Physiological and transcriptomic responses of Lanzhou Lily (Lilium davidii, var. unicolor) to cold stress.

Low temperature induces changes in plants at physiological and molecular levels, thus affecting growth and development. The Lanzhou lily (Lilium davidii, var. unicolor) is an important medicinal plant with high economic value. However, the molecular mechanisms underlying its photosynthetic and antioxidation responses to low temperature still remain poorly understood. This study subjected the Lanzhou lily to the two temperatures of 20°C (control) and 4°C (low temperature) for 24 h. Physiological parameters related to membrane integrity, photosynthesis, antioxidant system, and differentially expressed genes were investigated. Compared with control, low temperature increased the relative electrical conductivity by 43.2%, while it decreased net photosynthesis rate, ratio of variable to maximal fluorescence, and catalase activity by 47.3%, 10.1%, and 11.1%, respectively. In addition, low temperature significantly increased the content of soluble protein, soluble sugar, and proline, as well as the activity of superoxide dismutase and peroxidase. Comparative transcriptome profiling showed that a total of 238,109 differentially expressed genes were detected. Among these, 3,566 were significantly upregulated while 2,982 were significantly downregulated in response to low temperature. Gene Ontology enrichment analysis indicated that in response to low temperature, the mostly significantly enriched differentially expressed genes were mainly involved in phosphorylation, membrane and protein kinase activity, as well as photosynthesis, light harvesting, light reaction, and alpha,alpha-trehalose-phosphate synthase activity. Kyoto Encyclopedia of Genes and Genomes enrichment analysis also indicated that the most significantly enriched pathways involved ribosome biogenesis in eukaryotes, phenylalanine metabolism, circadian rhythm, porphyrin and chlorophyll metabolism, photosynthesis of antenna proteins, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, the expression patterns of 10 randomly selected differentially expressed genes confirmed the RNA-Seq results. These results expand the understanding of the physiological and molecular mechanisms underlying the response of the Lanzhou lily to low temperature stress.

Climatic niche characteristics of native and invasive Lilium lancifolium.

One of the topics currently under discussion in biological invasions is whether the species' climatic niche has been conserved or, alternatively, has diverged during invasions. Here, we explore niche dynamic processes using the complex invasion history model of Lilium lancifolium, which is the first tested case of a native species (Korea) with two hypothesized spatial (regional and intercontinental) and temporal arrivals: (1) as an archaeophyte in East Asia (before AD 1500); and (2) as a neophyte in Europe, North America, Australia, and New Zealand (after AD 1500). Following a niche examination through both environmental and geographical spaces, the species in the archaeophyte range has apparently filled the ancestral native niche and, rather, would have increased it considerably. The species as a neophyte shows a closer climatic match with the archaeophyte range than with the native one. This pattern of niche similarity suggests that the neophyte range was probably colonized by a subset of archaeophyte propagules adapted to local climate that promoted the species' establishment. Overall, niche conservatism is proposed at each colonization step, from native to archaeophyte, and from archaeophyte to neophyte ranges. We detected signals of an advanced invasion stage within the archaeophyte range and traces of an early introduction stage in neophyte ranges.

Scent chemistry is key in the evolutionary transition between insect and mammal pollination in African pineapple lilies.

Volatile emissions may play a key role in structuring pollination systems of plants with morphologically unspecialised flowers. Here we test for pollination by small mammals in Eucomis regia and investigate whether its floral scent differs markedly from fly- and wasp-pollinated congeners and attracts mammals. We measured floral traits of E. regia and made comparisons with insect-pollinated congeners. We observed floral visitors and examined fur and faeces of live-trapped mammals for pollen. We determined the contributions of different floral visitors to seed set with selective exclusion and established the breeding system with controlled pollination experiments. Using bioassays, we examined whether mammals are attracted by the floral scent and are effective agents of pollen transfer. Eucomis regia differs from closely related insect-pollinated species mainly in floral scent, with morphology, colour and nectar properties being similar. We found that mice and elephant-shrews pollinate E. regia, which is self-incompatible and reliant on vertebrates for seed production. Mammals are strongly attracted to the overall floral scent, which contains unusual sulphur compounds, including methional (which imparts the distinctive potato-like scent and which was shown to be attractive to small mammals). The results highlight the important role of scent chemistry in shifts between insect and mammal pollination systems.

Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant.

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L(-1) BA and 0.2 mg L(-1) NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L(-1) BA and 0.5 mg L(-1) NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot-root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.

Hydrogen peroxide affects ion channels in lily pollen grain protoplasts.

Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model - protoplasts obtained from lily pollen grains at the early germination stage - to reveal the effect of H2 O2 on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS-sensitive currents on the pollen grain plasma membrane: the hyperpolarisation-activated calcium current, which is strongly enhanced by H2 O2 , and the outward potassium current, which is modestly enhanced by H2 O2 . We used low concentrations of H2 O2 that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining.

Lily Cultivars Have Allelopathic Potential in Controlling Orobanche aegyptiaca Persoon.

As a devastating holoparasitic weed, Orobanche aegyptiaca Persoon. (Egyptian broomrape) causes serious damage to agricultural production and threatens economic development, which has raised widespread concern. The present study was conducted to determine whether lilies have the potential to be used as 'trap crops' for controlling O. aegyptiaca Persoon. In the experiments, the ability of three popular lily cultivars (Lilium Oriental hybrids 'Sorbonne', Lilium LA (Longiflorum hybrids x Asiatic hybrids) hybrids 'Ceb Dazzle', and Lilium Longiflorum hybrids (L. formosanum x L. longiflorum) 'L. formolongo') to induce O. aegyptiaca Persoon. seed germination was assessed. Parts of the three lily cultivars, including the rhizosphere soil and underground and above-ground organs, all induced "suicidal germination" of parasitic O. aegyptiaca Persoon. seed at four growth stages. Specifically, Sorbonne and Ceb Dazzle behaved with similar allelopathy, and the bulb, scale leaf and aerial stem exhibited stronger allelopathic effects on O. aegyptiaca Pers. germination compared to other organs. Aqueous L. formolongo leaf extracts may contain more stable, effective stimulants given that they induced the highest germination rate at 76.7% even though the extracts were serially diluted. We speculate that these organs may be advantageous in further isolating and purifying economical active substances that can be substitutes for GR24. These results indicate that lilies have the potential to be used as a trap crops or can be processed into green herbicide formulations that can be applied in agriculture production to rapidly deplete the seed bank of O. aegyptiaca Persoon. parasitic weeds in soil.

Pollination increases ethylene production in Lilium hybrida cv. Brindisi flowers but does not affect the time to tepal senescence or tepal abscission.

In many species, pollination induces a rapid increase in ethylene production, which induces early petal senescence, petal abscission, or flower closure. Cross-pollination in Lilium hybrida cv. Brindisi resulted in a small increase in flower ethylene production. In intact plants and in isolated flowers, pollination had no effect on the time to tepal senescence or tepal abscission. When applied to closed buds of unpollinated flowers, exogenous ethylene slightly hastened the time to tepal senescence and abscission. However, exogenous ethylene had no effect when the flowers had just opened, i.e. at the time of pollination. Experiments with silver thiosulphate, which blocks the ethylene receptor, indicated that endogenous ethylene had a slight effect on the regulation of tepal senescence and tepal abscission, although only at the time the tepals were still inside buds and not in open flowers. Low ethylene-sensitivity after anthesis therefore explains why pollination had no effect on the processes studied.

The plant stigma exudate: a biochemically active extracellular environment for pollen germination?

During sexual reproduction, pollen performance is greatly influenced by the female tissues. The stigma exudate, i.e., the extracellular secretion that covers the stigma outermost surface, has been usually regarded as a reservoir of water, secondary metabolites, cell wall precursors and compounds that serve as energy supply for rapid pollen tube growth. In an attempt to identify the proteins present in the stigma secretome, we performed a large-scale analysis in two species (Lilium longiflorum and Olea europaea) following a proteomic-based approach. The resulting data strongly suggest that the stigma exudate is not a mere storage site but also a biochemically active environment with a markedly catabolic nature. Thus, this secretion may modulate early pollen tube growth and contribute to the senescence of stigma after pollination. In addition, a putative cross-talk between genetic programs that regulate stress/defense and pollination responses in the stigma is also suggested. The stigma exudate might also functionally diverge between species on the basis on their ecology and the biochemical, morphological and anatomical features of their stigmas. Unexpectedly, we identified in both exudates some intracellular proteins, suggesting that a mechanism other than the canonical ER-Golgi exocytic pathway may exist in the stigma and contribute to exudate secretion.

LlSR28 is involved in pollen germination by affecting filamentous actin dynamics.

Alternative splicing plays important roles in gene regulation and contributes to protein complexity. Previous studies suggest that alternative splicing exists in members of the villin/gelsolin/fragmin superfamily. In this study, a serine/argine-rich (SR) protein cDNA with 28 kDa protein (LlSR28) was isolated from a lily (Lilium longiflorum) expression library. Protein domain analysis showed that LlSR28 had similar structures to Arabidopsis SR45 (AtSR45), and LlSR28 could complement the phenotype of loss of AtSR45 function. Therefore, overexpression of LlSR28 and AtSR45 mutant (atsr45-1) were used in the following experiments. Overexpression of LlSR28 in Arabidopsis completely inhibited pollen germination. In contrast, the pollen germination of atsr45-1 was earlier than that of wild-type. In addition, pollen of atsr45-1 contained less F-actin at the corresponding hydration stage during pollen germination compared to that of wild-type. Alternative splicing analysis showed that Arabidopsis villin1 (AtVLN1) transcript encoding the full-length protein was increased, and that encoding the truncated protein was decreased in atst45-1. Moreover, the mRNA expression level of other actin-binding proteins (ABPs) abundant in Arabidopsis pollen was also changed in atsr45-1. In conclusion, we hypothesize that LlSR28 alters F-actin dynamics probably through its alternative splicing activities to affect directly or indirectly the alternative splicing of AtVLN1 and the expression of different ABPs, which then affects the pollen germination.

A biomechanical model of anther opening reveals the roles of dehydration and secondary thickening.

Understanding the processes that underlie pollen release is a prime target for controlling fertility to enable selective breeding and the efficient production of hybrid crops. Pollen release requires anther opening, which involves changes in the biomechanical properties of the anther wall. In this research, we develop and use a mathematical model to understand how these biomechanical processes lead to anther opening. Our mathematical model describing the biomechanics of anther opening incorporates the bilayer structure of the mature anther wall, which comprises the outer epidermal cell layer, whose turgor pressure is related to its hydration, and the endothecial layer, whose walls contain helical secondary thickening, which resists stretching and bending. The model describes how epidermal dehydration, in association with the thickened endothecial layer, creates forces within the anther wall causing it to bend outwards, resulting in anther opening and pollen release. The model demonstrates that epidermal dehydration can drive anther opening, and suggests why endothecial secondary thickening is essential for this process (explaining the phenotypes presented in the myb26 and nst1nst2 mutants). The research hypothesizes and demonstrates a biomechanical mechanism for anther opening, which appears to be conserved in many other biological situations where tissue movement occurs.

An osmotic model of the growing pollen tube.

Pollen tube growth is central to the sexual reproduction of plants and is a longstanding model for cellular tip growth. For rapid tip growth, cell wall deposition and hardening must balance the rate of osmotic water uptake, and this involves the control of turgor pressure. Pressure contributes directly to both the driving force for water entry and tip expansion causing thinning of wall material. Understanding tip growth requires an analysis of the coordination of these processes and their regulation. Here we develop a quantitative physiological model which includes water entry by osmosis, the incorporation of cell wall material and the spreading of that material as a film at the tip. Parameters of the model have been determined from the literature and from measurements, by light, confocal and electron microscopy, together with results from experiments made on dye entry and plasmolysis in Lilium longiflorum. The model yields values of variables such as osmotic and turgor pressure, growth rates and wall thickness. The model and its predictive capacity were tested by comparing programmed simulations with experimental observations following perturbations of the growth medium. The model explains the role of turgor pressure and its observed constancy during oscillations; the stability of wall thickness under different conditions, without which the cell would burst; and some surprising properties such as the need for restricting osmotic permeability to a constant area near the tip, which was experimentally confirmed. To achieve both constancy of pressure and wall thickness under the range of conditions observed in steady-state growth the model reveals the need for a sensor that detects the driving potential for water entry and controls the deposition rate of wall material at the tip.

Sucrose accelerates flower opening and delays senescence through a hormonal effect in cut lily flowers.

Sugars are generally used to extend the vase life of cut flowers. Such beneficial effects have been associated with an improvement of water relations and an increase in available energy for respiration by floral tissues. In this study we aimed at evaluating to what extent (i) endogenous levels of sugars in outer and inner tepals, androecium and gynoecium are altered during opening and senescence of lily flowers; (ii) sugar levels increase in various floral tissues after sucrose addition to the vase solution; and (iii) sucrose addition alters the hormonal balance of floral tissues. Results showed that endogenous glucose levels increased during flower opening and decreased during senescence in all floral organs, while sucrose levels increased in outer and inner tepals and the androecium during senescence. Sucrose treatment accelerated flower opening, and delayed senescence, but did not affect tepal abscission. Such effects appeared to be exerted through a specific increase in the endogenous levels of sucrose in the gynoecium and of glucose in all floral tissues. The hormonal balance was altered in the gynoecium as well as in other floral tissues. Aside from cytokinin and auxin increases in the gynoecium; cytokinins, gibberellins, abscisic acid and salicylic acid levels increased in the androecium, while abscisic acid decreased in outer tepals. It is concluded that sucrose addition to the vase solution exerts an effect on flower opening and senescence by, among other factors, altering the hormonal balance of several floral tissues.

Osmoregulation in Lilium pollen grains occurs via modulation of the plasma membrane H+ ATPase activity by 14-3-3 proteins.

To allow successful germination and growth of a pollen tube, mature and dehydrated pollen grains (PGs) take up water and have to adjust their turgor pressure according to the water potential of the surrounding stigma surface. The turgor pressure of PGs of lily (Lilium longiflorum) was measured with a modified pressure probe for simultaneous recordings of turgor pressure and membrane potential to investigate the relation between water and electrogenic ion transport in osmoregulation. Upon hyperosmolar shock, the turgor pressure decreased, and the plasma membrane (PM) hyperpolarizes in parallel, whereas depolarization of the PM was observed with hypoosmolar treatment. An acidification and alkalinization of the external medium was monitored after hyper- and hypoosmotic treatments, respectively, and pH changes were blocked by vanadate, indicating a putative role of the PM H(+) ATPase. Indeed, an increase in PM-associated 14-3-3 proteins and an increase in PM H(+) ATPase activity were detected in PGs challenged by hyperosmolar medium. We therefore suggest that in PGs the PM H(+) ATPase via modulation of its activity by 14-3-3 proteins is involved in the regulation of turgor pressure.

The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen.

As a first step in understanding the membrane-related dynamics during pollen grain germination and subsequent tube growth, the changes in protein abundance of membrane and membrane-associated proteins of 5 different membrane/organelle fractions were studied at physiologically important stages (0, 10, 30, 60, and 240 min) of Lilium longiflorum pollen in vitro culture. Proteins of each fraction and time point were identified by 'shot-gun' proteomics (LC-MS/MS). Analysis of more than 270 identified proteins revealed an increase in the abundance of proteins involved in cytoskeleton, carbohydrate and energy metabolism, as well as ion transport before pollen grain germination (10-30 min), whereas proteins involved in membrane/protein trafficking, signal transduction, stress response and protein biosynthesis decreased in abundance during this time. Proteins of amino acids and lipids/steroids metabolism, proteolysis, transcription, cell wall biosynthesis as well as nutrient transport showed a time-independent abundance profile. These spatiotemporal patterns were confirmed by immunodetection of specific proteins of the cellular processes membrane/protein trafficking and ion transport. Our results reveal major protein rearrangements at endomembranes and the plasma membrane before and as the pollen grains start tube growth. The spatiotemporal protein abundance changes correlate with the underlying developmental and physiological processes of the germinating pollen grain.

[Polyspermism and antipodal fertilization in Lilium (Tourn.) L. species].

It has been shown that several pollen tubes can penetrate into the embryo sac on the source side of the antipodal apparatus. One of the pair of sperms of additional pollen tube copulates with the upper antipodal, the second sperm copulates with the lower antipodal or rarely penetrates in the central cell. The process of fertilization was accomplished by the phase of nuclei morphological similarity characteristic ofsyngamy (by postmitotic type of fertilization according to Gerasimova-Navashina). A directional growth of additional pollen tubes involves a specifically differentiation of antipodal cells that imitates the egg cell.

Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts.

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.

The LLA23 protein translocates into nuclei shortly before desiccation in developing pollen grains and regulates gene expression in Arabidopsis.

We have isolated the LLA23 gene in the pollen of Lilium longiflorum. The LLA23 gene encodes an ASR (named after abscisic acid, stress and ripening) protein that has a nuclear localization sequence at the C terminus. The gene is interrupted by one single intron and possesses a long 5'-untranslated region. Southern blots of lily genomic DNA indicated that LLA23 is a member of a small gene family. We examined the link between LLA23 location and the desiccation that naturally occurs in developing anthers using immunogold labeling. When pollen reached maturity, a significant increase in LLA23 labeling was observed in the nuclei of both vegetative and generative cells from 10- to 12-cm buds and thereafter. This clearly demonstrates that a marked increase in LLA23 translocation from the cytoplasm to both nuclei of pollen grains occurs in 12-cm buds, a stage shortly before the commencement of desiccation during anther development. In addition, microarray analysis showed that 410 (206 up-regulated and 204 down-regulated) genes have altered expression in LLA23-overexpressing plants. Quantitative PCR analysis confirmed the changes in mRNA levels observed in our microarray analysis. This genome-wide overview of gene expression supports the theory that LLA23 acts as a regulator.

Cytoplasmic channels and their association with plastids in male meiocytes of tobacco, onion and lily.

The ultrastructures of male meiocytes in tobacco, onion and lily were studied to elucidate the interaction between cytoplasmic channels (CCs) and plastids. Before meiosis, the male sporogenous cells had identically thickened cell walls (CWs) traversed by typical plasmodesmata (PDs). After entering meiosis, their CWs became uneven in thickness and 80-500nm aperture CCs were formed. Simultaneously, plastids or plastid-like bodies (PLBs) differing in size and morphology assembled at one or both ends of the CCs. These plastids and PLBs commonly orientated their sharper ends to face the CCs and were co-orientated on the axial line crossing the CC. Such pairs of plastids were often interconnected through the CC by thin (50-100nm) threads emanating from their membranes. Sometimes, plastids or PLBs extended directly from one side of a CW to the other, forming a bridge via the CC. In some cases, several plastids formed bridges between cells via one common CC. This is the first report that clearly demonstrates an intercellular continuum of, or communication between, plastids in male plant meiocytes.

The distribution of membrane-bound 14-3-3 proteins in organelle-enriched fractions of germinating lily pollen.

Proteins of the 14-3-3 family show a broad range of activities in plants, depending on their localisation in different cellular compartments. Different organelle membranes of pollen grains and pollen tubes of Lilium longiflorum Thunb. were separated simultaneously using optimised discontinuous sucrose density centrifugation. The obtained organelle-enriched fractions were identified as vacuolar, Golgi, endoplasmic reticulum and plasma membranes, according to their marker enzyme activities, and were assayed for membrane-bound 14-3-3 proteins by immunodetection. 14-3-3 proteins were detected in the cytoplasm as well as in all obtained organelle fractions but were also released into the extracellular medium. In pollen grains, much more plasma membrane-bound 14-3-3 proteins were detected than in the PM-enriched fraction of pollen tubes, whereas the level of Golgi- and ER-associated 14-3-3 proteins was similar in pollen grains and tubes. This shift in the localisation of membrane-associated 14-3-3 proteins is probably correlated with a change in the major function of 14-3-3 proteins, e.g., perhaps changing from initiating pollen grain germination by activation of the PM H +-ATPase to recruitment of membrane proteins via the secretory pathway during tube elongation.

Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube.

The actin cytoskeleton plays a crucial role in the growth and polarity of the pollen tube. Due to inconsistencies in the conventional preservation methods, we lack a unified view of the organization of actin microfilaments, especially in the apical domain, where tip growth occurs. In an attempt to improve fixation methods, we have developed a rapid freeze-whole mount procedure, in which growing pollen tubes (primarily lily) are frozen in liquid propane at -180 degrees C, substituted at -80 degrees C in acetone containing glutaraldehyde, rehydrated, quenched with sodium borohydride, and probed with antibodies. Confocal microscopy reveals a distinct organization of actin in the apical domain that consists of a dense cortical fringe or collar of microfilaments starting about 1-5 microm behind the extreme apex and extending basally for an additional 5-10 microm. In the shank of the pollen tube, basal to the fringe, actin forms abundant longitudinal filaments that are evenly dispersed throughout the cytoplasm. We have also developed an improved ambient-temperature chemical fixation procedure, modified from a protocol based on simultaneous fixation and phalloidin staining. We removed EGTA, elevated the pH to 9, and augmented the fixative with ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS). Notably, this protocol preserves the actin cytoskeleton in a pattern similar to that produced by cryofixation. These procedures provide a reproducible way to preserve the actin cytoskeleton; employing them, we find that a cortical fringe in the apex and finely dispersed longitudinal filaments in the shank are consistent features of the actin cytoskeleton.