Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus.

Myrosinase (EC 3.2.3.1) is a glucosinolate-degrading enzyme mainly found in special idioblasts, myrosin cells, in Brassicaceae. This two-component system of secondary products and degradative enzymes is important in plant-insect interactions. Immunocytochemical analysis of Arabidopsis localized myrosinase exclusively to myrosin cells in the phloem parenchyma, whereas no myrosin cells were detected in the ground tissue. In Brassica napus, myrosinase could be detected in myrosin cells both in the phloem parenchyma and in the ground tissue. The myrosin cells were similar in Arabidopsis and B. napus and were found to be different from the companion cells and the glucosinolate-containing S-cells present in Arabidopsis. Confocal laser scanning immunomicroscopy analysis of myrosin cells in B. napus embryos showed that the myrosin grains constitute a continuous reticular system in the cell. These findings indicate that in the two species studied, initial cells creating the ground tissue have different potential for making idioblasts and suggest that the myrosinase-glucosinolate system has at least partly different functions. Several myrosinases in B. napus extracts are recovered in complex together with myrosinase-binding protein (MBP), and the localization of MBP was therefore studied in situ. The expression of MBP was highest in germinating seedlings of B. napus and was found in every cell except the myrosin cells of the ground tissue. Rapid disappearance of the MBP from the non-myrosin cells and emergence of MBP in the myrosin cells resulted in an apparent colocalization of MBP and myrosinase in 7-d-old seedlings.     

Induction of myrosinase gene expression and myrosinase activity in radish hypocotyls by phototropic stimulation

The role of myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) in the phototropic response in radish hypocotyls was investigated. Unilateral illumination with blue light abruptly up-regulated the activity of myrosinase, which releases bioactive 4-methylthio-3-butenyl isothiocyanate (MTBI) from inactive 4-methylthio-3-butenyl glucosinolate (MTBG), in the illuminated halves of radish hypocotyls 10 min after onset of phototropic stimulation, peaking after 30 min and decreasing thereafter. The myrosinase activity in the shaded halves also increased, but was significantly lower than that in the illuminated halves. Furthermore, whether blue light illumination induces myrosinase gene expression was studied. Northern blotting analysis indicated that myrosinase mRNA levels were increased markedly in unilaterally illuminated hypocotyls, reaching maximum signal intensity within 10 min after onset of blue illumination, declining nearly to the control level thereafter. These results suggested that phototropic stimulation promotes myrosinase gene expression and myrosinase activity in the illuminated side, resulting in the conversion of inactive MTBG to active MTBI and simultaneously producing more active raphanusanins, causing a phototropic response.     

核桃试管不定根的组织学研究

以核桃品种‘新早丰’试管嫩茎为试材,采用二步生根法诱导生根,对其试管苗不定根发生发育过程进行了解剖学研究。结果表明:核桃试管嫩茎内未发现潜伏根原基;诱导生根后,不定根原基起源于形成层,特别是髓射线正对的形成层部分,属于诱生根原基型;不定根上的侧根起源于中柱鞘细胞。核桃试管嫩茎不定根的发育过程可分为4个阶段:(1)形成层细胞分裂;(2)转变为分生组织细胞群(即根原始细胞);(3)细胞群发育成可见的根原基; (4)根原基内细胞继续分裂分化形成根尖的外形,其内发育出维管束,并向外生长,穿过皮层,突破茎表皮。在组织培养条件下长出的不定根内部解剖构造为典型的初生构造,移栽后68 d出现次生构造。另外,试管苗根毛出现与否及其发育状况受基质理化性质的影响,即生态条件可以改变组织发生及其形状。     

Differences in aquaporin levels among cell types of radish and measurement of osmotic water permeability of individual protoplasts

We investigated tissue- and cell-specific accumulation of radish aquaporin isoforms by immunocytochemical analysis. In taproots, the plasma membrane aquaporins (RsPIP1 and RsPIP2) were accumulated at high levels in the cambium, while the tonoplast aquaporin (RsTIP) was distributed in all tissues. The three isoforms were highly accumulated in the central cylinder of seedling roots and hypocotyls, and rich in the vascular tissue of the petiole of mature plants. The results suggest that RsPIP1 and RsPIP2 are abundant in the cells surrounding the sieve tube of the radish plant. The swelling rate of protoplasts in a hypotonic solution was determined individually by a newly established method to compare the osmotic water permeability of different cell types. All cells of the cortex and endodermis in seedlings showed a high water permeability of more than 300 microm s(-1). There was no marked difference in the values between the root endodermis and cortex protoplasts, although the RsPIP level was lower in the cortex than in the endodermis. This inconsistency suggests two possibilities: (1) a low level of aquaporin is enough for high water permeability and (2) the water channel activity of aquaporin in the tissues is regulated individually. The uneven distribution of aquaporins in tissues is discussed along with their physiological roles.     

Histological study of callus formation and root regeneration from mung bean (Vigna radiata W.)

We have established a reproducible culture system for callus formation and root development from juvenile stem segments of mung bean(Vigna radiata). In particular, we have studied the influence of plant growth regulators. Induction of calli from young stem explants was very effective on MS inorganic salts supplemented with 0.5 mg/L 2,4-D and 1.0 mg/L kinetin. In regenerating adventitious roots from callus tissues, we found that a combination of 0.75 mg/L NAA, 1.5 mg/L kinetin, and MS salts resulted in 20% efficiency. Histological examination showed that callus tissues originated from out-growths of the cambium rings through de-novo meristematic activity. Those rings were localized outside the vascular cambium. Adventitious roots that developed from root primordia originated from the center of the Callus masses. These primordia produced tracheid-like cells, which then became meristemoid cells for the cambium. Newly formed adventitious roots had the typical tetrarche actinostele type.     

心里美萝卜营养生长期肉质根过氧化物酶同工酶谱及组化分析

过氧化物酶［peroxidase,POD,EC 1. 11. 1. 7 (X)］为一类多基因家族的同工酶,其聚合形式对其功能具有重要影响．心里美萝卜(Raphanus sativus var.L) 肉质根富含花青素和POD等．本文采用不同的电泳技术、组化定位和活性测定研究了心里美萝卜营养生长期肉质根中POD在不同时期和不同组织中的表达、分布特点及同工酶的组成和性质．结果显示,心里美萝卜营养生长期肉质根的皮和肉中POD活性均先升高后降低,分别在播种后第40 d和60 d出现峰值,30 d时皮中POD活性高于肉,之后则相反,酶活性最高时同工酶数目也最多,不同时期,皮和肉中同工酶种类不同;皮中POD同工酶由5个单体和4个二聚体组成,而肉中由5个二聚体或2个单体、2个二聚体和1个四聚体组成,均以酸性同工酶为主;组化定位显示POD主要分布在肉质根的周皮、形成层、木射线、木质部导管和肉的初生木质部等的细胞壁附近．从上述结果得出结论： 酸性POD同工酶在植物体中以多种形式存在．     

Phototropic stimulation induces the conversion of glucosinolate to phototropism-regulating substances of radish hypocotyls

The distribution of natural growth inhibitors, the raphanusanins (isomers of 3-(methylthio)methylene-2-pyrrolidinethione) and their precursors (4-methylthio-3-butenyl glucosinolate (MTBG) and 4-methylthio-3-butenyl isothiocyanate (MTBI), between illuminated and shaded halves of radish hypocotyls during phototropic curvature was analyzed using a physicochemical assay. Phototropic stimulation rapidly decreased MTBG content, and abruptly increased contents of MTBI and raphanusanins in the illuminated halves of radish hypocotyls within 30 min after the onset of unilateral illumination. Content in the shaded halves was similar to that in dark controls. When MTBG, MTBI, and raphanusanins at endogenous levels were applied unilaterally to etiolated hypocotyls, MTBI and raphanusanins caused hypocotyls to bend but MTBG showed no activity. Blue illumination promoted myrosinase (thioglucosidase) activity, which releases MTBI from MTBG, in hypocotyls after 10 min, although enzyme activity in dark controls did not change. These results suggest that phototropic stimulation promotes myrosinase activity in the illuminated side of radish hypocotyls, releasing bioactive MTBI from inactive MTBG and simultaneously producing bioactive raphanusanins.     

Role of CLE41 Peptide in the Development of Root Storage Parenchyma in Species of the Genus <Emphasis Type="Italic">Raphanus</Emphasis> L.

CLE peptides (CLAVATA3/ENDOSPERM SURROUNDING REGION) are signal molecules or plant peptide hormones that play an important role in regulation of development of various meristems governing the expression of WOX (WUSCHEL-RELATED HOMEOBOX) genes. In particular, CLE peptides belonging to a small TDIF (Tracheary Element Differentiation Inhibitory Factor) group are responsible for the operation of gene WOX4 controlling the development of cambium and the conducting system. We looked into the role of CLE41 peptide from the TDIF group in the development of storage root in two species of the genus Raphanus: cultivated radish (Raphanus sativus var. radicula Pers.) that is a popular root crop with a storage root and its ancestor wild radish (Raphanus raphanistrum L.) where storage parenchyma of the root is poorly developed. It was shown that overexpression of gene RsCLE41 and plant treatment with exogenous peptide CLE41 influenced the development of cambium and xylem in the roots of R. sativus and R. raphanistrum and affected expression of the genes from different groups. One could say that peptide CLE41 activates expression of the genes whose homologues in arabidopsis play a key role in the maintenance of cambium (RsWOX4, RsWOX14, RsHAM4, and RsCYCD3). In the storage root of radish, peptide CLE41 activates proliferation of cambium cells reducing the amount of one of the xylem’s elements (lignified parenchyma). The obtained results point to an important role of CLE41 in the development of storage root in radish.     

Epithiospecifier protein activity in broccoli: the link between terminal alkenyl glucosinolates and sulphoraphane nitrile

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs (non-catalytic cofactors of myrosinase) promote the formation of epithionitriles from terminal alkenyl glucosinolates and as recent evidence suggests, simple nitriles at the expense of isothiocyanates. The ratio of ESP activity to myrosinase activity is crucial in determining the proportion of these nitriles produced on hydrolysis. Sulphoraphane, a major isothiocyanate produced in broccoli seedlings, has been found to be a potent inducer of phase 2 detoxification enzymes. However, ESP may also support the formation of the non-inductive sulphoraphane nitrile. Our objective was to monitor changes in ESP activity during the development of broccoli seedlings and link these activity changes with myrosinase activity, the level of terminal alkenyl glucosinolates and sulphoraphane nitrile formed. Here, for the first time, we show ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. These activity changes paralleled changes in myrosinase activity and terminal alkenyl glucosinolate content. There is a significant relationship between ESP activity and the formation of sulforaphane nitrile in broccoli seedlings. The significance of these findings for the health benefits conferred by eating broccoli seedlings is briefly discussed.     

萝卜对土生空团菌菌丝生长的影响

纯培养条件下, 测定了十字花科植物萝卜(Raphanus sativus L.)种子、幼苗、根系分泌物及幼苗提取物对土生空团菌(Cenococcum geophilum Fr. (Cg))菌株CgSO1、CgSB2、CgO5、SPOP2 和Cg5#菌株生长的影响。结果表明供试Cg菌株与萝卜种子共培养, 或将萝卜根系分泌物和幼苗提取物加入到培养基中, 均促进了Cg菌丝生长。高温灭菌处理使萝卜根系分泌物和幼苗提取物对Cg菌株的促生作用更强, 而高温灭菌后的萝卜幼苗段对菌株生长影响不大。其中经高温灭菌处理的幼苗水提取物对5菌株的促生作用最大, CgSO1、CgSB2、CgO5、SPOP2和Cg5#每菌落的菌丝干重分别达到: 54.8、45.8、63.9、41.2和50.5 mg。     

Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.     

The myrosinase-glucosinolate system, its organisation and biochemistry

The myrosinase-glucosinolate system is involved in a range of biological activities affecting herbivorous insects, plants and fungi. The system characteristic of the order Capparales includes sulphur-containing substrates, the degradative enzymes myrosinases, and cofactors. The enzyme-catalyzed hydrolysis of glucosinolates initially involves cleavage of the thioglucoside linkage, yielding D-glucose and an unstable thiohydroximate- O -sulphonate that spontaneously rearranges, resulting in the production of sulphate and one of a wide range of possible reaction products. The products are generally a thiocyanate, isothiocyanate or nitrile, depending on factors such as substrate, pH or availability of ferrous ions. Glucosinolates in crucifers exemplify components that are often present in food and feed plants and are a major problem in the utilization of products from the plants. Toxic degradation products restrict the use of cultivated plants, e.g. those belonging to the Brassicaceae. The myrosinase-glucosinolate system may, however, have several functions in the plant. The glucosinolate degradation products are involved in defence against insects and phytopathogens. and potentially in sulphur and nitrogen metabolism and growth regulation. The compartmentalization of the components of the myrosinase-glucosinolate system and the cell-specific expression of the myrosinase represents a unique plant defence system. In this review, we summarize earlier results and discuss the organisation and biochemistry of the myrosinase-glucosinolate system.     

Microautoradiographic localisation of a glucosinolate precursor to specific cells in Brassica napus L. embryos indicates a separate transport pathway into myrosin cells

The in-situ localisation of a desulpho-glucosinolate precursor has been studied by microautoradiography of cryo-sections from immature seeds and pods of the high-glucosinolate Brassica napus L. cv. Argentine collected 23 days after pollination. After feeding with the tritium-labelled glucosinolate precursor [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid, embryo radicles, cotyledons and pod-wall were frozen in liquid nitrogen. Cryotome sections were freeze-dried and coated with nuclear emulsion autoradiographic film. A distinct pattern of radioactivity derived from the glucosinolate precursor was found in specific cells in both radicle and cotyledons. In contrast, the labelling in pod walls was not cell specific, but general at the inner side of the pod wall. The results show that the glucosinolate/desulphoglucosinolate was localised in specific cells, in a pattern resembling that of myrosin cells known to contain myrosinase (EC 3.2.3.1). In addition [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to immature seeds and pods of B. napus and a quantitative incorporation into 2-hydroxy-3-butenylglucosinolate and 3-butenyl-glucosinolate was observed. When [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to 4-day-old seedlings the label was taken up by all tissues. We propose a model in which glucosinolate/desulphoglucosinolates are transported to myrosin cells to participate in the myrosinase-glucosinolate multifunctional defence system.     

欧美杂种山杨微扦插不定根发生过程的解剖学研究

采用石蜡切片技术,以欧美杂种山杨插穗基部茎段为实验材料,连续解剖观察插穗不定根发生发育过程,分析根原基发生部位与扦插生根的关系。结果显示:欧美杂种山杨插穗不定根的发生过程分为4个时期,为根原基诱导期,不定根起始期、表达期和伸长生长期。根原基诱导期维管形成层产生具有分生组织特点的薄壁细胞;不定根起始期,维管形成层及附近的薄壁细胞脱分化,形成不定根原基发端细胞;不定根表达期,根原基发端细胞不断分裂成具有方向性的根原基,根原基穿过韧皮射线和皮层,向皮孔方向发展;不定根伸长生长期,根原基从皮孔伸出,其内部的维管系统开始发育,形成不定根。研究认为,欧美杂种山杨为皮部诱导生根类型,不定根原基起源于维管形成层区,起源部位单一,扦插难生根。     

Sulphate can induce differential expression of thioglucoside glucohydrolases (myrosinases)

Thioglucoside glucohydrolase (EC 3.2.3.1; myrosinase) hydrolyses glucosinolates and thereby liberates glucose and sulphur and nitrogen compounds. To examine the hypothesis that the myrosinase-glucosinolate system is influenced by environmental factors, the effect of sulphate on the expression of myrosinases was examined. On examining different plant organs at various stages, it was observed that sulphate induces a differential expression of myrosinase polypeptides in plants ofSinapis alba L. (white mustard). Specific myrosinase polypeptides, dependent on sulphate in the growth medium, were detected on immunoblots. Without sulphate a maximum of three polypeptides was detected in buds, two in cotyledons and one in stems and roots. In plants cultured on medium with sulphate up to four polypeptides could be observed in cotyledons, five polypeptides in buds, two in stems and one in roots. Expression of myrosinases was, in general, high in plants cultured on a medium supplemented with sulphate. In floweringS. alba plants, sulphate-starved plants showed a higher expression of myrosinase in cotyledons and stems compared to plants fed with sulphate. Sulphate-fed plants had a high expression in inflorescences and roots. The organ- and time-specific induction of the myrosinase expression is discussed in relation to sulphate metabolism and availability of sulphate under normal conditions of cultivation and in relation to protection of Brassicaceae species. This is the first evidence for a specific induction of individual myrosinase proteins.     

Enhancement of secondary xylem cell proliferation by Arabidopsis cyclin D overexpression in tobacco plants

Secondary xylem is composed of daughter cells produced by the vascular cambium in the stem. Cell proliferation of the secondary xylem is the result of long-range cell division in the vascular cambium. Most xylem cells have a thickened secondary cell wall, representing a large amount of biomass storage. Therefore, regulation of cell division in the vascular cambium and differentiation into secondary xylem is important for biomass production. Cell division is regulated by cell cycle regulators. In this study, we confirm that cell cycle regulators influence cell division in the vascular cambium in tobacco. We produced transgenic tobacco that expresses Arabidopsis thaliana cyclin D2;1 (AtcycD2;1) and AtE2Fa-DPa under the control of the CaMV35S promoter. Each gene is a positive regulator of the cell cycle, and is known to influence the transition from G1 phase to S phase. AtcycD2;1-overexpressing tobacco had more secondary xylem cells when compared with control plants. In order to evaluate cell division activity in the vascular cambium, we prepared a Populus trichocarpa cycB1;1 (PtcycB1;1) promoter containing a destruction box motif for ubiquitination and a β-glucuronidase-encoding gene (PtcycB1;1pro:GUS). In transgenic tobacco containing PtcycB1;1pro:GUS, GUS staining was specifically observed in meristem tissues, such as the root apical meristem and vascular cambium. In addition, mitosis-monitoring plants containing AtcycD2;1 had stronger GUS staining in the cambium when compared with control plants. Our results indicated that overexpression of AtcycD enhances cell division in the vascular cambium and increases secondary xylem differentiation in tobacco. Key message We succeeded in inducing cell proliferation of cambium and enlargement of secondary xylem region by AtcycD overexpression. We also evaluated mitotic activity in cambium using cyclin-GUS fusion protein from poplar.     

远志根的形态发生及组织化学研究

应用植物解剖学方法对远志(Potygda tenuiflia Willd.)根的发生和发育过程,以及1 a生与2 a生根的结构进行了比较观察,还应用组织化学方法对远志根储藏物质及主要药用成分积累部位进行了研究.结果表明:远志的药用部位为其主根,发育过程包括原分生组织、初生分生组织、初生结构和次生结构4个发育阶段.原分生组织来源于胚根,由3群原始细胞组成,具有典型分生组织的细胞学特征;初生分生组织包括根冠原、表皮原、皮层原和中柱原;初生结构由表皮、皮层和维管柱组成,初生木质部为二原型;次生生长主要是依靠维管形成层和木栓形成层的活动来完成.木栓形成层由中柱鞘细胞恢复分裂能力而形成,并且产生多层栓内层薄壁细胞.2 a生远志根的基本结构与1 a生的基本相同,只是栓内层增加至10层以上.远志根的储藏物质主要是脂类物质及少量的多糖.远志皂苷积累在远志根的薄壁细胞中,而山酮类化合物主要分布在根的木栓形成层、栓内层薄壁细胞和次生韧皮部中.     

The fate, distribution and biological performance of insecticide residues in vegetable crops following seedling treatment

Carrot, cauliflower and radish seedlings raised in sand culture and treated with aqueous suspensions containing chlorfenvinphos, diazinon, iodofenphos or triazophos were planted into the field in 1982 and 1983 to compare the performance against cabbage root fly and carrot fly of these treatments with that of bow-wave applications of granular formulations at sowing. Initial residue concentrations were up to 4100 mg kg^-1 in the seedlings at planting. Thereafter the amounts of insecticides per root system declined. Residue concentrations in mature carrot and radish roots were smaller when plants had been treated as seedlings rather than by bow-wave application. Chlorfenvinphos was the most effective insecticide against both pests. Amounts of insecticide applied in transplanted seedlings were < 5% of the amounts applied by the bow-wave method but short-term control of cabbage root fly on radish was similar. Carrot fly damage was reduced by 60% by some of the seedling treatments after 16 wk in 1982 but in 1983 they were less effective. Pot experiments with chlorfenvinphos showed that most of the insecticide in the seedlings was transferred rapidly into the surrounding soil and it was concluded that the bioactivity resulted mainly from uniform distribution of the released insecticide between and around individual plants.     

Root growth inhibition and ultrastructural changes in radish root tips after treatment with aqueous extracts of Fallopia japonica and F. ×bohemica rhizomes

Allelopathic compounds released by invasive alien plants can suppress the growth of plants in their vicinity. The aim of this study was to investigate changes in tissue and cell structure in roots of radish seedlings treated with 10% aqueous extracts of rhizomes from the invasive knotweeds Fallopia japonica and F. ×bohemica. After 7 days of growth without and with aqueous extracts from these rhizomes, the anatomical and ultrastructural changes in the radish seedling roots were analyzed with light and transmission electron microscopy, and hydrogen peroxide was localized with diaminobenzidine, to define oxidative stress. The roots of radish seedlings treated with the knotweed extracts were shorter and thicker, due to the shorter and wider shapes of their cortex cells, which were organized in more columns than the control roots. There were signs of cell damage and oxidative stress in the root cap cells, and to a lesser extent in the meristematic zone. As well as the irregularly shaped nuclei and plasma membrane detached from the cell wall, the most prominent ultrastructural effects in the root cap cells of these aqueous rhizome extracts were the ring-shaped form of the mitochondria and large endoplasmic reticulum bodies. Excessive vacuolization was seen for the cells of the root apical meristem.