Theory and Practice in Measuring In-Vitro Extensibility of Growing Plant Cell Walls.

This chapter summarizes four extensometer techniques for measuring cell wall extensibility in vitro and discusses how the results of these methods relate to the concept and ideal measurement of cell wall extensibility in the context of plant cell growth. These in-vitro techniques are particularly useful for studies of the molecular basis of cell wall extension. Measurements of breaking strength, elastic compliance and plastic compliance may be informative about changes in cell wall structure, whereas measurements of wall stress relaxation and creep are sensitive to both changes in wall structure and wall-loosening processes, such as those mediated by expansins and some lytic enzymes. A combination of methods is needed to obtain a broader view of cell wall behavior and properties connected with the concept of cell wall extensibility .

Assay of Plasma Membrane H+-ATPase in Plant Tissues under Abiotic Stresses.

Plasma membrane (PM) H+-ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H+-ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.

Variation in cucumber (Cucumis sativus L.) fruit size and shape results from multiple components acting pre-anthesis and post-pollination.

MAIN CONCLUSION: Morphological, QTL, and gene expression analyses indicate variation in cucumber fruit size and shape results from orientation, timing, and extent of cell division and expansion, and suggest candidate gene factors. Variation in cucumber (Cucumis sativus L.) fruit size and shape is highly quantitative, implicating interplay of multiple components. Recent studies have identified numerous fruit size and shape quantitative trait loci (QTL); however, underlying factors remain to be determined. We examined ovary and fruit development of two sequenced cucumber genotypes with extreme differences in fruit size and shape, Chinese Long '9930' (CL9930), and pickling type 'Gy14'. Differences were observed in several independent factors that can influence size and shape: ovule number, rate and period of cell division in longitudinal and cross section in ovaries and fruit, timing and rate of fruit expansion in length and diameter, and cell shape. Level and timing of expression of select fruit growth stage marker genes and candidate fruit size gene homologs associated with cucumber fruit size and shape QTL were examined from 5-day pre-anthesis to 20-day post-pollination. Our results indicate that variation in fruit size and shape results from differences in cell number and shape in longitudinal and cross section, driven in turn by differences in orientation, timing, and duration of cell division and expansion, both pre- and post-anthesis, and suggest candidate genes contributing to determination of cucumber fruit size and shape.

Isolation and functional characterization of CsLsi1, a silicon transporter gene in Cucumis sativus.

Cucumber (Cucumis sativus) is a widely grown cucurbitaceous vegetable that exhibits a relatively high capacity for silicon (Si) accumulation, but the molecular mechanism for silicon uptake remains to be clarified. Here we isolated and characterized CsLsi1, a gene encoding a silicon transporter in cucumber (cv. Mch-4). CsLsi1 shares 55.70 and 90.63% homology with the Lsi1s of a monocot and dicot, rice (Oryza sativa) and pumpkin (Cucurbita moschata), respectively. CsLsi1 was predominantly expressed in the roots, and application of exogenous silicon suppressed its expression. Transient expression in cucumber protoplasts showed that CsLsi1 was localized in the plasma membrane. Heterologous expression in Xenopus laevis oocytes showed that CsLsi1 evidenced influx transport activity for silicon but not urea or glycerol. Expression of cucumber CsLsi1-mGFP under its own promoter showed that CsLsi1 was localized at the distal side of the endodermis and the cortical cells in the root tips as well as in the root hairs near the root tips. Heterologous expression of CsLsi1 in a rice mutant defective in silicon uptake and the over-expression of this gene in cucumber further confirmed the role of CsLsi1 in silicon uptake. Our results suggest that CsLsi1 is a silicon influx transporter in cucumber. The cellular localization of CsLsi1 in cucumber roots is different from that in other plants, implying the possible effect of transporter localization on silicon uptake capability.

Impedance Flow Cytometry: A Novel Technique in Pollen Analysis.

INTRODUCTION: An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen. METHOD: Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays. RESULTS: Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population. CONCLUSION: The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.

Phloem transcriptome signatures underpin the physiological differentiation of the pedicel, stalk and fruit of cucumber (Cucumis sativus L.).

Cucumber is one of the most important vegetables grown worldwide due to its important economic and nutritional value. The cucumber fruit consists morphologically of the undesirable stalk and the tasty fruit; however, physiological differentiation of these two parts and the underlying molecular basis remain largely unknown. Here we characterized the physiological differences among the pedicel, stalk and fruit, and compared the respective phloem transcriptomes using laser capture microdissection coupled with RNA sequencing (RNA-Seq). We found that the pedicel was characterized by minor cell expansion and a high concentration of stachyose, the stalk showed rapid cell expansion and high raffinose accumulation, and the fruit featured transition from cell division to cell expansion and high levels of monosaccharides. Analyses of transcriptome data indicated that cell wall- and calcium ion binding-related genes contributed to the cell expansion in the pedicel and stalk, whereas genes implicated in cell cycle and hormone actions regulated the transition from cell division to cell expansion in the fruit. Differential sugar distribution in these three phloem-connected tissues resulted from tissue-specific sugar metabolism and transport. Enrichment of transcription factors in the stalk and fruit may facilitate nutrient accumulation in these sink organs. As such, phloem-located gene expression partially orchestrated physiological differentiation of the pedicel, stalk and fruit in cucumber. In addition, we identified 432 cucumber-unique genes and five phloem markers guiding future functional studies.

Mechanistic study of programmed cell death of root border cells of cucumber (Cucumber sativus L.) induced by copper.

Programmed cell death (PCD) in root border cells (RBCs) induced by Copper (Cu) has been little studied. This study explored whether Cu induced PCD in RBCs of cucumber or not and investigated the possible mechanisms. The results showed that the percentage of apoptotic and necrotic RBCs increased with increasing concentration of Cu treatment. A quick burst of ROS in RBCs was detected, while mitochondrial membrane potential (ΔΨm) decreased sharply with Cu treatment. Caspase-3 like protease activity showed a tendency of increase with Cu treatment. The potential of Cu to induce PCD in RBCs of cucumber was first proved. Our results showed that ROS generation and mitochondrial membrane potential loss played important roles in Cu-induced caspase-3-like activation and PCD in RBCs of cucumber, which provided new insight into the signaling cascades that modulate Cu phytotoxicity mechanism.

The role of putrescine in the regulation of proteins and fatty acids of thylakoid membranes under salt stress.

Polyamines can alleviate the inhibitory effects of salinity on plant growth by regulating photosynthetic efficiency. However, little information is available to explain the specific mechanisms underlying the contribution of polyamines to salt tolerance of the photosynthetic apparatus. Here, we investigated the role of putrescine (Put) on the photosynthetic apparatus of cucumber seedlings under salt stress. We found that NaCl stress resulted in severe ion toxicity and oxidative stress in cucumber chloroplasts. In addition, salinity caused a significant increase in the saturated fatty acid contents of thylakoid membranes. Put altered unsaturated fatty acid content, thereby alleviating the disintegration of thylakoid grana lamellae and reducing the number of plastoglobuli in thylakoid membranes. BN-PAGE revealed Put up-regulated the expression of ATP synthase, CP47, D1, Qb, and psbA proteins and down-regulated CP24, D2, and LHCII type III in NaCl-stressed thylakoid membranes. qRT-PCR analysis of gene expression was used to compare transcript and protein accumulation among 10 candidate proteins. For five of these proteins, induced transcript accumulation was consistent with the pattern of induced protein accumulation. Our results suggest that Put regulates protein expression at transcriptional and translational levels by increasing endogenous polyamines levels in thylakoid membranes, which may stabilise photosynthetic apparatus under salt stress.

Colletotrichum orbiculare Regulates Cell Cycle G1/S Progression via a Two-Component GAP and a GTPase to Establish Plant Infection.

Morphogenesis in filamentous fungi depends on appropriate cell cycle progression. Here, we report that cells of the cucumber anthracnose fungus Colletotrichum orbiculare regulate G1/S progression via a two-component GAP, consisting of Budding-uninhibited-by-benomyl-2 (Bub2) and Byr-four-alike-1 (Bfa1) as well as its GTPase Termination-of-M-phase-1 (Tem1) to establish successful infection. In a random insertional mutagenesis screen of infection-related morphogenesis, we isolated a homolog of Saccharomyces cerevisiae, BUB2, which encodes a two-component Rab GAP protein that forms a GAP complex with Bfa1p and negatively regulates mitotic exit. Interestingly, disruption of either Co BUB2 or Co BFA1 resulted in earlier onset of nuclear division and decreased the time of phase progression from G1 to S during appressorium development. S. cerevisiae GTPase Tem1p is the downstream target of the Bub2p/Bfa1p GAP complex. Introducing the dominant-negative form of Co Tem1 into Co bub2Δ or Co bfa1Δ complemented the defect in G1/S progression, indicating that Co Bub2/Co Bfa1 regulates G1/S progression via Co Tem1. Based on a pathogenicity assay, we found that Co bub2Δ and Co bfa1Δ reduced pathogenesis by attenuating infection-related morphogenesis and enhancing the plant defense response. Thus, during appressorium development, C. orbiculare Bub2/Bfa1 regulates G1/S progression via Co Tem1, and this regulation is essential to establish plant infection.

Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber.

BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade consists of three types of reversibly phosphorylated kinases, namely, MAPK, MAPK kinase (MAPKK/MEK), and MAPK kinase kinase (MAPKKK/MEKK), playing important roles in plant growth, development, and defense response. The MAPK cascade genes have been investigated in detail in model plants, including Arabidopsis, rice, and tomato, but poorly characterized in cucumber (Cucumis sativus L.), a major popular vegetable in Cucurbitaceae crops, which is highly susceptible to environmental stress and pathogen attack. RESULTS: A genome-wide analysis revealed the presence of at least 14 MAPKs, 6 MAPKKs, and 59 MAPKKKs in the cucumber genome. Phylogenetic analyses classified all the CsMAPK and CsMAPKK genes into four groups, whereas the CsMAPKKK genes were grouped into the MEKK, RAF, and ZIK subfamilies. The expansion of these three gene families was mainly contributed by segmental duplication events. Furthermore, the ratios of non-synonymous substitution rates (Ka) and synonymous substitution rates (Ks) implied that the duplicated gene pairs had experienced strong purifying selection. Real-time PCR analysis demonstrated that some MAPK, MAPKK and MAPKKK genes are preferentially expressed in specific organs or tissues. Moreover, the expression levels of most of these genes significantly changed under heat, cold, drought, and Pseudoperonospora cubensis treatments. Exposure to abscisic acid and jasmonic acid markedly affected the expression levels of these genes, thereby implying that they may play important roles in the plant hormone network. CONCLUSION: A comprehensive genome-wide analysis of gene structure, chromosomal distribution, and evolutionary relationship of MAPK cascade genes in cucumber are present here. Further expression analysis revealed that these genes were involved in important signaling pathways for biotic and abiotic stress responses in cucumber, as well as the response to plant hormones. Our first systematic description of the MAPK, MAPKK, and MAPKKK families in cucumber will help to elucidate their biological roles in plant.

Fruit removal increases root-zone respiration in cucumber.

BACKGROUND AND AIMS: Many attempts have been made to avoid the commonly observed fluctuations in fruit initiation and fruit growth in crop plants, particularly in cucumber (Cucumis sativus). Weak sinks of the fruit have been assumed to result in low sink/source ratios for carbohydrates, which may inhibit photosynthesis. This study focuses on the effects of low sink-source ratios on photosynthesis and respiration, and in particular root-zone respiration. METHODS: Mature fruit-bearing cucumber plants were grown in an aerated nutrient solution. The root containers were designed as open chambers to allow measurement of CO2 gas exchange in the root zone. A similar arrangement in a gas-exchange cuvette enabled simultaneous measurements of CO2 exchange in the shoot and root zones. KEY RESULTS: Reducing the sinks for carbohydrates by removing all fruit from the plants always resulted in a doubling of CO2 exchange in the root zone within a few hours. However, respiration of the shoot remained unaffected and photosynthesis was only marginally reduced, if at all. CONCLUSIONS: The results suggest that the increased level of CO2 gas exchange in the root zone after removing the carbon sinks in the shoot is due primarily to the exudation of organic compounds by the roots and their decomposition by micro-organisms. This hypothesis must be tested in further experiments, but if proved correct it would make sense to include carbon leakage by root exudation in cucumber production models. In contrast, inhibition of photosynthesis was measurable only at zero fruit load, a situation that does not occur in cucumber production systems, and models that estimate production can therefore ignore (end-product) inhibition of photosynthesis.

Single-copy gene-based chromosome painting in cucumber and its application for chromosome rearrangement analysis in Cucumis.

Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single-copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome-specific gene probes. This single-copy gene-based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis-aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross-species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research.

Evidence of programmed cell death induced by reconditioning after cold stress in cucumber fruit and possible involvement of ethylene.

BACKGROUND: Cucumber fruit is susceptible to chilling injury (CI), which could be accelerated significantly with subsequent shelf-life. This type of CI culminates in deterioration of organs and eventually leads to cell death. In this study, evidence of programmed cell death (PCD), involving cell death induced by cold stress, was investigated in cucumber. Harvested cucumber (Cucumis sativus L. cv. Zhexiu-1) fruits were stored at 2 °C for 3, 6 or 9 days and subsequently transferred to 20 °C for 2 days. RESULTS: Significant cell death acceleration was observed upon reconditioning after 9 days' cold stress when the hallmark of PCD - DNA laddering - was clearly observed. Further evidence of nuclear DNA cleavage was confirmed by the in situ TdT-mediated dUTP nick end labeling (TUNEL) assay. Chromatin condensation and nucleus distortion were observed by nuclear staining of DPI. Ethylene burst was observed upon reconditioning after 9 days of consecutive cold stress. CONCLUSION: The features of PCD process induced by reconditioning after cold stress in cucumber fruit may be mainly attributed to ethylene burst.

Characterization and expression profiling of cucumber kinesin genes during early fruit development: revealing the roles of kinesins in exponential cell production and enlargement in cucumber fruit.

Rapid cell division and expansion in early fruit development are important phases for cucumber fruit yield and quality. Kinesin proteins are microtubule-based motors responsible for modulating cell division and enlargement. In this work, the candidate kinesin genes involved in rapid cell division and expansion during cucumber fruit development were investigated. The morphological and cellular changes during early fruit development were compared in four cucumber genotypes with varied fruit size. The correlation between the expression profiles of cucumber kinesin genes and cellular changes in fruit was investigated. Finally, the biochemical characteristics and subcellular localizations of three candidate kinesins were studied. The results clarified the morphological and cellular changes during early cucumber fruit development. This study found that CsKF2-CsKF6 were positively correlated with rapid cell production; CsKF1 and CsKF7 showed a strongly positive correlation with rapid cell expansion. The results also indicated that CsKF1 localized to the plasma membrane of fast-expanding fruit cells, that CsKF2 might play a role in fruit chloroplast division, and that CsKF3 is involved in the function or formation of phragmoplasts in fruit telophase cells. The results strongly suggest that specific fruit-enriched kinesins are specialized in their functions in rapid cell division and expansion during cucumber fruit development.

Unisexual cucumber flowers, sex and sex differentiation.

Sex is a universal phenomenon in the world of eukaryotes. Attempts have been made to understand regulatory mechanisms for plant sex determination by investigating unisexual flowers. The cucumber plant is one of the model systems for studying how sex determination is regulated by phytohormones. A systematic investigation of the development of unisexual cucumber flowers is summarized here, and it is suggested that the mechanism of the unisexual flower can help us to understand how the process leading to one type of gametogenesis is prevented. Based on these findings, we concluded that the unisexual cucumber flowers is not an issue of sex differentiation, but instead a mechanism for avoiding self-pollination. Sex differentiation is essentially the divergent point(s) leading to heterogametogenesis. On the basis of analyses of sex differentiation in unicellular organisms and animals as well as the core process of plant life cycle, a concept of "sexual reproduction cycle" is proposed for understanding the essential role of sex and a "progressive model" for future investigations of sex differentiation in plants.

Size-topology relations in packings of grains, emulsions, foams, and biological cells.

Particulate packings in 3D are used to study the effects of compression and polydispersity on the geometry of the tiling in these systems. We find that the dependence of the neighbor number on cell size is quasilinear in the monodisperse case and becomes nonlinear above a threshold polydispersity, independent of the method of creation of the tiling. These size-topology relations can be described by a simple analytical theory, which quantifies the effects of positional disorder in the monodisperse case and those of size disorder in the polydisperse case and is applicable in two and three dimensions. The theory thus gives a unifying framework for a wide range of amorphous systems, ranging from biological tissues, foams, and bidisperse disks to compressed emulsions and granular matter.

Effects of short term iron citrate treatments at different pH values on roots of iron-deficient cucumber: a Mössbauer analysis.

Alkaline pH values and bicarbonate greatly reduce the mobility and uptake of Fe, causing Fe deficiency chlorosis. In the present work, the effects of pH and bicarbonate on the uptake and accumulation of Fe in the roots of cucumber were studied by Mössbauer spectroscopy combined with physiological tests and diaminobenzidine enhanced Perls staining. Mössbauer spectra of Fe-deficient cucumber roots supplied with 500 µM (57)Fe(III)-citrate at different pH values showed the presence of an Fe(II) and an Fe(III) component. As the pH was increased from 4.5 to 7.5, the root ferric chelate reductase (FCR) activity decreased significantly and a structural change in the Fe(III) component was observed. While at pH 4.5 the radial intrusion of Fe reached the endodermis, at pH 7.5, Fe was found only in the outer cortical cell layers. The Mössbauer spectra of Fe-deficient plants supplied with Fe(III)-citrate in the presence of bicarbonate (pH 7.0 and 7.5) showed similar Fe components, but the relative Fe(II) concentration compared to that measured at pH values 6.5 and 7.5 was greater. The Mössbauer parameters calculated for the Fe(II) component in the presence of bicarbonate were slightly different from those of Fe(II) alone at pH 6.5-7.5, whereas the FCR activity was similarly low. Fe incorporation into the root apoplast involved only the outer cortical cell layers, as in the roots treated at pH 7.5. In Fe-sufficient plants grown with Fe(III)-citrate and 1mM bicarbonate, Fe precipitated as granules and was in diffusely scattered grains on the root surface. The "bicarbonate effect" may involve a pH component, decreasing both the FCR activity and the acidification of the apoplast and a mineralization effect leading to the slow accumulation of extraplasmatic Fe particles, forming an Fe plaque and trapping Fe and other minerals in biologically unavailable forms.

A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases.

Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this "tethered network" model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-ß-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.

The origin and composition of cucurbit "phloem" exudate.

Cucurbits exude profusely when stems or petioles are cut. We conducted studies on pumpkin (Cucurbita maxima) and cucumber (Cucumis sativus) to determine the origin and composition of the exudate. Morphometric analysis indicated that the exudate is too voluminous to derive exclusively from the phloem. Cold, which inhibits phloem transport, did not interfere with exudation. However, ice water applied to the roots, which reduces root pressure, rapidly diminished exudation rate. Sap was seen by microscopic examination to flow primarily from the fascicular phloem in cucumber, and several other cucurbit species, but primarily from the extrafascicular phloem in pumpkin. Following exposure of leaves to 14CO2, radiolabeled stachyose and other sugars were detected in the exudate in proportions expected of authentic phloem sap. Most of this radiolabel was released during the first 20 s. Sugars in exudate were dilute. The sugar composition of exudate from extrafascicular phloem near the edge of the stem differed from that of other sources in that it was high in hexose and low in stachyose. We conclude that sap is released from cucurbit phloem upon wounding but contributes negligibly to total exudate volume. The sap is diluted by water from cut cells, the apoplast, and the xylem. Small amounts of dilute, mobile sap from sieve elements can be obtained, although there is evidence that it is contaminated by the contents of other cell types. The function of P-proteins may be to prevent water loss from the xylem as well as nutrient loss from the phloem.

Characterization of an ethylene-inducible, calcium-dependent nuclease that is differentially expressed in cucumber flower development.

• Production of unisexual flowers is an important mechanism that promotes cross-pollination in angiosperms. We previously identified primordial anther-specific DNA damage and organ-specific ethylene perception responsible for the arrest of stamen development in female flowers, but little is known about how the two processes are linked. • To identify potential links between the two processes, we performed suppression subtractive hybridization (SSH) on cucumber (Cucumis sativus L.) stamens of male and female flowers at stage 6, with stamens at stage 5 of bisexual flowers as a control. • Among the differentially expressed genes, we identified an expressed sequence tag (EST) encoding a cucumber homolog to an Arabidopsis calcium-dependent nuclease (CAN), designated CsCaN. Full-length CsCaN cDNA and the respective genomic DNA sequence were cloned and characterized. The CsCaN protein exhibited calcium-dependent nuclease activity. CsCaN showed ubiquitous expression; however, increased gene expression was detected in the stamens of stage 6 female flowers compared with male flowers. As expected, CsCaN expression was ethylene inducible. It was of great interest that CsCaN was post-translationally modified. • This study demonstrated that CsCaN is a novel cucumber nuclease gene, whose DNase activity is regulated at multiple levels, and which could be involved in the primordial anther-specific DNA damage of developing female cucumber flowers.