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Vitamin B₆ is an essential nutrient in the human diet derived primarily from plant sources. While it is well established as a cofactor for numerous metabolic enzymes, more recently, vitamin B₆ has been implicated as a potent antioxidant. The de novo vitamin B₆ biosynthesis pathway in plants has recently been unraveled and involves only two proteins, PDX1 and PDX2. To provide more insight into the effect of the compound on plant development and its role as an antioxidant, we have overexpressed the PDX proteins in Arabidopsis, generating lines with considerably higher levels of the vitamin in comparison with other recent attempts to achieve this goal. Interestingly, it was possible to increase the level of only one of the two catalytically active PDX1 proteins at the protein level, providing insight into the mechanism of vitamin B₆ homeostasis in planta. Vitamin B₆ enhanced lines have considerably larger vegetative and floral organs and although delayed in pre-reproductive development, do not have an altered overall morphology. The vitamin was observed to accumulate in seeds and the enhancement of its levels was correlated with an increase in their size and weight. This phenotype is predominantly a consequence of embryo enlargement as reflected by larger cells. Furthermore, plants that overaccumulate the vitamin have an increased tolerance to oxidative stress providing in vivo evidence for the antioxidant functionality of vitamin B₆. In particular, the plants show an increased resistance to paraquat and photoinhibition, and they attenuate the cell death response observed in the conditional flu mutant., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

The active endogenous dTph1 system of the Petunia hybrida mutator line W138 has been used in several forward-genetic mutant screens that were based on visible phenotypes such as flower morphology and color. In contrast, defective symbiotic phosphate (P(i)) transport in mycorrhizal roots of Petunia is a hidden molecular phenotype as the symbiosis between plant roots and fungi takes place below ground, and, while fungal colonization can be visualized histochemically, P(i) transport and the activity of P(i) transporter proteins cannot be assessed visually. Here, we report on a molecular approach in which expression of a mycorrhiza-inducible bi-functional reporter transgene and insertional mutagenesis in Petunia are combined. Bi-directionalization of a mycorrhizal P(i) transporter promoter controlling the expression of two reporter genes encoding firefly luciferase and GUS allows visualization of mycorrhiza-specific P(i) transporter expression. A population of selectable transposon insertion mutants was established by crossing the transgenic reporter line with the mutator W138, from which the P(i)transporter downregulated (ptd1) mutant was identified, which exhibits strongly reduced expression of mycorrhiza-inducible P(i) transporters in mycorrhizal roots.

Vitamin B6 is an essential coenzyme for numerous metabolic enzymes and is a potent antioxidant. In plants, very little is known about its contribution to viability, growth and development. The de novo pathway of vitamin B6 biosynthesis has only been described recently and involves the protein PDX1 (pyridoxal phosphate synthase protein). Arabidopsis thaliana has three homologs of PDX1, two of which, PDX1.1 and PDX1.3, have been demonstrated as functional in vitamin B6 biosynthesis in vitro and by yeast complementation. In this study, we show that the spatial and temporal expression patterns of PDX1.1 and PDX1.3, investigated at the transcript and protein level, largely overlap, but PDX1.3 is more abundant than PDX1.1. Development of single pdx1.1 and pdx1.3 mutants is partially affected, whereas disruption of both genes causes embryo lethality at the globular stage. Detailed examination of the single mutants, in addition to those that only have a single functional copy of either gene, indicates that although these genes are partially redundant in vitamin B6 synthesis, PDX1.3 is more requisite than PDX1.1. Developmental distinctions correlate with the vitamin B6 content. Furthermore, we provide evidence that in addition to being essential for plant growth and development, vitamin B6 also plays a role in stress tolerance and photoprotection of plants.

Solanaceous species are among the >200 000 plant species worldwide forming a mycorrhiza, that is, a root living in symbiosis with soil-borne arbuscular-mycorrhizal (AM) fungi. An important parameter of this symbiosis, which is vital for ecosystem productivity, agriculture, and horticulture, is the transfer of phosphate (Pi) from the AM fungus to the plant, facilitated by plasma membrane-spanning Pi transporter proteins. The first mycorrhiza-specific plant Pi transporter to be identified, was StPT3 from potato [Nature414 (2004) 462]. Here, we describe novel Pi transporters from the solanaceous species tomato, LePT4, and its orthologue StPT4 from potato, both being members of the Pht1 family of plant Pi transporters. Phylogenetic tree analysis demonstrates clustering of both LePT4 and StPT4 with the mycorrhiza-specific Pi transporter from Medicago truncatula [Plant Cell, 14 (2002) 2413] and rice [Proc. Natl Acad. Sci. USA, 99 (2002) 13324], respectively, but not with StPT3, indicating that two non-orthologous mycorrhiza-responsive genes encoding Pi transporters are co-expressed in the Solanaceae. The cloned promoter regions from both genes, LePT4 and StPT4, exhibit a high degree of sequence identity and were shown to direct expression exclusively in colonized cells when fused to the GUS reporter gene, in accordance with the abundance of LePT4 and StPT4 transcripts in mycorrhized roots. Furthermore, extensive sequencing of StPT4-like clones and subsequent expression analysis in potato and tomato revealed the presence of a close paralogue of StPT4 and LePT4, named StPT5 and LePT5, respectively, representing a third Pi transport system in solanaceous species which is upregulated upon AM fungal colonization of roots. Knock out of LePT4 in the tomato cv. MicroTom indicated considerable redundancy between LePT4 and other Pi transporters in tomato.

A cDNA encoding Pht2;1 from potato, a new member of the plant Pht2 gene family of low-affinity orthophosphate (Pi) transporters, was isolated. The expression pattern of the corresponding gene as well as its ortholog from Arabidopsis was analyzed and the encoded proteins were localized in the two plants. Pht2;1 expression is strongly upregulated by light in potato and Arabidopsis leaf tissue. RNA gel blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), promoter/GUS, and protein/green fluorescent protein (GFP) fusion studies, respectively, indicate that the gene is expressed in both auto- and heterotrophic tissues and its encoded protein is localized to the plastids. The similar patterns of Pht2;1 gene regulation in potato and Arabidopsis prompted us to screen publicly available gene expression data from 228 Arabidopsis oligonucleotide microarrays covering 83 different experimental conditions. Modulation of Pht2;1 transcript levels was overall moderate, except for a limited number of experimental conditions where Pht2;1 mRNA concentrations varied between 2- and 3.7-fold. Overall, these analyses suggest involvement of the Pht2;1 protein in cell wall metabolism in young, rapidly growing tissues, independent of other Pi transporters such as the high-affinity Solanum tuberosum Pi transporter 1 (StPT1). Cluster analysis allowed identification of colinear or antiparallel expression profiles of a small set of genes involved in post-translational regulation, and photosynthetic carbon metabolism. These data give clues about the possible biological function of Pht2;1 and shed light on the complex web of interactions in which Pht2;1 could play a role.

12-Oxophytodienoate reductases (OPRs) belong to a family of flavin-dependent oxidoreductases. With two new tomato isoforms reported here, three OPRs have now been characterized in both tomato and Arabidopsis. Only one of these isoforms (OPR3) participates directly in the octadecanoid pathway for jasmonic acid biosynthesis, as only OPR3 reduces the 9S,13S-stereoisomer of 12-oxophytodienoic acid, the biological precursor of jasmonic acid. The subcellular localization of OPRs was analyzed in tomato and Arabidopsis. The OPR3 protein and activity were consistently found in peroxisomes where they co-localize with the enzymes of beta-oxidation which catalyze the final steps in the formation of jasmonic acid. The octadecanoid pathway is thus confined to plastids and peroxisomes and, in contrast to previous assumptions, does not involve the cytosolic compartment. The expression of tomato (Lycopersicon esculentum,Le) OPR3 was analyzed in the context of defense-related genes using a microarray comprising 233 cDNA probes. LeOPR3 was found to be up-regulated after wounding with induction kinetics resembling those of other octadecanoid pathway enzymes. In contrast to the induction of genes for wound response proteins (e.g. proteinase inhibitors), the accumulation of octadecanoid pathway transcripts was found to be more rapid and transient in wounded leaves, but hardly detectable in unwounded, systemic leaves. Consistent with the expression data, OPDA and JA were found to accumulate locally but not systemically in the leaves of wounded tomato plants. The transcriptional activation of the octadecanoid pathway and the accumulation of JA to high levels are, thus not required for the activation of defense gene expression in systemic tissues.

An ABC-transporter of Arabidopsis thaliana exhibiting high sequence similarity to the human (MRP1) and yeast (YCF1) glutathione-conjugate transporters has been analysed and used to complement a cadmium-sensitive yeast mutant (DTY168) that also lacks glutathione-conjugate transport activity. Comparison of the hydrophobicity plots of this A. thaliana MRP-like protein with MRP1 and YCF1 demonstrates that the transmembrane domains are conserved, even at the N-terminus where sequence identity is low. Cadmium resistance is partially restored in the complemented ycf1 mutant, and glutathione-conjugate transport activity can be observed as well. The kinetic properties of the A. thaliana MRP-like protein (AtMRP3) are very similar to those previously described for the vacuolar glutathione-conjugate transporter of barley and mung bean. Furthermore, a hitherto undescribed ATP-dependent transport activity could be correlated with the gene product, i.e. vesicles isolated from the complemented yeast, but not from DTY168 or the wild type, take up the chlorophyll catabolite Bn-NCC-1. The results indicate that the product of the MRP-like gene of A. thaliana is capable of mediating the transport of the two different classes of compounds.

The three aromatic amino acids phenylalanine, tyrosine, and tryptophan are synthesized in the plastids of higher plants. There is, however, biochemical evidence that a cytosolic isoform exists of the enzyme catalysing the first step of that branch of the pathway which is specific for the synthesis of phenylalanine and tyrosine, i.e. chorismate mutase (CM). We now report on the isolation of a cDNA clone encoding a cytosolic CM isozyme from Arabidopsis thaliana that was identified by complementing a CM-deficient Escherichia coli strain. The deduced amino acid sequence of this isozyme was 50% identical to that of a previously isolated plastidic CM, and 41% identical to that of yeast CM. The organ-specific expression patterns of the two CM genes were rather similar, but only the gene encoding the plastidic isozyme was elicitor- and pathogen-inducible. The plastidic CM expressed in E. coli was activated by tryptophan and inhibited by phenylalanine and tyrosine, whereas the cytosolic isozyme was insensitive. The existence of a cytosolic CM isozyme implies that either a cytosolic pathway (partial or complete) for the biosynthesis of phenylalanine and tyrosine exists, or that prephenate, originating from chorismate in the cytosol, is utilized for the synthesis of metabolites other than these two aromatic amino acids.

The bean grp1.8 gene is specifically expressed in vascular tissue. Monomers and multimers of a 28 bp regulatory element of the grp1.8 promoter (vs-1) specifically activated both the -82 CaMV 35S and the -76/grp1.8 minimal promoters in vascular tissue of transgenic tobacco plants. vs-1 partially overlaps with a negative regulatory element in the grp1.8 promoter that is necessary for restriction of gene expression to vascular tissue. Nuclear extracts from tobacco and tomato cells contain a factor that binds to vs-1 in vitro. To study the molecular basis of xylem-specific expression mediated by the vs-1 promoter element, a gene was isolated from tomato encoding a protein that binds to vs-1 in vitro. This protein, designated VSF-1, contains a bZIP motif close to the C-terminus. Mutated vs-1 elements were no longer bound by VSF-1 and also failed to activate the minimal -82 CaMV 35S promoter in vivo. Transient expression of VSF-1 in protoplasts stimulated vs-1 dependent activation of the -76/grp1.8 minimal promoter. Binding studies and use of a polyclonal antiserum against VSF-1 provided further evidence that vs-1 is a potential in vivo target site, as VSF-1 was a part of the observed complex formed between vs-1 and nuclear protein extract. vs-1 does not contain the 5'-ACGT-3' core sequence that is part of known plant bZIP protein binding sites or another palindromic sequence. Based on the unusual binding specificity and a characteristic amino acid sequence in the bZIP domain we propose that VSF-1 and the partially homologous PosF21, a bZIP protein from Arabidopsis, belong to a new family of plant bZIP proteins.

The primary transcript of one of the two chorismate synthase genes (LeCS2) of tomato is differentially processed due to an alternative splicing of the third intron. A novel observation was made when the abundances of the two LeCS2-specific transcripts in different organs were analysed. The ratio of these two transcripts differs in RNA populations from different organs. Possible explanations for this finding and its potential physiological impact for plant metabolism are discussed.

Full-length cDNA clones encoding shikimate kinase (EC 2.7.1.71), an enzyme of the central section of the shikimate pathway, have been isolated from tomato (Lycopersicon esculentum L., cv. UC82b). The open reading frame has the capacity to encode a peptide of 300 amino acids. The in-vitro synthesized peptide catalysed the phosphorylation of shikimate thus confirming the identity of the isolated cDNA clones. The N-terminal portion of the deduced amino acid sequence resembles known chloroplast-specific transit peptides. The existence of such a transit peptide was proven by the uptake of the in-vitro synthesized peptide as well as its processing by isolated chloroplasts. Multiple sites of polyadenylation were observed in shikimate kinase mRNAs. The results of Northern and Southern blot analyses are consistent with the existence of only one shikimate kinase gene per haploid genome in tomato. These results are discussed with respect to the dual pathway hypothesis of the shikimate pathway in higher plants.