吕应堂
植物发育分子生物学和植物信号转导分子机理。
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- 姓名:吕应堂
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学术头衔:
博士生导师, 国家杰出青年科学基金获得者
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学科领域:
植物学
- 研究兴趣:植物发育分子生物学和植物信号转导分子机理。
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11
吕应堂, Wei Hua‡, Lei Zhang‡, Shuping Liang‡, Russell L. Jones§, and Ying-Tang Lu‡¶
Vol. 279, No. 30, Issue of July 23, pp. 31483-31494, 2004,-0001,():
-1年11月30日
A tobacco calcium/calmodulin-binding protein kinase (NtCBK1) was isolated and identified. The predicted NtCBK1 protein has 599 amino acids, an N-terminal kinase domain, and shares high homology with other calmodulin (CaM)-related kinases. Whereas NtCBK1 phosphorylates itself and substrates such as histone IIIS and syntide-2 in the absence of CaM, its kinase activity can be stimulated by tobacco CaMs. However, unlike another tobacco protein kinase designated NtCBK2, NtCBK1 was not differentially regulated by the different CaM isoforms tested. The CaM-binding domain of NtCBK1 was located between amino acids 436 and 455, and this domain was shown to be necessary for CaM modulation of kinase activity. RNA in situ hybridization showed that NtCBK1 was highly regulated in the transition to flowering. Whereas NtCBK1 mRNA was accumulated in the shoot apical meristem during vegetative growth, its expression was dramatically decreased in the shoot apical meristem after floral determination, and in young flower primordia. The expression of NtCBK1 was up-regulated to high levels in floral organ primordia. Fluctuations in NtCBK1 expression were verified by analysis of tobacco plants expressing green fluorescent protein under the control of the NtCBK1 promoter, suggesting a role of NtCBK1 in the transition to flowering. This conclusion was confirmed by overexpressing NtCBK1 in transgenic tobacco plants, where maintenance of high levels of NtCBK1 in the shoot apical meristem delayed the switch to flowering and extended the vegetative phase of growth. Further work indicated that overexpression of NtCBK1 in transgenic tobacco did not affect the expression of NFL, a tobacco homologue of the LFY gene that controls meristem initiation and floral structure in tobacco. In addition, the promotion of tobacco flowering time by DNA demethylation cannot be blocked by the overexpression of NtCBK1.
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【期刊论文】Characterization of a Novel Calcium/Calmodulin-Dependent Protein Kinase from Tobacco1[w]
吕应堂, Li Ma, Shuping Liang, Russell L. Jones, and Ying-Tang Lu*
Plant Physiol. Vol. 135, 2004, 1280-1293,-0001,():
-1年11月30日
A cDNA encoding a calcium (Ca21)/calmodulin (CaM)-dependent protein kinase (CaMK) from tobacco (Nicotiana tabacum), NtCaMK1, was isolated by protein-protein interaction-based screening of a cDNA expression library using 35S-labeled CaM as a probe. The genomic sequence is about 24.6 kb, with 21 exons, and the full-length cDNA is 4.8 kb, with an open reading frame for NtCaMK1 consisting of 1,415 amino acid residues. NtCaMK1 has all 11 subdomains of a kinase catalytic domain, lacks EF hands for Ca21-binding, and is structurally similar to other CaMKs in mammal systems. Biochemical analyses have identified NtCaMK1 as aCa21/CaMKsinceNtCaMK1phosphorylated itself and histone IIIs as substrate only in the presence ofCa21/CaMwith aKm of 44.5 mM and a Vmax of 416.2 nM min21 mg21. Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 22.1mM and aVmax of 644.1 nMmin21mg21 when assayed in the presence of Ca21/CaM. Once the autophosphorylation of NtCaMK1 was initiated, the phosphorylated form displayed Ca21/CaM-independent behavior, as many other CaMKs do. Analysis of the CaM-binding domain (CaMBD) in NtCaMK1 with truncated and site-directed mutated forms defined a stretch of 20 amino acid residues at positions 913 to 932 as the CaMBD with high CaM affinity (Kd 5 5 nM). This CaMBD was classified as a 1-8-14 motif. The activation of NtCaMK1 was differentially regulated by three tobacco CaM isoforms (NtCaM1, NtCaM3, and NtCaM13). While NtCaM1 and NtCaM13 activated NtCaMK1 effectively, NtCaM3 did not activate the kinase.
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吕应堂, Ying WANG, Shuping LIANG, Qi-Guang XIE and Ying-Tang LU
Biochem. J. (2004) 383, 73-81 (Printed in Great Britain),-0001,():
-1年11月30日
An AtCRK1 [Arabidopsis thaliana CDPK (Ca2+-dependent protein kinase)-related protein kinase 1] has been characterized molecularly and biochemically. AtCRK1 contains the kinase catalytic domain and a CaM (calmodulin)-binding site. Our results demonstrated thatAtCRK1couldbindCaMinaCa2+-dependent manner. This kinase phosphorylated itself and substrates such as histone IIIS and syntide-2 in a Ca2+-independent manner and the activitywas stimulated by several CaM isoforms through its CaMbinding domain. This domain was localized within a stretch of 39 amino acid residues at positions from 403 to 441 with Kd=67 nM for CaM binding. However, the stimulation amplification of the kinase activity of AtCRK1 by different CaM isoforms was similar.
Arabidopsis thaliana,, autophosphorylation,, calmodulin,, capillary electrophoresis,, Ca2+, -dependent protein-kinase (, CDPK), -related protein kinase (, CRK), .,
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吕应堂, ng Lu∗
Journal of Chromatography A, 1061(2004)99-104,-0001,():
-1年11月30日
A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues has been described. This method is based on the derivatization of ACC with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), and separation and quantification of the resulting FQ–ACC derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). Our results indicated that ACC derivatized with FQ could be well separated from other interfering amino acids using 20mM borate buffer (pH 9.35) containing 40mM sodium dodecyl sulfate and 10mM Brij 35. The linearity of ACC was determined in the range from 0.05 to 5 Mwith a correlation of 0.9967. The concentration detection limit for ACC was 10nM (signal-to-noise=3). The sensitivity and selectivity of this described method allows the analysis of ACC in crude apple extracts without extra purification and enrichment procedure.
Derivatization, 1-Aminocyclopropane-1-carboxylic acid, 3-(, 2-Furoyl), quinoline-2-carboxaldehyde, Micellar electrokinetic chromatography
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吕应堂, Xin Liu, Ye-Qin Hu, Li Ma, Ying-Tang Lu∗
Journal of Chromatography A, 1049(2004)237-242,-0001,():
-1年11月30日
A sensitive analytical protocol for determining phosphoamino acids using capillary electrophoresis coupled with laser-induced fluorescence detection has been developed. The technique involved the derivatization of the phosphoamino acids with fluorescent reagent 5-(4,6-dichloros- triazin-2-ylamino)fluorescein (DTAF) and the analyses of the derivatives by micellar electrokinetic chromatography with laser induced fluorescence detection (MEKC-LIF). Different variables that affect derivatization (DTAF concentration, pH, temperature and time) and separation (kind of surfactant, pH and concentration of buffer) were studied. The baseline separation of three phosphoamino acids could be obtained in less than 11 min with good reproducibility. There was a linear relationship between the peak area of the analyte and its concentration, with correlation coefficients in the range of 0.9979-0.9997. The concentration detection limits (signal to noise=3) with respect to each single phosphoamino acid were in the range of 0.5-1nM. The developed method was successfully applied for the determination of phosphoamino acids in the hydrolyzed phosphorylated protein samples.
Derivatization, MEKC, 5-(, 4,, 6-Dichloro-s-triazin-2-ylamino), fluorescein, Phosphoamino acids
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【期刊论文】Calmodulin-binding protein kinases in plants
吕应堂, Lei Zhang and Ying-Tang Lu
TRENDS in Plant Science Vol.8 No.3 March 2003, 123-127,-0001,():
-1年11月30日
Many calmodulin-binding protein kinases have been isolated from plants. Plant calmodulin-binding protein kinases are novel protein kinases that differ from calcium-dependent protein kinases in many important respects. Calmodulin-binding protein kinases are likely to be crucial mediators of responses to diverse endogenous and environmental cues in plants. In this update, we review the structure, regulation, expression and possible functions of plant calmodulin-binding protein kinases.
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吕应堂, Wei HUA, Shuping LIANG and Ying-Tang LU
Biochem. J. (2003) 376, 291-302 (Printed in Great Britain),-0001,():
-1年11月30日
A calcium (Ca2+)/calmodulin (CaM)-binding protein kinase (CBK) from tobacco (Nicotiana tabaccum), NtCBK2, has been characterized molecularly and biochemically. NtCBK2 has all 11 conserved subdomains of the kinase-catalytic domain and a CaM-binding site as shown by other kinases, including Ca2+-dependent protein kinase and chimaeric Ca2+/CaM-dependent protein kinases. However, this kinase does not contain an EF-hand motif for Ca2+ binding, and its activity was not regulated by Ca2+.Whereas NtCBK2 phosphorylated both itself and other substrates, such as histone IIIS and syntide-2, in a Ca2+/CaM-independent manner, as also shown by OsCBK, a CaM-binding protein kinase from rice (Oryza sativa), the kinase activity of NtCBK2 was greatly stimulated by Ca2+/CaM, whereas that of OsCBK was not. By molecular dissection analyses, the CaM-binding domain of NtCBK2 has been localized in a stretch of 30 amino acid residues at residue positions 431-460 as a 1-5-10 protein motif. Three tobacco CaM isoforms (NtCaM1, NtCaM3 and NtCaM13) used in the present study have been shown to bind to NtCBK2, but with different dissociation constants (Kds), as follows: NtCaM1, 55.7 nM; NtCaM3, 25.4 nM; and NtCaM13, 19.8 nM, indicating that NtCBK2 has a higher affinity for NtCaM3 and NtCaM13 than for NtCaM1. The enzymic activity of NtCBK2 was also modulated differently by various CaM isoforms. Whereas the phosphorylation activity of NtCBK2 was shown by assay to be enhanced only≈2-3-fold by the presence of NtCaM1, the activity could be amplified up to 8–9-fold by NtCaM3 or 10-11-fold by NtCaM13, suggesting that NtCaM3 and NtCaM13 are better activators than NtCaM1 for NtCBK2.
calmodulin-binding protein kinase (, CBK), ,, capillary electrophoresis,, dissociation constant,, phosphorylation,, tobacco.,
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吕应堂, Xin Liu, Li Ma, Ya-Wei Lin, Ying-Tang Lu∗
Journal of Chromatography A, 1021(2003)209-213,-0001,():
-1年11月30日
A novel method based on capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF) was developed for the determination of abscisic acid (ABA), which is an essential phytohormone during plant growth and development. ABA was labeled with 8-aminopyrene-1,3,6-trisulfonate via reductive amination in presence of acetic acid and sodium cyanoborohydride. The derivatization yield was maximized by optimizing several derivatization parameters including derivatization reagent concentration, reaction temperature and time. The conjugate was separated and quantitated by CE–LIF. The linearity of ABA was determined in the range from 0.1 to 10µmol l−1 with a correlation of 0.9979. The derivatization limit of detection for ABA was found to be 56 fmol (corresponding to the concentration of 2.8×10−8mol l−1). The detection limit for ABA was 5.5 amol for an injection volume of 5nl. As a preliminary application, the proposed methodwas successfully applied to determining trace amount ofABAin the crude extracts of tobacco without extra purification and enrichment procedure and showed a better selectivity and sensitivity than those conventional methods used in determination of ABA.
Derivatization,, electrophoresis, Tobacco, Plant materials, Abscisic acid, Plant hormones, Aminopyrenetrisulfonate
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吕应堂, Xin Liu, Li-Xiang Yang, Ying-Tang Lu*
Journal of Chromatography A, 998(2003)213-219,-0001,():
-1年11月30日
Micellar electrokinetic capillary chromatography with laser-induced fluorescence detection was applied to the determination of biogenic amines including putrescine, histamine, cadaverine, tyramine, tryptamine, 2-phenylethylamine, spermine, and spermidine. A fluorogenic derivatization reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde, was successfully used to fluorescently label these biogenic amines. Different variables that affect derivatization (derivatization reagent concentration, reaction time and temperature) and separation (buffer concentration, addition of organic modifiers and sodium deoxycholate concentration) were studied. The linearities within concentration ranges of up to two orders of magnitudes were achieved for those species with correlation coefficients from 0.9967 to 0.9992. The detection limits (signal to noise53) of 210 21 biogenic amines can reach 5310mol l, which are equivalent to or better than the detection limits obtained by other analytical methods of biogenic amines. As a preliminary application, this method has been successfully employed to determine biogenic amines in the extract of tobacco leaf.
Derivatization,, electrophoresis, Biogenic amines, 3-(, 2-Furoyl), quinoline-2-carboxaldehyde
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吕应堂, Lei ZHANG*, Bi-Feng LIU*, Shuping LIANG*, Russell L. JONES† and Ying-Tang LU*
Biochem. J. (2002) 368, 145-157 (Printed in Great Britain),-0001,():
-1年11月30日
A Ca2+/calmodulin (CaM)-binding protein kinase from rice (Oryza sativa), OsCBK, has been characterized that lacks Ca2+-binding EF hands and has Ca2+/CaM-independent autophosphorylation and substrate-phosphorylation activity. OsCBK has all 11 subdomains of a kinase catalytic domain and a putative CaM-binding domain, and shares high identity with Ca3+-dependent-protein-kinase (CDPK')-related protein kinases in plants. OsCBK bound CaM in a Ca2+-dependent manner as previously reported for Ca2+}calmodulin-dependent protein kinases in animals, but autophosphorylation and phosphorylation of histone IIIs were Ca2+/CaM-independent. Surface plasmon resonance analysis showed that OsCBK specifically bound CaM with high affinity (KD=30nM). Capillary electrophoresis showed that phosphorylation of OsCBK occurred on serine and threonine residues. These data show that OsCBK is a serine/threonine protein kinase that binds Ca2+/CaM, but whose enzymic activity is independent of Ca2+/CaM. In situ hybridization showed that OsCBK is expressed in reproductive and vegetative tissues of rice and shows temporal and spatial changes during plant growth and development. OsCBK is highly expressed in zones of cell division and it is particularly abundant in sporogenous cells of the anther at meiosis.
autophosphorylation,, calcium-dependent protein kinase (, CDPK), ,, capillary electrophoresis (, CE), ,, in situ hybridization,, surface plasmon resonance (, SPR), .,
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