Figure 1 - uploaded by Elena Del Campillo
Content may be subject to copyright.
Photographs showing bean flower buds and bean flower reproductive organs. A, Bean flower bud about 2 to 3 mm long (vy), 4 to 6 mm long (in), 8 to 10 mm long (ba), and open flower (of). B, Pistil of bean flower bud at ba stage of development. The left arrow indicates the place where the pistil was cut to separate the stigma with the stylar neck from the style. The right arrow
Source publication
The occurrence of enzymes associated with bean leaf abscission was investigated in bean (Phaseolus vulgaris) flower reproductive organs in which catabolic cell wall events are essential during anther and pistil development. Cellulase activity was detected in high levels in both pistil and anthers of bean flowers before anthesis. Sodium dodecyl sulf...
Contexts in source publication
Context 1
... analyze the regulation of the cellulose protein in anthers and stigma during flower development, bean flowers were collected at the three previously described stages of bud development (Fig. 1A). Reproductive organs from open flow- ers were not collected as they were dried and contaminated with pollen. At the vy stage, both the anther and the stigma of a bean flower are fully differentiated but immature. In- stead, at the ba stage, both reproductive organs have reached maturity. Anthers and stigma fractions were collected as previously described (Fig. 1, B and C). Proteins were ex- tracted, resolved on SDS-PAGE, and immunoblotted with 9.5 cellulose antibody. Gels were loaded on an equal fresh weight basis. Figure 2B illustrates that 9.5 cellulose protein accumulated in the anther in a developmentally regulated manner, changing from very low levels at a very young stage to high levels at an intermediate and mature stage of anther development. Figure 2B also shows that, in the stigma frac- tion (which also included the stylar neck), cellulose protein was present at similar levels during all developmental stages ...
Context 2
... analyze the regulation of the cellulose protein in anthers and stigma during flower development, bean flowers were collected at the three previously described stages of bud development (Fig. 1A). Reproductive organs from open flow- ers were not collected as they were dried and contaminated with pollen. At the vy stage, both the anther and the stigma of a bean flower are fully differentiated but immature. In- stead, at the ba stage, both reproductive organs have reached maturity. Anthers and stigma fractions were collected as previously described (Fig. 1, B and C). Proteins were ex- tracted, resolved on SDS-PAGE, and immunoblotted with 9.5 cellulose antibody. Gels were loaded on an equal fresh weight basis. Figure 2B illustrates that 9.5 cellulose protein accumulated in the anther in a developmentally regulated manner, changing from very low levels at a very young stage to high levels at an intermediate and mature stage of anther development. Figure 2B also shows that, in the stigma frac- tion (which also included the stylar neck), cellulose protein was present at similar levels during all developmental stages ...
Context 3
... bean abscission zones, along with 9.5 cellulose, ethylene induces the accumulation of PRPs, including enzymes such as f/-1,3-glucanase and chitinase (1, 3, 11). The ethylene- induced fl-1,3-glucanase and chitinase are basic proteins with molecular masses of 36 and 33 kD, respectively, and both accumulate to high levels in bean abscission zones before tissue fracture (3, 11, 24). Figure 3 shows proteins from bean abscission zones exposed to ethylene and from organs of bean flowers (ba) subjected to SDS-PAGE followed by im- munoblotting with antibodies raised against ethylene-in- duced f3-1,3-glucanase (basic i3-1,3-glucanase) and basic chi- tinase. Figure 3A illustrates that basic fl-1,3-glucanase (36 was also detected in anthers at both developmental stages, but the levels appeared to be higher in very young anthers. (3-1,3-glucanase activity was also detected in stigma + stylar neck but was undetectable in the ovary and the style. Overall, the levels of j3-1,3-glucanase activity in the anther were much lower than the levels found in the abscission zone after exposure to ethylene. The differ- ences in protein content between anthers (17 mg/g fresh weight) and abscission zone (2 mg/g fresh weight) made the differences in 3-1,3-glucanase specific activity more ...
Context 4
... with an affinity for ,B-1,3-glucan were selectively removed from bean abscission zone and bean anther extracts by addition of pachyman (18). The suspensions were incu- bated at 0°C for 1 h. After centrifugation and sequential washes with a high salt buffer at pH 8.0 and 5.0, respectively, the proteins bound to the insoluble substrates were (26). Figure 1A shows bean flower buds collected at various stages of development. In vy, the bud was 2 to 3 mm long, the style was linear and short, and the anthers were translucent. At (in), the bud was 4 to 6 mm long, the style was longer and curved in the apical portion, and the anthers were off-white. In the oldest stage of the bud, just (ba), the bud was 8 to 10 mm long, the style was long and coiled, the surface of the stigma revealed a sticky exudate (16), and the anthers contained many pollen grains and were ruptured or close to rupture. The developmental stages in and ba are comparable to those described by Webster et al. (26) in which in corresponded to green stage buds and ba corresponded to white stage buds. Between vy stage and anthesis, the pistil (Fig. 1B) and the surrounding filaments that support the anthers (Fig. 1C) extend greatly, forming a tightly coiled structure within the bud. When (of), the style length was about 17 mm. Pollination took place after anther dehiscence but before the flower was open (26). To analyze cellulose activity, total proteins were isolated from the different flower organs collected at the ba stage. The pistil was cut into two parts indicated by arrows in Figure 1B. One cut was made just above the ovary and separated the ovary from the style. The second cut was made 3 to 4 mm below the stigma and separated the stigma and the distal portion of the style (stylar neck) from the rest of the style. The mean length of the style between the arrows was 10 to 12 mm. The anthers were separated from the supporting filaments at the position indicated by the arrow in Figure ...
Context 5
... with an affinity for ,B-1,3-glucan were selectively removed from bean abscission zone and bean anther extracts by addition of pachyman (18). The suspensions were incu- bated at 0°C for 1 h. After centrifugation and sequential washes with a high salt buffer at pH 8.0 and 5.0, respectively, the proteins bound to the insoluble substrates were (26). Figure 1A shows bean flower buds collected at various stages of development. In vy, the bud was 2 to 3 mm long, the style was linear and short, and the anthers were translucent. At (in), the bud was 4 to 6 mm long, the style was longer and curved in the apical portion, and the anthers were off-white. In the oldest stage of the bud, just (ba), the bud was 8 to 10 mm long, the style was long and coiled, the surface of the stigma revealed a sticky exudate (16), and the anthers contained many pollen grains and were ruptured or close to rupture. The developmental stages in and ba are comparable to those described by Webster et al. (26) in which in corresponded to green stage buds and ba corresponded to white stage buds. Between vy stage and anthesis, the pistil (Fig. 1B) and the surrounding filaments that support the anthers (Fig. 1C) extend greatly, forming a tightly coiled structure within the bud. When (of), the style length was about 17 mm. Pollination took place after anther dehiscence but before the flower was open (26). To analyze cellulose activity, total proteins were isolated from the different flower organs collected at the ba stage. The pistil was cut into two parts indicated by arrows in Figure 1B. One cut was made just above the ovary and separated the ovary from the style. The second cut was made 3 to 4 mm below the stigma and separated the stigma and the distal portion of the style (stylar neck) from the rest of the style. The mean length of the style between the arrows was 10 to 12 mm. The anthers were separated from the supporting filaments at the position indicated by the arrow in Figure ...
Context 6
... with an affinity for ,B-1,3-glucan were selectively removed from bean abscission zone and bean anther extracts by addition of pachyman (18). The suspensions were incu- bated at 0°C for 1 h. After centrifugation and sequential washes with a high salt buffer at pH 8.0 and 5.0, respectively, the proteins bound to the insoluble substrates were (26). Figure 1A shows bean flower buds collected at various stages of development. In vy, the bud was 2 to 3 mm long, the style was linear and short, and the anthers were translucent. At (in), the bud was 4 to 6 mm long, the style was longer and curved in the apical portion, and the anthers were off-white. In the oldest stage of the bud, just (ba), the bud was 8 to 10 mm long, the style was long and coiled, the surface of the stigma revealed a sticky exudate (16), and the anthers contained many pollen grains and were ruptured or close to rupture. The developmental stages in and ba are comparable to those described by Webster et al. (26) in which in corresponded to green stage buds and ba corresponded to white stage buds. Between vy stage and anthesis, the pistil (Fig. 1B) and the surrounding filaments that support the anthers (Fig. 1C) extend greatly, forming a tightly coiled structure within the bud. When (of), the style length was about 17 mm. Pollination took place after anther dehiscence but before the flower was open (26). To analyze cellulose activity, total proteins were isolated from the different flower organs collected at the ba stage. The pistil was cut into two parts indicated by arrows in Figure 1B. One cut was made just above the ovary and separated the ovary from the style. The second cut was made 3 to 4 mm below the stigma and separated the stigma and the distal portion of the style (stylar neck) from the rest of the style. The mean length of the style between the arrows was 10 to 12 mm. The anthers were separated from the supporting filaments at the position indicated by the arrow in Figure ...
Context 7
... with an affinity for ,B-1,3-glucan were selectively removed from bean abscission zone and bean anther extracts by addition of pachyman (18). The suspensions were incu- bated at 0°C for 1 h. After centrifugation and sequential washes with a high salt buffer at pH 8.0 and 5.0, respectively, the proteins bound to the insoluble substrates were (26). Figure 1A shows bean flower buds collected at various stages of development. In vy, the bud was 2 to 3 mm long, the style was linear and short, and the anthers were translucent. At (in), the bud was 4 to 6 mm long, the style was longer and curved in the apical portion, and the anthers were off-white. In the oldest stage of the bud, just (ba), the bud was 8 to 10 mm long, the style was long and coiled, the surface of the stigma revealed a sticky exudate (16), and the anthers contained many pollen grains and were ruptured or close to rupture. The developmental stages in and ba are comparable to those described by Webster et al. (26) in which in corresponded to green stage buds and ba corresponded to white stage buds. Between vy stage and anthesis, the pistil (Fig. 1B) and the surrounding filaments that support the anthers (Fig. 1C) extend greatly, forming a tightly coiled structure within the bud. When (of), the style length was about 17 mm. Pollination took place after anther dehiscence but before the flower was open (26). To analyze cellulose activity, total proteins were isolated from the different flower organs collected at the ba stage. The pistil was cut into two parts indicated by arrows in Figure 1B. One cut was made just above the ovary and separated the ovary from the style. The second cut was made 3 to 4 mm below the stigma and separated the stigma and the distal portion of the style (stylar neck) from the rest of the style. The mean length of the style between the arrows was 10 to 12 mm. The anthers were separated from the supporting filaments at the position indicated by the arrow in Figure ...
Context 8
... with an affinity for ,B-1,3-glucan were selectively removed from bean abscission zone and bean anther extracts by addition of pachyman (18). The suspensions were incu- bated at 0°C for 1 h. After centrifugation and sequential washes with a high salt buffer at pH 8.0 and 5.0, respectively, the proteins bound to the insoluble substrates were (26). Figure 1A shows bean flower buds collected at various stages of development. In vy, the bud was 2 to 3 mm long, the style was linear and short, and the anthers were translucent. At (in), the bud was 4 to 6 mm long, the style was longer and curved in the apical portion, and the anthers were off-white. In the oldest stage of the bud, just (ba), the bud was 8 to 10 mm long, the style was long and coiled, the surface of the stigma revealed a sticky exudate (16), and the anthers contained many pollen grains and were ruptured or close to rupture. The developmental stages in and ba are comparable to those described by Webster et al. (26) in which in corresponded to green stage buds and ba corresponded to white stage buds. Between vy stage and anthesis, the pistil (Fig. 1B) and the surrounding filaments that support the anthers (Fig. 1C) extend greatly, forming a tightly coiled structure within the bud. When (of), the style length was about 17 mm. Pollination took place after anther dehiscence but before the flower was open (26). To analyze cellulose activity, total proteins were isolated from the different flower organs collected at the ba stage. The pistil was cut into two parts indicated by arrows in Figure 1B. One cut was made just above the ovary and separated the ovary from the style. The second cut was made 3 to 4 mm below the stigma and separated the stigma and the distal portion of the style (stylar neck) from the rest of the style. The mean length of the style between the arrows was 10 to 12 mm. The anthers were separated from the supporting filaments at the position indicated by the arrow in Figure ...
Citations
... These enzymes then promote hydration of pollen by degrading the pollen coat [77]. In addition, the accumulation of cellulase increases in pollen as pollen maturation progresses [78]. In Arabidopsis, EXOCYST SUBUNIT EXO70 FAMILY PROTEIN A1 (EXO70A1), an exocyst complex subunit in stigma, was shown to be required for the pollen hydration [79]. ...
To thrive on the earth, highly sophisticated systems to finely control reproductive development have been evolved in plants. In addition, deciphering the mechanisms underlying the reproductive development has been considered as a main research avenue because it leads to the improvement of the crop yields to fulfill the huge demand of foods for the growing world population. Numerous studies revealed the significance of ROS regulatory systems and carbohydrate transports and metabolisms in the regulation of various processes of reproductive development. However, it is poorly understood how these mechanisms function together in reproductive tissues. In this review, we discuss mode of coordination and integration between ROS regulatory systems and carbohydrate transports and metabolisms underlying reproductive development based on the hitherto findings. We then propose three mechanisms as key players that integrate ROS and carbohydrate regulatory systems. These include ROS-dependent programmed cell death (PCD), mitochondrial and respiratory metabolisms as sources of ROS and energy, and functions of arabinogalactan proteins (AGPs). It is likely that these key mechanisms govern the various signals involved in the sequential events required for proper seed production.
... According to these two criteria, 11 of the selected differentially regulated shading-responsive genes are regarded as being involved in the process of fruitlet abscission. Six of these genes are also documented as being related to the shedding of plant organs,including CHI [26,27,61], FFU [62], GH3 [63], SAMDC [64], MYB [28] and PMEI [65]. The remaining five genes (AS, ACL, TNL1, UBCE2 and LL-DAP-AT) have not yet been reported in the literature to be involved in organ shedding. ...
Litchi (Litchi chinensis Sonn.) is one of the most important fruit trees cultivated in tropical and subtropical areas. However, a lack of transcriptomic and genomic information hinders our understanding of the molecular mechanisms underlying fruit set and fruit development in litchi. Shading during early fruit development decreases fruit growth and induces fruit abscission. Here, high-throughput RNA sequencing (RNA-Seq) was employed for the de novo assembly and characterization of the fruit transcriptome in litchi, and differentially regulated genes, which are responsive to shading, were also investigated using digital transcript abundance(DTA)profiling.
More than 53 million paired-end reads were generated and assembled into 57,050 unigenes with an average length of 601 bp. These unigenes were annotated by querying against various public databases, with 34,029 unigenes found to be homologous to genes in the NCBI GenBank database and 22,945 unigenes annotated based on known proteins in the Swiss-Prot database. In further orthologous analyses, 5,885 unigenes were assigned with one or more Gene Ontology terms, 10,234 hits were aligned to the 24 Clusters of Orthologous Groups classifications and 15,330 unigenes were classified into 266 Kyoto Encyclopedia of Genes and Genomes pathways. Based on the newly assembled transcriptome, the DTA profiling approach was applied to investigate the differentially expressed genes related to shading stress. A total of 3.6 million and 3.5 million high-quality tags were generated from shaded and non-shaded libraries, respectively. As many as 1,039 unigenes were shown to be significantly differentially regulated. Eleven of the 14 differentially regulated unigenes, which were randomly selected for more detailed expression comparison during the course of shading treatment, were identified as being likely to be involved in the process of fruitlet abscission in litchi.
The assembled transcriptome of litchi fruit provides a global description of expressed genes in litchi fruit development, and could serve as an ideal repository for future functional characterization of specific genes. The DTA analysis revealed that more than 1000 differentially regulated unigenes respond to the shading signal, some of which might be involved in the fruitlet abscission process in litchi, shedding new light on the molecular mechanisms underlying organ abscission.
... Three and five genes encoding the GH9 and GH17 are also found in the pistil tissues, respectively. Twenty years ago, these two enzyme activities were assayed in pistils and anthers of bean and they were linked to the cell wall disruption occurring during the release of the pollen grain from the anther and during the penetration of the pollen tube through the stigma [194]. Recently, and despite its low abundance in the pollen tube cell wall, it was shown that cellulose plays a crucial role by influencing the diameter of in vitro-grown pollen tubes [195]. ...
The pollen tube is a fast tip-growing cell carrying the two sperm cells to the ovule allowing the double fertilization process and seed setting. To succeed in this process, the spatial and temporal controls of pollen tube growth within the female organ are critical. It requires a massive cell wall deposition to promote fast pollen tube elongation and a tight control of the cell wall remodeling to modify the mechanical properties. In addition, during its journey, the pollen tube interacts with the pistil, which plays key roles in pollen tube nutrition, guidance and in the rejection of the self-incompatible pollen. This review focuses on our current knowledge in the biochemistry and localization of the main cell wall polymers including pectin, hemicellulose, cellulose and callose from several pollen tube species. Moreover, based on transcriptomic data and functional genomic studies, the possible enzymes involved in the cell wall remodeling during pollen tube growth and their impact on the cell wall mechanics are also described. Finally, mutant analyses have permitted to gain insight in the function of several genes involved in the pollen tube cell wall biosynthesis and their roles in pollen tube growth are further discussed.
... In a previous study, high chitinase expression was observed in the roots of Sundew [41]. Conversely, chitinase genes are expressed in the roots and flowers of tobacco [42], A. thaliana [43] and beans [44]. These organs share characteristics that make them particularly prone to pathogen attack. ...
Brassica is an important vegetable group worldwide that is impacted by biotic and abiotic stresses. Molecular biology techniques offer the most efficient approach to address these concerns. Inducible plant defense responses include the production of pathogenesis-related (PR) proteins, and chitinases are very important PR proteins. We collected 30 chitinase like genes, three from our full-length cDNA library of Brassica rapa cv. Osome and 27 from Brassica databases. Sequence analysis and comparison study confirmed that they were all class I-V and VII chitinase genes. These genes also showed a high degree of homology with other biotic stress resistance-related plant chitinases. An organ-specific expression of these genes was observed and among these, seven genes showed significant responses after infection with Fusarium oxysporum f.sp. conglutinans in cabbage and sixteen genes showed responsive expression after abiotic stress treatments in Chinese cabbage. BrCLP1, 8, 10, 17 and 18 responded commonly after biotic and abiotic stress treatments indicating their higher potentials. Taken together, the results presented herein suggest that these chitinase genes may be useful resources in the development of stress resistant Brassica.
... Some evidences for involvement of hydrolytic enzymes in anther PCD has been presented. Several researchers remarked that during anther maturation TA56 thiol proteinase [13], cellulase [2], ubiquitin and/or ubiquitinated protein [16], serine palmitoyltransferase level [31] and vacuolar processing enzyme (VPE) which share several structural properties with animal caspase-1 [10] increased suggesting a role for multiple proteolytic systems during cell death. Lathyrus undulatus Boiss. ...
Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.
... High level expression of chitinase was noticed in roots of Sundew [38]. On the other hand, chitinase genes are expressed in roots and flowers of tobacco [39], Arabidopsis thaliana [40] and bean [41]. These organs share characteristics that make them particularly prone to pathogen attack. ...
Brassica is a very important vegetable group because of its contribution to human nutrition and consequent economic benefits. However, biotic stress is a major concern for these crops and molecular biology techniques offer the most efficient of approaches to address this concern. Chitinase is an important biotic stress resistance-related gene. We identified three genes designated as Brassica chitinase like protein (BrCLP1), BrCLP2 and BrCLP3 from a full-length cDNA library of Brassica rapa cv. Osome. Sequence analysis of these genes confirmed that BrCLP1 was a class IV chitinase, and BrCLP2 and BrCLP3 were class VII chitinases. Also, these genes showed a high degree of homology with other biotic stress resistance-related plant chitinases. In expression analysis, organ-specific expression of all three genes was high except BrCLP1 in all the organs tested and BrCLP2 showed the highest expression compared to the other genes in flower buds. All these genes also showed expression during all developmental growth stages of Chinese cabbage. In addition, BrCLP1 was up-regulated with certain time of infection by Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage plants during microarray expression analysis. On the other hand, expression of BrCLP2 and BrCLP3 were increased after 6 h post inoculation (hpi) but decreased from 12 hpi. All these data suggest that these three chitinase genes may be involved in plant resistance against biotic stresses.
... Various members of the chitinase family play a role in plant defence but it is now generally accepted that some chitinases and chitinase-like genes have roles in plant growth and development (Kasprzewska 2003). Accumulation of cellulase and chitinase and related proteins in the bean abscission zone and anthers and pistils, as well as the high levels of chitinase transcripts found in young leaves, stems, flowers and pollen (Neale et al. 1990; Campillo and Lewis 1992; Patil and Widholm 1997 ) suggest that these proteins may be important in cell wall disruption and loosening in rapidly growing tissues. There is good evidence for chitinolytic enzymes participating in cell wall remodelling and daughter cell separation in fungi (Kasprzewska 2003). ...
The resurrection grass Sporobolus stapfianus Gandoger can rapidly recover from extended periods of time in the desiccated state (water potential equilibrated to 2% relative humidity) (Gaff and Ellis, Bothalia 11:305-308 1974; Gaff and Loveys, Transactions of the Malaysian Society of Plant Physiology 3:286-287 1993). Physiological studies have been conducted in S. stapfianus to investigate the responses utilised by these desiccation-tolerant plants to cope with severe water-deficit. In a number of instances, more recent gene expression analyses in S. stapfianus have shed light on the molecular and cellular mechanisms mediating these responses. S. stapfianus is a versatile research tool for investigating desiccation-tolerance in vegetative grass tissue, with several useful characteristics for differentiating desiccation-tolerance adaptive genes from the many dehydration-responsive genes present in plants. A number of genes orthologous to those isolated from dehydrating S. stapfianus have been successfully used to enhance drought and salt tolerance in model plants as well as important crop species. In addition to the ability to desiccate and rehydrate successfully, the survival of resurrection plants in regions experiencing short sporadic rainfall events may depend substantially on the ability to tightly down-regulate cell division and cell wall loosening activities with decreasing water availability and then grow rapidly after rainfall while water is plentiful. Hence, an analysis of gene transcripts present in the desiccated tissue of resurrection plants may reveal important growth-related genes. Recent findings support the proposition that, as well as being a versatile model for devising strategies for protecting plants from water-loss, resurrection plants may be a very useful tool for pinpointing genes to target for enhancing growth rate and biomass production.
... Upon N-tertninal sequencing, the proteins were found to have homology to chitinases, PR-1 type, PR-2 type iP -1,3-glucanase) and PR-5 type (tbaumatin-like) ptoteins. Such ptoteins were also found to accumulate in bean anthet\s and pistils during flower ab.scission (del Campillo & Lewis 1992b). del Catnpillo & Lewis (1992b) pt-oposed that the PR ptoteins were either involved in the cell separation process itself or protected the plant frotn pathogenic attack. ...
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.
... The TA56 thiol endopeptidase mRNA accumulation pattern reflects the sequential degeneration of the circular cell cluster and stomium—TA56 mRNA accumulates first in the circular cell cluster and then in the stomium (Fig. 11), and these events are under precise transcriptional control (Koltunow et al. 1990; Beals and Goldberg 1997 ). Other hydrolytic enzymes (e.g., cellulase ) have been shown to be present in anthers just prior to dehiscence (del Campillo and Lewis 1992; Lashbrook et al. 1994; Neelam and Sexton 1995 ). In striking contrast with results obtained from tapetal cell ablation, targeted ablation of either the circular cell cluster and stomium or the stomium alone with a TA56/barnase gene late in anther development leads to anthers that fail to dehisce (Beals and Goldberg 1997). ...
Dehiscence is the terminal step in anther development that releases pollen grains from the wall of each theca at a specific site between the two locules. In tobacco, two groups of cells—the circular cell cluster and the stomium—are required for anther dehiscence and define the position at which pollen is released. The processes responsible for the differentiation of the circular cell cluster and the stomium from cells in specific anther regions are unknown. Nor is it understood what initiates the programmed degeneration of these cell types that ultimately is responsible for pollen release from the anther. We characterized stomium and circular cell cluster differentiation and degeneration using both light and transmission electron microscopy throughout anther development, from the emergence of stamen primordia to anther dehiscence at flower opening. We observed that histological changes within primordium L1 and L2 cells destined to become the stomium and circular cell cluster occur at the same time after the differentiation of surrounding locule regions. Sub-epidermal cells that differentiate into the circular cell cluster divide, enlarge, and generate vacuoles with calcium oxalate crystals prior to any detectable changes in pre-stomium epidermal cells. Differentiation and division of cells that generate the stomium occur after cell degeneration initiates in the circular cell cluster. Prior to dehiscence, the stomium consists of a small set of cytoplasmically dense cells that are easily distinguished from their larger, highly vacuolate epidermal neighbors. Plasmodesmata connections within and between cells of the stomium and circular cell cluster were observed at different developmental stages, suggesting that these cells communicate with each other. Circular cell cluster and stomium cell death is programmed developmentally and occurs at different times. Degeneration of the circular cell cluster occurs first, contributes to the formation of a bilocular anther, and generates the site of anther wall breakage. The stomium cell death process is complete at flower opening and provides an opening for pollen release from each theca. We used laser capture microdissection and real-time quantitative reverse-transcription polymerase chain reactions to demonstrate that stomium cells can be isolated from developing anthers and studied for the presence of specific mRNAs. Our data suggest that a cascade of unique gene expression events throughout anther development is required for the dehiscence program, and that the differentiation of the stomium and circular cell cluster in the interlocular region of the anther probably involves cell signaling processes.
... Twenty Sambucus abscission-related clones were isolated from a differential screen, and sequencing revealed that the majority encoded PR proteins, including a PR-1 protein, class I chitinase, proteinase inhibitors, and a metallothionein-like protein, all of which were matched by clones from the Cnr library. The specific function of PR proteins in this process is unclear, although it has been proposed that they are either involved in the cell separation process itself or protect the plant from pathogenic attack (delCampillo and Lewis, 1992). Our current work on cloning the gene at the Cnr locus is aimed at allowing us to place the gene product in a framework that will describe the molecular regulation of ripening and permit connections between the downstream events in the cell wall and the regulatory factors controlling ripening. ...
The Colorless non-ripening (Cnr) mutation in tomato (Solanum lycopersicum) results in mature fruits with colorless pericarp tissue showing an excessive loss of cell adhesion (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour [1999] Plant Physiol 120: 383-390). This pleiotropic mutation is an important tool for investigating the biochemical and molecular basis of cell separation during ripening. This study reports on the changes in enzyme activity associated with cell wall disassembly in Cnr and the effect of the mutation on the program of ripening-related gene expression. Real-time PCR and biochemical analysis demonstrated that the expression and activity of a range of cell wall-degrading enzymes was altered in Cnr during both development and ripening. These enzymes included polygalacturonase, pectinesterase (PE), galactanase, and xyloglucan endotransglycosylase. In the case of PE, the protein product of the ripening-related isoform PE2 was not detected in the mutant. In contrast with wild type, Cnr fruits were rich in basic chitinase and peroxidase activity. A microarray and differential screen were used to profile the pattern of gene expression in wild-type and Cnr fruits. They revealed a picture of the gene expression in the mutant that was largely consistent with the real-time PCR and biochemical experiments. Additionally, these experiments demonstrated that the Cnr mutation had a profound effect on many aspects of ripening-related gene expression. This included a severe reduction in the expression of ripening-related genes in mature fruits and indications of premature expression of some of these genes in immature fruits. The program of gene expression in Cnr resembles to some degree that found in dehiscence or abscission zones. We speculate that there is a link between events controlling cell separation in tomato, a fleshy fruit, and those involved in the formation of dehiscence zones in dry fruits.
