Effects of Cytokinin and Light on Polyamines during the Greening Response of Cucumber Cotyledons

The cucumber cotyledon greening test was used as a model systemto study possible relationships between cytokinins and polyaminesduring the greening process. When cytokinin was applied to dark-growncotyledons, large increases in chlorophyll and putrescine levelswere observed. However, inhibition of putrescine biosynthesiswith D-arginine and difluoromethylarginine did not affect chlorophyllproduction, and applied polyamines proved inhibitory to greening.Addition of 50 mM K^$ to the cytokinin treatments increased chlorophyllsynthesis but caused a marked reduction in putrescine levels.These results indicate that the large increase in putrescinecontent that derives from cytokinin treatment of the cotyledonsis not essential for the cytokinin-induced greening response. ¹Present address: Crop Science Department, University of Guelph,Guelph, Ontario, Canada, NIG 2W1. ²Present address: Agrogen Biotechnologies Inc., 520W. 6th Ave.,Vancouver, B.C., Canada, V5Z 4H5. (Received June 29, 1987; Accepted October 9, 1987)     

Chloroplast Biogenesis: XX. Accumulation of Porphyrin and Phorbin Pigments in Cucumber Cotyledons during Photoperiodic Greening

A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3.     

Plastid Development in Pisum sativum Leaves during Greening : II. Post-Translational Uptake by Plastids as an Indicator System to Monitor Changes in Translatable mRNA for Nuclear-Encoded Plastid Polypeptides

When isolated pea plastids are incubated with translation products of poly(A⁺) mRNA they specifically import precursor molecules of plastid polypeptides. Etioplasts and chloroplasts import the same polypeptides from identical translation products, and, the imported polypeptides can be well resolved by two-dimensional gel electrophoresis. Therefore, the posttranslational uptake system using isolated chloroplasts can monitor changes in the abundance of translatable plastid-targeted messages. Poly(A⁺) mRNA was isolated from peas at various times during greening and analyzed by this technique. (a) After 48 hours of illumination of dark-grown plants, the relative portion of nuclear encoded messages for plastid targeted proteins had increased by a factor of 2. The percentage of polypeptides recovered in the stroma fraction increased from about 50 to 65%. (b) More than 140 imported polypeptide species could be detected in fluorograms of two-dimensional gels, most of which could be identified throughout the time course of greening. At least 37 imported polypeptides decreased and 36 increased in relative abundance during greening of darkgreen plants. (c) In most cases, where differences in translatable messages were seen between dark- and light-grown plants, they were accompanied by parallel changes in polypeptide abundance.     

Elimination of the Far-Red-Induced Lag Phase of Chlorophyll a Accumulation by a Brief Light in Pharbitis nil Cotyledons

In dark-grown Pharbitis nil seedlings, far-red light (FR) irradiationof 48 h or less promotes Chl a accumulation in the first 2-hof a subsequent white light (WL) period, without a lag phaseof Chl a accumulation. However, continuous FR irradiation of72 h or more, causes the so-called FR-induced lag phase. A 5-minWL given 4 h before the onset of the continuous WL promotesChl a accumulation irrespective of the length of the precedingFR irradiation period, if a 4-h dark period is inserted betweenthe 5-min WL and continuous WL. This suggests that the effectof the brief WL is independent of and additive to the effectof the preceding FR irradiation, although the effect of theFR irradiation changes from promotive to inhibitory with anincrease in the irradiation period. Red light (R) is more active than blue light (B) in this brieflight effect. The R effect is reversed by subsequent exposureto FR when the period of the preceding FR irradiation is 24h, but not when it is 72 h. The relative effectiveness of Bto R increases after prolonged FR irradiation. (Received August 6, 1986; Accepted March 12, 1987)     

Changes in Phosphorylation Status of Wheat Plastid Polypeptides as Influenced by Light, Calcium and Cyclic AMP

The polypeptides of etioplast and chloroplast fractions, purified on Percoll discontinuous gradient, were phosphorylated in vitro using (γ-³²P)ATP, resolved by SDS-PAGE and autoradiographed. In general, about 15-18 phosphopolypeptides in the range of 14-150 kD were distinctly visible in autoradiograms of both organelle fractions with varying degree of radiolabel incorporation. Although short-term irradiation with red or far-red light did not have any significant effect on phosphorylation status of etioplast polypeptides, in vivo irradiation with 1 h white light, followed by in vitro phosphorylation, decreased phosphorylation of a 116 kD polypeptide and increased the phosphorylation of polypeptides of 38 kD and a doublet around 20 kD. Strikingly, the phosphorylation status of 116 kD etioplast polypeptide was adversely affected by Ca²⁺ as well, and this phosphopolypeptlde was not distinctly visible in the autoradiogram of the chloroplast fraction proteins. However, in vitro phosphorylation of 98, 57 and 50 kD polypeptides of both etioplast and chloroplast fractions was found to be Ca²⁺ dependent. Unlike Ca²⁺, 3′,5′-cyclic AMP down-regulated the phosphorylation of several polypeptides of both etioplasts and chloroplasts, including 98 and 50 kD, and up-regulated the phosphorylation of 32 and 57 kD polypeptides. The significance of these observations on changes in phosphoprotein profile of etioplasts and chloroplasts, as influenced by light, Ca²⁺ and cyclic nucleotides, has been discussed.     

Mechanism of Benzyladenine-Induced Stimulation of the Synthesis of 5-Aminolevulinic Acid in Greening Cucumber Cotyledons: Benzyladenine Increases Levels of Plastid tRNAGlu

The mechanism of the stimulatory effect of a cytokinin, namely,benzyladenine (BA), on the synthesis of 5-aminolevulinic acid(ALA) in cucumber cotyledons was studied. The rate of synthesisof ALA by plastids isolated from BA-treated cotyledons was twicethat by plastids from untreated controls. Western blot analysisof stromal proteins showed that BA did not affect the levelof glutamyl-tRNA synthetase or of glutamate l-semialdehyde (GSA)aminotransferase. Analysis of free amino acids revealed thatBA did not increase the level of glutamate in the stroma. However,the amount of total plastidic RNA was doubled in BA-treatedcotyledons. Northern blot analysis showed that the level ofplastid tRNA^Glu was increased by treatment with BA to the sameextent as that of another plastid tRNA, reflecting an increasein total plastidic RNA. The rate of formation of glutamyl-tRNAwas also doubled in plastids from BA-treated cotyledons. Theresults indicate that stimulation of the synthesis of ALA byBA is due to an increased level of tRNA^Glu in plastids. (Received June 6, 1993; Accepted November 26, 1993)     

Effects of Light Quality and Norflurazon on the Formation of Plastid Pigments in Cotyledons of Pinus sylvestris

The effects of different light qualities and a special inhibitor of carotenoid biosynthesis on formation of plastid pigments of cotyledons of Pinus sylvestris were studied. The experimental results indicate: 1. The rate of synthesis of carotenoids in far-red light is relatively higher than that of chlorophylls, on the contrary in red light the rate of chlorophyll synthesis is higher. 2. When biosynthesis of carotenoids is inhibited, in white light the rate of total chlorophyll synthesis reduced with similar proportion. Accumulation of chlorophyll, however, is relatively much more than that of carotenoids. The highest molar ratio of chlorophyll/carotenoids is approximately 10.0. This implicates that chlorophyll and carotenoid synthesis proceed with certain independence. 3. After 4h exposure of strong white light of 9 day-old pine seedlings grown with 10-5 mol 1-1 norflurazon in farred light, contents of carotenoids and total, chlorophyll of cotyledons increase. Chlorophyll a biosynthesis promoted by light is higher than photooxidation of the pigment.     

Accumulation of New Polypeptides in Ri T-DNA-Transformed Roots of Tomato (Lycopersicon esculentum) during the Development of Vesicular-Arbuscular Mycorrhizae

Root-inducing transferred-DNA (Ri T-DNA)-transformed roots of tomato (Lycopersicon esculentum) were in vitro inoculated with surface-sterilized vesicular-arbuscular mycorrhizal leek root pieces. About 1 week after inoculation, the infection of the transformed root culture by the fungal endophyte was confirmed by photonic microscopy. Total proteins were extracted from the mycorrhizal roots and analyzed by two-dimensional polyacrylamide gel electrophoresis. Control gels were run with proteins extracted from noninoculated roots mixed with purified intraradical vesicles and extraradical hyphae. Comparison of the resulting patterns revealed the presence of two polypeptides with estimated apparent masses of 24 and 39 kDa that were detected only in infected roots. Polypeptides with similar migration parameters were not detected in roots challenged with spore extracts, suggesting that the accumulation of the polypeptides was directly linked to root colonization by the fungus rather than to induction by fungus-derived elicitors.     

Identity of a Group of 13.5-20 kDa Polypeptides Whose Apparent Abundance Decreases during Plastid Development

The identity of a group of 13.5–20 kDa polypeptides whoseabundance decreases during the greening of intermittent-lightgrown barley seedlings was investigated. During greening therelative abundance of the 13.5–20 kDa polypeptides wasinversely related to that of LHC II, which had led others tosuggest a role of these polypeptides in the assembly of theLHC II and/or chloroplast development. The smallest 13.5 kDapolypeptide was identified as histone H4 by N-terminal sequencingof an internal peptide fragment produced by CNBr cleavage. Theentire group of 13.5–20 kDa polypeptides was thereafterverified to be nuclear histones by their similar mobility onSDS-PAGE to that of barley histones and immunoreactivity toyeast histone anti bodies. Their presence results from contaminationof plastid preparations by nucleosomes. Our results unequivocallysubstantiate earlier suggestions of others that polypeptidesoften found to contaminate immature plastids were of nuclearorigin. Methods to reduce or remove the histone contaminationwithout reduction in yield of thylakoids were developed so thattrue changes in the polypeptide content of thylakoids can bestudied during plastid development. (Received December 19, 1991; Accepted August 15, 1992)     

Plastid Development in Pisum sativum Leaves during Greening : I. A Comparison of Plastid Polypeptide Composition and in Organello Translation Characteristics

Changes in plastid polypeptide composition during greening of etiolated peas were investigated by two-dimensional gel electrophoresis. One hundred of the more than 250 polypeptides which could be detected upon silver staining were followed during plastid development. Thirty-nine polypeptides decreased in abundance on a per organelle basis. Twentythree of the 46 polypeptides which increased in abundance upon greening could be identified as proteins of the thylakoid membrane. The changes in proteins observed during greening of etiolated leaves corresponded largely to those observed during normal leaf expansion. The origin of some of the polypeptides was traced back by comparing the two-dimensional gels of plastid proteins with in organello translation products and with polypeptides which had been synthesized in vitro from poly(A⁺) mRNA preparations and posttranslationally imported by chloroplasts. Some polypeptides were specifically identified in two-dimensional gels by Western blot analysis.     

Photoregulation of the Greening Process of Wheat Seedlings Grown in Red Light*

Wheat seedling grown with their shoot bottom exposed to red light (400 μmol m⁻² s⁻¹) either with constant illumination or light-dark cycles did not accumulate chlorophyll. This near-etiolation response was manifested by a critical threshold intensity of red light and did not need continuous illumination. The inhibition of the greening process resulted from reduced synthesis of glutamate-1-semialdehyde and consequent reduction in tetrapyrrole precursor 5-aminolevulinic acid. Red light perceived by the shoot bottom down regulated the protein and/or gene expression of enzymes involved in the biosynthesis of tetrapyrroles. The contents of endogenous cytokinins, i.e., isopentenyl-adenosine and dihydrozeatinriboside, were reduced in seedlings grown in red light having their shoot bottom exposed. Application of exogenous cytokinin and its analogue to roots of seedlings grown in red light reversed the down regulation of the greening process. The reversal of red-light-induced near-etiolation morphogenesis by far-red (200 μmol m⁻² s⁻¹) or blue (25 μmol m⁻² s⁻¹) light suggests that it could be a very high red-irradiance response of phytochrome, in the meristematic layers of the shoot bottom, that works in concert with blue light receptor(s). This work was supported by a competitive grant from the Department of Science and Technology, Govt. of India (DST/SP/SO/A-49/95) to BCT. Suchi Sood Varsha Gupta: Equal contributors     

Correlated Changes in the Activity,Amount of Protein,and Abundance of Transcript of NADPH:Protochlorophyllide Oxidoreductase and Chlorophyll Accumulation during Greening of Cucumber Cotyledons

Changes in the activity and abundance of NADPH:protochlorophyllide oxidoreductase (NPR) and the abundance of mRNA encoding it were examined during the greening of 5-d-old etiolated cucumber cotyledons under continuous illumination. To measure NPR activity in the extracts from fully greened tissues, we have developed an improved method of assay. Upon exposure of etiolated cotyledons to light, NPR activity decreased rapidly within the first 2 h of exposure. Thereafter, enzymatic activity increased transiently, reaching a submaximum level at 12 h, and decreased slowly. The level of immunodetectable NPR protein followed the same pattern of changes during 96 h of greening as observed for NPR activity. The NPR mRNA in etiolated cotyledons disappeared quickly in the 1st h of irradiation. However, the level of mRNA increased thereafter to reach 3-fold or more of the dark level at 12 h and then decreased. The changes in the activity, protein level, and mRNA level after the first rapid decreases corresponded chronologically and nearly paralleled the increase in the rate of chlorophyll accumulation. These findings suggest that the greening of cucumber cotyledons is regulated basically by the level of NPR protein without activation or repression of enzymatic activity and that NPR mRNA increased by light maintains the level of enzyme protein necessary for greening.     

BIOGENESIS OF CHLOROPLAST MEMBRANES : II. Plastid Differentiation during Greening of a Dark-Grown Algal Mutant (Chlamydomonas reinhardi)

Dark-grown cells of the y-1 mutant of Chlamydomonas reinhardi contain a partially differentiated plastid lacking the photosynthetic lamellar system. When exposed to the light, a rapid synthesis of photosynthetic membranes occurs accompanied by synthesis of chlorophyll, lipids, and protein and extensive degradation of the starch reserve. The process is continuously dependent on illumination and is completed within 6–8 hr in the absence of cell division. Photosynthetic activity (O₂ evolution, Hill reaction, NADP photo-reduction, and cytochrome f photooxidation) parallels the synthesis of pigment and membrane formation. During the greening process, only slight changes occur in the levels of soluble enzymes associated with the photosynthetic process (RuDP-carboxylase, NADP-linked G-3-P dehydrogenase, alkaline FDPase (pH 8)) as compared with the dark control. Also cytochrome f concentration remains almost constant during the greening process. The kinetics of the synthesis of chlorophyll, formation of photosynthetic membranes, and the restoration of photosynthetic activity suggest that the membranes are assembled from their constituents in a single-step process.     

Abundance of the Major Chloroplast Polypeptides during Development and Ripening of Tomato Fruits: An Immunological Study

During maturation and ripening of tomato (Lycopersicon esculentum, cv Tamar) fruits, there are differential changes in the steady state levels of chloroplast proteins. Western blot analysis indicated that with the exception of the core polypeptide of photosystem I (PSI) (subunit I) the whole complex disappears during the transition of chloroplast to chromoplast. The amounts of the core polypeptide of photosystem II (PSII) (43 kilodaltons) and the light harvesting chlorophyll protein complex increase during maturation and decrease thereafter. In contrast, the 33 kilodalton subunit of PSII is found at the highest levels from the early recorded stages and decreases gradually until late stages of ripening. The level of cytochrome f decreases slowly during the maturation and ripening process, whereas the Rieske protein of the same complex disappears at a faster rate. There are also differential changes in the subunits of the chloroplast coupling factor·ATPase complex; α and β subunits increase during maturation, whereas the level of the γ subunit is already maximal at the earliest recorded stage of development and depleted thereafter. The two subunits of the ribulose-1,5 bisphosphate carboxylase increase in abundance during chloroplast maturation and gradually disappear after the transition from chloroplast to chromoplast. However, there are substantial differences in the rates of increase and disappearance of the large and small subunits of this enzyme. This imbalance is attributed to different regulation of nuclear and chloroplast gene expression. In addition, the steady state levels of chloroplastic superoxide dismutase and phosphoenolpyruvate carboxylase have been followed. Both enzymes reach their maxima at the final stages of ripening. This increase coincides with the climacteric rise of CO₂ release.     

The Abolition of the Lag Phase in Greening Cucumber Cotyledons by Exogenous delta-Aminolevulinic Acid

Etiolated cucumber cotyledons treated with δ-aminolevulinic acid accumulated protochlorophyllide which was phototransformable to chlorophyll (ide). The phototransformation process in the δ-aminolevulinic acid-treated tissue was markedly temperature-dependent, consistent with the view that this protochlorophyllide must combine with the holochrome apoenzyme before phototransformation can occur.     

Phylogeny of Dinoflagellate Plastid Genes Recently Transferred to the Nucleus Supports a Common Ancestry with Red Algal Plastid Genes

It is generally accepted that peridinin-containing dinoflagellate plastids are derived from red alga, but whether they are secondary plastids equivalent to plastids of stramenopiles, haptophytes, or cryptophytes, or are tertiary plastids derived from one of the other secondary plastids, has not yet been completely resolved. As secondary plastids, plastid gene phylogeny should mirror that of nuclear genes, while incongruence in the two phylogenies should be anticipated if their origin was as tertiary plastids. We have analyzed the phylogeny of plastid-encoded genes from Lingulodinium as well as that of nuclear-encoded dinoflagellate homologues of plastid-encoded genes conserved in all other plastid genome sequences. Our analyses place the dinoflagellate, stramenopile, haptophyte, and cryptophyte plastids firmly in the red algal lineage, and in particular, the close relationship between stramenopile plastid genes and their dinoflagellate nuclear-encoded homologues is consistent with the hypothesis that red algal-type plastids have arisen only once in evolution.     

Glyceraldehyde 3-Phosphate Dehydrogenases and Glyoxylate Reductase: I. Their Regulation Under Continuous Red and Far Red Light in the Cotyledons of Sinapis alba L

The development of NADP- and NAD-dependent glyceraldehyde 3-phosphate dehydrogenase and NADH-specific glyoxylate reductase was followed in Sinapis alba cotyledons grown in the dark or under continuous red and far red light. All three enzyme activities are promoted by light, continuous far red light being more than twice as effective as continuous red light. The activities of the NADP-glyceraldehyde 3-phosphate dehydrogenase and glyoxylate reductase increase in the far red light from 36 to 96 hours. They remain constant until at least 120 hours after sowing and are respectively 11 and 6 times higher than the maximum dark activities. Contrary to this, the activity of the NAD-glyceraldehyde 3-phosphate dehydrogenase is scarcely more than doubled under continuous far red irradiation relative to its maximal dark level, and its time course curve is displaced along the time axis, with the activity increasing between 24 and 72 hours after sowing.