Transcriptome Analysis Reveals Differential Gene Expression and a Possible Role of Gibberellins in a Shade-Tolerant Mutant of Perennial Ryegrass.

The molecular basis behind shade tolerance in plants is not fully understood. Previously, we have shown that a connection may exist between shade tolerance and dwarfism, however, the mechanism connecting these phenotypes is not well understood. In order to clarify this connection, we analyzed the transcriptome of a previously identified shade-tolerant mutant of perennial ryegrass (Lolium perenne L.) called shadow-1. shadow-1 mutant plants are dwarf, and are significantly tolerant to shade in a number of environments compared to wild-type controls. In this study, we treated shadow-1 and wild-type plants with 95% shade for 2 weeks and compared the transcriptomes of these shade-treated individuals with both genotypes exposed to full light. We identified 2,200 differentially expressed genes (DEGs) (1,096 up-regulated and 1,104 down-regulated) in shadow-1 mutants, compared to wild type, following exposure to shade stress. Of these DEGs, 329 were unique to shadow-1 plants kept under shade and were not found in any other comparisons that we made. We found 2,245 DEGs (1,153 up-regulated and 1,092 down-regulated) in shadow-1 plants, compared to wild-type, under light, with 485 DEGs unique to shadow-1 plants under light. We examined the expression of gibberellin (GA) biosynthesis genes and found that they were down-regulated in shadow-1 plants compared to wild type, notably gibberellin 20 oxidase (GA20ox), which was down-regulated to 3.3% (96.7% reduction) of the wild-type expression level under shade conditions. One GA response gene, lipid transfer protein 3 (LTP3), was also down-regulated to 41.5% in shadow-1 plants under shade conditions when compared to the expression level in the wild type. These data provide valuable insight into a role that GA plays in dwarfism and shade tolerance, as exemplified by shadow-1 plants, and could serve as a guide for plant breeders interested in developing new cultivars with either of these traits.

A Novel Two-Step Method for Screening Shade Tolerant Mutant Plants via Dwarfism.

When subjected to shade, plants undergo rapid shoot elongation, which often makes them more prone to disease and mechanical damage. Shade-tolerant plants can be difficult to breed; however, they offer a substantial benefit over other varieties in low-light areas. Although perennial ryegrass (Lolium perenne L.) is a popular species of turf grasses because of their good appearance and fast establishment, the plant normally does not perform well under shade conditions. It has been reported that, in turfgrass, induced dwarfism can enhance shade tolerance. Here we describe a two-step procedure for isolating shade tolerant mutants of perennial ryegrass by first screening for dominant dwarf mutants, and then screening dwarf plants for shade tolerance. The two-step screening process to isolate shade tolerant mutants can be done efficiently with limited space at early seedling stages, which enables quick and efficient isolation of shade tolerant mutants, and thus facilitates development of shade tolerant new cultivars of turfgrasses. Using the method, we isolated 136 dwarf mutants from 300,000 mutagenized seeds, with 65 being shade tolerant (0.022%). When screened directly for shade tolerance, we recovered only four mutants from a population of 150,000 (0.003%) mutagenized seeds. One shade tolerant mutant, shadow-1, was characterized in detail. In addition to dwarfism, shadow-1 and its sexual progeny displayed high degrees of tolerance to both natural and artificial shade. We showed that endogenous gibberellin (GA) content in shadow-1 was higher than wild-type controls, and shadow-1 was also partially GA insensitive. Our novel, simple and effective two-step screening method should be applicable to breeding shade tolerant cultivars of turfgrasses, ground covers, and other economically important crop plants that can be used under canopies of existing vegetation to increase productivity per unit area of land.

Isolation of prostrate turfgrass mutants via screening of dwarf phenotype and characterization of a perennial ryegrass prostrate mutant.

Prostrate turf varieties are desirable because of their increased low mowing tolerance, heat resistance, traffic resistance and ground coverage compared with upright varieties. Mutation breeding may provide a powerful tool to create prostrate varieties, but there are no simple, straightforward methods to screen for such mutants. Elucidation of the molecular basis of the major 'green revolution' traits, dwarfism and semi-dwarfism, guided us to design a simple strategy for isolating dwarf mutants of perennial ryegrass (Lolium perenne L.). We have shown that gamma-ray-mediated dominant dwarf mutants can be easily screened for at the three-leaf stage. About 10% of dwarf mutant lines also displayed a prostrate phenotype at mature stages (>10 tillers). One prostrate line, Lowboy I, has been characterized in detail. Lowboy I had significantly shorter canopy, leaf blade and internode lengths compared with wild type. Lowboy I also exhibited greater tolerance to low mowing stress than wild type. Exogenous gibberellic acid (GA) restored Lowboy I to a wild-type phenotype, indicating that the dwarf and prostrate phenotypes were both due to GA deficiency. We further showed that phenotypes of Lowboy I were dominant and stably inherited through sexual reproduction. Prostrate turfgrass mutants are difficult to screen for because the phenotype is not observed at young seedling stages, therefore our method represents a simple strategy for easily isolating prostrate mutants. Furthermore, Lowboy I may provide an outstanding germplasm for breeding novel prostrate perennial ryegrass cultivars.