植物体内的胼胝质

胼胝质(callose)是一种以β-1，3-键结合的葡聚糖。在植物的筛管代谢、配子体发育等生命活动中发挥着重要的调节作用，其合成、分解直接关系植物正常的生长代谢过程，因此，胼胝质的代谢是植物研究中的重要内容之一。     

CalS7 encodes a callose synthase responsible for callose deposition in the phloem

It has been known for more than a century that sieve plates in the phloem in plants contain callose, a β-1,3-glucan. However, the genes responsible for callose deposition in this subcellular location have not been identified. In this paper we examine callose deposition patterns in T-DNA insertion mutants (cs7) of the Callose Synthase 7 (CalS7) gene. We demonstrated here that the CalS7 gene is expressed specifically in the phloem of vascular tissues. Callose deposition in the phloem, especially in the sieve elements, was greatly reduced in cs7 mutants. Ultrastructural analysis of developing sieve elements revealed that callose failed to accumulate in the plasmodesmata of incipient sieve plates at the early perforation stage of phloem development, resulting in the formation of sieve plates with fewer pores. In wild-type Arabidopsis plants, callose is present as a constituent polysaccharide in the phloem of the stem, and its accumulation can also be induced by wounding. Callose accumulation in both conditions was eliminated in mature sieve plates of cs7 mutants. These results demonstrate that CalS7 is a phloem-specific callose synthase gene, and is responsible for callose deposition in developing sieve elements during phloem formation and in mature phloem induced by wounding. The mutant plants exhibited moderate reduction in seedling height and produced aberrant pollen grains and short siliques with aborted embryos, suggesting that CalS7 also plays a role in plant growth and reproduction.     

Magnesium induces neuronal apoptosis by suppressing excitability

In clinical obstetrics, magnesium sulfate (MgSO₄) use is widespread, but effects on brain development are unknown. Many agents that depress neuronal excitability increase developmental neuroapoptosis. In this study, we used dissociated cultures of rodent hippocampus to examine the effects of Mg⁺⁺ on excitability and survival. Mg⁺⁺-induced caspase-3-associated cell loss at clinically relevant concentrations. Whole-cell patch-clamp techniques measured Mg⁺⁺ effects on action potential threshold, action potential peak amplitude, spike number and changes in resting membrane potential. Mg⁺⁺ depolarized action potential threshold, presumably from surface charge screening effects on voltage-gated sodium channels. Mg⁺⁺ also decreased the number of action potentials in response to fixed current injection without affecting action potential peak amplitude. Surprisingly, Mg⁺⁺ also depolarized neuronal resting potential in a concentration-dependent manner with a +5.2 mV shift at 10 mM. Voltage ramps suggested that Mg⁺⁺ blocked a potassium conductance contributing to the resting potential. In spite of this depolarizing effect of Mg⁺⁺, the net inhibitory effect of Mg⁺⁺ nearly completely silenced neuronal network activity measured with multielectrode array recordings. We conclude that although Mg⁺⁺ has complex effects on cellular excitability, the overall inhibitory influence of Mg⁺⁺ decreases neuronal survival. Taken together with recent in vivo evidence, our results suggest that caution may be warranted in the use of Mg⁺⁺ in clinical obstetrics and neonatology.     

Brefeldin A induces callose formation in onion inner epidermal cells

Summary The antibiotic fungal toxin brefeldin A (BFA) causes synthesis of additional cell wall material in adult differentiated onion inner epidermal cells at concentrations of 5–30 g/ml. This tertiary wall contains callose and is layered on the secondary cellulosic wall in a time- and dose-dependent manner. Initially, callose is found in pit fields in the form of small vesicular patches. With time and dose, depositions grow in size and form large plugs invaginating into the cell, where the adjacent cytoplasm forms bulky accumulations and contains many organelles including endomembranes. Within the cytoplasm, BFA exerts the characteristic morphological effects on the secretory system including changes of the Golgi stacks, formation of large vesicles, and proliferation of dilated cisternae of the endoplasmic reticulum. Higher concentrations of BFA (60 g/ml) lead to disintegration of the Golgi apparatus; they have no effects on the cell wall, no callose synthesis occurs. We conclude from these observations that BFA has two independent targets in onion cells. BFA acts on the plasma membrane, hence operating as an elicitor of plant defense reactions and thus activates callose synthesis. BFA acts also on the membranes of the secretory system and influences budding and fusion of vesicles at the endoplasmic reticulum and at the dictyosomes. These two mechanisms occur in parallel, suggesting that the secretory system still can play its presumed role in callose synthesis. Only when dictyosomes are completely disintegrated, no more callose is formed.Abbreviations BFA Brefeldin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum - GS glucan synthetase Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

A monoclonal antibody recognizes a 65 kDa higher plant membrane polypeptide which undergoes cation-dependent association with callose synthase in vitro and co-localizes with sites of high callose deposition in vivo

Summary A monoclonal antibody (MAb) capable of immobilizing detergent-solubilized UDP-glucose: (13)--glucan (callose) synthase activity from higher plants has been selected and characterized. On Western blots this MAb recognizes a polypeptide of about 65 kDa found in membranes isolated from a variety of plant sources. The polypeptide recognized by this MAb does not appear to bind the substrate UDP-glucose, and evidence is presented which indicates that this polypeptide associates with the enzyme complex in a cation-dependent manner under conditions where the callose synthase assumes a larger size. Indirect immunofluorescence localization with this MAb was positive with sieve plates of cucumber (Cucumis sativus) seedlings, and with plasmodesmata of onion (Allium cepa) epidermal cells, both being sites of localized, stress-induced callose deposition.Abbreviations BSA bovine serum albumine - DMSO dimethylsulf-oxide - DTT dithiothreitol - FITC fluorescein isothiocyanate - HB Hepes buffer - HBS Hepes buffer plus 0.15 M NaCl - IgG immuno-globulin G - MAb monoclonal antibody - MSB microtubule stabilizing buffer - NP 40 Nonidet P 40 - PBS phosphate buffered saline - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - UDP uridine diphosphate - UV ultraviolet     

Study on metal-triggered callose deposition in roots of maize and soybean

Callose plays important roles in a variety of processes of plant development, and/or in a response to a range of biotic and abiotic stresses. In the current work we have studied and compared the effect of lead, cadmium and arsenic on accumulation of newly formed callose deposits in the roots of maize and soybean. We observed formation of characteristic callose deposits in the root cell walls, probably associated with plasmodesmata, depending on the type of metal and the plant species investigated. Further, the callose turnover was analysed by measuring of total callose content as well as activities of total β-(1,3)-glucanases in roots. The latter enzymes are responsible for callose depletion, and their possible role during metal stress has previously been proposed. However, neither of these biochemical values appeared to be sufficiently reliable for scoring the altered callose turnover (including local deposits) in plant tissue. The microscopical observations are discussed in light of the biochemical data obtained.     

Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death

The AvrPtoB type III effector protein is conserved among diverse genera of plant pathogens suggesting it plays an important role in pathogenesis. Here we report that Pseudomonas AvrPtoB acts inside the plant cell to inhibit programmed cell death (PCD) initiated by the Pto and Cf9 disease resistance proteins and, remarkably, the pro-apoptotic mouse protein Bax. AvrPtoB also suppressed PCD in yeast, demonstrating that AvrPtoB functions as a cell death inhibitor across kingdoms. Using truncated AvrPtoB proteins, we identified distinct N- and C-terminal domains of AvrPtoB that are sufficient for host recognition and PCD inhibition, respectively. We also identified a novel resistance phenotype, Rsb, that is triggered by an AvrPtoB truncation disrupted in the anti-PCD domain. A Pseudomonas syringae pv. tomato DC3000 strain with a chromosomal mutation in the AvrPtoB C-terminus elicited Rsb-mediated immunity in previously susceptible tomato plants and disease was restored when full-length AvrPtoB was expressed in trans. Thus, our results indicate that a type III effector can induce plant susceptibility to bacterial infection by inhibiting host PCD.     

The timely deposition of callose is essential for cytokinesis in Arabidopsis

The primary plant cell wall is laid down over a brief period of time during cytokinesis. Initially, a membrane network forms at the equator of a dividing cell. The cross-wall is then assembled and remodeled within this membrane compartment. Callose is the predominant luminal component of the nascent cross-wall or cell plate, but is not a component of intact mature cell walls, which are composed primarily of cellulose, pectins and xyloglucans. Widely accepted models postulate that callose comprises a transient, rapid spreading force for the expansion of membrane networks during cytokinesis. In this study, we clone and characterize an Arabidopsis gene, MASSUE / AtGSL8 , which encodes a putative callose synthase. massue mutants are seedling-lethal and have a striking cytokinesis-defective phenotype. Callose deposition was delayed in the cell plates of massue mutants. Mutant cells were occasionally bi- or multi-nucleate, with cell-wall stubs, and we frequently observed gaps at the junction between cross-walls and parental cell walls. The results suggest that the timely deposition of callose is essential for the completion of plant cytokinesis. Surprisingly, confocal analysis revealed that the cell-plate membrane compartment forms and expands, seemingly as far as the parental wall, prior to the appearance of callose. We discuss the possibility that callose may be required to establish a lasting connection between the nascent cross-wall and the parental cell wall.     

A comprehensive view on organ-specific callose synthesis in wheat (Triticum aestivum L.): glucan synthase-like gene expression, callose synthase activity, callose quantification and deposition.

Callose ((1,3)-beta-glucan) is important during basic developmental processes of plants, but only little is known about the regulation of callose biosynthesis on molecular level. Growing evidence indicates that glucan synthase-like (GSL) genes in higher plants are involved in callose synthesis. We analyzed the expression of eight GSL genes (TaGSL) as well as callose synthase activity and total callose content in the stem, leaf blade and spike of wheat (Triticum aestivum L.). Organ-specific expression of six TaGSL genes and strong differences in expression levels were detected by quantitative real-time PCR. Differences were also determined in callose synthase (EC 2.4.1.34) activity and total amount of callose in the examined organs. Aniline blue staining in tissue sections localized callose depositions. These results allow a comprehensive reflection of callose regulation, considering gene expression, enzyme activity and enzyme product quantification as well as localization. Our data suggests that callose synthesis is highly regulated by a combination of GSL genes, which are involved either in general or in organ-specific developmental processes.     

Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.     

DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals

Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway.     

Sangivamycin induces apoptosis by suppressing Erk signaling in primary effusion lymphoma cells

Sangivamycin, a structural analog of adenosine and antibiotic exhibiting antitumor and antivirus activities, inhibits protein kinase C and the synthesis of both DNA and RNA. Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients and HIV-infected homosexual males. PEL cells are derived from post-germinal center B cells, and are infected with KSHV. Herein, we asked if sangivamycin might be useful to treat PEL. We found that sangivamycin killed PEL cells, and we explored the underlying mechanism. Sangivamycin treatment drastically decreased the viability of PEL cell lines compared to KSHV-uninfected B lymphoma cell lines. Sangivamycin induced the apoptosis of PEL cells by activating caspase-7 and -9. Further, sangivamycin suppressed the phosphorylation of Erk1/2 and Akt, thus inhibiting activation of the proteins. Inhibitors of Akt and MEK suppressed the proliferation of PEL cells compared to KSHV-uninfected cells. It is known that activation of Erk and Akt signaling inhibits apoptosis and promotes proliferation in PEL cells. Our data therefore suggest that sangivamycin induces apoptosis by inhibiting Erk and Akt signaling in such cells. We next investigated whether sangivamycin, in combination with an HSP90 inhibitor geldanamycin (GA) or valproate (valproic acid), potentiated the cytotoxic effects of the latter drugs on PEL cells. Compared to treatment with GA or valproate alone, the addition of sangivamycin enhanced cytotoxic activity. Our data thus indicate that sangivamycin may find clinical utility as a novel anti-cancer agent targeting PEL.     

Long-term chemical castration induces depressive symptoms by suppressing serotonin expression in rats

Androgen deprivation therapy, also known as chemical castration, has been used as an adjunct to psychotherapy for sex offenders. Goserelin and bicalutamide are drugs used for chemical castration. Serotonin (5-hydroxytryptamine, 5-HT) is a key neurotransmitter involved in mood changes, such as depression. We investigated the effects of surgical and chemical castration on depressive symptoms in rats. Surgical castration was performed through a bilateral orchiectomy. Bicalutamide was administrated orally once a day for 84 consecutive days. Goserelin acetate was implanted subcutaneously into the anterior abdominal wall, and this implantation was repeated 3 times at 28-day intervals. Testosterone levels were detected by enzyme-linked immunosorbent assays. Sexual behaviors were analyzed by measuring mount latency, mount frequency, intromission latency, and intromission frequency. The forced swimming test was performed to evaluate rats’ depression status. To detect 5-HT and tryptophan hydroxylase (TPH)-positive cells in the dorsal raphe, immunohistochemistry for 5-HT and TPH and western blotting for 5-HT1A receptors and TPH were performed. Surgical castration and goserelin decreased testosterone levels and suppressed sexual behaviors. However, bicalutamide did not inhibit sexual behaviors, although it reduced testosterone levels to a limited extent. Both surgical and chemical castration induced depression in rats. The expression of 5-HT, TPH, and 5-HT1A receptors in the dorsal raphe was significantly decreased by both surgical castration and chemical castration via bicalutamide and goserelin. The present results showed that surgical and chemical castration for 12 weeks induced a depressive state in rats by inhibiting serotonergic function through 5-HT1A receptors.     

Movement of plant viruses is delayed in a beta-1,3-glucanase-deficient mutant showing a reduced plasmodesmatal size exclusion limit and enhanced callose deposition

Susceptibility to virus infection is decreased in a class I beta-1,3-glucanase (GLU I)-deficient mutant (TAG4.4) of tobacco generated by antisense transformation. TAG4.4 exhibited delayed intercellular trafficking via plasmodesmata of a tobamovirus (tobacco mosaic virus), of a potexvirus (recombinant potato virus X expressing GFP), and of the movement protein (MP) 3a of a cucumovirus (cucumber mosaic virus). Monitoring the cell-to-cell movement of dextrans and peptides by a novel biolistic method revealed that the plasmodesmatal size exclusion limit (SEL) of TAG4.4 was also reduced from 1.0 to 0.85 nm. Therefore, GLU I-deficiency has a broad effect on plasmodesmatal movement, which is not limited to a particular virus type. Deposition of callose, a substrate for beta-1,3-glucanases, was increased in TAG4.4 in response to 32 degrees C treatment, treatment with the fungal elicitor xylanase, and wounding, suggesting that GLU I has an important function in regulating callose metabolism. Callose turnover is thought to regulate plasmodesmatal SEL. We propose that GLU I induction in response to infection may help promote MP-driven virus spread by degrading callose.     

Ants suppressing plant pathogens: a review

Ant–plant mutualisms are usually regarded as driven by ants defending plants against herbivores in return for plant‐produced food rewards and housing. However, ants may provide additional services. In a review of published studies on ant–pathogen–plant interactions, we investigated whether ants’ extensive hygiene measures, including the use of ant‐produced antibiotics, extend to their host plants and reduce plant pathogen loads. From 30 reported species combinations, we found that the presence of ants lead to reduced pathogen levels in 18 combinations and to increased levels in 6. On average, ants significantly reduced pathogen incidence with 59%. This effect size did not differ significantly from effect sizes reported from meta‐analyses on herbivore protection. Thus, pathogen and herbivore protection could be of equal importance in ant–plant mutualisms. Considering the abundance of these interactions, ecological impacts are potentially high. Furthermore, awareness of this service may stimulate the development of new measures to control plant diseases in agriculture. It should be noted, though, that studies were biased toward tropical ant–plant symbioses and that the literature in the field is limited at present. Future research on plant pathogens is needed to enhance our understanding of ant–plant mutualisms and their evolution.     

Translocation blockage by sieve plate callose

Summary Axial translocation in 2-week-old cotton plants was inhibited by heating 4 cm of intact hypocotyl for 15 min by means of a 40–45° water jacket. A 1-cm jacket did not retard translocation, and temperatures below 40° had no effect. Translocation continued to be inhibited for at least 3 hours following heat treatment. After 6 hours, rates were equal to or above normal. Maximum amounts of callose were deposited on sieve plates after the heat treatment, but callose was noticeably diminished within 6 hours after heating and reduced to virtually normal levels within 2 days. Growth measurements, plasmolytic tests, vital staining, and visual observations revealed no evidence of injury in plants heated at 45°. Pore constriction from increased amounts of callose on sieve plates appears to be an effect of heating. Increased resistance due to such constriction may be an important factor in blockage of basipetal phloem translocation.Work supported in part by National Science Foundation Grant GB-2941. This material is abstracted from a dissertation presented in 1967 by R. B. McNairn to the Graduate Division, University of California at Davis in partial fulfillment of the requirements for the Ph. D. degree.All temperatures in this paper are in degrees centigrade (°C)