Luigi R. Ceci

Effects of a mustard trypsin inhibitor expressed in different plants on three lepidopteran pests

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco, arabidopsis and oilseed rape lines have been evaluated against three different lepidopteran insect pests. 1. Plutella xylostella (L.) larvae were the most sensitive to the ingestion of MTI-2. The inhibitor expressed at high levels in arabidopsis plants caused rapid and complete mortality. High mortality and significantly delayed larval development were also detectable in oilseed rape expressing MTI-2 at lower levels. 2. Mamestra brassicae (L.) larvae were sensitive only at high MTI-2 expression level, as obtained in transgenic tobacco and arabidopsis, whereas no effects were observed for larvae fed on plants showing relatively low expression levels such as those of oilseed rape lines. 3. Feeding bioassays with Spodoptera littoralis (Boisduval) larvae were carried out using the same oilseed rape lines, showing that at these low expression levels no mortality was observed although a delay in larval development did occur. The levels of insect gut proteolytic activities of the larvae still alive at the end of a 7 day feeding bioassay were usually higher than in the controls, but no new proteinases were expressed in any case. The combined results described in this paper demonstrate altogether the relevance of a case-by-case analysis [target insects and proteinase inhibitor (PI) level of expression in planta] in a PI-based strategy for plant protection.

Genome walking in eukaryotes

Genome walking is a molecular procedure for the direct identification of nucleotide sequences from purified genomes. The only requirement is the availability of a known nucleotide sequence from which to start. Several genome walking methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies, including the recent coupling with next generation sequencing technologies. This review focuses on the use of genome walking strategies in several aspects of the study of eukaryotic genomes. In a first part, the analysis of the numerous strategies available is reported. The technical aspects involved in genome walking are particularly intriguing, also because they represent the synthesis of the talent, the fantasy and the intelligence of several scientists. Applications in which genome walking can be employed are systematically examined in the second part of the review, showing the large potentiality of this technique, including not only the simple identification of nucleotide sequences but also the analysis of large collections of mutants obtained from the insertion of DNA of viral origin, transposons and transfer DNA (T‐DNA) constructs. The enormous amount of data obtained indicates that genome walking, with its large range of applicability, multiplicity of strategies and recent developments, will continue to have much to offer for the rapid identification of unknown sequences in several fields of genomic research.

Very rapid phosphorylation kinetics suggest a unique role for Lhcb2 during state transitions in Arabidopsis

Light-harvesting complex II (LHCII) contains three highly homologous chlorophyll-a/b-binding proteins (Lhcb1, Lhcb2 and Lhcb3), which can be assembled into both homo- and heterotrimers. Lhcb1 and Lhcb2 are reversibly phosphorylated by the action of STN7 kinase and PPH1/TAP38 phosphatase in the so-called state-transition process. We have developed antibodies that are specific for the phosphorylated forms of Lhcb1 and Lhcb2. We found that Lhcb2 is more rapidly phosphorylated than Lhcb1: 10 sec of ‘state 2 light’ results in Lhcb2 phosphorylation to 30% of the maximum level. Phosphorylated and non-phosphorylated forms of the proteins showed no difference in electrophoretic mobility and dephosphorylation kinetics did not differ between the two proteins. In state 2, most of the phosphorylated forms of Lhcb1 and Lhcb2 were present in super- and mega-complexes that comprised both photosystem (PS)I and PSII, and the state 2-specific PSI–LHCII complex was highly enriched in the phosphorylated forms of Lhcb2. Our results imply distinct and specific roles for Lhcb1 and Lhcb2 in the regulation of photosynthetic light harvesting.

Characterization of recombinant mustard trypsin inhibitor 2 (MTI2) expressed in Pichia pastoris.

The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C‐terminal amino acids of the precursor form on the inhibitor activity, the C‐terminal precursor and the mature protein were both expressed. A third His‐tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 40–160 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine β‐trypsin compared to the precursor and His‐tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut proteases.

Cystatins, Serpins and other Families of Protease Inhibitors in Plants

Plant protease inhibitors (PIs) are generally small proteins present in high concentrations in storage tissues (tubers and seeds), and to a lower level in leaves. Even if most of them are active against serine and cysteine proteases, PIs active against aspartic proteases and carboxypeptidases have also been identified. Inhibitors of serine proteases are further classifiable in several families on the basis of their structural features. They comprise the families known as Bowman-Birk, Kunitz, Potato I and Potato II, which are the subject of review articles included in this special issue. In the present article we aim to give an overview of other families of plant PIs, active either against serine proteases or other class of proteases, describing their distribution, activity and main structural characteristics.

Conservation of the organization of the mitochondrial nad3 and rps12 genes in evolutionarily distant angiosperms

The organization of the genesnad3 andrps12 has been investigated in the mitochondrial genome of two dicotyledonous plants —Helianthus andMagnolia — and one monocotyledonous plant (Allium). These plants all contain a completerps12 gene downstream of thenad3 gene. This arrangement is thus highly conserved within angiosperms. The two genes are co-transcribed and the transcript is modified at several positions by RNA editing of the C to U-type, thus confirming that both genes encode functional proteins. Some 26, 35 and 27 editing events have been identified in the PCR-derivednad3-rps12 cDNA population from sunflower,Magnolia and onion, respectively. Editing of thenad3-rps12 transcript is thus more extensive inMagnolia than in the other angiosperms so far investigated and radically changes the genomically encoded polypeptide sequence. A novel species-specific codon modification was observed inMagnolia. Several homologous sites show differences in editing pattern among plant species. A C-to-U alteration is also found in the non-coding region separating thenad3 andrps12 genes in sunflower. The PCR-derived cDNA populations from thenad3-rps12 loci analysed were found to be differently edited. In addition the plant species show marked variations in the completeness of RNA editing, with only theMagnolia nad3 mRNA being edited fully.

Analysis of mustard trypsin inhibitor-2 gene expression in response to developmental or environmental induction

Abstract. Transcription analysis of a mustard (Sinapis alba L.) serine proteinase inhibitor gene revealed identical 5′ termini of mRNAs synthesized during seed maturation and chemical or wounding induction. Polyadenylation of mRNAs on multiple or single sites differentiated gene expression, increasing the availability of stable mRNAs during seed maturation compared with chemical and wounding induction. Expression of the β-glucuronidase (GUS)-encoding region of the UidA reporter gene, detected under the control of deleted segments of the region flanking on the 5′ side the mti-2 gene, identified a stretch of about 520 bp essential for gene expression. The presence in this region of two ABRE motifs is relevant for plant response to gene induction. Expression of GUS was detectable under different induction stimuli in several organs such as seedlings and leaves and was active to varying extents in the vascular tissues and meristem.

Evolution of the nad3-rps12 Gene Cluster in Angiosperm Mitochondria: Comparison of Edited and Unedited Sequences

We have analyzed thenad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms.

Location of a single tRNA-His gene on the master chromosome of sunflower mitochondrial DNA

Abstract A highly purified tRNA fraction isolated from sunflower mitochondria was used to probe a restriction fragment library of sunflower mitochondrial DNA. Approximately 20 different clones were detected. The 0.2-kbp HindIII insert of recombinant plasmid pHD500 was sequenced revealing the presence of a tRNA-His gene with 97% similarity to the equivalent of maize mitochondria and chloroplast. The single copy gene appears to be located at least 30 kbp distant from regions of high homology with cpDNA in the mitochondrial genome. This observation and other evidence (analysis of flanking regions and hybridization to cpDNA) suggest that the gene probably does not belong to a chloroplast insertion. The possible expression of the tRNA-His gene is discussed.

A tRNA Val(GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed

A tRNAVal (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNAVal gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNAVal is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNAVal species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.