Magnesium deficiency results in accumulation of carbohydrates and amino acids in source and sink leaves of spinach

Accumulation of assimilates in source leaves of magnesium‐deficient plants is a well‐known feature. We had wished to determine whether metabolite concentrations in sink leaves and roots are affected by magnesium nutrition. Eight‐week‐old spinach plants were supplied either with a complete nutrient solution (control plants) or with one lacking Mg (deficient plants) for 12 days. Shoot and root fresh weights and dry weights were lower in deficient than in control plants. Mg concentrations in deficient plants were 11% of controls in source leaves, 12% in sink leaves and 26% in roots, respectively. As compared with controls, increases were found in starch and amino acids in source leaves and in sucrose, hexoses, starch and amino acids in sink leaves, whereas they were only slightly enhanced in roots. In phloem sap of magnesium‐deficient and control plants no differences in sucrose and amino acid concentrations were found. To prove that sink leaves were the importing organs they were shaded, which did not alter the response to magnesium deficiency as compared with that without shading. Since in the shaded sink leaves the photosynthetic production of metabolites could be excluded, those carbohydrates and amino acids that accumulated in the sink leaves of the deficient plants must have been imported from the source leaves. It is concluded that in magnesium‐deficient spinach plants the growth of sink leaves and roots was not limited by carbohydrate or amino acid supply. It is proposed that the accumulation of assimilates in the source leaves of Mg‐deficient plants results from a lack of utilization of assimilates in the sink leaves.     

Role of ethylene in senescence of watercress leaves

Detached watercress leaves showed a rapid senescence rate as compared with other herbs. It was therefore of interest to investigate the role of ethylene in the rapid senescence of watercress leaves, and So estimate the efficacy of various inhibitors of elhylene synthesis (aminoethoxyvinylglycine, AVG) and action (CO₂, Ag⁺) in retarding senescence processes. The progress of senescence in watercress bunches (leaves attached to cut stems) and in detached leaves was estimated by measuring the rate of chlorophyll (Chl) loss, proteolysis and lipid oxidation. Evidence is presented showing that application of 11% CO₂ to watercress bunches in a flow-through system had a long-lasting effect on senescence, exhibited by highly efficient retardation of all the senescence processes tested. On the other hand, application of AVG (O.1 m M ) or Ag-(30 μ M ) to detached leaves affected Chl loss much more than prciteolysis. These results suggest that the senescence-retarding activity of CO₂ cannot be attributed solely to its action as an anli-ethylene agent and that not all senescence-associated processes are regulated by ethylene.     

Effect of long-term H2S fumigation on photosynthesis in spinach. Correlation between CO2 fixation and chlorophyll a fluorescence

Spinach plunts (Spinacia oleracea L. cv. Monosa) were exposed to air with and without 0.25 μl l^-1 H₂S. Effects of H₂S exposure for up to 18 days on photosynthesis, dark respiration and on chlorophyll a fluorescence were studied. Dark respiration was not affected by H₂S fumigation. Photosynthetic CO₂ fixation decreased linearly with time in both control and fumigated plants. The rate of decrease in CO₂ fixation was faster in the fumigated plants; after 14 days of exposure the fumigated plants showed a decrease in CO₂ fixation of 23%äs compared with the control plants. The H₂S-induced decrease in CO₂ fixation was accompanied by a decrease in quenching of the chlorophyll fluorescence. The most characteristic change in chlorophyll fluorescence was a decreased difference between maximum and steady-state fluorescence [(P-T)/P), suggesting a reduced efficiency in the use of photochemical energy in photosynthesis. Differences in CO₂ fixation were more pronounced whcn measured at high light intensity; the maximum rate of CO₂ fixation at light saturation decreased significantly with time in the H₂S-exposed plants; after 14 days of H₂S exposure a decrease of more than 70% was noted. The decrease in CO₂ fixation could not be attributed to a decreased chlorophyll content; on the contrary, chlorophyll content even slightly increased during fumigation. The initial increase in CO₂ fixation rate with increasing light intensity was also reduced by prolonged H₂S fumigation, indicating an effect of H₂S fumigation on photosynthetic electron transport. Finally, the phytotoxicity of H₂S is discusscd in relation to the H₂S-induced changes in photosynthetic CO₂ fixation and chlorophyll a fluorescence, and the effect of H₂S on leaf development observed in earlier studies.     

Molecular cloning,characterization and expression of cDNA encoding phosphoserine aminotransferase involved in phosphorylated pathway of serine biosynthesis from spinach

Phosphoserine aminotransferase (PSA) catalyzes the conversion of phosphohydroxypyruvate to phosphoserine in the phosphorylated pathway of serine biosynthesis. A cDNA clone encoding PSA was isolated from the cDNA library of spinach (Spinacia oleracea L.) green leaves. Determination of the nucleotide sequence revealed the presence of an open reading frame encoding 430 amino acids, exhibiting 38-50% homology with the amino acid sequences of bacterial, yeast and animal PSA. It contains an N-terminal extension of ca. 60 amino acids in addition to the sequences from other organisms. The general features of plastidic transit peptide are observed in this N-terminal sequence, suggesting the plastid localization of the PSA protein encoded by this cDNA. The bacterial expression of the cDNA could functionally rescue the auxotrophy of serine in the serC- mutant, Escherichia coli KL282. The enzymatic activity of PSA was demonstrated in vitro in the extracts of E. coli over-expressing the cDNA. Southern blot analysis indicated the presence of a couple of related genes (Psa) in the spinach genome. RNA blot hybridization suggested the preferential expression of the Psa gene in the roots of green seedlings and in the suspension cells cultured under a dark condition.     

Photoinhibition at chilling temperatures and effects of freezing stress on cold acclimated spinach leaves in the field. A fluorescence study

The role of high light stress in a natural environment was studied on spinach plants ( Spinacia oleracea L. cv. Wolter) grown in the field during the winter season. Fluorescence induction (at 293 K and 77 K) of leaves was used to characterize the stress effects. Night frost with minimum temperatures between – 1.5°C and –7.5°C (i.e. above the'frost killing point'at ca. –11.5°C) led to impaired photosynthesis. This was seen as increased initial fluorescence (F_o), decreased ratio of variable to maximum fluorescence (F_V/F_M) and lowered rates of O₂ evolution. The freezing injury was reversible within several frostless days. Exposure to high light (about 900 mol m_–2 s_–1) at chilling temperatures in the field caused photoinhibition, manifested as decreased variable fluorescence (F_V) and F_V/F_M ratio without changes in F_O. The photoinhibitory fluorescence quenching was not stronger after frost than after frostless nights; synergism between light stress and preceding freezing stress was not observed. Fluorescence induction signals at 77 K showed that F_V of photosystems I and II decreased to the same extent, indicating increased thermal deactivation of excited chlorophyll. Photoinhibition was fully reversible at +4°C within 1 h in low light, but only partially in moderate light. Preceding night frosts did not affect the recovery. The photoinhibition observed here is regarded as a protective system of thermal dissipation of excess light energy.     

Isolation of plasma membrane and tonoplast fractions from spinach leaves by preparative free-flow electrophoresis and effect of photoinduction

A membrane fraction enriched in plasma membrane and tonoplast vesicles was isolated from green leaves of Spinacia oleracea L. and subjected to subfractionation by free-flow electrophoresis. The most electronegative membrane vesicle fraction collected after the free-flow electrophoretic separation was identified as derived from tonoplast, while the least electronegative fraction was identified as derived from plasma membrane. The identification of the fractions was based on membrane morphology, and on the presence or absence of biochemical markers. The plasma membrane fraction was enriched in thick (9–11 nm) membranes which bound N-1-naphthylphthalamic acid (NPA), and reacted with phosphotungstic acid at low pH on thin sections for electron microscopy. The tonoplast fraction was enriched in vesicles with 7–9 nm thick membranes that neither bound NPA nor reacted with phosphotungstic acid at low pH. Both the plasma membrane and the tonoplast fraction were about 90% pure, with a cross-contamination of not more than 2%. Membrane vesicles originating from dictyosomes, endoplasmic reticulum, mitochondria, plastids, or peroxisomes contaminated the plasma membrane and the tonoplast fractions by a few % only. In leaves of photoinduced plants (24 h light period), the plasma membranes were thicker than in control leaves (8 h light, 16 h dark). The plasma membrane fraction obtained from photo-induced leaves by free-flow electrophoresis retained this increase in thickness, showing not only that photoinduction alters plasma membrane structure, but also that this change is stable to isolation.     

Freezing injury in spinach leaf tissue: Effects on water-soluble proteins, protein-sulfhydryl and water-soluble non-protein-sulfhydryl groups

Freezing of spinach leaf discs ( Spinacia aleracea L. cv. Estivato) resulted in an irreversible and parallel loss of protein-sulfhydryl (SH) and water-soluble protein. This decrease was inversely related to the increase in freezing injury as determined by the loss of electrolytes from the tissue after thawing. Loss of proteins and protein-SH occurred during freezing of the tissue and was not enhanced by thawing. The parallel decreases in content of soluble proteins and SH groups make it impossible to determine whether oxidation of protein-SH groups is the primary step in decline of protein content. During freezing the content of non-protein-SH compounds, mainly glutathione (GSH), was decreased to a lesser extent than that of protein-SH. Contrary to protein-SH, the levels of non-protein-SH declined substantially after thawing. The data indicate that GSH is not directly involved in protection of soluble proteins against freezing-induced denaturation.     

Lipid composition of plasma membranes from barley leaves and roots, spinach leaves and cauliflower inflorescences

Highly purified plasma membranes (PM) were obtained from barley (Hordeum vulgare L. cv. Kristina) leaves and roots, spinach (Spinacia oleracea L. cv. Viking II) leaves, and cauliflower (Brassica oleracea) inflorescences by partitioning in an aqueous polymer two-phase system. The sterol and polar lipid composition of the PM, including the fatty acid composition of the glycerolipids, was determined. Dominating lipids were free sterols, glucocerebroside, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), although large variations in content were observed between the PM of the different species and organs. Thus, the spinach leaf PM contained only 7% (mol %) free sterol compared to over 30% free sterol in the other PM analysed, with the barley root PM as the other extreme (57% free sterol). On the other hand, sterol derivatives were more abundant in the spinach leaf PM, containing 13% acylated sterol glycosides. Cerebroside constituted 16% of the lipids in the barley leaf PM but only 3% in cauliflower. The phospholipids PC and PE ranged from 25 and 24%, respectively, in the spinach leaf PM to 8 and 7%, respectively, in the barley root PM. As a result of the large variations in sterol and phospholipid content, the ratio of free sterol to phospholipid varied from 2.2 in the barley root PM to only 0.1 in the spinach leaf PM. Sitosterol, campesterol and stigmasterol were the completely dominating sterols in the barley and cauliflower PM, whereas the unique sterol composition of spinach was dominated by spinasterol. Palmitic (16:0), linoleic (18:2) and linolenic (18:3) acid were the major glycerolipid fatty acids. The fatty acid composition of the barley root PM was the most saturated (44% 16:0, 13% 18:3), whereas that of the cauliflower PM was the most unsaturated (21% 16:0,42% 18:3). Thus, very large variations were observed in both total lipid and fatty acid composition of the PM investigated, which represent both mono— and dicotyledons, as well as both photosynthetic and non-photosynthetic tissue. The consequences of this large diversity in composition of the lipid bilayer for the function of integral PM proteins are discussed.     

Compartmentation of phenylacetic and cinnamic acid synthesis in spinach

Applying labelled phenylalanine or tyrosine to purified intact spinach chloroplasts, only the corresponding phenylacetic acids but not the cinnamic acids could be detected. The addition of mercaptoethanol or dl -dithiothreitol and the variation of light conditions had only a slight effect. However, cinnamic acids could be found together with phenylacetic acids in leaf homogenates indicating the presence of phenylalanine and/or tyrosine ammonia lyase outside the spinach chloroplasts. Similar results were obtained with barley leaf homogenates, where cinnamic acids were the main products. Reviewing recent findings on amino acid synthesis in spinach leaves, it may be concluded that the synthesis of aromatic amino acids is restricted to the chloroplast, whereas the metabolism of secondary aromatic compounds is predominantly localized outside the chloroplasts.     

Comparison of properties of leaf and root glutathione reductases from spinach

Glutathione reductase (GR; EC 1.6.4.2) was purified from spinach roots (rGR) to homogeneity in terms of SDS-PAGE, and its properties were compared with those of the enzyme from spinach leaves (IGR). The two enzymes had similar native molecular (118000) and subunit masses (58000) and immunochemical properties, but different pH optima (ca pH 7.8 for IGR, ca pH 7.2 for rGR) and amino acid compositions. Peptide maps of two GRs showed that they differed from each other. The N-terminal amino acid of the IGR was glycine and that of the rGR was blocked. The partial amino acid sequence of the N-terminal region of the IGR was determined to the 11 th residue and it was found that the sequence of 8 amino acids of the IGR had 100% homology with that of the putative chloroplast GR from Arabidopsis and pea.     

Promotive effect of jasmonates on the senescence of detached maize leaves

Promotion of senescence of detached maize leaves by jasmonates was investigated. Senescence of detached maize leaves was promoted by linolenic acid, the precursor of biosynthesis of jasmonic acid, and retarded by inhibitors of lipoxygenase, the first enzyme in the biosynthetic pathway of jasmonic acid. Results support a role of endogenous jasmonates in the regulation of senescence of detached maize leaves. Silver thiosulfate, an inhibitor of ethylene action, was found to inhibit methyl jasmonate, linolenic acid- and abscisic acid-promoted senescence of detached maize leaves. It seems that jasmonate-promoted senescence is mediated through an increase in ethylene sensitivity in detached maize leaves.Abbreviations ABA abscisic acid - MJ methyl jasmonate - STS silver thiosulfate     

Salicylic acid inhibits the biosynthesis of ethylene in detached rice leaves

The effects of salicylic acid (SA) on ethylene biosynthesis in detached rice leaves were investigated. SA at pH 3.5 effectively inhibited ethylene production within 2 h of its application. It inhibited the conversion of ACC to ethylene, but did not affect the levels of ACC and conjugated ACC. Thus, the inhibitory effect of SA resulted from the inhibition of both synthesis of ACC and the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - SA salicylic acid     

Effect of one night with "low light'on uptake, reduction and storage of nitrate in spinach

During the night, shoot nitrate concentration in spinach (Spinacia oleracea L. cv. Vroeg Reuzenblad) increased due to increased uptake of nitrate by the roots. When the plants were subjected to a one night “low light’period at 35 μmol m^?2 s^?1, the shoot nitrate concentration did not increase and was reduced by 25% compared to control plants in the dark. The major contribution to this decrease was located in the leaf blades, where the nitrate concentration was decreased by 60%, while the petiole nitrate concentration decreased by only 9%. Nitrate accumulated in the leaf blade vacuoles during a dark night, but this was not the case during the “low light’period. This decrease in vacuolar nitrate concentration, compared to control plants in the dark, was not caused by increased amounts of leaf blade nitrate reductase (NR; EC 1.6.6.1). During a “low light’night period, the cytoplasmic soluble carbohydrate concentration was increased compared to the control plants in the dark. Calculations showed in situ NR activity to be higher than in the control plants in the dark. This increase in NR activity, however, was not large enough to account for the total difference found in the shoot nitrate concentration. Net uptake of nitrate by the roots was increased during the initial hours of the dark night, while vacuolar nitrate concentration in the leaf blades increased at the same time. During the “low light’night period, however, net uptake of nitrate by the roots did not increase, and vacuolar nitrate concentration did not change. We conclude that nitrate uptake by the roots and vacuolar nitrate concentration in the leaf blades are tightly coupled. The decreased shoot nitrate concentration is mainly caused by a reduction in net uptake of nitrate by the roots. During the “low light’night period, carbohydrates and malic acid partly replaced vacuolar nitrate. A “low light’period one night prior to harvest provides a valuable tool to reduce shoot nitrate concentrations in spinach grown in greenhouses in the winter months.     

Cysteine, γ-glutamyl-cysteine and glutathione contents of spinach leaves as affected by darkness and application of excess sulfur. II. Glutathione accumulation in detached leaves exposed to H2S in the absence of light is stimulated by the supply of glycine to the petiole

When illuminated leaf discs and detached leaves of spinach ( Spinacia oleracea L. cv. Estivato) were exposed to 0.4 and 0.25 μl 1^-1 H₂S, respectively, pool sizes of cysteine and glutathione increased. In the dark, apart from these compounds, the level of γ-glutamyl-cysteine also increased. Incubation of leaf discs with 1.0 m M buthionine sulfoximine (BSO) resulted in the accumulation of cysteine only, both in the light and in darkness. When glycine was supplied to the petioles of detached leaves exposed to H₂S in the dark, the accumulation of glutathione was stimulated, while γ-glutamyl-cysteine accumulation was prevented completely. Glycolate and glyoxylate, precursors of glycine in the glycolate pathway, had nearly the same effect as glycine. Although other amino acids were apparently taken up equally well as glycine when supplied to the petiole, they were much less effective, or not effective at all, in restoring glutathione synthesis in the dark. These results provide evidence, that H₂S-induced glutathione accumulation in spinach leaves in the dark is limited by the availability of glycine, giving rise to the accumulation of the metabolic precursor γ-glutamyl-cysteine.     

Enzymes of nitrogen mobilization in detached leaves of Lolium temulentum during senescence

During the senescence of Lolium temulentum leaf sections in the dark, asparagine and glutamine accumulated as the level of soluble protein declined. During the first 3–4 days after detachment, when the rate of protein loss was maximal, a four-fold increase in acid protease activity (EC 3.4.4.?) occurred. Subsequently this activity was replaced by proteases with a higher pH optimum. There was also a pronounced and continued activation of glutamate dehydrogenase (EC 1.4.1.2) during senescence. Glutamate pyruvate transaminase (EC 2.6.1.2), benzoylarginine-p-nitroanilide hydrolase (EC 3.4.?.?) and leucyl-p-nitroanilide hydrolase (EC 3.4.1.1) declined from high initial activities after 3–4 days. Glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1) was fairly stable although a marked increase occurred in the activity of one of two major GOT isoenzymes over the first two days. Glutamine synthetase (EC 6.3.1.2) was highly active in non-senescent leaves but fell sharply during the first three days of senescence. Little asparagine synthetase (EC 6.3.1.1) was detected. The role of these enzymes in the nitrogen metabolism of senescent detached leaves is discussed.     

The effects of external electric field on the electron transport system of spinach chloroplasts

Since the thylakoid membranes of an active chloroplast are constantly exposed to the electric fields generated by the electron transport system inside the membranes, we have studied the effects of pretreating chloroplasts of spinach ( Spinacia oleracea L.) leaves with an external AC (alternating current) electric field on their electron transport system. It was found that a few minutes electric field pretreatment (333 V cm^-1 across chloroplast samples), especially at low frequency, irreversibly inhibited the activity of photosystem II (PSII), but under certain conditions, stimulated that of photosystem I (PSI). From the measurements of fluorescence from PSII, we ascribe the inhibition to a lesion close to its reaction center P680, leading to increased dissipation of excitation energy to heat. The effect on PSI was investigated by the reduction of its reaction center, P700 by various artificial donors. We suggest that the stimulative effect can be attributed to a positive shift of the surface charge density of thylakoid membranes that brings about an increase in the accessibility of exogenous electronegative donors.     

Glycinebetaine biosynthesis and its control in detached secondary leaves of spinach

In secondary leaves from spinach plants pretreated in vermiculite for 24 h with 300 mM NaCl, glycinebetaine accumulated at a rate of circa 0.16 mol 100 g^-1 Chl d^-1 (2 mol g^-1 FW d^-1), about three times the rate of control plants. The soluble carbohydrate and free amino acid contents did not increase significantly following salinisation until after 4 d when the relative growth rate also decreased. Leaf proline levels remained very low throughout the experimental period. K⁺ on a tissue water basis remained constant at 200 mM while Cl^- and Na⁺ levels increased linearly to reach 175 and 100 mM respectively after 5 d of saline treatment. The osmotic pressure of leaf tissue also increased from 300 to 500 mosmol kg^-1. These experimental conditions were considered suitable to study glycinebetaine biosynthesis and its induction by salinity in the absence of marked growth inhibition or metabolic disturbance. Radioactive labelled [¹⁴C]serine, ethanolamine and choline (all 1 mol, 13.3 MBq in 10 l) were fed to detached secondary leaves via the petiole 24 h after the exposure of plants to salt. The rate of isotope incorporation into water soluble products, lipids and residue was measured over a further 24 h. The major metabolic fate of exogenous [¹⁴C]choline and [¹⁴C]ethanolamine was incorporation into glycinebetaine while less ¹⁴C-label was found in phosphatidyl choline and phosphatidyl ethanolamine. Incorporation rates were identical in control and salinised leaves and were adequate to account for observed values of glycinebetaine accumulation previously reported in spinach. In contrast the labelling of glycinebetaine from [¹⁴C]serine was twice as great in salinated plants as in the controls. These results, together with short term labelling experiment with [¹⁴C]ethanolamine using leaf slices, were consistent with the formation of glycinebetaine via serine, ethanolamine and its methylated derivatives to choline with some control being exerted at the serine level. However a flux through the phosphorylated intermediates is not excluded.From a consideration of these results and the published data on barley subjected to water stress (Hanson and Scott, 1980 Plant Physiol. 66, 342–348) there appear to be significant differences in the biosynthetic pathways in spinach and barley.Abbreviations BHT butylated hydroxytoluerte (2,6-di-tert-butyl-4-methylphenol) - C₁ one-carbon fragment - 1,2DG diglyceride moiety - DW day weight - MCW methanol-chloroform-water (12:5:1, by vol.) - PA phosphatidic acid - PC phosphatidyl choline - PMME phosphatidyl monomethylethanolamine - PDME phosphatidyl dimethylethanolamine - PE phosphatidyl ethanolamine - PPO 2,5-diphenyloxazole - POPOP 1,4-bis(5-phenyloxazoyl) benzene     

Influence of a cyanobacterial crude extract containing microcystin-LR on the physiology and antioxidative defence systems of different spinach variants

Plants can be contaminated with cyanobacterial toxins during spray irrigation of lake water containing toxic cyanobacteria. Here, long-term effects of cyanobacterial crude extract (containing microcystin-LR) on the growth and physiology of different spinach (Spinacia oleracea) variants under semifield conditions were investigated. Changes in antioxidative enzyme activities, and in glutathione, ascorbate and tocopherol contents were investigated to assess the reaction of the antioxidative defence systems in spinach to toxin exposure. In addition to severe morphological effects, such as growth inhibition and chlorosis, the generation of oxidative stress was observed at the cellular level. In response to the negative effects of oxidative stress, plants stimulated an antioxidative system consisting of an enzyme network with superoxide dismutases, peroxidases, catalases, glutathione S-transferases and glutathione reductases, as well as a set of low-molecular-weight antioxidants, including glutathione, ascorbate and tocopherols. Exposure of spinach to cyanobacterial crude extract affected germination, growth and morphology, as well as antioxidative response parameters. Different variants of the same plant reacted in different ways to certain toxicants.     

Profiles of photosynthetic oxygen-evolution within leaves of Spinacia oleracea

Oxygen evolution was measured from mesophyll tissues in spinach leaves using a photoacoustic technique. The photosynthetic capacity of individual cell layers was measured by directing microscopic beams of light, 40 μm wide, to cells exposed within a leaf cross section. The resulting profile for oxygen-evolution potential was relatively flat, indicating a uniform capacity for photosynthesis in leaf mesophyll tissues. Two experimental approaches were used to estimate the photosynthetic performance of individual mesophyll cell layers when white light was applied to the adaxial leaf surface. These experiments indicated that oxygen was produced relatively uniformly across the mesophyll and that oxygen evolution increased with irradiance of the white light applied to the leaf surface. The measured profiles for oxygen evolution and capacity are flatter than previous measurements of profiles of fixed carbon and estimates of profiles for absorbed light within spinach leaves.     

Role of light in the in vivo and in vitro synthesis of spinach thioredoxin f

Upon continuous illumination of dark-grown spinach ( Spinacia oleracea L. cv. Winter Giant) seedlings, the thioredoxin f (Td f) content (ELISA) showed a steep rise, which can be evaluated after 3 and 36 h illumination as 3 times and 10 times the dark value, respectively. These figures correspond to 0.03% and 0.1% of total soluble protein, which means a higher biosynthetic rate for Td f compared to the average of total proteins in the earlier steps of plant development. After 40-50 h light the Td f level reached its highest value which remained stable for an additional 40 h and then decreased. Pulse-chase in vivo experiments with [³⁵S]-methionine also showed this sharp increase of Td f in the dark-light transition. From the pattern of decay of [³⁵S]-labelled Td f, a half-life of 7 h was determined for this chloroplast protein. In vitro translation experiments with poly(A)-mRNA isolated from illuminated young spinach seedlings, coupled to a wheat-germ synthesizing system, showed the appearance of a labelled fraction of ca 19 kDa molecular mass, recognizable by a specific Td f antiserum. When intact spinach chloroplasts were added to the translation assay medium, and then illuminated, the 19 kDa band disappeared, with a parallel increase of an internalized 13 kDa labelled polypeptide, also recognized by the Td f antiserum. These results are good evidence for a nuclear-coded synthesis of a Td f precursor, which travels through the chloroplast envelope, leaving the functional protein inside the organelle after the loss of a 6 kDa transit peptide.