Rice CYP734A cytochrome P450s inactivate brassinosteroids in Arabidopsis.

Endogenous brassinosteroid concentrations are an important target for optimizing the growth of crop plants because these hormones influence yield and stress tolerance. The CYP734A subfamily of cytochrome P450 enzymes has been shown to inactivate brassinosteroid hormones in Arabidopsis and tomato. Rice has three genes for CYP734A enzymes whose expression appears to be up-regulated by exogenous brassinolide. The amino acids predicted to be in the active site of the rice enzymes vary when compared with the Arabidopsis protein sequence, suggesting that there could be differences in their ability to inactivate the hormone. We have cloned three CYP734A rice genes and expressed them in Arabidopsis to assess their efficacy as brassinosteroid-inactivating enzymes. We found that incorrect transcript splicing can complicate the expression of monocot genomic clones in a eudicot. However, the Arabidopsis system allowed us to characterize an atypical splice variant in one of the rice genes. cDNA clones produced high levels of expression and conferred the brassinosteroid inactivation phenotype. This study shows that Arabidopsis is a useful heterologous system for testing plant genes predicted to act in biochemical pathways that are conserved between monocots and eudicots.

Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids.

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A subfamily inactivate brassinosteroids through C-26 hydroxylation, the biochemical activity of CYP72C1 is unknown. Because CYP734A1 and CYP72C1 in Arabidopsis diverge more than brassinosteroid inactivating P450s in other plants, this study examines the structure and biochemistry of each enzyme. Three-dimensional models were generated to examine the substrate binding site structures and determine how they might affect the function of each P450. These models have indicated that the active site of CYP72C1 does not contain several conserved amino acids typically needed for substrate hydroxylation. Heterologous expression of these P450s followed by substrate binding analyses have indicated that CYP734A1 binds active brassinosteroids, brassinolide and castasterone, as well as their upstream precursors whereas CYP72C1 binds precursors more effectively. Seedling growth assays have demonstrated that the genetic state of CYP734A1, but not CYP72C1, affected responsiveness to high levels of exogenous brassinolide supporting our observations that CYP72C1 acts on brassinolide precursors. Although there may be some overlap in their physiological function, the distinct biochemical functions of these proteins in Arabidopsis has significant potential to fine-tune the levels of different brassinosteroid hormones throughout plant growth and development.

Utility of a Phylogenetic Perspective in Structural Analysis of CYP72A Enzymes from Flowering Plants.

Plant adaptation to external pressures depends on functional diversity in cytochrome P450 (CYP) enzymes. CYPs contain structural domains necessary for the characteristic P450 fold that allows monooxygenation, but they also have great variation in substrate binding affinity. Plant genomes typically contain hundreds of CYPs that contribute to essential functions and species-specific metabolism. The CYP72A subfamily is conserved in angiosperms but its contribution to physiological functions is largely unknown. With genomic information available for many plants, a focused analysis of CYP subfamily diversity is important to understand the contributions of these enzymes to plant evolution. This study examines the extent to which independent gene duplication and evolution have contributed to structural diversification of CYP72A enzymes in different plant lineages. CYP72A genes are prevalent across angiosperms, but the number of genes within each genome varies greatly. The prevalence of CYP72As suggest that the last common ancestor of flowering plants contained a CYP72A sequence, but gene duplication and retention has varied greatly for this CYP subfamily. Sequence comparisons show that CYP72As are involved in species-specific metabolic functions in some plants while there is likely functional conservation between closely related species. Analysis of structural and functional domains within groups of CYP72As reveals clade-specific residues that contribute to functional constraints within subsets of CYP72As. This study provides a phylogenetic framework that allows comparisons of structural features within subsets of the CYP72A subfamily. We examined a large number of sequences from a broad collection of plant species to detect patterns of functional conservation across the subfamily. The evolutionary relationships between CYPs in plant genomes are an important component in understanding the evolution of biochemical diversity in plants.

High sensitivity proteomics assisted discovery of a novel operon involved in the assembly of photosystem II, a membrane protein complex.

Photosystem II (PSII) is a large membrane protein complex that performs the water oxidation reactions of photosynthesis in cyanobacteria, algae, and plants. The unusual redox reactions in PSII often lead to damage, degradation, and reassembly of this molecular machine. To identify novel assembly factors, high sensitivity proteomic analysis of PSII purified from the cyanobacterium Synechocystis sp. PCC 6803 was performed. This analysis identified six PSII-associated proteins that are encoded by an operon containing nine genes, slr0144 to slr0152. This operon encodes proteins that are not essential components of the PSII holocomplex but accumulate to high levels in pre-complexes lacking any of the lumenal proteins PsbP, PsbQ, or PsbV. The operon contains genes with putative binding domains for chlorophylls and bilins, suggesting these proteins may function as a reservoir for cofactors needed during the PSII lifecycle. Genetic deletion of this operon shows that removal of these protein products does not alter photoautotrophic growth or PSII fluorescence properties. However, the deletion does result in decreased PSII-mediated oxygen evolution and an altered distribution of the S states of the catalytic manganese cluster. These data demonstrate that the proteins encoded by the genes in this operon are necessary for optimal function of PSII and function as accessory proteins during assembly of the PSII complex. Thus, we have named the products of the slr0144-slr0152 operon Pap (Photosystem II assembly proteins).

Physcomitrella patens and Ceratodon purpureus, mosses as model organisms in photosynthesis studies.

With the discovery of targeted gene replacement, moss biology has been rapidly advancing over the last 10 years. This study demonstrates the usefulness of moss as a model organism for plant photosynthesis research. The two mosses examined in this study, Physcomitrella patens and Ceratodon purpureus, are easily cultured through vegetative propagation. Growth tests were conducted to determine carbon sources suitable for maintaining heterotrophic growth while photosynthesis was blocked. Photosynthetic parameters examined in these plants indicated that the photosynthetic activity of Ceratodon and Physcomitrella is more similar to vascular plants than cyanobacteria or green algae. Ceratodon plants grown heterotrophically appeared etiolated in that the plants were taller and plastids did not differentiate thylakoid membranes. After returning to the light, the plants developed green, photosynthetically active chloroplasts. Furthermore, UV-induced mutagenesis was used to show that photosynthesis-deficient mutant Ceratodon plants could be obtained. After screening approximately 1000 plants, we obtained a number of mutants, which could be arranged into the following categories: high fluorescence, low fluorescence, fast and slow fluorescence quenching, and fast and slow greening. Our results indicate that in vivo biophysical analysis of photosynthetic activity in the mosses can be carried out which makes both mosses useful for photosynthesis studies, and Ceratodon best sustains perturbations in photosynthetic activity.

Homologs of plant PsbP and PsbQ proteins are necessary for regulation of photosystem ii activity in the cyanobacterium Synechocystis 6803.

The mechanism of oxygen evolution by photosystem II (PSII) has remained highly conserved during the course of evolution from ancestral cyanobacteria to green plants. A cluster of manganese, calcium, and chloride ions, whose binding environment is optimized by PSII extrinsic proteins, catalyzes this water-splitting reaction. The accepted view is that in plants and green algae, the three extrinsic proteins are PsbO, PsbP, and PsbQ, whereas in cyanobacteria, they are PsbO, PsbV, and PsbU. Our previous proteomic analysis established the presence of a PsbQ homolog in the cyanobacterium Synechocystis 6803. The current study additionally demonstrates the presence of a PsbP homolog in cyanobacterial PSII. Both psbP and psbQ inactivation mutants exhibited reduced photoautotrophic growth as well as decreased water oxidation activity under CaCl(2)-depleted conditions. Moreover, purified PSII complexes from each mutant had significantly reduced activity. In cyanobacteria, one PsbQ is present per PSII complex, whereas PsbP is significantly substoichiometric. These findings indicate that both PsbP and PsbQ proteins are regulators that are necessary for the biogenesis of optimally active PSII in Synechocystis 6803. The new picture emerging from these data is that five extrinsic PSII proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV, are present in cyanobacteria, two of which, PsbU and PsbV, have been lost during the evolution of green plants.