Carbonic anhydrases （CAs） are zinc metalloenzymes that catalyze the interconversion of CO2 and HCO3- and are ubiquitous in nature. Higher plants contain three evolutionarily distinct CA families, ...αCAs, 13CAs, and γCAs, where each family is represented by multiple isoforms in all species. Alternative splicing of CA transcripts appears common; consequently, the number of functional CA isoforms in a species may exceed the number of genes. CAs are expressed in numerous plant tissues and in different cellular locations. The most prevalent CAs are those in the chloroplast, cytosol, and mitochondria. This diversity in location is paralleled in the many physiological and biochemical roles that CAs play in plants. In this review, the number and types of CAs in C3, C4, and crassulacean acid metabolism （CAM） plants are considered, and the roles of the α and yCAs are briefly discussed. The remainder of the review focuses on plant βCAs and includes the identification of homologs between species using phylogenetic approaches, a consideration of the inter- and intracellular localization of the proteins, along with the evidence for alternative splice forms. Current understanding of βCA tissue-specific expression patterns and what controls them are reviewed, and the physiological roles for which βCAs have been implicated are presented.
Summary
Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and ...water use efficiency. To create a two‐cell metabolic prototype for an NADP‐malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP‐malate dehydrogenase, pyruvate orthophosphate dikinase and NADP‐malic enzyme from Zea mays, driven by cell‐preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13CO2 labelling demonstrated a 10‐fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP‐malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.
Abstract
C4 photosynthesis concentrates CO2 around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait evolved independently in ...>60 angiosperm lineages. Its evolution can be investigated in genera such as Flaveria (Asteraceae) that contain species representing intermediate stages between C3 and C4 photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO2 release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO2-concentrating cycle between phosphoenolpyruvate carboxylase in the mesophyll and decarboxylases in the bundle sheath, and relocation of Rubisco to the latter. We have profiled 52 metabolites in nine Flaveria species and analysed 13CO2 labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C3–C4 intermediates and a switch towards a malate/pyruvate shuttle in C4-like species. The malate/pyruvate shuttle increases from C4-like to complete C4 species, accompanied by a rise in ancillary organic acid pools. Our findings support current models and uncover further modifications of metabolism along the evolutionary path to C4 photosynthesis in the genus Flaveria.
Metabolite profiling and 13CO2labelling studies of Flaveriaspecies on the spectrum between C3and C4photosynthesis reveal progressive re-wiring of photorespiration, nitrogen metabolism, carbon concentration shuttles, and the Calvin–Benson cycle.
Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of a methyl group from methyltetrahydrofolate to L-homocysteine (Hcy) without using an intermediate methyl carrier. Although ...MetE displays no detectable sequence homology with cobalamin-dependent methionine synthase (MetH), both enzymes require zinc for activation and binding of Hcy. Crystallographic analyses of MetE from T. maritima reveal an unusual dual-barrel structure in which the active site lies between the tops of the two (betaalpha)(8) barrels. The fold of the N-terminal barrel confirms that it has evolved from the C-terminal polypeptide by gene duplication; comparisons of the barrels provide an intriguing example of homologous domain evolution in which binding sites are obliterated. The C-terminal barrel incorporates the zinc ion that binds and activates Hcy. The zinc-binding site in MetE is distinguished from the (Cys)(3)Zn site in the related enzymes, MetH and betaine-homocysteine methyltransferase, by its position in the barrel and by the metal ligands, which are histidine, cysteine, glutamate, and cysteine in the resting form of MetE. Hcy associates at the face of the metal opposite glutamate, which moves away from the zinc in the binary E.Hcy complex. The folate substrate is not intimately associated with the N-terminal barrel; instead, elements from both barrels contribute binding determinants in a binary complex in which the folate substrate is incorrectly oriented for methyl transfer. Atypical locations of the Hcy and folate sites in the C-terminal barrel presumably permit direct interaction of the substrates in a ternary complex. Structures of the binary substrate complexes imply that rearrangement of folate, perhaps accompanied by domain rearrangement, must occur before formation of a ternary complex that is competent for methyl transfer.
• Background and Aims A common response of wetland plants to flooding is the formation of aquatic adventitious roots. Observations of aquatic root growth are widespread; however, controlled studies ...of aquatic roots of terrestrial herbaceous species are scarce. Submergence tolerance and aquatic root growth and physiology were evaluated in two herbaceous, perennial wetland species Cotula coronopifolia and Meionectes brownii. • Methods Plants were raised in large pots with 'sediment' roots in nutrient solution and then placed into individual tanks and shoots were left in air or submerged (completely or partially). The effects on growth of aquatic root removal, and of light availability to submerged plant organs, were evaluated. Responses of aquatic root porosity, chlorophyll and underwater photosynthesis, were studied. • Key Results Both species tolerated 4 weeks of complete or partial submergence. Extensive, photosynthetically active, aquatic adventitious roots grew from submerged stems and contributed up to 90 % of the total root dry mass. When aquatic roots were pruned, completely submerged plants grew less and had lower stem and leaf chlorophyll a, as compared with controls with intact roots. Roots exposed to the lowest PAR (daily mean 4·7 ± 2·4 µmol m⁻² s⁻¹) under water contained less chlorophyll, but there was no difference in aquatic root biomass after 4 weeks, regardless of light availability in the water column (high PAR was available to all emergent shoots). • Conclusions Both M. brownii and C. coronopifolia responded to submergence with growth of aquatic adventitious roots, which essentially replaced the existing sediment root system. These aquatic roots contained chlorophyll and were photosynthetically active. Removal of aquatic roots had negative effects on plant growth during partial and complete submergence.
Leaf photosynthesis (A) is limited by mesophyll conductance (gm), which is influenced by both leaf structure and the environment. Previous studies have indicated that the upper bound for gm declines ...as leaf dry mass per area (LMA, an indicator of leaf structure) increases, extrapolating to zero at a LMA of about 240 g m⁻². No data exist on gm and its response to the environment for species with LMA values higher than 220 g m⁻². In this study, laboratory measurements of leaf gas exchange and in vivo chlorophyll a fluorescence were used concurrently to derive estimates of gm in seven species of the Australian sclerophyllous genus Banksia covering a wide range of LMA (130-480 g m⁻²). Irradiance and CO₂ were varied during those measurements to gauge the extent of environmental effects on gm. A significant decrease of gm with increasing LMA was found. gm declined by 35-60% in response to increasing atmospheric CO₂ concentrations at high irradiance, with a more variable response (0-60%) observed at low irradiance, where gm was, on average, 22% lower than at high irradiance at ambient CO₂ concentrations. Despite considerable variation in A and LMA between the Banksia species, the CO₂ concentrations in the intercellular air spaces (Ci, 262±5 μmol mol⁻¹) and in the chloroplasts (Cc, 127±4 μmol mol⁻¹) were remarkably stable.
Mycorrhiza formation represents a significant carbon (C) acquisition alternative for orchid species, particularly those that remain achlorophyllous through all life stages. As it is known that orchid ...mycorrhizas facilitate nutrient transfer (most notably of C), it has not been resolved if C transfer occurs only after lysis of mycorrhizal structures (fungal pelotons) or also across the mycorrhizal interface of pre‐lysed pelotons. We used high‐resolution secondary ion mass spectrometry (nanoSIMS) and labelling with enriched ¹³CO₂ to trace C transfers, at subcellular scale, across mycorrhizal interfaces formed by Rhizanthella gardneri, an achlorphyllous orchid. Carbon was successfully traced in to the fungal portion of orchid mycorrhizas. However, we did not detect C movement across intact mycorrhizal interfaces up to 216 h post ¹³CO₂ labelling. Our findings provide support for the hypothesis that C transfer from the mycorrhizal fungus to orchid, at least for R. gardneri, likely occurs after lysis of the fungal peloton.
Abstract
C
4
photosynthesis is a remarkable complex trait, elucidations of the evolutionary trajectory of C
4
photosynthesis from its ancestral C
3
pathway can help us better understand the generic ...principles of the evolution of complex traits and guide the engineering of C
3
crops for higher yields. Here, we used the genus
Flaveria
that contains C
3
, C
3
–C
4
, C
4
-like and C
4
species as a system to study the evolution of C
4
photosynthesis. We first mapped transcript abundance, protein sequence and morphological features onto the phylogenetic tree of the genus
Flaveria
, and calculated the evolutionary correlation of different features; we then predicted the relative changes of ancestral nodes of those features to illustrate the major events during the evolution of C
4
photosynthesis. We found that gene expression and protein sequence showed consistent modification patterns in the phylogenetic tree. High correlation coefficients ranging from 0.46 to 0.9 among gene expression, protein sequence and morphology were observed. The greatest modification of those different features consistently occurred at the transition between C
3
-C
4
species and C
4
-like species. Our results show highly coordinated changes in gene expression, protein sequence and morphological features, which support evolutionary major events during the evolution of C
4
metabolism.
•The location of most carbonic anhydrase activity differs between C3 and C4 plants.•Chloroplast transit peptide loss changed a Flaveria C3 carbonic anhydrase to a C4 enzyme.•Control of C4 carbonic ...anhydrase gene expression was modified from that of C3 orthologs.•Maize carbonic anhydrase activity is not essential at ambient CO2 concentrations.
During the evolution of C4 photosynthesis, the intracellular location with most carbonic anhydrase (CA) activity has changed. In Flaveria, the loss of the sequence encoding a chloroplast transit peptide from an ancestral C3 CA ortholog confined the C4 isoform to the mesophyll cell cytosol. Recent studies indicate that sequence elements and histone modifications controlling the expression of C4-associated CAs were likely present in the C3 ancestral chromatin, enabling the evolution of the C4 pathway. Almost complete abolishment of maize CA activity yields no obvious phenotype at ambient CO2 levels. This contrasts with results for Flaveria CA mutants, and has opened discussion on the role of CA in the C4 carbon concentrating mechanism.
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