Elsevier

Plant Science

Volume 160, Issue 5, April 2001, Pages 951-959
Plant Science

Differential induction of Orobanche seed germination by Arabidopsis thaliana

https://doi.org/10.1016/S0168-9452(01)00331-4Get rights and content

Abstract

Parasitic plants, including the root holoparasites Orobanche spp., cause devastating damage to crops worldwide. Arabidopsis thaliana (L.) is widely used an amenable model for the study of plant biology, including plant–pathogen interactions. Bringing the two plants together in a controlled system will enable the study of the molecular and genetic basis involved in host-parasitic plant interactions and should provide tools for the detection of genes responsible for incompatibility and resistance responses. The objective of this study was to screen Arabidopsis lines for reduced germination of Orobanche seeds. A 96-cell well bioassay was developed to test the potential of lines, ecotypes and mutants of Arabidopsis to induce germination of Orobanche. Screening of 50 A. thaliana ecotypes did not reveal non-inducing ecotypes. Screening of 13 000 A. thaliana fast neutron mutated M2 plants detected 94 non-inducing mutant plants of which 34 were rescued, self pollinated, and M3 seeds collected. M3 seedlings from five lines were reduced in their ability to induce germination. In a separate assay, we determined that the reduced germination rates corresponded with reduced distance from the roots at which germination occurred. While further studies are necessary to determine the segregation of low germination phenotypes, these lines might prove useful for studying the genetic basis of variation in germination stimulant production in A. thaliana.

Introduction

Parasitic plants account for approximately 1% of extant angiosperm species and are represented in 22 plant families [1]. Several of the parasitic species are important agricultural weeds, particularly parasitic weeds in the closely related families Scrophulariaceae and Orobanchaceae. While genetic host resistance is generally considered a critical component of integrative pest management, there is currently little known about genetic resistance against parasitic weeds. The parasitism of plants by other plants is a multi-step process requiring signal exchanges between both players in the association [2]. The elimination or modification of host factors required by the parasite may prove a useful strategy for engineering resistance against these agricultural pests. This study was directed toward identifying host genes in Arabidopsis that contribute towards its susceptibility towards Orobanche. The family Orobanchaceae includes 14 genera of chlorophyll-lacking root holoparasites of which the most important genus is Orobanche, known in its common name as broomrape. Among the different Orobanche species, O. ramosa and the very similar O. aegyptiaca have the widest host range and inflict heavy damages to numerous important agricultural crops such as tomato, potato, tobacco, eggplant, faba bean, vetch, lentil, peanut, carrot, celery, parsley, sunflower and Brassica spp. [3], [4].

The Orobanche lifecycle is highly specialized for parasitism [5], [6]. Seeds germinate in the soil only after a preconditioning period of moist and suitable temperatures, and only in response to a specific chemical germination stimulant exuded by host plant roots, ensuring that only seeds within the host root rhizosphere will germinate. The parasite seedling radicle grows only a few mm and must contact a host root to ensure its existence. Upon contact with the host root, the radicle develops a specialized parasitic-plant organ, the haustorium, which adheres to the root, penetrates the epidermis and cortex tissues of the root, and ultimately establishes connections to the host vascular system [7], [8], [9]. Through these connections the parasite draws water and its nutritional needs from the host. This connection leads to the swelling of a parasite tubercle followed by a floral shoot that emerges above the soil surface. The plants rapidly develop flowers that produce capsules containing hundreds of thousands of tiny seeds per plant [10], [11].

One of the first stages in plant parasitism that can be targeted for genetic control is seed germination, a highly complex procedure in Orobanche and other obligate root parasites. Because the seeds are so small, once they have germinated they must rapidly attach to a host plant root before the seed resources are exhausted, typically within a few days. One of the parasites strategies for ensuring the presence of a potential host root is to require chemical signals released by the host roots, called xenognosins, for germination [12]. This allows the seeds to germinate within a few millimeters of a suitable host root [3]. Specific xenognosins identified for Striga germination include three tetracyclic sesquiterpenes: strigol isolated from cotton, a non-host of Orobanche and Striga [13], sorgolactone, released by Sorghum bicolor [14] and alectrol, identified in Vigna unguiculata roots [15]. An additional Striga-germinating xenognosin, the hydroquinone sorgoleone, was also detected in roots of S. bicolor [16]. Strigol and its synthetic analogue GR24 are widely used to induce germination of Striga and Orobanche seeds in laboratory studies. Recently an Orobanche minor xenognosin named orobanchol has been identified in the roots of Trifolium pratense [17]. However despite recent progress, the number, identities and mode of action of most xenognosins remain unknown [18].

In at least one case, resistance to the parasitic weed Striga results from the low germination stimulant production by host roots. The sorghum cultivar 1S-7777 is more resistant to Striga apparently because of the presence of a single recessive mutation in germination stimulant production [3], [19]. There is little information on mechanisms of tolerance or resistance to Orobanche due to low germination stimulant production by host roots [20]. Racovitza [21] found a wide range of O. ramosa susceptibility and germination stimulant production in tobacco cultivars in Romania. The most resistant tobacco cultivar ‘Joiner’ showed relatively low exudation of germination stimulant. Alders and Pieters [22] report reduced production of germination stimulants as one of the possible mechanisms for the low number of O. crenata spikes on resistant faba bean cultivars. Hershenhorn et al. [23] report that tolerant pepper varieties stimulate low germination of O. aegyptiaca.

Arabidopsis thaliana (L.) Heyhn is a small annual plant belonging to the mustard family (Cruciferea or Brassicaceae) found throughout temperate regions of the world in Europe, Asia and North America [24]. Arabidopsis is an attractive model organism for plant genetic studies because of its small size, the lack of repetitive DNA in its genome, and because of the number of genetic variants assembled by workers around the world. The eminent release of the entire Arabidopsis genomic sequence further advances its potential as a model organism (TAIR (2000) TAIR: The Arabidopsis Information Resource. NCGRI and Carnegie Institute of Washington. http://www.arabidopsis.org/index.html). Bringing A. thaliana and Orobanche plants together in a controlled system enables the study of physiological, molecular and genetic basis involved in host-parasitic plant interactions, thus providing practical tools for the detection of genes responsible for incompatibility and resistance responses.

In earlier studies we have demonstrated the susceptibility of A. thaliana ‘Columbia’ to O. aegyptiaca, O. ramosa and O. minor [25]. While there are multiple developmental stages in the parasitism of Arabidopsis by Orobanche that could be targeted for mutagenesis, in this manuscript we present results screening Arabidopsis for defective Orobanche germination. Our hypothesis was that inactivation of host genes responsible for synthesis, modification, or release of germination factors by Arabidopsis by mutagenesis will result in loss of parasite seed germination potential. The objectives of the present study were to screen Arabidopsis ecotypes and mutants for differential germination induction of O. ramosa and O. aegyptiaca seeds.

Section snippets

Arabidopsis

Seeds of wild type and Fast Neutron M2 populations of A. thaliana Columbia with the gl1 marker were purchased from Lehle Seeds (Round Rock, Texas) and stored at room temperature. The Arabidopsis ecotypes seeds were obtained from the Arabidopsis Biological Resource Center at The Ohio State University (ABRC (1999) Arabidopsis Biological Resource Center. http://aims.cps.msu.edu/aims/).

Orobanche

O.ramosa L. seeds were collected in 1998 from flowering plants parasitizing a tomato field near Courtland

Stimulation of Orobanche seed germination by A. thaliana seedlings

In previous studies we revealed that O. ramosa and O. aegyptiaca seeds germinate in the presence of factors released from A. thaliana roots [25]. In the 96-well plate assays, O. ramosa and O. aegyptiaca seeds began to germinate within 3 days of plating with 7-day-old A. thaliana seedlings. Germination reached a maximum rate after 7–10 days (Fig. 3). By 2 weeks, germination rates for O. ramosa and O. aegyptiaca were typically 40–50% and 60–70%, respectively. In contrast, the spontaneous

Acknowledgements

This research was supported by Postdoctoral Award No. FI-272-98 from BARD, The United States-Israel Binational Agricultural Research and Development Fund. We thank Dr Neil Willits from the statistical laboratory, division of statistics at UC Davis for assistance with the statistical analysis.

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