Hu Sun, Inken Thiemann, Nils Schmidt, Johannes Kromdijk, Tracey Lawson, Martin Hagemann, Stefan Timm
Summary Photorespiration is traditionally viewed as a limitation to photosynthetic efficiency. However, it is mandatory for safeguarding the Calvin–Benson–Bassham cycle from inhibitory byproducts through Rubisco‐mediated oxidative misfire and is tightly integrated with primary metabolism. Whether photorespiratory metabolism directly regulates guard cell function and stomatal behavior remains a matter of intense debate. We manipulated the photorespiration‐restricted pathway entry enzyme 2‐phosphoglycolate phosphatase (PGLP1) specifically in Arabidopsis thaliana (Arabidopsis) guard cells and assessed effects on growth, photosynthesis, carbohydrate allocation, cell‐specific H 2 O 2 accumulation, and stomatal traits under photorespiratory conditions, including exogenous 2‐phosphoglycolate (2‐PG) feeding. Altered guard cell PGLP1 protein expression consistently affected plant growth, photosynthetic performance, and stomatal movement. PGLP1 perturbation induced guard cell‐specific starch and H 2 O 2 accumulation patterns, both of which are central components involved in driving optimal stomatal behavior. Further, altered stomatal size was observed, a phenotype that was recapitulated by external 2‐PG application to wild‐type plants. Efficient photorespiratory metabolism is essential for proper guard cell function and acclimation to changing CO 2 : O 2 ratios. Our findings uncover a direct metabolic link between photorespiration and stomatal behavior, revealing an unexpected role for this ancient pathway in controlling gas exchange, photosynthesis, and potentially plant productivity and resilience.