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1.
J Exp Bot ; 74(12): 3651-3666, 2023 06 27.
Article in English | MEDLINE | ID: mdl-36987927

ABSTRACT

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (ßca5) missing the plastid carbonic anhydrase ßCA5. Neither DCAKO nor ßca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3- concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3- uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. Furthermore, the ßca5 plant bioassay used in this study is the first system for testing HCO3- transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.


Subject(s)
Carbonic Anhydrases , Chlamydomonas reinhardtii , Bicarbonates/metabolism , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Carbon Dioxide/metabolism , Chloroplasts/metabolism , Photosynthesis , Plants/metabolism , Carbonic Anhydrases/genetics , Carbonic Anhydrases/metabolism
2.
Front Plant Sci ; 13: 1019938, 2022.
Article in English | MEDLINE | ID: mdl-36714729

ABSTRACT

Spliceostatin C (SPC) is a component of a bioherbicide isolated from the soil bacterium Burkholderia rinojensis. The chemical structure of SPC closely resembles spliceostatin A (SPA) which was characterized as an anticancer agent and splicing inhibitor. SPC inhibited the growth of Arabidopsis thaliana seedlings with an IC50 value of 2.2 µM. The seedlings exposed to SPC displayed a significant response with decreased root length and number and inhibition of gravitropism. Reverse transcriptase semi-quantitative PCR (RT-sqPCR) analyses of 19 selected genes demonstrated the active impact of SPC on the quality and quantity of transcripts that underwent intron rearrangements as well as up or down expression upon exposure to SPC. Qualitative and quantitative proteomic profiles identified 66 proteins that were significantly affected by SPC treatment. Further proteomics data analysis revealed that spliceostatin C induces hormone-related responses in Arabidopsis seedlings. In silico binding studies showed that SPC binds to a pocket between the SF3B3 and PF5A of the spliceosome.

3.
Elife ; 72018 11 14.
Article in English | MEDLINE | ID: mdl-30426925

ABSTRACT

In Chlamydomonas the different stages of the Calvin-Benson cycle take place in separate locations within the chloroplast.


Subject(s)
Chloroplasts , Photosynthesis , Chlamydomonas , Chlamydomonas reinhardtii , Ribulose-Bisphosphate Carboxylase
4.
Plant Sci ; 268: 11-17, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29362079

ABSTRACT

Carbonic anhydrases (CAs) are enzymes that catalyze the interconversion of CO2 and HCO3-. In nature, there are multiple families of CA, designated with the Greek letters α through θ. CAs are ubiquitous in plants, algae and photosynthetic bacteria, often playing essential roles in the CO2 concentrating mechanisms (CCMs) which enhance the delivery of CO2 to Rubisco. As algal CCMs become better characterized, it is clear that different types of CAs are playing the same role in different algae. For example, an α-CA catalyzes the conversion of accumulated HCO3- to CO2 in the green alga Chlamydomonas reinhardtii, while a θ-CA performs the same function in the diatom Phaeodactylum tricornutum. In this review we argue that, in addition to its role of delivering CO2 for photosynthesis, other metabolic roles of CA have likely changed as the Earth's atmospheric CO2 level decreased. Since the algal and plant lineages diverged well before the decrease in atmospheric CO2, it is likely that plant, algae and photosynthetic bacteria all adapted independently to the drop in atmospheric CO2. In light of this, we will discuss how the roles of CAs may have changed over time, focusing on the role of CA in pH regulation, how CAs affect CO2 supply for photosynthesis and how CAs may help in the delivery of HCO3- for other metabolic reactions.


Subject(s)
Carbonic Anhydrases/metabolism , Photosynthesis , Plants/enzymology , Biocatalysis , Carbon Dioxide/metabolism , Isoenzymes/metabolism
5.
J Exp Bot ; 68(14): 3879-3890, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28633328

ABSTRACT

The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii.


Subject(s)
Algal Proteins/genetics , Carbon/metabolism , Chlamydomonas reinhardtii/genetics , Chloroplast Proteins/genetics , Photosynthesis , Algal Proteins/metabolism , Carbon Compounds, Inorganic/metabolism , Chlamydomonas reinhardtii/metabolism , Chloroplast Proteins/metabolism , Up-Regulation
6.
Plant Methods ; 13: 22, 2017.
Article in English | MEDLINE | ID: mdl-28392829

ABSTRACT

BACKGROUND: Random insertional mutagenesis of Chlamydomonas reinhardtii using drug resistance cassettes has contributed to the generation of tens of thousands of transformants in dozens of labs around the world. In many instances these insertional mutants have helped elucidate the genetic basis of various physiological processes in this model organism. Unfortunately, the insertion sites of many interesting mutants are never defined due to experimental difficulties in establishing the location of the inserted cassette in the Chlamydomonas genome. It is fairly common that several months, or even years of work are conducted with no result. Here we describe a robust method to identify the location of the inserted DNA cassette in the Chlamydomonas genome. RESULTS: Insertional mutants were generated using a DNA cassette that confers paromomycin resistance. This protocol identified the cassette insertion site for greater than 80% of the transformants. In the majority of cases the insertion event was found to be simple, without large deletions of flanking genomic DNA. Multiple insertions were observed in less than 10% of recovered transformants. CONCLUSION: The method is quick, relatively inexpensive and does not require any special equipment beyond an electroporator. The protocol was tailored to ensure that the sequence of the Chlamydomonas genomic DNA flanking the random insertion is consistently obtained in a high proportion of transformants. A detailed protocol is presented to aid in the experimental design and implementation of mutant screens in Chlamydomonas.

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