8.seminar(1)

Co2 role

1. ROLE OF ABA FOR STOMATAL REGULATION UNDER ELEVATED CO 2 ALOK KUMAR CHOURASIYA AKSHAY S. SAKHARE SUDHIR KUMAR DIVISION OF PLANT PHYSIOLOGY ICAR - INDIAN AGRICULTURAL RESEARCH INSTITUTE 1

2. OUTLINE Introduction Case study Results Conclusions Future prospects 2

3. Introduction Elevated atmospheric carbon dioxide concentration (elevated CO 2 ) is a major component of climate change. The present and ongoing elevation in [CO 2 ] not only causes global warming, but also affects the physiology and development of plants. The global surface temperature is projected to rise 2.6–4.8 C by the end of this century, according to RCP8.5 (IPCC, 2013). Climate change, including elevated CO2, rising temperatures, and altered precipitation patterns have markedly affected terrestrial ecosystem structure and function, carbon and water balance, and crop productivity. 3

4. At present atmospheric levels of CO 2 , C4 plants are more efficient at photosynthesis than C3 but at elevated levels of atmospheric CO2 the efficiencies of C3 plants (in photosynthesis rate and water use) are as good as or better than C4 plants. CO 2 acts as an environmental signal that regulates stomatal movements. Elevated [CO 2 ] reduces the stomatal conductance and transpiration and improves water use efficiency. High CO2 concentrations reduce stomatal aperture, whereas low concentrations trigger stomatal opening. 4

5. Two models have been considered: ( i ) CO 2 elevation enhances ABA concentrations and/or early ABA signalling in guard cells to induce stomatal closure. (ii) CO 2 signalling merges with ABA at OST1/SnRK2.6 protein kinase activation. The plant hormone Abscisic acid (ABA) mediates seed dormancy, controls seedling development and triggers tolerance to abiotic stresses, including drought. ABA also reduces the stomatal conductance in the drought conditions to maintain water in the plants. CO 2 elevation and the plant hormone Abscisic acid (ABA) both induce rapid stomatal closure. 5

6. CO 2 Signal Perception: The Role of Guard Cells Guard-Cell CO 2 -Response Mechanisms- Several genes have been identified and characterized that function in Arabidopsis guard cells in CO 2 -mediated control of stomatal movements, these encode proteins. These components like carbonic anhydrases β CA1 and β CA4, the protein kinases MPK12, MPK4, HT1, CBC1 and CBC2, OST1 and GHR1, the S-type and R-type anion channels AtSLAC1 and AtALMT12/QUAC1, and a MATE-type transporter RHC1. carbonic anhydrases accelerate the catalysis of CO 2 molecules to bicarbonate (HCO 3 – ) and protons. HT1 kinase is important for regulation of stomatal movements in response to CO2. 6

7. Possible stomatal response mechanisms controlling guard cells (GC) under elevated CO 2 Xu et al ., 2016 7

8. Zhang et al., 2018 Connections represent positive regulation (green arrows) and negative regulation (red blocks) of high [CO 2 ]-induced stomatal closing. Regulatory pathways are predicted to be direct (lines) or are unknown and remain to be further investigated (dashed lines). Simplified model for CO 2 signal transduction in regulation of stomatal movements . 8

9. Guard cell ion channel functions and ABA-induced signal transduction across the plasma membrane and vacuolar membrane of guard cells Abscisic acid, CO 2 , and Ca 2+ signaling in stomata Kim T. H.et al., 2010 9

10. Schematic model of ABA signal transduction mechanisms in guard cells Munemasa S.et al., 2015 10

11. ABA-induced stomatal closure 11

12. Schroeder et al., 2011 12

13. Plants respond to elevated CO 2 via carbonic anhydrases, that mediate stomatal closing. Bicarbonate functions as a small molecule activator of SLAC1 anion channels in guard cells. Downstream of the OST1 protein kinase there is evidence that the guard cell elevated [CO 2 ]-signalling pathway converges with the guard cell ABA-signalling pathway . Schroeder et al., 2011 13

14. This model indicates that CO 2 induced stomatal closure and reduction in stomatal density requires the ABA and ABA signaling pathways Either it is connected directly or indirectly. 14

15. Case study 15

16. Results 16

17. Stomatal closure induced by elevated [CO 2 ] is inhibited by reactive oxygen species (ROS) scavengers Tiron and Tempol . Elevated [CO 2 ] stimulates an increase in guard cell H2DCFDA fluorescence that is blocked in the presence of Tempol or Tiron . Elevated [CO 2 ]-induced stomatal closure is disrupted in the rbohD rbohF mutant. Elevated [CO 2 ] stimulates an increase in wild- type guard cell H2DCFDA fluorescence but results in decreased fluorescence rbohD rbohF guard cells. Representative images showing fluorescence of rbohD rbohF and wild-type guard cells under ambient and elevated (1,000 ppm) [CO 2 ] The stomatal density response to elevated (1,000 ppm) [CO 2 ] requires ROS signaling via NADPH oxidases RbohF and RbohD . Mean stomatal density of wild-type leaves was significantly reduced when grown under 1,000 ppm [CO 2 ] in comparison to ambient [CO 2 ] Stomatal Response to Elevated [CO 2 ] requires generation of Reactive Oxygen Species (ROS) in Guard cells 17

18. Mean stomatal aperture was significantly reduced in wild-type stomata treated with 800 ppm CO 2 compared with treatment with ambient CO 2 , but this response was disrupted in pyr1 pyl1 pyl4 and pyr1 pyl1pyl2 pyl4. Exposure to elevated [CO 2 ] fails to stimulate an increase in guard cell H2DCFDA fluorescence in ABA receptor mutants. Mean fluorescence was significantly higher in wild-type stomata treated with 800 ppm [CO 2 ] compared with treatment with ambient [CO 2 ] but did not increase in pyr1 pyl1 pyl4 and pyr1 pyl1 pyl2 pyl4. Stomatal Response to Elevated [CO 2 ] Requires the PYR/RCAR ABA Receptors 18

19. Mean stomatal aperture is significantly reduced in wild-type stomata treated with 1,000 ppm CO 2 but this response is disrupted in nced3, nced5. Exposure to elevated [CO 2 ] does not induce a significant reduction in stomatal conductance in nced3 nced5. Mean stomatal conductance was significantly reduced in leaves of wild-type plants exposed to 1,000 ppm [CO 2 ] compared to ambient [CO 2 ], but not in nced3, nced5. Exposure to elevated [CO 2 ] fails to stimulate an increase in guard cell H2DCFDA fluorescence in nced3 nced5. Mean fluorescence was significantly higher in wild-type stomata treated with 1,000 ppm [CO 2 ] compared with treatment with ambient [CO 2 ] but did not increase in nced3, nced5. Stomatal Aperture Response to Elevated [CO 2 ] Requires ABA Biosynthesis in Guard Cells 19

20. Mean stomatal density of wild-type leaves was significantly reduced when grown under 1,000 ppm [CO 2 ] in comparison to when grown at ambient [CO 2 ] but was not reduced in pyr1 pyl1 pyl2 pyl4 at elevated [CO 2 ]. Stomatal densities of nced3 nced5 and aba3 were significantly higher than wild-type when grown under either ambient or elevated [CO 2 ] 1,000 ppm) and did not reduce when grown at elevated [CO 2 ]. Stomatal densities of MYB60pro::ABA3 or SPCHpro ::NCED3-YFP were not significantly different to wild-type when grown under ambient [CO 2 ] but reduced significantly when grown at elevated [CO 2 ]. Tracing of epidermal impressions to illustrate the difference in stomatal densities between wild-type and nced3 nced5 leaves following growth at 1,000 ppm [CO 2 ]. The scale bar represents 100 mm. The Stomatal Density Response to [CO 2 ] Requires ABA Perception and Biosynthesis 20

21. Conclusions The elevated [CO 2 ]-induced closure and reductions in stomatal density require the generation of reactive oxygen species (ROS), thereby adding a new common element to these signalling pathways. It is also shown that the PYR/RCAR family of ABA receptors and ABA itself are required in both responses. ABA in guard cells or their precursors is sufficient to mediate the [CO 2 ]-induced stomatal density response. It is suggested that at least some of the effects of [CO 2 ] on stomata result from its ability to access the guard cell ABA signalling pathway through the intermediacy of ABA. Overall, these analyses suggest that it is the sensitivity to or precise localization of the ABA rather than total foliar ABA concentrations per se that are responsible for the stomatal density response to changes in [CO 2 ]. Finally, the dependency of stomatal [CO 2 ] signalling on ABA suggests that the ABA pathway is, in evolutionary terms, likely to be ancestral. 21

22. Future prospects It will be interesting in future work to investigate the question of synthesis versus sensitivity. Although key genetic components have been recently identified, many of the cellular signalling, biochemical, and interaction mechanisms remain to be elucidated. Further research can be aimed at identification of CO 2 and HCO 3 – sensors and the precise interactions among the newly recognized components in the CO 2 signal transduction network. Insights from such studies can further lead to new approaches for engineering crop plants to adapt to climate change. 22

23. Thank You 23