No difference in the regulation pattern of calcium on ethylene biosynthesis between wild-type and never-ripe tomato fruit at mature green stage

This work investigated how calcium regulates the ethylene biosynthesis in the fruits of wild-type tomato (Lycopersicon esculentum L.) and their ethylene receptor never-ripe (Nr) mutants. In Nr tomato, the ethylene perception was blocked. When both materials were treated with calcium, the content of 1-aminocyclopropane-1-carboxylic acid (ACC)/malonyl-ACC and the activity of ACC oxidase (ACO) in tomato fruit discs increased, whereas the production of ethylene, content of malondialdehyde, and membrane permeability decreased. Calcium treatment did not affect the activity of ACC synthase, which is the first committed step in the ethylene biosynthesis pathway. The expression of LeACO1 in mature green fruit was inhibited significantly by calcium treatment in wild-type and Nr tomatoes, but the expression of LeACS2, the key ACC synthase gene in ethylene synthesis during tomato fruit maturing, was not affected. These results revealed that the effect of calcium on ethylene biosynthesis in tomato mature green fruit was independent of ethylene perception. The results also revealed that the targeting step of calcium preventing ethylene production was located at the ACC conversion to ethylene, by means of inhibiting ACC availability for ACO through enhancing cell membrane integrity and by means of preventing LeACO1 gene expression. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 60–67. The text was submitted by the authors in English.     

Developmental regulation of peach ACC oxidase promoter--GUS fusions in transgenic tomato fruits

A genomic DNA sequence (PpACO1) encoding 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from peach (Prunus persica L. Batsch cv. Loring) was isolated. It has four exons interrupted by three introns and 2.9 kb of flanking region 5' of the translational start codon. Previous work with the cDNA demonstrated that accumulation of the peach ACO message correlated with increasing amounts of ethylene synthesized by the fruit as they ripened. To identify regulatory elements in the peach ACC oxidase gene, chimeric fusions between 403, 610, 901, 1319, 2141, and 2919 bp of the 5' flanking region of the PpACO1 sequence and the beta-glucuronidase (GUS) coding sequence were constructed and used to transform tomato (Lycopersicon esculentum [Mill] cv. Pixie). Fruits from the various promoter lines were analysed for GUS expression by histochemical GUS staining, GUS quantitative enzyme activity determination, and measuring the relative amounts of GUS mRNA. Constructs with the smallest promoter of 403 bp had significant GUS expression in fruit, but not in other tissues, indicating the presence of a region that affects tissue-specific expression. An increase in GUS expression was observed with promoters longer than 901 bp, indicating an enhancer region between -1319 and -901. The full-length promoter of 2919 bp directed GUS expression in the green stage of fruit development, and increased GUS expression as fruit matured, indicating a regulatory region between -2919 and -2141 that controls the temporal expression of the gene in fruit. Only the full-length promoter sequence demonstrated responsiveness to ethylene.     

Molecular characterization and tissue localization of sensory neuron membrane protein from Chinese honey bee, Apis cerana cerana (Hymenoptera: Apidae)

Sensory neuron membrane protein (SNMP) is an olfactory receptor with photoaffinity analogs, capable of binding the pheromone membrane protein receptor deduced from receptor membrane protein with the pheromone–pheromone binding protein complex. However, this hypothesis has not yet been experimentally verified. In this experiment, the cDNA sequence encoding an open reading frame (ORF) of the SNMP gene AccSNMP1 (GenBank, KC012595) was cloned from Chinese honey bee, Apis cerana cerana Fabricius. Results from sequence analysis showed that this gene is 1,563 bp long, and that the ORF encodes 520 amino acids with a predicted molecular weight of 58.02 kDa, and has a theoretical isoelectric point of 5.83. Furthermore, there are two putative transmembrane domains. Multiple sequence alignment indicated that the AccSNMP1 gene from A. cerana cerana had different degrees of identity with the corresponding genes in nineteen other insects at the amino acid level. Phylogenetic analysis of the aligned sequences showed that A. cerana cerana is closely related to Apis mellifera Linnaeus and Bombus impatiens Cresson. Its distribution in tissues, as quantified using real-time RT-PCR, indicated that AccSNMP1 is highly expressed in the antennae and legs of A. cerana cerana, and there was a significant difference (p < 0.05) in gene expression between those tissues and tissues in the thorax, abdomen, snout, and head (not including antennae). Western blotting also confirmed the existence in the antennae of AccSNMP1 with an M _W of 58.0 kDa, which is the same as the expected value of 58.02 kDa. An immunohistochemistry study showed that AccSNMP1 is expressed in the trichoid sensilla of A. cerana cerana antenna. Therefore, the results of this study provide the basis for further studies of the function of SNMP from A. cerana cerana.     

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984 bp and contained an open reading frame of 600 bp, which encoded a 200 amino acid protein with a molecular weight of 21.83 kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant.     

A novel tomato mutant,Solanum lycopersicum elongated fruit1 (Slelf1), exhibits an elongated fruit shape caused by increased cell layers in the proximal region of the ovary

Genes controlling fruit morphology offer important insights into patterns and mechanisms determining organ shape and size. In cultivated tomato (Solanum lycopersicum L.), a variety of fruit shapes are displayed, including round-, bell pepper-, pear-, and elongate-shaped forms. In this study, we characterized a tomato mutant possessing elongated fruit morphology by histologically analyzing its fruit structure and genetically analyzing and mapping the genetic locus. The mutant line, Solanum lycopersicum elongated fruit 1 (Slelf1), was selected in a previous study from an ethylmethane sulfonate-mutagenized population generated in the background of Micro-Tom, a dwarf and rapid-growth variety. Histological analysis of the Slelf1 mutant revealed dramatically increased elongation of ovary and fruit. Until 6 days before flowering, ovaries were round and they began to elongate afterward. We also determined pericarp thickness and the number of cell layers in three designated fruit regions. We found that mesocarp thickness, as well as the number of cell layers, was increased in the proximal region of immature green fruits, making this the key sector of fruit elongation. Using 262 F₂ individuals derived from a cross between Slelf1 and the cultivar Ailsa Craig, we constructed a genetic map, simple sequence repeat (SSR), cleaved amplified polymorphism sequence (CAPS), and derived CAPS (dCAPS) markers and mapped to the 12 tomato chromosomes. Genetic mapping placed the candidate gene locus within a 0.2?Mbp interval on the long arm of chromosome 8 and was likely different from previously known loci affecting fruit shape.     

Expression and regulation of the ethylene receptor PpERS gene during pear fruit development and following salicylic acid treatment

A gene encoding an ethylene receptor protein was isolated from pear (Pyrus pyrifolia). This gene, designated PpERS (GenBank accession No. KC517482), was 1,918 bp in length with an open reading frame encoding a protein of 638 amino acids that shared high similarity with another pear ethylene receptor protein PpERS1, and two apple ethylene receptor proteins MdERS and MdERS1. The PpERS was grouped into the ETR1 subfamily of ethylene receptor based on its conserved domain and phylogenetic status. The PpERS gene contained five exons interrupted by four introns. Quantitative RT-PCR indicated that PpERS was differentially expressed in pear tissues and predominantly expressed in petals, shoots, anthers, and 160 days after full bloom fruit. The PpERS expression was regulated during fruit development. In addition, the PpERS gene expression was regulated by salicylic acid (SA) and ethylene in fruit. The results indicated that PpERS might participate in ethylene and SA signaling transduction during pear fruit development.     

Cloning, Expression, and Characterization of Iron Superoxide Dismutase in Sonneratia alba, a Highly Salt Tolerant Mangrove Tree

In this study, a novel iron superoxide dismutase (FeSOD) gene from Sonneratia alba was cloned and then expressed in Escherichia coli Rosetta-gami, designated as SaFeSOD. The DNA sequence of SaFeSOD contained a 786-bp open reading frame which encodes a 261 amino-acid protein of 30.0 kDa. The 651-bp fragment coding for putative mature SaFeSOD was amplified and inserted into pET15b for expression. This recombinant SaFeSOD was subsequently isolated by Ni-trap column protein purification system. The apparent molecular mass of the purified enzyme was 25 kDa on SDS-PAGE. In comparison with FeSODs from other plant species, all iron-binding sites (His 27, His 80, Asp 164 and His 168) of SaFeSOD were conserved. SaFeSOD was found to have good pH stability in the pH range of 3.5–9.5 at 25 °C after 1 h incubation and was relatively stable and showed 78 % activity when incubated in 50 °C for 1 h. Quantitative real-time PCR experiments demonstrated that SaFeSOD was expressed in leaf, stem, flower, fruit and root tissues with the highest expression in leaf tissues.     

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.