Factors previously shown to promote leaf lesion development in Les1 plants are genetic background (i.e. W23), temperatures below 30 C, wounding (Hoisington et al. 1982 Devel. Biol. 93:381) and 5% propanol (Ray & Walbot 1984 MNL 58:190). I've now found that light is a strong promoter of lesion formation. In Les1/Les1 seedlings the absence or great reduction of light inhibits lesions from forming at temperatures below 30 C, and at wound sites. A 5% propanol solution appears not to be an effective inducer of lesions in young seedlings at the light intensities used in these experiments.
Les1 wt1 homozygotes from an F2 population were scored by the wt1 phenotype before lesion expression began. Because Les1 is 1 m.u. from wt1 (Hoisington, this issue) very few of the wt1 seedlings will fail to be homozygous for Les1. Lesions began to appear on leaf #1 2 to 4 days after seedling emergence and, spreading down the leaf from tip to base, covered the entire leaf by 7 to 9 days under the growth conditions used (25 C, 14h days with 310 or 600 microEinsteins/m2/sec; 20 C nights). When a section of the first leaf was covered with a reflective opaque mask before or at the beginning of lesion formation, lesions seldom developed in the covered area. In most cases covering the leaf also prevented preexisting lesions from expanding and prevented lesions from forming around wound sites. This inhibition of lesion formation continued as long as the masks were left in place, even when the non-covered portions of the leaf had become completely necrotic due to lesion development. The masks were 1.5 x 4 cm. provided a 2-3 mm air space around the covered section, were coated with aluminum foil to reflect most of the radiant heat, and had a matte black finish on their leaf-side surfaces to reduce any reflected light entering through the open ends. They were held in place with a paper clip or with rubber bands.
Once lesions had formed proximally to the covered sections removal of the masks resulted in the exposed areas becoming densely covered with lesions or, more often the case, completely necrotic within 24 h. Re-covering of the exposed areas at various intervals after exposure demonstrated that lesions can be inhibited by about 50%, when recovered within 4 h. Shorter intervals have not yet been studied. Re-covering after 6 h had no effect on subsequent lesion development. The same results were found when the entire plant was placed in the dark after 4 or 6 h exposure. This indicates that a certain amount of light is required for lesion initiation to occur but the subsequent necrosis occurs in the dark. I do not yet know if darkness is an absolute requirement for extensive necrosis to occur. I have observed lesions developing during the course of a day but they were always few and small.
In the instances where lesions did develop under the opaque masks they were small and few in number or, if large, were limited to the midrib region or the edge of the covered area. Whether these lesions are due to light leaks or to certain physiological or genetic conditions is not yet known.
The absence of a lesion-gradient and the rapid appearance of lesions in the uncovered areas indicates that the developmental or physiological signal(s) that determine tip-to-base lesion development can occur in the dark. The severity of lesion formation in the uncovered areas was greater than that in the surrounding leaf and was usually marked by total necrosis, not descrete lesions. Perhaps the factors that normally limit lesion initiation or expansion, or both, in the leaf require exposure to light for a certain length of time to become functional. Another possibility is that the factors that limit lesion initiation or expansion, or both, are induced by lesion necrosis. Without any light to trigger necrosis all of the cells become set (preinitiated?) to form lesions since there are no signals to prevent them from doing otherwise. If this latter possibility is what is actually occurring then lesion limitation appears to be a type of hypersensitive response.
Preliminary experiments using colored filters in place of opaque masks seemed to indicate that lesion development is related to general photosynthetic activity. Yet this relationship is not a straightforward one since lesions form quite well in the white sectors of wd1, ring-9 (Wd, C-I) or j1 plants (D. Hoisington, this issue).
The tentative conclusions of these observations are that lesion-forming potential and lesion necrosis are (usually) light independent while lesion initiation is (usually) light dependent. Is, then, Les1 a light regulated gene? The next set of experiments will try to answer this question.
Craig Echt
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