Expression of a CO2-permeable aquaporin enhances mesophyll conductance in the C4 species Setaria viridis
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Curated by eLife
Evaluation Summary:
The conductance of CO2 into the chloroplast from the intercellular airspace is a key limitation to rates of net photosynthesis. Despite its importance, past work has been contradictory in what does and does not affect this mesophyll conductance. This paper takes a unique and multi-pronged approach to resolving the mechanisms of mesophyll conductance and proposing a transgenic approach for increasing it in C4 plants.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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- Plant Biology (eLife)
Abstract
A fundamental limitation of photosynthetic carbon fixation is the availability of CO 2 . In C 4 plants, primary carboxylation occurs in mesophyll cytosol, and little is known about the role of CO 2 diffusion in facilitating C 4 photosynthesis. We have examined the expression, localization, and functional role of selected plasma membrane intrinsic aquaporins (PIPs) from Setaria italica (foxtail millet) and discovered that SiPIP2;7 is CO 2 -permeable. When ectopically expressed in mesophyll cells of Setaria viridis (green foxtail), SiPIP2;7 was localized to the plasma membrane and caused no marked changes in leaf biochemistry. Gas exchange and C 18 O 16 O discrimination measurements revealed that targeted expression of SiPIP2;7 enhanced the conductance to CO 2 diffusion from the intercellular airspace to the mesophyll cytosol. Our results demonstrate that mesophyll conductance limits C 4 photosynthesis at low p CO 2 and that SiPIP2;7 is a functional CO 2 permeable aquaporin that can improve CO 2 diffusion at the airspace/mesophyll interface and enhance C 4 photosynthesis.
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Evaluation Summary:
The conductance of CO2 into the chloroplast from the intercellular airspace is a key limitation to rates of net photosynthesis. Despite its importance, past work has been contradictory in what does and does not affect this mesophyll conductance. This paper takes a unique and multi-pronged approach to resolving the mechanisms of mesophyll conductance and proposing a transgenic approach for increasing it in C4 plants.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Counter to what one would expect, the effects of introducing the aquaporins seem to be more on Vmax than on Km for CO2, and it is unclear why that would be. Does this not rule out the proposed mechanism?
A more serious issue is that no details were given about how the transformant lines were selected. This has the potential to negate the claims of the paper. The Materials and Methods are so terse as to be unhelpful on this important point. Out of many, only three "positives" and one azygous transformant line were investigated. Given that past work of this type from other labs has fallen into the trap of "selecting for the desired results", it would be crucial to include both descriptions of how these were chosen as well as more detailed statistical comparisons with randomly-chosen transformants, …
Reviewer #1 (Public Review):
Counter to what one would expect, the effects of introducing the aquaporins seem to be more on Vmax than on Km for CO2, and it is unclear why that would be. Does this not rule out the proposed mechanism?
A more serious issue is that no details were given about how the transformant lines were selected. This has the potential to negate the claims of the paper. The Materials and Methods are so terse as to be unhelpful on this important point. Out of many, only three "positives" and one azygous transformant line were investigated. Given that past work of this type from other labs has fallen into the trap of "selecting for the desired results", it would be crucial to include both descriptions of how these were chosen as well as more detailed statistical comparisons with randomly-chosen transformants, non-transformed lines to show that the phenotypic effects can be reasonably attributed to the expression of aquaporins.
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Reviewer #2 (Public Review):
Strengths:
Past work looking at mesophyll conductance has approached the problem mainly using the same set of tools (transgenic transformation combined with a fluorescence or isotope-based estimate of mesophyll conductance) in C3 plants. This study adds confirmatory work with yeast to study the functional role of the transformed PIP in facilitating CO2 diffusion in a C4 species. They also examined the C4 transgenics using oxygen isotope approaches that have been recently developed to tackle the difficulty of measuring mesophyll conductance in a C4 system. This isotope approach is validated by comparing it to an independent method based on gas exchange alone.
Another strength in this work is that the lines selected for further analysis were selected based on the strength of the transgene expression and not a …
Reviewer #2 (Public Review):
Strengths:
Past work looking at mesophyll conductance has approached the problem mainly using the same set of tools (transgenic transformation combined with a fluorescence or isotope-based estimate of mesophyll conductance) in C3 plants. This study adds confirmatory work with yeast to study the functional role of the transformed PIP in facilitating CO2 diffusion in a C4 species. They also examined the C4 transgenics using oxygen isotope approaches that have been recently developed to tackle the difficulty of measuring mesophyll conductance in a C4 system. This isotope approach is validated by comparing it to an independent method based on gas exchange alone.
Another strength in this work is that the lines selected for further analysis were selected based on the strength of the transgene expression and not a "self-fulfilling" phenotype such as improved photochemical efficiency. This is important since in many other studies investigating improved photosynthetic capacity, large numbers of transgenic plants are screened using approaches that would preferentially select for improved photosynthesis that could have arisen through a pleiotropic effect such as where the insert occurred. By selecting based on transgene expression, then phenotyping photosynthesis, the group more effectively ties the transgene to the phenotype. Their point could be improved if they better highlighted the relationship between the protein abundance of the transgene and the strength of a phenotype.
Weaknesses
While there is compelling support for the role of aquaporins in mesophyll conductance in C3 plants, there are also many studies where no difference is seen. The paper could be improved by discussing this work as well and attempting to rectify it with their study's results.
It is not clear why the transgenics have higher photosynthetic rates under elevated CO2. It would be expected that if mesophyll conductance was the only thing altered, that the benefit to the photosynthesis would be most obvious in the initial portion of the A-Ci curve and then decrease as CO2 becomes saturating. This could be discussed more in depth if there is a ready explanation.
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Reviewer #3 (Public Review):
The potential of using aquaporins to engineer and improved mesophyll conductance and, consequently, an improved photosynthetic efficiency has long been mooted. In C3 plants there are data to support this approach (although nt without some controversy). In this paper the authors set out to test the hypothess that engineering aquaporin expression in a plant with C4 photosynthesis might also function to improve mesophyll conductance.
Using Setaria as the platform for engineering, they first performed a very careful analysis of genes encoding a type of aquaporinm the plasma membrane intrinsiv proteins (PIPs). This involved functional analaysis of CO2 and water permeability in yeast engineered to express the various PIP genes from setaria. This provided convincing evidence that at least one of the PIPs functioned …
Reviewer #3 (Public Review):
The potential of using aquaporins to engineer and improved mesophyll conductance and, consequently, an improved photosynthetic efficiency has long been mooted. In C3 plants there are data to support this approach (although nt without some controversy). In this paper the authors set out to test the hypothess that engineering aquaporin expression in a plant with C4 photosynthesis might also function to improve mesophyll conductance.
Using Setaria as the platform for engineering, they first performed a very careful analysis of genes encoding a type of aquaporinm the plasma membrane intrinsiv proteins (PIPs). This involved functional analaysis of CO2 and water permeability in yeast engineered to express the various PIP genes from setaria. This provided convincing evidence that at least one of the PIPs functioned to enhance CO2 flux. This gene was then used to generate transgenic setaria plants in which expression of the selected PIPs gene was targetted towards the leaf mespohyll. This was achieved, with subsequent analaysis of leaf physiology supporting the conclusion that the expresison of the PIPs gene did lead to an enhancement of carbon assimilation rate and that this was at least partially related to an increased measurement of mesophyll conductance.
The results extend the work performed on C3 plants by showing that in C4 plants mesophyll conductance can be enginnered by increased aquaporin expression, with the increased rate of carbon assimilation and, thus, the potential for improving crop yields in future work.
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