Chloroplast 2010: A Database for Large-Scale Phenotypic Screening of Arabidopsis Mutants    

Abstract

Large-scale phenotypic screening presents challenges and opportunities not encountered in typical forward or reverse genetics projects. We describe a modular database and laboratory information management system that was implemented in support of the Chloroplast 2010 Project, an Arabidopsis (Arabidopsis thaliana) reverse genetics phenotypic screen of more than 5,000 mutants (http://bioinfo.bch.msu.edu/2010_LIMS; www.plastid.msu.edu). The software and laboratory work environment were designed to minimize operator error and detect systematic process errors. The database uses Ruby on Rails and Flash technologies to present complex quantitative and qualitative data and pedigree information in a flexible user interface. Examples are presented where the database was used to find opportunities for process changes that improved data quality. We also describe the use of the data-analysis tools to discover mutants defective in enzymes of leucine catabolism (heteromeric mitochondrial 3-methylcrotonyl-coenzyme A carboxylase [At1g03090 and At4g34030] and putative hydroxymethylglutarylcoenzyme A lyase [At2g26800]) based upon a syndrome of pleiotropic seed amino acid phenotypes that resembles previously described isovaleryl coenzyme A dehydrogenase (At3g45300) mutants. In vitro assay results support the computational annotation of At2g26800 as hydroxymethylglutaryl-coenzyme A lyase.

PHOTOSYSTEM II PROTEIN33, a Protein Conserved in the Plastid Lineage, Is Associated with the Chloroplast Thylakoid Membrane and Provides Stability to Photosystem II Supercomplexes in Arabidopsis

“…A mutant of At1g71500 was identified as part of the Chloroplast 2010 functional genomics pipeline (Lu et al, 2008(Lu et al, , 2011a(Lu et al, , 2011bAjjawi et al, 2010). Over 5,200 Arabidopsis homozygous T-DNA lines were screened for altered Chl fluorescence in plants grown under a photosynthetic photon flux density (PPFD) of either 100 or 1,000 mmol photons m 22 s 21 .…”

“…GreenCut identifies proteins found only in photosynthetic organisms, and it is likely that many of them are involved in biochemical processes associated with the structure, assembly, or function of the photosynthetic apparatus and the chloroplast that houses it (Merchant et al, 2007;Karpowicz et al, 2011). The Chloroplast 2010 Project was a large-scale reverse-genetic mutant screen in which thousands of homozygous Arabidopsis (Arabidopsis thaliana) transfer DNA (T-DNA) insertion lines were analyzed for defects in the rise and decay kinetics of Chl fluorescence (Lu et al, 2008(Lu et al, , 2011a(Lu et al, , 2011bAjjawi et al, 2010).…”

“…Two complementary approaches (Merchant et al, 2007;Lu et al, 2008Lu et al, , 2011bAjjawi et al, 2010) that utilize phylogenomics (GreenCut) and large-scale phenotypic mutant screening (Chloroplast 2010 Project; http://www. plastid.msu.edu/) were employed by our groups to discover novel plant proteins with roles in photosynthesis.…”

Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents

“…Recently, it was reported that loss-of-function mutations in genes encoding branchedchain amino acid catabolic enzymes (isovaleryl-CoA dehydrogenase, methylcrotonyl-CoA carboxylase a-and b-subunits, and hydroxymethylglutaryl-CoA lyase) cause overaccumulation of branched-chain amino acids in seeds (Gu et al, 2010;Lu et al, 2011b). Moreover, it is not uncommon for feedback-insensitive mutants of an upstream enzyme to accumulate downstream products.…”

“…In an attempt to identify functions for chloroplast proteins and mechanisms for processes occurring in the chloroplast, thousands of Arabidopsis transfer DNA (T-DNA) lines of nuclear-encoded, chloroplasttargeted genes were analyzed for a variety of phenotypes, including leaf and seed free amino acid contents, using an HPLC-tandem mass spectrometry method (Lu et al, 2008(Lu et al, , 2011a(Lu et al, , 2011b(Lu et al, , 2011cAjjawi et al, 2010;Bell et al, 2012). This screen identified a homozygous mutant (SALK_082155) in the Columbia-0 (Col-0; CS60000) background with high-leaf Thr content (Supplemental Fig.…”