plant Mol Biol, 51: 773-787
3. Roelfsema MR, Hedrich R. (2005) New Phytol. 167:665-691
Main S&T results/foregrounds
The two main objectives proposed in the project, i.e. the identification of molecular determinants of AtKC1 activity and the physiological impacts of AtKC1 activity on whole plant transpiration, were addressed in parallel. Regarding the molecular determinants of AtKC1 activity, during the previous stage of the scientist in the host lab, molecular determinants involved in the ER retention of AtKC1 were identified. Briefly, the strategy relied on exchanging sequences between AtKC1 (retained in the ER) and KAT2 (Shaker subunit that is targeted to the plasma membrane (PM)). Chimeric channels were transiently expressed in tobacco protoplasts to assess their subcellular localization. As a result, precise sequences regulating the subcellular localisation of AtKC1 and KAT2 were characterised. These results have led to a manuscript (in press) and two contributions to international conferences (see dissemination of foreground section below). Such work was finished during the first months of the fellowship. On the other hand, search for interacting partners of AtKC1 by the split-ubiquitin system (SUS) yielded less promising results during the initial stage of the project. Interaction of putative candidate proteins identified with AtKC1 as a bait could not be further confirmed (putative false positives?). However, interaction of AtKC1 with other Shaker subunits, with which AtKC1 was previously shown to interact and also regulated (Pilot et al. 2003, Jeanguenin et al. 2011), seemed consistent. Thus, we decided to explore whether these Shaker partners participated together with AtKC1 in the control of plant transpiration. For that purpose, establishment of double mutant lines, exhibiting loss of function of AtKC1 and another Shaker subunit, was carried out. We succeeded to produce two different types of double KO mutants (atkc1 KO plus other Shaker subunit KO). For both of them, no interaction was observed between the phenotype of atkc1 mutant plants and that of the other Shaker subunit in comparison to the double mutant phenotype. Both types of double mutant plants behaved as atkc1 KO mutant plants, which points to a dominant/specific effect of AtKC1 in the regulation of stomatal opening.
With respect to the second objective, physiological impacts of AtKC1 activity on whole plant and guard cell physiology, preliminary experiments were performed with the aim to determine whether AtKC1 activity in guard cells regulated stomatal opening. They relied on characterisation of transgenic atkc1 KO mutant plants expressing AtKC1 only in guard cells (cell-specific complementation). The latter was achieved by fusing a guard-cell specific promoter to the AtKC1 gene in the transgene. Interestingly, these plants behaved similarly to atkc1 KO mutant plants and thus suggested that AtKC1 activity is required in other leaf cell types, probably epidermal cells, to display proper stomatal opening. Then cell-specific expression of AtKC1 in different leaf cell types (other than guard cells) in which native AtKC1 is expressed (namely trichomes, hydathodes and epidermal cells; Pilot et al. 2003), was started. Promoter sequences expected to drive cell-specific expression were fused to the AtKC1 cDNA and introduced in atkc1 mutant plants. In parallel, in other set of experiments, plants expressing cell-specific promoters fused to the reporter gene GUS (β-glucuronidase) were obtained to confirm the cell-specific expression of these promoters. While these plants were amplified in the greenhouse, experiments on atkc1 mutant plants to further characterize the mutant phenotypes were carried out. Whole plant transpiration experiments on adult plants were performed on sealed pots under a constant water regime. In agreement with the previous results obtained in the host lab with epidermis peels, mutant plants displayed larger transpiration rates during light and dark periods. These experiments provided us with quantitative data about the contribution of AtKC1 activity to total biomass and water use efficiency. Additionally, putative effect of auxins on the stomatal movements of atkc1 mutant plants (hypothesis (iii)) was tested on atkc1 mutant epidermal peels and was ruled out. Taking into consideration all these results, hypothesis (i) and (iii) seemed less certain and we focused further research on testing hypothesis (ii), especially by the characterization of plants expressing AtKC1 in a cell-specific manner.
Cell-specific expression of AtKC1 in leaf cells in which AtKC1 is expressed (namely trichomes, hydathodes and epidermal cells, besides guard cells; Pilot et al. 2003), was assessed. First, site-directed expression by these promoters in the corresponding cell types was confirmed in transgenic plants expressing a cell-specific promoter::GUS fusion in which GUS staining was observed in the expected cell types. Second, with respect atkc1 KO plants expressing cell-specific promoter::AtKC1 fusions, results in independent experiments showed that expression of AtKC1 in epidermal cells (achieved by two different promoters) allowed the corresponding atkc1 KO complemented plants to behave like wild type plants with respect stomatal aperture values. These results gave strong support to the hypothesis based on the role of the backpressure of epidermal cells on guard cells. To gain insights in the contribution of epidermal cells to stomatal aperture, the pressure probe technique was initially employed. However, epidermal cells have appeared to be not thick enough for the pressure probe methodology and values obtained could not be unequivocally attributable to epidermal cells. Then, alternative tests on epidermal peels were performed. Such tests relied on the use of different external concentration of osmolytes to induce changes in stomatal aperture values. As a result, behavior of atkc1 KO epidermal peels was markedly sensitive to external osmolytes in comparison to that of wild-type epidermal peels and evidenced a partial loss of mechanical resistance (provided by epidermal cells) of atkc1 KO guard cells to open and close. Finally, electophysiological recordings with single-barreled microelectrodes were carried out on epidermal cells from whole wild-type and atkc1 KO leaves. Results showed significant differences in membrane polarization of epidermal cells from each genotype. Thus, we hypothesize that a change in membrane polarization leads to altered K+ accumulation on epidermal cells and thereby giving rise to a loss of backpressure on guard cells. It is worth to note that such backpressure depends on the activity of the Shaker regulatory subunit AtKC1 in epidermal cells.
Potential impact
Understanding of the mechanisms involved in the control of plant transpiration is basic to develop new strategies aiming at improving plant water use efficiency. In the present project, the role of a Shaker regulatory subunit, AtKC1, in the regulation of plant transpiration by aiding epidermal cells to maintain backpressure on guard cells has been assessed. The latter mechanism has been described during the past decades in several plant species (Roelfsema and Hedrich, 2005) but the acting molecular entities have remained elusive. Such a discovery allows access to biotechnological approaches based on transgenic plants with improved performance. With this regard, since epidermal cell backpressure tunes stomatal aperture during both night and day periods, according to the transpiration defects of atkc1 mutant plants, it is thus a very attractive target mechanism to improve water use efficiency during the whole plant life. Moreover, since epidermal cell backpressure is present in several plant species, it is likely that the information gained on the AtKC1 contribution to stomatal opening by maintaining backpressure can be translatable to crops.
As dissemination of foreground activities, two articles have been accepted for publication and a third one based on the contribution of AtKC1 in plant transpiration is in preparation. Moreover, two contributions to international conferences have been made (see section A).
References
1. Jeanguenin L, Alcon C, Duby G, Boeglin M, Chérel I, Gaillard I, Zimmermann S, Sentenac H, Véry AA. (2011) Plant J 67:570-582.
2. Pilot G, Gaymard F, Mouline K, Cherel I, and Sentenac H (2003) Plant Mol Biol, 51: 773-787
3. Roelfsema MR, Hedrich R. (2005) New Phytol. 167:665-691