Carbonic anhydrases (CAs) are zinc-metalloenzymes that catalyze the interconversion of CO₂ and $HCO3−$⁠. In heterotrophic organisms, CAs provide $HCO3−$ for metabolic pathways requiring a carboxylation step. Arabidopsis (Arabidopsis thaliana) has 14 α- and β-type CAs, two of which are plastid CAs designated as βCA1 and βCA5. To study their physiological properties, we obtained knock-out (KO) lines for βCA1 (SALK_106570) and βCA5 (SALK_121932). These mutant lines were confirmed by genomic PCR, RT-PCR, and immunoblotting. While βca1 KO plants grew normally, growth of βca5 KO plants was stunted under ambient CO₂ conditions of 400 µL L⁻¹; high CO₂ conditions (30,000 µL L⁻¹) partially rescued their growth. These results were surprising, as βCA1 is more abundant than βCA5 in leaves. However, tissue expression patterns of these genes indicated that βCA1 is expressed only in shoot tissue, while βCA5 is expressed throughout the plant. We hypothesize that βCA5 compensates for loss of βCA1 but, owing to its expression being limited to leaves, βCA1 cannot compensate for loss of βCA5. We also demonstrate that βCA5 supplies $HCO3−$ required for anaplerotic pathways that take place in plastids, such as fatty acid biosynthesis.

LCIA 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 E. coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (βca5) missing the plastid carbonic anhydrase βCA5. Both DCAKO and βca5 cannot 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 βca5 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.

Long-standing growth/defence theories state that the production of defence compounds come at a direct cost to primary metabolism when resources are limited. However, such trade-offs are inherently difficult to quantify. We compared the growth and nitrogen partitioning in wild type Sorghum bicolor (L.) Moench, which contains the cyanogenic glucoside dhurrin, with unique mutants that vary in dhurrin production. The totally cyanide deficient 1 (tcd1) mutants do not synthesise dhurrin at all whereas mutants from the adult cyanide deficient class 1 (acdc1) have decreasing concentrations as plants age. Sorghum lines were grown at three different concentrations of nitrogen. Growth, chemical analysis, physiological measurements and expression of key genes in biosynthesis and turnover were determined for leaves, stems and roots at four developmental stages. Nitrogen supply, ontogeny, tissue type and genotype were all important determinants of tissue nitrate and dhurrin concentration and turnover. The higher growth of acdc1 plants strongly supports a growth/defence trade-off. By contrast, tcd1 plants had slower growth early in development, suggesting that dhurrin synthesis and turnover may be beneficial for early seedling growth rather than being a cost. The relatively small trade-off between nitrate and dhurrin suggests these may be independently regulated.

Sorghum bicolor (L.) Moench produces the nitrogen-containing natural product dhurrin that provides chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide gas. Drought can increase dhurrin in shoot tissues to concentrations toxic to livestock. As dhurrin is also a remobilizable store of reduced nitrogen and plays a role in stress mitigation, reductions in dhurrin may come at a cost to plant growth and stress tolerance. Here, we investigated the response to an extended period of water limitation in a unique EMS-mutant adult cyanide deficient class 1 (acdc1) that has a low dhurrin content in the leaves of mature plants. A mutant sibling line was included to assess the impact of unknown background mutations. Plants were grown under three watering regimes using a gravimetric platform, with growth parameters and dhurrin and nitrate concentrations assessed over four successive harvests. Tissue type was an important determinant of dhurrin and nitrate concentrations, with the response to water limitation differing between above and below ground tissues. Water limitation increased dhurrin concentration in the acdc1 shoots to the same extent as in wild-type plants and no growth advantage or disadvantage between the lines was observed. Lower dhurrin concentrations in the acdc1 leaf tissue when fully watered correlated with an increase in nitrate content in the shoot and roots of the mutant. In targeted breeding efforts to down-regulate dhurrin concentration, parallel effects on the level of stored nitrates should be considered in all vegetative tissues of this important forage crop to avoid potential toxic effects.

Cyanogenic glucosides are specialized metabolites produced by over 3000 species of higher plants from more than 130 families. The deployment of cyanogenic glucosides is influenced by biotic and abiotic factors in addition to being developmentally regulated, consistent with their roles in plant defense and stress mitigation. Despite their ubiquity, very little is known regarding the molecular mechanisms that regulate their biosynthesis. The biosynthetic pathway of dhurrin, the cyanogenic glucoside found in the important cereal crop sorghum (Sorghum bicolor (L.) Moench), was described over 20 years ago, and yet no direct regulator of the biosynthetic genes has been identified. To isolate regulatory proteins that bind to the promoter region of the key dhurrin biosynthetic gene of sorghum, SbCYP79A1, yeast one-hybrid screens were performed. A bait fragment containing 1204 base pairs of the SbCYP79A1 5′ regulatory region was cloned upstream of a reporter gene and introduced into Saccharomyces cerevisiae. Subsequently, the yeast was transformed with library cDNA representing RNA from two different sorghum developmental stages. From these screens, we identified SbGATA22, an LLM domain B-GATA transcription factor that binds to the putative GATA transcription factor binding motifs in the SbCYP79A1 promoter region. Transient assays in Nicotiana benthamiana show that SbGATA22 localizes to the nucleus. The expression of SbGATA22, in comparison with SbCYP79A1 expression and dhurrin concentration, was analyzed over 14 days of sorghum development and in response to nitrogen application, as these conditions are known to affect dhurrin levels. Collectively, these findings suggest that SbGATA22 may act as a negative regulator of SbCYP79A1 expression and provide a preliminary insight into the molecular regulation of dhurrin biosynthesis in sorghum.