Fast and abundant in vitro spontaneous haustorium formation in root hemiparasitic plant Pedicularis kansuensis Maxim. (Orobanchaceae)

Haustorium formation is the characteristic feature of all parasitic plants and a vital process for successful parasitism. Previous investigations on haustorium initiation and development are constricted to induced processes by host-derived signals or synthetic analogs. Spontaneous haustorium formation in the absence of host signals, a process representing an early stage in the evolution of parasitic plants, remains largely unexplored. Lack of fast and frequent formation of spontaneous haustoria greatly hinders full understanding of haustorium formation in root hemiparasites. In this study, seedlings of Pedicularis kansuensis Maxim., a facultative root hemiparasitic species in Orobanchaceae observed to produce many spontaneous haustoria, were grown in autoclaved water agar in the absence of any known haustoriuminducing stimulants. We aimed to test the temporal and developmental pattern of spontaneous haustorium formation. Also, effects of sucrose supply and root contact on spontaneous haustorium formation were tested. Spontaneous haustoria were observed starting from six days after germination, much earlier than previously reported root hemiparasites. A majority of the spontaneous haustoria formed on lateral roots. Percentage of seedlings with spontaneous haustoria was 28.8% when grown on water agar plates, with a mean of four haustoria per seedling two weeks after germination. Haustorium formation by seedlings grown in water agar amended with 2% sucrose was more than twice of those without sucrose amendment. Singly grown seedlings were able to develop spontaneous haustoria at similar levels as those grown with another conspecific seedling. In view of the fast and abundant formation of spontaneous haustoria, P. kansuensis may be developed as an excellent experimental system in future investigations for unraveling endogenous regulation of haustorium initiation and development in root hemiparasitic plants.     

Phytochrome Participation in Induction of Haustoria in Cuscuta japonica, a Holoparasitic Flowering Plant

Seedlings of Cuscuta japonica pasted on an acrylic plate developedmany haustoria in response to far-red light after pre-irradiationwith white light. The effect of far-red light was cancelledcompletely by red light irradiated immediately after the far-redlight. In order to elucidate the photoreceptor(s) for photocontrolin the induction of haustoria, action spectra for the photo-inductionand its reversion were determined in the wavelength region from340 nm to 800 nm using the Okazaki Large Spectrograph. The action spectrum for the induction of haustoria had a pronouncedpeak at 740 nm and a small peak at 420 nm, while the actionspectrum for reversal of the induction had a pronounced peakat 660 nm and a small peak at 380 nm. These results indicatethat phytochrome is involved in the photocontrol of inductionof haustoria in Cuscuta japonica. Considering the far-red /redreversibility, it was suggested that phytochrome B is the photorecepter.This is the first reliable evidence of phytochome participationon development of haustoria in parasitic plants. ⁴Present address: Graduate School of Science Division of BiologicalScience, Nagoya University, Nagoya, 464-01 Japan.     

Haustoria of Cuscuta japonica, a Holoparasitic Flowering Plant, Are Induced by the Cooperative Effects of Far-Red Light and Tactile Stimuli

When seedlings of Cuscuta japonica were grown with Vigna radiata(the host plant) in a flower pot for 6 d under white light andthen irradiated with far-red or blue light (ca. 6 µmolphotons m^–2 s^–1), the seedlings parasitized V. radiata.However, no parasitism of the seedlings was observed under redor white light or in darkness. The parasitic behavior of seedlingsof C. japonica was observed even if an acrylic rod was usedas a substitute for the host plant. Upon incubation under far-redlight, the seedling twined tightly around the rod and developedhaustoria towards it. Haustoria also developed when apical andsubapical regions of seedlings were held between two glass platesthat were about 0.7 mm apart and were irradiated with far-redlight. However, no haustoria were induced by either the holdor irradiation alone. These results indicate that parasitismof Cuscuta japonica is controlled by the cooperative effectsof two physical signals, far-red light and appropriate tactilepressure. Our findings suggest that parasitism by the genusCuscuta involves a novel strategy. (Received April 10, 1996; Accepted August 21, 1996)     

田野菟丝子寄生薇甘菊的形态解剖学研究

采用扫描电镜及光镜观察田野菟丝子——薇甘菊茎的全寄生系统建立的过程.根据对吸器发生发育的观察,将其发育过程划分为4个阶段:接触反应、吸附作用、侵入生长及维管系统的建立.田野菟丝子的寄生吸器由吸器原基发育而来;当田野菟丝子成功吸附到可寄生寄主时,两者间的接触面会积累一些分泌物;吸器原基继续发育为内生原基,侵入寄主植物后单...     

The response of Cuscuta planiflora seedlings to red and far-red, blue light and end-of-day irradiations

Irradiation of excised stem segments from de-etiolated seedlings of Cuscuta planiflora for 24 h with mixtures of red and far-red light with red to far-red ratios between 0.02 to 1.0 enhanced coiling and formation of prehaustoria. Maximum number of prehaustoria were recorded when red:far-red was near 0.1. Coiling and prehaustoria were observed whenever estimated in vivo Pfr/Ptotal at photoequilibrium was between 0.06 and 0.67. Irradiation of excised stem segments from white light grown seedlings with 12 h blue light also promoted coiling and prehaustoria formation after another 38 h in darkness. Coiling and prehaustoria were not observed in segments pulsed with 10 min red light at the end of 12 h in blue light. Coiling and prehaustoria were observed after photoreversible end-of-day far-red/red/far-red pulses but not after red/far-red/red pulses. A far-red pulse may not reverse inhibition by end-of-day red pulse when far-red is given more than 12 h after the red pulse.     

The early stages of mildew colony development on susceptible oats

The chronology of primary infection by Erysiphe graminis f. sp. avenae on detached leaves of a susceptible host, and growth patterns of the primary haustorium and secondary hyphae, proved similar to those of wheat and barley mildew found by other workers. The timing of formation and subsequent growth of the primary haustorium was not affected by the light regime, but the formation of subsequent haustoria was highly synchronous under an alternating dark/light cycle, and far less so under continuous illumination. Five days after inoculation almost twice as many secondary and tertiary haustoria were formed per colony under the dark/light treatment than under continuous light. Because of the synchronous formation of haustoria subsequent to the primary, haustoria selected at random from leaves of susceptible host cultivars showed a bimodal distribution in length, the less well developed tertiary haustoria being distinguished from earlier formed primary and secondary haustoria. There was also a significant positive correlation between length and the number of digitate processes/haustorium. The energy required to produce one secondary haustorium was calculated to be equivalent to that required to produce approximately 4–7 hyphal cells.     

Influence of carbohydrates on the induction of haustoria of the cowpea rust fungusin vitro

Rust fungi are obligate plant parasites whose successful parasitism appears to depend on the formation of haustoria within living plant cells. To test the hypothesis that the lack of haustorium formation in the absence of a living cell reflects a need for a simple “signal” released during attempted invasion, various carbohydrates were applied toin vitro-formed infection structures ofUromyces vignae just prior to haustorial mother cell (HMC) formation. Up to 6% of HMCs formed haustoria after treatment with arabinose, mannose, xylose, sucrose, or xylan. Other monosaccharides, oligosaccharides, and polysaccharides were less or not effective. Sugar mixtures induced haustorium frequencies of up to 10%. Timing of the application of inducing chemicals was critical. The data indicate that haustorium formation does not require the presence of a living plant cell and may be triggered by plant products at the time of penetration peg development.     

Haustorium Formation and Cell Wall Appositions in Susceptible and Partially Resistant Wheat and Barley Seedlings Infected with Wheat Leaf Rust

Phase contrast light microscopy observations of wheat and barley seedlings infected with wheat leaf rust spores suggested that cell wall appositions are structural barriers against haustorium formation leading to abortion of infection structures. Nearly equal numbers of cell wall appositions per infection structure were detected in seedlings of susceptible and partially resistant wheat genotypes. Differences between susceptible and partially resistant genotypes became evident after the first haustorium had been formed. This again indicates the presence of a post-haustorial effect of partial resistance. Some factors influencing nutrient uptake are discussed. Wheat leaf rust colonies hardly formed haustoria in barley seedlings, the few not aborted infection structures were accompanied by cell collapse. The mechanisms of partial resistance in wheat and barley to their respective leaf rust fungi seem different, but their non-host reactions appear similar.     

蔗糖对列当科两种根部半寄生植物吸器发生的促进作用

吸器是寄生植物的特征器官,研究影响其发生的因素,有助于了解寄生关系的建立和调控过程。该研究以两种列当科(Orobanchaceae)根部半寄生植物甘肃马先蒿(Pedicularis kansuensis)和松蒿(Phtheirospermum japonicum)为材料,通过皿内培养试验,分析了蔗糖、DMBQ(2,6-二甲氧基-对-苯醌,一种高效的列当科根部半寄生植物吸器诱导化合物)和寄主植物诱导下两种根部半寄生植物吸器发生情况。结果表明：(1)蔗糖显著促进两种根部半寄生植物吸器发生,无寄主存在时,2%蔗糖处理使甘肃马先蒿和松蒿吸器发生率分别提高39.9%和20.2%。(2)蔗糖明显提升寄主植物对两种根部半寄生植物的吸器诱导水平,添加蔗糖后,寄主诱导的甘肃马先蒿单株吸器数和具木质桥的吸器比例分别增加5.7个/株和17.9%,松蒿吸器发生率和具木质桥的吸器比例分别提升76.7%和16.2%。(3)蔗糖对松蒿吸器发生的促进作用与已知吸器诱导化合物DMBQ相当,均能诱导50%以上的植株产生吸器。(4)培养基中添加4%蔗糖对两种根部半寄生植物的吸器诱导效果最好,其中甘肃马先蒿吸器发生率为56%...     

In Vitro Haustorium Development in Roots and Root Cultures of the Hemiparasitic Plant Triphysaria Versicolor

Parasitic plants in the Orobanchaceae invade host plant roots through root organs called haustoria. Parasite roots initiate haustorium development when exposed to specific secondary metabolites that are released into the rhizosphere by host plant roots. While molecular approaches are increasingly being taken to understand the genetic mechanism underlying these events, a limitation has been the lack of a transformation system for parasitic plants. Since the haustorium development occurs in roots of Orobanchaceae, root cultures may be suitable material for transient or stable transformation experiments. To this end, root cultures were obtained from explants, and subsequently calluses, from the hemiparasitic plant Triphysaria versicolor. The cultured roots retained their competence to form haustoria when exposed to host roots, host root exudates, or purified haustorium-inducing factors. The root culture haustoria invaded host roots and initiated a vascular continuity between the parasite and host roots. The ontogeny of haustoria development on root cultures was indistinguishable from that on seedlings roots. Root cultures should provide useful material for molecular studies of haustorium development.     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.     

Proteomic analysis of blue light-induced twining response in Cuscuta australis

The parasitic plant Cuscuta australis (dodder) invades a variety of species by entwining the stem and leaves of a host and developing haustoria. The twining response prior to haustoria formation is regarded as the first sign for dodders to parasitize host plants, and thus has been the focus of studies on the host-parasite interaction. However, the molecular mechanism is still poorly understood. In the present work, we have investigated the different effects of blue and white light on the twining response, and identified a set of proteins that were differentially expressed in dodder seedlings using a proteomic approach. Approximately 1,800 protein spots were detected on each 2-D gel, and 47 spots with increased or decreased protein levels were selected and analyzed with MALDI-TOF-MS. Peptide mass fingerprints (PMFs) obtained for these spots were used for protein identification through cross-species database searches. The results suggest that the blue light-induced twining response in dodder seedlings may be mediated by proteins involved in light signal transduction, cell wall degradation, cell structure, and metabolism.     

Morphological and anatomical features of Cuscuta pedicellata and C. campestris

Observations on morphological and anatomical features of Cuscuta pedicellata and, to a lesser extent, C. campestris have been made at different stages of their life-cycles. The seedling is filiform with a bulbous radicular end which collapses early. The simple shoot apex is green and photosentitive. Initial "nutation" is followed by "creeping" over the substrate. Host plants less than about three weeks old seem not to be attacked by Cuscuta seedlings. When haustorial contact is established the shoot apex differentiates into buds and scale leaves. The chlorophyll disappears and stomata, which are lacking in the seedling, develop. The apical growth rate increases strongly by frequent cell division and - elongation. This is reflected anatomically by the formation of "cell families" in rows. Weakening and flowering of the host plants are followed by flowering of the parasite. Greening of stem segments of Cuscuta occurs during formation of haustoria and after death of the host. Stem anatomy is characterized by a tripartite cortex in which the middle layer consists of thin-walled angular cells. Characteristic are also the voluminuous intercellular spaces, the cortical and perivascular laticifers, and the central position of the small vascular bundles. Observations on self-parasitism were made. The epidermis of the haustorial cushion elongates and the cytoplasm is mainly positioned towards the host. Pseudo-intercellular spaces are formed between the epidermal cells. The haustorium connects the phloem of the two plants, but vascular elements of any kind have not been observed.     

Metabolite changes with induction of Cuscuta haustorium and translocation from host plants

Cuscuta is a stem holoparasitic plant without leaves or roots, parasitizing various types of host plants and causing major problems for certain crops. Cuscuta is known as a generalist and, thus, must have unique parasite strategies to cope with different host plants. For elucidating metabolic responses and mechanisms of parasitization, metabolomic approaches using GC/MS were applied. We compared five stages of Cuscuta japonica: early stage seedlings, with far red light (FR) cue, with contact signal, haustorium induced seedlings by both signals and adult plant parasites on host plants. Sugars, amino acids, organic acids, nucleic acids, and polyols were identified from the polar phase fraction. The apical part contained metabolite profiles different from the haustorium induced part or the basal part. Amino acid and some organic acids were up-regulated for haustorium induction but decreased after parasitization. After attachment to different host plants, metabolite profiles of Cuscuta japonica changed dramatically due to the absorption of specific host plant metabolites such as pinitol. Cuscuta seedlings attached to pinitol rich host plants contained more pinitol and showed different profiles from those attached to plants having less or lacking pinitol.     

氮磷供给对两种马先蒿根系形态建成的影响

采用溶液培养法在无寄主伴生的条件下对三色马先蒿（Pedicularis tricolor）和大王马先蒿（P. rex）进行培养,分别分析了不同氮磷供给水平（标准Long Ashton营养液、缺氮、缺磷、2倍氮、2倍磷）及不同氮素形态（硝态氮和铵态氮）对两种马先蒿根系形态建成的影响。结果表明：1）氮磷供给水平对两种马先蒿的主根长、侧根长、侧根数量以及吸器发生的影响存在种间差异,缺氮和缺磷均能促进三色马先蒿主根和侧根的伸长生长及吸器的产生,而相同条件下大王马先蒿根系生长受到抑制,且没有吸器产生;2）氮素形态对两种马先蒿根系形态建成及生长发育存在影响：1倍硝态氮的处理中,三色马先蒿的根系生长最好,生物量积累最多,相同条件下大王马先蒿根系生长和生物量积累与标准营养液处理无差异,而仅供应铵态氮对二者的生长不利。此外,发现三色马先蒿的吸器产生不需要机械刺激。初步推测,两种马先蒿的吸器产生机制或进程存在差异,氮和磷可能参与了吸器发生过程的调控。     

Photocontrol of anthocyanin formation in turnip seedlings

Summary The insertion of a dark period during irradiation with far-red resulted in the occurrence of a second lag-phase in the synthesis of anthocyanin when the seedlings were returned to light. The length of this second lag-phase was similar, to the first. It is suggested that the action of far-red light may not involve gene activation but rather the formation of substrate(s); the lag-phase may arise from ratelimiting steps in the synthesis of anthocyanin pigment.     

檀香吸器维管组织的个体发育

为了解檀香吸器维管组织的发育过程,采用激光共聚焦显微镜、光学显微镜和透射电镜观察檀香吸器维管组织的个体发育。结果表明,檀香维管组织的分化分为两个时期:入侵前和入侵后。吸器维管组织发育始于盘状吸器时期,起源于吸器基部具有分生能力的细胞,后分为两束。侵入前无向顶的分化,处于吸器基部。侵入后随吸管深入寄主根与寄主根维管束连通,形成具有吸收功能的维管组织。成熟吸器维管组织呈倒烧瓶结构,仅处于吸器烧瓶核心两边,由木质部组成而无韧皮部。檀香的吸器维管组织发育有两个因素诱导,一个是遗传因素,另一个为寄主。这些为檀香半寄生性特性研究提供了形态解剖学基础。     

Structural evaluations of zygotic embryos and seedlings of the macaw palm (<Emphasis Type="Italic">Acrocomia aculeata</Emphasis>, Arecaceae) during in vitro germination

Acrocomia aculeata is an oil producing tropical palm tree with exceptional potential for producing biofuel. As the propagation of this species is often difficult because of its pronounced seed dormancy, the present work examined the morphology and the anatomy of zygotic embryos and seedlings during in vitro germination. Embryos were put in MS media supplemented with organic compounds and cultivated in the dark at 30°C for 20 days. The dry weights, lengths, and diameters of the cotyledonary petioles, haustoria, roots, ligules, and leaf sheaths of embryos obtained from mature seeds and seedlings removed from culture were measured every 2 days; anatomical and histochemical evaluations were performed on embryos and seedlings removed from culture after 2, 5, 8, 10, 12, and 15 days. Elongation of the embryo axis was observed to initiate after 2 days. Elongation of the cotyledonary petiole was observed starting on the fifth day; this is a morphological indication of germination that is associated with the formation of starch and raphides as well as the differentiation of tracheary elements. The growth of the cotyledon is due to increases in cell volumes as well as the development of a meristematic band peripheral to the haustorium. In spite of the fact that the radicle is less differentiated than the plumule, radicular development is precocious and the root emerges first, indicating the absence of morphological dormancy. Atrophy of the haustorium and the accumulation of phenolic compounds in subepidermal cell layers occur due to culturing conditions.