Proteins Involved in the Interaction of Potato Tubers with Pectobacterium atrosepticum: a Proteomic Approach to Understanding Partial Resistance

Potato can be severely affected by various pathogens, including Pectobacterium atrosepticum, the cause of bacterial soft rot on tubers and of blackleg on stems. To date, no complete resistance to P. atrosepticum is available, so that only cultivars exhibiting partial resistance can be found. The mechanistic basis of this type of resistance is still poorly understood. A proteomic approach was thus developed to identify pathways specifically activated during the interaction between potato tubers and P. atrosepticum. Protein profiles on silver‐stained gels in the 5–8 pH range were obtained from healthy and infected tubers from two cultivars differing for resistance level and analyzed by 2‐DE and nano‐LC‐MS/MS. Thirteen proteins were differentially up‐regulated in the partially resistant cv. Kerpondy; by contrast, no significant differences in protein profiles of inoculated and control tubers were observed in the susceptible cv. Bintje. Mass spectrometry and database searching showed that these proteins are involved in energetic metabolism (glyceraldehyde‐3‐phosphate dehydrogenase, 2‐phosphoglycerate dehydratase or enolase, fructose biphosphate aldolase and ATPase α subunit), cytoskeleton structure (actin), protein catabolism (cysteine protease inhibitor) and patatins or patatin precursors. Their involvement in defence responses of cv. Kerpondy to P. atrosepticum is discussed. Proteomic appears as an efficient approach to have insight into the mechanisms and pathways leading to potato resistance against P. atrosepticum.     

Expanded bed adsorption for recovery of patatin from crude potato juice

An expanded bed adsorption process was used to isolate patatin possessing esterase activity, from a crude juice of potato tubers. Patatin is the major storage protein of potato tubers and is released in ample amounts in the processing effluent during starch milling. We employed mixed mode affinity resins, where the binding depends primarily on the pH, and is almost independent of the ionic strength. From a library of mixed mode chemistries involving both charged and hydrophobic functions, we screened for ligands with binding specificity for patatin. The dynamic binding capacity of two high density (1.45–1.5 g ml^-1) patatin-binding agarose-glass resins in response to change of linear velocity (85–230 cm h^-1) was tested in packed (25 ml) and expanded (250 ml) column modes. The column operation included a loading step at low expansion; H/H_o~1.2. Adsorption from crude juice at pH 7.5, retained patatins up to a breakthrough level of 50%. The eluate fraction at pH 3.5, now effectively stripped from the pigments, provided a 2.5-fold enzyme enrichment and produced 4 g protein per cycle. Column productivity was 122 kAU L^-1 h^-1. The study, using potato juice as model feedstock, demonstrated the feasibility of expanded bed-recovery of potentially valuable proteins from plant biomass.This revised version was published online in October 2005 with corrections to the Cover Date.     

TILLING to detect induced mutations in soybean

Background

Potato is a staple food in the diet of the world's population and also being used as animal feed. Compared to other crops, however, potato tubers are relatively poor in the essential amino acid, methionine. Our aim was to increase the methionine content of tubers by co-expressing a gene involved in methionine synthesis with a gene encoding a methionine-rich storage protein in potato plants.

Results

In higher plants, cystathionine γ-synthase (CgS) is the first enzyme specific to methionine biosynthesis. We attempted to increase the methionine content of tubers by expressing the deleted form of theArabidopsis CgS (CgS _Δ90), which is not regulated by methionine, in potato plants. To increase the incorporation of free methionine into a storage protein theCgS _Δ90was co-transformed with the methionine-rich15-kD β-zein. Results demonstrated a 2- to 6-fold increase in the free methionine content and in the methionine content of the zein-containing protein fraction of the transgenic tubers. In addition, in line with higher methionine content, the amounts of soluble isoleucine and serine were also increased. However, all of the lines with high level of CgS_Δ90 expression were phenotypically abnormal showing severe growth retardation, changes in leaf architecture and 40- to 60% reduction in tuber yield. Furthermore, the colour of the transgenic tubers was altered due to the reduced amounts of anthocyanin pigments. The mRNA levels of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first step of anthocyanin synthesis, were decreased.

Conclusion

Ectopic expression of CgS_Δ90 increases the methionine content of tubers, however, results in phenotypic aberrations in potato. Co-expression of the 15-kD β-zein with CgS_Δ90 results in elevation of protein-bound methionine content of tubers, but can not overcome the phenotypical changes caused by CgS_Δ90 and can not significantly improve the nutritional value of tubers. The level ofPAL mRNA and consequently the amount of anthocyanin pigments are reduced in the CgS_Δ90 transgenic tubers suggesting that methionine synthesis and production of anthocyanins is linked.     

Immunocytochemical localization of patatin,the major glycoprotein in potato (Solanum tuberosum L.) tubers

Patatin is a family of glycoproteins with an apparent molecular weight of 40 kDa. The protein is synthesized as a pre-protein with a hydrophobic signal sequence of 23 amino acids. Using different immunocytochemical methods we determined the tissue-specific as well as subcellular localization of the patatin protein. Since antibodies raised against patatin showed crossreactivity with glycans of other glycoproteins, antibodies specific for the protein portion of the glycoprotein were purified. Using these antibodies for electron-microscopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanum tuberosum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained detectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in parenchyma cells of potato tubers. The same distribution was observed for the esterase activity in potato tubers.Abbreviations PHA phytohemagglutinin - TFMS trifluoromethanesulfonic acid     

Extensive post-translational processing of potato tuber storage proteins and vacuolar targeting

Potato tuber storage proteins were obtained from vacuoles isolated from field-grown starch potato tubers cv. Kuras. Vacuole sap proteins fractionated by gel filtration were studied by mass spectrometric analyses of trypsin and chymotrypsin digestions. The tuber vacuole appears to be a typical protein storage vacuole absent of proteolytic and glycolytic enzymes. The major soluble storage proteins included 28 Kunitz protease inhibitors, nine protease inhibitors 1, eight protease inhibitors 2, two carboxypeptidase inhibitors, eight patatins and five lipoxygenases (lox), which all showed cultivar-specific sequence variations. These proteins, except for lox, have typical endoplasmic reticulum (ER) signal peptides and putative vacuolar sorting determinants of either the sequence or structure specific type or the C-terminal type, or both. Unexpectedly, sap protein variants imported via the ER showed multiple molecular forms because of extensive and unspecific proteolytic cleavage of exposed N- and C-terminal propeptides and surface loops, in spite of the abundance of protease inhibitors. Some propeptides are potential novel vacuolar targeting peptides. In the insoluble vacuole fraction two variants of phytepsin (aspartate protease) were identified. These are most probably the processing enzymes of potato tuber vacuolar proteins. Database Proteome data have been submitted to the PRIDE database under accession number 17707.     

Natural diversity of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) invertases

Background  

Invertases are ubiquitous enzymes that irreversibly cleave sucrose into fructose and glucose. Plant invertases play important roles in carbohydrate metabolism, plant development, and biotic and abiotic stress responses. In potato (Solanum tuberosum), invertases are involved in 'cold-induced sweetening' of tubers, an adaptive response to cold stress, which negatively affects the quality of potato chips and French fries. Linkage and association studies have identified quantitative trait loci (QTL) for tuber sugar content and chip quality that colocalize with three independent potato invertase loci, which together encode five invertase genes. The role of natural allelic variation of these genes in controlling the variation of tuber sugar content in different genotypes is unknown.     

Induction and accumulation of major tuber proteins of potato in stems and petioles

A family of immunologically identical glycoproteins with apparent molecular weights of approximately 40,000 are among the major tuber proteins of potato (Solanum tuberosum L.). These proteins, as purified by ion-exchange and affinity chromatography, have been given the trivial name `patatin.' To determine if patatin can be used as a biochemical marker to study the process of tuberization, its amount was measured in a variety of tissues by rocket immunoelectrophoresis and by enzyme-linked immunosorbent assay (ELISA).

Patatin comprises 40 to 45% of the soluble protein in tubers regardless of whether they are formed on underground stolons or from axillary buds of stem cuttings. Under normal conditions, patatin is present in only trace amounts, if at all, in leaves, stems, or roots of plants which are either actively forming tubers or which have been grown under long days to prevent tuberization. However, if tubers and axillary buds are removed, patatin can accumulate in stems and petioles. This accumulation occurred without any obvious tuber-like swelling and would occur even under long days. In all tissues containing large amounts of patatin, the other tuber proteins were also found as well as large amounts of starch.

     

The use of fluorescent reporter protein tagging to study the interaction between Root‐Knot Nematodes and Soft Rot Enterobacteriaceae

The study of plant parasitic nematodes such as Meloidogyne spp. and their interactions with phytopathogenic bacteria remains underexplored. One of the challenges towards establishing such interactions is the dependence on symptom development as a measure of interaction. In this study, mCherry was employed as a reporter protein to investigate the interaction between the soft rot Enterobacteriaceae (SRE) Pectobacterium carotovorum subsp. brasiliensis (Pcb) and root‐knot nematode (M. incognita). Pectobacterium carotovorum subsp. brasiliensis was transformed with pMP7604 generating Pcb_mCherry strain. This strain was shown to attach to the surface coat of M.incognita J2 at the optimum temperature of 28°C. This suggests that RKN juveniles may play a role in disseminating Pcb in soils that are heavily infested with Pcb. The presence of RKN juveniles was shown to play a role in introducing Pcb_mCherry into potato tubers potentially acting as a source of latent tuber infections.

Significance and Impact of the Study

This study uses fluorescent reporter protein tagging as a tool to demonstrate the interaction between root‐knot nematode (Meloidogyne incognita) and the soft rot Enterobacteriacea (Pectobacterium carotovorum subsp. brasiliensis). Introduction of Pectobacterium through wounds generated by second‐stage juveniles (J2) into potato tubers was demonstrated. These results suggest that RKN juveniles can facilitate latent infection of potato tubers in the soil. These findings have important implications in the management of RKN and SRE in seed potato production. Furthermore, this tool can be used to study other nematode–bacteria interactions that have not been previously studied.     

Effects of single and mixed inoculation with two arbuscular mycorrhizal fungi in two different levels of phosphorus supply on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers

Aims

This study aimed to determine the effect of arbuscular mycorrhizal (AM) fungi and phosphorus (P) supply levels on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers.

Methods

Two commercial AM fungal isolates of Glomus intraradices (IFP Glintra) and Glomus mosseae (IFP Glm) which differ in their life cycles were used. Sweet potato plants were grown in a horizontal split-root system that consisted of two root compartments. A root-free fungal compartment that allowed the quantification of mycelial development was inserted into each root compartment. The two root compartments were inoculated either with the same or with different AM isolates, or remained free of mycorrhizal propagules. Each fungal treatment was carried out in two P supply levels.

Results

In the low P supply level, mycorrhizal colonization significantly increased β-carotene concentrations in sweet potato tubers compared with the non-mycorrhizal plants. Glomus intraradices appeared to be more efficient in increasing β-carotene concentrations than G. mosseae. Dual inoculation of the root system with the two mycorrhizal fungi did not result in a higher increase in tuber β-carotene concentrations than inoculation with the single isolates. Improved P nutrition led to higher plant tuber biomass but was not associated with increased β-carotene concentrations.

Conclusions

The results indicate a remarkable potential of mycorrhizal fungi to improve β-carotene concentrations in sweet potato tubers in low P fertilized soils. These results also suggest that β-carotene metabolism in sweet potato tubers might be specifically activated by root mycorrhizal colonization.     

Proteomic analysis of the organic matrix of the abalone <Emphasis Type="Italic">Haliotis asinina</Emphasis> calcified shell

Background  

The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised.     

The three-dimensional distribution of minerals in potato tubers

Background and Aims

The three-dimensional distributions of mineral elements in potato tubers provide insight into their mechanisms of transport and deposition. Many of these minerals are essential to a healthy human diet, and characterizing their distribution within the potato tuber will guide the effective utilization of this staple foodstuff.

Methods

The variation in mineral composition within the tuber was determined in three dimensions, after determining the orientation of the harvested tuber in the soil. The freeze-dried tuber samples were analysed for minerals using inductively coupled plasma-mass spectrometry (ICP-MS). Minerals measured included those of nutritional significance to the plant and to human consumers, such as iron, zinc, copper, calcium, magnesium, manganese, phosphorus, potassium and sulphur.

Key Results

The concentrations of most minerals were higher in the skin than in the flesh of tubers. The potato skin contained about 17 % of total tuber zinc, 34 % of calcium and 55 % of iron. On a fresh weight basis, most minerals were higher in tuber flesh at the stem end than the bud end of the tuber. Potassium, however, displayed a gradient in the opposite direction. The concentrations of phosphorus, copper and calcium decreased from the periphery towards the centre of the tuber.

Conclusions

The distribution of minerals varies greatly within the potato tuber. Low concentrations of some minerals relative to those in leaves may be due to their low mobility in phloem, whereas high concentrations in the skin may reflect direct uptake from the soil across the periderm. In tuber flesh, different minerals show distinct patterns of distribution in the tuber, several being consistent with phloem unloading in the tuber and limited onward movement. These findings have implications both for understanding directed transport of minerals in plants to stem-derived storage organs and for the dietary implications of different food preparation methods for potato tubers.     

Cloning, expression, purification and characterization of patatin, a novel phospholipase A.

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.     

Long-distance transport of <Emphasis Type="SmallCaps">L</Emphasis>-ascorbic acid in potato

Background  

Following on from recent advances in plant AsA biosynthesis there is increasing interest in elucidating the factors contributing to the L-ascorbic acid (AsA) content of edible crops. One main objective is to establish whether in sink organs such as fruits and tubers, AsA is synthesised in situ from imported photoassimilates or synthesised in source tissues and translocated via the phloem. In the current work we test the hypothesis that long-distance transport is involved in AsA accumulation within the potato tuber, the most significant source of AsA in the European diet.     

Effects of Elicitors on the Accumulation of Proteinase Inhibitors in Injured Potato Tubers

The time course of accumulation and the composition of proteinase-inhibiting proteins in diffusates from potato tubers treated with elicitors such as salicylic, jasmonic, and arachidonic acids were studied. The 40-kDa reserve protein patatin and the chymotrypsin inhibitors, among which proteins of 24.6, 22.0, and 16.0 kDa were prevalent, accumulated in diffusates from potato tubers. Jasmonic and arachidonic acids activated the accumulation of the chymotrypsin inhibitors in tubers in response to the injury stress, whereas salicylic acid inhibited this process. The effects of jasmonic and arachidonic acids increased when their concentrations decreased to 10^–6M. Salicylic acid inhibited this process. The data suggest an important role of the lipoxygenase metabolism in signal transduction of the anti-injury defense system in dormant potato tubers.     

Cytosolic N-terminal arginine-based signals together with a luminal signal target a type II membrane protein to the plant ER

Background  

In eukaryotic cells, the membrane compartments that constitute the exocytic pathway are traversed by a constant flow of lipids and proteins. This is particularly true for the endoplasmic reticulum (ER), the main "gateway of the secretory pathway", where biosynthesis of sterols, lipids, membrane-bound and soluble proteins, and glycoproteins occurs. Maintenance of the resident proteins in this compartment implies they have to be distinguished from the secretory cargo. To this end, they must possess specific ER localization determinants to prevent their exit from the ER, and/or to interact with receptors responsible for their retrieval from the Golgi apparatus. Very few information is available about the signal(s) involved in the retention of membrane type II protein in the ER but it is generally accepted that sorting of ER type II cargo membrane proteins depends on motifs mainly located in their cytosolic tails.     

Display of wasp venom allergens on the cell surface of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast Saccharomyces cerevisiae preserve their native allergenic properties and whether the yeast native surface glycoproteins interfere with IgE binding. We chose to use the major allergens from the common wasp Vespula vulgaris venom: phospholipase A1, hyaluronidase and antigen 5 as the model.