Dynamic changes in calcium signals during root gravitropism.

Gravitropism is a vital mechanism through which plants adapt to their environment. Previous studies indicated that Ca2+ may play an important role in plant gravitropism. However, our understanding of the calcium signals in root gravitropism is still largely limited. Using a vertical stage confocal and transgenic Arabidopsis R-GECO1, our data showed that gravity stimulation enhances the occurrence of calcium spikes and increases the Ca2+ concentration in the lower side of the root cap. Furthermore, a close correlation was observed in the asymmetry of calcium signals with the inclination angles at which the roots were oriented. The frequency of calcium spikes on the lower side of 90°-rotated root decreases rapidly over time, whereas the asymmetric distribution of auxin readily strengthens for up to 3 h, indicating that the calcium spikes, promoted by gravity stimulation, may precede auxin as one of the early signals. In addition, the root gravitropism of starchless mutants is severely impaired. Correspondingly, no significant increase in calcium spike occurrence was observed in the root caps of these mutants within 15 min following a 90° rotation, indicating the involvement of starch grains in the formation of calcium spikes. However, between 30 and 45 min after a 90° rotation, asymmetric calcium spikes were indeed observed in the root of starchless mutants, suggesting that starch grains are not indispensable for the formation of calcium spikes. Besides, co-localization analysis suggests that the ER may function as calcium stores during the occurrence of calcium spikes. These findings provide further insights into plant gravitropism.

Adaptive evolution of the enigmatic Takakia now facing climate change in Tibet.

The most extreme environments are the most vulnerable to transformation under a rapidly changing climate. These ecosystems harbor some of the most specialized species, which will likely suffer the highest extinction rates. We document the steepest temperature increase (2010-2021) on record at altitudes of above 4,000 m, triggering a decline of the relictual and highly adapted moss Takakia lepidozioides. Its de-novo-sequenced genome with 27,467 protein-coding genes includes distinct adaptations to abiotic stresses and comprises the largest number of fast-evolving genes under positive selection. The uplift of the study site in the last 65 million years has resulted in life-threatening UV-B radiation and drastically reduced temperatures, and we detected several of the molecular adaptations of Takakia to these environmental changes. Surprisingly, specific morphological features likely occurred earlier than 165 mya in much warmer environments. Following nearly 400 million years of evolution and resilience, this species is now facing extinction.

Early evolution of wing scales prior to the rise of moths and butterflies.

Although scales are a defining and conspicuous feature of moths and butterflies (Lepidoptera),1-3 their earliest evolution predates the group but is shrouded by a dearth of fossil evidence. Herein, we report two new species in mid-Cretaceous Kachin amber, representing lineages closely related to Lepidoptera: one represents the extinct Tarachoptera, with dense scales on the fore- and hindwings, while the other is an early lineage of caddisflies, with a hindwing covered by a single layer of angustifoliate scales. A novel phylogenetic analysis of 174 morphological characters and 73 extant and fossil representatives of Mecopterida demonstrates a monophyletic origin of scales in the common ancestor of Tarachoptera, Trichoptera, and Lepidoptera; that Tarachoptera are monophyletic but their scale morphology is plesiomorphic for the whole group; and that scales were lost early in caddisfly evolution before reappearing multiple times within the clade. Collectively, these fossils provide clarity into the origin and early evolution of scales before their diversification among the moths and butterflies.

Gravity induces asymmetric Ca2+ spikes in the root cap in the early stage of gravitropism.

Gravitropism is an important strategy for the adaptation of plants to the changing environment. Previous reports indicated that Ca2+ participated in plant gravity response. However, present information on the functions of Ca2+ in plant gravitropism was obtained mainly on coleoptiles, hypocotyls, and petioles, little is known about the dynamic changes of Ca2+ during root gravitropism. In the present study, the transgenic Arabidopsis thaliana R-GECO1 was placed horizontally and subsequently vertically on a refitted Leica SP8 laser scanning confocal microscopy with a vertical stage. Real-time observations indicated that gravistimulation induced not only an increase in the Ca2+ concentration, but also an accelerated occurrence of Ca2+ sparks in the root cap, especially in the lower side of the lateral root cap, indicating a strong tie between Ca2+ dynamics and gravistimulation during the early stage of root gravity response.

Latrunculin B facilitates gravitropic curvature of Arabidopsis root by inhibiting cell elongation, especially the cells in the lower flanks of the transition and elongation zones.

Gravitropism plays a critical role in the growth and development of plants. Previous reports proposed that the disruption of the actin cytoskeleton resulted in enhanced gravitropism; however, the mechanism underlying these phenomena is still unclear. In the present study, real-time observation on the effect of Latrunculin B (Lat B), a depolymerizing agent of microfilament cytoskeleton, on gravitropism of the primary root of Arabidopsis was undertaken using a vertical stage microscope. The results indicated that Lat B treatment prevented the growth of root, and the growth rates of upper and lower flanks of the horizontally placed root were asymmetrically inhibited. The growth of the lower flank was influenced by Lat B more seriously, resulting in an increased differential growth rate between the upper and lower flanks of the root. Further analysis indicated that Lat B affected cell growth mainly in the transition and elongation zones. Briefly, the current data revealed that Lat B treatment inhibited cell elongation, especially the cells in the lower flanks of the transition and elongation zones, which finally manifested as the facilitation of gravitropic curvature of the primary root.

Light Deprivation-Induced Inhibition of Chloroplast Biogenesis Does Not Arrest Embryo Morphogenesis But Strongly Reduces the Accumulation of Storage Reserves during Embryo Maturation in Arabidopsis.

The chloroplast is one of the most important organelles found exclusively in plant and algal cells. Previous reports indicated that the chloroplast is involved in plant embryogenesis, but the role of the organelle during embryo morphogenesis and maturation is still a controversial question demanding further research. In the present study, siliques of Arabidopsis at the early globular stage were enwrapped using tinfoil, and light deprivation-induced inhibition of the chloroplast biogenesis were validated by stereomicroscope, laser scanning confocal microscope and transmission electron microscope. Besides, the effects of inhibited chloroplast differentiation on embryogenesis, especially on the reserve deposition were analyzed using periodic acid-Schiff reaction, Nile red labeling, and Coomassie brilliant blue staining. Our results indicated that tinfoil enwrapping strongly inhibited the formation of chloroplasts, which did not arrest embryo morphogenesis, but markedly influenced embryo maturation, mainly through reducing the accumulation of storage reserves, especially starch grains and oil. Our data provide a new insight into the roles of the chloroplast during embryogenesis.

Combined Cytological and Transcriptomic Analysis Reveals a Nitric Oxide Signaling Pathway Involved in Cold-Inhibited Camellia sinensis Pollen Tube Growth.

Nitric oxide (NO) as a signaling molecule plays crucial roles in many abiotic stresses in plant development processes, including pollen tube growth. Here, the signaling networks dominated by NO during cold stress that inhibited Camellia sinensis pollen tube growth are investigated in vitro. Cytological analysis show that cold-induced NO is involved in the inhibition of pollen tube growth along with disruption of the cytoplasmic Ca(2+) gradient, increase in ROS content, acidification of cytoplasmic pH and abnormalities in organelle ultrastructure and cell wall component distribution in the pollen tube tip. Furthermore, differentially expressed genes (DEGs)-related to signaling pathway, such as NO synthesis, cGMP, Ca(2+), ROS, pH, actin, cell wall, and MAPK cascade signal pathways, are identified and quantified using transcriptomic analyses and qRT-PCR, which indicate a potential molecular mechanism for the above cytological results. Taken together, these findings suggest that a complex signaling network dominated by NO, including Ca(2+), ROS, pH, RACs signaling and the crosstalk among them, is stimulated in the C. sinensis pollen tube in response to cold stress, which further causes secondary and tertiary alterations, such as ultrastructural abnormalities in organelles and cell wall construction, ultimately resulting in perturbed pollen tube extension.

Different heavy metals have various effects on Picea wilsonii pollen germination and tube growth.

Heavy metal pollution has became one of the realistic matters of globality. Previous reports indicated that heavy metals could significantly inhibit pollen germination and tube growth. In the present study, comparative studies on the effects of different heavy metals (As, Hg, Cd, Cr and Cu) on in-vitro picea wilsonii pollen germination and tube growth were carried out. Microscopic evaluation revealed that different heavy metals had various degree of toxicity on P. wilsonii pollen tube development. As showed the most toxic effects on pollen germination, which was followed by Hg and Cd, while Cr and Cu showed relatively lower toxicity. Besides, pollen tubes showed varying shapes in response to different heavy metal stress. Pollen tubes treated with Cd, Hg and As were usually characterized by irregularly increasing diameters and swelling tips with distinct cytoplasimic vacuolation. On the other hand, except for the slightly increased diameters, no obvious abnormal shape were observed in tubes treated with Cr or Cu. Lyso-Tracker Green staining indicated that only Cd-treated pollen tubes showed numerous vacuole-like acidic organelles, though cytoplasmic vacuolization were also observed in pollen tubes treated with Hg and A. In brief, our data indicated that different heavy metals have various effects on Picea wilsonii pollen germination and tube growth, and that in-vitro pollen culture might be used as a competent system for biomonitoring of air pollution.

OsSEC24, a functional SEC24-like protein in rice, improves tolerance to iron deficiency and high pH by enhancing H(+) secretion mediated by PM-H(+)-ATPase.

Iron is abundant in the soil, but its low solubility in neutral or alkaline soils limits its uptake. Plants can rely on rhizosphere acidification to increase iron solubility. OsSEC27p was previously found to be a highly up-regulated gene in iron-deficient rice roots. Here, pH-dependent complementation assays using yeast mutants sec24Δ/SEC24 and sec27Δ/SEC27 showed that OsSEC27 could functionally complement SEC24 but not SEC27 in yeast; thus, it was renamed as OsSEC24. We found that OsSEC24-transgenic tobacco plants increased the length and number of roots under iron deficiency at pH 8.0. To explore how OsSEC24 confers tolerance to iron deficiency, we utilized transgenic tobacco, rice and rice protoplasts. H(+) flux measurements using Non-invasive Micro-test Technology (NMT) indicated that the transgenic OsSEC24 tobacco and rice enhanced H(+) efflux under iron deficiency. Conversely, the application of plasma membrane PM-H(+)-ATPase inhibitor vanadate elucidated that H(+) secretion increased by OsSEC24 was mediated by PM-H(+)-ATPase. OsPMA2 was used as a representative of iron deficiency-responsive PM-H(+)-ATPases in rice root via RT-PCR analysis. In transgenic rice protoplasts OsPMA2 was packaged into OsSEC24 vesicles after export from the ER through confocal-microscopy observation. Together, OsSEC24 vesicles, along with PM-H(+)-ATPases stimulate roots formation under iron deficiency by enhancing rhizosphere acidification.

Cadmium stress disrupts the endomembrane organelles and endocytosis during Picea wilsonii pollen germination and tube growth.

As one of the most severe pollutants, cadmium has been reported to be harmful to plant cells, but the effects of cadmium on gymnosperm pollen germination and tube growth and the mechanism of this involvement are still unclear. Here, we report that cadmium not only strongly inhibited P. wilsonii pollen germination and tube growth, but also significantly altered tube morphology in a dose-dependent manner. Time-lapse images obtained with a laser scanning confocal microscope revealed that endocytosis was dramatically inhibited by cadmium stress. Further investigation with ER-Tracker dye indicated that cadmium stress reduced the number of the Golgi apparatus, and induced dilation of ER. Additionally, Lyso-Tracker staining showed that cadmium distinctly promoted the formation of acidic organelles in pollen tubes, likely derived from the dilated ER. Taken together, our studies indicated that P. wilsonii pollens were highly susceptible to cadmium stress, and that cadmium stress strongly inhibited pollen germination and tube growth by disrupting the endomembrane organelles, inhibiting endo/exocytosis, and forming acidic vacuoles, resulting in swollen tube tips and irregularly broadened tube diameters. These findings provide a new insight into the effects of cadmium toxicity on the tip growth of pollen tubes.

Different degree in proteasome malfunction has various effects on root growth possibly through preventing cell division and promoting autophagic vacuolization.

The ubiquitin/proteasome pathway plays a vital role in plant development. But the effects of proteasome malfunction on root growth, and the mechanism underlying this involvement remains unclear. In the present study, the effects of proteasome inhibitors on Arabidopsis root growth were studied through the analysis of the root length, and meristem size and cell length in maturation zone using FM4-64, and cell-division potential using GFP fusion cyclin B, and accumulation of ubiquitinated proteins using immunofluorescence labeling, and autophagy activity using LysoTracker and MDC. The results indicated that lower concentration of proteasome inhibitors promoted root growth, whereas higher concentration of inhibitors had the opposite effects. The accumulation of cyclin B was linked to MG132-induced decline in meristem size, indicating that proteasome malfunction prevented cell division. Besides, MG132-induced accumulation of the ubiquitinated proteins was associated with the increasing fluorescence signal of LysoTracker and MDC in the elongation zone, revealing a link between the activation of autophagy and proteasome malfunction. These results suggest that weak proteasome malfunction activates moderate autophagy and promotes cell elongation, which compensates the inhibitor-induced reduction of cell division, resulting in long roots. Whereas strong proteasome malfunction induces severe autophagy and disturbs cell elongation, resulting in short roots.

Lead stress disrupts the cytoskeleton organization and cell wall construction during Picea wilsonii pollen germination and tube growth.

Lead is a widespread pollutant and has been reported to inhibit pollen tube development, but the mechanism of toxicity involved remains unclear. Here, we report that lead stress significantly prevented Picea wilsonii pollen germination and tube growth and also dramatically altered the tube morphology in a concentration-dependent manner. Fluorescence labeling with JIM 5 (anti-acidic pectin antibody) and Calcofluor white revealed the lead-induced decline of acidic pectin and cellulose, especially in the subapical region. Decolorized aniline blue staining showed the marked accumulation of callose in the apical and subapical regions of lead-treated tubes. Fluorescence labeling with Alexa Fluor 568 phalloidin and anti-tubulin antibody revealed that the distribution of the cytoskeleton in P. wilsonii pollen grains and tubes were developmentally regulated and that lead disturbed the cytoskeleton organization, especially in the shank of the pollen tubes. Taken together, our experiments revealed a link between the dynamics of cytoskeleton organization and the process of P. wilsonii pollen tube development and also indicated that lead disturbed the cytoskeleton assembly and, consequently, cell wall construction. These findings provide new insights into the mechanism of lead toxicity in the tip growth of pollen tubes.

Mitochondrial dynamics and its responds to proteasome defection during Picea wilsonii pollen tube development.

Tip growth of pollen tubes is essential for higher plant sexual reproduction and has been proposed to be highly regulated by the ubiquitin/proteasome pathway (UPP). The dynamics of mitochondria and the functions of the UPP on mitochondrial dynamics during pollen tube development are still poorly understood. In the present study, using real-time laser scanning and transmission electron microscope, it was revealed that mitochondria in Picea wilsonii, are either ellipsoid or filamentous with various lengths. Time-lapse images indicated that the two forms of mitochondria interconvert frequently through opposite process of fusion and fission. Examination of mitochondrial morphology during four key stages of in vitro pollen tube development revealed a link between mitochondrial remodeling and the process of pollen tube elongation. We also report that MG132, a specific proteasome inhibitor, not only strongly disturbed the mitochondrial remodeling but also significantly reduced mitochondrial membrane potential during pollen tube development. This finding provides new insight into the function of the proteasome in tip growth of pollen tubes.

Imaging of dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy.

Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.

Roles of the ubiquitin/proteasome pathway in pollen tube growth with emphasis on MG132-induced alterations in ultrastructure, cytoskeleton, and cell wall components.

The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner, while hardly similar effects were detected when cysteine-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanate-phalloidin and beta-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.