Philippe Delavault

Structure-Activity Relationship Studies of Strigolactone-Related Molecules for Branching Inhibition in Garden Pea: Molecule Design for Shoot Branching

Initially known for their role in the rhizosphere in stimulating the seed germination of parasitic weeds such as the Striga and Orobanche species, and later as host recognition signals for arbuscular mycorrhizal fungi, strigolactones (SLs) were recently rediscovered as a new class of plant hormones involved in the control of shoot branching in plants. Herein, we report the synthesis of new SL analogs and, to our knowledge, the first study of SL structure-activity relationships for their hormonal activity in garden pea (Pisum sativum). Comparisons with their action for the germination of broomrape (Phelipanche ramosa) are also presented. The pea rms1 SL-deficient mutant was used in a SL bioassay based on axillary bud length after direct SL application on the bud. This assay was compared with an assay where SLs were fed via the roots using hydroponics and with a molecular assay in which transcript levels of BRANCHED1, the pea homolog of the maize TEOSINTE BRANCHED1 gene were quantified in axillary buds only 6 h after application of SLs. We have demonstrated that the presence of a Michael acceptor and a methylbutenolide or dimethylbutenolide motif in the same molecule is essential. It was established that the more active analog 23 with a dimethylbutenolide as the D-ring could be used to control the plant architecture without strongly favoring the germination of P. ramosa seeds. Bold numerals refer to numbers of compounds.

Defense Gene Expression Analysis of Arabidopsis thaliana Parasitized by Orobanche ramosa

ABSTRACT The infection of Arabidopsis thaliana roots with the obligate parasite Orobanche ramosa represents a useful model for a study of the molecular events involved in the host plant response to a parasitic plant attack. To avoid analysis problems due to the subterranean development of O. ramosa, we developed two in vitro co-culture systems: O. ramosa seedlings infesting Arabidopsis plantlet roots and callus tissues. We were then able to investigate the expression patterns of some host plant genes selected among genes known to be involved in metabolic pathways and resistance mechanisms activated during several plant-pathogen interactions including ethylene, isoprenoid, phenylpropanoid, and jasmonate biosynthesis pathways, oxidative stress responses, and pathogenesis-related proteins. Molecular analyses were carried out using polymerase chain reaction amplification methods allowing semiquantitative evaluation of transcript accumulation during early (first hours) and late (15 days) stages of infestation, in whole roots or parts close to the parasite attachment site. In A. thaliana, O. ramosa induced most of the general response signaling pathways in a transient manner even before its attachment to A. thaliana roots. However, no salicylic acid-dependent defense is observed because no activation of systemic acquired resistance markers is detectable, whereas genes, co-regulated by jasmonate and ethylene, do display enhanced expression.

Identification by suppression subtractive hybridization and expression analysis of Arabidopsis thaliana putative defence genes during Orobanche ramosa infection

A suppression subtractive hybridization strategy was used to identify genes that were induced 2 h after infection of Arabidopsis thaliana by broomrape (Orobanche ramosa) seedlings. Among these genes, the expression of twelve was analysed from the first hour to the seventh day of this compatible interaction by cDNA blot analyses. The twelve genes showed a transient expression, which occurred in seven cases as early as the first or second hour of interaction and ended 2 or 3 h later. Most of the proteins encoded by these genes are already known to be involved in different A. thaliana response pathways to pathogen attack. The first two genes to be induced (Rc kinase and ACaM5) encoded a receptor and a calmodulin, and could be involved in signal transduction. The two following genes encoding a sucrose carrier (SUC1) and a protein with a pectin methylesterase inhibitor domain were found to be overexpressed; their roles are consistent with plant defence emergence. The gst1, gst11 and peptide methionine sulfoxide reductase genes, which were turned on early, are known to play a role in the detoxification of reactive oxygen species. The accumulation of mRNAs for lox1, a latex allergen and a myrosinase binding protein could be related to a jasmonate dependent pathway, while genes for a class III peroxidase and a caffeoyl-CoA 3-O-methyltransferase, both likely to be involved in cell wall reinforcement, were also upregulated.

Nitrogen and carbon relationships between the parasitic weed Orobanche foetida and susceptible and tolerant faba bean lines.

The parasitic weed Orobanche foetida (Poiret) is an emergent agronomical problem on faba bean in Tunisia. The Tunisian breeding programs for faba bean resistance to O. foetida have produced several tolerant lines including the line XBJ90.03-16-1-1-1, which limits both parasite attachments to the host roots and growth of the attached parasites. The present study aims to provide a better understanding of the nutritional relationships between the parasite and this tolerant line in comparison with the susceptible Bachaar genotype. Phloem saps of faba bean were harvested using phloem exudation experiments. The major organic compounds potentially transferred from both faba bean genotypes to the parasite were identified as sucrose, raffinose, stachyose, citrate, malate, asparagine (ASN), aspartate (ASP), glutamine, glutamate, serine, alanine and GABA. However, the phloem exudates of the tolerant line were highly deficient in nitrogen when compared to that of the susceptible line. When attached to roots of the tolerant line, the parasite displayed limited activities of soluble invertases in tubercles, and especially in shoots, suggesting that the low performance of the broomrapes attached to the tolerant line resulted from a reduced capacity to utilize the host-derived carbohydrates. On the other hand, the mechanisms involved in the osmotic adjustment and primary metabolism of the parasite did not differ significantly according to the host genotype: mineral cations, especially potassium and calcium, predominated as the major osmotically-active compounds in both tubercles and shoots; shoots accumulated preferentially hexoses as organic solutes although tubercles accumulated preferentially starch and soluble amino acids, especially ASP and ASN. This suggests an important role for a glutamine-dependent asparagine synthetase (EC 6.3.5.4) in the N metabolism of the parasite.

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway.

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.

Divergent evolution of two plastid genes, rbcL and atpB, in a non-photosynthetic parasitic plant

Plastid DNA (ptDNA) regions for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubiso) (rbcL) and the β-subunit of ATP synthase (atpB) genes of the holoparasite Lathraea clandestina L. were sequenced. These regions were obtained by cloning either a Bam HI endonuclease generated fragment from the Lathraea ptDNA or polymerase chain reaction (PCR) amplified products. The Lathraea ptDNA contains the entire sequence for the rbcL gene which shares 94.5% homology with the Nicotiana tabacum gene, whereas atpB is maintained as a pseudogene. The intergenic region between divergently transcribed rbcL and atpB genes is shorter (758 bp) in L. clandestina plastid genome in comparison with N. tabacum (823 bp), however they have a noticeable similarity, mainly in the rbcL 5′-upstream region. A low level of the rbcL gene transcription was detected whereas no atpB transcripts were found in Lathraea. The plasmid rbcL gene of the hemiparasite Melampyrum pratense and the autotroph Digitalis purpurea both from the Scrophulariaceae were cloned by PCR amplification and then sequenced. The L. clandestina rbcL gene is highly homologous to the M. pratense and D. purpurea genes. The data indicate that the evolution of the plastid atpB-rbcL region was different in parasites from the Scrophulariaceae and Orobanchaceae families.

The rbcL gene from the non-photosynthetic parasite Lathraea clandestina is not transcribed by a plastid-encoded RNA polymerase

Abstract In the plastome of the obligate root-parasitic plant, Lathraea clandestina, the rbcL gene has been maintained and is expressed, despite the reduced size and gene content of the plastid genome. Some of the plastid genes involved in translation (e. g. transfer RNAs, ribosomal RNAs and ribosomal proteins) have been sequenced and still appear to code for functional ribosomal components. Indeed, the 16S rRNA and rpl20 genes are expressed whilst other necessary tRNA and ribosomal protein-encoding genes have probably been deleted or truncated. Although obtained by PCR, the four rpo genes for Escherichia coli-like plastid encoded RNA polymerase appear to be pseudogenes. Nevertheless, the rbcL gene, with a “–10, –35” prokaryotic-like promoter, is still transcribed. In contrast to photosynthetic plants, rbcL transcripts in Lathraea are larger in their 5′ region and cover the prokaryotic-like promoter. The transcription initiation site is located near the ATG start codon of the atpB pseudogene. Similarity to non-consensus E. coli-like plastid promoters suggests that rbcL transcription is driven by a nuclear-encoded RNA polymerase.

Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel

Highlight This study demonstrates for the first time that DNA demethylation, an epigenetic mechanism, can control parasitic plant seed response to the strigolactones, a new class of plant hormone.

Invertases involved in the development of the parasitic plant Phelipanche ramosa: characterization of the dominant soluble acid isoform, PrSAI1

Phelipanche ramosa L. parasitizes major crops, acting as a competitive sink for host photoassimilates, especially sucrose. An understanding of the mechanisms of sucrose utilization in parasites is an important step in the development of new control methods. Therefore, in this study, we characterized the invertase gene family in P. ramosa and analysed its involvement in plant development. Invertase-encoded cDNAs were isolated using degenerate primers corresponding to highly conserved regions of invertases. In addition to enzyme assays, gene expression was analysed using real-time quantitative reverse transcriptase-polymerase chain reaction during overall plant development. The dominant isoform was purified and sequenced using electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS). Five invertase-encoded cDNAs were thus characterized, including PrSai1 which encodes a soluble acid invertase (SAI). Of the five invertases, PrSai1 transcripts and SAI activity were dominant in growing organs. The most active invertase corresponded to the PrSai1 gene product. The purified PrSAI1 displayed low pI and optimal pH values, specificity for β-fructofuranosides and inhibition by metallic ions and competitive inhibition by fructose. PrSAI1 is a typical vacuolar SAI that is actively involved in growth following both germination and attachment to host roots. In addition, germinated seeds displayed enhanced cell wall invertase activity (PrCWI) in comparison with preconditioned seeds, suggesting the contribution of this activity in the sink strength of infected roots during the subsequent step of root penetration. Our results show that PrSAI1 and, possibly, PrCWI constitute good targets for the development of new transgenic resistance in host plants using proteinaceous inhibitors or silencing strategies.

The obligate root parasite Orobanche cumana exhibits several rbcL sequences.

Orobanche species characterization using plastid sequences as molecular markers revealed that O. cumana contains at least two distinct rbcL sequences: one similar in size to the truncated rbcL pseudogene from O. cernua, a closely related species, and another with a size comparable to that of rbcL plastid genes from autotrophic plants. In this work, the nucleotide sequences of these two copies are reported and analysed. The organization of the O. cumana plastid genome was investigated using a long-distance PCR strategy in order to determine their localization. Because of the non-plastid localization of the rbcL larger copy, Southern blot and PCR chromosome-walking experiments were carried out to better characterize this transferred sequence and to identify its localization. Then the mode of multiple transfer of genetic information from plastid to nucleus and the concomitant plastid sequence disorganization and migration during parasitic plant evolution are discussed.