Cross-Species Identification of Mendel's I Locus

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Science  05 Jan 2007:
Vol. 315, Issue 5808, pp. 73
DOI: 10.1126/science.1132912


A key gene involved in plant senescence, mutations of which partially disable chlorophyll catabolism and confer stay-green leaf and cotyledon phenotypes, has been identified in Pisum sativum, Arabidopsis thaliana, and Festuca pratensis by using classical and molecular genetics and comparative genomics. A stay-green locus in F. pratensis is syntenically equivalent to a similar stay-green locus on rice chromosome 9. Functional testing in Arabidopsis of a homolog of the rice candidate gene revealed (i) senescence-associated gene expression and (ii) a stay-green phenotype after RNA interference silencing. Genetic mapping in pea demonstrated cosegregation with the yellow/green cotyledon polymorphism (I/i) first reported by Gregor Mendel in 1866.

We identified gene homologs in Pisum sativum (pea), Arabidopsis thaliana, and Festuca pratensis (meadow fescue), mutations of which partially disable plant senescence. The biochemical properties and map location of this gene in pea indicate that it is the same locus that determines yellow (I) and green (i) cotyledon color, as originally described by Mendel in his seminal paper (1). Staygreen (the gene sgr), the indefinite retention of greenness in senescing leaves and cotyledons, is a genetic variant that interferes with the normal expression of senescence. Biochemical (2, 3) (Fig. 1A) and genetic analyses of sgr from pea and F. pratensis (4) show that it segregates as a discrete phenotype with equivalents in maize, sorghum, and rice (5, 6). By using the Lolium-Festuca gene introgression system and extrapolating from genetic synteny, we show that F. pratensis sgr on chromosome (C)5 maps to a position corresponding to sgr on rice C9 (4, 6, 7). Fine mapping in Festuca delimits this to a region equivalent to circa 200 kb of the rice genome containing 30 annotated gene models, including a predicted senescence-inducible chloroplast-specific stay-green protein (4, 7, 8).

Fig. 1.

(A) In mutant and silenced genotypes, inhibition (X) of the ring-opening step between (top) pheophorbide and (bottom) red chlorophyll catabolite in the chlorophyll breakdown pathway (3) leads to retention of greenness in senescing leaves. (B) RNAi silencing of Arabidopsis At4g22920 causes a stay-green phenotype. (Left) Degradation of chlorophyll in control (black) and two independently RNAi-silenced genotypes (gray and white) during dark-induced, detached leaf senescence. Error bars indicate standard deviation. (Right) Wild-type (top) and stay-green (bottom) leaf phenotypes of Arabidopsis after 5 days of dark-induced, detached-leaf senescence. (C) (Left) Northern analysis using RNA extracted from senescing leaves of wild-type (lanes 1 and 3) and stay-green (lanes 2 and 4) pea plants. (Right) Wild-type (top) and stay-green (bottom) pea cotyledons illustrating Mendel's I and i phenotypes, respectively.

We investigated the temporal and tissue-specific expression patterns of the most similar Arabidopsis gene model, At4g22920, through the Genevestigator Meta-Analyzer database (9). These patterns indicate that At4g22920 is up-regulated in days 45 to 50 during maximal senescence in the Arabidopsis life cycle (fig. S1A) and that the strongest expression of this gene occurs in senescent leaves, although it is also detected in seeds, petals, and sepals. RNA interference (RNAi) was also used to silence the expression of At4g22920 in Arabidopsis, resulting in plants with leaves exhibiting prolonged chlorophyll retention upon dark incubation (Fig. 1B), phenotypically equivalent to sgr F. pratensis. Thus, alleles of F. pratensis and rice homologs of Arabidopsis At4g22920 likely underlie the monocot stay-green phenotypes.

In order to explore the parallels between Mendel's pea cotyledons and the stay-green leaf phenotypes in Arabidopsis, Festuca, and rice, we genetically mapped the pea homolog of sgr in two different pea populations segregating for cotyledon color polymorphism (I and i). No recombination between I and sgr was observed in 104 progeny, reflecting a theoretical maximum distance between sgr and I of 0.48 cM (fig. S1B). Northern analysis in pea indicates that sgr has reduced expression in mutant senescing leaves when compared with that of wild type (Fig. 1C).

This report characterizes a rare example of an unbroken chain from gene sequence to trait through biochemical, cell biological, and physiological definitions of phenotype, in which genomics and functional analysis in model species have played a critical role. This study results in the identification of a gene that plays a fundamental role in chlorophyll catabolism during plant senescence. In addition, it suggests that the cotyledon color trait described by Mendel reflects allelic variation in a pea gene, homologs of which are responsible for the stay-green phenotype in both dicots and monocots.

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Materials and Methods

Fig. S1

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