Effect of Chitosan on Systemic Viral Infection and Some Defense Responses in Potato Plants

The development and the possible mechanism of the chitosan-induced resistance to viral infection were investigated in potato plants. The plants were sprayed with a solution of chitosans (1 mg/ml) with the mol wt of 3, 36, and 120 kD. After 1, 2, 3, or 4 days, the treated leaves were cut off and mechanically infected with the potato virus X (PVX). The disks cut out from the inoculated leaves were used for determining virus accumulation, callose content, and ribonuclease and -1,3-glucanase activities. In another set of experiments, the plants were infected with PVX within 1, 4, or 8 days after chitosan treatment, and the number of systemically infected plants was determined. It was found that, a day after treatment, the plants acquired a resistance to viral infection. The disks from the chitosan-treated leaves, as compared to the control, accumulated less amount of virus. The chitosan treatment also significantly decreased the number of systemically infected plants as compared to the control. After 2–3 days, the resistance disappeared or even gave way to an increased susceptibility to the infection; subsequently, the resistance increased again. The extent of the resistance correlated with the callose content and the level of ribonuclease activity observed on the infection day. The resistance towards the infection with PVX is probably mediated by the callose and ribonuclease induction. The cultivation of test-tube potato plants from the cuttings previously infected with PVX on the chitosan-containing nutrient medium did not eradicate the viral infection from the plants.     

Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris

The effect of regurgitant from Leptinotarsa decemlineata Say larvae on wound-induced responses was studied using two plant species, Solanum tuberosum L. and Phaseolus vulgaris L. Wounding of one leaf of intact S. tuberosum plants differentially affected ethylene production and activities of peroxidase and polyphenol oxidase. Only polyphenol oxidase activity was stimulated by wounding in both wounded and systemic leaves. Peroxidase activity was not affected by wounding. Wounding caused only a transient increase of ethylene production from wounded leaves. The application of regurgitant to wound surfaces stimulated ethylene production as well as activities of peroxidase and polyphenol oxidase in both wounded and systemic leaves. Wounding significantly enhanced ethylene production and polyphenol oxidase activity in wounded and systemic leaves of P. vulgaris . The application of regurgitant caused an amplification of ethylene production, peroxidase activity, and polyphenol oxidase activity, in both wounded and systemic leaves of bean plants. Several substances were tested for their role as possible endogenous signals in P. vulgaris . Hydrogen peroxide and methyl jasmonate appeared as potential local and systemic signals of ethylene formation in wounded bean plants. Local ethylene production in leaf discs was differentially affected by the regurgitant application in potato versus bean plants. While all tested concentrations of regurgitant caused stimulation of ethylene formation from potato leaf discs, ethylene production was completely inhibited by increasing concentrations of the regurgitant in bean leaf discs. Our data present evidence that ethylene may play an important role in the interaction between plants and herbivores at the level of recognition of a particular herbivore leading to specific induction of signalling cascades.     

Variations of Phenoloxidase Activities as a Consequence of Stresses that Induce Resistance to Fusarium Wilt of Tomato

The variations over 7–8 day of peroxidase (PO) and polyphenoloxidase (PPO) activity have been investigated in tomato plants the roots of which had been subjected to stresses (heat, chloroform and a non-pathogenic form of Fusarium oxysporum) that induce resistance to Fusarium oxysporum f. sp. lycopersici. All treatments induced increase of PO and PPO activity that reached a maximum 3 days after the treatments in leaves, 4 days in stem and roots and were higher in leaves than in other parts. Activity decreased to levels for the control plants after 8 days. Inoculation with Fusarium oxysporum f. sp. lycopersici further stimulated PO and PPO activity in all treated plants over that caused by the treatments alone. Again, activity of treated plants was lower than in controls 7 days after inoculation. It is concluded that 1. increased PO and PPO activity in tomato is a systemic response to cellular injury caused in the root by heat, chloroform and non-pathogenic Fusarium oxysporum, 2. these treatments do no prevent the pathogen from interacting with the plants and inducing further enzyme increase, 3. treated plants react more strongly to the challenge inoculation than untreated plants.     

Microbial populations,fungal species diversity and plant pathogen levels in field plots of potato plants expressing theBacillus thuringiensis var.tenebrionis endotoxin

The environmental release of genetically engineered (transgenic) plants may be accompanied by ecological effects including changes in the plant-associated microflora. A field release of transgnic potato plants that produce the insecticidal endotoxin ofBacillus thuringiensis var.tenebrionis (Btt) was monitored for changes in total bacterial and fungal populations, fungal species diversity and abundance, and plant pathogen levels. The microflora on three phenological stages of leaves (green, yellow and brown) were compared over the growing season (sample days 0, 21, 42, 63 and 98) for transgenic potato plants, commercial Russet Burbank potato plants treated with systemic insecticide (Di-Syston) and commercial Russet Burbank potato plants treated with microbialBtt (M-Trak). In addition, plant and soil assays were performed to assess disease incidence ofFusarium spp.,Pythium spp.,Verticillium dahliae, potato leaf roll virus (PLRV) and potato virus Y (PVY). Few significant differences in phylloplane microflora among the plant types were observed and none of the differences were persisent. Total bacterial populations on brown leaves on sample day 21 and on green leaves on sample day 42 were significantly higher on the transgenic potato plants. Total fungal populations on gree leaves on sample day 63 were significantly different among the three plant types; lowest levels were on the commerical potato plants treated with systemic insecticide and highest levels were on the commercial potato plants treated with microbialBtt. Differences in fungal species assemblages and diversity were correlated with sampling dates, but relatively consistent among treatments.Alternaria alternata, a common saprophyte on leaves and in soil and leaf litter, was the most commonly isolated fungus species for all the plant treatments. Rhizosphere populations of the soilborne pathogensPythium spp.,Fusarium spp. andV. dahliae did not differ between the transgenic potato plants and the commercial potato plants treated with systemic insecticide. The incidence of tuber infection at the end of the growing season by the plant pathogenV. dahliae was highest for the transgenic potato plants but this difference was related to longer viability of the transgenic potato plants. This difference in longevity between the transgenic potato plants and the commercial + systemic insecticide potato plants also made comparison of the incidence of PVY and PLRV problematic. Our results indicate that under field conditions the microflora of transgenicBtt-producing potato plants differed minimally from that of chemically and microbially treated commerical potato plants.     

Effects of Daylength on Endogenous Gibberellins in Solanum andigena III. Gibberellin Production by the Leaves

Using an agar diffusion technique, it was found that leaves from potato plants growing under long days produced more gibberellin-like substances than did leaves from plants growing under short days. Short day plants irradiated with red light during the long dark period and harvested during the ensuing light period, contained levels of gibberellin-like substances approximating those found in long day grown plants. Red irradiation during the long dark period also resulted in an increase in gibberellin production in short day plants. Four zones of gibberellin-like activity (A, B, C, D) were separated by thin layer chromatography in extracts from potato leaves. Red light treatment reduced the levels of peak D and brought about a concomitant increase in the levels of peak A.     

Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning

Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content. To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer. Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels. Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning. In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type. However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content. Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance. Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period. Old source leaves accumulate up to 100-fold more soluble sugars than control leaves. This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%). Transgenic potato plants showed a less dramatic change in their growth behaviour. Plants were slightly reduced in size, with stems more highly branched. Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.     

Induction of early blight resistance in tomato by <Emphasis Type="Italic">Quercus infectoria</Emphasis> gall extract in association with accumulation of phenolics and defense-related enzymes

Treatment of tomato leaves with aqueous extract (0.5%) of the galls of Quercus infectoria significantly reduced infection from subsequent inoculation with Alternaria solani, the tomato early blight pathogen. When the leaves were challenge-inoculated with A. solani 3 d after application of Q. infectoria gall extract (QIGE), the percent defoliation decreased from 33.6 to 7.3. Two to three day pre-treatment with QIGE reduced the percent defoliation by 77 percent. The biochemical responses of tomato plants to QIGE were also studied. In tomato plants treated with QIGE, phenolic content increased rapidly, reached the maximum at 2 d after treatment. Phenylalanine ammonia-lyase (PAL) activity increased significantly from 1 d after treatment and the maximum enzyme activity was recorded 2 d after treatment at which period a 3-fold increase in PAL activity was observed when compared to the control. Peroxidase (PO) activity was also significantly increased 1 d after treatment and the maximum activity was reached 2 d after treatment. Peroxidase isozyme analysis indicated that PO-1 was increased dramatically in tomato leaves 1 d after treatment and maintained at the same level throughout the experimental period of 6 d. When tomato leaves were treated with QIGE, a two-fold increase in chitinase and β-1,3-glucanase activities was recorded 2 and 3 d respectively, after treatment. The enhanced activities of defense-related enzymes and elevated levels of phenolics in QIGE-treated tomato plants between 1 and 3 d after treatment suggest that these induced biochemical defenses may be involved in the suppression of early blight by QIGE.     

Accumulation of vitamin E in potato (Solanum tuberosum) tubers

Vitamin E (tocopherol) is a powerful antioxidant essential for human health and synthesized only by photosynthetic organisms. The effects of over-expression of tocopherol biosynthetic enzymes have been studied in leaves and seeds, but not in a non-photosynthetic, below-ground plant organ. Genetic and molecular approaches were used to determine if increased levels of tocopherols can be accumulated in potato (Solanum tuberosum L.) tubers through metabolic engineering. Two transgenes were constitutively over-expressed in potato: Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana homogentisate phytyltransferase (At-HPT). α-Tocopherol levels in the transgenic plants were determined by high-performance liquid chromatography. In potato tubers, over-expression of At-HPPD resulted in a maximum 266% increase in α-tocopherol, and over-expression of At-HPT yielded a 106% increase. However, tubers from transgenic plants still accumulated approximately 10- and 100-fold less α-tocopherol than leaves or seeds, respectively. The results indicate that physiological and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato tubers. Studying regulation and induction of tocopherol biosynthesis should reveal approaches to more effectively engineer crops with enhanced tocopherol content.     

Effect of freezing and thawing on the distribution of lysosomal hydrolases in leaves of Solanum tuberosum L.

Density-gradient ultracentrifugation techniques showed that freezing and thawing of potato leaves resulted in a change in the density of subcellular particles containing acid phosphatase and acid ribonuclease (RNase). Gel filtration experiments were used to characterise the molecular forms of the hydrolases associated with the various cell fractions. Freezing and thawing promoted a release of a portion of the complement of acid phosphatase and RNase from the lysosomes to the supernatant fluid fraction of cell homogenates. The freezing treatment appeared to activate latent lysosomal RNase.     

Ribonuclease activity in roots of soybean seedlings subjected to chilling stress and recovery

Many developmental and physiological changes, including alterations of enzyme activities, occur in plants under low temperature stress. In this study the total ribonuclease activity was determined in crude extracts from root tips of soybean seedlings germinated at 25 °C, subjected to chilling conditions (10°C) and recovered at optimal temperature (25°C). Measurements of RNase activity were performed every 24 hours starting from the third to the 10-th day of growth. We found that chilling caused a considerable increase in ribonuclease activity (in comparison with the control), with an activity peak on the fourth day of the cold treatment. The enzyme activity in root extracts of the plants recovered after cold stress decreased along with the time of recovery. No differences were found in approximate molecular weight (35 kDa) and pH optimum (6.0) for ribonucleases extracted from control and chilled soybean roots.     

Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.     

Zebra chip disease incidence on potato is influenced by timing of potato psyllid infestation, but not by the host plants on which they were reared

Abstract  The Zebra chip (ZC) syndrome is an emerging disease of potato and a major threat to the potato industry. The potato psyllid, Bactericerca cockerelli (Sulc) is believed to be a vector of the ZC pathogen, which is now thought to be Candidatus Liberibacter, a bacterium. To further understand the relationship between potato psyllid infestation and ZC disease expression, healthy potato plants at different growth stages (4, 6 and 10 weeks after germination) were exposed separately to potato psyllids that were separately reared on four solanaceous hosts plants (potato, tomato, eggplant or bell pepper) for more than 1 year. ZC symptoms, leaf rates and total nonstructural carbohydrate accumulation in leaves and tubers of healthy and psyllid-infested plants were monitored and recorded. Typical ZC symptoms were observed in leaves and tubers of all plants exposed to potato psyllids regardless of the host plant on which they were reared. This was also accompanied by significant reductions in net photosynthetic rate. Caged potato plants without exposure to potato psyllids (uninfested controls) did not show any ZC symptom in both foliage and in harvested tubers. Foliage damage and ZC expression were most severe in the potato plants that were exposed to potato psyllids 4 weeks after germination compared to plants infested at later growth stages. Tubers from potato psyllid-infested plants had significantly higher levels of reducing sugars (glucose) and lower levels of starch than those in healthy plants, indicating that potato psyllid infestation interfered with carbohydrate metabolism in either leaves or tubers, resulting in ZC expression.     

Molecular cloning and characterisation of cDNAs complementary to mRNAs from wounded potato (Solanum tuberosum) tuber tissue

Five cDNA clones complementary to mRNAs representing different abundances and responses to wounding have been isolated from a library of Sau 3A fragments in the bacteriophage M13 mp8. These were characterised by hybrid-release translation and hybridisation to RNA blots. The levels of RNA complementary to two of the clones show a marked increase during the 24h after wounding, one shows a small increase and two show no appreciable changes except that caused by a general increase in the total amount of polyadenylated RNA per microgram of total RNA which increases 2.5-fold during the same period. The would-induced RNAs are not induced in diluted suspension-culture cells, but RNA complementary to each clone is present in varying levels in stems, leaves and roots of intact potato plants.Abbreviations cDNA complementary DNA - poly(A) polyadenosine - poly(A)⁺ RNA polyadenylated RNA - poly(U) polyuridine     

Ribonuclease activity of stressed tomato leaflets

Homogenates of leaflets of desiccated tomato plants show increased ribonuclease activity compared to homogenates of turgid controls. Much of this increase is independent of changes in translocation to and from the leaflet. Interruption of translocation through living cells by detachment of leaflets or steam damage to the petiolules produces increased ribonuclease activity, but this activity is increased further when excised leaflets are allowed to wilt. Increases in ribonuclease often parallel or precede increases in the soluble nitrogen content. Further increases in activity occur when excised leaves become yellow. Exposure of leaflets to CO₂-free air has little effect on activity at low-light intensity (120 ft-c). These results suggest that water stress directly affected ribonuclease activity at the cellular level.     

A comparative study of two RNase isozyme activities inNicotiana glutinosa leaves

Effects of some chemicals on the activities of two ribonuclease isozymes, M and F, in the leaves ofNicotiana glutinosa were investigated. CHX and AMD suppressed the increase of M isozyme activity induced with mock-inoculation. On the other hand, CHX suppressed slightly the increase of F izozyme activity induced with TMV-inoculation, but AMD and 5-FU stimulated it.     

Roots and leaves display contrasting oxidative response during salt stress and recovery in cowpea

In this study, we compare some antioxidative responses of leaves and roots associated to growth reduction in cowpea plants (Vigna unguiculata) during short-term salt stress and recovery. The salt treatment was imposed (200 mM NaCl) for six consecutive days and the salt withdrawal after 3 d. The salt treatment caused an almost complete cessation in the relative growth rate of both leaves and roots. Although NaCl withdrawal has induced an intense reduction in the Na(+) content from the leaves and roots, the growth recovery was slight, after 3 d. The leaf lipid peroxidation was increased in salt-stressed plants and slightly reduced in recovered plants after 3 d. Surprisingly, in the salt-stressed roots it decreased markedly after 3 d treatment and in the pre-stressed/recovered roots it was restored to levels near to the control. In leaves, catalase (CAT) activity showed a rapid and prominent decrease after 1 d of NaCl treatment and salt withdrawal had no effect on its recovery. In contrast, the root CAT activity was not changed by effects of both NaCl and salt withdrawal, over time interval. Leaf superoxide dismutase (SOD) activity did not change in all treatments, whereas in roots it significantly decreased after 3 d of salt treatment and recovered after NaCl withdrawal. Contrasting to the other enzymes, the guaiacol-peroxidase activity increased in leaves and roots, reaching almost 200% of control values and it significantly decreased in both organs from the pre-stressed/recovered plants. In conclusion, cowpea roots and leaves present distinct mechanisms of response to lipid peroxidation and CAT and SOD activities during salt stress and recovery. However, these responses and/or the oxidative damages caused by reactive oxygen species were not related with the growth reduction.     

Studies on the chromatin of barley leaves during senescence

1. The activity of soluble ribonuclease and deoxyribonuclease first declined during senescence, but later increased during advanced stages of senescence. 2. Young leaves had very low ribonuclease or deoxyribonuclease activity associated with the chromatin, but the activity of these enzymes increased progressively during senescence until the leaves died. 3. No significant changes in the composition of chromatin from first seedling leaves of barley plants during aging (from 7 to 25 days) were noted. 4. The amount of RNA synthesized by chromatin in vitro declined as the leaf aged. However, if the loss of RNA due to chromatin-associated ribonuclease was taken into account, the RNA-synthesizing activity of chromatin from senescing (15-16-day-old) leaves appeared to be somewhat higher than that of chromatin from young (7-8-day-old) leaves. In leaves at the terminal stages of senescence (23 days old) the estimates of RNA synthesis by chromatin could not be made owing to complications created by high nuclease activities. 5. It is suggested that senescence may be triggered by a decline in some hormonal factor in leaves, and that the resulting production of chromatin-associated deoxyribonuclease and ribonuclease in increasing proportions may progressively cause increased degradation of DNA and newly synthesized RNA, so that ultimately the cellular functions are impaired and the cells die.