Marie-Michèle Cordonnier-Pratt

Transcriptional Profiling of Sorghum Induced by Methyl Jasmonate, Salicylic Acid, and Aminocyclopropane Carboxylic Acid Reveals Cooperative Regulation and Novel Gene Responses

We have conducted a large-scale study of gene expression in the C4 monocot sorghum (Sorghum bicolor) L. Moench cv BTx623 in response to the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA), and the ethylene precursor aminocyclopropane carboxylic acid. Expression profiles were generated from seedling root and shoot tissue at 3 and 27 h, using a microarray containing 12,982 nonredundant elements. Data from 102 slides and quantitative reverse transcription-PCR data on mRNA abundance from 171 genes were collected and analyzed and are here made publicly available. Numerous gene clusters were identified in which expression was correlated with particular signaling compound and tissue combinations. Many genes previously implicated in defense responded to the treatments, including numerous pathogenesis-related genes and most members of the phenylpropanoid pathway, and several other genes that may represent novel activities or pathways. Genes of the octadecanoic acid pathway of jasmonic acid (JA) synthesis were induced by SA as well as by MeJA. The resulting hypothesis that increased SA could lead to increased endogenous JA production was confirmed by measurement of JA content. Comparison of responses to SA, MeJA, and combined SA+MeJA revealed patterns of one-way and mutual antagonisms, as well as synergistic effects on regulation of some genes. These experiments thus help further define the transcriptional results of cross talk between the SA and JA pathways and suggest that a subset of genes coregulated by SA and JA may comprise a uniquely evolved sector of plant signaling responsive cascades.

The phytochrome gene family in tomato includes a novel subfamily

Data presented here define five tomato phytochrome genes (PHY) and indicate the existence of additional PHY in the tomato genome. Portions of each gene, encoding amino acids 203 through 315 in a consensus amino acid sequence, were amplified by polymerase chain reaction. Four of these genes, PHYA, PHYB1, PHYB2 and PHYE, are members of previously identified PHY subfamilies, while the fifth, PHYF, is identified as a member of a new PHY subfamily. PHYA, PHYB1, PHYB2 and PHYE fragments encode amino acid sequences that share 88% to 98% sequence identity with their Arabidopsis counterparts. The PHYF fragment, however, encodes a polypeptide that shares only 65% to 74% sequence identity with previously identified Arabidopsis phytochromes. A phylogenetic analysis suggests that PHYF arose soon after, or perhaps prior to, the origin of angiosperms. This analysis leads to the prediction that PHYF might be widespread among angiosperms, including both monocotyledons and dicotyledons. Each of the five tomato PHY is expressed as a transcript of sufficient size to encode a full-length phytochrome apoprotein. Two PHYF transcripts, 4.4 and 4.7 kb in length, have been detected in 9-day-old light-grown seedlings, consistent with either multiple transcription start sites or differential processing. Analyses of genomic Southern blots hybridized with radiolabelled RNA probes derived from the five tomato PHY, as well as Arabidopsis PHYC, indicate that the tomato genome contains as many as 9 to 13 PHY. The tomato PHY family is apparently not only different from, but also larger than, the PHY family presently described for Arabidopsis.

A Functional Genomics Investigation of Allelochemical Biosynthesis in Sorghum bicolor Root Hairs

Sorghum is considered to be one of the more allelopathic crop species, producing phytotoxins such as the potent benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8′,11′,14′-pentadecatriene]-p-benzoquinone) and its analogs. Sorgoleone likely accounts for much of the allelopathy of Sorghum spp., typically representing the predominant constituent of Sorghum bicolor root exudates. Previous and ongoing studies suggest that the biosynthetic pathway for this plant growth inhibitor occurs in root hair cells, involving a polyketide synthase activity that utilizes an atypical 16:3 fatty acyl-CoA starter unit, resulting in the formation of a pentadecatrienyl resorcinol intermediate. Subsequent modifications of this resorcinolic intermediate are likely to be mediated by S-adenosylmethionine-dependent O-methyltransferases and dihydroxylation by cytochrome P450 monooxygenases, although the precise sequence of reactions has not been determined previously. Analyses performed by gas chromatography-mass spectrometry with sorghum root extracts identified a 3-methyl ether derivative of the likely pentadecatrienyl resorcinol intermediate, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3′-position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity. An expressed sequence tag data set consisting of 5,468 sequences selected at random from an S. bicolor root hair-specific cDNA library was generated to identify candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Quantitative real time reverse transcription-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from the expressed sequence tag data set have led to the identification of a novel O-methyltransferase highly and predominantly expressed in root hairs (designated SbOMT3), which preferentially utilizes alk(en)ylresorcinols among a panel of benzene-derivative substrates tested. SbOMT3 is therefore proposed to be involved in the biosynthesis of the allelochemical sorgoleone.

The mapping of phytochrome genes and photomorphogenic mutants of tomato.

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2j) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1.

Sequence Analysis of Bacterial Artificial Chromosome Clones from the Apospory-Specific Genomic Region of Pennisetum and Cenchrus

Apomixis, asexual reproduction through seed, is widespread among angiosperm families. Gametophytic apomixis in Pennisetum squamulatum and Cenchrus ciliaris is controlled by the apospory-specific genomic region (ASGR), which is highly conserved and macrosyntenic between these species. Thirty-two ASGR bacterial artificial chromosomes (BACs) isolated from both species and one ASGR-recombining BAC from P. squamulatum, which together cover approximately 2.7 Mb of DNA, were used to investigate the genomic structure of this region. Phrap assembly of 4,521 high-quality reads generated 1,341 contiguous sequences (contigs; 730 from the ASGR and 30 from the ASGR-recombining BAC in P. squamulatum, plus 580 from the C. ciliaris ASGR). Contigs containing putative protein-coding regions unrelated to transposable elements were identified based on protein similarity after Basic Local Alignment Search Tool X analysis. These putative coding regions were further analyzed in silico with reference to the rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes using the resources at Gramene (www.gramene.org) and Phytozome (www.phytozome.net) and by hybridization against sorghum BAC filters. The ASGR sequences reveal that the ASGR (1) contains both gene-rich and gene-poor segments, (2) contains several genes that may play a role in apomictic development, (3) has many classes of transposable elements, and (4) does not exhibit large-scale synteny with either rice or sorghum genomes but does contain multiple regions of microsynteny with these species.

Absolute quantification of five phytochrome transcripts in seedlings and mature plants of tomato {Solanum lycopersicum L.)

Described here are the first quantitative measurements of absolute amounts of mRNAs transcribed from individual members of a phytochrome gene (PHY) family. The abundances of PHY mRNAs were determined for dry seed and for selected organs of greenhouse-grown tomato (Solanum lycopersicum L.) seedlings and mature plants. With a PhosphorImager, absolute amounts of PHY A, PHYB1, PHYB2, PHYE and PHYF transcripts were measured with reference to standard curves prepared from mRNA fragments synthesized in vitro. Methodology was developed permitting the use of polymerase chain reaction (PCR)-generated probes derived from a highly conserved region of PHY, obviating the necessity to clone cDNAs and to isolate probes derived from their 3′ non-coding regions. In dry seeds, PHYB1 mRNA appeared to be most abundant (4–5 μmol/mol mRNA) while in all other instances PHYA mRNA predominated. In seedlings, PHYB1, PHYB2, PHYE, and PHYF mRNAs were most abundant in the shoot (25–87 μmol/mol mRNA) while PHYA mRNA was most abundant in the root (325 umol/mol mRNA). In adult plants, the levels of PHYA, PHYB1 and PHYE mRNAs were relatively uniform among different organs (approx. 100, 75, and 10 μmol/ mol mRNA, respectively). In contrast, PHYB2 and PHYF were expressed preferentially in ripening fruits (35 and 47 μmol/mol mRNA, respectively), indicative of a possible role in fruit ripening for the phytochromes they encode. In general, the order of decreasing abundance of the five mRNAs for both seedlings and mature plants was PHYA, PHYB1, PHYE, PHYB2 and PHYF. Based upon observations that relatively modest changes in the extent of PHY expression result in changes in phenotype, the differential expression of each of the five tomato PHY described here is predicted to impact upon the spatial expression of biological activity of each phytochrome.

Photomorphogenic mutants of tomato

Photomorphogenesis of tomato is being studied with the aid of mutants which are either modified in their photoreceptor composition or in their signal transduction chain(s). Several mutants affecting the phytochrome family of photoreceptors, some of which appear deficient for specific genes encoding phytochrome apoproteins have been isolated. In addition, other mutants, including transgenic lines overexpressing phytochrome A, exhibit exaggerated photomorphogenesis during de-etiolation. Anthocyanin biosynthesis and plastid development are being used as model systems for the dissection of the complex interactions among photomorphogenic photoreceptors and to elucidate the nature of their transduction chains.

Tomato contains two differentially expressed genes encoding B-type phytochromes, neither of which can be considered an ortholog of Arabidopsis phytochrome B

Tomato (Solanum lycopersicon L.) contains two B-type phytochrome genes (PHYB1 and PHYB2). Fragments of these two PHYB were cloned following amplification by the polymerase chain reaction of a portion of their relatively well conserved 5′ coding regions. Polypeptides encoded by these gene fragments exhibit 90% sequence identity. These two PHYB are independently expressed in organ-specific fashion. In mature plants, PHYB2 mRNA is most abundant in fruit and PHYB1 mRNA in expanded leaves. A phylogenetic analysis fails to establish which tomato PHYB is orthologous to either Arabidopsis PHYB or PHYD, the latter being a second B-type phytochrome. Instead, this analysis indicates that following the divergence of the Solanaceae and Brassicaceae from one another, a PHYB gene duplicated independently in each lineage. Consequently, Arabidopsis PHYB mutants cannot be considered strictly equivalent to the tomato tri mutants, which appear to be mutated at the PHYB1 locus. Similarly, other putative PHYB mutants might not be equivalent to those described for Arabidopsis and tomato. This situation complicates efforts to determine ‘PHYB function’ because there might be no one answer to this question.

Toward Sequencing the Sorghum Genome: A U.S. National Science Foundation-Sponsored Workshop Report

Members of the worldwide sorghum (Sorghum spp.) community, including private sector and international scientists as well as community representatives from closely related crops such as sugarcane (Saccharum spp.) and maize (Zea mays), met in St. Louis, Missouri, on November 9, 2004, to lay the groundwork for future advances in sorghum genomics and, in particular, to coordinate plans for sequencing of the sorghum genome. Key developments that made this workshop timely included advances in knowledge of the sorghum genome that provide for the development of a genetically anchored physical map to guide sequence assembly and annotation, the growing role of the sorghum genome as a nucleation point for comparative genomics of diverse tropical grasses including many leading crops, and the need for dramatically increased sorghum production to sustain human populations in many regions where its inherent abiotic stress tolerance makes it an essential staple. This report reviews current knowledge of the sorghum genome, a community-endorsed schema for integrating this knowledge into a finished sequence, and early plans for translating the sequence into sustained advances to benefit a worldwide group of stakeholders.

Molecular analysis of tri-mutant alleles in tomato indicates the Tri locus is the gene encoding the apoprotein of phytochrome B1

Four monogenic recessive tomato (Lycopersicon esculentum Mill.) mutants at the temporarily red light-Insensitive (tri) locus (tri1, tri2in the genetic background breeding line GT; tri3, tri4in the genetic background cultivar Moneymaker) were studied. These mutants had slightly longer hypocotyls under white light than the wild type (WT). Western-blot analysis showed that the tri1mutant was deficient in a relatively lightstable phytochrome apoprotein (116 kDa) that was recognized in the WT by an antibody to tobacco phytochrome B; tri2had a 166-kDa band reduced in abundance; and tri2and tri4had bands reduced in molecular mass, approx. 105 and 95 kDa, respectively. These patterns were also found in light-grown plants. Northern-blot analysis for PHYB1 mRNA showed for tri2a transcript approx. 2 kb larger, for tri4, a transcript of WT size, but much reduced in abundance and for tri1and tri3transcripts equivalent in size and abundance to WT. In these mutants the transcripts of other members of the tomato phytochrome gene family (PHYA, PHYB2, PHYE, PHYF) were indistinguishable in size and abundance from WT. Thus, it appears that the tri locus specifically affects PHYB1 gene expression. Unlike phytochrome-B mutants in other plants, de-etiolated seedlings of the tri mutants exhibited normal responses to end-of-day far-red (EODFR) light and supplementary far-red light during the day. Since the holophytochromes of types B1 and B2 (phyB1 and phyB2) are closely related, it is proposed that there might be redundancy between them for these responses.