The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, β-CA1 and β-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δβ-ca1 and Δβ-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δβ-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δβ-ca1ca5 plants normalized at 9,000 ppm CO2. Leaves of Δβ-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δβ-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δβ-ca1ca5 seedling germination and development is negatively affected at ambient CO2. Transgenes expressing full-length β-CA1 and β-CA5 proteins complemented the Δβ-ca1ca5 mutation but inactivated (ΔZn-βCA1) and cytoplasm-localized (Δ62-βCA1) forms of β-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-βCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δβ-ca1 and Δβ-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.
The circadian clock regulates temporal metabolic activities, enabling organisms to adapt to cyclic environmental changes, but how it affects lipid metabolism in plants is poorly understood. Our previous finding showed that the central clock transcription factors LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) increased seed oil contents. Here we investigated the molecular and metabolic mechanism underlying the LHY and CCA1 regulated oil accumulation. Triacylglycerol (TAG) accumulation in Arabidopsis developing seeds increased in LHY-overexpressing (LHY-OE) and decreased in lhycca1 plants compared to wild-type (WT). Metabolic tracking indicated that fatty acids (FAs) of major lipid precursors for TAG production increased more rapidly in LHY-OE and slowly in lhycca1 than in WT, suggesting that LHY enhanced FA synthesis. Transcript analysis revealed that the expression of genes involved in FA synthesis, including beta-ketoacyl-ACP synthase III (KASIII), was oppositely changed in developing seeds of LHY/CCA1-OEs and lhycca1. Chromatin immunoprecipitation, electrophoretic mobility shift, and transactivation assays indicated that LHY directly bound and activated the promoter of KASIII. Furthermore, phosphatidic acid, a metabolic precursor to TAG, inhibited LHY binding to KASIII promoter elements. Our data reveal a new regulatory mechanism by the core clock regulators for storage lipid production during plant seed development.
Background Non-invasive reporter systems are powerful tools to query physiological and transcriptional responses in organisms. For example, fluorescent and bioluminescent reporters have revolutionized cellular and organismal assays and have been used to study plant responses to abiotic and biotic stressors. Integrated, cooled charge-coupled device (CCD) camera systems have been developed to image bioluminescent and fluorescent signals in a variety of organisms; however, these integrated long-term imaging systems are expensive. Results We have developed self-assembled systems for both growing and monitoring plant fluorescence and bioluminescence for long-term experiments under controlled environmental conditions. This system combines environmental growth chambers with high-sensitivity CCD cameras, multi-wavelength LEDs, open-source software, and several options for coordinating lights with imaging. This easy-to-assemble system can be used for short and long-term imaging of bioluminescent reporters, acute light-response, circadian rhythms, delayed fluorescence, and fluorescent-protein-based assays in vivo. Conclusions We have developed two self-assembled imaging systems that will be useful to researchers interested in continuously monitoring in vivo reporter systems in various plant species.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.