NADPH oxidase inhibitor diphenyleneiodonium and reduced glutathione mitigate ethephon-mediated leaf senescence,H2O2 elevation and senescence-associated gene expression in sweet potato (Ipomoea batatas)

Ethephon, an ethylene releasing compound, promoted leaf senescence, H₂O₂ elevation, and senescence-associated gene expression in sweet potato. It also affected the glutathione and ascorbate levels, which in turn perturbed H₂O₂ homeostasis. The decrease of reduced glutathione and the accumulation of dehydroascorbate correlated with leaf senescence and H₂O₂ elevation at 72 h in ethephon-treated leaves. Exogenous application of reduced glutathione caused quicker and significant increase of its intracellular level and resulted in the attenuation of leaf senescence and H₂O₂ elevation. A small H₂O₂ peak produced within the first 4 h after ethephon application was also eliminated by reduced glutathione. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, delayed leaf senescence and H₂O₂ elevation at 72 h, and its influence was effective only within the first 4 h after ethephon treatment. Ethephon-induced senescence-associated gene expression was repressed by DPI and reduced glutathione at 72 h in pretreated leaves. Leaves treated with l-buthionine sulfoximine, an endogenous glutathione synthetase inhibitor, did enhance senescence-associated gene expression, and the activation was strongly repressed by reduced glutathione. In conclusion, ethephon-mediated leaf senescence, H₂O₂ elevation and senescence-associated gene expression are all alleviated by reduced glutathione and NADPH oxidase inhibitor DPI. The speed and the amount of intracellular reduced glutathione accumulation influence its effectiveness of protection against ethephon-mediated effects. Reactive oxygen species generated from NADPH oxidase likely serves as an oxidative stress signal and participates in ethephon signaling. The possible roles of NADPH oxidase and reduced glutathione in the regulation of oxidative stress signal in ethephon are discussed.     

Cloning a peanut resveratrol synthase gene and its expression in purple sweet potato

A resveratrol synthase gene was cloned from the peanut plant (Arachis hypogaea) by RT-PCR and was transformed into purple sweet potato (Ipomoea batatas) by Agrobacterium-mediated transformation. Stem sections were infected with bacterial solution of OD₆₀₀ = 0.4 for 20 min and then cocultured for 2 days. Infected explants were cultured on MS media containing 50 mg/l kanamycin, 0.02 mg/l NAA and 1 mg/l 6-BA for bud induction or containing 75 mg/l kanamycin, 1.0 mg/l NAA and 0.1 mg/l 6-BA for root formation. The bud and root induction rates were 37.5 and 25.0%, respectively. 105 regenerated plants were obtained, with 11 positive plants by PCR and Southern blotting analyses. A high level of resveratrol glucoside (340 μg/g dry weight), but no resveratrol, was detected in the transformed plants by HPLC. This study also provides a stable genetic transformation and plant regeneration method for metabolic modification of purple sweet potato.     

Cloning and expression analysis of a gene that shows developmental regulation upon tuberization in potato

An Arabidopsis cDNA clone encoding a DNA-binding protein, RAP-1, was isolated by southwestern screening of an Escherichia coli cDNA expression library. The protein contains a bHLH DNA-binding domain and is homologous to R proteins, regulating anthocyanin biosynthesis. RAP-1 binds to the sequence CACNTG. It is encoded by a single gene, which is expressed to high levels in root and stem and to low levels in leaf and flower. No expression could be detected in siliques. Rap-1 does not correspond to one of the known loci involved in anthocyanin biosynthesis, since it is located at a different map position. In contrast to the maize R protein Lc, RAP-1 did not induce anthocyanin biosynthesis in pea cotyledons. Thus, RAP-1 is a novel member of the bHLH class of DNA-binding proteins.     

Trypanosoma cruzi calmodulin: Cloning, expression and characterization

We have cloned and expressed calmodulin (CaM) from Trypanosoma cruzi, for the first time, to obtain large amounts of protein. CaM is a very well conserved protein throughout evolution, sharing 100% amino acid sequence identity between different vertebrates and 99% between trypanosomatids. However, there is 89% amino acid sequence identity between T. cruzi and vertebrate CaMs. The results demonstrate significant differences between calmodulin from T. cruzi and mammals. First, a polyclonal antibody developed in an egg-yolk system to the T. cruzi CaM recognizes the autologous CaM but not the CaM from rat. Second, it undergoes a larger increase in the α-helix content upon binding with Ca²⁺, when compared to CaM from vertebrates. Finally, two classic CaM antagonists, calmidazolium and trifluoperazine, capable of inhibiting the action of CaM in mammals when assayed on the plasma membrane Ca²⁺ pump, showed a significant loss of activity when assayed upon stimulation with the T. cruzi CaM.     

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl₂ which delayed loss of chlorophyll but not that of photochemical efficiency.     

Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1.

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.     

Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting

Calmodulin (CaM) is a highly conserved calcium (Ca²⁺) binding protein that transduces Ca²⁺ signals into downstream effects influencing a range of cellular processes, including Ca²⁺ homeostasis. The present study explores CaM expression when Ca²⁺ homeostasis is challenged during the mineralization cycle of the freshwater crayfish (Procambarus clarkii). In this paper we report the cloning of a CaM gene from axial abdominal crayfish muscle (referred to as pcCaM). The pcCaM mRNA is ubiquitously expressed but is far more abundant in excitable tissue (muscle, nerve) than in any epithelia (gill, antennal gland, digestive) suggesting that it plays a greater role in the biology of excitation than in epithelial ion transport. In muscle cells the pcCaM was colocalized on the plasma membrane with the Ca²⁺ ATPase (PMCA) known to regulate intracellular Ca²⁺ through basolateral efflux. While PMCA exhibits a greater upregulation in epithelia (than in non-epithelial tissues) during molting stages requiring transcellular Ca²⁺ flux (pre- and postmolt compared with intermolt), expression of pcCaM exhibited a uniform increase in epithelial and non-epithelial tissues alike. The common increase in expression of CaM in all tissues during pre- and postmolt stages (compared with intermolt) suggests that the upregulation is systemically (hormonally) mediated. Colocalization of CaM with PMCA confirms physiological findings that their regulation is linked.     

Identification and functional characterization of a leucine-rich repeat receptor-like kinase gene that is involved in regulation of soybean leaf senescence

We report here the cloning and characterization of a soybean receptor-like kinase (RLK) gene, designated GmSARK (Glycine max senescence-associated receptor-like kinase), which is involved in regulating leaf senescence. The conceptual protein product of GmSARK contains typical domains of LRR receptor-like kinases: a cytoplasmic domain with all the 11 kinase subdomains, a transmembrane domain and an extracelullar domain containing 9 Leucine-Rich Repeat (LRR) units that may act as a receptor. The expression of GmSARK in soybean leaves was up-regulated in all the three tested senescence systems: senescing cotyledons, dark-induced primary leaf senescence and the natural leaf senescence process after florescence. Furthermore, the RNA interference (RNAi)-mediated knocking-down of GmSARK dramatically retarded soybean leaf senescence. A more complex thylakoid membrane system, higher foliar level of chlorophyll content and a very remarkable delay of senescence-induced disintegration of chloroplast structure were observed in GmSARK-RNAi transgenic leaves. A homolog of maize lethal leaf-spot 1 gene, which has been suggested to encode a key enzyme catalyzing chlorophyll breakdown, was isolated and nominated Gmlls1. The expression level of Gmgtr1 gene, which encodes a key enzyme of chlorophyll synthesis, was also analyzed. It was found that Gmlls1 was up-regulated and Gmgtr1 was down-regulated during senescence in wild-type soybean leaves. However, both of the up-regulation of Gmlls1 and down-regulation of Gmgtr1 were retarded during senescence of GmSARK-RNAi transgenic leaves. In addition, over-expression of the GmSARK gene greatly accelerated the senescence progression of CaMV 35S:GmSARK transgenic plants. Taken together, these results strongly suggested the involvement of this LRR-RLK in regulation of soybean leaf senescence, maybe via regulating chloroplast development and chlorophyll accumulation. Multiple functions of GmSARK besides its regulation of leaf senescence were also discussed. Electronic Supplementary Material Supplementary material is available for this article at Rui Gan, Peng-Li Li and Yuan-Yuan Ma contributed equally to this work.     

Salicylic acid has a role in regulating gene expression during leaf senescence

Leaf senescence is a complex process that is controlled by multiple developmental and environmental signals and is manifested by induced expression of a large number of different genes. In this paper we describe experiments that show, for the first time, that the salicylic acid (SA)-signalling pathway has a role in the control of gene expression during developmental senescence. Arabidopsis plants defective in the SA-signalling pathway (npr1 and pad4 mutants and NahG transgenic plants) were used to investigate senescence-enhanced gene expression, and a number of genes showed altered expression patterns. Senescence-induced expression of the cysteine protease gene SAG12, for example, was conditional on the presence of SA, together with another unidentified senescence-specific factor. Changes in gene expression patterns were accompanied by a delayed yellowing and reduced necrosis in the mutant plants defective in SA-signalling, suggesting a role for SA in the cell death that occurs at the final stage of senescence. We propose the presence of a minimum of three senescence-enhanced signalling factors in senescing leaves, one of which is SA. We also suggest that a combination of signalling factors is required for the optimum expression of many genes during senescence.     

Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.     

Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts

A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0–5.0×10⁶ were obtained following 4–6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 10⁵ protoplasts/ml was optimal for subsequent division. An initial division frequency of 12–15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μm), and zeatin (2.3 μm). Colonies consisting of 100–200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1–2 mm in diameter) were formed after an additional 4–6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μm) and benzylaminopurine (2.2 μm). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μm gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation. Received: 10 October 1997 / Revision received: 10 February 1998 / Accepted: 2 March 1998