Expression of Mitochondrial Gene Fragments within the Tapetum Induce Male Sterility by Limiting the Biogenesis of the Respiratory Machinery in Transgenic Tobacco

Plant mitochondrial genomes （mtDNAs） are large and undergo frequent recombination events. A common phenotype that emerges as a consequence of altered mtDNA structure is cytoplasmic-male sterility （CMS）. The molecular basis for CMS remains unclear, but it seems logical that altered respiration activities would result in reduced pollen production. Analysis of tobacco （Nicotiana tabacum） mtDNAs indicated that CMS-associated loci often contain fragments of known organellar genes. These may assemble with organellar complexes and thereby interfere with normal respiratory functions. Here, we analyzed whether the expression of truncated fragments of mitochondrial genes （i.e. atp4, cox1 and rps3） may induce male sterility by limiting the biogenesis of the respiratory machinery, cDNA fragments corresponding to atp4f, coxlfand rps3f were cloned in-frame to a mitochondrial localization signal and a C-termini HA-tag under a tapetum-specific promoter and introduced to tobacco plants by Agrobacterium-mediated transformation. The constructs were then analyzed for their effect on mitochondrial activity and pollen fertility. Atp4f, Coxlf and Rps3f plants demonstrated male sterility phenotypes, which were tightly correlated with the expression of the recombinant fragments in the floral meristem. Fractionation of native organellar extracts showed that the recombinant ATP4f-HA, COXlf-HA and RPS3f-HA proteins are found in large membrane-associated particles. Analysis of the respiratory activities and protein profiles indicated that organellar complex I was altered in Atp4f, Coxlfand Rps3f plants.

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant- specific protein involved in organeUar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the rash I phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppres- sion lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein-protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochon- dria results in 7%-10% of plants altered in leaf morphology, heat tolerance, and mitochondrlal genome sta- bility. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH 1. MSH1 deple- tion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.