Potato 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.

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.

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.

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.

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.

Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

Adenine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. It was previously reported that decreased expression of plastidial adenylate kinase, catalysing the interconversion of ATP and AMP to ADP, leads to increased adenylate pools and starch content in transgenic potato tubers. However, the underlying mechanisms were not elucidated. Here, it is shown that decreased expression of plastidial adenylate kinase in growing tubers leads to increased rates of respiratory oxygen consumption and increased carbon fluxes into starch. Increased rates of starch synthesis were accompanied by post-translational redox-activation of ADP-glucose pyrophosphorylase (AGPase), catalysing the key regulatory step of starch synthesis in the plastid, while there were no substantial changes in metabolic intermediates or sugar levels. A similar increase in post-translational redox-activation of AGPase was found after supplying adenine to wild-type potato tuber discs to increase adenine nucleotide levels. Results provide first evidence for a link between redox-activation of AGPase and adenine nucleotide levels in plants.

Novel Inhibitors of Phenylalanine Ammonia-lyase: 5-Aryl-1, 3, 4-oxathiazol-2-ones

A series of 5‐aryl‐1,3,4‐oxathiazol‐2‐ones was synthesized and tested for inhibition against phenylalanine ammonia‐lyase (PAL) derived from the yeast Rhodotorula glutinis. All compounds inhibited PAL at 100 µM. Of the compounds tested, 5‐(4‐nitrophenyl)‐1,3,4‐oxathiazol‐2‐one (19) was the most effective, showing IC50 value of 2.5 µM, which was 2 times stronger than that of N‐(aminooxy) acetyl‐2,5‐dichloroaniline (Z302), a known PAL inhibitor. Compound 19 behaved as a noncompetitive inhibitor of PAL. When potato tuber disks were treated with 19 in the presence of laminarin, a β ‐1,3‐glucooligosaccharide elicitor, the amount of trans‐cinnamic acid diminished after 48 hr, being consistent with an effective inhibition of PAL. Most of the compounds inhibited the growth of lettuce seedlings at 100‐500 µM. However, no correlation was observed between the ability of 5‐aryl‐1,3,4‐oxathiazol‐2‐ones to inhibit PAL derived from R. glutinis and growth inhibitory activity against lettuce seedlings.

An Important Pool of Sucrose Linked to Starch Biosynthesis is Taken up by Endocytosis in Heterotrophic Cells

We have recently shown the occurrence of endocytic sucrose uptake in heterotrophic cells. Whether this mechanism is involved in the sucrose-starch conversion process was investigated by comparing the rates of starch accumulation in sycamore cells cultured in the presence or absence of the endocytic inhibitors wortmannin and 2-(4-morpholynyl-)-8-phenyl-4H-1 benzopyran-4-1 (LY294002). These analyses revealed a two-phase process involving an initial 120 min wortmannin- and LY294002-insensitive starch accumulation period, followed by a prolonged phase that was arrested by the endocytic inhibitors. Both wortmannin and LY294002 led to a strong reduction of the intracellular levels of both sucrose and the starch precursor molecule, ADPglucose. No changes in maximum catalytic activities of enzymes closely linked to starch and sucrose metabolism occurred in cells cultured with endocytic inhibitors. In addition, starch accumulation was unaffected by endocytic inhibitors when cells were cultured with glucose. These results provide a first indication that an important pool of sucrose incorporated into the cell is taken up by endocytosis prior to its subsequent conversion into starch in heterotrophic cells. This conclusion was substantiated further by experiments showing that sucrose-starch conversion was strongly prevented by both wortmannin and LY294002 in both potato tuber discs and developing barley endosperms.

Enhancement of natural disease resistance in potatoes by chemicals

The mechanism involved in systemic acquired resistance (SAR) can be non-specifically induced in susceptible plants. In response to pathogens, plants' natural defence mechanisms include the production of lignin and phytoalexins and the induction of plant enzymes. The aim of this research was to study the induction of SAR mediated by the chemical activator DL-3-aminobutyric acid (BABA) and the fungicide fosetyl-aluminium in potato cultivars with different levels of resistance against Phytophthora infestans (Mont) de Bary. To study the chemical induction of the resistance, the foliage of several potato cultivars was sprayed with BABA, fosetyl-aluminium or water (as a control treatment). After 3 days the foliage was inoculated with P. infestans. Seven days after inoculation, development of disease symptoms in the foliage was assessed. In postharvest tuber samples, evidence for enhancement of the defence response was evaluated by measuring the protein content of several hydrolytic enzymes as well as the phenol and phytoalexin content. The highest level of protection against late blight was observed when the chemicals were applied at early stages of crop development. An increase in resistance to late blight was also detected in tubers after harvest. There was also an increase in the protein level of beta-1,3-glucanase and aspartic protease as well as in the phenol and phytoalexin content of potato tuber discs obtained from postharvest tubers of treated plants. Thus the protective effect seemed to persist throughout the whole crop cycle. This treatment may offer the possibility of controlling both foliage and tuber blight and could have a major impact in reducing over-winter survival of P. infestans in tubers.

Inhibition of trans-Cinnamate 4-Hydroxylase by 4-Amino-5-aryl-2,3-dihydro-3H-1,2,4-triazole-3-thiones

A number of 4-amino-5-aryl-2,3-dihydro-3H-1,2,4-triazole-3-thiones were synthesized and tested for inhibitory activity against trans-cinnamate 4-hydroxylase (C4H) from Populus kitakamiensis, which was expressed in yeast. Of the compounds tested, 4-amino-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (1) was the most effective, having an IC50 value of 0.32 µM. Replacement of the phenyl group with heterocycles or substituted phenyl groups drastically reduced the activity. Compound 1 behaved as a mixed-inhibitor of C4H. When potato tuber disks were treated with 1 (0.2–20 ng/disk) in the presence of laminarin, a β-1,3-glucooligosaccharide elicitor, trans-cinnamic acid was accumulated at levels 3 times higher than in disks treated with laminarin alone. © Pesticide Science Society of Japan

Evidence that SNF1-related kinase and hexokinase are involved in separate sugar-signalling pathways modulating post-translational redox activation of ADP-glucose pyrophosphorylase in potato tubers.

We recently discovered that post-translational redox modulation of ADP-glucose pyrophosphorylase (AGPase) is a powerful new mechanism to adjust the rate of starch synthesis to the availability of sucrose in growing potato tubers. A strong correlation was observed between the endogenous levels of sucrose and the redox-activation state of AGPase. To identify candidate components linking AGPase redox modulation to sugar supply, we used potato tuber discs as a model system. When the discs were cut from growing wild-type potato tubers and incubated for 2 h in the absence of sugars, redox activation of AGPase decreased because of a decrease in internal sugar levels. The decrease in AGPase redox activation could be prevented when glucose or sucrose was supplied to the discs. Both sucrose uptake and redox activation of AGPase were increased when EDTA was used to prepare the tuber discs. However, EDTA treatment of discs had no effect on glucose uptake. Feeding of different glucose analogues revealed that the phosphorylation of hexoses by hexokinase is an essential component in the glucose-dependent redox activation of AGPase. In contrast to this, feeding of the non-metabolisable sucrose analogue, palatinose, leads to a similar activation as with sucrose, indicating that metabolism of sucrose is not necessary in the sucrose-dependent AGPase activation. The influence of sucrose and glucose on redox activation of AGPase was also investigated in discs cut from tubers of antisense plants with reduced SNF1-related protein kinase activity (SnRK1). Feeding of sucrose to tuber discs prevented AGPase redox inactivation in the wild type but not in SnRK1 antisense lines. However, feeding of glucose leads to a similar activation of AGPase in the wild type and in SnRK1 transformants. AGPase redox activation was also increased in transgenic tubers with ectopic overexpression of invertase, containing high levels of glucose and low sucrose levels. Expression of a bacterial glucokinase in the invertase-expressing background led to a decrease in AGPase activation state and tuber starch content. These results show that both sucrose and glucose lead to post-translational redox activation of AGPase, and that they do this by two different pathways involving SnRK1 and an endogenous hexokinase, respectively.

Oospore Formation by Phytophthora infestans in Potato Tubers

ABSTRACT The ability of Phytophthora infestans, the causal agent of potato and tomato late blight, to produce oospores in potato tuber tissue was studied in the field and under laboratory conditions. In 1998 and 2000 field experiments, the canopy of potato cvs. Alpha and Mondial, respectively, were coinoculated with A1 + A2 sporangia of the fungus, and the infected tubers collected at harvest were examined for the presence of oospores. In 1998, only 2 of 90 infected tubers had oospores, whereas none of the 90 tubers examined in 2000 had any oospores. In the latter experiment, infected tubers kept in storage up to 12 weeks after harvest had no oospores. Artificial co-inoculations of whole tubers with A1 + A2 sporangia resulted only rarely in the formation of oospores inside the tubers. Co-inoculations of potato tuber discs taken from dormant tubers 0 to 16 weeks after harvest failed to support any oospore production, whereas discs taken from sprouting tubers of >/=18 weeks after harvest allowed oospores to form. Tuber discs showed enhanced oospore formation when treated before inoculation with either sugars, amino acids, casein hydrolysate, beta-sitosterol, or chloroethylphosphonic acid. In contrast, reducing airflow into the petri dishes where potato tuber discs were incubated reduced the number of oospores produced. The number of oospores produced in tuber tissue was lower compared with that in leaf tissue regardless of the origin of isolates used. The data show that the ability of Phytophthora infestans to produce oospores in potato tuber tissue is very limited and increases with tuber aging.

Cadmium and oxidative stress influence on phytochelatin synthase activity in potato tuber

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl2 induced biosynthesis of phytochelatin synthase (PCS). The intensity of this process depended on the concentration of cadmium ions (0.01 – 1 mmol·dm−3), time and cadmium resistance of tissues. In more resistant tissues, PCS activity was much higher and PCS was more resistant to oxidative stress. It seems that these tissues possessed more efficient cadmium detoxification system.

Soybean Kunitz, C-II and PI-IV inhibitor genes confer different levels of insect resistance to tobacco and potato transgenic plants

In modern, highly intensive agriculture, the control of insect pests is basically achieved with the appli- cation of chemical pesticides. Heavy reliance on this sole strategy is associated with several drawbacks, and the de- velopment of alternative or complementary methods to chemical control is desirable. In this work, three soybean genes (KTi3, C-II and PI-IV) coding for serine proteinase inhibitors were isolated by PCR and transferred to Agro- bacterium tumefaciens EHA 105, which in turn was used for transforming tobacco leaf and potato tuber discs. Bio- chemical assays confirmed that transgenic plants synthe- sized serine proteinase inhibitors; rates of expression varied among plants. The level of insect resistance (tested with Spodoptera littoralis Boisduval) was particularly high in tobacco, where many plants caused the death of all larvae. In potatoes, larval mortality was much less fre- quently achieved, but the results were still encouraging in that larval weight gain was reduced by 50% in the pres- ence of adequate amounts of inhibitor. When 8-day-old larvae were fed different KTi 3 -expressing tobacco plants, a highly significant (P<0.01) correlation was observed be- tween inhibitor content and larval live weight. Larval weight gain was found to be dependent on midgut proteo- lytic activity. On the basis of the evidence collected, it is suggested that further work is required to identify more specific inhibitors for the main proteinases of the target in- sect.

Beta-1,3-glucooligosaccharide induced activation of four enzymes responsible for N-p-coumaroyloctopamine biosynthesis in potato (Solanum tuberosum cv.) tuber tissue.

Potato tuber disks, when treated with laminarin, a beta-1,3-glucooligosaccharide from Laminaria digitata, accumulate a hydroxycinnamoyl amide compound, N-p-coumaroyloctopamine (p-CO). The biosynthesis of p-CO was investigated by feeding experiments, in order to show that the precursors of N-p-coumaroyl and octopamine moieties of p-CO are L-phenylalanine and L-tyrosine, respectively. The treatment of potato tuber tissue with laminarin resulted in elevated activities of four enzymes which are putatively involved in p-CO biosynthesis: phenylalanine ammonia lyase (PAL; EC 4.3.1.5), 4-hydroxycinnamic acid:CoA ligase (4CL; EC 6.2.1.12), hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25). Among these, the response of TyrDC was specific to laminarin treatment, thus indicating that the regulation of TyrDC activity is critical for the accumulation of p-CO in potato tuber tissue.