构树形成层活动中内源IAA的变化及其结合蛋白的研究

构树(Broussonetiapapyrifera(L.)Vent.)形成层活动周期中用酶联免疫吸附法测定了形成层区域的内源IAA浓度的变化,并用自行发展的免疫荧光检测方法测定了由此处生活细胞制得的原生质体中IAA结合蛋白的分布。结果表明,形成层旺盛形成未成熟木质部和未成熟韧皮部时期,内源IAA急剧增加,当这些细胞分化成熟时IAA浓度降低,并维持在一定范围内。IAA结合蛋白主要分布于质膜、胞质、核膜及核质中     

烯效唑对水稻幼苗内源IAA含量的影响

研究了烯效唑对3~H-IAA色氨酸合成3~H-IAA的效率及对IAA氧化酶活性的影响,以探讨烯效唑延缓植物生长的作用机理。结果表明,烯效唑对水稻(Oryza sativa L.)幼苗生长的控制效应与其降低内源IAA含量有关,烯效唑浸种处理降低水稻幼苗内源IAA含量有两条途径,其一是提高水稻幼苗IAA氧化酶活性,增强内源IAA的氧化;其二是阻抑内源IAA的合成。烯效唑除阻抑内源赤霉素的生物合成而延缓作物生长外,通过降低内源IAA水平也可能是其延缓作物生长的一个原因。     

GA3对杂交稻“粒间顶端优势”及灌浆期间籽粒内源IAA的影响

以杂交稻协优 413为材料 ,研究了外源GA3 对籽粒内源IAA影响。观察到开花后其优势粒比劣势粒灌浆启动早 ,优势粒灌浆势下降后 ,劣势粒开始灌浆 ,且优势粒灌浆势峰值比劣势粒高 ,这种优劣势粒的灌浆异步现象是一种“粒间顶端优势” ,即劣势粒的灌浆启动迟 ,与优势粒对劣势粒的抑制有关。籽粒灌浆期间优势粒内源IAA含量增加早于劣势粒 ,优势粒内源IAA峰值也高于劣势粒 ;优劣势粒灌浆势变化趋势与优劣势粒内源IAA含量水平的变化趋势相似 ,“粒间顶端优势”似为内源IAA所调节。于初穗时喷施外源GA3,“放大”了单穗中优劣势粒灌浆异步效应 ,优势粒灌浆势更强 ,劣势粒更弱 ,即外源GA3 加强“粒间顶端优势”现象。在试验中还看到初穗期喷施 10～ 40mg/L浓度的GA3 溶液 ,随浓度提高 ,优势粒内源IAA含量增加 ,劣势粒IAA含量更低 ,这种变化似与施用GA3、使优势粒对IAA吸收更多有关     

IBA和NAA对山杜英组培苗生根过程中内源IAA、ABA含量变化的影响

本文研究山杜英组培苗生根过程中内源IAA、ABA含量变化规律。结果表明,培养基添加IBA和NAA后,在生根过程中内源IAA、ABA含量变化类似,根点出现前内源IAA、ABA含量一直上升,根点出现后含量开始下降,产生愈伤组织时两种处理的IAA/ABA分别是2.526和3.226。在不添加外源生长素情况下,内源IAA含量一直维持在较低水平,而内源ABA含量一直呈现上升趋势,IAA/ABA始终都在1.211以下。     

不同浓度外源IAA处理对杉木茎部基因表达的影响

为了揭示吲哚-3-乙酸(Indole-3-acetic acid,IAA)参与杉木木材发育调控的遗传机制,文章分别以0、3mg.IAA/g.lanolin处理不同阶段的杉木截顶茎秆作为驱动方(Driver)和测试方(Tester),利用抑制消减杂交技术(Suppression substractive hybridization,SSH),对其中差异表达的目的基因进行了分离和克隆。共获得332个Unigenes,其潜在的功能分别涉及到细胞组织和生物合成、发育进程调控、电子传递、逆境应答以及信号传导等方面;进一步地表达鉴定发现ClHIRA、ClPGY1和ClARF4等集中于茎部近轴区域表达的基因,能够积极地响应外源IAA刺激的维管形成层分裂和管胞分化活动;而ClSMP1、ClTCTP1和ClTRN2等集中于茎部远轴区域表达的基因,则在转录水平上对外源IAA的处理水平及近轴次生维管的发育变化表现出负相关的关系。这一结果表明特异性定位的发育基因对木材形成组织中内源IAA水平变化的差异性识别和响应很可能是生长素参与林木维管形成层次生发育调节的重要分子机制。     

乙酰胆碱对管花肉苁蓉种子萌发及其内源IAA和ABA含量的影响

检测不同浓度乙酰胆碱对管花肉苁蓉种子发芽率和内源吲哚乙酸(IAA)和脱落酸(ABA)含量影响的结果表明:适宜浓度(1.0~3.0mmol·L~(-1))的乙酰胆碱可促进肉苁蓉种子萌发,并改变内源IAA和ABA含量。1.0mmol·L~(-1)乙酰胆碱是促进肉苁蓉种子萌发的临界点。     

杨树试管苗嫩茎生根过程中内源IAA的免疫金银定位

生长素类物质在木本植物生根过程中发挥重要作用。杨树生根与生长素的关系及生根过程中内源激素的变化已有大量报道,而生根过程中生长素的组织定位分析则尚未见报道。该文应用免疫化学分析方法对741杨（Populus alba×（P.davidiana×P.simonii）×P.tomentosa）嫩茎生根过程中内源IAA在组织中的分布进行了研究。结果显示,741杨的嫩茎在无外源激素的1/2MS培养基上诱导10天后可生根,14天后生根率达100%。诱导前,嫩茎基部组织中几乎没有IAA信号;诱导8天后,嫩茎基部维管组织中有大量的IAA积累,而且中部的维管组织中也有明显的IAA信号（主要分布在韧皮部和维管形成层）;10天后,形成不定根原基,此时IAA主要分布在根原基;12天后,根原基分化成不定根并突破表皮,IAA在不定根中的分布主要集中在根尖和中柱。该文对741杨的嫩茎生根过程中IAA的组织分布特点及运输途径进行了讨论。     

杨树试管苗嫩茎生根过程中内源IAA的免疫金银定位

生长素类物质在木本植物生根过程中发挥重要作用。杨树生根与生长素的关系及生根过程中内源激素的变化已有大量报道, 而生根过程中生长素的组织定位分析则尚未见报道。该文应用免疫化学分析方法对741杨(Populus alba × (P. davidiana × P. simonii) × P. tomentosa)嫩茎生根过程中内源IAA在组织中的分布进行了研究。结果显示, 741杨的嫩茎在无外源激素的1/2MS培养基上诱导10天后可生根, 14天后生根率达100%。诱导前, 嫩茎基部组织中几乎没有IAA信号; 诱导8天后, 嫩茎基部维管组织中有大量的IAA积累, 而且中部的维管组织中也有明显的IAA信号(主要分布在韧皮部和维管形成层); 10天后, 形成不定根原基, 此时IAA主要分布在根原基; 12天后, 根原基分化成不定根并突破表皮, IAA在不定根中的分布主要集中在根尖和中柱。该文对741杨的嫩茎生根过程中IAA的组织分布特点及运输途径进行了讨论。     

砀山酥梨不同程度缺铁叶片生长素抑制蛋白基因表达分析

以‘砀山酥梨’为材料,采用酶联免疫分析法(ELISA),测定叶片中内源IAA的含量.依据已构建的缺铁叶片SSH文库中生长素抑制蛋白(ARP)基因片段的序列信息,应用RACE技术克隆其cDNA全长,通过实时荧光定量(qRT-PCR)技术,分析ARP基因的相对表达量.结果表明:(1)ARP基因cDNA全长为707 bp,其中开放阅读框为351 bp,编码116个氨基酸,推测的蛋白质分子量为12.82 kD;该蛋白可能定位于微体,属于非分泌型、非跨膜蛋白类,并具有ARP基因家族的保守结构域.(2)在不同程度缺铁叶片中ARP基因的表达量存在差异,随着缺铁程度的增加,表达量显著升高,同时叶片中内源IAA含量逐渐降低.据此推测,ARP基因可能负反馈调节缺铁黄化叶片中IAA的水平,从而调控叶片的生长发育.     

荷兰芹胚性愈伤组织诱导及胚状体发生与内源IAA和ABA关系的初步研究

分析了荷兰芹胚性愈伤组织发生的条件, 对Co²⁺作用下胚状体形成与培养物内源IAA和ABA 的关系做了研究。结果表明, 荷兰芹下胚轴胚性发生能力随其相对位置而异, 自下而上逐渐提高。提高2, 4-D 浓度有利于胚性愈伤组织的诱导, 水解酪蛋白对胚性能力的表达没有显著影响。在胚状体发生过程中, 添加Co²⁺显著降低培养物内源IAA 和ABA 水平, 并提高胚状体诱导率。其中对照组IAA 有一个峰值, ABA 有两个峰值;实验组ABA 变化趋势与对照组相似, 而IAA 则始终没有峰值出现。Co²⁺对IAA 和ABA 的抑制机制可能有所不同。     

Indole-3-Acetic Acid Controls Cambial Growth in Scots Pine by Positional Signaling

The vascular cambium produces secondary xylem and phloem in plants and is responsible for wood formation in forest trees. In this study we used a microscale mass-spectrometry technique coupled with cryosectioning to visualize the radial concentration gradient of endogenous indole-3-acetic acid (IAA) across the cambial meristem and the differentiating derivatives in Scots pine (Pinus sylvestris L.) trees that had different rates of cambial growth. This approach allowed us to investigate the relationship between growth rate and the concentration of endogenous IAA in the dividing cells. We also tested the hypothesis that IAA is a positional signal in xylem development (C. Uggla, T. Moritz, G. Sandberg, B. Sundberg [1996] Proc Natl Acad Sci USA 93: 9282–9286). This idea postulates that the width of the radial concentration gradient of IAA regulates the radial number of dividing cells in the cambial meristem, which is an important component for determining cambial growth rate. The relationship between IAA concentration in the dividing cells and growth rate was poor, although the highest IAA concentration was observed in the fastest-growing cambia. The radial width of the IAA concentration gradient showed a strong correlation with cambial growth rate. The results indicate that IAA gives positional information in plants.     

The Resumption of Cambial Activity and the Change of Peroxidase Isozymes in Broussonetia papyrifera

Five Broussonetia papyrifera (L.) Vent. trees were selected in a natural stand located on the campus of Peking University, Beijing, China. The trees were ca. 5-6 years old, 3-4 m tall,and had diameters of about 3 cm measured 1.2 m above ground level. They were samplied at monthly intervals between January 28 and March 25, then at ten-day intervals between March 25 and May 20,1991. On each occasion, one 3-year-old shoot was cut from the tree. Two blocks (about 1 cm ×1 cm) contained peridern,phloem,cambium and wood with more than one annual ring were cut from every shoot,fixed in FAA,and then were prepared for anatomical studies. And on each occasion,7 layers of tissues (from periderm to mature xylem)were scraped off from the shoots and 100 mg of separate tissues were randomly extracted in 0.1 ml of 20% sucrose. The extracts were used for isoelectric-focusing in polyacrylamide gel slabs (85 mm × 60 mm × 1 mm). Benziding and odianisidine was used as substrate. After electrophoresis the gel slabs were placed in the substrate buffer until the isozyme bands were visible. Owing to the ring-porous structure of the wood of Broussonetia papyrifera, the cambial activity was comparable with that in the most ring-porous dicots. The cambium activity started about ten days before bud sprouting. On April 4,the dormant cambial zone consisted of ca. 4 cell layers. The trees did not sprout until April 16,but ca. 2 cell layers of immature xylem and phloem were formed concomitantly. Ten days later, 8-9 cell layers of xylem and ca. 5 cell layers of phloem were formed. The formation of immature phloem cells continued to increase slowly between April 4 and May 20, whereas that of immature xylem cells increased rapidly between April 4 and April 26,and then decreased between April 26 and May 20. It was suggested that differentiation of immature xylem into mature xylem lasted ca. 10 days,whereas that of immature phloem into mature one lasted ca. 20 days. There were totally 6 peroxidase isozyme bands in dormant cambial region and functional phloem. Variation of zymogram in cambial region occurred before cambial activity activated which is followed by more or less minor changes of bands in all other tissues. These indicated that several significant changes were related to the level of endogenous IAA and differentiation of vascular tissues.     

Auxin Stimulation of Cambial Activity in Pinus silvestris I. The Differential Cambial Response

Isolated stem segments of Pinus silvestris L. produce new xylem in sterile culture for 5 weeks if sucrose and IAA are present in the medium. The response of cambium varies in the course of the season and along the tree stem. The cambium is more sensitive in spring and in the stem portion closer to tree apex than later in the season and closer to the stem base. Spring initiation of cambial activity in adult pine trees under natural conditions could not be correlated with any consistent concentration gradient of natural auxin extracted from the cambial region. Thus, the relation between concentration of auxin and the activity of cambium is complex and involves changes of cambial responsivity. Interaction with gibberellic acid or kinetin and changing concentration of sucrose were studied during the season, but none of these substances alone appeared to be responsible for the observed variation in cambial response to auxin.     

The Effect of Applied Growth Hormones on Cambial Division and the Differentiation of the Cambial Derivatives

When indole-3-acetic acid (IAA) is applied to woody shoots cambialdivision is stimulated and the cambial derivatives differentiateto produce xylem tissue. When gibberellic acid (GA) is applied,cambial division occurs but the resultant derivatives on thexylem side of the cambium remain undifferentiated. The relativelevels of applied IAA and GA are important in determining whethermainly xylem or phloem tissue is produced. High IAA/low GA concentrationsfavour xylem formation, whereas low IAA/high GA concentrationsfavour phloem production. The new phloem tissue produced asa result of hormone treatment is fully differentiated, containingsieve elements and sieve plates. IAA is important in promotingthe elongation of the cambial derivatives to produce xylem vesseland fibre elements, though in the case of xylem fibres appliedGA causes further elongation. IAA is an important factor indetermining vessel diameter in the ring-porous species Robiniapseudacacia, high levels of applied IAA giving wide springwood-typevessels and low levels giving narrow ‘summerwood’vessels.     

Auxin Stimulation of Cambial Activity in Pinus silvestris II. Dependence upon Basipetal Transport

Natural auxin content has been determined in the cambial region of large Pinus silvestris L. trees at various dates during the year. The tissue was collected from the stem of intact or ring-barked trees and from stumps remaining after the trees were cut down at breast height in early summer or late autumn. No seasonal decrease of concentration of the extractable auxin in the cambial region could be detected. Decapitation or ring-barking produced severe reduction in auxin content and arrested cambial division. In the next season the auxin level and the cambial activity remained completely depressed. It is concluded that without tissue continuity in the region external to xylem and without basipetal supply of substances, no mechanism operated by roots or remaining stem tissue near the tree base can ensure a high level of auxin in the cambial region or activate and maintain the cambial division. The activity of extracted pine auxin was found not to be identical with the stimulatory potential of authentic IAA determined by standard bioassays. The possibility of interaction with other extracted substances is discussed.     

Epicormic Bud Development in Quercus robur L. Studies of Endogenous IAA, ABA, IAA Polar Transport and Water Potential in Cambial Tissues10

Seasonal measurements of IAA,³ made using GC-MS, ⁴ indicatedthat in Q. robur the spring initiation of cambial activity andonset of visible bud outgrowth in the canopy is preceded byan increase in cambial region IAA. The effects of notch-girdlescut into the bole indicated that IAA in the cambial region laterwas present in separate physiological pools, with only the polar-transportedfraction affecting epicormic bud outgrowth. The stage in thespring when the epicormic buds grew out coincided with an increaseboth in cambial region IAA and in the capacity of cambial explantsfor IAA polar transport. Thus the stimulus needed by the epicormicbuds to overcome inhibition by polar-transported IAA appearedto be self-generated. The observed effects of exogenous hormoneson epicormic bud outgrowth from stem explants indicated thatthis stimulus might be cytokinin. The seasonal changes detectedin cambial region ABA³ were consistent with a role for stress-inducedABA in the induction of epicormic bud dormancy after canopydevelopment during the summer. No consistent effects of standthinning on cambial region IAA, ABA, water potentials or watercontents were detected, although polar transport of exogenousIAA by cambial region explants removed in the spring was reducedby thinning. Key words: Epicormic buds, cambium, hormones     

Tracheid production in response to changes in the internal level of indole-3-acetic Acid in 1-year-old shoots of scots pine

Different concentrations of indole-3-acetic acid (IAA) were applied in lanolin to 1-year-old shoots of Pinus sylvestris (L.) in a manner known to stimulate cambial activity. The internal concentration of free IAA was measured at a distance below the application point by combined gas chromatography-selected ion monitoring-mass spectrometry using [¹³C₆]IAA as a quantitative internal standard, and related to the production of tracheids at the same site. The experiment was performed with: (a) debudded cuttings, where the major source of endogenous IAA, the apical buds, were replaced with exogenous IAA, and (b) intact, attached shoots, where endogenous IAA was supplemented by applying IAA around the circumference of the shoot. In both experimental systems, an increase in the internal IAA level was positively related to increased tracheid production. It was also demonstrated that the concentration of internal IAA measured at the sampling site was comparable with endogenous IAA levels found in intact control shoots, and that a wide range of applied IAA concentrations was associated with a relatively small range of internal IAA levels.     

Level of endogenous indole-3-acetic acid in the stem of Pinus sylvestris in relation to the seasonal variation of cambial activity

Abstract. Gas chromatography – selected ion monitoring – mass spectrometry was used to measure the level of indole-3-acetic acid (IAA) in the cambial region at the top and bottom of the branchless portion of the main stem of three large Scots pine trees, at weekly intervals from 28 April to 13 July. During this period, the cambium reactivated from the dormant state and entered its 'grand' period of xylem and phloem production, which was monitored by microscopy. The total amount of IAA (ng cm⁻²) increased steadily from 28 April until late June, and thereafter remained constant. In contrast, the concentration of IAA (ng g⁻¹ fresh weight) was high at the start of cambial reactivation, declined when the number of differentiating tracheids began to increase, and then rose as the number of cells decreased. The timing and magnitude of the changes in xylem and phloem production and in IAA level were similar at the two sampling positions. It is concluded that the seasonal changes in cambial activity in the conifer stem cannot be ascribed simply to a fluctuation in the level of endogenous IAA in the cambial region.     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.