Understanding CO₂ diffusion in C₄ plants: An investigation of CO₂ permeable aquaporins and carbonic anhydrase in the C₄ grass Setaria viridis
Date
2017
Authors
Osborn, Hannah Louise
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Abstract
The productive yield of key C₄ crops must increase in the future
to meet the demands of an increasing global population. We are
therefore endeavouring to improve the availability of CO₂ for
photosynthesis, one of the fundamental limitations to
photosynthetic carbon fixation. The initial steps of CO₂
assimilation in leaf mesophyll cells involve the diffusion of CO₂
from the intercellular airspace to the mesophyll cytosol
(mesophyll conductance). This involves CO₂ passing through the
liquid phase and the plasma membrane, a process believed to be
both passive and possibly facilitated by protein pores, known as
aquaporins. Within the cytosol of mesophyll cells, carbonic
anhydrase (CA) catalyses the hydration of CO₂ to HCO₃- which PEP
Carboxylase uses in the first CO₂ fixation step of C₄
photosynthesis. Here, I have examined the role of CO₂ permeable
aquaporins and CA from a C₄ photosynthesis perspective using the
model monocot species Setaria viridis (Foxtail millet).
CO₂ permeable aquaporins have been demonstrated to increase CO₂
diffusion in C3 plants. However, to date very little is known
about the role of CO₂ permeable aquaporins in the highly
efficient and specialised C₄ photosynthetic pathway. After
bioinformatic identification of all twelve Setaria PIPs (plasma
membrane intrinsic proteins) I first used yeast as a heterologous
expression system to confirm plasma membrane localisation and
determine CO₂ permeability of the plasma membrane using CO₂
triggered intracellular acidification on a stopped flow
spectrophotometry. This in vitro approach identified SiPIP2;7 as
a putative CO₂ permeable aquaporin, adding a third CO₂ pore to
the list of C₄ plant aquaporins characterised to date. I also
examined the effect of PIP1 and PIP2 co-expression and found
improved localisation to the plasma membrane but no improvement
to CO₂ permeability compared to the single PIP1s.
The effects of modifying CA activity in C₄ photosynthesis was
examined in planta. I silenced the major leaf CA in Setaria
viridis in three independent, stably transformed lines. At low
CO₂ a strong correlation between photosynthetic assimilation rate
and CA hydration rates was observed in the transformed lines,
which have as little as 13% of wild type CA activity.
Significantly, no visual phenotype or photosynthetic effect was
observed in the transformed lines at ambient CO₂. C¹⁸O¹⁶O isotope
discrimination was used to estimate the mesophyll conductance to
CO₂ diffusion from the intercellular air space to the mesophyll
cytosol in control plants, which allowed us to calculate CA
activities in the mesophyll cytosol. These results indicated that
CA is not rate limiting for C₄ photosynthesis in S. viridis under
current atmospheric conditions.
We conclude that CO₂ permeable aquaporins and CA activity are
factors with variable importance to CO₂ diffusion in C₄
photosynthesis, with both factors becoming rate limiting under
extreme environmental conditions that result in low intercellular
CO₂ such as drought stress.
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Photosynthesis, setaria viridis, aquaporins, carbonic anhydrase, C4 plants
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