The noctuid, Helicoverpa armigera, is the most important crop pest worldwide. We evaluated the biological activity of soybean trypsin inhibitor and plant lectins against this pest to identify toxin genes for deployment through transgenic plants. Of the seven plant lectins tested, chickpea and snowdrop lectins showed marked antibiotic effects in terms of insect survival and development. Larval survival was lower in artificial diet impregnated with soybean trypsin inhibitor (49%), and snowdrop (64%) and chickpea (65%) lectins compared to untreated control diet (90%). Pupal weight was adversely affected by chickpea lectin (272.6 mg) compared as to untreated control (335.4 mg). Lower pupation and/or adult emernence (<50%) was observed in diets impregnated with soybean trypsin inhibitor and chickpea, snowdrop and peanut lectins as compared to 90% pupation/adult emergence on untreated control diet. Soybean trypsin inhibitor, and lectins from snowdrop, peanut, and chickpea can be considered for deployment through transgenic plants for the management of H. armigera.
Salicylic acid (SA)-induced protein kinase (SIPK) in Nicotiana tabacum L. is activated in response to various stimuli. We assessed the response of SIPK in N. benthamiana. leaves to ultraviolet-C (UV-C) and SA stress. Infiltration of Agrobacterium tumefaciens EHA105 cells containing pBE2113-GST-SIPK into leaves gave transient production of glutathione-S transferase (GST)-fused SIPK. Irradiation of agroinfiltrated leaves with UV-C (254 nm) activated GST-SIPK and endogenous protein kinases with a molecular mass of about 45 kDa, close to that of SIPK simultaneously. These results suggest that SIPK is involved in stress signalling in Nicotiana plants under UV-C irradiation.
Wild watermelon plants are drought and excess light stress-tolerant despite carrying out normal C3-type photosynthesis. In this study, a system was established for the genetic transformation of wild watermelons. Adventitious shoots were induced efficiently from cotyledon explants on medium containing 4 mg l−1 of benzyladenine. The explants were infected with Agrobacterium carrying a plasmid containing selectable marker genes, nptII (neomycin phosphotransferase), hpt (hygromycin phosphotransferase), and a reporter gene gusA (β-glucuronidase). A β-Glucuronidase (GUS) histochemical assay showed that kanamycin was more effective than hygromycin as the selective agent for transformation. The presence of T-DNA in the regenerated shoots was confirmed by amplification of the transgene using polymerase chain reaction. Southern blot analysis revealed stable integration of the transgene in the T1 progeny. The system presented here will provide an experimental basis for molecular studies of wild watermelon genes, and thus facilitate an understanding of their contribution to stress tolerance in this plant.
Cadmium (Cd) is a highly toxic element for living organisms. Its toxicity and the thereof by calcium (Ca) in 2-week-old Arabidopsis seedlings were studied. The seedlings were treated with Cd at concentrations of 50–500 µM for 24 h. Retardation of root growth was evident through increased cell death at the root elongation zone. Supplementing with 30 mM Ca restored root elongation in the presence of up to 200 µM Cd, resulting in up to a 1/3 decrease in Cd content in the seedlings compared to Arabidopsis treated with Cd alone. Ca was able to alleviate Cd toxicity, presumably through competition for metal ion influx. These results provide us with information regarding plant ion uptake systems and metal ion toxicities.
Adenosine phosphosulfate (APS) reductase is one of key enzymes in the sulfur assimilation pathway in higher plants, catalyzing the formation of adenosine 5′-phosphosulfate from sulfate and ATP. In order to improve sulfur uptake capacity of water spinach (Ipomea aquatica), a plant which commonly grows wild in Southern Asia and has good potential for sequestration of environmental pollutants like sulfuric compounds, an Arabidopsis gene (APR1), encoding a plastid-resident APS reductase, was introduced into cut cotyledons via Agrobacterium-mediated transformation. Among 267 regenerated shoots initially obtained from 2,119 cotyledon explants, two were found to efficiently express the introduced gene and could be grown to maturity. APS reductase activity in leaves was estimated to be over 2-fold the wild-type level. Upon cultivation in the presence of 2 mM sodium sulfate, a 2.5-fold higher sulfate uptake was observed in comparison with wild-type plants. When grown in the presence of toxic levels of sulfide or cadmium, they showed a higher tolerance with increased fresh weight as compared with controls. These results suggest that transcription from the introduced gene indeed strengthened the sulfur assimilation pathway, and that the generated plants may be practically useful for phytoremediation.
The role of brassinolide (BR) in acid-induced cell wall loosening was studied using lkb dwarf pea (Pisum sativum L.), a BR mutant. Cell wall loosening at pH 4.5 was analyzed by creep measurements of internode segments from lkb and LKB (wild type) seedlings stored in 50% glycerol at −15°C (G-segments). Although cell wall extensibility (φ) was not significantly different between LKB and lkb, the yield threshold (y) was distinctly lower in LKB than in lkb, suggesting that some load-bearing bonds in the cell wall of LKB are more easily broken than in lkb. In G-segments from BR-treated, growth stimulated lkb seedlings y was lowered, and the pattern of the creep rate change was similar to that of LKB. BR applied in vitro to lkb G-segments also lowered y. In heat-treated LKB G-segments no acid-induced cell wall loosening was observed. However, it was restored by addition of a crude cell wall protein fraction. In heat-treated lkb G-segments, acid-induced cell wall loosening could be observed only in the presence of both the protein fraction and BR. It is suggested that BR is necessary for acid-induced cell wall loosening and that BR acts as a modulator of the activity of wall loosening proteins which are limiting the yield threshold.
The nonmevalonate pathway produces isopentenyl diphosphate (IPP) in plastids, as does the mevalonate pathway present in the cytosol in higher plants. IPP is a precursor of an abundant array of isoprenoids, including pigments essential for photosynthesis. Two high-chlorophyll-fluorescence mutants, isp1-1 and isp1-2, in which the ispD gene was partially inactivated, were characterzed. The ispD gene encodes 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, which functions in the third step of the nonmevalonate pathway in plastids. In mutant seedlings cultured at 50 µmol photons m−2 s−1, the photosynthetic electron transport activity and chlorophyll content were reduced. The phenotype was partially suppressed in seedlings cultured at a relatively high light intensity of 300 µmol photons m−2 s−1. These results suggest that the full activity of the nonmevalonate pathway is essential for photo-acclimation, particularly to low light conditions.
We profiled metabolic patterns of transgenic rice organs and tissue by Fourier-transform ion cyclotron mass spectrometry (FT-MS) to reveal effects of the over-expression of YK1 possessing high homology with maize HC-toxin reductase gene. Comparison of metabolic patterns revealed that compositions of organ- or tissue-specific metabolites were not significantly varied between the control and the YK1 rice, where expression levels of some metabolites were altered. Proteome analysis of cultured cells over-expressing YK1 showed the up-regulation of several stress-related proteins such as osmotin-like protein and osr40c1. Thus, alteration of metabolites as well as proteins may contribute to multiple stress tolerances in transgenic YK1 rice.
We investigated the effects of a four-day treatment with 0.3 mM glutathione (GSH) and 10 mM O-acetyl-L-serine (OAS), negative and positive regulators of sulfur-responsive gene expression, respectively, alone or in combination, on the sulfur-responsive gene expression in ten-day-old Arabidopsis thaliana plants. We determined the relationship between the concentrations of GSH, OAS and sulfate, and expression of sulfur-responsive genes in rosette leaves. The concentrations of GSH and OAS correlated negatively and positively with expression of genes, respectively, suggesting that GSH and OAS independently affect expression of sulfur-responsive genes.
Arabidopsis cDNA microarray analysis was performed for 5,722 genes using leaf, stem, root and flower mRNA as targets. We identified 55, 22, 21, and 131 genes expressed specifically in leaves, stems, flowers and roots, respectively. Statistical cluster analysis revealed that the roots were the most distant and the leaves and flowers the most related organs with regards to gene expression. K-means clustering revealed that the clusters for leaf-specific genes tended to mainly have genes for plastid-localized proteins, while the cluster for predominantly root-specific genes was mainly secretory proteins.
Buchanania lanzan (Spreng) is an endemic but vulnerable plant from the tropical region of India. A tissue culture technique for the rapid clonal multiplication of B. lanzan was developed. The decoated seeds were cultured on MS medium enriched with various concentrations of auxins and cytokinins alone or in combination. Combinations of Benzyl amino purine (BAP) and napthalene acetic acid (NAA) were found to be superior to BAP and Indole butyric acid (IBA). Murashige-Skoog (MS) medium supplemented with 22.2 µM of BAP and 5.37 µM of NAA promoted formation of the maximum number of shoots. Furthermore, MS medium containing 23.3 µM kinetin induced profuse rooting of the initiated shoots. We propose here that multiple shoot formation is one of the effective techniques for rapid clonal multiplication.
Shoot explants cut from aseptically grown Ficus thunbergii plants were irradiated with 12C5+, 12C6+ and 4He2+ ion beams of 10 to 200 Gray (Gy). After irradiation, the explants were allowed to regenerate. The regeneration frequency (RF), or percent explants bearing regenerated shoots to the total number of explants irradiated, decreased in a dose-dependent manner. Morphological changes in leaves and stems were observed in regenerants. The morphological change frequency (MF), or percent regenerants bearing morphologically changed shoots to the total number of regenerants peaked at 15 to 25% at the dose of each ion beam that gave an RF of about 80% (except for 4He2+). A negative correlation was observed between the linear energy transfer (LET) and ion beam dose both in RF and MF. The present results strongly suggest that ion beam irradiation can induce mutations in the genome of F. thunbergii.
Maize Hm1 gene encodes a NAD(P)H-dependent HC-toxin reductase, which detoxify HC-toxin produced by fungus Cochliobolus carbonum (Meeley and Walton 1991 Plant Phys 97: 1080). Measurements of ion leakage indicated that H2O2 treatment of a recessive mutant (hm1) of maize resulted in accelerated death in excised leaves. Furthermore, an hm1 maize showed quantitative decrease of NAD(H) level. Thus, the Hm1 gene may confer other functions related to ROS stress tolerance.