Tuber Discs Research Articles

Laboratory Efficacy of Essential Oils Against Pectobacterium carotovorum Subsp. carotovorum and Pectobacterium atrosepticum Causing Soft Rot of Potato Tubers

AbstractThe antibacterial activity of essential oils (EOs) from Carum carvi, Cinnamomum zeylanicum, Cuminum cyminum, Eugenia caryophyllus, Foeniculum vulgare, Melaleuca alternifolia, Mentha × piperita, Origanum vulgare, Rosmarinus officinalis and Thymus vulgaris was tested against Pectobacterium carotovorum subsp. carotovorum (Pcc) and Pectobacterium atrosepticum (Pa), which cause soft rot of potato tubers. In disc diffusion, minimum inhibitory concentration and minimum bactericidal concentration (MBC) tests, cinnamon EO was found to be most effective against both bacteria. The inhibition zones ranged from 20.46 to 29.58 mm for a concentration of 100 μL/mL. The minimum inhibitory concentration was 0.5 μL/mL, and MBC was between 0.5 and 5 μL/mL. The higher sensitivity of bacteria was manifested in clove (Pcc and Pa), mint (Pcc), oregano (Pa) and thyme (Pa) EOs. Rosemary EO was the least effective. The results of the in vivo test were not entirely consistent with those of the in vitro tests. The most significant antibacterial effect was achieved with mint EO. The treatment of potato tuber discs with mint EO at a concentration of 3 μL/mL for Pcc and 3–10 μL/mL for Pa was 100% effective. The efficacy of the essential oils of caraway (5–10 μL/mL), thyme (10 μL/mL) and oregano (5 μL/mL) also ranged from 95.7 to 99.7%. Based on the results of the in vivo test, it may be recommended that mint EO and potentially caraway, oregano and thyme EOs be further tested for pickling potato tubers against bacteria of the genus Pectobacterium.

Modulatory role of nitric oxide in wound healing of potato tubers

IntroductionWounding of potato (Solanum tuberosum L.) tubers affects the postharvest storage qualities and marketability. To mitigate wound-related losses, it is necessary to accelerate wound healing (WH) responses of potato tubers. While role of nitric oxide (NO) in healing of wounded biological tissues is well known, its impact on WH responses of potato tubers has not been examined thoroughly. The aim of this study was to elucidate the potential modulatory role of NO in WH response of potato tubers by determining the activity of key enzymes associated with NO biosynthesis.MethodsUsing a model wounding system, tuber tissue discs were extracted from mini tubers of cv. Russet Burbank to examine their WH responses. Tuber discs were treated with NO related chemicals, such as NO scavenger, nitric oxide synthase (NOS) inhibitor, and NO donors. The effect of these treatments on in vitro production of NO, activity of nitrate reductase, NOS, and phenylalanine ammonia-lyase were determined. The accumulation of suberin polyphenolics (SPP) in wounded parenchyma cells, an indicator of early suberization process, was analyzed histologically.ResultsTreatment of tuber tissues with the higher dose of an NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) at the time of wounding completely ceased SPP accumulation between 0 h to 3 d after wounding. Increased production of NO and enhanced activity of NOS were also observed immediately after wounding of the tuber tissues. The results suggest that NO is a key biological modulator in WH responses of potato tubers and could be optimized as a potential exogenous treatment to counter wound-related losses.

Первое сообщение о Cephalotrich um asper ulum как патогене картофеля в России

During the phytosanitary inspection of a lot of stored potatoes of cv. Zhukovskiy ranniy in the Orel region of Russia, tubers showed signs of lesions in the form of local darkening of the flesh without apparent softening and fungal structures on the surface (coremia). A pure culture of the pathogen was isolated and the morphological characteristics of the conidial sporulation structures in the extracted isolate were described; the average sizes of vegetative mycelium, conidia, conidiophores and coremia were given. Pathogenicity testing of the isolate on leaves and tuber discs showed symptoms in the form of localised chlorosis and necrosis. Subsequent identification of the isolate by PCR and sequencing showed that the isolate analysed was most closely related to the species Cephalotrichum asperulum of the genus Cephalotrichum. The resulting sequence was deposited in GenBank (accession number ON364353). Although these fungi are described in the literature as endophytic, this study emphasises their parasitic effect on the potato plant with certain semeiologies. This is probably the first report of C. asperulum causing local necrosis of potato leaves and tubers during storage in Russia.

The Plastidial Glucan Phosphorylase Affects the Maltooligosaccharide Metabolism in Parenchyma Cells of Potato (Solanum tuberosum L.) Tuber Discs.

Maltodextrin metabolism is thought to be involved in both starch initiation and degradation. In this study, potato tuber discs from transgenic lines containing antisense constructs against the plastidial and cytosolic isoforms of α-glucan phosphorylase and phosphoglucomutase were used to evaluate their influences on the conversion of externally supplied glucose-1-phosphate into soluble maltodextrins, as compared to wild-type potato tubers (Solanum tuberosum L. cv. Desiree). Relative maltodextrin amounts analyzed by capillary electrophoresis with laser-induced fluorescence revealed that tuber discs could immediately uptake glucose-1-phosphate and use it to produce maltooligosaccharides with a degree of polymerization of up to 30, as opposed to tubers repressing the plastidial glucan phosphorylase. The results presented here support previous indications that a specific transporter for glucose-1-phosphate may exist in both the plant cells and the plastidial membranes, thereby allowing a glucose-6-phosphate-independent transport. Furthermore, it confirms that the plastidial glucan phosphorylase is responsible for producing longer maltooligosaccharides in the plastids by catalyzing a glucosyl polymerization reaction when glucose-1-phosphate is available. All these findings contribute to a better understanding of the role of the plastidial phosphorylase as a key enzyme directly involved in the synthesis and degradation of glucans and their implication on starch metabolism.

A tissue culture system for callus formation and plant regeneration using tuber discs of Dioscorea tokoro Makino, a pharmaceutical yam species

Yam (Dioscorea sp.) is a recalcitrant plant species with respect to plant regeneration in tissue culture. We report here on callus formation and plantlet regeneration in the pharmaceutical yam species D. tokoro Makino. Explants of leaves, nodes, internodes, roots, and tuber discs of three lines of D. tokoro were cultured on media containing the synthetic auxins 2,4-dichlorophenoxyacetic acid or picloram at 10 µM. Explants of nodes, roots, and tuber discs formed calluses at high frequency in all lines we examined, regardless of type of auxin added to the media. However, explants of leaves and internodes rarely formed calluses. Some calluses derived from tuber discs differentiated shoots through the formation of bud-like structures by culture on media with or without 10 µM 6-benzyladenine. The T-1110 line showed the highest shoot formation efficiency. The regenerants from the calluses derived from tuber discs successfully acclimatized to a greenhouse, grew normally and ultimately formed tubers in soil.

Optimization of Hairy Root Induction in Solanum tuberosum

An efficient protocol for hairy root induction in Solanum tuberosum was established using Agrobacterium rhizogenes. Transformation of Desiree was more efficient than with Shepody or Purple Majesty. Transformation efficiency of Desiree tuber discs (97% transformation efficiency) was higher in comparison to root (67% transformation efficiency), stem (61% transformation efficiency) and leaf (14% transformation efficiency) explants. Bacterial density, culture parameters, explant type, and genotype influenced the transformation efficiency, as well as the growth of hairy roots. Hairy root transformation efficiency in explants treated with acetosyringone was enhanced from 72 to 89% in Purple Majesty tubers and 56 to 80% in roots during co-cultivation. The growth rate of hairy roots from tuber discs was about 5 times greater than that of hairy roots from leaves. Tuber hairy roots were highly resistant to kanamycin in the absence of NPT II. This study describes the efficient generation of hairy roots in different potato tissues and cultivars that can provide a fast method to generate transformed tissue or be used for production of secondary metabolites.

Genotypic and Phenotypic Characterization of Streptomyces Species Causing Potato Common Scab in Uruguay.

Isolation and characterization of common scab (CS) pathogen Streptomyces spp. from Uruguayan potato tubers and soil samples were done in response to significant economic losses due to CS on potato in autumn 2010. Seventy of the 331 isolates were classified as pathogenic owing to their ability to induce necrosis on tuber disks and stunting of radish seedling. Streptomyces spp. causing CS on potato in Uruguay were found to represent a range of different species by virtue of their diverse morphological and physiological traits as well as rep-PCR, rpoB phylogenetic analysis, and multi-locus sequences analysis. We identified isolates primarily as Streptomyces scabiei, S. acidiscabies, and S. europaeiscabiei. However, some of the pathogenic isolates still remain to be identified at the species level. This highlights the need for improved methods for discrimination among pathogenic Streptomyces species. The presence of Streptomyces pathogenicity island (PAI) genes was analyzed, including genes encoding for thaxtomin synthetase (txtA, txtB), tomatinase (tomA), and a necrosis protein (nec1). Among the isolates that were pathogenic, 50% contained the four pathogenicity genes, 33% had an atypical composition of PAI marker genes, and 17% did not contain any genes. The absence of the genes reported to be involved in thaxtomin biosynthesis (txtA, txtB) was confirmed by whole-genome sequencing of two representative strains of this group. This finding suggests the participation of other virulence factors in plant pathogenicity.

Expression and functional characterization of the Agrobacterium VirB2 amino acid substitution variants in T-pilus biogenesis, virulence, and transient transformation efficiency.

Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease by transferring transferred DNA (T-DNA) into the plant genome. The translocation process is mediated by the type IV secretion system (T4SS) consisting of the VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. Here, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2−) and tumorigenesis on tomato stems (Vir+ or Vir−), the mutants could be classified into three groups: ExB2−/Vir−, ExB2−/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2−/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation. Interestingly, although the five T-pilus−/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency.

Cadmium-induced oxidative stress in potato tuber

Short-term treatment of tuber discs of potato (<em>Solanum tuberosum</em> L.) with cadmium chloride elevated the concentration of active oxygen species (<sup>.</sup>O<sup>-</sup><sub>2</sub>, H<sub>2</sub>0<sub>2</sub>) and activated the antioxidative system. Two cultivars, Bintje and Bzura, susceptible and tolerant, respectively, to cadmium were examined. In more tolerant, control tissues the activity of ascorbic acid peroxidase (AAP) and catalase (CAT) was higher than in the sensitive ones. During first hours of stress, the inhibition of superoxide dismutase (SOD), CAT and AAP was observed and it comes from inactivation of enzymes by cadmium ions. A subsequent activity increase of the enzymes aroused earlier in tolerant tissues. It seems therefore, that tolerant tissues possess a more efficient antioxidative system.

Agrobacteria lacking ornithine lipids induce more rapid tumour formation

Ornithine lipids (OLs) are phosphorus-free membrane lipids that are widespread among Gram-negative bacteria. Their basic structure consists of a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of ornithine and a second fatty acyl group ester-linked to the 3-hydroxy position of the first fatty acid. It has been shown that OLs can be hydroxylated within the amide-linked fatty acyl moiety, the secondary fatty acyl moiety or within the ornithine moiety. These modifications have been related to increased stress tolerance and symbiotic proficiency in different organisms such as Rhizobium tropici or Burkholderia cenocepacia. Analysing the membrane lipid composition of the plant pathogen Agrobacterium tumefaciens we noticed that it forms two different OLs. In the present work we studied if OLs play a role in stress tolerance and pathogenicity in A. tumefaciens. Mutants deficient in the OLs biosynthesis genes olsB or olsE were constructed and characterized. They either completely lack OLs (ΔolsB) or only form the unmodified OL (ΔolsE). Here we present a characterization of both OL mutants under stress conditions and in a plant transformation assay using potato tuber discs. Surprisingly, the lack of agrobacterial OLs promotes earlier tumour formation on the plant host.

Biocontrol of black scurf on potato by seed tuber treatment with Pythium oligandrum

The biological control activity of Pythium oligandrum against black scurf of potato caused by Rhizoctonia solani AG-3 was evaluated in field experiments after treatment of potato seed tubers with P. oligandrum. Seed tubers infected with black scurf sclerotia were dipped for a few seconds in a suspension of 103, 104 or 105mL−1P. oligandrum oospores and were then air-dried. Each level of P. oligandrum-treatment significantly reduced the disease rates of stolon at a level similar to that achieved by chemical control. When P. oligandrum populations adherent to the surface of seed tubers were determined, oospore counts on tubers treated with 104 or 105oosporesmL−1 were about 540/cm2 or about 22,000/cm2 just after dipping and decreased to about 170/cm2 or 2900/cm2 after a 3-week incubation, respectively. Confocal laser scanning microscopic observation with an immuno-enzymatic staining procedure showed that P. oligandrum hyphae had colonized the sclerotia and established close contact by coiling around the R. solani hyphae present on the surface of seed tubers, in a manner similar to that observed in the dual-culture test. Quantification of R. solani DNA by PCR indicated that the R. solani population was reduced on the seed tubers treated with P. oligandrum compared to untreated tubers. Furthermore, the ability of P. oligandrum to induce resistance against black scurf was determined using a potato tuber disk assay. Treatment of tuber disks with the cell wall protein fraction of P. oligandrum enhanced the expression of defense-related genes such as 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, lipoxygenase and basic PR-6 genes, and reduced disease severity upon challenge with R. solani compared with untreated controls. These results suggest that biocontrol mechanisms employed by P. oligandrum against black scurf involve both mycoparasitism and induced resistance.

Coordinate expression of AOS genes and JA accumulation: JA is not required for initiation of closing layer in wound healing tubers

Wounding induces a series of coordinated physiological responses essential for protection and healing of the damaged tissue. Wound-induced formation of jasmonic acid (JA) is important in defense responses in leaves, but comparatively little is known about the induction of JA biosynthesis and its role(s) in tuber wound-healing. In this study, the effects of wounding on JA content, expression of JA biosynthetic genes, and the involvement of JA in the initiation of closing layer formation in potato tubers were determined. In addition, the role of abscisic acid (ABA) in wound-induced JA accumulation was examined. The basal JA content in non-wounded tuber tissues was low (<3 ng g −1 FW). Two hours after wounding, the JA content increased by >5-fold, reached a maximum between 4 and 6 h after wounding, and declined to near-basal levels thereafter. Tuber age (storage duration) had little effect on the pattern of JA accumulation. The expressions of the JA biosynthetic genes ( StAOS2, StAOC, and StOPR3) were greatly increased by wounding reaching a maximum 2–4 h after wounding and declining thereafter. A 1-h aqueous wash of tuber discs immediately after wounding resulted in a 94% inhibition of wound-induced JA accumulation. Neither JA treatment nor inhibition of JA accumulation affected suberin polyphenolic accumulation during closing layer development indicating that JA was not essential for the initiation of primary suberization. ABA treatment did not restore JA accumulation in washed tuber tissues suggesting that leaching of endogenous ABA was either not involved or not solely involved in this loss of JA accumulation by washing. Collectively, these results indicate that JA is not required for the induction of processes essential to the initiation of suberization during closing layer development, but do not exclude the possibility that JA may be involved in other wound related responses.

Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker

The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.

ABA or cadmium induced phytochelatin synthesis in potato tubers

Short-term treatment of potato (Solanum tuberosum L.) tuber discs with CdCl2 solution elevated both the StPCS1 transcript level, phytochelatin synthase (PCS) activity and contents of phytochelatin (PC) and abscisic acid (ABA). Similar effects but less marked were noticed after treatment of tuber tissues with ABA solution. Cd-treatment increased also cysteine (CYS) content but did not change glutathione content. If ABA treatment preceded Cd-treatment, the elevation of CYS and PC contents were limited. The data suggest the participation of ABA in the regulation of PCS.

Glucose 1‐phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules

Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [(14)C]glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.

Inhibition of crown-gall tumorigenesis with plant extracts

Measurement of the inhibition of crown-gall tumors on potato discs is an antitumor bioassay method for detection of anti-tumor compounds from higher plants. In this study, after surface sterilization, tuber discs were co-cultivated with Agrobacterium tumefaciens B6S3 for two days. The discs were then inoculated on MS media with the crude extracts obtained from different parts of various plants – Allium sativum L. (Liliaceae), Salvia verbenaca L., Rosmarinus officinalis L., Ocimum basiculum L., Lavandula stoechas L. (Lamiaceae), Althaea cannabina L. (Malvaceae), Petroselinum sativum Mill. (Apiaceae), Pelargonium radicula L. (Geraniaceaea), Juglans regia L. (Juglandaceae), Platanus orientalis L. (Platanaceae), Laurus nobilis L. (Lauraceae), Ranunculus ficaria L. (Ranunculaceae), and Abies equi-trojani Aschers. et Sint. Ex Boiss (Pinaceae). Tumors appeared after 10-15 days, and tumor inhibition was detected only in six plant extracts of 13 (A. sativum, R. officinalis, P. orientalis, L. nobilis, R. ficaria, and A. equi-trojani) when compared to control material. Constitutive tumor inhibition activities were higher in these plant extracts (63.5%, 56.1%, 61.7%, 54.6%, 69.7%, and 57.9% at 0 min; 58.8%, 54.6%, 58.8%, 48.4%, 62.6%, and 51.6% for 15 min after bacterial inoculation).

Inhibition of 2-Oxoglutarate Dehydrogenase in Potato Tuber Suggests the Enzyme Is Limiting for Respiration and Confirms Its Importance in Nitrogen Assimilation

The 2-oxoglutarate dehydrogenase complex constitutes a mitochondrially localized tricarboxylic acid cycle multienzyme system responsible for the conversion of 2-oxoglutarate to succinyl-coenzyme A concomitant with NAD(+) reduction. Although regulatory mechanisms of plant enzyme complexes have been characterized in vitro, little is known concerning their role in plant metabolism in situ. This issue has recently been addressed at the cellular level in nonplant systems via the use of specific phosphonate inhibitors of the enzyme. Here, we describe the application of these inhibitors for the functional analysis of the potato (Solanum tuberosum) tuber 2-oxoglutarate dehydrogenase complex. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-oxoglutarate dehydrogenase complex, displaying greater inhibitory effects than a diethyl ester of SP, a phosphono ethyl ester of SP, or a triethyl ester of SP. Incubation of potato tuber slices with the inhibitors revealed that they were adequately taken up by the tissue and produced the anticipated effects on the in situ enzyme activity. In order to assess the metabolic consequences of the 2-oxoglutarate dehydrogenase complex inhibition, we evaluated the levels of a broad range of primary metabolites using an established gas chromatography-mass spectrometry method. We additionally analyzed the rate of respiration in both tuber discs and isolated mitochondria. Finally, we evaluated the metabolic fate of radiolabeled acetate, 2-oxoglutarate or glucose, and (13)C-labeled pyruvate and glutamate following incubation of tuber discs in the presence or absence of either SP or the carboxy ethyl ester of SP. The data obtained are discussed in the context of the roles of the 2-oxoglutarate dehydrogenase complex in respiration and carbon-nitrogen interactions.

Alterations in cytosolic glucose-phosphate metabolism affect structural features and biochemical properties of starch-related heteroglycans.

The cytosolic pools of glucose-1-phosphate (Glc-1-P) and glucose-6-phosphate are essential intermediates in several biosynthetic paths, including the formation of sucrose and cell wall constituents, and they are also linked to the cytosolic starch-related heteroglycans. In this work, structural features and biochemical properties of starch-related heteroglycans were analyzed as affected by the cytosolic glucose monophosphate metabolism using both source and sink organs from wild-type and various transgenic potato (Solanum tuberosum) plants. In leaves, increased levels of the cytosolic phosphoglucomutase (cPGM) did affect the cytosolic heteroglycans, as both the glucosyl content and the size distribution were diminished. By contrast, underexpression of cPGM resulted in an unchanged size distribution and an unaltered or even increased glucosyl content of the heteroglycans. Heteroglycans prepared from potato tubers were found to be similar to those from leaves but were not significantly affected by the level of cPGM activity. However, external glucose or Glc-1-P exerted entirely different effects on the cytosolic heteroglycans when added to tuber discs. Glucose was directed mainly toward starch and cell wall material, but incorporation into the constituents of the cytosolic heteroglycans was very low and roughly reflected the relative monomeric abundance. By contrast, Glc-1-P was selectively taken up by the tuber discs and resulted in a fast increase in the glucosyl content of the heteroglycans that quantitatively reflected the level of the cytosolic phosphorylase activity. Based on (14)C labeling experiments, we propose that in the cytosol, glucose and Glc-1-P are metabolized by largely separated paths.

Regulatory involvement of abscisic acid in potato tuber wound-healing

Rapid wound-healing is crucial in protecting potato tubers from infection and dehydration. Wound-induced suberization and the accumulation of hydrophobic barriers to reduce water vapour conductance/loss are principal protective wound-healing processes. However, little is known about the cognate mechanisms that effect or regulate these processes. The objective of this research was to determine the involvement of abscisic acid (ABA) in the regulation of wound-induced suberization and tuber water vapour loss (dehydration). Analysis by liquid chromatography-mass spectrometry showed that ABA concentrations varied little throughout the tuber, but were slightly higher near the periderm and lowest in the pith. ABA concentrations increase then decrease during tuber storage. Tuber wounding induced changes in ABA content. ABA content in wound-healing tuber discs decreased after wounding, reached a minimum by 24 h, and then increased from the 3rd to the 7th day after wounding. Wound-induced ABA accumulations were reduced by fluridone (FLD); an inhibitor of de novo ABA biosynthesis. Wound-induced phenylalanine ammonia lyase activity was slightly reduced and the accumulation of suberin poly(phenolics) and poly(aliphatics) noticeably reduced in FLD-treated tissues. Addition of ABA to the FLD treatment restored phenylalanine ammonia lyase activity and suberization, unequivocally indicating that endogenous ABA is involved in the regulation of these wound-healing processes. Similar experiments showed that endogenous ABA is involved in the regulation of water vapour loss, a process linked to wax accumulation in wound-healing tubers. Rapid reduction of water vapour loss across the wound surface is essential in preventing desiccation and death of cells at the wound site; live cells are required for suberization. These results unequivocally show that endogenous ABA is involved in the regulation of wound-induced suberization and the processes that protect surface cells from water vapour loss and death by dehydration.

Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System inAgrobacterium tumefaciens

Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant- and animal-pathogenic bacteria.