Large-Scale Reverse Genetics in Arabidopsis: Case Studies from the Chloroplast 2010 Project      

Abstract

Traditionally, phenotype-driven forward genetic plant mutant studies have been among the most successful approaches to revealing the roles of genes and their products and elucidating biochemical, developmental, and signaling pathways. A limitation is that it is time consuming, and sometimes technically challenging, to discover the gene responsible for a phenotype by map-based cloning or discovery of the insertion element. Reverse genetics is also an excellent way to associate genes with phenotypes, although an absence of detectable phenotypes often results when screening a small number of mutants with a limited range of phenotypic assays. The Arabidopsis Chloroplast 2010 Project (www.plastid.msu.edu) seeks synergy between forward and reverse genetics by screening thousands of sequence-indexed Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants for a diverse set of phenotypes. Results from this project are discussed that highlight the strengths and limitations of the approach. We describe the discovery of altered fatty acid desaturation phenotypes associated with mutants of At1g10310, previously described as a pterin aldehyde reductase in folate metabolism. Data are presented to show that growth, fatty acid, and chlorophyll fluorescence defects previously associated with antisense inhibition of synthesis of the family of acyl carrier proteins can be attributed to a single gene insertion in Acyl Carrier Protein4 (At4g25050). A variety of cautionary examples associated with the use of sequence-indexed T-DNA mutants are described, including the need to genotype all lines chosen for analysis (even when they number in the thousands) and the presence of tagged and untagged secondary mutations that can lead to the observed phenotypes.

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

“…All are in the Col-0 ecotype (Alonso et al, 2003;Woody et al, 2007). Homozygosity was confirmed by PCR as described by Ajjawi et al (2010).…”

“…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.…”