Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : II. Formation from Glutamate

Intact plastids from greening maize (Zea mays L.) leaves converted [¹⁴C]glutamate and [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA). Glutamate appeared to be the immediate precursor of ALA, while KG was first converted to glutamate, as shown by the effect of various inhibitors of amino acid metabolism. Plastids from greening leaves contained markedly higher activity as compared with etioplasts or chloroplasts. The synthesis of ALA by intact plastids was light dependent. The enzyme system resides in the stroma of plastids or may be lightly bound to membranes. The solubilized system showed maximal activity around pH 7.9 and required Mg²⁺, ATP, and NADPH although dependence on the latter was not clear-cut. A relatively high level of activity could be extracted from etioplasts. Maximal activity was obtained from plastids of leaves which had been illuminated for 90 minutes, after which activity declined sharply. The enzyme system solubilized from plastids also catalyzed the conversion of putative glutamate 1-semialdehyde to ALA in a reaction which was not dependent on the addition of an amino donor.

The system in maize greatly resembled the one which had been reported from barley. It is suggested that this system is the one responsible for the biosynthesis of ALA destined for chlorophyll formation.

     

The Chloroplast Min System Functions Differentially in Two Specific Nongreen Plastids in Arabidopsis thaliana

The nongreen plastids, such as etioplasts, chromoplasts, etc., as well as chloroplasts, are all derived from proplastids in the meristem. To date, the Min system members in plants have been identified as regulators of FtsZ-ring placement, which are essential for the symmetrical division of chloroplasts. However, the regulation of FtsZ-ring placement in nongreen plastids is poorly understood. In this study, we investigated the division site placement of nongreen plastids by examining the etioplasts as representative in Arabidopsis Min system mutants. Surprisingly, the shape and number of etioplasts in cotyledons of arc3, arc11 and mcd1 mutants were similar to that observed in wild-type plants, whereas arc12 and parc6 mutants exhibited enlarged etioplasts that were reduced in number. In order to examine nongreen plastids in true leaves, we silenced the ALB3 gene in these Min system mutant backgrounds to produce immature chloroplasts without the thylakoidal network using virus induced gene silencing (VIGS). Interestingly, consistent with our observations in etioplasts, enlarged and fewer nongreen plastids were only detected in leaves of parc6 (VIGS-ALB3) and arc12 (VIGS-ALB3) plants. Further, the FtsZ-ring assembled properly at the midpoint in nongreen plastids of arc3, arc11 and mcd1 (VIGS-ALB3) plants, but organized into multiple rings in parc6 (VIGS-ALB3) and presented fragmented filaments in arc12 (VIGS-ALB3) plants, suggesting that division site placement in nongreen plastids requires fewer components of the plant Min system. Taken together, these results suggest that division site placement in nongreen plastids is different from that in chloroplasts.     

An ultrastructural study of maize leaf etioplasts throughout their entire life-cycle

Summary The entire life-cycle of maize leaf etioplasts has been followed. Prolamellar bodies with different types of tubular membrane arrangement can be found in the juvenile stages of the organelles, while in mature etioplasts nearly all the prolamellar bodies exhibit an hexagonal ring arrangement, which, by optical diffraction, appears to be the most regular and compact possible.The prothylakoid membranes also undergo changes during organelle differentiation, and their different organization and arrangement produce a clear dimorphism between the etioplasts of mesophyll and bundle sheath cells.In senescent etioplasts the prothylakoids are more affected, while the prolamellar bodies appear rather stable, also in situations where protochlorophyll(ide) content is very low. The formation of clusters of osmiophilic globules is coupled with the breakdown of the etioplast membranes.     

Light-mediated Oxygen Uptake Measured in Wheat Etioplasts

An in vitro O₂ assay was used to measure early response of wheat (Triticum aestivum L.) etioplasts to light. A transient photoinducible O₂ uptake occurred when dark-grown etioplasts were initially exposed to light. The rate of inducible O₂ consumption was dependent on both the intensity of light and the quantity of organelle protein present. Higher light intensities resulted in greater O₂ utilization per minute, and a greater quantity of organelle protein in the sample resulted in an increased rate of O₂ uptake under the same light intensity conditions. Experiments with various plant tissues as well as with mitochondrial respiratory inhibitors indicated that etioplasts are the organelles responsible for the photoinduced O₂ uptake phenomenon. A preliminary action spectrum study revealed that wavelengths 640 to 680 nm resulted in maximum O₂ uptake. This indicated the presence of an etioplast red light receptor pigment which induces O₂ uptake in etioplasts.     

Appearance of Membrane-bound Iron-Sulfur Centers and the Photosystem I Reaction Center during Greening of Barley Leaves

Dark-grown barley (Hordeum vulgare) etioplasts were examined for their content of membrane-bound iron-sulfur centers by electron paramagnetic resonance spectroscopy at 15K. They were found to contain the high potential iron-sulfur center characterized (in the reduced state) by an electron paramagnetic resonance g value of 1.89 (the “Rieske” center) but did not contain any low potential iron-sulfur centers. Per mole of cytochrome f, dark-grown etioplasts and fully developed chloroplasts had the same content of the Rieske center. During greening of etioplasts under continuous light, low potential bound iron-sulfur centers appear. In addition, the photosystem I reaction center, as measured by the photooxidation of P700 at 15K, also became functional; during greening the appearance of a photoreducible low potential iron-sulfur center paralleled the appearance of P700 photoactivity.     

Light-induced structural changes during incubation of isolated maize etioplasts

Summary Etioplasts were isolated from leaves of 9-day-old etiolated maize (Zea mays L.) seedlings and incubated in a relatively simple medium in light and in the dark. During the first 5 h no changes occurred in the fine structure of the isolated etioplasts in the dark. In light the size of the prolamellar bodies decreased and significantly more plastid sections without prolamellar bodies were counted. The total length of the thylakoids per plastid section increased, but there was no evidence for bi- and polythylakoid formation. It is concluded that light induces the structural transformation of the prolamellar body membranes into primary thylakoids also in isolated etioplasts.     

Studies on a Maize Mutant Sensitive to Low Temperature II. Chloroplast Structure, Development, and Physiology

In the Zea mays L. mutant M11 grown in the dark at 15°, the ultrastructure of the etioplast is abnormal. The pigment content of the etioplasts is reduced but the in vivo absorption characteristics suggest that the normal protochlorophyll (ide)-holochrome is present. The lowered synthetic ability of the etioplasts is not primarily due to a reduced complement of plastid ribosomes. The plastids of mutant M11 grown in the light at 15° contain little pigment, are markedly deficient in ribosomes and their ultrastructure is abnormal. In mutant M11 grown at 15°, an extreme sensitivity of the plastid membranes to light was observed.     

Ultrastructural changes in isolated plastids

Summary Etioplasts were isolated from maize leaves and the changes in their ultrastructure were followed in light and in darkness for several hours. It has been shown that the regular crystalline structures of prolamellar bodies, present after the isolation in darkness, disappear after 30 to 60 minutes of illumination, and long straight tubules appear within prolamellar bodies. Their appearance is influenced by the molarity of the isolation medium used, by light intensity, duration of illumination and by the temperature at which the isolates are kept. Long tubules appear, however, also in isolated etioplasts incubated for several hours in complete darkness.In isolates illuminated for 2–3 hours long tubules disappear again, and prolamellar bodies produced eventually consist of irregularly connected short tubules. In prolamellar bodies, regions with regular and very dense arrangement of tubules sometimes develop at this stage. The thylakoids (usually perforated) are now arranged concentrically in the plastids. True grana or poly-thylakoids can never be found in isolated etioplasts, not even when the etioplasts have been illuminated for 6 hours or more (up to 24).The present investigations have indicated that in isolated etioplasts in light, tubular elements, which build up the prolamellar bodies, cannot normally be transformed into thylakoids as is the case with intact tissue.The survival of isolated etioplasts is limited at present, and for this reason changes in their fine structure could be followed successfully for as long as 6 hours (in light at 15 °C), although a certain percentage of plastids survive up to 24 hours.     

Loss or retention of chloroplast DNA in maize seedlings is affected by both light and genotype

We examined the chloroplast DNA (cpDNA) from plastids obtained from wild type maize (Zea mays L.) seedlings grown under different light conditions and from photosynthetic mutants grown under white light. The cpDNA was evaluated by real-time quantitative PCR, quantitative DNA fluorescence, and blot-hybridization following pulsed-field gel electrophoresis. The amount of DNA per plastid in light-grown seedlings declines greatly from stalk to leaf blade during proplastid-to-chloroplast development, and this decline is due to cpDNA degradation. In contrast, during proplastid-to-etioplast development in the dark, the cpDNA levels increase from the stalk to the blade. Our results suggest that DNA replication continues in the etioplasts of the upper regions of the stalk and in the leaves. The cpDNA level decreases rapidly, however, after dark-grown seedlings are transferred to light and the etioplasts develop into photosynthetically active chloroplasts. Light, therefore, triggers the degradation of DNA in maize chloroplasts. The cpDNA is retained in the leaf blade of seedlings grown under red, but not blue light. We suggest that light signaling pathways are involved in mediating cpDNA levels, and that red light promotes replication and inhibits degradation and blue light promotes degradation. For five of nine photosynthetic mutants, cpDNA levels in expanded leaves are higher than in wild type, indicating that nuclear genotype can affect the loss or retention of cpDNA.     

Adenosine triphosphatase of bean plastids: its properties and site of formation

Extracts of bean (Phaseolus vulgaris L.) etioplasts and chloroplasts contain a dithiothreitol-activated Ca²⁺-dependent adenosine triphosphatase which is inhibited by Dio-9. The chloroplast and etioplast enzymes have identical R_F values upon disc gel electrophoresis. Optimum extraction of the enzyme from either plastid preparation is accomplished with 1 mm ethylenediamine tetraacetic acid. Photophosphorylation capacity can be partially restored to depleted chloroplast preparations by addition of either the chloroplast or etioplast extract. These results suggest that the adenosine triphosphatase from etioplasts and chloroplasts represents a modified coupling factor for photophosphorylation.     

Etioplast Development in Dark-grown Leaves of Zea mays L

The ultrastructure of etioplasts and the acyl lipid and the fatty acid composition of sequential 2-centimeter sections cut from the base (youngest) to the top (oldest) of nonilluminated 5-day-old etiolated leaves of Zea mays L., and the acyl lipid and fatty acid composition of the etioplasts isolated from them have been investigated. There is a 2.5-fold increase in the size of the plastids from the base to the tip of the leaf, and an increase both in the size of the prolamellar body and in the length of lamellae attached to it. The etioplasts in the bundle sheath and mesophyll cells of the older, but not the younger leaf tissue, are morphologically distinct. The monogalactosyl and digalactosyldiglycerides, phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol were the only detectable acyl lipids in the isolated etioplast fractions. Together with phosphatidylethanolamine these were also the major acyl lipids in the whole leaf sections. With increasing age of the leaf tissue, increases occurred in two of the major plastid lipids, monogalactosyldiglyceride and phosphatidylglycerol, while the levels of essentially nonplastid lipids remained constant or declined slightly. The monogalactosyldiglyceride to digalactosyldiglyceride ratio increased from 0.4 to 1.1 in the tissue sections of increasing age and from 0.7 to 1.2 in the etioplasts isolated from them. Similarly, the galactolipid to phospholipid ratio increased from 0.8 to 1.4 in the tissue and from 0.5 to 4.5 in the isolated plastids. In the latter, the proportions of phosphatidylglycerol (as a per cent of total phospholipid) increased from 20 to 41% with increasing age of plastids.

Linolenic acid was the major fatty acid in the total lipid of each of the etioplast fractions, but it was only the major fatty acid in the total lipid of the oldest leaf tissue. Its proportion in both total lipid extracts and individual lipids increased with age. The trans Δ³ hexadecenoic acid was absent from all lipids. The protochlorophyllide content of the tissue increased with age. The results are discussed in relation to the use of illuminated etiolated leaves for studying chloroplast development.

     

Prolamellar body and saponins: Avenacosides are not constituents of Avena etioplasts

Etiolated Avena leaf cells were homogenized, then fractionated into four fractions in the presence of salts by differential centrifugation, and intact etioplasts were prepared by Percoll or sucrose density gradient centrifugation. Using thin layer chromatography steroidal saponins, avenacoside A and B were found in the leaf cells and heavy cell fractions which were rich in other cell structures besides intact etioplasts, but not detected in the purified etioplasts. We concluded that saponins are not constituents of the prolamellar bodies in etioplasts.     

δ-Aminolevulinate dehydratase from Raphanus sativus cotyledons

Phytochrome induces δ-aminolevulinate dehydratase (ALAD) activity in radish seedling cotyledons under continuous far red light. Analysis of the enzymatic activity in etioplasts vs total activity shows a constant ALAD level in these organelles (10 %) in etiolated seedlings. In far red irradiated seedlings, the percentage of enzyme detected into etioplasts increases up to 45 % of the total. Comparative kinetic studies of ALAD activity detected in the cytoplasm and the etioplasts indicate an increase in both compartments with a maximum value reached respectively at 96 and 120 hr from sowing. Treatment with cycloheximide shows a very fast abolition of cytoplasmic ALAD activity which is always correlated to an etioplast decrease with a time shift of ca 24 hr. Erythromycin acts only on the cytoplasmic level of ALAD, and only for far red irradiated seedlings, with an increase of activity twice the level detected in untreated ones. This unexpected effect is discussed.     

Chloroplast maintenance and partial differentiation in vitro

Tissue homogenates, etioplasts, and developing chloroplasts were prepared from cucumber (Cumucis sativus L.) cotyledons in tris-sucrose. They were incubated aerobically in the dark or in the light at pH 7.7 in the presence or absence of a cofactor mixture containing coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate. These cofactors were previously shown to be essential for protochlorophyll and chlorophyll biosynthesis. Ultrastructural changes were monitored by electron microscopy. The following observations were made. (a) Crude homogenates contained agents which degraded etioplasts and developing chloroplasts. (b) Added cofactors were essential for the maintenance of the membrane structure; they were also implicated in the transformation of the prolamellar body in the absence and presence of light. (c) Light pretreatment of the cotyledons improved the maintenance of the developing chloroplast membranes during subsequent in vitro incubation. (d) In the presence of the cofactors, grana formation appeared to take place in the absence of nuclear-cytoplasmic control.     

Compartmentation of Aldolase Isoforms in Maize Leaves

The leaves of maize seedlings contain two principal isozymesof fructose 1,6-bisphosphate aldolase (E.C. 4.1.2.13 [EC] ), one chloroplasticand one cytosolic (Gasperini and Pupillo, 1982). Mesophyll protoplastswere separated from bundle sheath (BS) strands of both light-grownand dark-grown maize leaves. Aldolase isozymes were separatedfrom extracts of chloroplasts, etioplasts, protoplasts and BSstrands by column isoelectric focusing. The major isozyme ofgreen leaves (pI 4.2) was exclusively in BS chloroplasts, andthere was no evidence of other isozymes occurring in BS tissue.The cytosolic isozyme (pI 6.7) was present in protoplasts ofmesophyll cells, where it may limit the synthesis of hexose-phosphates(estimated activity of 9.4 µmol h^–1 g^–1 fr.wt.) together with lower activities of an acidic form (pI 4.6).Etiolated leaves contained significant amounts of the pI 6.7isozyme in both mesophyll and BS cells, but also minor activitiesof one or more acidic forms with pI values of 4.4–4.7(average pI 4.6) which appear to be located partly in BS etioplasts.The main developmental events for maize leaf aldolase afterillumination were a moderate decrease of cytosolic isozyme (pI6.7) which disappears from the BS within hours and a large,gradual increase of the BS plastid isozyme (pI 4.2). The isoformwith a pI 4.6 also increased rapidly to a low, steady activityin greening mesophyll protoplasts. Key words: C₄, fructose 1,6-bisphosphate, aldolase, Zea mays     

Expression of protein complexes and individual proteins upon transition of etioplasts to chloroplasts in pea (Pisum sativum)

The protein complexes of pea (Pisum sativum L.) etioplasts,etio-chloroplasts and chloroplasts were examined using 2D BlueNative/SDS–PAGE. The most prominent protein complexesin etioplasts were the ATPase and the Clp and FtsH proteasecomplexes which probably have a crucial role in the biogenesisof etioplasts and chloroplasts. Also the cytochrome b₆f (Cytb₆f) complex was assembled in the etioplast membrane, as wellas Rubisco, at least partially, in the stroma. These complexesare composed of proteins encoded by both the plastid and nucleargenomes, indicating that a functional cross-talk exists betweenpea etioplasts and the nucleus. In contrast, the proteins andprotein complexes that bind chlorophyll, with the PetD subunitand the entire Cyt b₆f complex as an exception, did not accumulatein etioplasts. Nevertheless, some PSII core components suchas PsbE and the luminal oxygen-evolvong complex (OEC) proteinsPsbO and PsbP accumulated efficiently in etioplasts. After 6h de-etiolation, a complete PSII core complex appeared with40% of the maximal photochemical efficiency, but a fully functionalPSII was recorded only after 24 h illumination. Similarly, thecore complex of PSI was assembled after 6 h illumination, whereasthe PSI–light-harvesting complex I was stably assembledonly in chloroplasts illuminated for 24 h. Moreover, a batteryof proteins responsible for defense against oxidative stressaccumulated particularly in etioplasts, including the stromaland thylakoidal forms of ascorbate peroxidase, glutathione reductaseand PsbS.     

Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity of Nuclei and Plastids from Etiolated Peas and their Response to Red and Far Red Light in Vivo

DNA-dependent RNA polymerase activity has been found in both the nuclei and etioplasts of dark-grown pea seedlings (Pisum sativum). Although these enzymes had similar over-all characteristics with respect to substrate, pH, and inhibitor responses, they could be distinguished by their different sensitivities to sonication.     

Localization of Galactolipid Biosynthesis in Etioplasts Isolated from Dark-Grown Wheat (Triticum aestivum L.)

Etioplasts were isolated from leaves of dark-grown wheat (Triticum aestivum L. var Starke II). Galactolipid biosynthesis was assayed in an envelope-rich fraction and in the fraction containing the rest of the etioplast membranes by measuring incorporation of ¹⁴C from uridine-diphospho[¹⁴C]galactose into monogalactosyl diacylglycerol and digalactosyl diacylglycerol. More than half of the galactolipid biosynthetic capability was found in the fraction of inner etioplast membranes. This fraction was subfractioned into fractions enriched in prolamellar bodies and membrane vesicles (prothylakoids), respectively. All membrane fractions obtained from etioplasts were able to carry out galactolipid biosynthesis, although the activity was very low in prolamellar body-enriched fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed markedly different polypeptide patterns between the different fractions. It is concluded that the capability of galactolipid biosynthesis of etioplasts probably is not restricted to the envelope, but is also present in the inner membranes of this plastid.     

Protein synthesis by isolated etioplasts and chloroplasts from pea and wheat and the effects of chloramphenicol and cycloheximide

Etioplasts capable of incorporating ¹⁴C-leucine into protein have been isolated from dark-grown pea and wheat plants. The requirements for leucine incorporation for etioplasts were similar to those for chloroplasts. An ATP-generating system, Mg²⁺, and GTP were required. The amino-acid-incorporation activity of etioplasts from wheat was comparable to that of chloroplasts on an RNA basis, whereas the activity of pea etioplasts was about 50% of the activity of pea chloroplasts. The incorporation of leucine into protein by etioplasts and chloroplasts from pea and wheat was inhibited by chloramphenicol, and to a slight extent by cycloheximide.