Chloroplast Development in Etiolated Peas: Reformation of Prolamellar Bodies in Red Light without Accumulation of Protochlorophyllide

Chloroplast development and accumulation of chlorophylls werestudied in etiolated peas (Pisum sativum L., cv. Green feast)exposed for 24 h to one of three intensities of red light orto a corresponding intensity of white light producing a similarhigh medium or low terminal chlorophyll content. Chloroplastdevelopment was assessed by counts of grana and partitions pergranum in electron micrographs. Chlorophylls were partitionedand protochlorophyllide measured in the unphytylated fraction.More sensitive estimates of protochlorophyllide were made byfluorescence. In the high- and medium-intensity red-light treatmentsconsiderable reformation of prolamellar bodies took place duringdevelopment. This occurred at a stage when no protochlorophyllidewould be detected. In the low-intensity treatments reformationwas accompanied by an accumulation of protochlorophyllide ata higher level than that obtaining in the dark.     

Herbivory and Calcium Concentrations Affect Calcium Oxalate Crystal Formation in Leaves ofSida (Malvaceae)

Calcium oxalate crystals have potential roles in plants as partof a defence mechanism against herbivores and/or in accumulatingexcess calcium. To date, these potential roles have been studiedindependently. In this experimental study the effects of calciumlevels and herbivory on the production of calcium oxalate crystals(i.e. druse, spherical crystal aggregates) were examined inseedlings of Sida rhombifolia. Seedlings were subjected to threecalcium levels (low, normal or high) and an artificial herbivorytreatment. Calcium levels and herbivory both affected densityof crystals in leaves. Leaves from seedlings grown in low calciumhad a greater crystal density than those grown in high calcium.Leaves from seedlings subjected to herbivory had a greater crystaldensity than those from seedlings not subjected to herbivory.This study provides additional evidence that calcium oxalatecrystal production depends not only on calcium levels but canalso be influenced by external pressures such as herbivory.In addition to their physiological role in plants, these resultssuggest that calcium oxalate crystals can also act as a defencemechanism against herbivores. Copyright 2001 Annals of BotanyCompany Calcium concentrations, calcium oxalate crystals, herbivory, Malvaceae, Sida rhombifolia     

Identification of the chlB Gene and the Gene Product Essential for the Light-Independent Chlorophyll Biosynthesis in the Cyanobacterium Plectonema boryanum

We cloned a 6.0-kb HindIII fragment from the cyanobacteriumPlectonema boryanum using the chloroplast chlB (ORF513) geneof the liverwort (Marchantia polymorpha) as a probe. An openreading frame (ORF508) encoding a polypeptide of 508 amino acidresidues was found within the nucleotide sequence of the 4,437-bpHindIII-EcoRV subfragment. The deduced amino acid sequence ofORF508 shows very high similarity to that encoded by the liverwortchlB gene (72.7%). A mutant, YFB14, in which ORF508 was inactivatedby the insertion of a kanamycin-resistance cartridge, was unableto synthesize chlorophyll, accumulating protochlorophyllidein darkness while synthesizing chlorophyll normally in the light.Thus, the chlB gene is the third gene that is essential forthe light-independent reduction of protochlorophyllide. Theother two genes are chlL and chlN, and the results suggest thatthe light-independent protochlorophyllide reductase consistsof at least three subunits, which are encoded by chlL, chlNand chlB. Using an antiserum prepared against a ChlB-6xHis fusionprotein expressed in Escherichia coli, we detected a proteinwith an apparent molecular weight of 58,000 in the membranefraction of the cyanobacterium. These results indicate thateither the cytoplasmic or thylakoid membranes could be the siteof the light-independent reduction of protochlorophyllide. (Received November 16, 1995; Accepted February 7, 1996)     

Photoconversion of protochlorophyll to chlorophyll a in a mutant of Chlorella regularis

Dark-grown cells of a mutant strain of Chlorella regularis containedchlorophyll a and protochlorophyll, phytyl ester of protochlorophyllide.Under illumination, protochlorophyll was quantitatively anddirectly converted into chlorophyll a. The photoconversion wasdependent on light intensity and temperature and proceeded ina cell-free preparation. The pathway of chlorophyll formation found in the mutant cellsis entirely different from that from protochlorophyllide byway of chlorophyllide a, which is generally observed in greenplants. ¹Present address: Division of Biology, Medical College of Miyazaki,Miyazaki 889-16, Japan. ²Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Ibaragi 300-21, Japan. (Received October 24, 1975; )     

Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in the Liverwort, Marchantia paleacea var. diptera

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase(EC 1.6.99.1 [EC] ) was isolated from suspension-cultured cells ofthe liverwort, Marchantia paleacea var. diptera. In contrastto the situation in most higher plants, the liverwort gene wasexpressed in a light-dependent manner. ²Present address: Department of Biological Science, Facultyof Science, Kumamoto University, Kurokami, Kumamoto, 860-8555Japan.     

The Effect of Calcium Starvation on Assimilate Partitioning and Mineral Distribution of the Phloem

Populus plants were grown in a medium lacking calcium and exposedto ¹⁴CO₂. In contrast to plants in the complete nutrient medium,the percentage amount of ¹⁴C-assimilates increased in the leavesof calcium-deficient plants and decreased in the stem and theroots. When plants were grown without potassium or magnesiumno differences in the amount of ¹⁴C-label occurred in comparisonwith plants in the complete nutrient medium. Translocation wasrecorded by microautoradiography. It was observed that considerableamounts of labelled photoassimilates were unloaded from thephloem in the middle part of the stem in plants of the completenutrient medium. In contrast, during calcium starvation ¹⁴C-labelwas restricted to the phloem of the stem. In addition, the concentrationsof magnesium and phosphorus showed a remarkable increase instem sieve tubes of calcium-deficient plants. When sieve tubesof source leaves from Populus, barley and maize were comparedwith those of sink leaves, the latter showed higher calciumconcentrations. The results suggest that calcium is a necessaryfactor in the regulation of phloem translocation. Key words: Calcium deficiency, phloem translocation, sieve element loading and unloading, X-ray microanalysis     

Chloroplast biogenesis 51 : modulation of monovinyl and divinyl protochlorophyllide biosynthesis by light and darkness in vitro

It is shown that the monovinyl and divinyl protochlorophyllide biosynthetic patterns of etiolated maize (Zea mays L.), and cucumber (Cucumis sativus L.) seedlings and of their isolated etiochloroplasts can be modulated by light and darkness as was shown for green photoperiodically grown plants (E. E. Carey, C. A. Rebeiz 1985 Plant Physiol. 79: 1-6). In etiolated corn and cucumber seedlings and isolated etiochloroplasts poised in the divinyl protochlorophyllide biosynthetic mode by a 2 hour light pretreatment, darkness induced predominantly the biosynthesis of monovinyl protochlorophyllide in maize and of divinyl protochlorophyllide in cucumber. When etiolated seedlings and their isolated etiochloroplasts were poised in the monovinyl protochlorophyllide biosynthetic mode by a prolonged dark-pretreatment, light induced mainly the biosynthesis of divinyl protochlorophyllide in both maize and cucumber.     

Separation and Characterization of Prolamellar Bodies and Prothylakoids from Squash Etioplasts

Intact etioplasts of squash cotyledons, which had been preparedby Percoll density gradient centrifugation, were ruptured hypotonicallyin the presence of deoxyribonuclease I then fractionated intoprolamellar bodies and prothylakoids by differential and Percolldensity gradient centrifugations. This procedure provided ahighly purified prolamellar body fraction that was composedmainly of a 36,000-dalton protein. This protein was identifiedas NADPH:protochIorophyllideoxidoreductase [Ikeuchi and Murakami(1982) Plant & Cell Physiol. 23: 1089]. The fraction alsohad a high content of protochlorophyllide that absorbed at 648nm and its NADPH:protochlorophyllide oxidoreductase had highactivity. When the fraction was illuminated, a chlorophyllidethat absorbed at 684–685 nm formed. In contrast, the prothylakoid fraction, which showed high activityfor the Ca²⁺-dependent ATPase of coupling factor 1, containedonly a small amount of the 36,000-dalton protein and showedvery low NADPH:protochlorophyllide oxidoreductase activity.The protochlorophyllide content of this fraction also was low,and the ratio of protochlorophyll to protochlorophyll(ide) high.The absorption peak in the prothylakoids was at 633–635nm, and after a brief illumination a chlorophyllide that absorbedat 672–673 nm formed. These results indicate that thephotoactive protochlorophyllide-NADPHreductase complex in etioplastsis concentrated in the prolamellar body and that the physicalstate of protochlorophyll(ide) in the prolamellar body differsfrom that of the prothylakoid. (Received April 28, 1982; Accepted November 15, 1982)     

The Dependence of Calcium Transport and Leaf Tipburn in Strawberry on Relative Humidity and Nutrient Solution Concentration

Strawberry plants were grown in controlled-environment cabinetswith different day-and-night relative humidities, in nutrientsolutions of different osmotic potential and different calciumconcentrations. Leaf calcium (% d. wt.) depended strongly on leaf age and waslowest and very sensitive to environment when the leaf was emergingfrom the bud. Calcium in the emergent leaf was greatest andtipburn least when plants were grown in humid nights (VPD usually< 100 Pa) and weak nutrient solutions (osmotic potentialabout –25 kPa). Such plants guttated freely. In contrastplants grown in dry nights (VPD, c. 600 Pa) never guttated,had small concentrations of calcium in emergent leaves and sufferedtipburn. The behaviour of plants transferred between humiditytreatments rapidly reflected the new conditions. Increasingthe osmotic potential of the nutrient increased tipburn anddecreased calcium in emergent leaves even though the nutrientcontained more calcium. When the calcium concentration in the emergent leaf exceeded0.07 per cent of d. wt, tipburn was never seen; below 0.05 percent tipburn was usually severe. These results suggest that pre-emerged, and therefore non-transpiring,leaves depend for their calcium on water flow arising from rootpressure at night. After leaf emergence, calcium intake intoleaves was promoted by dry days, indicating that calcium wasthen supplied by transpirational water flow. Humid nights, drydays and weak nutrient solutions minimize the risk of leaf tipburnin strawberry. Fragaria ananassa Duch., strawberry, tipburn, calcium transport, relative humidity, nutrient supply, guttation     

Characterization and Properties of a Protochlorophyllide Ester in Leaves of Dark Grown Barley with Geranylgeraniol as Esterifying Alcohol

The protochlorophyllide ester isolated from dark grown barley leaves was shown to contain geranylgeraniol as esterifying alcohol. No phytylester was found. The qualitative analyses were performed with combined gas chromatography-mass spec-trometry. Chromatographic separation and spectrofluorometric determination of the protochlorophyll and chlorophyll pigments before and after irradiation of the dark grown leaves with light flashes at 2°C showed that part of the protochlorophyllide ester was photoconverted to chlorophyll a.     

Temperature dependent reduction of protochlorophyllide in darkness followed by the assembly of active photosystems in pigment mutant C-2A' of Scenedesmus obliquus

In the wild type of Scenedesmus obliquus strain D3 grown heterotrophically, the chlorophyll biosynthesis and thus the reduction of protochlorophyllide to chlorophyllide takes place in darkness. However, in pigment mutant C-2A' of Scenedesmus obliquus only traces of protochlorophyllide are reduced under optimal growth conditions in darkness. By lowering the growth temperature from 33° to 15–25°C, protochlorophyllide can be reduced in darkness. At 20°C this process is about 10 times more active than at 33°C, but reaches only about 13% of the light-dependent chlorophyll biosynthesis. The chlorophylls synthesized at the lower temperatures are inserted into the pigment-protein complexes and photosystem I as well as photosys-tem II capacities are developed. The rate of light-independent protochlorophyllide reduction at lower temperatures is not limited by the enzyme PChlide-oxidoreductase itself, but rather by its substrate, being in turn limited by the amount of 5-amino levulinic acid (ALA) available.     

Effects of elevated carbon dioxide on three grass species from montane pasture II. Nutrient uptake, allocation and efficiency of use

Agrostis capillaris L.⁴ Festuca vivipara L. and Poa alpinaL.were grown in outdoor open-top chambers at either ambient (340µmol mol^–1) or elevated (680 µmol^–1)CO² for periods from 79 to 189 d. Under these conditions thereis increased growth of A. caplllarls and P. alpina, but reducedgrowth of F. vivipara. Nutrient use efficiency, nutrient productivity(total plant dry weight gain per unit of nutrient) and nutrientallocation of all three grass species were measured in an attemptto understand their individual growth responses further andto determine whether altered nutrient-use efficiencies and productivitiesenable plants exposed to an elevated atmospheric CO₂ environmentto overcome potential limitations to growth imposed by soilfertility. Total uptake of nutrients was, in general, greater in plantsof A. capillaris and P. alpina (with the exception of N andK in the latter) when grown at 680 µmol mol^–1 CO₂.In F. vivipara, however, uptake was considerably reduced inplants grown at the higher CO₂ concentration. Overall, a doubling of atmospheric CO₂ concentration had littleeffect on the nutrient use efficiency or productivity of A.capillaris. Reductions in tissue nutrient content resulted fromincreased plant growth and not altered nutrient use efficiency.In P. alpina, potassium, magnesium and calcium productivitieswere significantly reduced and photosynthetic nitrogen and phosphorususe efficiencies were doubled at elevated CO₂ with respect toplants grown at ambient CO₂ F. vivipara grown for 189 d showedthe most marked changes in nutrient use efficiency and nutrientproductivity (on an extracted dry weight basis) when grown atelevated CO₂, F. vivipara grown at elevated CO₂ however, showedlarge increases in the ratio of non-structural carbohydrateto nitrogen content of leaves and reproductive tissues, indicatinga substantial imbalance between the production and utilizationof assimilate. Key words: Nutrient, allocation, nutrient use efficiency, grasses, nutrient productivity, elevated CO₂, cliniate change     

Physiological disturbances caused by high rhizospheric calcium in the calcifuge Lupinus luteus

A detailed study of the calcifuge Lupinus Iuteus L. (yellowlupin) has been carried out in an attempt to explain its poorperformance in the presence of high concentrations of rhizosphenccalcium. Plants were grown on two different calcium regimes,1 or 15 mol m^– Ca and, after an establishment period,measurements were made of the rate of leaf extension, finallength of the leaflets and the leaf gas exchange. In addition,the distribution of calcium within the leaf tissue was investigated. At 15 mol m^–3 Ca, leaflet length at full expansion wasreduced as a consequence of reduced extension rate and a declinein cell wall extensibility. Transpiration in excised leaves,assayed gravimetrically, was significantly reduced in plantsgrown in high calcium. Similar results were also obtained fromgas exchange measurements. Analysis of A/C, curves indicatedthat in plants grown in high [Ca] there was a substantial reductionin net assimilation over a range of concentrations of CO₂ X-raymicroanalysis revealed that a large amount of cal cium deliveredin the xylem sap is retained in the mesophyll tissue, and mostof that reaching the epidermal tissue is not found in the guardcells but in the cells adjacent to them, which in this speciesare not anatomically distinct as ‘subsidiary’ cells. Key words: Calcium, calcifuge, Lupinus luteus, stomata, leaf growth     

Changes in the Distribution of Magnesium, Potassium, Calcium and Phosphorus during Growth of Cucurbita Seedlings

Ockenden, I. and Lott, J. N. A. 1988. Changes in the distributionof magnesium, potassium, calcium and phosphorus during growthof Cucurbita seedlings—J. exp. Bot. 39: 973–980. Etiolated seedlings of Cucurbita maxima cv. Warted Hubbard andCucurbita andreana, grown in distilled water, showed considerablegrowth. However, only a small percentage of the minerals storedin the large cotyledons was mobilized to the root-shoot axisof the etiolated seedlings. The proportion of minerals removedfrom the cotyledons to the root-shoot axis was about two-timeshigher in C. andreana than in C. maxima. When the seedlingswere grown in distilled water in the presence of light, thepercentage of cotyledon mineral content that was mobilized waslower than the amount mobilized during growth in the dark. Cotyledonsof light-grown seedlings that were given adequate nutrients,took up minerals from the medium. These fully-expanded and photosyntheticcotyledons had slightly more P and Mg than did the cotyledonsof the dry embryos but had increased their K content 5-foldand their Ca content 50-fold. Key words: Cucurbita, minerals, seeds, seedings, NAA     

Properties of Alkaline Phosphatase in Cucumber Roots Induced by Calcium Starvation

Calcium deficiency caused an increase in alkaline phosphataseactivity in cucumber roots [Matsumoto and Yamaya (1981) Plant& Cell Physiol. 22: 1137]. The activities of other hydrolasesincluding acid phosphatase, nucleases and proteases, however,were much less affected by the removal of calcium. Nucleosidedi- and triphosphates and inorganic pyrophosphate were effectivelyhydrolyzed by the induced alkaline phosphatase, whereas nucleosidemonophosphate-hydrolyzing activity was basically equal in theroots grown with either complete medium or a medium lackingcalcium. The alkaline phosphatase in cucumber roots was foundin fractions pelleting at 3,000 x g and in the 100,000 x g supernatant.The calcium-starved roots increased their alkaline phosphataseactivity in both fractions. Four isozyme bands of the alkalinephosphatase in the soluble fraction were separated by polyacrylamidegel electrophoresis. One of the isozyme bands showed a prominentincrease with the calcium deficiency, but not in the presenceof cycloheximide. (Received June 24, 1981; Accepted September 3, 1981)     

Separation of Protochlorophylls Esterified with Different Alcohols from Inner Seed Coats of Three Cucurbitaceae

Reversed-phase high-performance liquid chromatography was usedto separate protochlorophyllide esters isolated from inner seedcoats of three Cucurbitaceae. At least 17 protochlorophyllsesterified with different alcohols were separated on an octadecylsilica (ODS) column from purified pigments of pumpkin (Cucurbitamoschata) and balsam pear (Momordica charantica) seeds witha single elution using 100% methanol within 22 min. The separationwas done according to the numbers of carbon atoms in the esterifyingalcohol of the protochlorophyll molecule with high resolutionand reproducibility. Gas chromatographic analysis of the pigment-esterifyingalcohols confirmed the presence of a number of protochlorophyllsesterified with different alcohols. This method permits rapid,efficient and quantitative detection and identification of chlorophyllsin complex mixtures. (Received May 17, 1982; Accepted September 6, 1982)     

Transformation of Protochlorophyllide, Formed from Exogenous δ-Aminolevulinic Acid, in Continuous Light and in Flashlight

δ-Aminolevulinic acid supplied to dark grown isolated leaves or wheat causes an accumulation of protochlorophyllide which is only partly transformed to chlorophyllide α in continuous light At the same time a considerable photodestruction of both pigments takes place. By a suitable combinations of short lights flashes and dark periods it is possible, however, to obtain at least double the amount of the protochlorophyllide transformed without photodestruction. The transformation isshown to be dependent on the dark interval between the light flashes. Possible connections with the formation of the protein part of the protochlorophyllide holochrome are discussed.     

The Effect of Ions on Growth and Leaf Succulence of Atriplex hortensis var. cupreata

When plants of Atriplex hortensis var. cupreata were grown insolutions containing at least 100 mmoll^–1 of various saltsit was found that sodium was the ion most effective in stimulatingleaf succulence, irrespective of whether the anion was chloride,bromide or sulphate. Potassium was the next most effective ion.Magnesium and calcium were without effect. Dry weight productioncould be greatly reduced by sodium and potassium but stimulatedby magnesium. The results are compared with those previouslyreported for C₄ species of Atriplex.     

Temperature inducible protochlorophyllide reduction in darkness in a pigment mutant of Scenedesmus obliquus

Accumulation of chlorophyll and protochlorophyllide (PChlide) was followed during beterotrophic growth of the pigment mutant C-2A' of Scenedesmus obliquus L. in the darkness at 30 and 20°C. At 30°C the cells remained yellow with accumulation of protochlorophyllide, whereas they became green at 20°C with only traces of protochlorophyllide. The capacity of mutant cells to reduce PChlide to chlorophyllide (Chlide) in the dark with or without addition of 5-aminolevulinic acid as measured in isolated membranes, was high in cells grown at 20°C but negligible at 30°C. The high capacity to reduce PChlide created in cells growing at 20°C was only slightly diminished by exposure of cells to 38°C for 3 h. Mechanisms of temperature-sensitive chlorosis in algae and higher plants are discussed in relation to the results with pigment mutant C-2A' of Scenedesmus obliquus . It is assumed that either an activator of NADPH protochlorophyllide oxidoreductase (EC 1.6.99.1) or a different enzyme system can be activated by lower temperature as by light.