Regenerative Xylem in Inflorescence Stems of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

By inserting entomological needles into the lower parts of young inflorescence stems of three-month-old Arabidopsis thaliana (L.) Heynh var. Colombia plants, we studied the process of regenerative xylem production. Regenerative xylem was formed only in one- to two-day-old inflorescence stems but not in older ones. The regenerative vessels originated from re-differentiation of cortical parenchyma. To characterize the process of regenerative xylem formation, we conducted a histological study from the time of wounding to day 30 after wounding. In the first day after wounding the tissues showed no structural responses except for the wounding itself. After six days, regenerative vessel members were already differentiating in a basipetal pattern, forming a vascular bypass around the wound. Regenerative vessel member formation reached a maximal level on the twelfth day after wounding. Sixteen days after wounding the pith parenchyma started to become loose as if indicating tissue senescence. Altogether, vascular regeneration following wounding in inflorescence stems of Arabidopsis thaliana is similar to that in other dicotyledon plants. These findings provide the basis for the use of Arabidopsis thaliana as a model system to study the genetics, physiology and cell biology of wound healing and regenerative vascular tissue formation.     

The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis

Despite its importance in a variety of plant defense responses, our understanding of how jasmonic acid (JA) functions at the biochemical level is limited. Several amino acid conjugates of JA were tested for their ability to complement the JA-insensitive Arabidopsis thaliana mutant jar1-1. Unlike free JA, JA-Ile inhibited root growth in jar1-1 to the same extent as in the wild type, whereas JA-Val, JA-Leu, and JA-Phe were ineffective inhibitors in both genotypes. Thin-layer chromatography and gas chromatography-mass spectrometry (GC-MS) analysis of products produced in vitro by recombinant JAR1 demonstrated that this enzyme forms JA-amido conjugates with several amino acids, including JA-Ile. JA-Val, -Leu, -Ile, and -Phe were each quantified in Arabidopsis seedlings by GC-MS. JA-Ile was found at 29.6 pmole g(-1) fresh weight (FW) in the wild type but was more than sevenfold lower in two jar1 alleles. JA-Leu, -Val, and -Phe were present at only low levels in both genotypes. Expression of wild-type JAR1 in transgenic jar1-1 plants restored sensitivity to JA and elevated JA-Ile to the same level as in the wild type. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) conjugated to JA was also found in plant tissue at 18.4 pmole g(-1) FW. JA-ACC was determined not be an effective jasmonate root inhibitor, and surprisingly, was twofold higher in the mutants than in the wild type. This suggests that another JA-conjugating enzyme(s) is present in Arabidopsis. Synthesis of JA-ACC might provide a mechanism to coregulate the availability of JA and ACC for conversion to the active hormones JA-Ile and ethylene, respectively. We conclude that JAR1 is a JA-amino synthetase that is required to activate JA for optimal signaling in Arabidopsis. Plant hormone activation by conjugation to amino acids and the enzymes involved in their formation were previously unknown.     

Polymorphic markers <Emphasis Type="Italic">Val762Ala</Emphasis> and <Emphasis Type="Italic">Leu54Phe</Emphasis> of the <Emphasis Type="Italic">ADPRT1</Emphasis> gene associated with chronic glomerulonephritis in Russian patients from Moscow

A comparative analysis of allele and genotype distribution of polymorphic markers Val762Ala and Leu54Phe of ADPRT1 gene encoding poly(ADP-ribose)polymerase1 has been performed in chronic glomerulonephritis patients compared to normal controls. This has shown a significant difference in the ADPRT1 gene polymorphic marker Val762Ala allele and genotype frequency distribution between chronic glomerulonephritis patients and healthy controls (according to Fisher’s exact test). At the same time the allele and genotype frequency for a polymorphic marker Leu54Phe distribution did not show significant difference between these groups. Therefore, we have concluded that the ADPRT1 gene polymorphic marker Val762Ala is associated with the development of chronic glomerulonephritis in Russian patients of the Moscow region.     

Systemic induction of traumatic resin ducts and resin flow in Austrian pine by wounding and inoculation with<Emphasis Type="Italic"> Sphaeropsis sapinea</Emphasis> and<Emphasis Type="Italic"> Diplodia scrobiculata</Emphasis>

The potential role of the resin system in the response of Austrian pine (Pinus nigra Arn.) seedlings to mechanical injury and fungal infection was studied in greenhouse experiments. Anatomical observations were performed on 2-year-old plants wounded at collar level and inoculated with Sphaeropsis sapinea (Fr.: Fr.) Dyko & Sutton in Sutton or Diplodia scrobiculata (J. de Wet, B. Slippers & M. J. Wingfield, sp. nov.; sensu de Wet et al. 2003), two fungal pathogens that cause shoot blight and canker on conifers, and that are characterized by different levels of aggressiveness. Histological examination of host tissue taken from the stem at 0, 8, and 12 cm above the treatment site revealed significant treatment- and time-dependent effects on the course of locally and systemically induced traumatic resin duct (TRD) development. Occurrence of TRDs was observed after 4 days only in seedlings inoculated with D. scrobiculata. At 12 days, TRDs were present also in mock-inoculated controls. No TRDs appeared in seedlings inoculated with S. sapinea. However, S. sapinea caused loss of vacuolar phenolics, severe disruption of cambial tissue and invaded the host xylem quickly and apparently unimpeded, whereas D. scrobiculata was never detected in the host xylem. Five-year-old Austrian pines subjected to the same stem base treatments were used to determine the resin mass flowing from the stem 30 cm above the treatment sites. Wounding and/or inoculation induced a significant, 8.3-fold average increase in systemic resin flow over the untreated trees 3 weeks after basal treatment, suggesting that wounding is the sole prerequisite for systemic induction of resin flow. The results are discussed in the context of current disease resistance models.     

Identification and characterisation of <Emphasis Type="Italic">ssrA</Emphasis> in members of the <Emphasis Type="Italic">Helicobacter</Emphasis> genus

Bacterial ssrA encodes tmRNA that functions both as a tRNA and an mRNA to rescue the stalled ribosome on defective mRNAs. In this study, ssrA was identified in four gastric species of Helicobacters and four enterohepatic species of Helicobacters. The tag peptide of 14 amino acids encoded by ssrA showed a pattern of Val(1)Ala(13) in gastric species, a pattern of Ala(1)Val(13 )in enterohepatic species, in contrast to the pattern of Ala(1)Ala(13) in W. succinogenes and C. jejuni, which are closely related to helicobacters. Phylogenetic analysis and the patterns of the tag peptide suggest that the Helicobacter genus could be separated into two genera. High conservation of ssrA in H. pylori was observed. The annotated ORF HP0784 in H. pylori, which largely overlaps ssrA, is unlikely to be functional. H. pylori ssrA interestingly expressed a large and a small tmRNA molecule.     

Metabolism of polychlorinated dibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxins by cytochrome P450 BM-3 and its mutant

The metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 BM-3 from Bacillus megaterium and a mutant enzyme of it (AL₄V; Ala74Gly, Phe87Val, Leu188Gln triple mutant) was examined. Both purified enzymes metabolized 1-monochloro-, 2,3-dichloro-, and 2,3,7-trichloro-dibenzo-p-dioxin, but not 2,3,7,8-tetrachloro-dibenzo-p-dioxin. The mutant AL ₄V had 2–12 times higher activity than the wild-type P450 BM-3 towards polychlorinated dibenzo-p-dioxins. The products were hydroxylated at an unsubstituted position and/or showing migration of the chloride and were less toxic derivatives with lower than 10% toxicity of the original compounds.Revisions requested 26 August 2004; Revisions received 15 October 2004     

<Emphasis Type="Italic">CDKN2A</Emphasis> and <Emphasis Type="Italic">MC1R</Emphasis> variants found in Cypriot patients diagnosed with cutaneous melanoma

The prevalence of genetic variants associated to cutaneous melanoma (CM) has never been determined within Cypriot melanomas. This study evaluates the frequency of variants in cyclin-dependent kinase inhibitor 2A (CDKN2A) and melanocortin-1 receptor (MC1R) in 32 patients diagnosed with CM. Other characteristics and risk factors were also assessed. CDKN2A p.Ala148Thr was detected in three of 32 patients, while the control group revealed no variations within CDKN2A. MC1R screening in 32 patients revealed the following variations: p.Val60Leu in 11 patients, p.Arg142His in four patients, p.Thr314Thr in one patient, p.Arg160Trp in one patient, p.Val92Met/p.Thr314Thr in one patient and p.Val92Met/p.Arg142His/p.Thr314Thr in one patient. The control group revealed only p.Val60Leu (in 10 of 45 individuals), which is frequently found in general populations. Two unrelated patients carried CDKN2A p.Ala148Thr in combination with MC1R p.Arg142His, suggesting digenic inheritance that may provide evidence of different gene variants acting synergistically to contribute for CM development. This study confirms the presence of CDKN2A and MC1R variants among Cypriot melanomas and supports existing evidence of a role for these variants in susceptibility to melanoma.     

The macromolecule with antimicrobial activity synthesized by <Emphasis Type="Italic">Pseudoalteromonas luteoviolacea</Emphasis> strains is an <Emphasis Type="SmallCaps">l</Emphasis>-amino acid oxidase

Two purple pigmented bacterial strains, CPMOR-1 and CPMOR-2, have been newly isolated from the Mediterranean Sea. 16S RNA sequencing and phenotypic characteristics indicate that they belong to the species Pseudoalteromonas luteoviolacea. The synthesis of macromolecules with antimicrobial activity is a capacity described in many strains of this species although the nature of those macromolecules has not been reported up to now. The search for antimicrobial compounds in the two new strains described in this work shows that they synthesize a macromolecule with antimicrobial activity that can be inhibited by catalase, as it had been described in the type strain P. luteoviolacea NCIMB 1893(T). This work elucidates the nature of such macromolecule as a novel L-amino acid oxidase (LAO) with broad substrate specificity. The enzyme is most active with Met, Gln, Leu, Phe, Glu, and Trp. In growth media containing those amino acids, the hydrogen peroxide generated by the reaction catalyzed by the LAO mediates its antimicrobial activity.