Molecular identification and expression of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family.

Molecular characterization has been accomplished for five members of the peroxidase gene family in French bean. The most important of these, designated FBPI, corresponds to the isoform believed to be responsible for the apoplastic oxidative burst demonstrated by suspension-cultured cells in response to fungal elicitor. Identification was made by a complete match of six peptide sequences derived from the native protein to the translated sequence of the cDNA. Modelling of the surface structure in comparison with two other members of the peroxidase family did not reveal any unusual features which might account for its role in the oxidative burst. However, FBP1 when expressed in Pichia pastoris generated H2O2 using cysteine at pH 7.2, a specific property of the native protein when isolated from suspension-cultured cells. FBP1, together with other members of the family, were all induced in cell cultures by elicitor action although they all showed some expression in non-induced cultured cells. They were also expressed in all tissues examined with varying levels of intensity of detection in northern blots. This was confirmed by in situ hybridization and FBP1 expression was confirmed in tissues where it has been previously detected by immunolocalization methods. Assigning roles to individual peroxidases is an important goal and molecular identification of the oxidative burst peroxidase allows further exploration of the relative roles of the different systems involved in generating reactive oxygen species.

Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco.

A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast.

cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5'-untranslated region and a 140 bp 3'-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with a predicted Mr of 59229 and a pI of 8.8. It contains the conserved cysteine haem-binding site found in all cytochrome P450s. The protein encoded by this cDNA diverges however from other CYP73As in its N- and C-terminus and in four domains internally, so that overall sequence similarity is in the range 58-66%. Many clones contained an identical intron, which may be associated with a novel regulatory mechanism. Sequence similarity is sufficient for it to be classified as CYP73A15, although it is the least similar member of this family classified so far. The cDNA was expressed in yeast. Successful expression of cinnamate 4-hydroxylase activity required removal of the intron. High-level expression also required modification of the N-terminus to that of CYP73A1. Yeast did not process the intron at all and the leader sequence for A15 was not as compatible as that of A1. The mRNA for CYP73A15 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean but induction was more transient than that of phenylalanine ammonia-lyase (PAL). In contrast, induction in cells undergoing xylogenesis was much more coordinate with PAL. The cloned cDNA may represent a cinnamate 4-hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the majority of CYP73As cloned so far are involved.

Molecular cloning of the cDNA encoding a stress-inducible protein phosphatase 1 (PP1) catalytic subunit from French bean (Phaseolus vulgaris L.).

A cDNA showing high sequence similarity (> 70%) to plant protein phosphatase 1 catalytic subunit variants from other species has been isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. The clone appears to be a near full-length 1431 bp with a 172 bp 5'-untranslated region and a 317 bp 3'-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with predicted M(r) of 35,552. Alternatively an ATG situated to the 5' end of the putative start site would increase the protein size by 6 amino acids. The mRNA for Pvpp1 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean. The cloned cDNA represents one of the few examples of a gene product that is probably involved in dephosphorylation events arising after the initial responses to biotic stress.

Rapid changes in oxidative metabolism as a consequence of elicitor treatment of suspension-cultured cells of French bean (Phaseolus vulgaris L.).

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H2O2. The cells recover and start producing ATP immediately of H2O2 production. This indicates that the increased O2 uptake is primarily incorporated into active O2 species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M(r) 40,000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.

Cellular basis of the effects of gibberellin and the pro gene on stem growth in tomato.

Tomato (Lycopersicon esculentum Mill.) plants homozygous for the mutant pro gene, exhibiting the distinctive procera phenotype, appeared virtually identical to gibberellic acid (GA3)-treated isogenic normal plants. The pro gene and GA3 caused analogous increases in internode length, and in the length and number of cells in the outer cell layers of each internode. Internode number was also increased by pro and GA3 over the period of the experiment. Despite their greater length, the internodes of GA3-treated and pro plants reached their final size within a time period similar to that of internodes of untreated normal plants. The pro mutant itself was responsive to GA3, especially in the seedling stage, but the proportional increase in height seen in the later stages of growth was less than that of normal plants.