Overexpression of a developing xylem cDNA library in transgenic poplar generates high mutation rate specific to wood formation

We investigated feasibility of the Full‐length complementary DNA OvereXpression (FOX) system as a mutagenesis approach in poplar, using developing xylem tissue. The main goal was to assess the overall mutation rate and if the system will increase instances of mutants affected in traits linked to the xylem tissue. Indeed, we found a high mutation rate of 17.7%, whereas 80% of all mutants were significantly affected in cellulose, lignin and/or hemicellulose. Cell wall biosynthesis is a major process occurring during xylem development. Enrichment of mutants affected in cell wall composition suggests that the tissue source for the FOX library influenced the occurrence of mutants affected in a trait linked to this tissue. Additionally, we found that FLcDNAs from mutants affected in cell wall composition were homologous to genes known to be involved in cell wall biosynthesis and most recovered FLcDNAs corresponded to genes whose native expression was highest in xylem. We characterized in detail a mutant line with increased diameter. The phenotype was caused by a poplar homolog of LONELY GUY 1 (LOG1), which encodes an enzyme in cytokinin biosynthesis and significantly increased xylem proliferation. The causative role of LOG1 in the observed phenotype was further reaffirmed by elevated cytokinin concentration in the mutant and recapitulation overexpression experiment wherein multiple independent lines phenocopied the original FOX mutant. Our experiments show that the FOX approach can be efficiently used for gene discovery and molecular interrogation of traits specific to woody perennial growth and development.     

Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. `Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem–preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one α-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies. Received: 28 June 1999 / Accepted: 3 September 1999     

领春木茎次生木质部中导管穿孔板的变异

领春木Euptelea pleiosperma Hook. f. &; Thoms.隶属领春木科Eupteleaceae。该科为东亚特有的单型科,其系统位置一直颇有争议。本文对中国产领春木茎次生木质部中导管穿孔板的变异进行了观察,以期为它的系统位置提供进一步的解剖学证据。结果表明,领春木茎次生木质部中包括无明显穿孔板的管胞状导管和典型的导管两种类型。在无明显穿孔板的导管中,穿孔中的纹孔膜全部或部分消失,但穿孔无规则排列或聚集,不形成具典型的形态特征的穿孔板;在典型的导管中,穿孔板形态变异较大,包括几个类型:网状穿孔板(含麻黄式穿孔板)、网状和梯状混合型穿孔板、梯状穿孔板、梯状穿孔板向单穿孔板的过渡。在上述导管穿孔板类型中,只有梯状穿孔板的穿孔中可以观察到纹孔膜的残余。在领春木次生木质部中也观察到了端壁多穿孔板及侧壁穿孔板。根据观察结果,我们认为领春木次生木质部导管穿孔板的许多特征说明该科可能处于毛茛目中比较原始的系统位置。     

The regulation of cambial division and secondary xylem differentiation in xanthium by auxins and gibberellin

Cambial division continued in decapitated Xanthium plants without concomitant xylem fiber differentiation. The application of indoleacetic acid to these plants did not affect the production of cambial derivatives or induce xylem fiber differentiation. When naphthaleneacetic acid was applied either to the second internode or to the stump of a lateral shoot, xylem fiber differentiation was induced in the newly formed cambial derivatives on the xylem side of the cambium in the stem. When naphthaleneacetic acid was applied unilaterally, xylem fiber differentiation was restricted to that side of the stem in the first internode and hypocotyl. Naphthaleneacetic acid also enhanced the production of cambial derivatives. Gibberellic acid enhanced cambial derivative production but did not affect the differentiation of xylem fibers. Similar numbers of cambial derivatives were produced in some naphthaleneacetic acid-treated plants in which xylem fiber differentiation was induced and in gibberellic acid-treated plants which did not differentiate xylem. When naphthaleneacetic acid was applied 72 hours after decapitation, the oldest of the cambial derivatives on the xylem side failed to develop into fibers although younger cells did. These results suggest that auxin has its direct effect on the induction of xylem differentiation rather than the induction of divisions prerequisite to differentiation.     

Gravitational and hormonal control in secondary xylem formation of Japanese flowering cherry.

It has been reported that Japanese flowering cherry promotes the formation of tension wood in the upper side of the inclined stem, which induces the negative gravitropism. In the other hand, the plant under simulated micro-gravity conditions reduces the width of the secondary xylem and increases the density of the vessels, which means decreased the density of the fiber cells. The plant under simulated micro-gravity conditions showed that the inhibition of xylem development and the decreased supporting mechanism in the stem. In this study we examined that the effects of auxin and gibberellin in the plant under simulated micro-gravity conditions and inclined stimulus. The gibberellin treatment promotes the secondary xylem development under simulated microgravity conditions and inclined stimulus. The upper side of the inclined stem contents much higher levels of IAA and gibberellin A1(GA1) than the lower side of that.     

Regulatory effect of cytokinin on secondary xylem fiber formation in an in vivo system

The regulatory effect of cytokinin on the formation of secondary xylem fibers was studied in the hypocotyl of young Helianthus annuus L. plants. Positive correlation was found between the kinetin supplied (0.25-0.5 micrograms/gram) to the growth medium and the rate of fiber formation within and between the vascular bundles. Reducing the root originated cytokinin supply, either by root removal or by lowering the transpiration rate, diminished the number of newly formed secondary xylem fibers. This decrease was considerably reversed in the presence of 0.5 microgram/gram kinetin. Early pulse exposure of kinetin had a temporary promoting effect on fiber differentiation at low concentrations and a permanent inhibitory effect at high concentration.     

Tumor susceptibility gene 101 (TSG101) is a novel binding-partner for the class II Rab11-FIPs

The Rab11-FIPs (Rab11-family interacting proteins; henceforth, FIPs) are a family of Rab11a/Rab11b/Rab25 GTPase effector proteins implicated in an assortment of intracellular trafficking processes. Through proteomic screening, we have identified TSG101 (tumor susceptibility gene 101), a component of the ESCRT-I (endosomal sorting complex required for transport) complex, as a novel FIP4-binding protein, which we find can also bind FIP3. We show that α-helical coiled-coil regions of both TSG101 and FIP4 mediate the interaction with the cognate protein, and that point mutations in the coiled-coil regions of both TSG101 and FIP4 abrogate the interaction. We find that expression of TSG101 and FIP4 mutants cause cytokinesis defects, but that the TSG101-FIP4 interaction is not required for localisation of TSG101 to the midbody/Flemming body during abscission. Together, these data suggest functional overlap between Rab11-controlled processes and components of the ESCRT pathway.     

Cell-wall-bound oxidases from tobacco (Nicotiana tabacum) xylem participate in lignin formation

A band of cells closest to the cambium in the xylem of tobacco (Nicotiana tabacum L. cv. Samsun) stems oxidized 2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS), o-dianisidine and syringaldazine in the absence of exogenously added hydrogen peroxide. The oxidation was not prevented by catalase which suggests that the oxidation is not dependent on the production and utilisation of endogenous hydrogen peroxide by cell-wall peroxidases. Cell walls, isolated from tobacco xylem, also oxidized these substrates in the absence of added hydrogen peroxide. The cell walls consumed molecular oxygen whilst oxidizing a range of compounds including coniferyl alcohol. The substrate preference and sensitivity to inhibitors suggest the presence of laccasetype polyphenol oxidases (p-diphenol:O₂ oxidoreductase EC 1.14.18.1) which are covalently bound to the wall. The oxidation of coniferyl alcohol by the xylem cell walls was confirmed by assays based on the disappearance of coniferyl alcohol and was not affected by the presence of 500 units·mi^-1 catalase or Superoxide dismutase. Prolonged incubation of cell walls with coniferyl alcohol led to the production of a yellow-orange water-insoluble material that precipitated with the cell walls. Although a proportion of this material was soluble in methanol, the majority was tightly associated with the cell walls. These coloured cell walls had elevated lignin contents when assayed by the acetyl-bromide method. Fourier transforminfrared spectroscopic analysis of the coloured cell walls indicated that the increased lignin content is due to the deposition of guaiacyl-type lignin. Digestion of the xylem cell walls with Driselase, a mixture of fungal glycases, produced a wall residue that had a dramatically reduced ability to oxidize ABTS in the absence of added H₂O₂. However, oxidase activity could not be detected in the Driselase-solubilized extract, although small amounts of oxidase activity could be recovered from the Driselaseresistant wall residue by extraction in 3 M CaCl₂.Abbreviations ABTS 2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) - dl-DOPA 3-(3,4-dihydroxyphenyl)-alanine - FTIR Fourier transform infra-red - o-D o-dianisidine - o-pD o-phenylenediamine - SYR syringaldazine The authors acknowledge funding from the Scottish Office Agriculture and Food Department. They would like to thank Professor J.R. Hillman for his support, Dr. G.D. Lyon for his help and advice with the oxygen electrode and Mrs F. Carr for lignin determinations.     

The role of introns in repeat protein gene formation

Genes composed of tandem repetitive sequence motifs are abundant in nature and are enriched in eukaryotes. To investigate repeat protein gene formation mechanisms, we have conducted a large-scale analysis of their introns and exons. We find that a wide variety of repeat motifs exhibit a striking conservation of intron position and phase, and are composed of exons that encode one or two complete repeats. These results suggest a simple model of repeat protein gene formation from local duplications. This model is corroborated by amino acid sequence similarity patterns among neighboring repeats from various repeat protein genes. The distribution of one- and two-repeat exons indicates that intron-facilitated repeat motif duplication, in which the start and end points of duplication are located in consecutive intronic regions, significantly exceeds intron-independent duplication. These results suggest that introns have contributed to the greater abundance of repeat protein genes in eukaryotic versus prokaryotic organisms, a conclusion that is supported by taxonomic analysis.