Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria

In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2 was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to the E. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain PDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.     

Functional complementation between the PDX1 vitamin B6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli

The pathway for de novo vitamin B(6) biosynthesis has been characterized in Escherichia coli, however plants, fungi, archaebacteria, and most bacteria utilize an alternative pathway. Two unique genes of the alternative pathway, PDX1 and PDX2, have been described. PDX2 encodes a glutaminase, however the enzymatic function of the product encoded by PDX1 is not known. We conducted reciprocal transformation experiments to determine if there was functional homology between the E. coli pdxA and pdxJ genes and PDX1 of Cercospora nicotianae. Although expression of pdxJ and pdxA in C. nicotianae pdx1 mutants, either separately or together, failed to complement the pyridoxine mutation in this fungus, expression of PDX1 restored pyridoxine prototrophy to the E. coli pdxJ mutant. Expression of PDX1 in the E. coli pdxA mutant restored very limited ability to grow on medium lacking pyridoxine. We conclude that the PDX1 gene of the alternative B(6) pathway encodes a protein responsible for synthesis of the pyridoxine ring.     

Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses

Pyridoxine (vitamin B6) is a cofactor required by numerous enzymes in all cellular organisms. Plants are the major source of vitamin B6 for animals, yet the biosynthesis pathway and the function of vitamin B6 in plants are not well elucidated. In this study, an Arabidopsis pyridoxine synthase gene PDX1 was characterized and its in vivo functions were investigated. The PDX1 gene was expressed in all plant parts examined and its expression level was not significantly regulated by abiotic stress or the phytohormone abscisic acid. In roots, PDX1 was highly expressed in a defined region behind the root tips that undergoes rapid cell division. The PDX1 protein was mainly associated with the plasma membrane and endomembranes, implying a potential involvement of vitamin B6 in membrane function. To reveal the in vivo role of PDX1, Arabidopsis insertional mutants were isolated. Strikingly, the pdx1 knockout mutants were impaired in root growth and early seedling development. The stunted roots resulted from both reduced cell division and elongation. Supplementation of the growth media with pyridoxine or reintroduction of the wild-type PDX1 gene into the mutants completely restored the mutant growth, demonstrating that PDX1 is required for pyridoxine biosynthesis in planta. In addition to the developmental defects, pdx1 mutants are hypersensitive to osmotic stress and oxidative stress. These mutant seedlings had increased peroxidation of membrane lipids following UV treatment. Our study establishes a critical role of vitamin B6 in plant development and stress tolerance and suggests that vitamin B6 may represent a new class of antioxidant in plants.     

Revealing complexity and specificity in the activation of lipase-mediated oxylipin biosynthesis: a specific role of the Nicotiana attenuata GLA1 lipase in the activation of jasmonic acid biosynthesis in leaves and roots

The activation of enzymatic oxylipin biosynthesis upon wounding, herbivory and pathogen attack depends on the biochemical activation of lipases that make polyunsaturated fatty acids (PUFAs) available to lipoxygenases (LOXs). The identity and number of the lipases involved in this process remain controversial and they probably differ among plant species. Analysis of transgenic Nicotiana attenuata plants (ir-gla1) stably reduced in the expression of the NaGLA1 gene showed that this plastidial glycerolipase is a major supplier of trienoic fatty acids for jasmonic acid (JA) biosynthesis in leaves and roots after wounding and simulated herbivory, but not during infection with the oomycete Phytophthora parasitica (var. nicotianae). NaGLA1 was not essential for the developmental control of JA biosynthesis in flowers and for the biosynthesis of C(6) volatiles by the hydroperoxide lyase (HPL) pathway; however, it affected the metabolism of divinyl ethers (DVEs) early during infection with P. parasitica (var. nicotianae) and the accumulation of NaDES1 and NaLOX1 mRNAs. Profiling of lysolipids by LC-MS/MS was consistent with a rapid activation of NaGLA1 and indicated that this lipase utilizes different lipid classes as substrates. The results revealed the complexity and specificity of the regulation of lipase-mediated oxylipin biosynthesis, highlighting the existence of pathway- and stimulus-specific lipases.     

Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.     

Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.     

Electric Signaling and Pin2 Gene Expression on Different Abiotic Stimuli Depend on a Distinct Threshold Level of Endogenous Abscisic Acid in Several Abscisic Acid-Deficient Tomato Mutants

Experiments were performed on three abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) mutants, notabilis, flacca, and sitiens, to investigate the role of ABA and jasmonic acid (JA) in the generation of electrical signals and Pin2 (proteinase inhibitor II) gene expression. We selected these mutants because they contain different levels of endogenous ABA. ABA levels in the mutant sitiens were reduced to 8% of the wild type, in notabilis they were reduced to 47%, and in flacca they were reduced to 21%. In wild-type and notabilis tomato plants the induction of Pin2 gene expression could be elicited by heat treatment, current application, or mechanical wounding. In flacca and sitiens only heat stimulation induced Pin2 gene expression. JA levels in flacca and sitiens plants also accumulated strongly upon heat stimulation but not upon mechanical wounding or current application. Characteristic electrical signals evolved in the wild type and in the notabilis and flacca mutants consisting of a fast action potential and a slow variation potential. However, in sitiens only heat evoked electrical signals; mechanical wounding and current application did not change the membrane potential. In addition, exogenous application of ABA to wild-type tomato plants induced transient changes in membrane potentials, indicating the involvement of ABA in the generation of electrical signals. Our data strongly suggest the presence of a minimum threshold value of ABA within the plant that is essential for the early events in electrical signaling and mediation of Pin2 gene expression upon wounding. In contrast, heat-induced Pin2 gene expression and membrane potential changes were not dependent on the ABA level but, rather, on the accumulation of JA.The plant hormones ABA and JA play a predominant role in the conversion of environmental signals into changes in plant gene expression. An increase in endogenous ABA and JA levels precedes and is involved in Pin2 (proteinase inhibitor II) gene expression upon wounding (Peña-Cortés et al., 1989, 1991, 1995, 1996; Farmer and Ryan, 1992; Farmer et al., 1992). This increase in ABA and JA is not restricted to the tissue damaged directly but can also be detected in the nonwounded, systemically induced tissue (Peña-Cortés et al., 1989; Peña-Cortés and Willmitzer, 1995). The accumulation of ABA and JA have been described for several plant species, including potato, tomato, and tobacco (Sanchez-Serrano et al., 1991; Peña-Cortés and Willmitzer, 1995).Further evidence for the involvement of ABA and JA in wound-induced Pin2 gene expression was provided by a series of experiments in which potato plants were sprayed with ABA or JA and Pin2 mRNA accumulated in the absence of any wounding (Peña-Cortés et al., 1989; Hildmann et al., 1992). Both nonsprayed leaves and leaves that were sprayed directly showed increased Pin2 mRNA levels with a pattern identical to the one described for wounded plants (Peña-Cortés et al., 1988; Peña-Cortés and Willmitzer, 1995). Conclusive evidence for the involvement of ABA in wound-induced Pin2 activation was obtained from mutants impaired in ABA biosynthesis. Consequently, wound induction of Pin2 was not observed in the droopy mutant of potato or the sitiens mutant of tomato (Peña-Cortés et al., 1989). However, in these mutants treatment with ABA caused a return of the accumulation of Pin2 mRNA to levels normally found in wild-type plants upon wounding (Peña-Cortés et al., 1991).Like wounding, the application of electrical current was able to initiate ABA and JA accumulation in wild-type plants but not in ABA-deficient plants (Herde et al., 1996). These results suggested that, like wounding, electrical current requires the presence of ABA for the induction of Pin2 gene expression (Herde et al., 1996). In contrast to wounding and electrical current, burning of leaves activated Pin2 gene expression in sitiens mutants by directly triggering the biosynthesis of JA via an alternative pathway that is independent of endogenous ABA levels (Herde et al., 1996).To determine the endogenous levels of ABA that are sufficient to mediate electrical current-, heat-, and wound- induced Pin2 gene expression via electrical signals, we used several tomato mutants containing progressively reduced levels of ABA. The effects of these attenuated ABA levels on JA content and membrane potentials and the expression pattern of Pin2 genes were analyzed. Analysis of JA content was conducted to confirm the existence of an alternative pathway that is independent of endogenous ABA levels in the different ABA-deficient mutants.     

Enhancement of vitamin B6 levels in seeds through metabolic engineering

As a versatile cofactor for many enzymes catalyzing important biochemical reactions, vitamin B₆ is required for all cellular organisms. In contrast to bacteria, fungi and plants, which have the ability to synthesize vitamin B₆ de novo , animals have to take up the vitamin from their diet. Plants are the major source of vitamin B₆ for animals. The recent identification of vitamin B₆ biosynthetic enzymes PDX1 and PDX2 in plants makes it possible to regulate the biosynthesis of this important vitamin. In this study, we generated Arabidopsis plants overexpressing the PDX1 and/or PDX2 gene and used a liquid chromatography/mass spectrometry/mass spectrometry method to determine the levels of different forms of vitamin B₆ in these transgenic plants. It was found that expression of the PDX genes under control of the CaMV 35S promoter caused only a limited increase in pyridoxine contents in dry seeds but not in shoots or roots. When using the Arabidopsis seed-specific 12S promoter to drive the expression of the PDX genes, the levels of vitamin B₆ increased more than twofold in transgenic plants. Our work demonstrates that it is feasible to enhance vitamin B₆ content in seeds by metabolic engineering.     

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.     

Regulation of the Arabidopsis thaliana vitamin B6 biosynthesis genes by abiotic stress.

Vitamin B(6) (pyridoxine and its vitamers) plays an essential role as a co-factor for enzymatic reactions and has also recently been implicated in defense against cellular oxidative stress. The biosynthetic pathway was thoroughly characterized in Escherichia coli, however most organisms, including plants, utilize an alternate pathway involving two genes, PDX1 and PDX2. Arabidopsis thaliana contains one copy of PDX2, but three full-length copies of PDX1, one each on chromosomes 2, 3, and 5 (referred to as PDX1.1, PDX1.2, and PDX1.3, respectively). Phylogenetic analysis of the PDX1 homologues in A. thaliana showed that PDX1.1 and PDX1.3 clustered with the homologues from the other dicots, whereas PDX1.2 was more divergent, and did not cluster with either the dicots or monocots. Expression analysis using quantitative PCR showed that PDX1.1 and PDX1.3 were highly expressed in A. thaliana rosettes, while PDX1.2 showed only low level expression. All three PDX1 genes and PDX2 were responsive to abiotic stressors including high light, chilling, drought, and ozone, however, the response of PDX1.2 was disparate from that of the other PDX genes, showing a lessened response to high light, chilling, and drought, but an increased response to ozone. Green fluorescent protein fusion studies demonstrated that PDX2 localizes in the nucleus and membranes of cells, consistent with recent published data for PDX1. Insight into regulation of the biosynthetic genes during abiotic stress could have important applications in the development of stress-tolerant crops.     

Three polyphenol oxidases from hybrid poplar are differentially expressed during development and after wounding and elicitor treatment

The expression of three polyphenol oxidase (PPO; EC 1.10.3.1) genes was investigated in hybrid poplar ( Populus trichocarpa  ×  P. deltoides ). PtdPPO1 was previously isolated as a wound- and herbivore-inducible PPO (Constabel et al. Plant Physiol 124: 285–295, 2000), whereas PtdPPO2 and PtdPPO3 are two novel hybrid poplar PPO genes isolated from stem and root tissue, respectively. Sequence analysis revealed that the three PPOs were 60–66% identical at the amino acid level. Using gene-specific probes, the expression patterns of the three PPOs was investigated in various organs at different developmental stages. Under normal growing conditions, PtdPPO1 mRNA was absent from all organs tested, while PtdPPO2 was found to be expressed in mid-veins, petioles, stems and roots. PtdPPO3 was expressed only in roots. PtdPPO1 and PtdPPO2 were induced by mechanical wounding and methyl jasmonate, but in different tissues. Overall, the expression patterns suggest that the three PPO genes may have specialized physiological functions in hybrid poplar.     

Effect of wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in <Emphasis Type="Italic">Artemisia annua</Emphasis>

Abstract-Effects of mechanical wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in Artemisia annua leaves were investigated. HPLC-ELSD analysis indicated that there was a remarkable enhancement of the artemisinin content in 2 h after wounding treatment, and the content reached the maximum value at 4 h (nearly 50% higher than that in the control plants). The expression profile analysis showed that many important genes (HMGR, ADS, CPR, and CYP71AV1) involved in the artemisinin biosynthetic pathway were induced in a short time after wounding treatment. This study indicates that the artemisinin biosynthesis is affected by mechanical wounding. The possible mechanism of the control of gene expression during wounding is discussed.     

Wounding of Arabidopsis leaves causes a powerful but transient protection against Botrytis infection

Physical injury inflicted on living tissue makes it vulnerable to invasion by pathogens. Wounding of Arabidopsis thaliana leaves, however, does not conform to this concept and leads to immunity to Botrytis cinerea , the causal agent of grey mould. In wounded leaves, hyphal growth was strongly inhibited compared to unwounded controls. Wound-induced resistance was not associated with salicylic acid-, jasmonic acid- or ethylene-dependent defence responses. The phytoalexin camalexin was found to be involved in this defence response as camalexin-deficient mutants were not protected after wounding and the B. cinerea strains used here were sensitive to this compound. Wounding alone did not lead to camalexin production but primed its accumulation after inoculation with B. cinerea , further supporting the role of camalexin in wound-induced resistance. In parallel with increased camalexin production, genes involved in the biosynthesis of camalexin were induced faster in wounded and infected plants in comparison with unwounded and infected plants. Glutathione was also found to be required for resistance, as mutants deficient in γ-glutamylcysteine synthetase showed susceptibility to B. cinerea after wounding, indicating that wild-type basal levels of glutathione are required for the wound-induced resistance. Furthermore, expression of the gene encoding glutathione- S -transferase 1 was primed by wounding in leaves inoculated with B. cinerea . In addition, the priming of MAP kinase activity was observed after inoculation of wounded leaves with B . cinerea compared to unwounded inoculated controls. Our results demonstrate how abiotic stress can induce immunity to virulent strains of B. cinerea , a process that involves camalexin and glutathione.     

The extremely conserved pyroA gene of Aspergillus nidulans is required for pyridoxine synthesis and is required indirectly for resistance to photosensitizers.

Numerous disparate studies in plants, filamentous fungi, yeast, Archaea, and bacteria have identified one of the most highly conserved proteins (SNZ family) for which no function was previously defined. Members have been implicated in the stress response of plants and yeast and resistance to singlet oxygen toxicity in the plant pathogen Cercospora. However, it is found in some anaerobic bacteria and is absent in some aerobic bacteria. We have cloned the Aspergillus nidulans homologue (pyroA) of this highly conserved gene and define this gene family as encoding an enzyme specifically required for pyridoxine biosynthesis. This realization has enabled us to define a second pathway for pyridoxine biosynthesis. Some bacteria utilize the pdx pyridoxine biosynthetic pathway defined in Escherichia coli and others utilize the pyroA pathway. However, Eukarya and Archaea exclusively use the pyroA pathway. We also found that pyridoxine is destroyed in the presence of singlet oxygen, helping to explain the connection to singlet oxygen sensitivity defined in Cercospora. These data bring clarity to the previously confusing data on this gene family. However, a new conundrum now exists; why have highly related bacteria evolved with different pathways for pyridoxine biosynthesis?     

The tomato mutant spr1 is defective in systemin perception and the production of a systemic wound signal for defense gene expression

Wound-induced systemic expression of defensive proteinase inhibitor (PI) genes in tomato plants requires the action of systemin and its precursor protein prosystemin. Although it is well established that systemin induces PI expression through the octadecanoid pathway for jasmonic acid (JA) biosynthesis, relatively little is known about how systemin and JA interact to promote long-distance signaling between damaged and undamaged leaves. Here, this question was addressed by characterizing a systemin-insensitive mutant (spr1) that was previously identified as a suppressor of prosystemin-mediated responses. In contrast to JA biosynthetic or JA signaling mutants that lack both local and systemic PI expression in response to wounding, spr1 plants were deficient mainly in the systemic response. Consistent with this phenotype, spr1 plants exhibited normal PI induction in response to oligosaccharide signals that are thought to play a role in the local wound response. Moreover, spr1 abolished JA accumulation in response to exogenous systemin, and reduced JA accumulation in wounded leaves to approximately 57% of wild-type (WT) levels. Analysis of reciprocal grafts between spr1 and WT plants showed that spr1 impedes systemic PI expression by blocking the production of the long-distance wound signal in damaged leaves, rather than inhibiting the recognition of that signal in systemic undamaged leaves. These experiments suggest that Spr1 is involved in a signaling step that couples systemin perception to activation of the octadecanoid pathway, and that systemin acts at or near the site of wounding (i.e. in rootstock tissues) to increase JA synthesis to a level that is required for the systemic response. It was also demonstrated that spr1 plants are not affected in the local or systemic expression of a subset of rapidly induced wound-response genes, indicating the existence of a systemin-independent pathway for wound signaling.     

Jasmonate Signal Pathway in Arabidopsis

Jasmonates （JAs）, which include jasmonic acid and its cyclopentane derivatives are synthesized from the octadecanoid pathway and widely distributed throughout the plant kingdom. JAs modulate the expression of numerous genes and mediate responses to stress, wounding, insect attack, pathogen infection, and UV damage. They also affect a variety of processes in many plant developmental processes. The JA signal pathway involves two important events： the biosynthesis of JA and the transduction of JA signal. Several important Arabidopsis mutants in jasmonate signal pathway were described in this review.