Endonucleolytic activation directs dark-induced chloroplast mRNA degradation

Plastid mRNA stability is tightly regulated by external signals such as light. We have investigated the biochemical mechanism responsible for the dark-induced decrease of relative half-lives for mRNAs encoding photosynthetic proteins. Protein fractions isolated from plastids of light-grown and dark-adapted plants correctly reproduced an RNA degradation pathway in the dark that is downregulated in the light. This dark-dependent pathway is initiated by endonucleolytic cleavages in the petD mRNA precursor substrate proximal to a region that can fold into a stem–loop structure. Polynucleotide phosphorylase (PNPase) polyadenylation activity was strongly increased in the protein fraction isolated from plastids in dark-adapted plants, but interestingly PNPase activity was not required for the initiation of dark-induced mRNA degradation. A protein factor present in the protein fraction from plastids of light-grown plants could inactivate the endonuclease activity and thereby stabilize the RNA substrate in the protein fraction from plastids of dark-adapted plants. The results show that plastid mRNA stability is effectively controlled by the regulation of a specific dark-induced RNA degradation pathway.     

The Effect of Light and Inhibitors on Chloroplast and Cytoplasmic RNA Synthesis

Chloroplast RNA is synthesized in dark-grown radish cotyledons at about one-third the rate of that in the light. The synthesis, however, continues for longer in the dark and the percentage of chloroplast RNA can approach that in light-grown tissue. Light stimulates the synthesis and accumulation of both cytoplasmic and chloroplast RNA, but shows a 4-fold greater stimulation of the chloroplast RNA. Chloramphenicol, streptomycin and cycloheximide inhibit the synthesis of chloroplast RNA with little effect on cytoplasmic RNA. 5-Fluorouracil inhibits the synthesis of cytoplasmic more than chloroplast RNA. Synthesis of the 0.56 x 10(6) mol wt chloroplast RNA is inhibited much less than the other ribosomal RNA components by actinomycin D.     

Plastid stromules are induced by stress treatments acting through abscisic acid

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.     

The Effect of Red Irradiation on Plastid Ribosomal RNA Synthesis in Dark-grown Pea Seedlings

Dark-grown pea seedlings (Pisum sativum L.) were irradiated for a short period each day with low intensity red light (662 nm), red light immediately followed by far red light (730 nm), or far red light alone. Other plants were transferred to a white light regime (14 hours light/10 hours dark). There was no change in the amount of RNA in the tissue on a fresh weight basis after the various treatments. However, compared with dark-grown seedlings, those plants irradiated with red light showed an increase in the net RNA content per stem apex. In addition there was a two- to three-fold increase in ribosomal RNA of the etioplasts relative to the total ribosomal RNA. These increases were comparable to those found in plants grown in the white light regime. The changes were much smaller if the dark-grown plants were irradiated either with red light followed by far red light, or with far red light alone. Thus continuous light is not essential for the production of ribosomal RNA in plastids, and the levels of ribosomal RNA found in chloroplasts can also be attained in etioplasts of pea leaves in the dark provided the leaf phytochrome is maintained in its active form.     

Biochemical characteristics of thylakoid membranes in chloroplasts of dark-grown pine cotyledons

Japanese black pine (Pinus thunbergii) cotyledons were found to synthesize chlorophylls in complete darkness during germination, although the synthesis was not as great as that in the light. The compositions of thylakoid components in plastids of cotyledons grown in the dark and light were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of polypeptides and spectroscopic determination of membrane redox components. All thylakoid membrane proteins found in preparations from light-grown cotyledons were also present in preparations from dark-grown cotyledons. However, levels of photosystem I, photosystem II, cytochrome b_[ill]/f, and light-harvesting chlorophyll-protein complexes in dark-grown cotyledons were only one-fourth of those in light-grown cotyledons, on a fresh weight basis. These results suggest that the low abundance of thylakoid components in dark-grown cotyledons is associated with the limited supply of chlorophyll needed to assemble the two photosystem complexes and the light-harvesting chlorophyll-protein complex.     

Light Mediated Changes in the Chloroplasts of the Liverwort, Sphaerocarpos donnellii Aust

Dark-grown plants of Sphaerocarpos, incubated in a liquid medium containing sucrose and mineral salts, have a much lower chlorophyll and nitrogen content than do light-grown plants. Two minutes of red light per 12 hours is about two-thirds as effective in increasing chlorophyll and nitrogen content as is continuous white light. These red light-induced increases are mediated by phytochrome, as they are reversible by alternating exposures to red and far-red light. They appear to be related to differences in the ultrastructure of the chloroplasts. Plastids from dark-grown plants are full of starch and develop few lamellae, while light-grown plastids contain little starch and have many lamellae. The ultrastructural studies are supported by starch determinations which revealed a phytochrome-mediated decrease in starch content. The effect of white light in increasing the chlorophyll and nitrogen content above the level attained in red light-treated plants is not mediated by photosynthetic activity. These results are related to similar responses in other archegoniates and angiosperm seedlings.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

An enzyme-linked immunosorbent assay for phytochrome

A double-antibody sandwich, enzyme-linked immunosorbent assay has been developed for phytochrome in Avena sativa L. cv. Saladin. An immunoglobulin fraction of rabbit antiserum raised to 118 kdalton phytochrome was used with alkaline phosphatase as the enzyme label. The assay detected as little as 0.2 ng phytochrome in extracts of dark-grown plant material. No evidence for specific or non-specific measurement of proteins other than phytochrome was found. The assay detected phytochrome in extracts of Avena grown in the light. Dilution curves for light-grown phytochrome extracts had a reduced slope and saturated at a lower level of enzyme activity than those for dark extracts. These differences were not caused by an inhibitor in extracts from light-grown plants. Phytochromes from dark- and light-grown plants may be immunologically different.