Abstract
The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).
Highlights
Angiosperm seedlings that germinate in the dark etiolate
No Pchlide could be extracted from the fluorescent bands in this work; the extraction of Pchlide at the gel front indicated that Pchlide was released from POR during solubilization of the membrane and native PAGE analysis
Four levels of evidence indicate that light-harvesting-like protein 3 (Lil3) directly assembles with proteins regulating Chl biosynthesis (Fig 6)
Summary
Angiosperm seedlings that germinate in the dark etiolate. Proplastids develop into etioplasts and Protochlorophyllide (Pchlide), Pchlide oxidoreductase A (PORA) and thylakoid lipids accumulate [1, 2] in an inner membrane system of prolamellar bodies that develop a semicrystalline structure [3]. The first molecular indication of the plants morphological switch from skoto- to photo-morphogenesis is the light-dependent photoreduction of Pchlide to Chlide by POR (EC 1.3.1.33), and the accumulation of ChlGG and ChlPY after esterification by CHS (EC 2.5.1.62) [4,5,6,7]. Flat membrane sacs emerge that transform into the thylakoid containing the photosynthetic complexes [8].
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