The regulation from guaiacyl to syringyl lignin in the differentiating xylem of Robinia pseudoacacia

13C- and deuterium (D)-labeled ferulic acid and sinapic acid ([8-(13)C, 3-OCD3]-ferulic acid and [8-(13)C, 3,5-OCD3]-sinapic acid) were administered to robinia (Robinia pseudoacacia L.) shoots. To estimate the distribution of the label from administrated ferulic or sinapic acid, continuous 50-microm-thick tangential sections cut from the cambium of robinia were subjected to lignin chemical analysis by the DFRC method. Labeled ferulic acid was incorporated into guaiacyl and syringyl lignin. The incorporation of labeled ferulic acid into syringyl units was observed only in the later stage of lignification. Labeled sinapic acid was incorporated into syringyl lignin in the early stage and the later stage of lignification. In general, syringyl lignin was deposited in the later stage of cell wall lignification. Thus, the incorporation of sinapic acid to syringyl lignin in the early stage of lignification was abnormal. Taken together, the aromatic ring-modifying reactions (the conversion from guaiacyl to syringyl moiety, including the hydroxylation and methylation) were more important for the regulation of the sinapyl alcohol biosynthesis than the reducing reactions (the reduction of acids to alcohols) in the differentiating xylem.     

Temperature indirectly affects benthic microalgal diversity by altering effects of top–down but not bottom–up control

Understanding the ecological mechanisms that underlie species diversity decline in response to environmental change has become an urgent objective in current ecological research. Not only direct (lethal) effects on single species but also indirect effects altering biotic interactions between species within and across trophic levels comprise the driving force of ecosystem change. In an experimental marine benthic microalgae–grazer system we tested for indirect effects of moderate temperature change on algal diversity by manipulation of temperature, nutrient supply and grazer density. In our model system warming did not exert indirect effects on microalgal diversity via effects on resource competition. However, moderate warming strengthened consumer control and thereby indirectly affected algal community structure which ultimately resulted in decreased diversity. Only in low temperature and low nutrient regimes did the antagonizing mechanisms of bottom–up and top–down regulation establish a balancing effect on algal diversity within 29 days (corresponding to 15–29 algae generations). Effects of thermal habitat change did not appear before 9–18 algae generations, which points to the relevance of longer‐term experiments and ecological monitoring in order to separate transient biotic responses and subtle changes of community dynamics in consequence to global change.     

The overexpression of AtPrx37, an apoplastic peroxidase, reduces growth in Arabidopsis

Understanding peroxidase function in plants is difficult because of the lack of substrate specificity, the high number of genes and their diversity in structure. In the present study, the relative expression of 22 genes coding putative peroxidases (E.C 1.11.1.x) in Arabidopsis was studied. The relative expression of AtPrx37 showed a correlation with the cessation of growth in rosette leaves as well as with the growth capacity along the flower stem. Using AtPrx37::GUS construction, its expression was associated with the vascular bundles. Furthermore, the overexpression of AtPrx37 under the control of CaMV 35S promoter rendered a dwarf phenotype with smaller plants and delayed development. The plants overexpressing AtPrx37 also showed an increase in the amount of esterified phenolic material associated with their walls. A role in the growth cessation and phenolic cross-linking during lignin deposition is postulated.     

Dimorphic DNA variation in the anionic peroxidase gene AtPrx53 of Arabidopsis thaliana

The DNA polymorphism in the AtPrx53 gene which encodes anionic peroxidase was analyzed in 20 Arabidopsis thaliana accessions. There are two divergent sequence types (Col and Dj-like haplotypes) in the AtPrx53 gene that differ by 2 indel and 16 non-singleton nucleotide polymorphisms including 5 nucleotide polymorphic sites responsible for 4 deduced amino acid replacements. Two of the amino acid substitutions (Phe/Ser180and Asp/Asn270) could be responsible for the difference in electrophoretic mobility of AtPrx53 allozymes. One of them (Phe/Ser180) lies within the hypervariable region, indicating that this amino acid polymorphism is subjected to balancing selection. The revealed difference between deduced allozymes is related to the dimorphism in mobility of three major anionic peroxidase isoforms which according to previously established data encoded by AtPrx53 gene. The haplotype Col which included 12 accessions from three different continents is characterized by faster mobility of three isoforms in comparison with the Dj haplotype represented by eight accessions. There is a significant association between the haplotype and several developmental traits: leaf number, flowering time, main stem height etc. Lines of the Dj haplotype have shorter duration of vegetative stages and flower earlier than most of Col haplotype accessions. The reasons of this association are discussed.     

Peroxidase and not laccase is the enzyme responsible for cell wall lignification in the secondary thickening of xylem vessels inLupinus

Summary The post-exponential growth phase of lupin (Lupinus albus cv. Multolupa) hypocotyls is characterized by a strong deposition of lignins in the primary and secondary walls of the xylem vessels. Coinciding with this phenomenon, there is a clearly peroxidatic activity in both the primary cell walls and the outer-most layers of the secondary thickening of the xylem vessels, as demonstrated by 3,3-diaminobenzidine cytochemistry. This activity was completely inhibited by KCN and the removal of H₂O₂ and was not due to laccase since this enzyme shows an almost total inability to oxidize 3,3-diaminobenzidine both in the presence and in the absence of H₂O₂. The absence of laccase-like activities in cell walls of vascular cells was supported by the fact that cell wall proteins from vascular cells were only capable of oxidizing 3,3-diaminobenzidine and coniferyl alcohol in the presence of H₂O₂. These results support the idea of an exclusive role of peroxidase (and exclude any role for laccase) in lignin formation in the secondary thickening of xylem vessels inLupinus.     

Antisense and chemical suppression of the nonmevalonate pathway affects ent-kaurene biosynthesis in Arabidopsis

Transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) expressing the antisense AtMECT gene, encoding 2- C-methyl- D-erythritol 4-phosphate cytidylyltransferase, were generated to elucidate the physiological role of the nonmevalonate pathway for production of ent-kaurene, the latter being the plastidic precursor of gibberellins. In transformed plants pigmentation and accumulation of ent-kaurene were reduced compared to wild-type plants. Fosmidomycin, an inhibitor of 1-deoxy- D-xylulose 5-phosphate reductoisomerase (DXR), caused a similar depletion of these compounds in transgenic plants. These observations suggest that both AtMECT and DXR are important in the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate and that ent-kaurene is mainly produced through the nonmevalonate pathway in the plastid.     

Structural motifs of syringyl peroxidases predate not only the gymnosperm-angiosperm divergence but also the radiation of tracheophytes

* The most distinctive variation in the monomer composition of lignins in vascular land plants is that found between the two main groups of seed plants. Thus, while gymnosperm lignins are typically composed of guaiacyl (G) units, angiosperm lignins are largely composed of similar levels of G and syringyl (S) units. * However, and contrary to what might be expected, peroxidases isolated from basal (Cycadales and Ginkgoales) and differentially evolved (Coniferales and Gnetales) gymnosperms are also able to oxidize S moieties, and this ability is independent of the presence or absence of S-type units in their lignins. * The results obtained led us to look at the protein database to search for homologies between gymnosperm peroxidases and true eudicot S-peroxidases, such as the Zinnia elegans peroxidase. * The findings showed that certain structural motifs characteristic of eudicot S-peroxidases (certain amino acid sequences and beta-sheet secondary structures) predate the gymnosperm-angiosperm divergence and the radiation of tracheophytes, since they are found not only in peroxidases from basal gymnosperms, ferns and lycopods, but also in peroxidases from the moss Physcomitrella patens (Bryopsida) and the liverwort Marchantia polymorpha (Marchantiopsida), which, as typical of bryophytes, do not have xylem tissue nor lignins.     

The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana

The irregular xylem 2 (irx2) mutant of Arabidopsis thaliana exhibits a cellulose deficiency in the secondary cell wall, which is brought about by a point mutation in the KORRIGAN (KOR) beta,1-4 endoglucanase (beta,1-4 EGase) gene. Measurement of the total crystalline cellulose in the inflorescence stem indicates that the irx2 mutant contains approximately 30% of the level present in the wild type (WT). Fourier-Transform Infra Red (FTIR) analysis, however, indicates that there is no decrease in cellulose in primary cell walls of the cortical and epidermal cells of the stem. KOR expression is correlated with cellulose synthesis and is highly expressed in cells synthesising a secondary cell wall. Co-precipitation experiments, using either an epitope-tagged form of KOR or IRX3 (AtCesA7), suggest that KOR is not an integral part of the cellulose synthase complex. These data are supported by immunolocalisation of KOR that suggests that KOR does not localise to sites of secondary cell wall deposition in the developing xylem. The defect in irx2 plant is consistent with a role for KOR in the later stages of secondary cell wall formation, suggesting a role in processing of the growing microfibrils or release of the cellulose synthase complex.     

The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent (normal) program of lignification

Previous studies have indicated that the Arabidopsis thalianairregular xylem 4 (irx4) mutant is severely lignin-deficient, forming abnormal lignin from aberrant monomers. Studies of lignin structure in dwarfed cinnamoyl CoA reductase (CCR)-downregulated tobacco were also previously reported to incorporate feruloyl tyramine derivatives. The lignin in the Arabidopsis irx4 mutant was re-investigated at 6 weeks and at maturation (9 weeks). Application of (1)H, (13)C, 2D Heteronuclear Multiple Quantum Coherence and 2D Heteronuclear Multiple Bond Coherence spectroscopic analyses to the lignin-enriched isolates from both Arabidopsis wild-type (Ler) and the CCR-irx4 mutant at both developmental stages revealed that only typical guaiacyl/syringyl lignins were formed. For the irx4 mutant, the syringyl content at 6 weeks growth was lower, in accordance with a delayed but coherent program of lignification. At maturation, however, the syringyl/guaiacyl ratio of the irx4 mutant approached that of wild-type. There was no evidence for feruloyl tyramines, or homologues thereof, accumulating as a chemical signature in lignins resulting from CCR mutation. Nor were there any noticeable increases in other phenolic components, such as hydroxycinnamic acids. These findings were further confirmed by application of thioacidolysis, alkaline nitrobenzene oxidation and acetyl bromide analyses. Moreover, in the case of CCR downregulation in tobacco, there were no NMR spectroscopic correlations that demonstrated feruloyl tyramines being incorporated into the lignin biopolymers. This study thus found no evidence that abnormal lignin formation occurs when CCR activity is modulated.     

Waterlogging affects nitrogen transport in the xylem of soybean

Transfer of nodulated and non-nodulated plants grown in vermiculite to hydroponic culture without soil was used to study waterlogging and nitrogen transport in the xylem of soybean. Non-aeration, aeration or aeration with nitrogen gas were used to obtain different levels of oxygen in the culture solutions. Ureides, the principal form of nitrogen transport in nodulated plants, were considerably reduced in waterlogged plants or after transfer to water-culture, especially when not aerated or aerated with nitrogen gas. Aeration of the water-culture following a period of non-aeration allowed some recovery of ureides, as did the return of plants to drained vermiculite. Although smaller changes in the total amino acid fraction were observed for the different treatments, marked changes occurred in the composition depending on the treatment imposed. A high proportion of asparagine and low glutamine characterised non-nodulated plants grown on nitrate, or nodulated plants subsequently fed nitrate. A higher level of glutamine and lower level of asparagine characterised nodulated plants dependent on nitrogen fixation. High levels of aspartic acid characterised plants transferred to water-culture with aeration, especially in N-deficient solution, while alanine and serine were very prominent in non-aerated or hypoxic water-culture. These changes also occurred in non-nodulated plants and plants kept in vermiculite in a flooded condition. Some of the changes in transport were accompanied by similar changes in the free amino acid fraction of the roots. It is suggested that an alteration in asparagine metabolism may underlie the changes in amino acid transport in the xylem associated with waterlogging.     

Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.     

The consistency of element transformations affects the visibility but not the direction of illusory motion

Two experiments tested whether the consistency of element transformations affected perceptions of long-range apparent motion. Vertical lines were used to generate apparent motion against one of two different backgrounds, control and depth. Consistency was manipulated by changing the size of the vertical lines. In some displays, the size of the vertical lines remained constant during movement. In other displays, the size of the vertical lines changed during movement. Consistent movement occurred when the size manipulation was in agreement with the type of background used. In Experiment I, points of subject equality for the quality of motion relative to a standard display were measured. These PSEs indicated that consistent movement (e.g., line sizes held constant for control background displays) was more visible than inconsistent movement (e.g., line sizes constant for depth background displays). In Experiment II, a motion competition display was used to measure thresholds for perceived direction of motion. The depth background was used to make motion in one direction more consistent than motion in the opposite direction. However, no significant differences were noted between thresholds obtained in this condition and those obtained in a control condition. Thus the consistency of element transformations affected the quality of motion, but did not affect the perceived direction of motion. These results are consistent with Ullman's (The Interpretation of Visual Motion, MIT Press, 1979) two-component theory of apparent motion.     

The dioxygenase GIM2 functions in seed germination by altering gibberellin production in Arabidopsis

The phytohormones gibberellic acid (GA) and abscisic acid (ABA) antagonistically control seed germination. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germination ability need further investigation. Here, we identified the ABA‐insensitive gain‐of‐function mutant germination insensitive to ABA mutant 2 (gim2) by screening a population of XVE T‐DNA‐tagged mutant lines. Based on two loss‐of‐function gim2‐ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germination, as observed in transgenic lines overexpressing GIM2 (OE). We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2‐ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA‐ and/or ABA‐related genes were altered in both the gim2‐ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA₁₂. Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis.     

The gut bacterial community affects immunity but not metabolism in a specialist herbivorous butterfly

1. Plant tissues often lack essential nutritive elements and may contain a range of secondary toxic compounds. As nutritional imbalance in food intake may affect the performances of herbivores, the latter have evolved a variety of physiological mechanisms to cope with the challenges of digesting their plant‐based diet. Some of these strategies involve living in association with symbiotic microbes that promote the digestion and detoxification of plant compounds or supply their host with essential nutrients missing from the plant diet. In Lepidoptera, a growing body of evidence has, however, recently challenged the idea that herbivores are nutritionally dependent on their gut microbial community. It is suggested that many of the herbivorous Lepidopteran species may not host a resident microbial community, but rather a transient one, acquired from their environment and diet. Studies directly testing these hypotheses are however scarce and come from an even more limited number of species.
2. By coupling comparative metabarcoding, immune gene expression, and metabolomics analyses with experimental manipulation of the gut microbial community of prediapause larvae of the Glanville fritillary butterfly (Melitaea cinxia, L.), we tested whether the gut microbial community supports early larval growth and survival, or modulates metabolism or immunity during early stages of development.
3. We successfully altered this microbiota through antibiotic treatments and consecutively restored it through fecal transplants from conspecifics. Our study suggests that although the microbiota is involved in the up‐regulation of an antimicrobial peptide, it did not affect the life history traits or the metabolism of early instars larvae.
4. This study confirms the poor impact of the microbiota on diverse life history traits of yet another Lepidoptera species. However, it also suggests that potential eco‐evolutionary host‐symbiont strategies that take place in the gut of herbivorous butterfly hosts might have been disregarded, particularly how the microbiota may affect the host immune system homeostasis.

     

Arabidopsis peroxisomal malate dehydrogenase functions in beta-oxidation but not in the glyoxylate cycle

The aim was to determine the function of peroxisomal NAD⁺-malate dehydrogenase (PMDH) in fatty acid β-oxidation and the glyoxylate cycle in Arabidopsis. Seeds in which both PMDH genes are disrupted by T-DNA insertions germinate, but seedling establishment is dependent on exogenous sugar. Mutant seedlings mobilize their triacylglycerol very slowly and growth is insensitive to 2,4-dichlorophenoxybutyric acid. Thus mutant seedlings are severely impaired in β-oxidation, even though microarray analysis shows that β-oxidation genes are expressed normally. The mutant phenotype was complemented by expression of a cDNA encoding PMDH with either its native peroxisome targeting signal-2 (PTS2) targeting sequence or a heterologous PTS1 sequence. In contrast to the block in β-oxidation in mutant seedlings, [¹⁴C]acetate is readily metabolized into sugars and organic acids, thereby demonstrating normal activity of the glyoxylate cycle. We conclude that PMDH serves to reoxidize NADH produced from fatty acid β-oxidation and does not participate directly in the glyoxylate cycle.