拟南芥紫色酸性磷酸酶基因(AtPAPs)对磷饥饿的响应

根据拟南芥基因组测序所获得的信息,对拟南芥2号染色 7个可能的紫色酸性磷酸酶基因进行了cDNA克隆、测序及生物信息学分析,并对其在磷饥饿状态下转录水平的表达模式进行了研究,发现大部分的AtPAPs都是组成性表达的,只有AtPAP9,AtPAP10是诱导表达的,其中AtPAP9的转录产物是磷饥饿重新诱导的,而AtPAP10是磷饥饿诱导增加的。     

The efficiency of Arabidopsis thaliana (Brassicaceae) root hairs in phosphorus acquisition

Arabidopsis thaliana root hairs grow longer and denser in response to low-phosphorus availability. In addition, plants with the root hair response acquire more phosphorus than mutants that have root hairs that do not respond to phosphorus limiting conditions. The purpose of this experiment was to determine the efficiency of root hairs in phosphorus acquisition at high- and low-phosphorus availability. Root hair growth, root growth, root respiration, plant phosphorus uptake, and plant phosphorus content of 3-wk-old wild-type Arabidopsis (WS) were compared to two root hair mutants (rhd6 and rhd2) under high (54 mmol/m) and low (0.4 mmol/m) phosphorus availability. A cost-benefit analysis was constructed from the measurements to determine root hair efficiency. Under high-phosphorus availability, root hairs did not have an effect on any of the parameters measured. Under low-phosphorus availability, wild-type Arabidopsis had greater total root surface area, shoot biomass, phosphorus per root length, and specific phosphorus uptake. The cost-benefit analysis shows that under low phosphorus, wild-type roots acquire more phosphorus for every unit of carbon respired or unit of phosphorus invested into the roots than the mutants. We conclude that the response of root hairs to low-phosphorus availability is an efficient strategy for phosphorus acquisition.     

拟南芥ABA不敏感型突变体及其ABA结合特性

拟南芥的脱落醚（ABA）不敏感型突变体abi2,在对ABA的敏感性、气孔开度及种子休眠方面,与野生型有明显差异。通过3H－ABA与野生型对的亚细胞组分的结合分析,表明38000×g组分特异结合活性最高,结合最适温度为20℃,最适保温时间：20℃时为70min;0℃时为90min。由饱和曲线的Scatchard分析表明：abi2存在一种ABA结合位点,野生型有两种ABA结合位点。对3H－ABA结合的38000×g组分的SDS－PAGE电泳分析表明：野生型有3个结合活性峰,而abi2只有1个结合活性峰。     

干旱胁迫下拟南芥中H_2S与ABA信号关系研究

以野生型拟南芥(WT)、硫化氢(H_2S)合成酶缺失型突变体lcd、脱落酸(ABA)合成缺失型突变体aba1实生苗为材料,以0.3 mol·L^-1甘露醇模拟干旱胁迫,研究干旱胁迫对ABA含量、H_2S含量的影响,及其在拟南芥抵抗干旱胁迫中的作用及信号关系。结果显示:干旱胁迫显著提高LCD和ABA1基因相对表达以及H_2S含量,ABA含量;干旱胁迫显著抑制突变体lcd、aba1的种子萌发;干旱胁迫下,外施NaHS促进干旱胁迫下WT、lcd和aba1中內源H_2S的产生及上调LCD、ABA1基因相对表达,而外施ABA提高干旱胁迫下WT、aba1中H_2S含量及LCD、ABA1基因相对表达,但是对lcd中H_2S含量及LCD基因相对表达没有显著影响。研究结果表明,信号分子H_2S和ABA在拟南芥的干旱胁迫响应中发挥一定的作用,且H_2S位于ABA的下游参与调控拟南芥的信号过程。     

Cortical actin filaments in guard cells respond differently to abscisic acid in wild-type and abi1-1 mutant Arabidopsis

Cortical actin filaments in guard cells of Commelina communis L. show signal-specific organization during stomatal movements [S.-O. Eun and Y. Lee (1997) Plant Physiol 115: 1491–1498; S.-O. Eun and Y. Lee (2000) Planta 210: 1014–1017]. To study the roles of actin in signal transduction, it is advantageous to use Arabidopsis thaliana (L.) Heynh., an excellent model plant with numerous well-characterized mutants. Using an immunolocalization technique, we found that actin deployments in guard cells of A. thaliana were basically identical to those in C. communis: actin proteins were assembled into radial filaments under illumination, and were disassembled by ABA. In addition, we examined actin organization in an ABA-insensitive mutant (abi1-1) to test the involvement of protein phosphatase 2C (PP2C) in the control of actin structure. A clear difference was observed after ABA treatment, namely, neither stomatal closing nor depolymerization of actin filaments was observed in guard cells of the mutant. Our results indicate that PP2C participates in ABA-induced actin changes in guard cells. Received: 23 June 2000 / Accepted: 20 October 2000     

The phs1-3 mutation in a putative dual-specificity protein tyrosine phosphatase gene provokes hypersensitive responses to abscisic acid in Arabidopsis thaliana

The plant hormone abscisic acid (ABA) controls numerous physiological traits: dormancy and germination of seeds, senescence and resistance to abiotic stresses. In order to get more insight into the role of protein tyrosine phosphatase (PTP) in ABA signalling, we obtained eight homozygous T-DNA insertion lines in Arabidopsis thaliana PTP genes. One mutant, named phs1-3, exhibited a strong ABA-induced inhibition of germination as only 26% of its seeds germinated after 3 days instead of 92% for the Columbia (Col-0) line. Genetic and molecular analyses of phs1-3 showed that it bears a unique T-DNA insertion in the promoter of the gene and that the mutation is recessive. PHS1 expression in the mutant is about half that of the Col-0 line. The upregulation of two ABA-induced genes (At5g06760, RAB18) and the downregulation of two ABA-repressed genes (AtCLC-A, ACL) are enhanced in the phs1-3 mutant compared with the wild-type. The 'in planta' aperture of phs1-3 stomata is reduced and the inhibition of the light-induced opening of stomata by ABA is stronger in phs1-3 leaves than in Col-0 leaves. Finally, PHS1 expression is upregulated in the presence of ABA in both phs1-3 and Col-0 but more intensively in the mutant. Thus, phs1-3 is hypersensitive to ABA. Taken together, these results show that PHS1, which encodes a dual-specificity PTP, is a negative regulator of ABA signalling.     

Plant growth and phosphorus accumulation of wild type and two root hair mutants of Arabidopsis thaliana (Brassicaceae)

Arabidopsis thaliana root hairs grow longer and denser in response to low-phosphorus availability. We tested the hypothesis that wild-type Arabidopsis would acquire more phosphorus under phosphorus-limiting conditions than mutants that do not have the root hair response. The growth and phosphorus acquisition of wild-type Arabidopsis (WS) were compared to two root hair mutants (rhd6 and rhd2) under eight phosphorus treatments ranging from 0.4 mmol/m to 54 mmol/m phosphorus. At the lowest phosphorus treatment, all plants were small and showed severe phosphorus stress symptoms. At 1.5 mmol/m phosphorus, WS plants had greater shoot biomass, absolute growth rate, total phosphorus, and specific phosphorus absorption than the two root hair mutants. At the highest phosphorus treatment, there was no difference between genotypes in any of the parameters measured. We conclude that the response of increased root hair growth under low phosphorus availability in Arabidopsis is important in increasing phosphorus acquisition under phosphorus-limiting conditions.     

ABA-deficient (aba1) and ABA-insensitive (abi1-1, abi2-1) mutants of Arabidopsis have a wild-type stomatal response to humidity

In most plant species, a decrease in atmospheric humidity at the leaf surface triggers a decrease in stomatal conductance. While guard cells appear to respond to humidity‐induced changes in transpiration rate, as opposed to relative humidity or vapour pressure difference, the underlying cellular mechanisms for this response remain unknown. In the present set of experiments, abscisic acid (ABA)‐deficient (aba1) and ABA‐insensitive (abi1‐1 and abi2‐1) mutants of Arabidopsis thaliana were used to test the hypothesis that the humidity signal is transduced by changes in the flux or concentration of ABA delivered to the stomatal complex in the transpiration stream. In gas exchange experiments, stomatal conductance was as sensitive to changes in vapour pressure difference in aba1, abi1‐1 and abi2‐1 mutant plants as in wild‐type plants. These experiments appear to rule out an obligate role for either the concentration or flux of ABA or ABA conjugates as mediators of the guard cell response to atmospheric water potential. The results stand in contrast to the well‐established role of ABA in mediating guard cell responses to decreases in soil water potential.     

Induction of maize acid phosphatase activities under phosphorus starvation

Large variation in phosphorus-(P) acquisition efficiency exists among maize inbred and hybrid genotypes. Acid phosphatases are a type of enzyme that affects P acquisition and P-use efficiency in plants. The objectives of this research were (1) to characterize acid phosphatase activity in maize grown hydroponically under P starvation, and (2) to determine if there is differential induction of acid phosphatases in two maize genotypes previously characterized as P efficient (Mo17) and P inefficient (B73). B73 and Mo17 seedlings were grown hydroponically and both intracellular and secreted acid phosphatase activities were characterized. Fresh seedling weight of both genotypes decreased under P starvation, but percent fresh weight allocated to roots increased 14 days after P starvation in B73. Soluble protein concentration in shoots and roots was affected little, but secreted protein decreased by 40 and 20% in B73 and Mo17 seedlings grown without P for 14 days. Intracellular and secreted acid phosphate activity increased substantially in leaves and roots in B73 and Mo17 in response to P starvation. Secreted APase activity per unit protein increased 310 and 300% in B73 and Mo17, respectively, 7 days after P withdrawal. One of the minor isozymes identified on non-denaturing PAGE, was increased specifically in response to P starvation in both maize genotypes. The patterns and levels of change in APase activities in B73 and Mo17 were not sufficiently different to account for the diverse growth response of these genotypes in low-P conditions. The results suggest that APases may not be a major mechanism for scavenging or acquiring P and changes in APases may reflect a state of P stress in both varieties. Other factors such as root architecture, secretion of low-molecular weight carboxylates and microbial interactions might explain the difference between these two genotypes.     

Regulation of root hair density by phosphorus availability in Arabidopsis thaliana

We characterized the response of root hair density to phosphorus (P) availability in Arabidopsis thaliana. Arabidopsis plants were grown aseptically in growth media with varied phosphorus concentrations, ranging from 1 mmol m⁻³ to 2000 mmol m⁻³ phosphorus. Root hair density (number of root hairs per mm of root length) was analysed starting at 7 d of growth. Root hair density was highly regulated by phosphorus availability, increasing significantly in roots exposed to low-phosphorus availability. The initial root hairs produced by the radicle were not sensitive to phosphorus availability, but began to respond after 9 d of growth. Root hair density was about five times greater in low phosphorus (1 mmol m⁻³) than in high phosphorus (1000 mmol m⁻³) media. Root hair density decreased logarithmically in response to increasing phosphorus concentrations within that range. Root hair density also increased in response to deficiencies of several other nutrients, but not as strongly as to low phosphorus. Indoleacetic acid (IAA), the auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid (CMPA), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the ethylene synthesis inhibitor amino-oxyacetic acid (AOA) all increased root hair density under high phosphorus but had very little effect under low phosphorus. Low phosphorus significantly changed root anatomy, causing a 9% increase in root diameter, a 31% decrease in the cross-sectional area of individual trichoblasts, a 40% decrease in the cross-sectional area of individual atrichoblasts, and 45% more cortical cells in cross-section. The larger number of cortical cells and smaller epidermal cell size in low phosphorus roots increased the number of trichoblast files from eight to 12. Two-thirds of increased root hair density in low phosphorus roots was caused by increased likelihood of trichoblasts to form hairs, and 33% of the increase was accounted for by changes in low phosphorus root anatomy resulting in an increased number of trichoblast files. These results show that phosphorus availability can fundamentally alter root anatomy, leading to changes in root hair density, which are presumably important for phosphorus acquisition.     

拟南芥prr5突变体对ABA的响应

以拟南芥为材料,统计PRRs （pseudo-response regulators）突变体 prr5及其野生型经ABA处理后的萌发率、根长和NaCl处理后的萌发率,并采用实时定量PCR方法,对不同浓度ABA处理的拟南芥幼苗中的PRR5基因表达进行分析.结果表明：prr5突变体对ABA弱敏感,其种子萌发率比野生型显著或极显著增高,主根比野生型长,且PRR5基因表达受ABA抑制.同时,NaCl处理后,prr5的萌发率比野生型极显著增高.因此,推测prr5可能为ABA信号通路相关基因.     

Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2 , display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.
Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis . The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 m M . Stomatal apertures were increased by lowering external Ca²⁺ to 0.05 m M . The apertures of stomates incubated with 10 μ M ABA were not altered by changes in Ca²⁺ from 0.05 to 1.0 m M .
Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.     

Proteomic pattern-based analyses of light responses in Arabidopsis thaliana wild-type and photoreceptor mutants

Light critically affects the physiology of plants. Using two-dimensional gel electrophoresis, we used a proteomics approach to analyze the responses of Arabidopsis thaliana to red (660 nm), far-red (730 nm) and blue (450 nm) light, which are utilized by type II and type I phytochromes, and blue light receptors, respectively. Under specific light treatments, the proteomic profiles of 49 protein spots exhibited over 1.8-fold difference in protein abundance, significant at p <0.05. Most of these proteins were metabolic enzymes, indicating metabolic changes induced by light of specific wavelengths. The differentially-expressed proteins formed seven clusters, reflecting co-regulation. We used the 49 differentially-regulated proteins as molecular markers for plant responses to light, and by developing a procedure that calculates the Pearson correlation distance of cluster-to-cluster similarity in expression changes, we assessed the proteome-based relatedness of light responses for wild-type and phytochrome mutant plants. Overall, this assessment was consistent with the known physiological responses of plants to light. However, we also observed a number of novel responses at the proteomic level, which were not predicted from known physiological changes.     

Elevated CO2 enhances stomatal responses to osmotic stress and abscisic acid in Arabidopsis thaliana

A , carbon assimilation rate
ABA, abscisic acid
Ci , intercellular space CO₂ concentration
g , leaf conductance
WUE, water use efficiency

Carbon dioxide and abscisic acid (ABA) are two major signals triggering stomatal closure. Their putative interaction in stomatal regulation was investigated in well-watered air-grown or double CO₂-grown Arabidopsis thaliana plants, using gas exchange and epidermal strip experiments. With plants grown in normal air, a doubling of the CO₂ concentration resulted in a rapid and transient drop in leaf conductance followed by recovery to the pre-treatment level after about two photoperiods. Despite the fact that plants placed in air or in double CO₂ for 2 d exhibited similar levels of leaf conductance, their stomatal responses to an osmotic stress (0·16–0·24 MPa) were different. The decrease in leaf conductance in response to the osmotic stress was strongly enhanced at elevated CO₂. Similarly, the drop in leaf conductance triggered by 1 μ M ABA applied at the root level was stronger at double CO₂. Identical experiments were performed with plants fully grown at double CO₂. Levels of leaf conductance and carbon assimilation rate measured at double CO₂ were similar for air-grown and elevated CO₂-grown plants. An enhanced response to ABA was still observed at high CO₂ in pre-conditioned plants. It is concluded that: (i) in the absence of stress, elevated CO₂ slightly affects leaf conductance in A. thaliana ; (ii) there is a strong interaction in stomatal responses to CO₂ and ABA which is not modified by growth at elevated CO₂.     

Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability

Low phosphorus availability stimulates root hair elongation in many plants, which may have adaptive significance in soil phosphorus acquisition. We investigated the effect of low phosphorus on the elongation of Arabidopsis thaliana root hairs. Arabidopsis thaliana plants were grown in plant culture containing high (1000 mmol m^?3) or low (1 mmol m^?3) phosphorus concentrations, and root hair elongation was analysed by image analysis. After 15d of growth, low-phosphorus plants developed root hairs averaging 0.9 mm in length while high-phosphorus plants of the same age developed root hairs averaging 0.3 mm in length. Increased root hair length in low-phosphorus plants was a result of both increased growth duration and increased growth rate. Root hair length decreased logarithmically in response to increasing phosphorus concentration. Local changes in phosphorus availability influenced root hair growth regardless of the phosphorus status of the plant. Low phosphorus stimulated root hair elongation in the hairless axr2 mutant, exogenously applied IAA stimulated root hair elongation in wild-type high-phosphorus plants and the auxin antagonist CM PA inhibited root hair elongation in low-phosphorus plants. These results indicate that auxin may be involved in the low-phosphorus response in root hairs.     

拟南芥VSP2蛋白的重组表达及其酸性磷酸酶活性的测定

通过PCR扩增从拟南芥cDNA文库中得到VSP2蛋白的编码序列,将其构建到原核表达载体pET-22b上,并在大肠杆菌BL21菌株中实现高效可溶表达。经过Ni-NTA亲和层析一步纯化,获得电泳纯的重组VSP2蛋白。以pNPP为底物检测,该蛋白具有酸性磷酸酶活性,反应的最适pH值4.5,最适温度为45oC,Km值为26.2mM。重组VSP2蛋白表达量高,纯化后均一性好,适于蛋白晶体生长。