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.

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.

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.

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.

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.

Examination of the Abscission-Associated Transcriptomes for Soybean, Tomato, and Arabidopsis Highlights the Conserved Biosynthesis of an Extensible Extracellular Matrix and Boundary Layer.

Abscission zone (AZ) development and the progression of abscission (detachment of plant organs) have been roughly separated into four stages: first, AZ differentiation; second, competence to respond to abscission signals; third, activation of abscission; and fourth, formation of a protective layer and post-abscission trans-differentiation. Stage three, activation of abscission, is when changes in the cell wall and extracellular matrix occur to support successful organ separation. Most abscission research has focused on gene expression for enzymes that disassemble the cell wall within the AZ and changes in phytohormones and other signaling events that regulate their expression. Here, transcriptome data for soybean, tomato and Arabidopsis were examined and compared with a focus not only on genes associated with disassembly of the cell wall but also on gene expression linked to the biosynthesis of a new extracellular matrix. AZ-specific up-regulation of genes associated with cell wall disassembly including cellulases (beta-1,4-endoglucanases, CELs), polygalacturonases (PGs), and expansins (EXPs) were much as expected; however, curiously, changes in expression of xyloglucan endotransglucosylase/hydrolases (XTHs) were not AZ-specific in soybean. Unexpectedly, we identified an early increase in the expression of genes underlying the synthesis of a waxy-like cuticle. Based on the expression data, we propose that the early up-regulation of an abundance of small pathogenesis-related (PR) genes is more closely linked to structural changes in the extracellular matrix of separating cells than an enzymatic role in pathogen resistance. Furthermore, these observations led us to propose that, in addition to cell wall loosening enzymes, abscission requires (or is enhanced by) biosynthesis and secretion of small proteins (15-25 kDa) and waxes that form an extensible extracellular matrix and boundary layer on the surface of separating cells. The synthesis of the boundary layer precedes what is typically associated with the post-abscission synthesis of a protective scar over the fracture plane. This modification in the abscission model is discussed in regard to how it influences our interpretation of the role of multiple abscission signals.

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.

De novo Transcriptome Sequencing and Development of Abscission Zone-Specific Microarray as a New Molecular Tool for Analysis of Tomato Organ Abscission.

Abscission of flower pedicels and leaf petioles of tomato (Solanum lycopersicum) can be induced by flower removal or leaf deblading, respectively, which leads to auxin depletion, resulting in increased sensitivity of the abscission zone (AZ) to ethylene. However, the molecular mechanisms that drive the acquisition of abscission competence and its modulation by auxin gradients are not yet known. We used RNA-Sequencing (RNA-Seq) to obtain a comprehensive transcriptome of tomato flower AZ (FAZ) and leaf AZ (LAZ) during abscission. RNA-Seq was performed on a pool of total RNA extracted from tomato FAZ and LAZ, at different abscission stages, followed by de novo assembly. The assembled clusters contained transcripts that are already known in the Solanaceae (SOL) genomics and NCBI databases, and over 8823 identified novel tomato transcripts of varying sizes. An AZ-specific microarray, encompassing the novel transcripts identified in this study and all known transcripts from the SOL genomics and NCBI databases, was constructed to study the abscission process. Multiple probes for longer genes and key AZ-specific genes, including antisense probes for all transcripts, make this array a unique tool for studying abscission with a comprehensive set of transcripts, and for mining for naturally occurring antisense transcripts. We focused on comparing the global transcriptomes generated from the FAZ and the LAZ to establish the divergences and similarities in their transcriptional networks, and particularly to characterize the processes and transcriptional regulators enriched in gene clusters that are differentially regulated in these two AZs. This study is the first attempt to analyze the global gene expression in different AZs in tomato by combining the RNA-Seq technique with oligonucleotide microarrays. Our AZ-specific microarray chip provides a cost-effective approach for expression profiling and robust analysis of multiple samples in a rapid succession.

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.

Identification of defense-related genes newly-associated with tomato flower abscission.

The current abscission model suggests the formation of a post-abscission trans-differentiation of a protective layer as the last step of the process. The present report expands the repertoire of genes activated in the tomato flower abscission zone (AZ), which are likely to be involved in defense responses. We identified four different defense-related genes, including: Cysteine-type endopeptidase, α-Dioxygenase 1 (α-DOX1), HopW-1-1-Interacting protein2 (WIN2), and Stomatal-derived factor-2 (SDF2), that are newly-associated with the late stage of the abscission process. The late expression of these genes, induced at 8-14 h after flower removal when pedicel abscission was already in progress, was AZ-specific, and was inhibited by treatments that prevented pedicel abscission, including 1-methylcyclopropene pretreatment or IAA application. This information supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses.

Actomyosin mediates gravisensing and early transduction events in reoriented cut snapdragon spikes.

We investigated the involvement of the actomyosin network in the early events of the gravitropic response of cut snapdragon (Antirrhinum majus L.) spikes. The effects of the actin-modulating drug, cytochalasin D (CD) and/or the myosin inhibitor, 2,3-butanedione-2-monoxime (BDM) on amyloplast displacement, lateral auxin transport and consequently on stem bending were examined. The inhibitory effect on cytoskeleton integrity was studied by using indirect immunofluorescence double-labeling of actin and myosin. Our results demonstrate that no organizational changes in actin filaments occurred in cortical and endodermal cells of the stem bending zone during reorientation. These results suggest that actin depolymerization is not required for amyloplast sedimentation. Unlike the chloroplasts in the cortex, the amyloplasts in the endodermis were surrounded by actin and myosin, indicating that amyloplasts may be attached to the actin filaments via the motor protein, myosin. This suggests the involvement of myosin as part of the actomyosin complex in amyloplast movement in vertical as well as in reoriented stems. This suggestion was supported by the findings showing that: (a) BDM or CD disrupted the normal organization of actin either by altering characteristic distribution patterns of myosin-like protein in the cortex (BDM), or by causing actin fragmentation (CD); (b) both compounds inhibited the gravity-induced amyloplast displacement in the endodermis. Additionally, these compounds also inhibited lateral auxin transport across the stem and stem gravitropic bending. Our study suggests that during stem reorientation amyloplasts possibly remain attached to the actin filaments, using myosin as a motor protein. Thus, gravisensing and early transduction events in the gravitropic response of snapdragon spikes, manifested by amyloplast displacement and lateral auxin transport, are mediated by the actomyosin complex.

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.

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.

Changes in starch and inositol 1,4,5-trisphosphate levels and auxin transport are interrelated in graviresponding oat (Avena sativa) shoots.

This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.

Gravitropic bending and plant hormones.

Gravitropism is a complex multistep process that redirects the growth of roots and various above-ground organs in response to changes in the direction of the gravity vector. The anatomy and morphology of these graviresponding organs indicates a certain spatial separation between the sensing region and the responding one, a situation that strongly suggests the requirement of phytohormones as mediators to coordinate the process. The Cholodny-Went hypothesis suggested auxin as the main mediator of gravitropism. So far, ample evidence has been gathered with regard to auxin asymmetrical detection, polar and lateral transport involving influx and efflux carriers, response signaling pathway, and possible modes of action in differential cell elongation, supports its major role in gravitropism at least in roots. However, it is becoming clear that the participation of other hormones, acting in concert with auxin, is necessary as well. Of particular importance is the role of ethylene in shoot gravitropism, possibly associated with the modulation of auxin transport or sensitivity, and the key role implicated for cytokinin as the putative root cap inhibitor that controls early root gravitropism. Therefore, the major advances in the understanding of transport and signaling of auxin, ethylene, and cytokinin may shed light on the possibly tight and complicated interactions between them in gravitropism. Not much convincing evidence has been accumulated regarding the participation of other phytohormones, such as gibberellins, abscisic acid, brassinosteroids, jasmonates, and salicylic acid, in gravitropism. However, the emerging concept of cooperative hormone action opens new possibilities for a better understanding of the complex interactions of all phytohormones and their possible synergistic effects and involvement in the gravitropic bending process.