The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli. Received: 12 March 1998 / Accepted: 30 March 1998     

The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli.     

A Bacterial Indole-3-acetyl-L-aspartic Acid Hydrolase Inhibits Mung Bean (Vigna radiata L.) Seed Germination Through Arginine-rich Intracellular Delivery

Indole-3-acetyl-L-aspartic acid (IAA-Asp) is a natural product in many plant species and plays many important roles in auxin metabolism and plant physiology. IAA-Asp hydrolysis activity is, therefore, believed to affect plant physiology through changes in IAA metabolism in plants. We applied a newly discovered technique, arginine-rich intracellular delivery (AID), to deliver a bacterial IAA-Asp hydrolase into cells of mung bean (Vigna radiata) seeds and measured its effects on mung bean seed germination. IAA-Asp hydrolase inhibited seed germination about 12 h after the enzyme was delivered into cells of mung bean seeds both covalently and noncovalently. Mung bean seed germination was delayed by 36 h when the enzyme protein was noncovalently attached to the AID peptide and longer than 60 h when the enzyme protein was covalently attached to the AID peptide. Root elongation of mung bean plants was inhibited as much as 90% or 80%, respectively, when the IAA-Asp hydrolase was delivered with the AID peptide by covalent or noncovalent association. Further thin-layer chromatography analysis of plant extracts indicated that the levels of IAA increased about 12 h after treatment and reached their peak at 24 h. This result suggests that IAA-Asp hydrolase may increase IAA levels and inhibit seed germination of mung bean plants and that the AID peptide is a new, rapid, and efficient experimental tool to study the in vivo activity of enzymes of interest in plant cells.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.     

掌叶木种皮障碍及种子各部位内源抑制活性的研究

掌叶木(Handeliodendron bodinieri)是残遗于中国的稀有单种属植物,因人为破坏、生境特殊及自身特性的影响,资源稀少,被列为国家一级重点保护野生珍稀濒危植物。该研究以掌叶木种子为材料,研究了4种不同发芽条件下(带种皮、浓硫酸处理种皮、完全去除种皮、仅露出胚根)种子萌发性、种皮透水性、掌叶木果皮、假种皮、种皮和种仁四个部位不同浓度甲醇浸提物(0、3.125、6.25、12.5、25 mg/m L)对白菜种子萌发及幼苗生长的影响以及掌叶木各部位浸提物对种子萌发的影响。结果表明:(1)掌叶木种皮具有一定的透水性,为掌叶木种子的萌发提供必要的透水透气条件,不影响种子萌发前的水分吸收,但掌叶木种皮的机械阻碍、易霉变对种子的萌发影响较大。(2)掌叶木的果皮、假种皮、种皮和种仁甲醇浸提物对白菜种子的萌发和生长都有影响,尤其对白菜幼根的生长有较强的抑制作用,抑制强度依次是种仁果皮假种皮种皮,且随着浓度的升高,抑制作用增强。该研究结果揭示了掌叶木种子难发芽、发芽率低的原因,为掌叶木的人工扩繁和保护与利用奠定了基础。     

Lipid Accumulation during Canola Seed Germination in Response to Cinnamic Acid Derivatives

The objective of this research was to investigate how ferulic and p-coumaric acids affect lipid and fatty acid composition during canola (Brassica napus L.) seed germination. Data showed that both compounds increased total lipid and fatty acid contents in the cotyledons during germination. The largest accumulation in lipids occurred at 1.0 mM p-coumaric acid with an increase in all unsaturated fatty acids. The results suggest that allelochemicals interfere in canola seed germination by reducing lipid mobilization.     

Biocatalytic preparation of chiral epichlorohydrins using recombinant<Emphasis Type="Italic">Pichia pastoris</Emphasis> expressing epoxide hydrolase of<Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The use of enantioselective hydrolysis for preparing chiral epichlorohydrins was investigated using recombinantPichia pastoris with the enantioselective epoxide hydrolase ofRhodotorula glutinis. The rate of the recombinant epoxide hydrolase-catalyzed enantioselective hydrolysis of racemic epichlorohydrins was enhanced by the addition of 5% (v/v) Tween 20. Enantiopure (R)-epichlorohydrins with an enantiopurity of 100%ee and a yield of 26% were obtained within 5 min from 50 mM racemates.     

盐磷胁迫对木麻黄和台湾相思种子萌发及幼苗生长的影响

恶劣环境下,人工海防林因面临养分胁迫而经营困难。为探讨盐、磷胁迫对主要海防林树种木麻黄和台湾相思种子萌发及生长的影响,该研究分别用不同浓度的NaCl(盐)和KH₂PO₄(磷)溶液处理种子和浇灌幼苗,测定种子萌发和幼苗生长指标。结果表明：(1)高盐胁迫显著抑制种子萌发,对幼苗生长有一定影响,但两种植物影响程度不同;台湾相思种子萌发耐盐性高于木麻黄,前者相对盐害率最大值为23.03%,后者为89.15%;随着盐浓度增加,木麻黄和台湾相思种子的发芽率、发芽势、发芽指数和活力指数均降低,对应最大值分别为38.70%、34.67%、18.70、0.055和76.67%、62.22%、48.46、6.11。(2)两种植物的株高和根长随盐浓度增加而降低,木麻黄和台湾相思株高分别为12.29～6.01 mm和48.27～17.33 mm,根长分别为8.57～1.45 mm和33.41～5.88 mm;台湾相思根、茎、叶生物量及根冠比均随盐浓度的增加逐渐减小,木麻黄各处理差异较小。(3)台湾相思的种子和幼苗较木麻黄更耐低磷环境,二者最适磷浓度存在差异;木麻黄种...     

Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.     

黄土丘陵区草本群落演替中先锋种群茵陈蒿浸提液的化感作用

为探索自然演替中植被群落结构与化感作用之间的关系,以黄土丘陵区自然演替中先锋种群茵陈蒿为研究对象,通过种子萌发试验,分析演替初期优势种群茵陈蒿浸提液(甲醇浸提液和水浸提液)对自身以及伴生种种子萌发以及幼苗生长的影响,从化学生态学角度解释演替初期群落结构形成的原因。结果表明,茵陈蒿浸提液对3种伴生种(铁杆蒿、白羊草和达乌里胡枝子)的种子萌发均有显著化感作用,低浓度(0.002g/m L)时表现为促进或轻微的抑制作用,高浓度(0.2g/m L)时为较强的抑制作用;不同部位茵陈蒿浸提液的化感作用不同,相比之下,地上部的抑制作用更为强烈,其浸提液均抑制铁杆蒿、白羊草和达乌里胡枝子的种子萌发,尤其在浓度为0.2 g/m L时,达到完全抑制;根系甲醇浸提液对铁杆蒿种子萌发的抑制作用要强于同浓度下的水浸提液,而地上部水浸提液对受体植物根芽的作用大于甲醇浸提液。茵陈蒿对于自身具有一定的抑制作用,表现为高浓度(0.2和0.02 g/m L)的水和甲醇浸提液抑制自身种子萌发和幼苗生长。研究结果说明化感作用可能是该地区在演替前期形成以茵陈蒿为优势种植被群落的重要原因之一。创新点:从化学生态学的角度解释了黄土丘陵区植被演替初期植物之间的关系以及群落结构形成的原因,为认识自然恢复的内在机理提供了新的视角。     

Effects of Biological Soil Crusts on Seed Bank, Germination and Establishment of Two Annual Plant Species in the Tengger Desert (N China)

The presence of biological soil crusts can affect the germination and survival of vascular plants, but the reasons are not well investigated. We have conducted a field investigation and greenhouse experiments to test the effect of crusts on two desert annual plants, which occur on the stabilized dunes of the Tengger Desert in N China. The results showed that biological soil crusts negatively influenced the seed bank of Eragrostis poaeoides and Bassia dasyphylla. The important effect of biological soil crusts on seed germination and establishment were performed indirectly through reducing the amount of germinating seeds. Field investigation and experimental results with regard to the seed bank indicated that higher seedling density was found in disturbed crust soil and bare soil surface than in intact crust soils. Greenhouse experiments showed that the effects of biological soil crusts on germination and establishment of the two plants are not obvious in moist condition, while disturbed crusts are more favorable to seed germination in dry treatment. Significant differences in biomass were found between disturbed crust soil and bare soil. Survival and growth of the two annual plants were enhanced in both algal and moss crusts during the season of rainfall or in moist environment, but crusts did not affect seedling survival in the dry period. The small seeded E. poaeoides has higher germination than larger-seeded B. dasyphylla in crust soils, but B. dasyphylla has a relatively higher survival rate than E. poaeoides in crust soils.     

AtMKK1 and AtMPK6 are involved in abscisic acid and sugar signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis> seed germination

Abscisic acid (ABA) and sugars have been well established to be crucial factors controlling seed germination of Arabidopsis. Here we demonstrate that AtMKK1 and AtMPK6 are both critical signals involved in ABA and sugar-regulated seed germination. Wild type plants depended on stratification and after-ripening for seed germination, whereas this dependence on either stratification or after-ripening was not required for mutants of mkk1 and mpk6 as well as their double mutant mkk1 mpk6. While seed germination of wild type plants was sensitively inhibited by ABA and glucose, mkk1, mpk6 and mkk1 mpk6 were all strongly resistant to ABA or glucose treatments, and in contrast, plants overexpressing MKK1 or MPK6 were super-sensitive to ABA and glucose. Glucose treatment significantly induced increases in MKK1 and MPK6 activities. These results clearly indicate that MKK1 and MPK6 are involved in the ABA and sugar signaling in the process of seed germination. Further experiments showed that glucose was capable of inducing ABA biosynthesis by up-regulating NCED3 and ABA2, and furthermore, this up-regulation of NCED3 and ABA2 was arrested in the mkk1 mpk6 double mutant, indicating that the inhibition of seed germination by glucose is potentially resulted from sugar-induced up-regulation of the ABA level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nitrate,abscisic acid and gibberellin interactions on the thermoinhibition of lettuce seed germination

Germination of lettuce seeds has obvious thermoinhibition, but the mechanism for thermoinhibition of seed germination is poorly understood. Here, we investigated the interactions of nitrate, abscisic acid (ABA) and gibberellin on seed germination at high temperatures to understand further the mechanism for thermoinhibition of seed germination. Our results showed that lettuce (Lactuca sativa L. ‘Jianye Xianfeng No. 1’) seeds exhibited notable thermoinhibiton of germination at ≥17°C in darkness, and at ≥23°C in light, but the thermoinhibited seeds did not exhibit secondary dormancy. Thermoinhibition of seed germination at 23 or 25°C in light was notably decreased by 5 and 10 mM nitrate, and the stimulatory effects were markedly prevented by nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The sensitivity of seed germination to exogenous ABA increased with increasing temperature. Thermoinhibition of seed germination was markedly decreased by fluridone (an inhibitor of ABA biosynthesis) and GA₃, and was increased by diniconazole (an inhibitor of the ABA-catabolizing enzyme ABA 8′-hydroxylase) and paclobutrazol (an inhibitor of GA biosynthetic pathway). The effect of fluridone in decreasing thermoinhibition of seed germination was obviously antagonized by paclobutrazol, and that of GA₃ was notably added to by fluridone, and that of nitrate was antagonized by paclobutrazol, diniconazole and ABA and was added to by GA₃ and fluridone. Our data show that thermoinhibition of lettuce seed germination is decreased by nitrate in a NO-dependent manner, which is antagonized by ABA, diniconazole and paclobutrazol and added by fluridone.     

In vitro assay for 2,4-D resistance in transgenic cotton

Summary 2,4-Dichlorophenoxyacetic acid (2,4-D) resistant plants of transgenic cotton (Gossypium hirsutum L.) were produced using Agrobacterium tumefaciens containing a plasmid carrying the neomycin phosphotransferase II (npt II) and 2,4-D monooxygenase (tfd A) genes. An in vitro assay was performed to determine the sensitivity of seed germination, and the growth of seedlings of transgenic and non-transgenic cotton to various concentrations of kanamycin and 2,4-D. The results indicated that kanamycin caused the cotyledons of non-transgenic plants to turn white, but transgenic plants grew normally. Seed germination and seedling growth of non-transgenic plants were strongly inhibited by 2,4-D, but only slightly for transgenic plants. Transgenic plants and non-transgenic plants can be clearly distinguished by the use of 2 mg l⁻¹ 2,4-D in seed germination medium. There was a high correlation between the response of seed germination and the growth of seedlings to kanamycin or 2,4-D, based on the germination ration, albino ratio, dry weight or fresh weight. On this basis, we development a rapid method for identifying transgenic plants that has been verified in the field. These findings will allow identification of cotton transformants at an early stage of plant development, saving time and improving cultivars containing the 2,4-D resistance trait.     

巨尾桉叶片挥发物对三种植物种子萌发及幼苗生长的化感效应

为探讨人工巨尾桉林叶片挥发物对周边农作物的化感作用,采用不同质量新鲜巨尾桉叶片及由新鲜叶片提取的桉叶油对玉米、辣椒、西红柿等三种植物种子进行处理,观测其种子萌发和幼苗生长状况。结果表明:(1)在三种被测植物中,桉树叶片挥发物对玉米种子萌发影响最小,对辣椒影响最大;(2)当叶片用量小于200g时,桉树叶片挥发物对三种植物种子萌发影响不明显,当叶片用量达400g时,能完全抑制辣椒、西红柿种子萌发,并能极显著降低玉米种子萌发(P0.01);(3)玉米幼苗芽生长随叶片用量的增加呈现先促进后抑制现象,对芽高、鲜重、干重的促进和抑制均达到显著(P0.05)或极显著水平(P0.01);(4)当叶片用量小于或等于100g时,桉树叶片挥发物对辣椒、西红柿幼苗生长影响不明显,当用量达到200g时则能极显著抑制辣椒、西红柿幼苗生长;(5)桉叶油对三种测试植物的抑制效果与叶片自然挥发物相似,且效果更显著。     

Purification and properties of d-hydantoin hydrolase and N-carbamoyl-d-amino acid amidohydrolase from Flavobacterium sp. AJ11199 and Pasteurella sp. AJ11221

We characterized recombinant d-hydantoin hydrolase (DHHase) and N-carbamoyl-d-amino acid amidohydrolase (DCHase) from Flavobacterium sp. AJ11199 and Pasteurella sp. AJ11221. The DHHases from these two strains showed a wide range of hydrolytic activity for various 5-monosubstituted d-hydantoin compounds, including a very high level activity for d-hydantoin compounds corresponding to d-aromatic amino acids such as d-tryptophan d-phenylalanine and d-tyrosine. The DCHases, in turn, were capable of catalyzing the hydrolysis of various N-carbamoyl-d-amino acids (NCD-A.A.) corresponding to d-aliphatic and d-aromatic amino acids. The combination of these enzymes was found to be applicable for the production of various d-amino acids.     

芒和荻种子对复合盐碱胁迫的生理响应

土壤盐碱化在世界范围内普遍存在,日益严峻的盐碱化形势严重威胁着植物的生长发育。芒(Miscanthus sinensis)和荻(Miscanthus sacchariflora)作为能源植物具有良好的经济效益和生态效益,并且在城市园林中已得到广泛应用。该研究以引种自辽宁省本溪阿家岭的芒和哈尔滨市太阳岛的荻为对象,模拟我国东北大庆盐碱地的低(浓度1、2)、中(浓度3)、高(浓度4、5)浓度土壤环境,分别对芒和荻的种子进行复合盐碱胁迫处理,对芒和荻的种子萌发情况进行研究。结果表明：(1)复合盐碱胁迫处理下,芒种子的发芽率随着复合盐碱浓度的升高而降低,发芽势、活力指数、发芽指数、胚根长度、胚芽长度、胚根鲜重、胚芽鲜重和耐盐碱指数均先升高后降低;荻种子的发芽率、发芽势、活力指数、发芽指数、胚根长度、胚芽长度、胚根鲜重、胚芽鲜重和耐盐碱指数均随着复合盐碱浓度的升高而降低。(2)芒和荻的种子能够抵抗低、中浓度的复合盐碱胁迫处理,当高浓度的复合盐碱胁迫处理时,各项指标均下降明显,且芒种子的各项指标均优于荻种子,说明芒种子比荻种子更耐盐碱。该研究结果基本界定芒荻种子的复合盐碱耐受范围,为今后芒荻类能源植物的耐盐碱性筛选和在园林中的应用提供了理论依据。     

Seed bank persistence of clonal weeds in contrasting habitats: implications for control

The ability of weeds to form a seed bank is important for their population dynamics and management because it provides a refuge enabling reinvasion after established target plants have died. However, knowledge of the differential seed behaviour of individual species over multiple years and varying environmental conditions is surprisingly rare but necessary for effective control of diverse weed populations. We established a seed burial experiment in alpine habitats differing in management regime (i.e., forest, hay meadow and pasture) to determine whether seeds of the unpalatable perennial weeds, Veratrum album (white hellebore) and Gentiana lutea (yellow gentian) were able to delay germination and remain viable over 3 years. Our study shows that both species formed a short-term persistent seed bank; in the third-year, the soil seed banks of both species were nearly depleted, having declined to <5% of their original size. Both species had strikingly different germination strategies: G. lutea seeds mainly germinated in their first-year, whilst the majority of V. album seeds germinated in their second-year. The fraction of dormant G. lutea seeds increased with seed age, indicating that seeds remained viable after forgoing germination in the previous year. Habitat-specific differences in seed germination increased with seed age, with germination fractions being lowest in moist hay meadows. This suggests that the negative effects of anoxic conditions became more pronounced as seeds aged in hay meadows. Conversely, seed dormancy was equal among habitats. The absence of a long-term persistent seed bank has important implications for the management of both nuisance and endangered-plant populations. In the case of V. album and G. lutea, re-colonization of habitats from the seed bank is unlikely after established plants have been removed.     

授粉方式对铁皮石斛种质座果及种子萌发的影响

为进行铁皮石斛(Dendrobium officinale)的良种选育和种苗生产,对46个铁皮石斛种质进行人工授粉试验,研究了不同授粉方式的授粉成功率、座果率和种子的萌发能力。结果表明,4种授粉方式的授粉成功率明显不同,不同种质间的授粉成功率可达100%,且座果率普遍较高。只有31.3%的种质可以成功自花授粉,且座果率相差较大。同株异花授粉和种质内异株授粉的成功率和座果率均介于上述二者之间。授粉后所得种子的萌发率和萌发速度在4种授粉方式间也呈现与授粉成功率和座果率一致的趋势。这表明在铁皮石斛种质中存在自交不亲和现象,因此以不同产地来源的铁皮石斛种质为亲本进行人工授粉,可大大提高授粉的成功率和座果率,以及后代种子的萌发能力。     

Phosphatidylglycerol Degradation is One Crucial Reason for the Decrease of its Concentration in Wheat Leaves under Phosphate Deprivation Stress

Phosphatidylglycerol (PG) is of crucial importance for the organization and function of thylakoid membranes. The reason for a decrease of PG concentration in plants under phosphate deprivation stress still remains unclear. By comparing PG concentration and PG hydrolase activity of wheat leaves at different developmental stages, we show that when the first leaves are fully developed, the PG concentration and PG hydrolase activity in phosphate-deficient plants were similar to those of the controls. With age, there was a significant decrease in PG concentration combined with an increase in PG hydrolase activity for phosphate-deficient plants. The close correlation between the decrease in PG concentration and the increase in PG hydrolases activities suggests that PG hydrolysis was the most important factor responsible for the decrease in its concentration. The main hydrolysis products of PG are phosphatidic acid (PA), diacylglycerol (DAG) and free fatty acid (FFA). The application of an inhibitor, n-butanol, which blocks the formation of PA via the PLD pathway, to the in vitro enzyme reaction mixture may restrict PA and DAG production. Neomycin sulfate, a PLC inhibitor, was shown to exhibit significant inhibition in DAG generation. These results suggest that both PLD and PLC were responsible for PG degradation in the leaves of phosphate-deficient wheat. The possible role of PLA activity for PG degradation is also discussed.