87 kDa tomato cystatin exhibits properties of a defense protein and forms protein crystals in prosystemin overexpressing transgenic plants

Transgenic tomato plants (Lycopersicon esculentum) overexpressing the prosystemin transgene have been shown previously to accumulate a soluble 87 kDa cystatin constitutively. We report here that this protein can be found in a crystalline form which can be purified using a glycerol/sucrose gradient. Midgut homogenate of third-instar larvae of two coleopteran pest insects, Callosobruchus maculatus and Zabrotes subfasciatus, had their proteolytic activity content significantly inhibited by tomato cystatin (TC). In leaves of wild-type tomato plants, cystatin mRNA accumulated systemically in response to wounding, treatment with methyl jasmonate (MJ) and when supplied with systemin, corroborating the anti-herbivorous activity. Accumulation of cystatin mRNA occurred when plants were supplied with chitosan and oligogalacturonic acid fragments (OGA), suggesting an effect of TC against pathogens. Moreover, this protein reduced the growth of two fungi, Fusarium solani and Trichoderma viride in vitro. Taken together, the data reinforce a role for TC in defense response against pests or pathogens.     

Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants

A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculentum, cv Bonnie Best) that had been sprayed with methyl jasmonate. For seedlings, cystatin purification was accomplished using EDTA washing, KCI extraction, 70 degrees C heat treatment, ammonium sulfate fractionation and gel filtration chromatography. For mature plants, DEAE chromatography was also needed to separate a protease, hydrolysis products of cystatin and serine proteinase inhibitors from the intact cystatin. Purified tomato cystatin has a molecular weight (Mr) of 88 kDa, eight papain binding domains, is a non-competitive inhibitor of papain with K1 of 1.4 nM and is not a glycoprotein. Tryptic peptides (Mr 26, 13 kDa) and most chymotryptic peptides (Mr 33, 13 kDa) of tomato cystatin retain inhibitor activity. Amino acid analysis revealed no Cys; Asx, Glx, Gly, Ser accounted for almost half the residues and there was some homology with potato multicystatin. Activity is stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at > 60 degrees C (Ea = 92.5 kJ/mole). Extracts of mature plants treated with methyl jasmonate contain lower Mr cystatins that appear to result from the action of an endogenous 26 kDa protease on the 88 kDa inhibitor.     

Oligosaccharides that induce proteinase inhibitor activity in tomato plants cause depolarization of tomato leaf cells

Abstract. Two size ranges of oligosaccharide elicitors of pectic origin have been investigated for their effects on tomato plants. Both size ranges, with degrees of polymerization of 1–7 and 10–20 respectively, induced the accumulation of proteinase inhibitor (PI) activity in excised plants, and also induced changes in membrane potential of leaf mesophyll cells. The depolarizations were substantial, rapid, and reversible on removal of the elicitors. The effects are discussed in the context of early events in the signal transduction pathway linking oligosaccharides to changes in PI gene expression.     

Effects of soil flooding on leaf gas exchange of tomato plants

Carbon dioxide and water vapor exchange of tomato (Lycopersicon esculentum Mill. cv Rheinlands Ruhm) leaves were measured before and after 24 h of soil flooding to characterize both stomatal and nonstomatal responses to the stress. Leaf epidermal conductance to water vapor decreased by 47% after flooding, accompanied by an increase in the sensitivity of stomata to changes in CO₂ concentration. Assimilation rates under ambient conditions fell by 27%, and the inhibition could not be overcome by elevated CO₂ partial pressures. Stomatal conductance limited the assimilation rate to approximately the same degree both before and after flooding. The reduction in photosynthetic capacity was not due to a decrease in apparent quantum yield or to an increase in photorespiration. The results were analyzed according to a recent model of photosynthesis, and possible mechanisms underlying the flooding effect are discussed.     

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.Abbreviation PG polygalacturonase The authors of this paper are grateful to David Jackson of the John Innes Institute, Norwich, UK for his assistance with the in-situ hybridisation work. This research was supported by an Agricultural and Food Research Council Post-Doctoral award to J.E.T., and by a grant to D.G. from the Science and Engineering Research Council Biotechnology Directorate in association with ICI seeds. The work was carried out under Ministry of Agriculture, Food and Fisheries licences.     

Immunolocalization of 67 kDa elastin-binding protein in perinatal rat lungs

Summary 67 kDa elastin-binding protein (RL-67EBP) has been isolated from neonatal rat lungs by the use of an elastin-coupled affinity column, followed by elution with either lactose or synthetic elastin hexapeptide (VGVAPG), and immunohistochemistry has been used on perinatal rat lungs to determine the tissue localization of this protein. No immunoreactive structures occur in fetal lungs, or in the lungs of day-1 and-4 neonates. On day-7 after birth, immunoreactive cells appear in the subepithelial connective tissue of the intrapulmonary airways, from day-10 on, these cells become evenly distributed in the alveolar parenchyma. Occasionally, some cells occur in the alveolar air space, being free from the surface of the alveolar septum. Unpermeabilized cells obtained by bronchoalveolar lavage, show cell surface immunoreactivity, indicating that RL-67EBP is expressed on the surface membrane of the cells. From these findings, it is suggested that the immunoreactive cells are blood-borne monocytes, and that RL-67EBP may function as an elastin peptide receptor by which monocytes mobilize through interstitial connective tissue during their migration from blood to alveolar air space, where they eventually differentiate into alveolar macrophages.     

Effect of leaf nitrogen content of tomato plants on preference and performance of a leafmining fly

Summary In a study on intraspecific host plant acceptability, Liriomyza trifolii females that had previously been exposed to plants of high nitrogen content, showed a feeding and oviposition preference for plants of high nitrogen (Minkenberg and Fredrix 1989). Females showed a preference to feed and oviposit on the high middle leaves within plants. It was hypothesized that the preference between plants was related to a better performance of females and offspring on high nitrogen plants compared to low nitrogen plants. Different nitrogen dosages were applied to tomato plants, resulting in plants containing 3.4, 3.9, 4.6 or 4.9% leaf nitrogen. L. trifolii females responded to increased leaf nitrogen with significantly increased feeding and fecundity, longer oviposition periods, and higher feeding and oviposition rates. Their offspring on the same plants showed reduced developmental time, lower mortality and increased pupal size. Consequently, intrinsic rate of increase was positively linearly related to leaf nitrogen. Size of L. trifolii females appeared to be independent of fecundity, longevity and developmental time. Pupal length of males increased with increasing developmental time. These results indicate that L. trifolii is well adapted in its intraspecific host plant selection, because the ability to distinguish between plants with differences in leaf nitrogen content will directly lead to an increase in their fecundity, longevity and overall fitness. The implications of leaf nitrogen as a significant factor in the behavior and population dynamics of L. trifolii are discussed.     

Fungal pathogen-induced changes in the antioxidant systems of leaf peroxisomes from infected tomato plants

Peroxisomes, being one of the main organelles where reactive oxygen species (ROS) are both generated and detoxified, have been suggested to be instrumental in redox-mediated plant cell defence against oxidative stress. We studied the involvement of tomato (Lycopersicon esculentum Mill.) leaf peroxisomes in defence response to oxidative stress generated upon Botrytis cinerea Pers. infection. The peroxisomal antioxidant potential expressed as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (GSH-Px, EC 1.11.1.19) as well as the ascorbate-glutathione (AA-GSH) cycle activities was monitored. The initial infection-induced increase in SOD, CAT and GSH-Px indicating antioxidant defence activation was followed by a progressive inhibition concomitant with disease symptom development. Likewise, the activities of AA-GSH cycle enzymes: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) as well as ascorbate and glutathione concentrations and redox ratios were significantly decreased. However, the rate and timing of these events differed. Our results indicate that B. cinerea triggers significant changes in the peroxisomal antioxidant system leading to a collapse of the protective mechanism at advanced stage of infection. These changes appear to be partly the effect of pathogen-promoted leaf senescence.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

Abscisic acid root and leaf concentration in relation to biomass partitioning in salinized tomato plants

Salinization is one of the most important causes of crop productivity reduction in many areas of the world. Mechanisms that control leaf growth and shoot development under the osmotic phase of salinity are still obscure, and opinions differ regarding the Abscisic acid (ABA) role in regulation of biomass allocation under salt stress. ABA concentration in roots and leaves was analyzed in a genotype of processing tomato under two increasing levels of salinity stress for five weeks: 100 mM NaCl (S10) and 150 mM NaCl (S15), to study the effect of ABA changes on leaf gas exchange and dry matter partitioning of this crop under salinity conditions. In S15, salinization decreased dry matter by 78% and induced significant increases of Na⁺ and Cl⁻ in both leaves and roots. Dry matter allocated in different parts of plant was significantly different in salt-stressed treatments, as salinization increased root/shoot ratio 2-fold in S15 and 3-fold in S15 compared to the control. Total leaf water potential (Ψ_w) decreased from an average value of approximately −1.0 MPa, measured on control plants and S10, to −1.17 MPa in S15. In S15, photosynthesis was reduced by 23% and stomatal conductance decreased by 61%. Moreover, salinity induced ABA accumulation both in tomato leaves and roots of the more stressed treatment (S15), where ABA level was higher in roots than in leaves (550 and 312 ng g⁻¹ fresh weight, respectively). Our results suggest that the dynamics of ABA and ion accumulation in tomato leaves significantly affected both growth and gas exchange-related parameters in tomato. In particular, ABA appeared to be involved in the tomato salinity response and could play an important role in dry matter partitioning between roots and shoots of tomato plants subjected to salt stress.     

The contribution of tomato and alternative host plants to tomato leaf curl virus inoculum pressure in different areas of South India

Indian tomato leaf curl virus (ToLCV) (Geminiviridae: Sub-group III) was detected both in field-collected and laboratory-reared B. tabaci using a triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). ToLCV was detected in six of the 10 group samples of field collected B. tabaci. ToLCV was also identified in 13 weed species commonly found in Karnataka, both by symptom expression and TAS-ELISA. ToLCV from c. 61% of infected plants was transmitted successfully to tomato by B. tabaci. Tomato plots were planted at three locations on the University of Agricultural Sciences Campus, Bangalore. Indian tomato leaf curl virus disease (ToLCVD) incidence increased most rapidly when the tomato plot was situated adjacent to an older ToLCVD-infected tomato field. When the plots were positioned in a dryland or a wetland area, at least 500 m away from any infected tomato fields, the ToLCVD incidence increased less rapidly, although in all sites it was 100% by 11 wk after transplanting. The numbers of B. tabaci caught on yellow traps in all sites increased during weeks 1–3 after transplanting and thereafter remained at between 10–15 adults trap^-1 24 h^_1. Adult numbers recorded on tomato plants by direct counts remained approximately constant at 2–4 adults plant“”¹. Tomato fields were planted in three taluks (administrative areas) of Karnataka, that had different current and previous histories of tomato production. ToLCVD incidence increased most and least rapidly, respectively, in Kolar taluk where tomato is grown continuously and Doddaballapur tuluk where tomato was grown in the area for the first time. In Malur tuluk, where tomato was grown discontinuously (once a year), the incidence of ToLCVD increased at an intermediate rate. Weed host-plant species growing near the experimental sites had averages of between 1.5–10.0 B. tabaci nymphs per plant, whereas the tomato plants had only 0.3 nymphs per plant. The percentage parasitism of B. tabaci nymphs on tomato and weed species, respectively, was 0.7% and 2–6%. Nymphs and pupae were parasitised by an Encarsia sp. and Eretmocerus mundus Mercet. The relevance and implications of these findings for the epidemiology and management of ToLCVD in Karnataka State, South India is discussed.     

Effect of Pepsin–Trypsin In Vitro Gastro-Intestinal Digestion on the Antioxidant Capacities of Ultra-Filtrated Rice Bran Protein Hydrolysates (Molecular Weight > 10 kDa; 3–10 kDa,and < 3 kDa)

International Journal of Peptide Research and Therapeutics - The antioxidant capacities of rice bran protein hydrolysates (RBPH) obtained by in vitro gastro-intestinal (GI) digestion by...     

Evaluation of barrier plants for the cultural control of tomato yellow leaf curl disease

Barrier cropping plays an essential role in controlling insect pests and insect-transmitted diseases in cultural control. It has been proven efficient in suppressing the spread of nonpersistently transmitted viruses. For suppressing the spread of persistently transmitted viruses, barrier cropping is not considered an effective control strategy because barrier plants cannot act as a virus sink to purge the virus in the vector. However, few successful cases of barrier cropping suppressing the spread of persistently transmitted viruses have been reported. The objectives of the present study were to screen candidates (cucumber, okra, Chinese kale, soybean, and corn) for potential barrier plants to control tomato yellow leaf curl Thailand virus (TYLCTHV) and examine whether prefeeding on these plants can reduce the virus titer in its vector, Bemisia tabaci, thus reducing TYLCTHV transmission. The results revealed that nonviruliferous whiteflies preferred cucumber and okra to tomato, whereas viruliferous whiteflies preferred cucumber to tomato. Although prefeeding on cucumber, okra, and Chinese kale did not reduce the titer of TYLCTHV in viruliferous whiteflies, the vector transmission rate decreased after the whiteflies fed on Chinese kale. It implies that planting Chinese kale as a barrier plant for tomato cultivation may reduce the incidence of TYLCTHV. In addition, the preference to cucumber plants may reduce the incidence of whiteflies acquiring TYLCTHV from virus-infected tomato plants and of viruliferous whiteflies inoculating the virus into healthy tomato plants, thereby reducing the disease incidence. Further field trials of barrier cropping using the candidate plants are warranted.     

Interrelated responses of tomato plants and the leaf miner Tuta absoluta to nitrogen supply

Plant–insect interactions are strongly modified by environmental factors. This study evaluates the influence of nitrogen fertilisation on the tomato (Solanum lycopersicum L.) cv. Santa clara and the leafminer (Tuta absoluta (Meyrick), Lepidoptera: Gelechiidae). Greenhouse‐grown tomato plants were fed hydroponically on a complete nutrient solution containing either a high nitrogen concentration (HN) sustaining maximum growth or a low nitrogen concentration (LN) limiting plant growth. Insect‐free plants were compared with plants attacked by T. absoluta. Seven and 14 days after artificial oviposition leading to efficacious hatching and larvae development, we measured total carbon, nitrogen and soluble protein as well as defence compounds (phenolics, glycoalkaloids, polyphenol oxidase activity) in the HN versus LN plants. Only in the HN treatment did T. absoluta infestation slightly impair leaf growth and induce polyphenol oxidase (PPO) activity in the foliage. Neither the concentration of phenolic compounds and proteins nor the distribution of nitrogen within the plant was affected by T. absoluta infestation. In contrast, LN nutrition impaired T. absoluta‐induced PPO activity. It decreased protein and total nitrogen concentration of plant organs and enhanced the accumulation of constitutive phenolics and tomatine. Moreover, LN nutrition impaired T. absoluta development by notably decreasing pupal weight and lengthening the development period from egg to adult. Adjusting the level of nitrogen nutrition may thus be a means of altering the life cycle of T. absoluta. This study provides a comprehensive dataset concerning interrelated responses of tomato plants and T. absoluta to nitrogen nutrition.