Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.

BACKGROUND: Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking. RESULTS: To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised. CONCLUSIONS: Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

CLUMPED CHLOROPLASTS 1 is required for plastid separation in Arabidopsis.

We identified an Arabidopsis thaliana mutant, clumped chloroplasts 1 (clmp1), in which disruption of a gene of unknown function causes chloroplasts to cluster instead of being distributed throughout the cytoplasm. The phenotype affects chloroplasts and nongreen plastids in multiple organs and cell types, but is detectable only at certain developmental stages. In young leaf petioles of clmp1, where clustering is prevalent, cells lacking chloroplasts are detected, suggesting impaired chloroplast partitioning during mitosis. Although organelle distribution and partitioning are actin-dependent in plants, the actin cytoskeleton in clmp1 is indistinguishable from that in WT, and peroxisomes and mitochondria are distributed normally. A CLMP1-YFP fusion protein that complements clmp1 localizes to discrete foci in the cytoplasm, most of which colocalize with the cell periphery or with chloroplasts. Ultrastructural analysis revealed that chloroplasts within clmp1 clusters are held together by membranous connections, including thin isthmi characteristic of late-stage chloroplast division. This finding suggests that constriction of dividing chloroplasts proceeds normally in clmp1, but separation is impaired. Consistently, chloroplast size and number, as well as positioning of the plastid division proteins FtsZ and ARC5/DRP5B, are unaffected in clmp1, indicating that loss of CLMP1-mediated chloroplast separation does not prevent otherwise normal division. CLMP1-like sequences are unique to green algae and land plants, and the CLMP1 sequence suggests that it functions through protein-protein interactions. Our studies identify a unique class of proteins required for plastid separation after the constriction stage of plastid division and indicate that CLMP1 activity is also required for plastid distribution and partitioning during cell division.

New connections across pathways and cellular processes: industrialized mutant screening reveals novel associations between diverse phenotypes in Arabidopsis.

In traditional mutant screening approaches, genetic variants are tested for one or a small number of phenotypes. Once bona fide variants are identified, they are typically subjected to a limited number of secondary phenotypic screens. Although this approach is excellent at finding genes involved in specific biological processes, the lack of wide and systematic interrogation of phenotype limits the ability to detect broader syndromes and connections between genes and phenotypes. It could also prevent detection of the primary phenotype of a mutant. As part of a systems biology approach to understand plastid function, large numbers of Arabidopsis thaliana homozygous T-DNA lines are being screened with parallel morphological, physiological, and chemical phenotypic assays (www.plastid.msu.edu). To refine our approaches and validate the use of this high-throughput screening approach for understanding gene function and functional networks, approximately 100 wild-type plants and 13 known mutants representing a variety of phenotypes were analyzed by a broad range of assays including metabolite profiling, morphological analysis, and chlorophyll fluorescence kinetics. Data analysis using a variety of statistical approaches showed that such industrial approaches can reliably identify plant mutant phenotypes. More significantly, the study uncovered previously unreported phenotypes for these well-characterized mutants and unexpected associations between different physiological processes, demonstrating that this approach has strong advantages over traditional mutant screening approaches. Analysis of wild-type plants revealed hundreds of statistically robust phenotypic correlations, including metabolites that are not known to share direct biosynthetic origins, raising the possibility that these metabolic pathways have closer relationships than is commonly suspected.