David J. Simpson

Chlorophyll-proteins of thylakoids from wild-type and mutants of barley (Hordeum vulgare L.)

A sodium dodecyl sulphate-polyacrylamide gel electrophoresis system is described that resolves wild-type barley thylakoid components into ten chlorophyll-containing bands, plus free chlorophyll. Many of these bands have been characterised with respect to their absorption spectra and polypeptide composition. The identity and probable function of eight bands were established by comparison of the wild-type pattern with those of isolated light-harvesting chlorophylla/b-protein complex and a number of nuclear gene mutants of barley. It was found that the mutantchlorina-f2, which lacks chlorophyllb, contains only three chlorophylla-proteins designated Chla-P1, Chla-P2 and Chl3-P3 according to a new system of nomenclature proposed in this paper. Chla-P1 contains the reaction centre of photosystem I (P700) and with the aid of the mutantviridis-m29, Chla-P3 was deduced to be the most likely site of the reaction centre (P680) of photosystem II.Two chlorophylla/b-proteins were found, which differed in theira/b ratios and which are thought to play a role in light-harvesting or light-focusing. These were designated Chla/b-P1 and Chla/b-P2, the latter being found in multimeric complexes designated Chla/b-P2*, Chla/b-P2** and Chla/b-P2***, in order of increasing apparent molecular weight. In addition to these eight bands, there were two chlorophyll-containing bands in the wild-type pattern that were not Chla/b-P2 complexes and which were called Chl-P bands until further characterised.

A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants

We propose a nomenclature for the genes encoding the chlorophylla/b-binding proteins of the light-harvesting complexes of photosystem I and II. The genes encoding LHC I and LHC II polypeptides are namedLhca1 throughLhca4 andLhcb1 throughLhcb6, respectively. The proposal follows the general format recommended by the Commision on Plant Gene Nomenclature. We also present a table for the conversion of old gene names to the new nomenclature.

A homogenizer with replaceable razor blades for bulk isolation of active barley plastids

A modification of the cutting device of a kitchen homogenizer is described which allows the preparation of biochemically active greening barley plastids. The new cutting device consists of four easily replaceable razor blades. Intact plastids are isolated from the immature leaves of spinach or from greening barley leaves with a yield of 10% and from etiolated barley with a yield of 6%.

Freeze-fracture studies on barley plastid membranes. III. Location of the light-harvesting chlorophyll-protein

The thylakoids of chloroplasts from wild-type barley and the nuclear gene mutantchlorina-f2 were examined by freeze-fracturing and rotary shadowing. The advantages of rotary shadowing were: the shape of freeze-fracture particles was revealed, particles on membrane surfaces were seen in greater detail, estimation of particle density was more reliable, and measurements could be made of membrane thickness. When wild-type andchlorina-f2 thylakoid ultrastructure were compared, the most significant difference was the large reduction in the number of particles on the PFs face of the mutant. In addition, EFs particles were about 12% smaller in the mutant, whereas the appearance of the EFu, PFu and PSu was substantially the same as for wild-type thylakoids. These ultrastructural differences are correlated with the complete absence of the light-harvesting chlorophylla/b-protein fromchlorina-f2 thylakoids. Freeze-fracturing of purified light-harvesting complex in vesicles revealed particles similar in size and shape to those missing from the PFs face ofchlorina-f2 but present on the PFs face of wild-type. It is concluded that in wild-type granal thylakoids, the light-harvesting chlorophylla/b-protein is located in particles which are closely associated with EFs particles, but which cleave with the PFs face during freeze-fracturing.

Down-regulation of the PSI-F Subunit of Photosystem I (PSI) in Arabidopsis thaliana THE PSI-F SUBUNIT IS ESSENTIAL FOR PHOTOAUTOTROPHIC GROWTH AND CONTRIBUTES TO ANTENNA FUNCTION

The PSI-F subunit of photosystem I is a transmembrane protein with a large lumenal domain. The role of PSI-F was investigated in Arabidopsis plants transformed with an antisense construct of the psaF cDNA. Several plant lines with reduced amounts of the PSI-F subunit were generated. Many of the transgenic plants died, apparently because they were unable to survive without the PSI-F subunit. Plants with 5% of PSI-F were capable of photoautotrophic growth but were much smaller than wild-type plants. The plants suffered severely under normal growth conditions but recovered somewhat in the dark indicating chronic photoinhibition. Photosystem I lacking PSI-F was less stable, and the stromal subunits PSI-C, PSI-D, and PSI-E were present in lower amounts than in wild type. The lack of PSI-F resulted in an inability of light-harvesting complex I-730 to transfer energy to the P700 reaction center. In thylakoids deficient in PSI-F, the steady state NADP+reduction rate was only 10% of the wild-type levels indicating a lower efficiency in oxidation of plastocyanin. Surprisingly, the lack of PSI-F also gave rise to disorganization of the thylakoids. The strict arrangement in grana and stroma lamellae was lost, and instead a network of elongated and distorted grana was observed.

The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum

The ricinosome (synonym, precursor protease vesicle) is a novel organelle, found so far exclusively in plant cells. Electron microscopic studies suggest that it buds off from the endoplasmic reticulum in senescing tissues. Biochemical support for this unusual origin now comes from the composition of the purified organelle, which contains large amounts of a 45-kDa cysteine endoprotease precursor with a C-terminal KDEL motif and the endoplasmic reticulum lumen residents BiP (binding protein) and protein disulfide isomerase. Western blot analysis, peptide sequencing, and mass spectrometry demonstrate retention of KDEL in the protease proform. Acidification of isolated ricinosomes causes castor bean cysteine endopeptidase activation, with cleavage of the N-terminal propeptide and the C-terminal KDEL motif. We propose that ricinosomes accumulate during senescence by programmed cell death and are activated by release of protons from acidic vacuoles.

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment

Abstract. A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863–871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.).

Chlorophyll-proteins of two photosystem I preparations from maize

Two photosystem I (PSI) preparations were purified by non-denaturing SDS-PAGE or sucrose gradient ultracentrifugation and examined as to their chlorophyll-protein composition. In both preparations a minimum of two chlorophyll-proteins can be distinguished in addition to the 110 kDP-700 Chla-P1 complex. One (LHCI-730) is a chlorophyll-protein (Mr 40 kD) having a high chlorophylla/b ratio, and a major 77 K fluorescence peak at 730 nm. It consists of three polypeptides with apparent molecular weights of 21, 22.5 and 24 kD. Another chlorophyll-protein (LHCI-680) with a lower molecular weight (Mr 25 kD) fluoresces at 77 K with a maximum at 680 nm. This chlorophyll-protein has a high chlorophyllb content and two constituent polypeptides of 20 and 25 kD. Absorption, 77 K fluorescence and circular dichroism spectra of the PSI related chlorophyll-proteins are presented and compared with those of photosystem II chlorophyll-proteins from maize thylakoids. We propose a model for energy transfer in the photosystem I reaction centre with the following sequence: LHCI-730→LHCI-680 →Chla-P1. LHCI-680 acts as a connecting antenna which can also transfer energy from Chla/b-P2. This model was used to interpret the 77 K fluorescence emission from two barley mutants.

Macromolecular physiology of plastids XIV.Viridis mutants in barley: Genetic, fluoroscopic and ultrastructural characterisation

A sample of 42viridis mutants of barley has been localised by diallelic crosses to 32 nuclear genes. They have been grouped into five different categories on the basis of their fluorescence induction kinetics; this was used as a rapid method for the determination of their photosynthetic capacities. The mutants within each category were further characterised by low temperature fluorescence emission spectroscopy, chlorophyll content, visible fluorescence under UV light, viability, and chloroplast ultrastructure as seen by thin-section electron microscopy. The photosystem I-type mutants have a high initial fluorescence, variable fluorescence, but no fluorescence decline. They fluoresce under UV light and are seedling lethals, but in general have well-developed thylakoid systems. Photosystem II-type mutants, which have a high initial fluorescence, no variable fluorescence, and no fluorescence decline, fluoresce brightly under UV light and are all seedling lethals. Their chloroplast ultrastructure is characterised by giant grana, but as a group, their chlorophyll content is low. The nature of the photosynthetic defects of mutants in the third category is not known, but they are unusual in having a steady-state fluorescence lower than the initial fluorescence. Many of these mutants have a near-normal chloroplast ultrastructure, high chlorophyll content, and are viable under favourable conditions. The fourth group of mutants have fluorescence induction kinetics similar to those of the wild-type, and they are not considered to have major photosynthetic defects, as might be expected from the fact that many will survive under suitable growth conditions. The fifth category contained mutants with extremely low levels of chlorophyll under the growth conditions used.The low temperature fluorescence emission peak at long wavelengths is in part due to antennae chlorophyll of photosystem I. In the mutants deficient in photosystem I, this peak was present, but its wavelength was shifted from 739 nm (wild-type) to 729–731 nm. However, photosynthetically competent mutants with reduced amounts of chlorophyll also showed similar or greater wavelength shifts, and it is concluded that at present, fluorescence emission spectra can not be used to predict the nature of the photosynthetic defects. Similarly, it was not possible to discern a usable relationship between chloroplast ultrastructure and specific photosynthetic deficiencies.

Changes in the organization of stroma membranes induced by in vivo state 1-state 2 transition

Abstract Stroma lamellae were isolated from mesophyll thylakoids of maize plants exposed to dark (state 1) and high-intensity white light (state 2). Freeze-fracture electron microscopy established that the preparations consisted of small, spherical, right-side-out vesicles, and that the extent of contamination by grana appreassed lamellae was less than 0.1%. The state 1–state 2 transition resulted in a decrease in the chlorophyll a/b ratio of the isolated stroma lamellae due to lateral migration of light-harvesting chlorophyll a/b protein of Photosystem II (LHC II) from appressed grana lamellae. Fluorescence spectroscopy showed that this ‘mobile LHC II’ became functionally attached to the Photosystem I reaction centre, with a resulting 15% increase in antenna size, as determined by measurement of the chlorophyll/P-700 ratio and kinetics of P-700 photo-oxidation under light-limiting conditions. The mobile LHC II could be resolved by gel electrophoresis into four polypeptides with an approximate 1:1:1:1 stoichiometry. This differed significantly from the polypeptide composition of grana LHC II, which had a different stoichiometry, and an additional polypeptide of lower apparent molecular mass which did not become phospharylated. The freeze-fracture particle density data showed that the phosphorylated LHC II was not visible as discrete small particles, as found in appressed grana membranes, and may have become incorporated into the large PF particles of the stroma lamellae, which contain the Photosystem I reaction centre.