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Background  Carbonic anhydrase (CA) catalyzes the hydration of CO 2  in the first biochemical step of C 4  photosynthesis, and has been considered a potentially rate-limiting step when CO 2  availability within a leaf is low. Previous work in  Zea mays  (maize) with a double knockout of the two highest-expressed β-CA genes, CA1 and CA2, reduced total leaf CA activity to less than 3% of wild-type. Surprisingly, this did not limit photosynthesis in maize at ambient or higher CO 2 concentrations. However, the  ca1ca2  mutants exhibited reduced rates of photosynthesis at sub-ambient CO 2 , and accumulated less biomass when grown under sub-ambient CO 2  (9.2 Pa). To further clarify the importance of CA for C 4  photosynthesis, we assessed gene expression changes in wild-type,  ca1  and  ca1ca2  mutants in response to changes in  p CO 2  from 920 to 9.2 Pa.    Results  Leaf samples from each genotype were collected for RNA-seq analysis at high CO 2  and at two time points after the low CO 2  transition, in order to identify early and longer-term responses to CO 2  deprivation. Despite the existence of multiple isoforms of CA, no other CA genes were upregulated in CA mutants. Although photosynthetic genes were downregulated in response to low CO 2 , differential expression was not observed between genotypes. However, multiple indicators of carbon starvation were present in the mutants, including amino acid synthesis, carbohydrate metabolism, and sugar signaling. In particular, multiple genes previously implicated in low carbon stress such as asparagine synthetase, amino acid transporters, trehalose-6-phosphate synthase, as well as many transcription factors, were strongly upregulated. Furthermore, genes in the CO 2  stomatal signaling pathway were differentially expressed in the CA mutants under low CO 2 .    Conclusions  Using a transcriptomic approach, we showed that carbonic anhydrase mutants do not compensate for the lack of CA activity by upregulating other CA or photosynthetic genes, but rather experienced extreme carbon stress when grown under low CO 2 . Our results also support a role for CA in the CO 2  stomatal signaling pathway. This study provides insight into the importance of CA for C 4  photosynthesis and its role in stomatal signaling.    Electronic supplementary material  The online version of this article (10.1186/s12864-019-5522-7) contains supplementary material, which is available to authorized users.

Insights from transcriptome profiling on the non-photosynthetic and stomatal signaling response of maize carbonic anhydrase mutants to low CO2