Unconditional and conditional analysis of epistasis between tillering QTLs based on single segment substitution lines in rice.

Epistasis plays an important role in manipulating rice tiller number, but epistatic mechanism still remains a challenge. Here we showed the process of epistatic analysis between tillering QTLs. A half diallel mating scheme was conducted based on 6 single segment substitution lines and 9 dual segment pyramiding lines to allow the analysis of 4 epistatic components. Additive-additive, additive-dominance, dominance-additive, and dominance-dominance epistatic effects were estimated at 9 stages of development via unconditional QTL analysis simultaneously. Unconditional QTL effect (QTL cumulative effect before a certain stage) was then divided into several conditional QTL components (QTL net effect in a certain time interval). The results indicated that epistatic interaction was prevalent, all QTL pairs harboring epistasis and one QTL always interacting with other QTLs in various component ways. Epistatic effects were dynamic, occurring mostly within 14d and 21-35d after transplant and exhibited mainly negative effects. The genetic and developmental mechanism on several tillering QTLs was further realized and perhaps was useful for molecular pyramiding breeding and heterosis utilization for improving plant architecture.

Uncovering the functional residues of Arabidopsis isoprenoid biosynthesis enzyme HDS.

The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designated Rceh1-3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that of ceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing the ceh1 mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the corresponding Rceh lines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.