James S. Keddie

The Tomato Cf-2 Disease Resistance Locus Comprises Two Functional Genes Encoding Leucine-Rich Repeat Proteins

In plants, resistance to pathogens is frequently determined by dominant resistance genes, whose products are proposed to recognize pathogen-encoded avirulence gene (Avr) products. The tomato resistance locus Cf-2 was isolated by positional cloning and found to contain two almost identical genes, each conferring resistance to isolates of tomato leaf mould (C. fulvum) expressing the corresponding Avr2 gene. The two Cf-2 genes encode protein products that differ from each other by only three amino acids and contain 38 leucine-rich repeat (LRR) motifs. Of the LRRs, 20 show extremely conserved alternating repeats. The C-terminus of Cf-2 carries regions of pronounced homology to the protein encoded by the unlinked Cf-9 gene. We suggest that this conserved region interacts with other proteins involved in activating plant defense mechanisms.

The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves.

The defective chloroplasts and leaves‐mutable (dcl‐m) mutation of tomato was identified in a Ds mutagenesis screen. This unstable mutation affects both chloroplast development and palisade cell morphogenesis in leaves. Mutant plants are clonally variegated as a result of somatic excision of Ds and have albino leaves with green sectors. Leaf midribs and stems are light green with sectors of dark green tissue but fruit and petals are wild‐type in appearance. Within dark green sectors of dcl‐m leaves, palisade cells are normal, whereas in albino areas of dcl‐m leaves, palisade cells do not expand to become their characteristic columnar shape. The development of chloroplasts from proplastids in albino areas is apparently blocked at an early stage. DCL was cloned using Ds as a tag and encodes a novel protein of approximately 25 kDa, containing a chloroplast transit peptide and an acidic alpha‐helical region. DCL protein was imported into chloroplasts in vitro and processed to a mature form. Because of the ubiquitous expression of DCL and the proplastid‐like appearance of dcl‐affected plastids, the DCL protein may regulate a basic and universal function of the plastid. The novel dcl‐m phenotype suggests that chloroplast development is required for correct palisade cell morphogenesis during leaf development.

Plant inositol monophosphatase is a lithium-sensitive enzyme encoded by a multigene family.

myo-Inositol monophosphatase (IMP) is a soluble, Li(+)-sensitive protein that catalyzes the removal of a phosphate from myo-inositol phosphate substrates. IMP is required for de novo inositol synthesis from glucose 6-phosphate and for breakdown of inositol trisphosphate, a second messenger generated by the phosphatidylinositol signaling pathway. We cloned the IMP gene from tomato (LeIMP) and show that the plant enzyme is encoded by a small gene family. Three different LeIMP cDNAs encode distinct but highly conserved IMP enzymes that are catalytically active in vitro. Similar to the single IMP from animals, the activities of all three LeIMPs are inhibited by low concentrations of LiCl. LeIMP mRNA levels are developmentally regulated in seedlings and fruit and in response to light. Immunoblot analysis detected three proteins of distinct molecular masses (30, 29, and 28 kD) in tomato; these correspond to the predicted molecular masses of the LeIMPs encoded by the genes. Immunoreactive proteins in the same size range are also present in several other plants. Immunolocalization studies indicated that many cell types within seedlings accumulate LeIMP proteins. In particular, cells associated with the vasculature express high levels of LeIMP protein; this may indicate a coordinate regulation between phloem transport and synthesis of inositol. The presence of three distinct enzymes in tomato most likely reflects the complexity of inositol utilization in higher plants.

DCL is a plant-specific protein required for plastid ribosomal RNA processing and embryo development

The defective chloroplast and leaf-mutable (dcl-m) mutation of tomato blocks chloroplast differentiation in leaf mesophyll cells and a signaling system that appears to be required for morphogenesis of palisade cells during leaf growth. To dissect the function of DCL, mutants with stable dcl alleles (dcl-s) were generated and examined for their phenotype. DCL/dcl-s plant produce dcl-s/dcl-s seeds with embryos arrested at the globular stage of development. The levels of several chloroplast- and nuclear-encoded proteins are strongly reduced in dcl-m mutant leaf sectors without significant changes in their corresponding mRNAs. The 4.5S rRNA fails to be processed efficiently, however, suggesting that DCL has a direct or indirect function in rRNA processing or correct ribosome assembly. Accordingly, chloroplasts in dcl-m sectors are impaired in polysome assembly, which can explain the reduced accumulation of chloroplast-encoded proteins. These results suggest that DCL is required for chloroplast rRNA processing, and emphasize the importance of plastid function during embryogenesis.

A seed-specific Brassica napus oleosin promoter interacts with a G-box-specific protein and may be bi-directional

In Brassica napus, oleosins are expressed at high levels in the seed during the latter stages of embryo development. The cis-acting regulatory properties of an 872 bp promoter fragment of a B. napus oleosin gene were examined by analysis of β-glucuronidase (GUS) expression in transgenic tobacco plants containing an oleosin promoter-GUS transcriptional fusion. The reporter gene was expressed at high levels only in seeds, specifically in embryo and endosperm tissue and regulated throughout seed development. These data demonstrate that oleosin gene transcription is regulated in a tissue-specific and temporally regulated manner and clearly indicate that oleosin protein expression is co-ordinated primarily at the transcriptional level. Oleosin mRNA was shown to be abscisic acid (ABA) inducible and an ABA-response element in the oleosin promoter was shown to be bound by a protein factor in a sequence-specific manner. Sequence analysis of the oleosin promoter has identified several other putative cis-acting sequences which may direct oleosin gene expression. The presence of a large open reading frame in the bottom strand of the oleosin promoter (ORF2) which encodes a polypeptide similar to the ethylene-induced E4 gene of tomato is reported. A PCR-generated DNA probe containing the ORF2 sequence hybridised with a 1.4 kb transcript in total RNA extracts of a variety of tissues, including leaves and germinated seed cotyledons. This finding suggests that the oleosin gene promoter directs transcription in both directions. It is the first report of a bi-directional nuclear gene promoter in plants.In Brassica napus, oleosins are expressed at high levels in the seed during the latter stages of embryo development. The cis-acting regulatory properties of an 872 bp promoter fragment of a B. napus oleosin gene were examined by analysis of β-glucuronidase (GUS) expression in transgenic tobacco plants containing an oleosin promoter-GUS transcriptional fusion. The reporter gene was expressed at high levels only in seeds, specifically in embryo and endosperm tissue and regulated throughout seed development. These data demonstrate that oleosin gene transcription is regulated in a tissue-specific and temporally regulated manner and clearly indicate that oleosin protein expression is co-ordinated primarily at the transcriptional level. Oleosin mRNA was shown to be abscisic acid (ABA) inducible and an ABA-response element in the oleosin promoter was shown to be bound by a protein factor in a sequence-specific manner. Sequence analysis of the oleosin promoter has identified several other putative cis-acting sequences which may direct oleosin gene expression. The presence of a large open reading frame in the bottom strand of the oleosin promoter (ORF2) which encodes a polypeptide similar to the ethylene-induced E4 gene of tomato is reported. A PCR-generated DNA probe containing the ORF2 sequence hybridised with a 1.4 kb transcript in total RNA extracts of a variety of tissues, including leaves and germinated seed cotyledons. This finding suggests that the oleosin gene promoter directs transcription in both directions. It is the first report of a bi-directional nuclear gene promoter in plants.

Cloning and characterisation of an oleosin gene from Brassica napus

The sequence of an oleosin gene from Brassica napus has been determined. This gene contains a single intron of 437 bp and encodes a polypeptide of 195 amino acids. The oleosin gene product has an estimated molecular mass of 21.5 kDa and consists of a highly hydrophobic central domain flanked by relatively polar N- and C-terminal domains. The central domain is highly conserved between all oleosins sequenced to date and contains a run of periodically spaced leucine residues similar to that of a leucine-zipper motif. The gene has been shown to be expressed specifically in the embryo, maximally between 9 and 11 weeks after flowering, i.e. during the seed desiccation stage. Two transcriptional start sites have been mapped to -70 and -21 of the ATG and a putative ABA-responsive element and three repeated motifs have been identified in the promoter. These short promoter sequences could correspond to regulatory elements responsible for embryo-specific gene expression. Up to six genes exist in the oleosin gene family.

Sequence of an oleocin cDNA from Brassica napus

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived λgt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 pb and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3′ untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.