Analysis of plant phosphoproteins.

Chromatography supports to purify phosphorylated proteins (P-proteins) have become available recently, yet this has not been thoroughly investigated in the case of plant materials. In this study we used a commercial affinity matrix (Qiagen) and a test plant enzyme (phosphoenolpyruvate carboxylase PEPC). The malate test and gel blot experiments probed with a specific antibody (antiphosphorylated N-terminal domain) showed that the column efficiently binds P-PEPC from Sorghum with little or no contamination by non-P-PEPC. Similar results were obtained with the low-abundance PEPC of Arabidopsis leaves when a gel filtration step (Sephadex G-200) was performed prior to the chromatography. Three-dimensional mass spectrometry analysis of immunoprecipitated PEPC in Qiagen fractions confirmed this observation. Denaturing protein extraction by cold acetone/trichloroacetic acid of fixed material led to a complete, one-step separation of P-PEPC and non-P-PEPC. At a global scale, the column captured most of the (32)P-phosphate-labeled proteins in vivo (80%), the majority of which were subsequently found in the elution fraction (88%). This was also visualized by SDS-PAGE (1D and 2D gels) followed by Pro-Q diamond staining. Analysis of the P-protein fraction by 1D gels and liquid chromatography/tandem mass spectrometry allowed the identification of 250 proteins belonging to various functional categories. These results validate the method for in vitro/in vivo studies of native/denatured individual proteins/enzymes regulated by phosphorylation and for phosphorylome studies.

Stress Induction of Mitochondrial Formate Dehydrogenase in Potato Leaves

In higher plants formate dehydrogenase (FDH, EC 1.2.1.2.) is a mitochondrial, NAD-dependent enzyme. We previously reported that in potato (Solanum tuberosum L.) FDH expression is high in tubers but low in green leaves. Here we show that in isolated tuber mitochondria FDH is involved in formate-dependent O2 uptake coupled to ATP synthesis. The effects of various environmental and chemical factors on FDH expression in leaves were tested using the mitochondrial serine hydroxymethyltransferase as a control. The abundance of FDH transcripts is strongly increased under various stresses, whereas serine hydroxymethyltransferase transcripts decline. The application of formate to leaves strongly enhances FDH expression, suggesting that it might be the signal for FDH induction. Our experiments using glycolytic products suggest that glycolysis may play an important role in formate synthesis in leaves in the dark and during hypoxia, and in tubers. Of particular interest is the dramatic accumulation of FDH transcripts after spraying methanol on leaves, as this compound is known to increase the yields of C3 plants. In addition, although the steady-state levels of FDH transcript increase very quickly in response to stress, protein accumulation is much slower, but can eventually reach the same levels in leaves as in tubers.

Identification of a major soluble protein in mitochondria from nonphotosynthetic tissues as NAD-dependent formate dehydrogenase.

In many plant species, one of the most abundant soluble proteins (as judged by two-dimensional polyacrylamide gel electrophoresis) in mitochondria from nongreen tissues is a 40-kD polypeptide that is relatively scarce in mitochondria from photosynthetic tissues. cDNA sequences encoding this polypeptide were isolated from a lambda gt11 cDNA expression library from potato (Solanum tuberosum L.) by screening with a specific antibody raised against the 40-kD polypeptide. The cDNA sequence contains an open reading frame of 1137 nucleotides whose predicted amino acid sequence shows strong homology to an NAD-dependent formate dehydrogenase (EC 1.2.1.2) from Pseudomonas sp. 101. Comparison of the cDNA sequence with the N-terminal amino acid sequence of the mature 40-kD polypeptide suggests that the polypeptide is made as a precursor with a 23-amino acid presequence that shows characteristics typical of mitochondrial targeting signals. The identity of the polypeptide was confirmed by assaying the formate dehydrogenase activity in plant mitochondria from various tissues and by activity staining of mitochondrial proteins run on native gels combined with antibody recognition. The abundance and distribution of this protein suggest that higher plant mitochondria from various nonphotosynthetic plant tissues (tubers, storage roots, seeds, dark-grown shoots, cauliflower heads, and tissues grown in vitro) might contain a formate-producing fermentation pathway similar to those described in bacteria and algae.

Variation of the polypeptide composition of mitochondria isolated from different potato tissues.

The protein contents of mitochondria from different potato (Solanum tuberosum L.) tissues (tubers, dark-grown shoots, and green leaves) grown in a greenhouse or in vitro were compared by two-dimensional polyacrylamide gel electrophoresis. Two different methods were used: using the method that gave the highest resolution, an average number of 360 polypeptides was revealed on the mitochondrial patterns after silver staining. The mitochondrial protein patterns of etiolated tissues (tubers, dark-grown shoots) are roughly similar but distinct from those of green leaves. The four subunits of the glycine decarboxylase complex (involved in photorespiration) and a few other polypeptides are very abundant in green tissues, compared with nonphotosynthetic tissues. Conversely, some other polypeptides that are abundant in tubers and dark-grown shoots are hardly detectable in green leaf mitochondria. A rabbit antiserum was raised against a 40 kilodalton polypeptide that is among the most characteristic of these nonphotosynthetic tissue-specific polypeptides, and the N-terminal sequence of this polypeptide was determined. No effect of in vitro culture was observed on the protein composition of mitochondria isolated from differentiated tissues. However, the protein patterns of callus and cell suspension mitochondria are distinct from those of any differentiated tissues, although their basic pattern is clearly mitochondrial.

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones.

Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines of Nicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes, ms1, ms2 and ms3, while no changes can be seen in the mitochondrial genome. However, differences were found between the in organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.

Two dimensional analysis of chloroplast proteins from normal and cytoplasmic male sterile Brassica napus.

Stromal and thylakoid proteins isolated from normal (N) and cytoplasmic male sterile (cms) lines of Brassica napus have been compared using a two dimensional gel separation. It has been shown that: 1) stromal compartments of the two lines were very similar; 2) although there was extensive homology between protein maps of thylakoids isolated from the two lines, these could be distinguished by the spots corresponding to the ß subunits of the coupling factor CF1 from the ATPase complex.

Comparative macromolecular analysis of the cytoplasms of normal and cytoplasmic male sterile Brassica napus.

Chloroplast (cp) and mitochondrial (mt) compartments of normal (N) and cytoplasmic male sterile (cms) lines of Brassica napus have been characterized and compared on the basis of cp and mt DNA restriction enzyme analysis and in vitro protein synthesis by isolated mitochondria. Cytoplasmic male sterility of B. napus (rape) comes from cms Raphanus sativus (radish) through intergeneric crosses.Cp DNAs isolated from N and cms lines had distinct restriction patterns with Sal I, Kpn I and Sma I enzymes. The size of the two cp DNAs measured from the restriction patterns was found to be identical and of about 95 × 10(6) d. N and cms lines of B. napus were characterized by specific mt DNAs, as shown from Sal I, Kpn I, Pst I and Xho I cleavage patterns. The small number of well-separated restriction fragments obtained with Sal I enabled us to determine precisely mt DNA sizes. The values of 136.5 and 140.3 × 10(6) d, obtained from restriction patterns with N and cms DNAs respectively, are smaller than any of those previously obtained from studies on other genera. With molecular hybridization experiments, it was possible to distinguish N and cms lines by the different locations of rRNA genes on the cp and mt DNAs.Two lines of B. napus are characterized by specific mt translation products formed in isolated mitochondria.

Functional and Structural Organization of Chlorophyll in the Developing Photosynthetic Membranes of Euglena gracilis Z: II. CHARACTERISTICS OF THE LATE FORMATION OF ACTIVE PHOTOSYSTEM II REACTION CENTERS DURING FIRST STAGES OF GREENING.

During light-induced greening of dark-grown, nondividing Euglena gracilis Z, there is a delay of about 10 hours in the formation of active photosystem II (PSII) reaction centers compared to chlorophyll synthesis. Experiments with greening under different light intensities rule out the possibility that this delay results from a late induction of active PSII reaction center formation when a definite amount of chlorophyll is attained in the early greened cells. Experiments on greening after preillumination show that this delay does not originate in a long, light-induced formation of specific synthesizing machinery for reaction center components. Experiments with greening in the presence of streptomycin show that, when this inhibitor of protein synthesis by chloroplastic ribosomes is added to dark-grown, preilluminated cells or to cells already greened for 24 hours, the formation of active PSII reaction centers is inhibited after a time which depends on the light intensity used for greening. Under very low light intensity (150 lux), the addition of streptomycin to 24-hour greened cells does not prevent further development of functional chloroplasts. These observations lead to the conclusion that streptomycin-insensitive chloro-plast development occurs due to syntheses of cytoplasmic origin and of light-induced pools of components synthesized early by chloroplastic ribo-somes. Conformational changes requiring time may allow the insertion of components necessary for the reorganization of PSII reaction centers in the developing thylakoid after synthesis. This hypothesis accounts for the observed delay in PSII reaction center formation compared to chlorophyll synthesis.

Functional and Structural Organization of Chlorophyll in the Developing Photosynthetic Membranes of Euglena gracilis Z: III. SEQUENCE OF EVENTS LEADING TO THE FORMATION OF FUNCTIONAL PHOTOSYSTEM II PHOTOSYNTHETIC UNITS.

Greening cells of Euglena were transferred back to darkness at different stages of chloroplast development in the presence or absence of specific inhibitors of protein synthesis. The analysis of chloroplast components showed that: (a) cycloheximide or streptomycin does not significantly inhibit the formation in darkness of active photosystem II (PSII) reaction centers if added after the lag phase for chloroplast development; (b) a limited number of active reaction centers are formed in the dark, sufficient to increase PSII reaction center to chlorophyll ratios to values close to those found in fully greened cells; (c) these dark-formed reaction centers appear to be inserted in already constituted and complete light-harvesting antennae. These results complement previous ones and lead us to propose a model for a sequential formation of PSII photosynthetic units during greening of Euglena, whereby conformational changes requiring time would allow already synthesized components of PSII reaction centers to be inserted or reorganized as active photochemical complexes in association with previously formed light-harvesting antennae.