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Analysis of Flowering Time Control in Arabidopsis by Comparison of Double and Triple Mutants
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Three genetic pathways promote flowering of Arabidopsis under long photoperiods. These pathways are represented by the genes CO, FCA, and GA1, which act in the long-day, autonomous, and gibberellin pathways, respectively. To test whether these are the only pathways that promote flowering under long photoperiods, the co-2 fca-1 ga1-3 triple mutant was constructed. These plants never flowered under long- or short-day conditions, indicating that the three pathways impaired by these mutations are absolutely required for flowering under these conditions. The triple mutant background represents a "vegetative ground state" enabling the roles of single pathways to be described in the corresponding double mutants. The phenotypes of plants carrying all eight combinations of wild-type and mutant alleles at the three loci were compared under long- and short-day conditions. This analysis demonstrated that under long photoperiods the long-day pathway promoted flowering most effectively, whereas under short photoperiods the gibberellin pathway had the strongest effect. The autonomous pathway had a weak effect when acting alone under either photoperiod but appeared to play an important role in facilitating the promotion of flowering by the other two pathways. The vegetative phenotype of the triple mutant could be overcome by vernalization, suggesting that a fourth pathway promoted flowering under these conditions. These observations are discussed in light of current models describing the regulation of flowering time in Arabidopsis. 相似文献
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Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in
Blue-Light-Induced Shrinking of Arabidopsis
Hypocotyl
Protoplasts 总被引:6,自引:1,他引:5
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Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl− and K+. The postshrinking volume recovery is achieved by K+ and Cl− influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B. 相似文献
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The roles of phytochrome A (phyA), phytochrome B (phyB) and a putative blue-light (BL) photoreceptor (HY4) in the control of hypocotyl growth by natural radiation were investigated using phyA, phyB and hy4 mutants of Arabidopsis thaliana. Full sunlight inhibited hypocotyl growth to a larger extent in wild-type (WT) than in phyA, phyB and, particularly, hy4 seedlings. In WT seedlings, hypocotyl growth was promoted by selectively lowering BL irradiance, lowering red-light (R) plus far-red-light (FR) irradiance or lowering the R/FR ratio (which was achieved either by increasing FR or by reducing R). The effects of lowering BL were reduced in hy4 and exaggerated in phyA seedlings. The effects of lowering R+FR were reduced in phyA and exaggerated in hy4 seedlings. Neither phyB nor hy4 mutants responded to low R/FR ratios. Neighbouring plants reflecting FR without shading caused subtle reductions of the R/FR ratio. This signal promoted hypocotyl growth in WT but not in phyA, phyB or hy4 seedlings. Intermediate canopy shade produced similar effects in all genotypes. Under deep shade, de-etiolation was severely impaired in phyA seedlings, which died prematurely. Thus, the FR ‘high-irradiance reaction’ mediated by phyA could be important for seedling survival under dense canopies. 相似文献
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Jeff S. Isenberg Douglas S. Annis Michael L. Pendrak Malgorzata Ptaszynska William A. Frazier Deane F. Mosher David D. Roberts 《The Journal of biological chemistry》2009,284(2):1116-1125
Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells
via CD47. Because CD47 binding motifs are conserved in the C-terminal
signature domains of all five thrombospondins and indirect evidence has
implied CD47 interactions with other family members, we compared activities of
recombinant signature domains of thrombospondin-1, -2, and -4 to interact with
CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4
were less active than that of thrombospondin-1 for inhibiting binding of
radiolabeled signature domain of thrombospondin-1 or SIRPα
(signal-regulatory protein) to cells expressing CD47. Consistent with this
binding selectivity, the signature domain of thrombospondin-1 was more potent
than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP
synthesis in vascular smooth muscle cells and downstream effects on cell
adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary
vascular cells from thrombospondin-2-null mice lack enhanced basal and
NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on
NO/cGMP signaling could be detected only in thrombospondin-1-null cells.
Furthermore, tissue survival of ischemic injury and acute recovery of blood
flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore,
thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and
its limiting role in acute ischemic injury responses is not shared by
thrombospondin-2.Nitric oxide (NO) is a major mediator of intracellular and paracellular
signal transduction. NO preserves vascular health by minimizing the adhesion
of inflammatory cells to the vessel wall, limiting platelet activation, and
increasing blood vessel diameter and blood flow by relaxing vascular smooth
muscle cells (VSMC).3
These actions of NO are mediated by activating soluble isoforms of guanylate
cyclase (sGC) to increase cGMP levels, resulting in downstream activation of
cGMP-dependent protein kinases and ion channels
(1).Physiological NO/cGMP signaling is limited by several phosphodiesterases
that degrade cGMP and by thrombospondin-1 (TSP). TSP1 is a secreted protein
that is produced by vascular and inflammatory cells that regulates cellular
behavior by engaging several cell surface receptors. Recently we reported that
TSP1 potently blocks NO-stimulated prosurvival responses in endothelial and
VSMC (2,
3). TSP1 also plays a role in
promoting platelet thrombus formation and hemostasis by antagonizing the
antithrombotic activity of NO
(4). In all of these vascular
cells, picomolar concentrations of TSP1 are sufficient to block NO-stimulated
fluxes in cGMP by engaging its receptor CD47
(5). Nanomolar concentrations
of TSP1 further inhibit the same signaling pathway by inhibiting CD36-mediated
uptake of myristate into vascular cells
(6). In vivo, mice
lacking TSP1 demonstrate elevated basal tissue cGMP levels and greater
increases in regional blood flow in response to a NO challenge than wild type
controls (4). After an ischemic
insult, the absence of TSP1 or CD47 in transgenic mice is associated with
better maintenance of tissue perfusion and enhanced tissue survival.
Similarly, targeting TSP1 or CD47 using function blocking antibodies enhances
ischemic tissue perfusion and survival in wild type mice and pigs
(7,
8).TSP1 belongs to a family of five secreted glycoproteins that share an
evolutionarily conserved C-terminal signature domain
(9). TSP1 and TSP2 form a
distinct subfamily of trimeric proteins that exhibit similar anti-angiogenic
activities for endothelial cells in vitro and activities in
vivo to block tumor growth. Despite their similarities in structure, TSP1
and TSP2 have markedly different expression patterns after tissue injury, with
TSP1 being immediately expressed and maximal at day 3, whereas TSP2 was not
expressed until day 7 and was maximal 10 days after injury
(10). In addition, large
amounts of TSP1 but not TSP2 are stored in platelet α-granules and
released into the wound environment. Polymorphisms in TSP1 and TSP2 have been
linked to altered risk of premature myocardial infarction
(11,
12). A 3′-untranslated
region polymorphism in TSP2 is also associated with type 2 diabetes in men
(13). The molecular basis for
these associations is unclear.Less is known about the roles of the pentameric TSP3–5 in vascular
cells. TSP3 and TSP5 (also known as cartilage oligomeric matrix protein)
appear to serve their primary functions in bone development
(14,
15). However, a polymorphism
in TSP4 is associated with premature myocardial infarcts in certain
populations (11,
16,
17). A proatherogenic activity
for the A387P variant of TSP4 was proposed based on its differential ability
to modulate proliferation of endothelial and VSMC
(18). Cardiovascular functions
of TSP4 may also be linked to the high expression of TSP4 in heart
(19) and its altered
expression in that tissue during hypertensive heart failure
(20).The C-terminal domain of TSP1 is sufficient to mediate CD47-dependent
inhibition of cGMP signaling
(5). Of the two CD47 binding
VVM motifs identified in this domain of TSP1, the first is conserved among all
five TSPs, suggesting that CD47 binding could be a universal attribute of this
family (21). Based on
structural evidence that the VVM motifs may not be accessible
(22,
23), however, conservation of
VVM motifs may not be sufficient to predict CD47 binding. Uncertainty
regarding the location of the CD47 binding site in the G domain of TSP1
therefore limits interpretation of the known sequence homology to predict CD47
binding to other TSP family members.Although CD47 recognition of other TSPs has not been demonstrated
experimentally, a local deficiency of inflammation-associated T cell apoptosis
shared by TSP1-, CD47-, and TSP2-null mice is consistent with this hypothesis
(24). Furthermore, a
21-residue peptide from the C-terminal domain of TSP4 was found to decrease
human umbilical vein endothelial cell proliferation similar to the CD47
binding peptides from TSP1, although it lacks the VVM motif and no interaction
with CD47 was demonstrated
(25).To directly address whether other TSP family members can inhibit NO
responses and signaling in vascular cells, we now compare binding of
recombinant signature domains of TSP1, TSP2, and TSP4 to cell surface CD47 and
inhibition of NO-stimulated cell responses and cGMP signaling by these
domains. We also compared acute tissue blood flow and perfusion responses to
ischemic challenge in TSP1 and TSP2-null mice and cGMP responses in primary
cultures of vascular cells isolated from these mice. These studies clearly
demonstrate that CD47 selectively interacts with TSP1 and that the signature
domains of TSP2 and TSP4 are less potent inhibitors of NO signaling in
vascular cells in vitro. Furthermore, we show that the role of TSP1
to acutely limit recovery from ischemic injury in vivo is not shared
by TSP2. 相似文献
8.
Shanshan Yang Rebecca L. Murphy Daryl T. Morishige Patricia E. Klein William L. Rooney John E. Mullet 《PloS one》2014,9(8)
Light signaling by phytochrome B in long days inhibits flowering in sorghum by increasing expression of the long day floral repressors PSEUDORESPONSE REGULATOR PROTEIN (SbPRR37, Ma1) and GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (SbGHD7, Ma6). SbPRR37 and SbGHD7 RNA abundance peaks in the morning and in the evening of long days through coordinate regulation by light and output from the circadian clock. 58 M, a phytochrome B deficient (phyB-1, ma3R) genotype, flowered ∼60 days earlier than 100 M (PHYB, Ma3) in long days and ∼11 days earlier in short days. Populations derived from 58 M (Ma1, ma3R, Ma5, ma6) and R.07007 (Ma1, Ma3, ma5, Ma6) varied in flowering time due to QTL aligned to PHYB/phyB-1 (Ma3), Ma5, and GHD7/ghd7-1 (Ma6). PHYC was proposed as a candidate gene for Ma5 based on alignment and allelic variation. PHYB and Ma5 (PHYC) were epistatic to Ma1 and Ma6 and progeny recessive for either gene flowered early in long days. Light signaling mediated by PhyB was required for high expression of the floral repressors SbPRR37 and SbGHD7 during the evening of long days. In 100 M (PHYB) the floral activators SbEHD1, SbCN8 and SbCN12 were repressed in long days and de-repressed in short days. In 58 M (phyB-1) these genes were highly expressed in long and short days. Furthermore, SbCN15, the ortholog of rice Hd3a (FT), is expressed at low levels in 100 M but at high levels in 58 M (phyB-1) regardless of day length, indicating that PhyB regulation of SbCN15 expression may modify flowering time in a photoperiod-insensitive manner. 相似文献
9.
Ana Rita Nunes Lemes Camila Chiaradia Davolos Paula Cristina Brunini Crialesi Legori Odair Aparecido Fernandes Juan Ferré Manoel Victor Franco Lemos Janete Apparecida Desiderio 《PloS one》2014,9(10)
Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested. 相似文献
10.
Phenols are present in the environment and commonly in contact with humans and animals because of their wide applications in many industries. In a previous study, we reported that uridine diphosphate-glucose-dependent glucosyltransferase PtUGT72B1 from Populus trichocarpa has high activity in detoxifying trichlorophenol by conjugating glucose. In this study, more experiments were performed to determine the substrate specificity of PtUGT72B1 towards phenolic compounds. Among seven phenols tested, three were glucosylated by PtUGT72B1 including phenol, hydroquinone, and catechol. Transgenic Arabidopsis plants expressing the enzyme PtUGT72B1 showed higher resistance to hydroquinone and catechol but more sensitivity to phenol than wild type plants. Transgenic Pichia pastoris expressing PtUGT72B1 showed enhanced resistance to all three phenols. Compared with wild type Arabidopsis plants, transgenic Arabidopsis plants showed higher removal efficiencies and exported more glucosides of phenol, phenyl β-D-glucopyranoside, to the medium after cultured with the three phenols. Protein extracts from transgenic Arabidopsis plants showed enhanced conjugating activity towards phenol, hydroquinone and catechol. PtUGT72B1 showed much higher expression level in Pichia pastoris than in Arabidopsis plants. Kinetic analysis of the PtUGT72B1 was also performed. 相似文献
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Analysis of the Arabidopsis rsr4-1/pdx1-3 mutant reveals the critical function of the PDX1 protein family in metabolism, development, and vitamin B6 biosynthesis
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Wagner S Bernhardt A Leuendorf JE Drewke C Lytovchenko A Mujahed N Gurgui C Frommer WB Leistner E Fernie AR Hellmann H 《The Plant cell》2006,18(7):1722-1735
Vitamin B6 represents a highly important group of compounds ubiquitous in all living organisms. It has been demonstrated to alleviate oxidative stress and in its phosphorylated form participates as a cofactor in >100 biochemical reactions. By means of a genetic approach, we have identified a novel mutant, rsr4-1 (for reduced sugar response), with aberrant root and leaf growth that requires supplementation of vitamin B6 for normal development. Cloning of the mutated gene revealed that rsr4-1 carries a point mutation in a member of the PDX1/SOR1/SNZ (for Pyridoxine biosynthesis protein 1/Singlet oxygen resistant 1/Snooze) family that leads to reduced vitamin B6 content. Consequently, metabolism is broadly altered, mainly affecting amino acid, raffinose, and shikimate contents and trichloroacetic acid cycle constituents. Yeast two-hybrid and pull-down analyses showed that Arabidopsis thaliana PDX1 proteins can form oligomers. Interestingly, the mutant form of PDX1 has severely reduced capability to oligomerize, potentially suggesting that oligomerization is important for function. In summary, our results demonstrate the critical function of the PDX1 protein family for metabolism, whole-plant development, and vitamin B6 biosynthesis in higher plants. 相似文献
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Relation between TLR4/NF‐κB signaling pathway activation by 27‐hydroxycholesterol and 4‐hydroxynonenal,and atherosclerotic plaque instability
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Simona Gargiulo Paola Gamba Gabriella Testa Daniela Rossin Fiorella Biasi Giuseppe Poli Gabriella Leonarduzzi 《Aging cell》2015,14(4):569-581
It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll‐like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low‐density lipoproteins, the oxysterol 27‐hydroxycholesterol (27‐OH) and the aldehyde 4‐hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high‐risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27‐OH and HNE were found to enhance cell release of IL‐8, IL‐1β, and TNF‐α and to upregulate matrix metalloproteinase‐9 (MMP‐9) via TLR4/NF‐κB‐dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP‐9 upregulation, thus enhancing the release of this matrix‐degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP‐9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF‐κB downstream signaling. 相似文献
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Lrp4 is a multifunctional member of the low density lipoprotein-receptor gene family and a modulator of extracellular cell signaling pathways in development. For example, Lrp4 binds Wise, a secreted Wnt modulator and BMP antagonist. Lrp4 shares structural elements within the extracellular ligand binding domain with Lrp5 and Lrp6, two established Wnt co-receptors with important roles in osteogenesis. Sclerostin is a potent osteocyte secreted inhibitor of bone formation that directly binds Lrp5 and Lrp6 and modulates both BMP and Wnt signaling. The anti-osteogenic effect of sclerostin is thought to be mediated mainly by inhibition of Wnt signaling through Lrp5/6 within osteoblasts. Dickkopf1 (Dkk1) is another potent soluble Wnt inhibitor that binds to Lrp5 and Lrp6, can displace Lrp5-bound sclerostin and is itself regulated by BMPs. In a recent genome-wide association study of bone mineral density a significant modifier locus was detected near the SOST gene at 17q21, which encodes sclerostin. In addition, nonsynonymous SNPs in the LRP4 gene were suggestively associated with bone mineral density. Here we show that Lrp4 is expressed in bone and cultured osteoblasts and binds Dkk1 and sclerostin in vitro. MicroCT analysis of Lrp4 deficient mutant mice revealed shortened total femur length, reduced cortical femoral perimeter, and reduced total femur bone mineral content (BMC) and bone mineral density (BMD). Lumbar spine trabecular bone volume per total volume (BV/TV) was significantly reduced in the mutants and the serum and urinary bone turnover markers alkaline phosphatase, osteocalcin and desoxypyridinoline were increased. We conclude that Lrp4 is a novel osteoblast expressed Dkk1 and sclerostin receptor with a physiological role in the regulation of bone growth and turnover, which is likely mediated through its function as an integrator of Wnt and BMP signaling pathways. 相似文献
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Jianting Ma Xingguang Zhang Gang He Chunlin Yang 《Reproductive biology and endocrinology : RB&E》2017,15(1):83