Translatable mRNA changes in ethylene induced abscission zones of Phaseolus vulgaris (Red Kidney)

Abstract The wheat germ translation system was programmed with soluble RNA extracted from foliar abscission zones of Phaseolus vulgaris, These extracts were taken at various times after the induction of abscission. A translation product with a molecular weight of 42 kilodalton (kD) was only present after this treatment, though three other species 32, 27 and 17 kD increased substantially. The isozyme of cellulase with a pi of 9.5 could not be conclusively identified amongst the products though the 32 kD protein is probably chitinase. Comparison of the abscission zone translatable RNA with that from adjacent petiole and stem tissues showed the 17 kD protein developed in all these location. The 42, 32 and 27 kD bands were found predominantly in the zone and petiole.     

Temporal and spatial expression of mRNAs encoding pathogenesis-related proteins during ethylene-promoted leaflet abscission in Sambucus nigra*

Differential screening of a cDNA library generated from RNA extracted from ethylene-treated leaflet abscission zones of Sambucus nigra resulted in the isolation of 20 abscission-related clones. These clones could be grouped into seven families. Sequencing of members of these families revealed that the majority encoded pathogenesis-related (PR) proteins, and these could be identified by sequence homology as a polyphenol oxidase (PPO), a PR-1 type protein, a Chial type chitinase, a PR-4 type protein similar to the potato win peptides, a PR-6 type proteinase inhibitor, a Chia4 type chitinase and a metallothionein-like protein (Coupe, Taylor & Roberts 1995, Planta 197, 442–447). Northern analysis revealed that these mRNAs were not expressed in freshly excised material but accumulated primarily in the abscission zone tissue after 18 h of exposure to ethylene at a time when abscission of the leaflet explants had reached 70%. Expression of the PPO and the Chia4-type chitinase was ethylene-dependent while that of the PR-4 type was up-regulated in the abscission zone tissue in the absence of the gas. The characterization of these mRNAs and their encoded proteins is presented and their possible roles during abscission are discussed.     

Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.     

Role of polygalacturonase in bean leaf abscission

The role of polygalacturonase in leaf abscission was studied in explants of Phaseolus vulgaris L. cv. Red Kidney. Bean polygalacturonase was partially characterized and comparisons were made between the bean enzyme and previously reported higher plant polygalacturonases. Polygalacturonase isolated from bean leaf abscission zones has a pH optimum between 4.5 and 5.0 and hydrolyzed polygalacturonides in an exo-fashion. Activity was found to be higher with a deesterified substrate than with an esterified pectin. No correlation between polygalacturonase activity and abscission was observed. Activity remained virtually constant over the course of abscission in explants aged either in air or in ethylene. The enzyme was primarily localized in the abscission zone, however, indicating a possible involvement in the abscission process. A theoretical model which could explain the relationship between polygalacturonase and bean leaf abscission is discussed.     

Enhancement of RNA synthesis, protein synthesis, and abscission by ethylene

Ethylene stimulated RNA and protein synthesis in bean (Phaseolus vulgaris L. var. Red Kidney) abscission zone explants prior to abscission. The effect of ethylene on RNA synthesis and abscission was blocked by actinomycin D. Carbon dioxide, which inhibits the effect of ethylene on abscission, also inhibited the influence of ethylene on protein synthesis. An aging period appears to be essential before bean explants respond to ethylene. Stimulation of protein synthesis by ethylene occurred only in receptive or senescent explants. Treatment of juvenile explants with ethylene, which has no effect on abscission also has no effect on protein synthesis. Evidence in favor of a hormonal role for ethylene during abscission is discussed.     

Immunodetection and characterization of tomato endo-beta-1,4-glucanase Cel1 protein in flower abscission zones.

Tomato (Lycopersicon esculentum Mill.) endo-beta-1,4-glucanase Cel1 mRNA accumulation was previously correlated with abscission of flower explants. Cel1 antibodies were raised against a fusion protein encoding a portion of the Cel1 polypeptide and was shown to react specifically with three polypeptides with molecular masses ranging between 51 and 53 kD in flower abscission zones induced to abscise. All three polypeptides were clearly suppressed in two transgenic lines expressing an antisense Cel1 gene that specifically suppressed the accumulation of Cel1 mRNA, indicating that all three polypeptides are products of the Cel1 gene. Cel1 protein accumulation was correlated with flower abscission. Breakstrength and Cel1 protein content were also analyzed in flower explants, indicating that Cel1 protein accumulation is correlated with the final stages of flower shedding, which suggests that Cel1 is involved in the late stage of abscission. These results support the involvement of Cel1 in the abscission of flower explants and suggest that other hydrolase activities also participate in that process.     

Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves

Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens.     

Pectin esterase in relation to leaf abscission in coleus and phaseolus

Pectin esterase (PE) activities in abscission zones, other portions of leaves, and adjacent stem tissues were compared in attached leaves and abscissing petioles (previously debladed) of Coleus blumei Benth. and Phaseolus vulgaris L., cv. Canadian Wonder. Earlier findings of Osborne in bean were confirmed and changes in PE activity in coleus were shown to resemble those in bean in some respects. In both plants PE was lower in the distal portion of abscission zones of abscissing petioles than in that portion of attached leaves but this difference was not as large or as consistently clear-cut in coleus as in bean. The general level of PE activity was an order of magnitude lower and changes associated with abscission were smaller in coleus than in bean. Auxin treatment of debladed petioles of coleus prevented abscission and resulted in small increases in PE activity in abscission zones and most of the other regions sampled. The largest increase was observed in the stem tissue adjacent to the attached leaf opposite the debladed, auxin treated one.     

Anatomical and physiological effects of silver thiosulfate on ethylene-induced abscission inColeus

Petioles of expiants ofColeus blumei Benth. exposed to 20 μl/l ethylene abscised within 36 h. Pretreatment of expiants with 4 mM silver thiosulfate (STS) inhibited ethylene-induced abscission. Delaying treatment with STS reduced its effectiveness in retarding ethylene-promoted abscission, suggesting that some events leading to abscission are initiated during the first hours of ethylene treatment. Microscopic study of abscission zones of ethylene-treated expiants showed greatly increased amounts of rough endoplasmic reticulum, disruptions of the plasma membrane, and some cell separation in the region of the middle lamella. Pretreatment with STS prevented ethylene-induced reorganization of the endomembrane system and the subsequent middle lamellar dissolution.     

Ethylene-dependent and -independent pathways controlling floral abscission are revealed to converge using promoter::reporter gene constructs in the ida abscission mutant

The process of floral organ abscission in Arabidopsis thaliana can be modulated by ethylene and involves numerous genes contributing to cell separation. One gene that is absolutely required for abscission is INFLORESCENCE DEFICIENT IN ABSCISSION, IDA, as the ida mutant is completely blocked in abscission. To elucidate the genetic pathways regulating floral abscission, molecular markers expressed in the floral abscission zone have been studied in an ida mutant background. Using plants with promoter-reporter gene constructs including promoters of a novel FLORAL ABSCISSION ASSOCIATED gene (FAA) encoding a putative single-stranded binding protein (BASIL), chitinase (CHIT::GUS) and cellulase (BAC::GUS), it is shown that IDA acts in the last steps of the abscission process. These markers, as well as HAESA, encoding a receptor-like kinase, were unaffected in their temporal expression patterns in ida compared with wild-type plants; thus showing that different regulatory pathways are active in the abscission process. In contrast to BASIL, CHIT::GUS and BAC::GUS showed, however, much weaker induction of expression in an ida background, consistent with a reduction in pathogen-associated responses and a lack of total dissolution of cell walls in the mutant. IDA, encoding a putative secreted peptide ligand, and HAESA appeared to have identical patterns of expression in floral abscission zones. Lastly, to address the role of ethylene, IDA::GUS expression in the wild type and the ethylene-insensitive mutant etr1-1 was compared. Similar temporal patterns, yet restricted spatial expression patterns were observed in etr1-1, suggesting that the pathways regulated by IDA and by ethylene act in parallel, but are, to some degree, interdependent.     

Purification of a cellulase from kidney bean abscission zones

The purification of a cellulase isoenzyme with a pI of 9.5 from kidney bean abscission zones is described. An important step in the purification involved the adsorption of the cellulase isoenzyme onto an affinity column of CF-11 cellulose and the subsequent elution with cellobiose. Native and SDS polyacrylamide gel electrophoresis established that there was only one component in the purified cellulase samples. Antibodies raised against the purified pI 9.5 cellulase precipitated this isoenzyme from crude or purified solutions but did not cross react with pI 4.5 cellulase from 2,4-D-treated abscission zones. The antibody was shown to be monospecific by immunoelectrophoresis and by the fact that it precipitated only a single ¹⁴C-labeled protein from an abscission zone extract heavily labeled with ¹⁴C amino acids.     

小黑麦抗真菌蛋白组分的分离纯化和性质研究

以木霉为指示菌,小黑麦中饲237种子中的蛋白提取物经过分离纯化后,得到了3种主要的抗真菌蛋白组分,经酶活检测鉴定,分别是分子量为30.5 kD的ClassⅡ型几丁质酶,两种分子量为51kD和23 kD的β-1,3-葡聚糖酶。其中几丁质酶的最适反应pH为6.0,最适反应温度为37℃,测定的N末端氨基酸序列与大麦几丁质酶的有很高的同源性。在一定条件下,这3种蛋白组分都有较强的抗木霉活性,并且有明显的协同作用,同时它们对离体易感小麦叶片上白粉菌有很好的生长抑制作用。     

Identification of polypeptides specific to rachis abscission zone cells of Sambucus nigra

Specific polypeptides and antigenic determinants in abscission zone cells of the leaf rachis of Sambucus nigra L. (elderberry) were identified. Extracts from abscission zone tissue (OZ) and from ethylene-treated abscission zone tissue after separation (Zone) were probed, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological techniques, for unique peptide components absent from neighbouring non-zone mid-rachis tissue (MR). Ouchterlony immuno-diffusion revealed differences in the spectrum of antigenic determinants possessed by each tissue type when challenged with antiserum raised against OZ. Immuno-electrophoresis showed a basic polypeptide which is expressed preferentially in the abscission zone.
Immune-competition of OZ, MR and Zone extracts using immuno-affinity column chromatography has identified polypeptides of ca 34, ca 32, and ca 28 kDa which are (up to the limits of detection) abscission-cell specific. An antibody raised against the ca 34 kDa polypeptide recognises a peptide of ca 34 kDa present in OZ and Zone. This peptide is absent from MR.
These results suggest that the specific positional differentiation of ethylene-responsive target cells which constitute the leaf rachis abscission zone in S. nigra is accompanied by the expression of zone-cell-specific antigenic determinants which are not expressed by non-target neighbouring tissue.     

Calcium distribution in the abscission zone of bean leaves: electron microprobe x-ray analysis

The calcium content and distribution across the abscission zones of (2-chloroethyl) phosphonic acid-treated bean (Phaseolus vulgaris L. var. Contender) leaves were lower and not uniformly distributed as compared to the control. Calcium chloride-treated bean leaves had a higher calcium content, with more calcium localized in the potential abscission layer. Ethephon treatment promoted abscission in both debladed and nondebladed plants; there was a corresponding decrease in calcium in the abscission zone just prior to separation. Deblading of bean leaves under a calcium solution increased the calcium level in the abscission zone and delayed abscission.     

Endo‐1,4‐β‐glucanase gene expression and cell wall hydrolase activities during abscission in Valencia orange

The physiological and molecular events of ethylene‐induced abscission in mature fruit calyx, laminar and floral abscission zones of cv. Valencia orange were examined. Continuous exposure of fruit explants to 5 µl 1⁻¹ ethylene for 2 to 40 h resulted in marked increases in endo‐1,4‐β‐glucanase (cellulase) and polygalacturonase (PG) activities in calyx abscission zones. Two abscission‐related cellulases and one PG were found. The major peak of cellulase activity corresponded to a pI of 8.0 and molecular weight of 51 kDa, whereas the minor cellulase peak had a pI of 5.5. The abscission polygalacturonase had a pI of 5.5. Calyx abscission zone RNA was amplified with degenerate primers based on sequence of the purified Valencia orange calyx abscission cellulase, and cloned. The two partial cellulase cDNA clones were 59% identical at the nucleotide level. Genomic Southern analysis suggested that Valencia orange contained two groups of cellulase genes. A full‐length cDNA clone from each group was isolated from a cDNA library prepared from ethylene‐induced calyx abscission zone mRNA. Both genes were expressed in ethylene‐induced calyx, laminar and floral abscission zones, but were not expressed in non‐induced abscission zones or mature leaves treated with or without ethylene, young bark or young fruit of Valencia.     

Anatomical and physiological effects of silver thiosulfate on ethylene-induced abscission inColeus

Petioles of expiants ofColeus blumei Benth. exposed to 20 l/l ethylene abscised within 36 h. Pretreatment of expiants with 4 mM silver thiosulfate (STS) inhibited ethylene-induced abscission. Delaying treatment with STS reduced its effectiveness in retarding ethylene-promoted abscission, suggesting that some events leading to abscission are initiated during the first hours of ethylene treatment. Microscopic study of abscission zones of ethylene-treated expiants showed greatly increased amounts of rough endoplasmic reticulum, disruptions of the plasma membrane, and some cell separation in the region of the middle lamella. Pretreatment with STS prevented ethylene-induced reorganization of the endomembrane system and the subsequent middle lamellar dissolution.