The Fate of Chloroplast Proteins during Photooxidation in Carotenoid-Deficient Maize Leaves

Maize seedlings, treated with the herbicide norflurazon to produce a deficiency in carotenoid pigments, were grown in low-fluence-rate light. Under these conditions, which induced chlorophyll biosynthesis while minimizing photooxidation, carotenoid-deficient seedlings showed identical patterns of chloroplast protein accumulation compared with normal seedlings. Carotenoid pigments thus play no direct role in regulating the accumulation of chloroplast proteins. When shifted to high-fluence-rate light, chlorophyll was rapidly photooxidized in carotenoid-deficient seedlings. Chloroplast proteins showed varying degrees of sensitivity to photooxidation. The P-700 apoprotein of photosystem I was rapidly degraded. Most stromal and thylakoid proteins either decreased progressively in photooxidative conditions or appeared to be unaffected. The relative quantity of the light-harvesting chlorophyll a/b-binding protein of photosystem II increased significantly in the first few hours of high-fluence-rate light. It then appeared to be only minimally affected 18 hours after complete photooxidation of chlorophyll.     

Binding Proteins to Phytochrome A in Etiolated Pea Seedlings

In order to detect and characterize a putative receptor(s) fora signal from PhyA, proteins that bind to purified pea PhyAwere searched for in the crude extract of etiolated pea seedlingswith affinity chromatography. PhyA was coupled to the columnsubstrate either in P_R form (P_R column) or in red-irradiatedform (P_FR column). The coupled PhyA of both columns retainsits spectral reversibility between P_R and P_FR, although theirpeptide mapping by trypsin digestion suggests that the C-terminalhalf of PhyA in the P_FR column is partially fixed in P_FR structure.15 polypeptides were detected reproducibly in the elution fromthe P_FR column by silver-staining of SDS-PAGE. These 15 polypeptidesmay form two complexes judging from their elution profiles.Of the 15 polypeptides, the 6 major polypeptides have approximatemol wt of 80, 55, 53, 46, 40 and 35 kDa. On the other hand,only a trace amount of protein, which mainly consists of the46 kDa species, was eluted from P_R column, indicating the presenceof P_FR-specific BPs in the crude extract of etiolated pea seedlings.Of the 6 major polypeptides, the 40 kDa species binds to thePhyA in a photoreversible manner. (Received June 19, 1998; Accepted December 19, 1998)     

Effect of Chloramphenicol on Formation of Chloroplast Structure and Protein During Greening of Etiolated Leaves of Phaseolus vulgaris

Greening of leaves of Phaseolus vulgaris in the presence of chloramphenicol inhibits formation of A) total chloroplast protein, B) an easily extractable fraction removed during isolation of chloroplasts in isotonic media by differential centrifugation, and C) the insoluble lamellar fraction which remains after extracting osmotically shocked freeze-dried plastids. The inhibition of insoluble chloroplast protein formation is correlated with decreased formation of lamellae and increased formation of vesicular structures. In contrast, chloramphenicol increases the formation of a fraction not removed during differential centrifugation, but removed by water extraction after osmotic shock and freeze-drying of chloroplasts. Analysis of this fraction by electrophoresis and column chromatography, indicates that the increased accumulation of this protein fraction is largely due to accumulation of a protein which is normally present in this fraction in small quantities. It was suggested that this protein may be a precursor which is normally incorporated into the lamellae. The protein extracted from freeze-dried chloroplasts of chloramphenicol treated chloroplasts contains a smaller proportion of one or more proteins than a similar extract of untreated plastids. However, per plastid, no such difference exists.     

Phytochrome Control of Cell Wall-bound Hydroxyproline Content in Etiolated Pea Epicotyls

The red light inhibition of growth of the intact pea (Pisum sativum L. cv. Alaska) third internode was correlated with an increase in the content of cell wall-bound hydroxyproline. These changes were detected 3 hours after irradiation, and possibly at 1 hour. Far red light reversed the effects of red light. The iron chelator α,α′-dipyridyl reversed the red light effects on both growth and hydroxyproline content. Using segments incubated in vitro, no phytochrome-mediated change in hydroxyproline content could be observed, perhaps because of an overwhelming wounding response. If plants were irradiated in situ and grown for 8 hours before excision and incubation of segments, some enhancement of hydroxylation by red light was detectable both colorimetrically and radioisotopically. The red light inhibition of segment growth was reversed by α,α′-dipyridyl. These results are examined in reference to the role of extensin in normal and induced growth cessation.     

Phytochrome Control of Longitudinal Growth and Phytochrome Synthesis in Maize Seedlings

The active, far-red light absorbing, form of phytochrome was found to inhibit growth and phytochrome levels in the mesocotyl and coleoptile of 4- to 5.5-day-old seedlings of Zea mays L. Short, low-irradiance red or far-red light treatments were used to produce different proportions of active phytochrome at the end of highdirradiance white-light periods, which left different levels of total phytochrome in the plants. After light treatments which left relatively high levels of spectrophotometrically assayable phytochrome in the seedlings, apparent phytochrome synthesis in the subsequent dark period was low regardless of the proportions of each form of the pigment present at the beginning of the dark period. In light treatments producing relatively low levels of assayable phytochrome, levels of apparent phytochrome synthesis in both red and far-red treatments and differences between apparent synthesis in red and far-red treatments were maximal. No simple correlation was found between growth and apparent phytochrome synthesis. However, growth and total phytochrome levels were positively correlated in both organs. Using a subtractive method of correlation, in which only phytochrome effects were plotted, strong linear relationships between phytochrome levels or longitudinal growth and P_fr levels were found in those light treatments leaving greater than 8% of dark control levels of phytochrome in the tissues. Using this technique non-linear, inverse relationships between P_fr and apparent phytochrome synthesis was found, indicating that modes of phytochrome control over phytochrome synthesis and growth differ. Our results are consistent with the view that in vivo assays of “bulk’ phytochrome reflect levels and states of the physiologically active phytochrome fraction under our experimental conditions in maize.     

Phytochrome Control of the Level of Extractable Ribonuclease Activity in Etiolated Hypocotyls

AN increase in RNA degrading activity of hypocotyl homo-genates can be detected 6–10 h after transferring 5 day old, dark-grown seedlings of Lupinus albus L. to continuous white light¹. This increase is concomitant with the onset of inhibition of hypocotyl extension^{1, 2}. The ribonuclease component whose level of activity is affected by light treatment (tentatively identified as a ribonucleate nucleotido-2′-transferase (cyclizing) (EC 2.7.7.17)) is associated with the ribosomes (and polysomes)¹ in hypocotyl homogenates. This is in contrast to the bulk of RNA degrading activity in homogenates which is associated with the soluble fraction¹. Isolation of the ribosomal fraction therefore makes it possible to follow changes in the level of activity of this ribonuclease which would be too small to detect in unfractionated cell homogenates. The results reported here indicate that the photoconversion of the red-absorbing form of phytochrome (P_r) to the physiologically-active far-red-absorbing form (P_fr) leads to a specific increase in ribosome-bound ribonuclease activity.     

Nitrate Reductase Activity: Phytochrome Mediation of Induction in Etiolated Peas

THE extractable activity of nitrate reductase from higher plant leaves is inducible by light and shows, under natural growth conditions, a pattern of diurnal variation¹. Studies on the nature of light involvement have generally used the green leaf as experimental material, implying that photosynthesis supports the induction process^1,2. We have examined the role of light for the induction of nitrate reductase activity in the etiolated terminal buds of field peas (Pisum arvense cv. Century). Treatments consisted of brief exposure of intact plants to broad bands of light, followed by a period in darkness before extraction for enzyme assay. These light treatments exclude the possibility of photosynthesis as a process contributing to induction. Under these conditions, induction is shown to be reversibly controlled by red and far red light, an effect ascribable to the pigment phytochrome.     

豌豆黄化苗114kD光敏色素的纯化

以25℃暗室中培养7d的阿拉斯加豌豆黄化苗为材料,采用改进的Yamamoto等方法,制备光敏色素粗提取液,再经DEAE-Sephacel,Brushite以及DEAE-Agarose柱层析得到初步提纯的光敏色素。然后再通过豌豆光敏色素抗体mAP1-Sepharose 4B亲和层析得到了提纯的豌豆114 kD光敏色素,经SDS-PAG电泳检测为一条带,并具有类似于纯净光敏色素的吸收光谱。     

Identification of a Triterpenoid Saponin in Etiolated Pea Shoots as Phytochrome Killer

Phytochrome killer, a substance which instantaneously causesin vitro spectral denaturation of phytochrome, was isolatedin the pure state from the methanol extract of etiolated peashoots by Sephadex G-25, charcoal and silica gel chromatographies.Chemical and spectrometric (nuclear magnetic resonance, massand infra-red) analyses disclosed that this substance was identicalwith soyasaponin I, a triterpenoid saponin. Most of the phytochromekiller activity in the methanol extract was ascribable to soyasaponinI. The content of this saponin was 61 µg/g fr wt in theetiolated epicotyl tissue and 27 µg/g fr wt in the greenepicotyl tissue. (Received August 15, 1981; Accepted December 16, 1981)     

Phytochrome Control of Acid and Alkaline Phosphatases in Etiolated Shoot Apices of Sorghum bicolor

A short pulse of red light or continuous far-red light enhancedthe activities of acid and alkaline phosphatases over the valuesof the dark controls in 5-day-old etiolated seedlings of Sorghumbicolor. For 30 min after the red light pulse 100% of the red/far-redphotoreversibilities was maintained for the acid and 80% forthe alkaline phosphatases. Thereafter, the "photoreversibilityescape reaction" was fast, being completed within 180 min. Cycloheximideas well as 6-methyl purine markedly inhibited red light enhancementof the activities of the phosphatases, but chloramphenicol,lincomiycin and rifamycin SV were ineffective. In spite of photoregulationof both the phosphatases at the time of de novo synthesis, itappears that control of the acid and alkaline phosphatases maybe affected by two independent initial actions of phytochrome. ¹ Present address: Biologisches Institut II, University of Freiburg,FRG. (Received August 4, 1984; Accepted April 3, 1985)     

Ferredoxin-dependent Nitrite Reductase from Spinach Leaves

A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.

The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A₃₉₀: A₂₇₈ and A₆₇₃: A₃₉₀ were 0.61 and 0.37, respectively.

By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel.     

Differences in Photoresponse and Phytochrome Spectrophotometry Between Etiolated and De-etiolated Pea Stem Tissue

Morphologically similar pea plants having a 4-fold difference in spectrophoto-metrically detectable phytochrome can be produced by pretreatment of etiolated plants with red light (R) or with red and far-red light combined (RF). A search for response differences which could be ascribed to differences in phytochrome content has resulted only in the establishment of differences due to de-etiolation. Segments of etiolated plants differ from those of plants de-etiolated by R and RF pretreatments in 2 ways. Segments from etiolated plants appear to respond rapidly to the far-red absorbing form of phytochrome (P_FR), while segments from de-etiolated plants do not respond rapidly to P_FR. This statement is based upon 2 observations: (i) the red light induced growth inhibition in segments from etiolated plants rapidly escapes reversibility by far-red light, while with segments from R or RF pretreated plants, the red light effect is fully reversed by subsequent far-red light for up to 2 hr; and (ii) segments from etiolated plants were inhibited to a greater degree than were segments from RF pretreated plants when various photostationary state levels of P_FR were maintained for 30 or 90 min and then removed by photoconversion to P_R. The in vivo nonphotochemical transformation curves of the phytochrome of etiolated and RF pretreated plants appear to differ in 2 related respects: (i) the amount of phytochrome destroyed in de-etiolated tissue is greater than that in etiolated tissue, perhaps as a result of the fact that (ii) the rate and extent of apparent reversion of P_FR to P_R in etiolated tissue is about twice that in de-etiolated tissue.     

Chloroplast DNA in Expanding Spinach Leaves

The proportion of chloroplast DNA in total DNA from spinachleaves has been measured using the second order reassociationkinetics of a ³H-labelled chloroplast DNA probe in total DNAextracts. There was no significant difference between the proportionof chloroplast DNA in the basal and distal halves of 2 cm leavesand in the distal halves of 5, 8, and 10 cm leaves. The meanof all the observations was 21.1 ± 0.7%. There was littlechange in the average total DNA content of cells from any ofthe leaves but cells from larger leaves contained 130–170chloroplasts while cells from the basal half of 2 cm leavescontained about 20 chloroplasts which were smaller than thosefrom the larger leaves. Consequently the average number of copiesof the plastome per chloroplast in large leaves was about 30(5 x 10^–15 g DNA) and in the smaller chloroplasts in thebase of 2 cm leaves was 200 (32 x 10^–15 g DNA). Stainingwith the DNA fluorochrome 4, 6-diamidino-2 phenyl indole (DAPI)showed 10–15 plastid nucleoid areas in chloroplasts oflarger leaves, suggesting there are 2–3 copies of theplastome per plastid nucleoid.     

Regulation of Phytochrome Message Abundance in Root Caps of Maize : Spatial, Environmental, and Genetic Specificity

In many cultivars of maize (Zea mays L.) red light affects root development via the photomorphogenetic pigment phytochrome. The site of perception for the light is the root cap. In the maize cultivar Merit, we investigated phytochrome-mediated events in the cap. We established that the message encoded by the phyA1 gene was most abundant in dark-grown tissue and was asymmetrically distributed in the root cap, with greatest expression in the cells which make up the central columella core of the cap. Phytochrome message was negatively autoregulated in a specific region within the root cap. This autoregulation was sensitive to very-low-fluence red light, and thus was characterized as a phytochrome-mediated, very-low-fluence event. The kinetics of message reaccumulation in the dark were also examined and compared to the kinetics of the light requirement for root gravitropism in this cultivar. Similarly, the degree of autoregulation present in two other maize cultivars with different light requirements for gravitropic sensitivity was investigated. It appears that the Merit cultivar expresses a condition of hypersensitivity to phytochromemediated light regulation in root tissues. We conclude that phytochrome regulates many activities within the cap, but the degree to which these activities share common phytochrome-mediated steps in not known.