Background and Objectives Sorghum (Sorghum bicolor L. Moench) is a multipurpose crop with high potential for the bioeconomy. Ten sweet, grain and dual-purpose sorghum genotypes were compared with respect to sugar-related traits and cell wall composition. Findings The dual-purpose hybrids Ganymed, Zerberus, and Tarzan performed better with respect to plant height, cane weight, and juice volume, but the sweet genotype KIT1 produced the highest sugar concentration (degrees Brix) in the stem. Analytical sugar data demonstrate genotypic differences in terms of sugar composition and concentrations, with sucrose being dominant. The monosaccharide composition of the stem cell wall polysaccharides showed surprisingly little genotypic variability. Variations in contents of lignin and cell wall-bound hydroxycinnamate monomers were moderate. Conclusions Sweet, grain and dual-purpose sorghum genotypes differ widely in terms of sugar-related morphological parameters but are comparable with respect to their cell wall chemistry. Significance and Novelty The use of sorghum as a bioeconomy crop has mainly focused on the extraction of sugar for bioethanol production. However, besides cell wall polymers, the potential usage of hydroxycinnamates as platform molecules for the chemical industry may improve the valorization of the residues after sugar extraction. This application appears to be fairly independent of genotype, further increasing the potential of sorghum for the bioeconomy.

The transition to terrestrial plants was accompanied by a progressive loss of microtubule minus-end-directed dynein motors. Instead, the minus-end-directed class-XIV kinesins expanded considerably, likely related to novel functions. One of these motors, OsDLK (Dual Localisation Kinesin from rice), decorates cortical microtubules but moves into the nucleus in response to cold stress. This analysis of loss-of-function mutants in rice indicates that OsDLK participates in cell elongation during development. Since OsDLK harbours both a nuclear localisation signal and a putative leucin zipper, we asked whether the cold-induced import of OsDLK into the nucleus might correlate with specific DNA binding. Conducting a DPI-ELISA screen with recombinant OsDLKT (lacking the motor domain), we identified the Opaque2 motif as the most promising candidate. This motif is present in the promoter of NtAvr9/Cf9, the tobacco homologue of Cold-Box Factor 4, a transcription factor involved in cold adaptation. A comparative study revealed that the cold-induced accumulation of NtAvr9/Cfp9 was specifically quelled in transgenic BY−2 cells overexpressing OsDLK-GFP. These findings are discussed as a working model, where, in response to cold stress, OsDLK partitions from cortical microtubules at the plasma membrane into the nucleus and specifically modulates the expression of genes involved in cold adaptation.

Interaction between phytochromes and hormones is becoming one of the major issues in plant photophysiology. In this work, effects of defense‐related jasmonic acid (JA) on phytochrome A (phyA) were investigated by fluorescence spectroscopy making use of two JA biosynthesis mutants of rice: cpm2 with the inactivated gene allene oxide cyclase and hebiba with additional genes deleted. Constant far‐red light (FRc) mediated by phyA reduced its content in the wild type (WT) and mutants, and brought about domination of its light‐stable pool (phyA″) in WT and light‐labile pool (phyA′) in the mutants. Pulsed FRp was much less effective. This FR effect classifies as primarily HIR with a low fluence threshold; it comprises inhibition of phyA biosynthesis, stimulation of phyA″→phyA′ transformation and phyA′ destruction. In the mutants, phyA suppresses [Pchlide] under FRp (VLFR) and stimulates it under FRc (HIR); these effects are lacking in WT. Similarly, phyA suppresses roots'growth under FRp in the mutants but not in WT. These JA mutant features suggest that JA reduces the phyA functional activity primarily in its phyA″ form mediating HIR. This modulating JA action on phyA functions under FR limiting their extreme manifestations may have contributed to the evolutionary advances of the land plants.

BACKGROUND:Plant discrimination is of relevance for taxonomic, evolutionary, breeding and nutritional studies. To this purpose, evidence is reported to demonstrate TBP (Tubulin-Based-Polymorphism) as a DNA-based method suitable for assessing plant diversity.RESULTS:Exploiting one of the most valuable features of TBP, that is the convenient and immediate application of the assay to groups of individuals that may belong to different taxa, we show that the TBP method can successfully discriminate different agricultural species and their crop wild relatives within the Papilionoideae subfamily. Detection of intraspecific variability is demonstrated by the genotyping of 27 different accessions of Phaseolus vulgaris.CONCLUSIONS:These data illustrate TBP as a useful and versatile tool for plant genotyping. Since its potential has not yet been fully appreciated by the scientific community, we carefully report all the experimental details of a successful TBP protocol, while describing different applications, so that the method can be replicated in other laboratories.

Plants have the ability to alleviate the harmful effects caused by abiotic and biotic stress. Phytohormones play a very important role in the acclimation to these stresses. To study the role of jasmonate in the acclimation to osmotic stress, an ALLENE OXIDE CYCLASE (AOC) mutant of rice (cpm2), disrupted in the biosynthesis of jasmonic acid (JA), and its wild type (WT) background were employed to investigate their responses to osmotic stress caused by treatment with polyethylene glycol (PEG) 6000. WT showed tolerance to osmotic stress, correlated with a fast transient increase of JA and JA-isoleucine (JA-Ile) in the shoots prior to an increase in abscisic acid (ABA), followed by a second increase in jasmonates when exposing to osmotic stress during 24 h. In roots, the pattern of hormonal increase was similar, but the response appeared to be faster, and remained transient, also with respect to low levels of jasmonates upon continuing osmotic stress. The mutant, which was containing extremely low levels of jasmonates, was hypersensitive to the stress. However, ABA accumulated in both, shoots and roots of cpm2, to similar (but not equal) levels as those seen in the WT, demonstrating that the biosynthesis or catabolism of ABA in response to osmotic stress is at least partially independent of JA, but can be modulated by JA. Our results suggest that jasmonates operate in parallel, presumably synergistically, to ABA, and are indispensable for osmotic stress tolerance in rice.

Plants can use volatiles for remote suppression of competitors. Mints produce essential oils, which are known to affect the growth of other plants. We used a comparative approach to identify allelopathic compounds from different Mints (genus Mentha, but also including Cat Mint, Nepeta cataria, and Corean Mint, Agastache rugosa, belonging to sisters clades within the Mentheae) using the standard cress germination assay as readout. To understand the mechanism behind this allelopathic effect, we investigated the response of tobacco BY-2 cell lines, expressing GFP-tagged markers for microtubules and actin filaments to these essential oils. Based on the comparison between bioactivity and chemical components, we identified menthone as prime candidate for the allelopathic effect, and confirmed this bioactivity targeted to microtubules experimentally in both, plant cells (tobaccoBY-2), and seedlings (Arabidopsis thaliana). We could show that menthone disrupted microtubules and induced mortality linked with a rapid permeabilization (less than 15 min) of the plasma membrane. This mortality was elevated in a tubulin marker line, where microtubules are mildly stabilized. Our study paves the way for the development of novel bioherbicides that would be environmentally friendly.

In the present study, we used 12 genotypes of sorghum originated from different countries (five sweet, four grain and three forage). These different genotypes and types of sorghum were evaluated for the agro-morphological traits that are associated with the estimated sugar and bioethanol yield to estimate their phenotypic diversity. Analysis of variance showed significant differences between different types of sorghum for all the evaluated traits. Sweet sorghum genotypes, however, showed better performance with respect to all studied traits than the other genotypes. A positive significant correlation was observed between plant height, leaf number, leaf area, biomass yield, cane and bagasse yields, and the predicted bioethanol yield. Both, cluster and principal component analysis were performed to group the genotypes according to their agro-morphological and molecular similarity coefficients. For analytical approaches, the Iranian grain and forage genotypes clustered separately from the other genotypes. The clustering patterns obtained from the molecular dominant markers had higher discriminatory power than using morphological characters to separate sweet genotypes from the forage and grain sorghum ones. The results clearly indicated that sweet sorghum can be grown in Germany and maintains its superiority in biomass production and sugar yield over grain and forage sorghum types.

Polar auxin transport depends on the polar localization of auxin-efflux carriers. The cycling of these carriers between cell interior and plasma membrane depends on actin. The dynamic of actin not only affects auxin transport, but also changes the auxin-responsiveness. To study the potential link between auxin responsiveness and actin dynamics, we investigated developmental responses of the non-transformed BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cell line and the transgenic BY-2 strain GF11 (stably transformed BY-2 cells with a GFP-fimbrin actin-binding domain 2 construct). The developmental process was divided into three distinct stages: cell cycling, cell elongation and file disintegration. Several phenotypes were measured to monitor the cellular responses to different concentrations of exogenous natural auxin (Indole-3-acetic acid, IAA). We found that auxin stimulated and prolonged the mitotic activity, and delayed the exit from the proliferation phase. However, both responses were suppressed in the GF11 line. At the stationary phase of the cultivation cycle, auxin strongly accelerated the cell file disintegration. Interestingly, it was not suppressed but progressed to a more complete disintegration in the GF11 line. During the cultivation cycle, we also followed the organization of actin in the GF11 line and did not detect any significant difference in actin organization from untreated control or exogenous IAA treatment. Therefore, our findings indicate that the specific differences observed in the GF11 line must be linked with a function of actin that is not structural. It means that there is a sensory role of actin for auxin signaling.

The eukaryotic cytoskeleton is a complex and dynamic network that regulates important cellular functions and is driven by a large variety of forces or mechanical stimuli. Due to their mechanical rigidity, microtubules are able to transport such stimuli allowing integrating distant regions of a cell instantaneously. This is relevant for the response to pressure, gravity, or osmotic changes and during mechanotransduction, a critical process during many severe diseases such as deafness or cancer. So far, only equilibrium mechanical properties of single microtubules have been characterized. Since intra- and extracellular forces occur on a brought range of time scales, we fill this void by using an in vitro bottom-up approach to determine the frequency response of single microtubules and small networks thereof that mimic the basic cytoskeletal structure. We combine a label-free darkfield imaging technique with multiple time-shared optical tweezers to flexibly construct and force probe such networks with a well-defined, user-selected geometry over a broad frequency range. We report on a length dependent stiffening of individual microtubules above a physiologically relevant transition frequency between 1-30Hz due to the excitation of higher order bending modes. This increased transport efficiency for high frequencies displays a mechanical high-pass filter with a tunable cutoff frequency, e.g., allowing the cell to react to fluctuations at distant sites. Furthermore, we identify and relate different mechanical responses of different network geometries to different functions inside the cell. Triangular networks, for example, are sensitive to tiny imbalances and display a high stiffness even for low frequencies, resembling a load bearing scaffold protecting the nucleus. The mechanistic comparison between basic network geometries, the known cytoskeletal topologies and the general function of different cell lines will substantially strengthen our understanding of the function and structure of the cytoskeleton.

China is one of the most important centers of origin for Citrus genetic resources. Due to the high content of secondary metabolites, mining wild Chinese Citrus for novel medical applications is promising. In this study, extracts of Chinese wild species from different taxonomical groups were screened for potential effects on microtubules (MTs) in vitro. MT density as a readout for nucleation, and frequency distribution over MT lengths as a readout for elongation and decay were determined by quantitative image analysis via a standardized coverslip assay using fluorescently labelled neurotubulin. Extract from peels of Citrus ichangensis Swing. strongly increased the density of MTs; whereas, extract from peels of Citurs limon (L.) Burm.f. exerted the opposite effect. Extract from pulp of Citrus limonia Osbeck promoted MT elongation, and in addition induced a small population of very long MTs. These data suggest that wild Chinese Citrus harbour compounds that act specifically on different aspects of MT nucleation, elongation, and decay.

Plants can acquire freezing tolerance in response to cold but non-freezing temperatures. To efficiently activate this cold acclimation, low temperature has to be sensed and processed swiftly, a process that is linked with a transient elimination of microtubules. Here, we address cold-induced microtubules elimination in a grapevine cell line stably expressing a green fluorescent protein fusion of Arabidopsis TuB6, which allows to follow their response invivo and to quantify this response by quantitative image analysis. We use time-course studies with several specific pharmacological inhibitors and activators to dissect the signalling events acting upstream of microtubules elimination. We find that microtubules disappear within 30 min after the onset of cold stress. We provide evidence for roles of calcium influx, membrane rigidification, and activation of NAD(P)H oxidase as factors in signal susception and amplification. We further conclude that a G-protein in concert with a phospholipase D convey the signal towards microtubules, whereas calmodulin seems to be not involved. Moreover, activation of jasmonate pathway in response to cold is required for an efficient microtubule response. We summarize our findings in a working model on a complex signalling hub at the membrane-cytoskeleton interphase that assembles the susception, perception and early transduction of cold signals.

Nuclear migration and positioning are crucial for the morphogenesis of plant cells. We addressed the potential role of nuclear positioning for polarity induction using an experimental system based on regenerating protoplasts, where the induction of a cell axis de novo can be followed by quantification of specific regeneration stages. Using overexpression of fluorescently tagged extranuclear (perinuclear actin basket, kinesins with a calponin homology domain (KCH)) as well as intranuclear (histone H2B) factors of nuclear positioning and time-lapse series of the early stages of regeneration, we found that nuclear position is no prerequisite for polarity formation. However, polarity formation and nuclear migration were both modulated in the transgenic lines, indicating that both phenomena depend on factors affecting cytoskeletal tensegrity and chromatin structure. We integrated these findings into a model where retrograde signals are required for polarity induction. These signals travel via the cytoskeleton from the nucleus toward targets at the plasma membrane.

Plants employ two layers of defence that differ with respect to cell death: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). In our previous work, we have comparatively mapped the molecular events in a cell system derived from the wild American grape Vitis rupestris, where cell death-independent defence can be triggered by PAMP flg22, whereas the elicitor Harpin activates a cell death-related ETI-like response. Both defence responses overlapped with respect to early events, such as calcium influx, apoplastic alkalinisation, oxidative burst, mitogen-activated protein kinase (MAPK) signalling, activation of defence-related genes and accumulation of phytoalexins. However, timing and amplitude of early signals differed. In the current study, we address the role of jasmonates (JAs) as key signalling compounds in hypersensitive cell death. We find, in V. rupestris, that jasmonic acid and its bioactive conjugate jasmonoyl-isoleucine (JA-Ile) rapidly accumulate in response to flg22 but not in response to Harpin. However, Harpin can induce programmed cell death, whereas exogenous methyl jasmonate (MeJA) fails to do so, although both signals induce a similar response of defence genes. Also in a second cell line from V. vinifera cv. 'Pinot Noir', where Harpin cannot activate cell death and where flg22 fails to induce JA and JA-Ile, defence genes are activated in a similar manner. These findings indicate that the signal pathway culminating in cell death must act independently from the events culminating in the accumulation of toxic stilbenes.

Understanding the mechanism by which plants sense, signal and respond to salinity stress is of great interest to plant biologists. In stress signalling, often the same molecules are involved in both damage-related and adaptive events. To dissect this complexity, we compared the salinity responses of two grapevine cell lines differing in their salinity tolerance. We followed rapid changes in the cellular content of sodium and calcium, apoplastic alkalinisation and slower responses in the levels of jasmonic acid, its active isoleucine conjugate and abscisic acid, as well as of stilbenes. Differences in timing and sensitivity to either the lanthanoid Gd or exogenous calcium provide evidence for an adaptive role of early sodium uptake through non-selective cation channels acting upstream of Ca(2+) and H(+) fluxes. We find a correlation of salt sensitivity with unconstrained jasmonate (JA) signalling, whereas salt adaptation correlates with tight control of jasmonic acid and its isoleucine conjugate, accompanied by accumulation of abscisic acid and suppression of stilbenes that trigger defence-related cell death. The data are discussed by a model where efficient fine-tuning of JA signalling determines whether cells will progress towards adaptation or programme cell death.

Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP2) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus.

Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently-tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage-like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.

Plasmopara viticola is the causal agent of grapevine downy mildew and is among the most important diseases in viticulture. It originates from North America, where it coevolved with wild Vitis species. Beginning in the 1870s it turned into a global epidemic that has been causing severe yield losses. It is generally believed that a single species is causing downy mildew on a large variety of economically important cultivars. Here we report, based on one nuclear and two mitochondrial markers, that isolates from vineyards in the United States fall into three highly distinct phylogenetic lineages. One of these contains European strains and affects Vitis vinifera cultivars, while the other two lineages affect also other species of Vitis. The divergence between these lineages is high, and, judging from the genetic variation in other Plasmopara lineages, might reflect distinct species. Due to the potentially significant implications for quarantine regulations and resistance breeding, detailed studies will be necessary to clarify whether these genetically distinct lineages occur outside of North America or are still confined there.

Fluorescence microscopy has developed into a key technology of the postgenomic era in biology, because it combines structural information with molecular specificity. However, the resolution of this approach is limited by bleaching and optical cross‐reference of the fluorescent labels. Fluorescent semiconductor quantum dots (QDs) provide excellent bleaching stability and tunable emission spectra, and therefore would be an excellent alternative to overcome these limitations. However, to apply them to cell biology, three challenges have to be met: bioconjugation to molecular probes that confer the specificity of the label, passage through the external barriers of the cell, and suppression of toxic side effects of the nanoparticles. In plant cells that are ensheathed by a cellulosic cell wall, these challenges are especially prominent. Moreover, plants are located at the start of the food chain and thus of high relevance for the ecotoxicological assessment of nanomaterials. We have therefore explored the application of nanoparticles to plant cell biology. We have first evaluated different strategies to visualize microtubules by QDs in vitro and in cellula. By using silica‐coated QDs coupled to anti‐tubulin antibodies we were able to image microtubules in tobacco BY‐2 cells by direct immunofluorescence making use of the superior bleaching stability of the nanoparticle label. To adapt this tool for in vivo imaging, we have successfully employed Trojan Peptoids as vehicles into living tobacco cells. We want to extend this strategy not only to use functionalized nanoparticles for life‐cell imaging, but also to adapt them as tool to manipulate intracellular architecture.