Role of the KNOTTED1-LIKE HOMEOBOX protein (KD1) in regulating abscission of tomato flower pedicels at early and late stages of the process.

The KNOTTED1-LIKE HOMEOBOX PROTEIN1 (KD1) gene is highly expressed in flower and leaf abscission zones (AZs), and KD1 was reported to regulate tomato flower pedicel abscission via alteration of the auxin gradient and response in the flower AZ (FAZ). The present work was aimed to further examine how KD1 regulates signaling factors and regulatory genes involved in pedicel abscission, by using silenced KD1 lines and performing a large-scale transcriptome profiling of the FAZ before and after flower removal, using a customized AZ-specific microarray. The results highlighted a differential expression of regulatory genes in the FAZ of KD1-silenced plants compared to the wild-type. In the TAPG4::antisense KD1-silenced plants, KD1 gene expression decreased before flower removal, resulting in altered expression of regulatory genes, such as epigenetic modifiers, transcription factors, posttranslational regulators, and antioxidative defense factors occurring at zero time and before affecting auxin levels in the FAZ detected at 4 h after flower removal. The expression of additional regulatory genes was altered in the FAZ of KD1-silenced plants at 4-20 h after flower removal, thereby leading to an inhibited abscission phenotype, and downregulation of genes involved in abscission execution and defense processes. Our data suggest that KD1 is a master regulator of the abscission process, which promotes abscission of tomato flower pedicels. This suggestion is based on the inhibitory effect of KD1 silencing on flower pedicel abscission that operates via alteration of various regulatory pathways, which delay the competence acquisition of the FAZ cells to respond to ethylene signaling.

Re-evaluation of the ethylene-dependent and -independent pathways in the regulation of floral and organ abscission.

Abscission is a developmental process with important implications for agricultural practices. Ethylene has long been considered as a key regulator of the abscission process. The existence of an ethylene-independent abscission pathway, controlled by the complex of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide and the HAESA (HAE) and HAESA-like2 (HSL2) kinases, has been proposed, based mainly on observations that organ abscission in ethylene-insensitive mutants was delayed but not inhibited. A recent review on plant organ abscission signaling highlighted the IDA-HAE-HSL2 components as the regulators of organ abscission, while the role of auxin and ethylene in this process was hardly addressed. After a careful analysis of the relevant abscission literature, we propose that the IDA-HAE-HSL2 pathway is essential for the final stages of organ abscission, while ethylene plays a major role in its initiation and progression. We discuss the view that the IDA-HAE-HSL2 pathway is ethylene independent, and present recent evidence showing that ethylene activates the IDA-HAE-HSL2 complex. We conclude that the ability of an organ to abscise is tightly linked to cell turgidity in the abscission zone, and suggest that lack of cell turgidity might contribute to the failure of floral organ abscission in the ida mutants.

A KNOTTED1-LIKE HOMEOBOX protein regulates abscission in tomato by modulating the auxin pathway.

A gene encoding a KNOTTED1-LIKE HOMEOBOX PROTEIN1 (KD1) is highly expressed in both leaf and flower abscission zones. Reducing the abundance of transcripts of this gene in tomato (Solanum lycopersicum) by both virus-induced gene silencing and stable transformation with a silencing construct driven by an abscission-specific promoter resulted in a striking retardation of pedicel and petiole abscission. In contrast, Petroselinum, a semidominant KD1 mutant, showed accelerated pedicel and petiole abscission. Complementary DNA microarray and quantitative reverse transcription-polymerase chain reaction analysis indicated that regulation of abscission by KD1 was associated with changed abundance of genes related to auxin transporters and signaling components. Measurement of auxin content and activity of a DR5::ß-glucuronidase auxin reporter assay showed that changes in KD1 expression modulated the auxin concentration and response gradient in the abscission zone.

Abscission of flowers and floral organs is closely associated with alkalization of the cytosol in abscission zone cells.

In vivo changes in the cytosolic pH of abscission zone (AZ) cells were visualized using confocal microscopic detection of the fluorescent pH-sensitive and intracellularly trapped dye, 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF), driven by its acetoxymethyl ester. A specific and gradual increase in the cytosolic pH of AZ cells was observed during natural abscission of flower organs in Arabidopsis thaliana and wild rocket (Diplotaxis tenuifolia), and during flower pedicel abscission induced by flower removal in tomato (Solanum lycopersicum Mill). The alkalization pattern in the first two species paralleled the acceleration or inhibition of flower organ abscission induced by ethylene or its inhibitor 1-methylcyclopropene (1-MCP), respectively. Similarly, 1-MCP pre-treatment of tomato inflorescence explants abolished the pH increase in AZ cells and pedicel abscission induced by flower removal. Examination of the pH changes in the AZ cells of Arabidopsis mutants defective in both ethylene-induced (ctr1, ein2, eto4) and ethylene-independent (ida, nev7, dab5) abscission pathways confirmed these results. The data indicate that the pH changes in the AZ cells are part of both the ethylene-sensitive and -insensitive abscission pathways, and occur concomitantly with the execution of organ abscission. pH can affect enzymatic activities and/or act as a signal for gene expression. Changes in pH during abscission could occur via regulation of transporters in AZ cells, which might affect cytosolic pH. Indeed, four genes associated with pH regulation, vacuolar H(+)-ATPase, putative high-affinity nitrate transporter, and two GTP-binding proteins, were specifically up-regulated in tomato flower AZ following abscission induction, and 1-MCP reduced or abolished the increased expression.

Programmed cell death occurs asymmetrically during abscission in tomato.

Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.

Reevaluation of ethylene role in Arabidopsis cauline leaf abscission induced by water stress and rewatering.

It was previously reported that cauline leaf abscission in Arabidopsis is induced by a cycle of water stress and rewatering, which is regulated by the complex of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), HAESA (HAE), and HAESA-LIKE2 (HSL2) kinases. However, the involvement of ethylene in this process was ruled out. Because this conclusion contradicts the well-established role of ethylene in organ abscission induced by a cycle of water stress and rewatering, our present study was aimed to reevaluate the possible involvement of ethylene in this process. For this purpose, we examined the endogenous ethylene production during water stress and following rewatering, as well as the effects of exogenous ethylene and 1-methylcyclopropene (1-MCP), on cauline leaf abscission of Arabidopsis wild type. Additionally, we examined whether this stress induces cauline leaf abscission in ethylene-insensitive Arabidopsis mutants. The results of the present study demonstrated that ethylene production rates increased significantly in cauline leaves at 4 h after rewatering of stressed plants and remained high for at least 24 h in plants water-stressed to 40 and 30% of system weight. Ethylene treatment applied to well-watered plants induced cauline leaf abscission, which was inhibited by 1-MCP. Cauline leaf abscission was also inhibited by 1-MCP applied during a cycle of water stress and rewatering. Finally, no abscission occurred in two ethylene-insensitive mutants, ein2-1 and ein2-5, following a cycle of water stress and rewatering. Taken together, these results clearly indicate that ethylene is involved in Arabidopsis cauline leaf abscission induced by water stress and rewatering. Our results show that ethylene is involved in Arabidopsis cauline leaf abscission induced by water stress and rewatering, similar to leaf abscission in other plants.

Microarray analysis of the abscission-related transcriptome in the tomato flower abscission zone in response to auxin depletion.

The abscission process is initiated by changes in the auxin gradient across the abscission zone (AZ) and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the molecular and biochemical basis of the increased AZ sensitivity to ethylene. We examined transcriptome changes in the tomato (Solanum lycopersicum 'Shiran 1335') flower AZ during the rapid acquisition of ethylene sensitivity following flower removal, which depletes the AZ from auxin, with or without preexposure to 1-methylcyclopropene or application of indole-3-acetic acid after flower removal. Microarray analysis using the Affymetrix Tomato GeneChip revealed changes in expression, occurring prior to and during pedicel abscission, of many genes with possible regulatory functions. They included a range of auxin- and ethylene-related transcription factors, other transcription factors and regulatory genes that are transiently induced early, 2 h after flower removal, and a set of novel AZ-specific genes. All gene expressions initiated by flower removal and leading to pedicel abscission were inhibited by indole-3-acetic acid application, while 1-methylcyclopropene pretreatment inhibited only the ethylene-induced expressions, including those induced by wound-associated ethylene signals. These results confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes resulting from auxin depletion. Our results shed light on the regulatory control of abscission at the molecular level and further expand our knowledge of auxin-ethylene cross talk during the initial controlling stages of the process.

A randomised placebo-controlled multicentre trial of intravenous semapimod HCl for moderate to severe Crohn's disease.

OBJECTIVE: Semapimod, a small molecule known to inhibit proinflammatory cytokine activity, was studied to determine the optimal dose necessary to achieve a response in patients with moderate to severe Crohn's disease (CD). METHODS: A randomised, double-blind, placebo-controlled trial (CD04) was carried out followed by an open-label extension study (CD05). The trial was conducted in international multicentre outpatient clinics and included patients with moderate to severe CD (Crohn's Disease Activity Index (CDAI) 250-400). Placebo was administered for 3 days; 60 mg semapimod intravenously for 1 day with placebo for 2 days; or 60 mg semapimod intravenously for 3 days. Participants who completed CD04 could participate in the open-label extension study, CD05, to receive up to five additional semapimod HCl 60 mg daily doses three times every 6-8 weeks. The main outcome measures were CDAI, Inflammatory Bowel Disease Questionnaire (IBDQ), Crohn's Disease Endoscopic Inflammation Score (CDEIS) and serum C-reactive protein (CRP) concentration. RESULTS: 152 patients were randomised in CD04. Responses for 1 and 3 day regimens were similar to placebo for CDAI (p=0.82), IBDQ (p=0.85), CDEIS (p=0.57) and CRP (p=0.40). The only noteworthy treatment-related safety finding was infusion reaction (phlebitis): 7.3, 34.8 and 62.7% for the placebo and 1 and 3 day semapimod treatment groups, respectively (p<0.001). In the open-label CD05 study (included=119 patients) a posthoc analysis showed that the mean CDAI improved in patients receiving 6 compared with < or = 3 cumulative doses (204.1+/-83 vs 251.4+/-103.05, p=0.006). CONCLUSIONS: Single and 3 day dosing of semapimod (< or = 180 mg) was ineffective for the treatment of moderate to severe CD. However, cumulative dosing > or = 360 mg was associated with decreased CDAI in a limited number of patients.

Characterization of Two Ethephon-Induced IDA-Like Genes from Mango, and Elucidation of Their Involvement in Regulating Organ Abscission.

In mango (Mangifera indica L.), fruitlet abscission limits productivity. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide acts as a key component controlling abscission events in Arabidopsis. IDA-like peptides may assume similar roles in fruit trees. In this study, we isolated two mango IDA-like encoding-genes, MiIDA1 and MiIDA2. We used mango fruitlet-bearing explants and fruitlet-bearing trees, in which fruitlets abscission was induced using ethephon. We monitored the expression profiles of the two MiIDA-like genes in control and treated fruitlet abscission zones (AZs). In both systems, qRT-PCR showed that, within 24 h, both MiIDA-like genes were induced by ethephon, and that changes in their expression profiles were associated with upregulation of different ethylene signaling-related and cell-wall modifying genes. Furthermore, ectopic expression of both genes in Arabidopsis promoted floral-organ abscission, and was accompanied by an early increase in the cytosolic pH of floral AZ cells-a phenomenon known to be linked with abscission, and by activation of cell separation in vestigial AZs. Finally, overexpression of both genes in an Atida mutant restored its abscission ability. Our results suggest roles for MiIDA1 and MiIDA2 in affecting mango fruitlet abscission. Based on our results, we propose new possible modes of action for IDA-like proteins in regulating organ abscission.

Expression Kinetics of Regulatory Genes Involved in the Vesicle Trafficking Processes Operating in Tomato Flower Abscission Zone Cells during Pedicel Abscission.

The abscission process occurs in a specific abscission zone (AZ) as a consequence of the middle lamella dissolution, cell wall degradation, and formation of a defense layer. The proteins and metabolites related to these processes are secreted by vesicle trafficking through the plasma membrane to the cell wall and middle lamella of the separating cells in the AZ. We investigated this process, since the regulation of vesicle trafficking in abscission systems is poorly understood. The data obtained describe, for the first time, the kinetics of the upregulated expression of genes encoding the components involved in vesicle trafficking, occurring specifically in the tomato (Solanum lycopersicum) flower AZ (FAZ) during pedicel abscission induced by flower removal. The genes encoding vesicle trafficking components included soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), SNARE regulators, and small GTPases. Our results clearly show how the processes of protein secretion by vesicle trafficking are regulated, programmed, and orchestrated at the level of gene expression in the FAZ. The data provide evidence for target proteins, which can be further used for affinity purification of plant vesicles in their natural state. Such analyses and dissection of the complex vesicle trafficking networks are essential for further elucidating the mechanism of organ abscission.

Raising the pH of the Pulsing Solution Improved the Acropetal Transport of NAA and 2,4-D and Their Efficacy in Reducing Floret Bud Abscission of Red Cestrum Cut Flowers.

The use of auxins to improve the vase life of cut flowers is very limited. Previous studies demonstrated that a pulse treatment of Red Cestrum (Cestrum elegans Schlecht.) cut flowers with 2,4-dichlorophenoxyacetic acid (2,4-D) significantly reduced floret bud abscission, whereas 1-naphthaleneacetic acid (NAA) was ineffective. This difference resulted, at least in part, from the higher acropetal transport capability of 2,4-D compared to that of NAA. The present research focused on examining the factors affecting the acropetal transport, and hence the efficacy of the two auxins in reducing floret bud abscission of Red Cestrum cut flowers. We assumed that the differential acropetal transport capability of the two auxins results from the difference in their dissociation constants (pKa), with values of 2.75 and 4.23 for 2,4-D and NAA, respectively, which affects their pH-dependent physicochemical properties. Thus, increasing the pH of the pulsing solution above the pKa of both auxins might improve their acropetal movement. Indeed, the results of the present research show that raising the pH of the pulsing solution to pH 7.0 and above improved the efficacy of the two auxins in reducing floret bud abscission, with a higher effect on 2,4-D than that on NAA. Raising the pH of the pulsing solution decreased the adsorption and/or uptake of the two auxins by the cells adjacent to the xylem vessels, leading to an increase in their acropetal transport. The high pH of the pulsing solution increased the dissociation and hence decreased the lipophilicity of the auxin molecules, leading to improved acropetal movement. This effect was corroborated by the significant reduction in their 1-octanol/water partition coefficient (K OW ) values with the increase in the pH. A significant increase in the CeIAA1 transcript level was obtained in response to 2,4-D pulsing at pH 7.0 and 8.25 and to NAA pulsing at pH 8.25, indicating that the acropetally transported auxins were taken up by the cells under these conditions. Our data suggest that raising the pH of the pulsing solution would significantly contribute to the increased efficacy of auxins in improving the vase life of cut flowers.

Ethylene Induces a Rapid Degradation of Petal Anthocyanins in Cut Vanda 'Sansai Blue' Orchid Flowers.

Ethylene plays a major role in the regulation of flower senescence, including in the ethylene-sensitive Vanda 'Sansai Blue' orchid flowers. This cut flower is popular in Thailand due to its light blue big size florets possessing a beautiful shape pattern. In the present study, we further examined the rapid ethylene-induced process of active anthocyanin degradation in cut Vanda 'Sansai Blue' flowers, which occurred much before detection of other typical senescence-related symptoms. For this purpose, the cut inflorescences were exposed to air (control), 1 or 10 µl L-1 ethylene for 24 h, or to 0.2 µl L-1 1-methylcyclopropene (1-MCP) for 6 h followed by 10 µl L-1 ethylene for 24 h at 21°C, and the effects of these treatments on various parameters were assayed. While the fading-induced effect of ethylene was not concentration-dependent in this range, the ethylene treatment significantly reduced the flower vase life in a concentration-dependent manner, further confirming the separation of the bleaching process from senescence. Exposure of the inflorescences to 1-MCP pre-treatment followed by 10 µl L-1 ethylene, recovered both inflorescence color and anthocyanin content to control levels. Quantification of total anthocyanin content, performed by HPLC analysis on the basis of cyanidin-3-glocuside equivalents, showed that ethylene reduced and 1-MCP recovered the anthocyanins profile in non-hydrolyzed anthocyanin samples of Vanda 'Sansai Blue' florets, assayed at half bloom and bloom developmental stages. The results showed that the ethylene-induced color fading, observed immediately after treatment, resulted from a significant reduction in the levels of the two main anthocyanidins, cyanidin and delphinidin, as well as of other anthocyanidins present in low abundance, but not from changes in the levels of flavonols, such as kaempferol. This anthocyanin degradation process seems to operate via ethylene-increased peroxidase activity, detected at the bud stage. Taken together, our results suggest that the ethylene-induced rapid color bleaching in petals of cut Vanda 'Sansai Blue' flowers is an outcome of in-planta anthocyanin degradation, partially mediated by increased peroxidase activity, and proceeds independently of the flower senescence process.

Silencing polygalacturonase expression inhibits tomato petiole abscission.

Virus-induced gene silencing (VIGS) was used as a tool for functional analysis of cell wall-associated genes that have been suggested to be involved in leaf abscission. Tobacco rattle virus is an effective vector for VIGS in tomato (Lycopersicon esculentum). Silencing was more efficient when the plants were grown at 22 degrees C than when they were grown at 20 degrees C or 25 degrees C. The photobleaching phenotype resulting from silencing phytoene desaturase varied, depending on cultivar, from barely visible to photobleaching of entire leaves. To study the function of abscission-related genes, a purple transgenic tomato line constitutively expressing the maize anthocyanin regulatory gene, Leaf colour (Lc) was used. Silencing Lc expression in this line resulted in reduced anthocyanin production (reversing the colour from purple to green), thus providing a convenient silencing 'reporter'. Silencing tomato abscission-related polygalacturonases (TAPGs), using a TAPG1 fragment, delayed abscission and increased break strength of the abscission zone in explants treated with 1 mul l(-1) ethylene. The abundance of TAPG1 transcripts in the green (silenced) abscission zone tissues was <1% of that in the purple (non-silenced) controls. As a highly homologous region was used for all five polygalacturonases it is assumed that the effect of delayed abscission is the result of silencing all the genes in this family. By contrast, silencing abscission-related expansins (LeEXP11 and LeEXP12) and endoglucanases (LeCEL1 and LeCEL2) had no discernible effect on break strength, even when the two endoglucanase genes were silenced concurrently. Simultaneous silencing of TAPG and LeCEL1 was no more effective than silencing TAPG alone. The data demonstrate the importance of TAPGs in the abscission of leaf petioles.

Differential effects of NAA and 2,4-D in reducing floret abscission in cestrum (Cestrum elegans) cut flowers are associated with their differential activation of Aux/IAA homologous genes.

BACKGROUND AND AIMS: A previous study showed that the relative effectiveness of 2,4-dichlorophenoxyacetic acid (2,4-D) compared with that of 1-naphthaleneacetic acid (NAA) in reducing floret bud abscission in cestrum (Cestrum elegans) cut flowers was due to its acropetal transport. The aim of the present study was to examine if the differential effect of these auxins on floret abscission is reflected in the expression of Aux/IAA genes in the floret abscission zone (AZ). METHODS: cDNAs were isolated by PCR-based cloning from the floret AZ of auxin-treated cut flowers. The expression patterns of the cDNAs in various tissues and the effect of indole-3-acetic acid (IAA), applied with or without cycloheximide, on their expression in the floret AZ were examined by northern blot analysis. The regulation of transcript accumulation in the floret AZ in response to NAA or 2,4-D was measured by real-time PCR during auxin pulsing of cut flowers and vase life, concomitantly with floret abscission. KEY RESULTS: Six isolated cDNAs were identified to represent Aux/IAA homologous genes, designated as Cestrum elegans (Ce)-IAA1 to Ce-IAA6. Four Ce-IAA genes were characterized as early auxin-responsive genes (ARGs), and two (Ce-IAA1 and Ce-IAA5) as late ARGs. Only Ce-IAA5 was AZ-specific in floret buds. A temporal regulation of Ce-IAA transcript levels in the floret AZ was found, with 2,4-D inducing higher expression levels than NAA in floret buds. These Ce-IAA expression levels were negatively correlated with floret abscission. CONCLUSIONS: The differential transport characteristics of NAA and 2,4-D in cestrum cut flowers were reflected in differential activation of the Ce-IAA genes identified in the floret AZ. Therefore, Aux/IAA genes can be used as molecular markers to measure auxin activity, which reflects free auxin level in the AZ. Two of the identified genes, Ce-IAA1 and Ce-IAA5, may also have a regulatory role in abscission.

Microtubule reorientation in shoots precedes bending during the gravitropic response of cut snapdragon spikes.

The microtubule reorientation during the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes was investigated. Using indirect immunofluorescence methods, we examined changes in microtubule orientation in the cortex, endodermis and pith tissues of the shoot bending zone, in response to gravistimulation. Our results show that dense microtubule arrays were visible throughout the cortical, endodermal and pith shoot tissues, and that the transverse orientation of the microtubules (perpendicular to the growth axis) was specifically associated with the shoot growing bending zone. Microtubules showed gravity-induced kinetics of changes in their orientation, which occurred only in the upper stem flank and preceded shoot bending. While this observation, that the gravity-induced microtubule orientation precedes bending, was previously reported only in special above-ground organs such as coleoptiles and hypocotyls, our present study is the first to show that such patterns of change occur in mature flowering shoots. These changes were exhibited first in the upper flank of the cortex and then in the upper flank of the endodermis. No changes in microtubule orientation were observed in the cortex or endodermis tissues of the lower flanks or in the pith, suggesting that these tissues continue to grow during shoot gravistimulation. Our results imply that microtubules may be involved in growth cessation of the upper shoot flank occurring during the gravitropic bending of snapdragon cut spikes.

Ethylene production and signaling in tomato (Solanum lycopersicum) pollen grains is responsive to heat stress conditions.

KEY MESSAGE: Tomato pollen grains have the capacity for ethylene production, possessing specific components of the ethylene-biosynthesis and -signaling pathways, being affected/responsive to high-temperature conditions. Exposure of plants to heat stress (HS) conditions reduces crop yield and quality, mainly due to sensitivity of pollen grains. Recently, it was demonstrated that ethylene, a gaseous plant hormone, plays a significant role in tomato pollen heat-tolerance. It is not clear, however, whether, or to what extent, pollen grains are dependent on the capacity of the surrounding anther tissues for ethylene synthesis and signaling, or can synthesize this hormone and possess an active signaling pathway. The aim of this work was (1) to investigate if isolated, maturing and mature, tomato pollen grains have the capacity for ethylene production, (2) to find out whether pollen grains possess an active ethylene-biosynthesis and -signaling pathway and characterize the respective tomato pollen components at the transcript level, (3) to look into the effect of short-term HS conditions. Results from accumulation studies showed that pollen, anthers, and flowers produced ethylene and HS affected differentially ethylene production by (rehydrated) mature pollen, compared to anthers and flowers, causing elevated ethylene levels. Furthermore, several ethylene synthesis genes were expressed, with SlACS3 and SlACS11 standing out as highly HS-induced genes of the pollen ethylene biosynthesis pathway. Specific components of the ethylene-signaling pathway as well as several ethylene-responsive factors were expressed in pollen, with SlETR3 (ethylene receptor; named also NR, for never ripe) and SlCTR2 (constitutive triple response2) being HS responsive. This work shows that tomato pollen grains have the capacity for ethylene production, possessing active ethylene-biosynthesis and -signaling pathways, highlighting specific pollen components that serve as a valuable resource for future research on the role of ethylene in pollen thermotolerance.

Proteomics of Heat-Stress and Ethylene-Mediated Thermotolerance Mechanisms in Tomato Pollen Grains.

Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however, not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1,355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate, and energy metabolism. Ethephon-pre-treatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle, and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin, and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research.

The Tomato Hybrid Proline-rich Protein regulates the abscission zone competence to respond to ethylene signals.

The Tomato Hybrid Proline-rich Protein (THyPRP) gene was specifically expressed in the tomato (Solanum lycopersicum) flower abscission zone (FAZ), and its stable antisense silencing under the control of an abscission zone (AZ)-specific promoter, Tomato Abscission Polygalacturonase4, significantly inhibited tomato pedicel abscission following flower removal. For understanding the THyPRP role in regulating pedicel abscission, a transcriptomic analysis of the FAZ of THyPRP-silenced plants was performed, using a newly developed AZ-specific tomato microarray chip. Decreased expression of THyPRP in the silenced plants was already observed before abscission induction, resulting in FAZ-specific altered gene expression of transcription factors, epigenetic modifiers, post-translational regulators, and transporters. Our data demonstrate that the effect of THyPRP silencing on pedicel abscission was not mediated by its effect on auxin balance, but by decreased ethylene biosynthesis and response. Additionally, THyPRP silencing revealed new players, which were demonstrated for the first time to be involved in regulating pedicel abscission processes. These include: gibberellin perception, Ca2+-Calmodulin signaling, Serpins and Small Ubiquitin-related Modifier proteins involved in post-translational modifications, Synthaxin and SNARE-like proteins, which participate in exocytosis, a process necessary for cell separation. These changes, occurring in the silenced plants early after flower removal, inhibited and/or delayed the acquisition of the competence of the FAZ cells to respond to ethylene signaling. Our results suggest that THyPRP acts as a master regulator of flower abscission in tomato, predominantly by playing a role in the regulation of the FAZ cell competence to respond to ethylene signals.

Mutational analysis of hMsh6 in Israeli HNPCC and HNPCC-like families.

Germline mutations in DNA mismatch repair (DNA-MMR) genes, mainly hMlh1 and hMsh2, underlie Hereditary Non-Polyposis Colorectal Cancer (HNPCC). Germline hMSH6 gene mutations have been reported in a small subset of HNPCC families. In the present study, ethnically diverse individuals with HNPCC and HNPCC-like features were genotyped for hMsh6 germline mutations using exon-specific PCR, DGGE, and DNA sequencing. The study encompassed 92 individuals representing 88 unrelated families who were previously analyzed for Msh2 and Mlh1 mutations: Jewish Ashkenazim (n = 44), non-Ashkenazim (n = 27), Israeli Moslem-Arab (n = 15), Druze (n=3), and Cypriot non-Jews (n = 3). Of the study population, 71 had colon cancer (CRC), mean age at diagnosis was 50.9+/-13.2 years (range 16-73 years), 5 had endometrial cancer (two with concurrent CRC), (mean 43.6+/-3.26 years, range 38-45 years), and unaffected individuals (n = 18) were first degree relatives within HNPCC families and were genotyped at a mean age of 48.3+/-11.7 years (range 30-69 years). Of the 92 individuals analyzed, none showed a truncating hMsh6 mutation, and 6 (6.6%) harbored one of three germline missense mutations: a previously reported one (V878A), and two novel mutations (V509A, S227I). The pathogenic significance of these three missense mutations is yet unclear. In addition, 5 polymorphisms were detected, 2 of which were novel. We conclude that the rate of pathogenic hMsh6 mutations in HNPCC families of Jewish and Mediterranean origin is low, and that mutations in other genes probably account for the phenotype in these families.

Flowering shoots of ornamental crops as a model to study cellular and molecular aspects of plant gravitropism.

Flowering shoots offer a very convenient and excellent model system for in-depth study of shoot gravitropism in regular stems rather than in special aboveground organs, showing how plants cope with the force of gravity on Earth and change in orientation. Regarding the emerging notion that roots and shoots execute their gravitropic bending by different mechanisms, the use of flowering shoots offers additional confirmation for the suggested shoot-sensing mechanisms initially found in Arabidopsis. As a part of confirming this mechanism, studying this unique model system also enabled elucidation of the sequence of events operating in gravity signalling in shoots. Hence, using the system of flowering shoots provided an additional dimension to our understanding of shoot gravitropism and its hormonal regulation, which has been less advanced than root gravitropism. This is particularly important since the term "shoots" includes various aboveground organs. Hence, unlike other aboveground organs such as pulvini, the asymmetric growth in response to change in shoot orientation is accompanied in cut ornamental spikes by a continuous growth process. This chapter provides an overview of the basic methods, specifically developed or adapted from other graviresponding systems, for determining the main components which play a key role in gravistimulation signalling in flowering shoots.