The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells

Transitory starch is stored during the day inside chloroplasts and then broken down at night for export. Recent data indicate that maltose is the major form of carbon exported from the chloroplast at night but its fate in the cytosol is unknown. An amylomaltase gene (malQ) cloned from Escherichia coli is necessary for maltose metabolism in E. coli. We investigated whether there is an amylomaltase in the cytosol of plant leaves and the role of this enzyme in plants. Two mutants of Arabidopsis thaliana (L) Heynh. were identified in which the gene encoding a putative amylomaltase enzyme [disproportionating enzyme 2, DPE2 (DPE1 refers to the plastid version of this enzyme)] was disrupted by a T-DNA insertion. Both dpe2-1 and dpe2-2 plants exhibited a dwarf phenotype and accumulated a large amount of maltose. In addition, dpe2 mutants accumulated starch and a water-soluble, ethanol/KCl-insoluble maltodextrin in their chloroplasts. At night, the amount of sucrose in dpe2 plants was lower than that in wild-type plants. These results show that Arabidopsis has an amylomaltase that is involved in the conversion of maltose to sucrose in the cytosol. We hypothesize that knocking out amylomaltase blocks the conversion from maltose to sucrose, and that the higher amount of maltose feeds back to limit starch degradation reactions in chloroplasts. As a result, dpe2 plants have higher maltose, higher starch, and higher maltodextrin but lower nighttime sucrose than wild-type plants. Finally, we propose that maltose metabolism in the cytosol of Arabidopsis leaves is similar to that in the cytoplasm of E. coli.Abbreviations F6P fructose 6-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - GTase glucanotransferase     

A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night

Maltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night. To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria. Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic. Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves. They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides. Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period. A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identified in leaves of wild-type plants. Its activity was sufficient to account for the rate of starch degradation. This activity was absent from dpe2 mutant plants. Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night. Its role is probably to metabolise maltose exported from the chloroplast. We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf.     

A maltose transporter from apple is expressed in source and sink tissues and complements the Arabidopsis maltose export-defective mutant

Prior to the cytosolic synthesis of transport sugars during transitory starch utilization, intermediate products of starch breakdown, such as maltose, must be exported from chloroplasts. Recent work in Arabidopsis indicates that a novel transporter mediates maltose transfer across the chloroplast inner envelope membrane. We cloned a gene from an apple cDNA library that is highly homologous with the Arabidopsis maltose transporter, MEX1. Expression levels of MdMEX determined by real-time PCR were low in the tips of growing shoots, higher in expanding leaves and maximal in mature leaves. Expression was also detected in fruits and roots, indicating a role for MdMEX in starch mobilization in sink tissues. The cDNA from apple was subcloned into an expression cassette between the cauliflower mosaic virus 35S promoter and the sGFP (green fluorescent protein) coding sequence. Plants of the Arabidopsis maltose excess1-1 mutant, which is homozygous for a defective MEX1 allele, were transformed with the 35S:MdMEX:GFP construct. Fluorescence of GFP was localized to chloroplasts, indicating that Arabidopsis recognized the predicted 55 amino acid chloroplast transit peptide in the apple protein. The phenotypes of several independently transformed lines were analyzed. The complemented plants were relieved of the severe stunting and chlorosis characteristic of mex1-1 plants. Furthermore, starch levels and concentrations of soluble sugars, leaf chlorophyll content and maximum quantum efficiency of PSII were restored to wild-type levels. MdMEX (Malus domestica maltose transporter) is the second member of the unique maltose transporter gene family.     

Leaves of the Arabidopsis?maltose exporter1?Mutant Exhibit a?Metabolic Profile with?Features of Cold Acclimation in the Warm

Background

Arabidopsis plants accumulate maltose from starch breakdown during cold acclimation. The Arabidopsis mutant, maltose excess1-1, accumulates large amounts of maltose in the plastid even in the warm, due to a deficient plastid envelope maltose transporter. We therefore investigated whether the elevated maltose level in mex1-1 in the warm could result in changes in metabolism and physiology typical of WT plants grown in the cold.

Principal Findings

Grown at 21 °C, mex1-1 plants were much smaller, with fewer leaves, and elevated carbohydrates and amino acids compared to WT. However, after transfer to 4 °C the total soluble sugar pool and amino acid concentration was in equal abundance in both genotypes, although the most abundant sugar in mex1-1 was still maltose whereas sucrose was in greatest abundance in WT. The chlorophyll a/b ratio in WT was much lower in the cold than in the warm, but in mex1-1 it was low in both warm and cold. After prolonged growth at 4 °C, the shoot biomass, rosette diameter and number of leaves at bolting were similar in mex1-1 and WT.

Conclusions

The mex1-1 mutation in warm-grown plants confers aspects of cold acclimation, including elevated levels of sugars and amino acids and low chlorophyll a/b ratio. This may in turn compromise growth of mex1-1 in the warm relative to WT. We suggest that elevated maltose in the plastid could be responsible for key aspects of cold acclimation.     

A critical role for disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in Arabidopsis

Disproportionating enzyme (D-enzyme) is a plastidial alpha-1,4-glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T-DNA insertion. While D-enzyme activity is eliminated in the homozygous dpe1-1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1-1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1-1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de-starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto-oligosaccharides occurs in dpe1-1 but not in wild type. These data show that D-enzyme is required for malto-oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1-1 suggests that malto-oligosaccharides affect an enzyme that attacks the starch granule, or that D-enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1-1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of malto-oligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme is in starch degradation.     

The role of cytosolic alpha-glucan phosphorylase in maltose metabolism and the comparison of amylomaltase in Arabidopsis and Escherichia coli

Transitory starch of leaves is broken down hydrolytically, making maltose the predominant form of carbon exported from chloroplasts at night. Maltose metabolism in the cytoplasm of Escherichia coli requires amylomaltase (MalQ) and maltodextrin phosphorylase (MalP). Possible orthologs of MalQ and MalP in the cytosol of Arabidopsis (Arabidopsis thaliana) were proposed as disproportionating enzyme (DPE2, At2g40840) and alpha-glucan phosphorylase (AtPHS2, At3g46970). In this article, we measured the activities of recombinant DPE2 and AtPHS2 proteins with various substrates; we show that maltose and a highly branched, soluble heteroglycan (SHG) are excellent substrates for DPE2 and propose that a SHG is the in vivo substrate for DPE2 and AtPHS2. In E. coli, MalQ and MalP preferentially use smaller maltodextrins (G(3)-G(7)) and we suggest that MalQ and DPE2 have similar, but nonidentical, roles in maltose metabolism. To study this, we complemented a MalQ(-) E. coli strain with DPE2 and found that the rescue was not complete. To investigate the role of AtPHS2 in maltose metabolism, we characterized a T-DNA insertion line of the AtPHS2 gene. The nighttime maltose level increased 4 times in the Atphs2-1 mutant. The comparison of maltose metabolism in Arabidopsis with that in E. coli and the comparison of the maltose level in plants lacking DPE2 or AtPHS2 indicate that an alternative route to metabolize the glucan residues in SHG exists. Other plant species also contain SHG, DPE2, and alpha-glucan phosphorylase, so this pathway for maltose metabolism may be widespread among plants.     

Ectopic maltase alleviates dwarf phenotype and improves plant frost tolerance of maltose transporter mutants

Maltose, the major product of starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves, exits the chloroplast via the maltose exporter1 MEX1. Consequently, mex1 loss-of-function plants exhibit substantial maltose accumulation, a starch-excess phenotype and a specific chlorotic phenotype during leaf development. Here, we investigated whether the introduction of an alternative metabolic route could suppress the marked developmental defects typical for mex1 loss-of-function mutants. To this end, we ectopically expressed in mex1  chloroplasts a functional maltase (MAL) from baker’s yeast (Saccharomyces cerevisiae, chloroplastidial MAL [cpMAL] mutants). Remarkably, the stromal MAL activity substantially alleviates most phenotypic peculiarities typical for mex1 plants. However, the cpMAL lines contained only slightly less maltose than parental mex1 plants and their starch levels were, surprisingly, even higher. These findings point to a threshold level of maltose responsible for the marked developmental defects in mex1. While growth and flowering time were only slightly retarded, cpMAL lines exhibited a substantially improved frost tolerance, when compared to wild-types. In summary, these results demonstrate the possibility to bypass the MEX1 transporter, allow us to differentiate between possible starch-excess and maltose-excess responses, and demonstrate that stromal maltose accumulation prevents frost defects. The latter insight may be instrumental for the development of crop plants with improved frost tolerance.

Expressing a yeast maltase in chloroplasts of the Arabidopsis maltose transporter mutant mex1 prevents the marked developmental defects typical for that mutant and enhances plant frost tolerance.     

Proteome analysis of chlorotic leaves of the Arabidopsis mex1 mutant defective in the mobilization of starch degradation products

Leaf starch synthesized during the day for transient storage of photoassimilated carbon is degraded the following night to support respiration and growth in plants. Maltose is a major product of starch degradation, and is exported to the cytosol through the maltose transporter (MEX1). The Arabidopsis mex1 mutant displays growth retardation and an exceptional chlorotic phenotype that is not observed in other mutants demonstrating defective starch synthesis or degradation. Consistent with the chlorotic phenotype, proteomic analysis revealed degeneration of the photosynthetic machinery in mex1, and the down-regulation of essential components for photosynthesis was also observed. The chlorosis observed in mex1 occurs during vegetative growth period under normal growth conditions, which is distinct from general senescence-induced chlorosis. No up-regulation of senescence-related genes was found in the proteomic analysis of mex1, suggesting that the chlorotic process occurring in mex1 is likely distinct from senescence-dependent processes. On the other hand, cellular processes needed to survive stress situations caused by the blocking of maltose export are induced in mex1 by up-regulation of stress-related proteins, such as a germin-like protein and glutathione S-transferase. The increased abundance of heat shock protein 93-V participating in chloroplast biogenesis and rubisco activase, a regulatory protein of photosynthesis, likely reflects an attempt by the mex1 mutant to maintain chloroplast function to survive stress conditions.     

Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana

Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (HIPs) are expected to exist.Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants.     

Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato

Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the first detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking StDPE2. In addition, they contained a range of malto-oligosaccharides from maltose to maltoheptaose. Plants repressed in both isoforms had chlorotic leaves and did not grow as well as either the controls or lines where only one of the isoforms was repressed. Examination of photosynthetic parameters suggested that this was most likely due to a decrease in carbon assimilation. The subcellular localisation of StDPE2 was re-addressed in parallel with DPE2 from Arabidopsis thaliana by transient expression of yellow fluorescent protein fusions in tobacco. No translocation to the chloroplasts was observed for any of the fusion proteins, supporting a cytosolic role of the StDPE2 enzyme in leaf starch metabolism, as has been observed for Arabidopsis DPE2. It is concluded that StDPE1 and StDPE2 have individual essential roles in starch metabolism in potato and consequently repression of these disables regulation of leaf malto-oligosaccharides, starch content and photosynthetic activity and thereby plant growth possibly by a negative feedback mechanism.     

An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis

Phytochelatins (PCs) are peptides that function in heavy-metal chelation and detoxification in plants and fungi. A recent study showed that PCs have the ability to undergo long-distance transport in a root-to-shoot direction in transgenic Arabidopsis (Arabidopsis thaliana). To determine whether long-distance transport of PCs can occur in the opposite direction, from shoots to roots, the wheat (Triticum aestivum) PC synthase (TaPCS1) gene was expressed under the control of a shoot-specific promoter (CAB2) in an Arabidopsis PC-deficient mutant, cad1-3 (CAB2TaPCS1/cad1-3). Analyses demonstrated that TaPCS1 is expressed only in shoots and that CAB2TaPCS1/cad1-3 lines complement the cadmium (Cd) and arsenic metal sensitivity of cad1-3 shoots. CAB2TaPCS1/cad1-3 plants exhibited higher Cd accumulation in roots and lower Cd accumulation in shoots compared to wild type. Fluorescence HPLC coupled to mass spectrometry analyses directly detected PC2 in the roots of CAB2:TaPCS1/cad1-3 but not in cad1-3 controls, suggesting that PC2 is transported over long distances in the shoot-to-root direction. In addition, wild-type shoot tissues were grafted onto PC synthase cad1-3 atpcs2-1 double loss-of-function mutant root tissues. An Arabidopsis grafting technique for mature plants was modified to obtain an 84% success rate, significantly greater than a previous rate of approximately 11%. Fluorescence HPLC-mass spectrometry showed the presence of PC2, PC3, and PC4 in the root tissue of grafts between wild-type shoots and cad1-3 atpcs2-1 double-mutant roots, demonstrating that PCs are transported over long distances from shoots to roots in Arabidopsis.     

Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression

We have developed a genetics-based phytoremediation strategy for arsenic in which the oxyanion arsenate is transported aboveground, reduced to arsenite, and sequestered in thiol-peptide complexes. The Escherichia coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite. Arabidopsis thaliana plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently hypersensitive to arsenate. Arabidopsis plants expressing the E. coli gene encoding gamma-glutamylcysteine synthetase (gamma-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type. However, plants expressing SRS1p/ArsC and ACT2p/gamma-ECS together showed substantially greater arsenic tolerance than gamma-ECS or wild-type plants. When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild type or plants expressing gamma-ECS or ArsC alone. This arsenic remediation strategy should be applicable to a wide variety of plant species.     

Do Polyamines Participate in the Long-Distance Translocation of Stress Signals in Plants?

Accumulation and ethylene-dependent translocation of free polyamines was studied in various organs, the phloem and xylem exudates of common ice plants (Mesembryanthemum crystallinum L.). Under normal conditions (23–25°C), spermidine predominated among polyamines. Cadaverine was found in old leaves, stems, and, in large quantities, in roots. The heat shock treatment (HS; 47°C, 2 h) of intact plant shoots induced intense evolution of ethylene from leaves but reduced the leaf content of polyamines. Under these conditions, the concentration of polyamines in roots, particularly of cadaverine, increased many times. The HS treatment of roots (40°C, 2 h) induced translocation of cadaverine to stems and putrescine to leaves. An enhanced polyamine content after HS treatment was also found in the xylem and phloem exudates. The exposure of detached leaves to ethylene led to a reduction in their putrescine and spermidine and accumulation of cadaverine, which implies the ethylene-dependent formation of cadaverine and a possible relation between the HS-induced translocation of this diamine to roots and the transient ethylene evolution by leaves. To validate this hypothesis, we compared the ethylene evolution rate and interorgan partitioning of cadaverine and other polyamines for two lines of Arabidopsis thaliana: the wild type (Col-0) and ein4 mutant with impaired ethylene reception. In plants grown in light at 20–21°C, the rate of ethylene evolution by rosetted leaves was higher in the mutant than in the wild type. The content of putrescine and spermidine was reduced in mutant leaves, whereas cadaverine concentration increased almost threefold compared with the wild type. In roots, cadaverine was found only in the wild type and not in the mutant line. Our data indicate the ethylene-dependent formation of cadaverine in leaves and possible involvement of cadaverine and ethylene in the long-distance translocation of stress (HS) signal in plants.     

Influence of prior Cd(2+) exposure on the uptake of Cd(2+) and other elements in the phytochelatin-deficient mutant, cad1-3, of Arabidopsis thaliana

In order to test the potential effect of prior exposure to different Cd concentrations on Cd uptake and accumulation, plants of Arabidopsis thaliana, including a phytochelatin-deficient mutant, cad1-3, and the wild type, were compared. For Cd uptake experiments, plants were grown for 1 week in nutrient solution containing different Cd concentrations (0, 0.05, 0.1, 0.25, 0.5, and 1.0 microM Cd(NO(3))(2)). Thereafter they were subjected to 0.5 microM Cd labelled with (109)Cd for 2 h. Uptake experiments with (109)Cd showed that the phytochelatin-deficient mutant cad1-3, accumulated less Cd than the wild type. Both a lower proportion and lower total amount of absorbed Cd were translocated to the shoot in cad1-3 plants compared to wild-type plants. Cadmium exposure also influenced the amounts of nutrients found, whereby after exposure to high Cd concentrations (0.5, 1.0 microM) during growth, cad1-3 roots contained less Fe, K, Mg, P, and S compared to roots of the wild type. In cad1-3 these elements decreased with increasing Cd concentration. The total Cd content in roots and shoots increased significantly with increasing Cd concentration during growth, although the increase was much less in cad1-3 plants. In time-dependent experiments of Cd uptake carried out between 15 and 120 min on plants not previously exposed to Cd, no significant difference in Cd accumulation between the mutant and wild type were found, although a smaller amount of Cd was translocated to the shoot in cad1-3 plants. The possibility that the differences in Cd accumulation in mutant and wild-type lines may be due to the cytosolic Cd regulation, which is inhibited by the complexation of Cd by phytochelatins, is discussed.     

Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana

Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2).In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids.In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.     

Effect of replacing nutrient potassium by sodium on uptake and distribution of sodium in tomato plants

Summary Tomato plants (Lycopersicon esculentum cv. Amberley Cross) were grown in a series of nutrient solutions in which 0, 60, 90, 95, 98 and 99 per cent of the K was replaced by Na. The plants selectively absorbed K relative to Na from the nutrient solutions and transported K to the shoots in preference to Na. In the nutrient solution having the highest K/Na ration most of the Na taken up by the plants accumulated in the roots, but as the K was progressively replaced by Na an increasing proportion of the total Na absorbed was transported to the leaves. Sodium was present at up to 2.4 per cent of the dry wt of whole, fully-expanded leaves without there being any apparent visual signs of damage or reduction in the rate of growth of the plants. On closer examination it was found that most of the Na transported to the leaves was excluded from the laminar tissue and accumulated in the adjacent petioles. The ability of the roots and petioles to retain large amounts of Na depended on an adequate supply of K to the plants.     

Phytochelatin synthase (PCS) protein is induced in Brassica juncea leaves after prolonged Cd exposure

Higher plants respond to cadmium exposure with the production of phytochelatins (PCn), small heavy metal binding peptides, which are synthesized from glutathione by phytochelatin synthase (PCS). The isolation of a PCS cDNA clone from Brassica juncea L. cv. Vitasso, a candidate species for phytoremediation, is reported here. CLUSTAL analysis revealed a close relationship of BjPCS1 with PCS proteins from Arabidopsis thaliana and Thlaspi caerulescens. BjPCS1 expressed as recombinant protein in E. coli had PCS activity in vitro that was activated by 50 microM Cu and 200 microM Cd to a similar extent. Immunoblot analysis with an antiserum directed against recombinant BjPCS1 showed constitutive PCS expression during plant development. As a percentage of the total protein, the expression was higher in the roots, internodes and petioles in comparison with the leaf tissue. When B. juncea plants were treated with 25 microM cadmium, PCn accumulated increasingly over a 6 d period. Levels in shoots were about 3-fold higher than in roots. Prolonged cadmium exposure caused a significant increase of PCS protein in leaves, whereas in roots PCS protein levels were not affected.     

Long-distance phloem transport of glucosinolates in Arabidopsis

Glucosinolates are a large group of plant secondary metabolites found mainly in the order Capparales, which includes a large number of economically important Brassica crops and the model plant Arabidopsis. In the present study, several lines of evidence are provided for phloem transport of glucosinolates in Arabidopsis. When radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) and sucrose were co-applied to the tip of detached leaves, both tracers were collected in the phloem exudates at the petioles. Long-distance transport of [(14)C]p-OHBG was investigated in wild-type and transgenic 35S::CYP79A1 plants, synthesizing high amounts of p-OHBG, which is not a natural constituent of wild-type Arabidopsis. In both wild-type and 35S::CYP79A1 plants, radiolabeled p-OHBG was rapidly transported from the application site into the whole plant and intact p-OHBG was recovered from different tissues. The pattern of distribution of the radioactivity corresponded to that expected for transport of photoassimilates such as sucrose, and was consistent with translocation in phloem following the source-sink relationship. Radiolabeled p-OHBG was shown to accumulate in the seeds of wild-type and 35S::CYP79A1 plants, where p-OHBG had been either exogenously applied or endogenously synthesized from Tyr in the leaves. p-OHBG was found in phloem exudates collected from cut petioles of leaves from both wild-type and 35S::CYP79A1 plants. Phloem exudates were shown to contain intact glucosinolates, and not desulphoglucosinolates, as the transport form. It is concluded that intact glucosinolates are readily loaded into and transported by the phloem.