菊芋耐性胁迫及种质保存研究进展

菊芋(Helianthus tuberous L.)属菊科向日葵属多年生草本植物,是重要的作物种质资源。国内外对菊芋已开展了生态、经济、能源及育种栽培等研究,近年来胁迫条件对菊芋的影响研究成为新的热点。菊芋是无性繁殖作物,目前对菊芋种质资源的保存主要采取田间圃位的形式,国外已经开展了试管苗保存和超低温保存等研究,而我国尚存在空白。本文着重从菊芋的胁迫耐性响应研究,包括干旱、盐碱及低温3个不同胁迫条件对田间性状、生理生化、蛋白、分子水平的研究,以及常规保存和离体保存等种质保存研究2个方面进行阐述,并指出目前存在的问题,为菊芋超低温保存和开发利用提供理论依据。     

Apoptosis-like DNA fragmentation in leaves and floral organs precedes their developmental senescence

ABSTRACT

We investigated the senescence of flag leaves of durum wheat (Triticum durum) during grain-filling, and of petal-like ray flowers of Jerusalem artichoke (Helianthus tuberosus) at anthesis. In both systems, we observe cleavage of DNA to high molecular weight fragments, followed by further degradation to nucleosomal fragments (laddering), a classical hallmark of apoptosis. We show that DNA fragmentation in such specialised leaves is triggered early in organ development, before the appearance of visual symptoms of senescence. Our observations support the hypothesis that senescence and cell death are part of the plant developmental program, activated by developmental cues.     

DNA and histone content of immature tracheary elements from cultured artichoke explants

Relative amounts of DNA and histone were determined by Feulgen microdensitometry and alkaline fast-green microdensitometry in differentiating tracheary elements in cultured explants of Jerusalem artichoke (Helianthus tuberosus L.) tubers. The absence of endopolyploidy in cultured artichoke tissue was confirmed, and the nuclei of tracheary elements were exclusively at the 2C level for both DNA and histones.     

Genetic diversity of worldwide Jerusalem artichoke (Helianthus tuberosus) germplasm as revealed by RAPD markers

Jerusalem artichoke (Helianthus tuberosus) is a wild relative of the cultivated sunflower (H. annuus); it is an old tuber crop that has recently received renewed interest. We used RAPD markers to characterize 147 Jerusalem artichoke accessions from nine countries. Thirty RAPD primers were screened; 13 of them detected 357 reproducible RAPD bands, of which 337 were polymorphic. Various diversity analyses revealed several different patterns of RAPD variation. More than 93% of the RAPD variation was found within accessions of a country. Weak genetic differentiation was observed between wild and cultivated accessions. Six groups were detected in this germplasm set. Four ancestral groups were found for the Canadian germplasm. The most genetically distinct accessions were identified. These findings provide useful diversity information for understanding the Jerusalem artichoke gene pool, for conserving Jerusalem artichoke germplasm, and for choosing germplasm for genetic improvement.     

Characterization of Mitotic Cycles Preceding Xylogenesis in Cultured Explants of Helianthus tuberosus L.

The number of mitotic cycles intervening between the transferof dormant Helianthus tuberosus (Jerusalem artichoke) tuberexplants to culture medium and the differentiation of the firsttracheary elements at 48 h was investigated by a pulse-labellingtechnique employing quantitative autoradiography. Silver-graincounts indicated that differentiation was preceded by threemitotic cycles Duration of the cell cycle phases were estimatedby a pulse-labelling method. From calculation of the phase durationsit was estimated that the first visible signs of tracheary elementdifferentiation occurred from 7–10 h after the last mitosis. Helianthus tuberosus L., Jerusalem artichoke, tracheary elements, cultured explants, tissue culture, mitotic cycles, cell cycle, tritiated thymidine, autoradiography     

菊芋组织培养快繁技术的建立

以菊芋带芽点的薯盘及带节的幼嫩茎段为外植体,MS为基本培养基,附加不同种类和浓度的生长调节物质,研究菊芋组织培养和快速繁殖的技术环节,最终筛选出最优技术参数组合,建立菊芋再生体系。试验结果表明：茎段外植体是理想的快速繁殖材料,正接（形态学下端向下）是最佳的接种方式。芽诱导最佳培养基为MS＋1.0mg·L-16-BA＋0.2mg·L-1IBA,诱导率为95%;继代增殖最适宜培养基为MS＋2.0mg·L-16-BA;壮苗培养最佳培养基为MS＋0.1mg·L-16-BA;生根适宜培养基为MS＋0.2mg·L-1NAA,生根率达100%;移栽成活率达95%,大田种植成活率达95%以上。     

Physiological responses of two Jerusalem artichoke cultivars to drought stress induced by polyethylene glycol

Physiological responses of two Jerusalem artichoke (Helianthus tuberosus L.) cultivars with different drought sensitivity to drought stress induced by polyethylene glycol (PEG) 6000 were investigated by characterizing water status, membrane lipid peroxidation, key antioxidant enzymes activity, and proline accumulation. It was observed that the drought-tolerant Jerusalem artichoke cv. Xiuyan maintained a relatively higher water status than the drought-sensitive cv. Yulin upon drought treatments. Meanwhile, lower levels of malondialdehyde (MDA) as well as higher levels of free proline occurred in cv. Xiuyan after 36 h drought treatments. Moreover, the activities of catalase (CAT) and superoxide dismutase (SOD) in cv. Xiuyan were higher than cv. Yulin after drought stress. These results indicated that drought sensitivities actually differ between Jerusalem artichoke cv. Xiuyan and cv. Yulin, and the cv. Xiuyan was more tolerant to drought stress caused by polyethylene glycol.     

Solubilization of Jerusalem artichoke tuber chromatin by 2,4-dichlorophenoxyacetic acid

Chromatin from the tuber of the Jerusalem artichoke (Helianthus tuberosus) was solubilized in 2,4-dichlorophenoxyacetic acid (2,4-D) solution (100 mM) at pH 7.0. This solubilization was much affected by the pH; below 6.0 less chromatin was solubilized. The elution pattern of the products on gel filtration with Sepharose 4B showed that the solubilization was caused by the dissociation of the DNA and associated proteins. The pattern of polyacrylamide gel electrophoresis of histones extracted from the chromatin solubilized by 2,4-D was quite different from those of histones extracted from the original chromatin or from NaCl (2.0M) solubilized chromatin. The F1 and F3 fractions seemed to be little affected by 2,4-D, but the F2a1, F2a2 and F2b fractions were greatly decreased. In addition, the ratios of histones and non-histone proteins to DNA changed considerably in 2,4-D solubilized chromatin in an inverse manner. None of these changes were observed with NaCl. which suggests that the behaviour of 2,4-D for the solubilization of chromatin differs substantially from that of NaCl.     

Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase

Cinnamic acid 4-hydroxylase (CA4H) was purified from microsomes of manganese-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. The three-step purification procedure involved solubilization and phase partitioning in Triton X-114, followed by chromatography on DEAE-Trisacryl and hydroxylapatite columns. Purification was monitored using carbon monoxide and type I substrate binding properties of the enzyme. The protein, purified to electrophoretic homogeneity, showed an Mr of about 57,000 on SDS-PAGE. Polyclonal antibodies raised against this protein selectively reacted with a 57-kDa polypeptide on Western blots of induced Jerusalem artichoke microsomes. The antibody selectively and strongly inhibited CA4H activity from several plant species.     

菊芋叶片提取物抑菌活性与化学成分的研究

为了开发新型植物源杀菌剂和充分利用菊芋(Helianthus tuberosus L.)资源,本文尝试用石油醚、乙醚、乙酸乙酯和水等4种溶剂对菊芋叶片进行平行提取,采用生长速率法测定菊芋叶片提取物对水稻纹枯菌(Rhizoc-tonia solaniKühn)、小麦赤霉菌[Gibberella zeae(Schw.)Petch]、番茄早疫菌[Alternaria solani(Ellis et Martin)Jones et Grour]和番茄灰霉菌(Botrytis cinerea Pers.)生长量的抑制活性;并通过试管法和滤纸法对菊芋叶片内化学成分进行初步预试。抑菌实验结果表明:(1)菊芋叶片各溶剂提取物处理与对照处理相比差异显著;(2)菊芋叶片水提取物处理与各有机溶剂提取物处理差异也基本显著;(3)菊芋叶片各溶剂提取物同浓度处理对水稻纹枯菌、番茄早疫菌和番茄灰霉菌抑制效果较好;(4)菊芋叶片乙酸乙酯提取物抑菌效果最为显著,浓度为20mg/mL时对番茄早疫菌和番茄灰霉菌已达到完全抑制,对水稻纹枯菌抑制率也达到77.91%。初步化学预试结果说明,菊芋叶片中含有蛋白质、氨基酸、还原糖类、有机酸、酚类和鞣质、黄酮类、内酯类、强心甙以及油脂等化学成分。     

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater

Aims: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. Methods and Results: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38°C for growth and inulinase production, and 35°C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l^?1, and the highest ethanol yield of 0·467, or 91·5% of the theoretical value of 0·511, was achieved for the slurry with a solid concentration of 200 g l^?1. Conclusions: Halophilic Jerusalem artichoke can be used for fuel ethanol production. Significance and Impact of the Study: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.     

萌芽菊芋块茎对盐碱土壤胁迫的生理响应

土壤盐碱化是影响全球农业生产和生态环境的重要问题。在农田、轻度盐碱草地和重度盐碱草地设置样地以块茎种植菊芋,次年5月块茎萌发阶段取块茎样品测定丙二醛、游离脯氨酸、可溶性糖含量以及抗氧化酶活性并进行蛋白质组学分析,分析了萌芽菊芋块茎对盐碱土壤胁迫的生理响应。0—20 cm土层的电导率(表征土壤可溶盐含量)表明从农田到轻度、重度盐碱草地土壤盐碱胁迫逐渐增强,丙二醛含量变化反映出菊芋块茎受害程度逐渐增加,并且基于游离脯氨酸的渗透调节能力也在逐渐增强。蛋白质组学分析结果显示与遗传信息加工相关的差异蛋白数量最多(占28.75%)且多为表达上调,意味着DNA复制和转录、蛋白质合成和折叠的相关蛋白在响应盐碱胁迫中发挥关键作用。碳水化合物及多糖代谢(占15%)、氨基酸代谢(占11.25%)以及能量代谢(占7.5%)相关的差异蛋白数量也较多,说明调节物质代谢平衡在萌芽菊芋块茎应对盐碱土壤胁迫过程中有重要作用。这些结果为揭示萌芽菊芋块茎适应盐胁迫的生理机制奠定了基础。     

Effect of Ethylene and Ethylene Precursors on Protein Phosphorylation and Xylogenesis in Tuber Explants of Helianthus tuberosus (L.)

Koritsas, V. M. 1988. Effect of ethylene and ethylene precursorson protein phosphorylation and xylogenesis in tuber explantsof Helianthus tuberosus (L.).J. exp. Bot. 39: 375–386. The role of ethylene in protein phosphorylation and xylem-celldifferentiation was studied in explant cultures of Jerusalemartichoke, Helianthus tuberosus (L.). Explants on xylem-inducingmedium growing at 25 °C developed more xylem elements andproduced more ethylene gas within 3 d of culture than controlcultures growing and developing at similar rates. L-methionineand 1-aminocdopropane-1-carboxylic acid (ACC) incorporated intoboth xylem-inducing and control media enhanced xylem differentiation,increased protein levels and stimulated protein kinase activityin the explants. Inhibitors of the ethylene biosynthetic pathway,cobalt nitrate and aminoethoxyinyl glycine (AVG), depressedxylem differentiation, protein phosphorylation and polypeptidesynthesis. Ethylene gas or ACC reversed the effects of the inhibitors.Ethylene production, protein phosphoryilation and xylem-celldifferentiation were all linked. Ethylene thus promotes xylem-celldifferentiation in Jerusalem artichoke explants, the processprobably being regulated by protein phosphorylation. Key words: Ethylene, phosphorylation, xylem differentiation, Jerusalem artichoke explants     

Feeding Jerusalem artichoke reduced skatole level and changed intestinal microbiota in the gut of entire male pigs

Different levels of dried Jerusalem artichoke were fed to entire male pigs 1 week before slaughter. The objective was to investigate the effect on skatole level in the hindgut and in adipose tissue, as well as the effect on microflora and short-chain fatty acids (SCFA) in the hindgut. Five experimental groups (n = 11) were given different dietary treatments 7 days before slaughtering: negative control (basal diet), positive control (basal diet + 9% chicory-inulin), basal diet + 4.1% Jerusalem artichoke, basal diet + 8.1% Jerusalem artichoke and basal diet + 12.2% Jerusalem artichoke. Samples from colon, rectum, faeces and adipose tissue were collected. Effect of dietary treatment on skatole, indole and androstenone levels in adipose tissue and on skatole, indole, pH, dry matter (DM), microbiota and SCFA in the hindgut was evaluated. Feeding increasing levels of Jerusalem artichoke to entire male pigs reduced skatole in digesta from colon and in faeces (linear, P < 0.01). There was also a tendency towards a decreased level of skatole in adipose tissue (linear, P = 0.06). Feeding Jerusalem artichoke decreased DM content in colon and faeces and pH in colon (linear, P < 0.01). Increasing levels of Jerusalem artichoke resulted in a reduced level of Clostridium perfringens in both colon and rectum (linear, P < 0.05) and a tendency towards decreased levels of enterobacteria in colon (linear, P = 0.05). Further, there was an increase in total amount of SCFA (linear, P < 0.05), acetic acid (linear, P < 0.05) and valerianic acid (linear, P < 0.01) in faeces. In conclusion, adding dried Jerusalem artichoke to diets for entire male pigs 1 week before slaughter resulted in a dose-dependent decrease in skatole levels in the hindgut and adipose tissue. The reduced skatole levels might be related to the decrease in C. perfringens and the increase in SCFA with subsequent reduction in pH.     

The Use of Fura-2 Fluorescence to Monitor the Movement of Free Calcium Ions into the Matrix of Plant Mitochondria (Pisum sativum and Helianthus tuberosus)

Purified mitochondria isolated from pea (Pisum sativum L. cv Alaska) stems and Jerusalem artichoke (Helianthus tuberosus L. cv OB1) tubers were loaded with the acetoxymethyl ester of the fluorescent Ca2+ indicator fura-2. This made possible the continuous monitoring of free [Ca2+] in the matrix ([Ca2+]m) without affecting the apparent viability of the mitochondria. Pea stem mitochondria contained an initial [Ca2+]m of approximately 60 to 100 nM, whereas [Ca2+]m was severalfold higher (400-600 nM) in mitochondria of Jerusalem artichoke tubers. At low extramitochondrial Ca2+ concentrations ([greater than or equal to]100 nM), there was an energy-dependent membrane potential increase in [Ca2+]m; the final [Ca2+]m was phosphate-dependent in Jerusalem artichoke but was phosphate-independent in pea stem mitochondria. The data presented indicate that (a) there is no absolute requirement for phosphate in Ca2+ uptake; (b) plant mitochondria can accumulate external free Ca2+ by means of an electrophoretic Ca2+ uniporter with an apparent affinity for Ca2+ (Km approximately 150 nM) that is severalfold lower than that measured by conventional methods (isotopes and Ca2+-sensitive electrodes); and (c) [Ca2+]m is within the regulatory range of mammalian intramitochondrial dehydrogenases.     

鹰嘴豆孢克鲁维酵母利用菊芋原料同步糖化与发酵生产乙醇

菊芋含有大量的菊粉多糖,且种植简单、产量高,是极具开发价值的替代玉米等粮食作物生产燃料乙醇的原料。文中研究了鹰嘴豆孢克鲁维酵母Y179利用菊芋原料同步糖化与发酵生产乙醇。鹰嘴豆孢克鲁维酵母Y179具有高效分泌菊粉酶的能力,摇瓶试验显示Y179酵母能够利用完全由菊芋原料配制而成的培养基良好生长并发酵产生乙醇。通气及温度对乙醇产量影响明显,相对厌氧环境对Y179酵母发酵产乙醇具有促进作用,30℃发酵温度相对37℃和42℃更有利于乙醇产量提高。种子液培养时间及接种量对乙醇产量影响较小。在5 L发酵罐中以10%(V/V)量接入预培养36 h的Y179种子液,发酵液完全由菊芋干粉配制而成,总糖含量22%(W/V),30℃不通气,300 r/min搅拌,发酵144 h时,乙醇浓度达到12.3%(V/V),糖醇转化效率86.9%,糖利用率大于93.6%。初步研究结果显示鹰嘴豆孢克鲁维酵母Y179在利用菊芋原料生产乙醇方面具有良好应用前景。