Pea leaf chloroplasts

LIGHT ACTIVATION OF MEMBRANE-BOUND FRUCTOSE-1.6-BISPHOSPHATASE FROM PEA LEAF CHLOROPLASTS. 

Isolated pea leaf chloroplasts v/ere devoid of m.itochondrial contamination as evaluated by Western blotting using specific anti-marker protein antibodies. 

Long-chain acylcarnitine + CoASH The presence of carnitine palmitoyltransferase (CPT) has been confirmed in pea leaf chloroplasts. It has been partially purified and some of its properties elucidated as previously published [3]. In brief, there is both an overt CPT activity (CPTo) bound to the outside (cytosol side) of the inner chloroplast envelope, and a latent CPT activity (GPTi), bound to the inside (stromal side) of the inner chloroplast envelope. This report concerns the possible function of these chloroplastic CPTs and their regulation in vivo. 

Anderson, L. E. Lim, T. and Park, K. (1974) Inactivation of pea leaf chloroplastic and cytoplasmic glucose 6-phosphate dehydrogenase by light and dithiothreitol. Plant Physiol. 53, 835-839. 

Few studies on the localization of the starch biosynthetic enzymes were done before 1978, when it was found that ADPGlc PPase was located exclusively in the chloroplast fraction in both spinach (Mares et al., 1978) and pea (Levi and Preiss, 1978). The first detailed study was done by Okita et al. (1979), in which spinach leaf chloroplasts were isolated either by differential centrifugation (Walker, 1971 see also later) or from protoplasts (Nishimura et al, 1976). These plastid preparations contained essentially all of the activity of the starch biosynthetic enzymes, ADPGlc PPase, starch synthase, and branching enzyme. Subsequently, in guard cells of Commelina communis, Robinson and Preiss (1987) showed that the starch biosynthetic enzymes were present exclusively in the chloroplast fraction. 

Foyer CH and Lelandais M (1996) A comparison ofthe relative rates of transport of ascorbate and glucose across the thylakoid, chloroplast and plasmalemma membranes of pea leaf mesophyll cells. J Plant Physiol 148 391-398 Foyer CH, Rowell J and Walker D (1983) Measurement ofthe ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157 239-244 Foyer CH, Furbank R, Harbinson J and Horton P (1990) The mechanisms contributing to photosynthetic control of electron transport by carbon assimilation in leaves. Photosynth Res 25 83-100... 

FIGURE 1. A - Interrelation of DP on P and S levels of induction and ferricyonide mediated electron transport of pea chloroplast at different light intensities. B - Interrelation of DP on S level of induction and steady-state chemges of light soattering at 520 nm of pea leaf. 

On the other hand, using fractionated pea leaf protoplasts and PercoU purified organelles, we have specifically localized HCS activity in three plant cell compartments [11]. Enzyme activity was mainly located in cytosol but significant activity was also identified in both chloroplast and mitochondrial soluble fractions (Table 2). 

Anderson, L.E. et al. (2005) Both chloroplastic and cytosolic phosphofructoaldolase isozymes are present in the pea leaf nucleus. Protoplasma 225,235-242... 

Fig. 2.17. Bottom left. Chloroplasts (P) in chlorenchyma of pea leaf. Note the large starch grains within the chloroplast (asterisks). IS, intercellular space V, vacuole. X 780. Right. Electron micrograph of a chloroplast of a leaf of spinach Spinacea oleracea). The chloroplast is surrounded by a double membrane (PM) and the internal membrane system is differentiated into grana (asterisks) and stroma lamellae (open arrows). Osmiophilic droplets (small black arrows) occur in the plastid stroma. The structure of the grana is shown in more detail in the inset top left) as are regions of continuity between the grana and stroma lamellae (large solid arrows). Key CM, cell membrane CW, cell wall SG, starch grain ...

According to Ladonin and Spesivtsev (1974), maize tolerant to chloro-5-triazines stores the triazine taken up in the cytoplasm and in the sensitive pea it is stored in the nucleus, chloroplasts and mitochondria. They assume that the triazines are thus incorporated into proteins and nucleic acids as antimetabolites during the biosynthesis of internuclear nucleic acids. This may also be one of the explanations of the different sensitivity of plants to triazines. Black currant is also tolerant to 4 ppm simazine. According to Shone and Wood (1972) triazine translocated into the leaves of black currant cannot get from the tissue system into the mesophyll. The simazine taken up remains in the leaf veins and does not enter the chloroplasts. 

The dual location of GS probably means that there is more than one form of the enzyme present in pea leaves since these may have different properties the characteristics worked out on total leaf GS (O Neal and Joy, 1974) may refer to either the chloroplastic or cytoplasmic form. Nevertheless the results indicate that chloroplastic GS is likely to have a low for ammonia (see also Stewart et al., this volume. Chapter 7). Recent studies have shown... 

Cysteine synthase has been purified from various plants and its properties examined in some detail (Giovanelli et al., this volume. Chapter 12). Several features of the enzyme are relevant to the problem of sulfide assimilation. The first of these concerns the subcellular localizaticxi of the enzyme in leaf tissue. In wheat, kidney bean and rape the enzyme is reported to be associated with the soluble fraction (Ascano and Nicholas, 1976 Masada ef al., 1975 Smith, 1972). However, in spinach, pea, and clover leaf tissue the enzyme is reported to be associated with intact chloroplasts (Fankhauser et al., 1976 Ng and Anderson, 1978a, 1979). Some possible explanaticms for these differences have been discussed by Ng and Anderson (1978a). For pea chloroplasts, however, the data in Fig. 1 show that in the absence of OAS, sulfite is reduced to sulfide in a light-dependent reaction but addition of OAS causes an immediate consumption of sulfide with the concomitant formatim of cysteine. 

The ability of pea chloroplasts to synthesize lysine, threonine and the metabolic intermediate homoserine (which is made in unusually high amounts in peas [8]) from p aspartate is highly dependent on the age of the plants from which th are prepared (Fig. 2). The rates of incorporation of [i ]aspartic acid into lysine, threonine, and homoserine were 2 to 10 times greater in plastids from 7 day old shoots than in those from 13 day old plants (Fig. 2). Moreover, when chloroplasts were isolated from each of four leaf stages of 12 day-old plants (see Fig. 3) those from the youngest leaves (stage 4) were 2 to 5 times more active in amino acid synthesis (Table 1) than those from the oldest leaves (stage 1). 

To determine whether HCS activity from pea leaves could be resolved into several forms by anion-exchange chromatography, extracts from pea leaves (crude leaf extract, chloroplast stroma, mitochondrial matrix and cytosol) were fractionated on a Mono Q HR 5/5 column [11]. We have observed that HCS activity from a crude extract could be resolved into two peaks. The minor peak, eluting at 50 mM NaCl represented approximately 10% of the total HCS activity and was detected in the chloroplast and mitochondrial fractions. The major peak (90% of the total activity), eluting at 140 mM NaCl was specific of the cytosolic fraction. These results strongly suggest the existence of two HCS isoforms, one localized in the cytosol and the other one in both chloroplasts and mitochondria. Thus, the occurrence of HCS isoforms in different cell compartments also suggest that the different biotin-dependent carboxylases present in plants are biotinylated in the cell compartment where these enzymes are active. 

The presence of GAT has been report barley [7] and pea [5] leaves and it v/as described in detail by Masterson experimental procedures and conclusi v/ere strongly criticized by Roughan not detect any CAT activity associat or indeed leaf homogenates, v/hich al not found in mitochondria, despite s contrary [2]. This v/ork describes th and properties of chloroplastic GAT. 

Roughan et air [6] were unable to detect CAT activity in leaf homogenates of spinach, amaranthus and pea, or in the purified chloroplasts of spinach and pea. The fact that they found it necessary to add CoASH to the medium for fatty acid synthesis would suggest that their chloroplasts were damaged. Intact chloroplasts should be self-sufficient in CoASH as it cannot cross the chloroplast membrane barrier [1]. Addition of an external, nonpenetrating CoASH should have no discernible effect upon fatty acid synthesis. If the chloroplasts were damaged then the soluble stromal CAT would have been lost during isolation. 

Chloroplasts were isolated from 14-day-old peas by grinding leaf tissue in the medium of Stokes and Walker (1971), and resuspended in the medium of Renger et at. (1976) Incubations with trypsin or chymotrypsin (Sigma types XIII, TCPK VII TLCK respectively) were at pH 7.2, temp. 23 C and were stopped by addition of Soya Bean trypsin inhibitor (Sigma). 

A rather different picture has emerged from studies of GS in pea, where five distinct GS polypeptides are differentially expressed in leaves, roots, and nodules (Tingey et ai, 1987). The predominant leaf GS is composed of a 44-kDa polypeptide localized to chloroplast stroma. Leaves also express, at a much lower level, a cytosoUc 38-kDa GS polypeptide. This 38-kDa polypeptide is the major form of GS in root cytosol. Roots and leaves also express, at a very reduced level, three 37-kDa GS polypeptides. The high GS activity that occurs as root nodules develop is accompanied by a striking increase in the three 37-kDa polypeptides. Nodules also express at low levels the 44- and 38-kDa GS polypeptides. Similar to Phaseolus, pea GS appears to be under multigene control. However, there does not appear to be a solely nodule-specific form of GS in pea as there is in Phaseolus. 

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