In 1975, Travis, Stewart and Wilson induced six chlorophyll-deficient mutant lines of the scarlet monkeyflower Mimulus cardinalis (now Erythranthe cardinalis) with the chemical mutagens hydroxylamine and N-methyl-N'-nitro-N-nitrosoguanidine. Segregation analysis assigned three lines to nuclear recessive genes and two to plastome (chloroplast-genome) inheritance, while one chlorotic line was sterile and could not be analyzed genetically; electron microscopy showed each line to carry a distinct defective chloroplast ultrastructure. The biochemical bases and molecular identities of the causative lesions have never been determined. The open problem is a causal biochemical and molecular account of this mutant panel: the affected enzymatic or assembly step behind each distinct ultrastructural phenotype, and the sequence-level lesion in each line and its location (plastome versus nuclear gene). Later literature has applied the same descriptive approach in other species and has resolved the corresponding general questions in model organisms, but no published work has returned to these mutants.
Background
Chlorophyll-deficient mutants of seed plants have been classical tools for dissecting chloroplast biogenesis since the work of Correns and Baur around 1909. Chloroplasts develop from plastids and contain an internal membrane system, the thylakoids (stacked regions called grana), where the photosynthetic pigment complexes reside; 'plastid ultrastructure' refers to the morphology of these membranes as seen by electron microscopy. Chloroplast biogenesis is controlled jointly by two genetic systems: the nuclear genome, whose mutations segregate in ordinary Mendelian fashion, and the plastome, the chloroplast's own DNA genome, whose mutations are usually inherited non-Mendelian (maternally, through the egg cell) and can produce variegated plants in which mutant (white or pale) and normal green plastids coexist. When a cell contains mixed plastid genotypes (heteroplasmy), random partitioning of plastids during somatic cell divisions can sort daughter cell lineages toward uniformly mutant or uniformly normal plastids — the classical phenomenon of somatic 'sorting-out' — which explains the sectoring of variegated leaves; why certain cell types, such as the guard cells that flank stomatal pores, sometimes retain normal plastids next to mutant mesophyll was historically unexplained. In the system studied here — the scarlet monkeyflower Mimulus cardinalis, a species now usually placed in the regenerated genus Erythranthe as Erythranthe cardinalis — Travis and Wilson (1972) first established survival curves and dominant mutant yields after treatment of Mimulus cardinalis seeds with hydroxylamine (NH2OH) and MNNG (N-methyl-N'-nitro-N-nitrosoguanidine, an alkylating agent that induces point mutations). Travis, Stewart and Wilson (1975) then isolated six chlorophyll-deficient mutant lines, analyzed their inheritance, and examined their plastids by electron microscopy. Three lines behaved as single nuclear recessive genes, two as maternally (plastome) inherited, and one chlorotic line was sterile and could not be crossed for genetic analysis; each of the six showed a unique defective chloroplast ultrastructure, and, based on plastid structure, the authors judged the genome and plastome mutations to be non-allelic or significantly different forms of the same function. The 1975 paper explicitly framed the mutant panel as material for future work: 'the study of the effects of specific biochemical blocks and the elucidation of developmental pathways leading to chloroplast biogenesis' and 'the interrelationship between the nucleic acid of the genome and the plastome' (paper Summary). At the time, no tools existed to identify either the affected enzymes and assembly factors or the DNA sequence changes underlying the plastome and nuclear mutations. Since 1975 the corresponding general questions have been answered in model organisms: chloroplast genomes have been sequenced, the enzymes of chlorophyll biosynthesis and thylakoid assembly have been cloned from barley, Arabidopsis and Chlamydomonas mutants, plastome lesions causing chlorophyll deficiency have been identified at sequence level (for example, independent mutations of the plastid infA gene, which encodes the chloroplast translation initiation factor IF1, in barley; Landau et al. 2007), and stochastic bottlenecks in plastid genome transmission during plant development have been demonstrated in tobacco (Lutz and Maliga 2008); the Mimulus genetics community, meanwhile, moved its resources to the congeneric model Mimulus guttatus, in which chlorophyll-deficient lethals were already being genetically analyzed by 1992 (Willis). What has never been done is the molecular and biochemical characterization of the original M. cardinalis mutant panel itself, and that is the open problem stated below.
The 1975 study assigned each line a segregation class and recorded a distinctive defective plastid ultrastructure for it, as follows:
| Line | Inheritance class | Defective plastid ultrastructure (Travis, Stewart and Wilson 1975) |
|---|---|---|
| viridis | plastome (maternal, non-Mendelian) | several parallel primary lamellar layers and up to six 'super-extended' grana running almost the full length of the plastid, with greatly reduced fretwork between grana, osmiophilic globuli present and starch grains absent |
| striata | plastome (maternal, non-Mendelian), variegated | two defective forms in white tissue — a 'dumbbell' cross-section and an amoeboid form containing a vacuole-like inclusion — both retaining the plastid's outer double membrane but with little or no lamellar organization beyond a small amount of stroma |
| xantha | nuclear recessive | three defective forms — a dense fused 'super-granum' formed by fusion of most thylakoids; plastids with only osmiophilic globuli in the surrounding matrix and an undulating outline, without identifiable thylakoids; and plastids with parallel lamellae extending about one-third of the plastid length and lacking grana |
| albina-MG | nuclear recessive | small plastids about the size of mitochondria, bounded by a dense matrix and showing only slight evidence of primary lamellar structure, as expected of an arrest very early in plastid development |
| albina-NA | nuclear recessive | amoeboid plastids with osmiophilic globuli, primary lamellae and occasional concentric (degenerative) thylakoid arrangements — a more advanced but still abnormal state than that of albina-MG |
| chlorotic | undetermined (sterile, so not analyzable by crossing) | near-normal internal chloroplast ultrastructure containing one or more abnormally large starch grains |
Problem Statement
For the six chemically induced chlorophyll-deficient mutant lines of Mimulus cardinalis (Erythranthe cardinalis) characterized genetically and ultrastructurally by Travis, Stewart and Wilson (1975) — three nuclear-recessive lines, two plastome-inherited lines, and one sterile chlorotic line, each with a distinct defective plastid ultrastructure — provide the causal characterization that the paper itself framed as future work: for each mutant line, (1) identify the specific biochemical block (the affected enzyme, assembly factor, or structural component) responsible for its unique defective chloroplast ultrastructure, and the step of the developmental pathway leading to chloroplast biogenesis at which it acts, stated at the granularity of a named stage of that pathway, such as early plastid development, chlorophyll biosynthesis, thylakoid assembly and grana stacking, or plastid gene expression, rather than a specific intermediate; and (2) identify the causative molecular lesion — the affected gene and the class of sequence-level change, read at the granularity of a base substitution, a small insertion or deletion (frameshift), or another named class — and the genome in which it lies, noting that the plastome-versus-nuclear class is already fixed by the 1975 segregation analysis for the five segregating lines and is to be newly established only for the sterile line. For each line, identify the best-supported candidate gene and lesion that its phenotype and inheritance implicate, together with the molecular and biochemical argument for them and the experiments or observations that would establish causativity. These components jointly constitute one objective: a genotype-to-phenotype causal chain from molecular lesion to biochemical block to plastid ultrastructure for the defined mutant panel of this species.
References
- Travis, D. M., Stewart, K. D., & Wilson, K. G. (1975). Nuclear and cytoplasmic chloroplast mutants induced by chemical mutagens in Mimulus cardinalis: Genetics and ultrastructure. Theoretical and Applied Genetics 46:67-77. DOI 10.1007/BF00281645. https://doi.org/10.1007/BF00281645
- Travis, D. M., & Wilson, K. G. (1972). Effects of hydroxylamine and N-Methyl-N'-Nitro-N-nitrosoguanidine in Mimulus cardinalis (Scrophulariaceae): Survival curves and dominant mutants. Theoretical and Applied Genetics 42:288-292. DOI 10.1007/bf00277720. https://doi.org/10.1007/bf00277720
- Travis, D. M. (1977). Molecular Properties of Chloroplast and Nuclear DNA from Mimulus cardinalis. Biochemie und Physiologie der Pflanzen 171:109-126. DOI 10.1016/s0015-3796(17)30281-0. https://doi.org/10.1016/s0015-3796(17)30281-0
- Vaughn, K. C., Wilson, K. G., & Stewart, K. D. (1978). Light-harvesting pigment-protein complex deficiency in Hosta (Liliaceae). Planta 143:275-278. DOI 10.1007/BF00391998. https://doi.org/10.1007/BF00391998
- Miller, P. D., Vaughn, K. C., & Wilson, K. G. (1980). Induction, ultrastructure, isolation, and tissue culture of chlorophyll mutants in carrot. In Vitro 16:823-828. DOI 10.1007/BF02619418. https://doi.org/10.1007/BF02619418
- Landau, A., Diaz Paleo, A., Civitillo, R., Jaureguialzo, M., & Prina, A. R. (2007). Two infA Gene Mutations Independently Originated from a Mutator Genotype in Barley. Journal of Heredity 98:272-276. DOI 10.1093/jhered/esm014. https://doi.org/10.1093/jhered/esm014
- Lutz, K. A., & Maliga, P. (2008). Plastid genomes in a regenerating tobacco shoot derive from a small number of copies selected through a stochastic process. The Plant Journal 56:975-983. DOI 10.1111/j.1365-313x.2008.03655.x. https://doi.org/10.1111/j.1365-313x.2008.03655.x
- Yang, D., Li, S., Li, M., Yang, X., Wang, W., Cao, Z., & Li, W. (2011). Physiological Characteristics and Leaf Ultrastructure of a Novel Chlorophyll-deficient chd6 Mutant of Vitis venifera Cultured in vitro. Journal of Plant Growth Regulation 31:124-135 (published online 2011; print issue 2012). DOI 10.1007/s00344-011-9225-9. https://doi.org/10.1007/s00344-011-9225-9
- Willis, J. H. (1992). Genetic analysis of inbreeding depression caused by chlorophyll-deficient lethals in Mimulus guttatus. Heredity 69:562-572. DOI 10.1038/hdy.1992.172. https://doi.org/10.1038/hdy.1992.172