Enzymatically derived aldouronic acids from Cryptomeria japonica arabinoglucuronoxylan

An arabinoglucuronoxylan was extracted from the holocellulose of sugi (Cryptomeria japonica) wood with 10% KOH and subjected to hydrolysis by partially purified xylanase fraction from a commercial cellulase preparation “Meicelase”. Neutral sugars liberated were analyzed by size exclusion chromatography showing the presence of xylooligosaccharides up to xylohexaose. Aldouronic acids liberated were purified by preparative anion exchange chromatography. Their structures were identified by monosaccharide analysis, comparison of their volume distribution coefficients (Dvs) with those of the authentic samples in anion exchange chromatography and ¹H and ¹³C NMR spectroscopy, resulting in the characterization of eight aldouronic acids including acids consisting of two 4-O-Me-α-D-GlcAp residues and 3-5 D-Xyl residues.

1.

Fr. 1-S1: (aldohexaouronic acid, MeGlcA³Xyl₅), O-β-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)-(1 → 2)]-O-β-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

2.

Fr. 1-S2: (aldopentaouronic acid, MeGlcA³Xyl₄), O-β-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)-(1 → 2)]-O-β-D-Xylp-(1 → 4)-O-β-Xylp-(1 → 4)-D-Xyl

3.

Fr. 2-S1: (aldotetraouronic acid, MeGlcA³Xyl₃), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

4.

Fr. 3-S1: (aldotetraouronic acid, GlcA³Xyl₃), O-(α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-Xylp-(1 → 4)-D-Xyl,

5.

Fr. 4-S1: (aldotriouronic acid, GlcA²Xyl₂), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-D-Xyl

6.

Fr. 4-S2: (MeGlc⁴MeGlcA³Xyl₅), O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

7.

Fr. 6-S1: (MeGlcA⁴MeGlcA³Xyl₄), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-[(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

8.

Fr. 7-S1: (MeGlcA³MeGlc²Xyl₃), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-[(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-D-Xyl

Fr. 4-S2 was a new acidic oligosaccharide. The distribution pattern of these vicinal uronic acid units along the D-xylan chain was discussed.     

Emission of isoprene from salt-stressed Eucalyptus globulus leaves

Eucalyptus spp. are among the highest isoprene emitting plants. In the Mediterranean area these plants are often cultivated along the seashore and cope with recurrent salt stress. Transient salinity may severely but reversibly reduce photosynthesis and stomatal conductance of Eucalyptus globulus leaves but the effect on isoprene emission is not significant. When the stress is relieved, a burst of isoprene emission occurs, simultaneously with the recovery of photosynthetic performance. Later on, photosynthesis, stomatal conductance, and isoprene emission decay, probably because of the onset of leaf senescence. Isoprene emission is not remarkably affected by the stress at different light intensities, CO(2) concentrations, and leaf temperatures. When CO(2) was removed and O(2) was lowered to inhibit both photosynthesis and photorespiration, we found that the residual emission is actually higher in salt-stressed leaves than in controls. This stimulation is particularly evident at high-light intensities and high temperatures. The maximum emission occurs at 40 degrees C in both salt-stressed and control leaves sampled in ambient air and in control leaves sampled in CO(2)-free and low-O(2) air. However, the maximum emission occurs at 45 degrees C in salt-stressed leaves sampled in CO(2)-free and low-O(2) air. Our results suggest the activation of alternative non-photosynthetic pathways of isoprene synthesis in salt-stressed leaves and perhaps in general in leaves exposed to stress conditions. The temperature dependence indicates that this alternative synthesis is also under enzymatic control. If this alternative synthesis still occurs in the chloroplasts, it may involve a thylakoid-bound isoprene synthase.     

New formylated phloroglucinol compounds from Eucalyptus globulus foliage

Two new acylphloroglucinols were isolated from the leaves of Eucalyptus globulus Labill and identified as macrocarpals P (1) and Q (2). Structural elucidations were carried out using conventional 1D and 2D NMR and mass spectrometry together with complementary techniques (UV and IR). Macrocarpal Q was a diastereoisomer of macrocarpal E (3), configuration of which was not precised. Simultaneous isolation of macrocarpals E and Q allowed to determine the configurations of both compounds. The diformylphloroglucinol (4) was also isolated as well as already known compounds grandinol, macrocarpals D, I, L, N, O and am-1.     

蓝桉果实中的五环三萜化学成分

从蓝桉果实乙醇提取物的乙酸乙酯部位分离得到7个五环三萜化合物,经理化和波谱分析鉴定为3β-乙酰基-乌索-11,12-烯-28,13β内酯(1)、桦木酮酸(2)、白桦脂酸(3)、2α-羟基白桦脂酸(4)、2α,3β-二羟基乌苏-12-烯-28-酸(5)、熊果酸(6)、3β-羟基-乌索-11,12-烯-28,13β内酯(7),其中化合物1,4,5和7系首次从该植物中分离得到。     

Somatic embryogenesis in Eucalyptus globulus

Somatic embryos were obtained from 1% of cotyledon pieces and hypocotyls of mature embryos of Eucalyptus globulus Labill. cultured on media containing a high concentration of picloram or IBA. 2,4-D and other synthetic auxins did not yield somatic embryos or embryogenic callus. Somatic embryos arose indirectly via callus, being visible after four months, and directly, where little callus or adventitious root initiation occurred. Somatic embryos, formed directly from explants, were visible within five weeks. Various structural abnormalities of somatic embryos were observed, especially after induction on media containing picloram. Only two out of fifteen somatic embryos showed hypocotyl and radical elongation, but plantlets did not develop further.     

Withanolide A series steroidal lactones from Eucalyptus globulus bark

A new steroidal lactone of the Withanolide A series has been isolated from the supercritical fluid extract of Eucalyptus globulus L. (bark) as a major component (I) along with a known structurally similar steroidal lactone as minor component (II). The structural identification of the new lactone was accomplished by different spectroscopic techniques viz. ¹H and ¹³C NMR, etc. The relative stereochemistry was unequivocally determined from the X-ray crystallography.     

Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill

A heteroxylan was isolated from Eucalyptus globulus wood by extraction of peracetic acid delignified holocellulose with dimethyl sulfoxide. Besides (1-->4)-linked beta-D-xylopyranosyl units of the backbone and short side chains of terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl residues (MeGlcA) in a 1:10 molar ratio, this hemicellulose contained galactosyl and glucosyl units attached at O-2 of MeGlcA originating from rhamnoarabinogalactan and glucan backbones, respectively. About 30% of MeGlcA units were branched at O-2. The O-acetyl-(4-O-methylglucurono)xylan showed an acetylation degree of 0.61, as determined by 1H NMR spectroscopy, and a weight-average molecular weight (M(w)) of about 36 kDa (P=1.05) as revealed from size-exclusion chromatography (SEC) analysis. About half of the beta-D-xylopyranosyl units of the backbone were found as acetylated moieties at O-3 (34 mol%), O-2 (15 mol%) or O-2,3 (6 mol%). Practically, all beta-D-xylopyranosyl units linked at O-2 with MeGlcA residues were 3-O-acetylated (10 mol%).     

Boron requirements for Eucalyptus globulus seedlings

Although boron (B) deficiency limits the productivity of eucalypts in plantations in many parts of the world, the concentrations of foliar B used in the diagnosis of B deficiency vary greatly among studies. There has been a lack of reliable diagnosis standards for B deficiency in Eucalyptus species. Therefore, the present study investigated the relationship between internal and external B concentrations and growth of Eucalyptus globulus, the main commercial temperate eucalypt species. Seedlings were grown in a B-buffered solution culture (Amberlite IRA 743) from 0.03 to 8.35 M B. Boron deficiency symptoms appeared at day 5 in the nutrient solution containing less than 0.27 M B. The external critical B concentrations, estimated for the growth of shoots and roots, were 1.08 and 0.99 M B, respectively. The internal critical B concentration range in the youngest fully expanded leaf (YFEL) for shoot growth was 12–16 mg B kg^–1 dry weight. The internal critical B concentrations estimated in the present study have been successfully used in the diagnosis of B deficiency in E. globulus trees up to three years of age in south-east Asia.     

Nitrogen-Fixing Bacteria in Eucalyptus globulus Plantations

Eucalypt cultivation is an important economic activity worldwide. In Portugal, Eucalyptus globulus plantations account for one-third of the total forested area. The nutritional requirements of this crop have been well studied, and nitrogen (N) is one of the most important elements required for vegetal growth. N dynamics in soils are influenced by microorganisms, such as diazotrophic bacteria (DB) that are responsible for biological nitrogen fixation (BNF), so the aim of this study was to evaluate and identity the main groups of DB in E. globulus plantations. Samples of soil and root systems were collected in winter and summer from three different Portuguese regions (Penafiel, Gavião and Odemira). We observed that DB communities were affected by season, N fertilization and moisture. Furthermore Bradyrhizobium and Burkholderia were the most prevalent genera in these three regions. This is the first study describing the dynamic of these bacteria in E. globulus plantations, and these data will likely contribute to a better understanding of the nutritional requirements of eucalypt cultivation and associated organic matter turnover.     

Genetic control of flowering time in Eucalyptus globulus ssp. globulus

Understanding the factors affecting variation in phenology within a species is important as flowering time constitutes one of the major barriers to gene flow. We studied the genetic and environmental control of flower initiation and anthesis time in E. globulus ssp. globulus. For 5 years, flower initiation and anthesis were monitored in a seed orchard containing clones of 63 genotypes from four different regions of the species’ natural distribution. Anthesis occurred over a long period each year, spanning as much as 9 months in 2008. This variation was under strong genetic control with little genotype by year interaction (broad-sense heritability, Ĥ ² = 0.78 ± 0.04). There were highly significant differences among regions; anthesis occurred earlier for Furneaux and Tasmania than Strzelecki and Otways each year. Surprisingly though, there was little variation in flower initiation time between regions and genotypes, and this was under weak genetic control (Ĥ ² = 0.06 ± 0.05). The average anthesis time in the orchard varied from year to year, and there was evidence that heat sum was a major driver of this environmental variation. Anthesis time is controlled by both genetic and environmental factors, with the responses to each being predictable to some extent, and unrelated to the timing of flower initiation.     

Supercritical carbon dioxide extraction of lipids from Eucalyptus globulus wood

Various typical lipid components of wood extractives have been isolated from Eucalyptus globulus wood by supercritical carbon dioxide modified with methanol. The influence of various extraction parameters on the yield and qualitative composition of the extracts have been studied. The extracts were analyzed by gas chromatography-mass spectrometry and compared with those obtained by Soxhlet extraction with acetone, the standard method for the determination of wood extractives. The qualitative and quantitative results obtained by both methods were in good agreement. The experimental planning to asses the influence of pressure, temperature and percentage of methanol and their interactions on the extraction efficiency was carried out with a factorial design, followed by multiple linear regression algorithm.     

Ellagic acid rhamnosides from the stem bark of Eucalyptus globulus

Four ellagic acid rhamnosides were isolated from the stem bark of Eucalyptus globulus. Their structures have been established on the basis of the analysis of their 1H NMR, 13C NMR, HMBC, IR and MS spectral data. The HMBC data of these compounds were most useful for their structure determinations, with these bring determined to be 3-O-methylellagic acid 3'-O-alpha-rhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-3'-O-acetylrhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-2'-O-acetylrhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-4'-O-acetylrhamnopyranoside, respectively. Their antioxidant activities were evaluated by measuring the inhibition of lipid peroxidation using rat liver microsomes, with IC50 values of 10.0-14.0 microg/ml.     

Effects of flooding on Eucalyptus camaldulensis and Eucalyptus globulus seedlings

Summary Flooding for up to 40 days induced morphological changes and reduced growth of 6-week-old Eucalyptus camaldulensis and Eucalyptus globulus seedlings. However, the specific responses to flooding varied markedly between these species and with duration of flooding. Both species produced abundant adventitious roots that originated near the tap root and original lateral roots, but only E. camaldulensis produced adventitious roots on submerged portions of the stem. Flooding induced leaf epinasty and reduced total dry weight increment of seedling of both species but growth of E. globulus was reduced more. In both species dry weight increment of shoots was reduced more than dry weight increment of roots, reflecting compensatory growth of adventitious roots. Adaptation to flooding appeared to be greater in E. camaldulensis than in E. globulus. the importance of formation of adventitious roots in flooding tolerance is emphasized.Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; CEPEC (Cacao Research Center), Bahia, Brazil; and BMBRAPA (Brazilian Research Institute), Brasilia, Brazil     

Development of SCAR molecular markers for early and late differentiation of Eucalyptus globulus ssp globulus from E. globulus ssp maidenii

The genus Eucalyptus includes over 700 species, some of which are the most widely planted hardwoods worldwide. Each species and subspecies of Eucalyptus present different characteristics regarding its wood quality and yield. This fact makes it very important to work with known species/subspecies so as to optimize handling and conservation of forest resources. Some of them are morphologically very similar, making it difficult to differentiate by simple observation. This is the case with Euclyptus globulus ssp globulus and E. globulus ssp maidenii, which can only be distinguished in the adult tree. These issues can be avoided using well-characterized seeds. This can be quite expensive, especially for the small growers. An alternative approach is to develop simple methods for the differentiation of the two subspecies. In this work, we develop a quick method, based on SCAR molecular markers derived from RAPD molecular markers, for the differentiation of the subspecies of E. globulus, in particular E. globulus ssp globulus and E. globulus ssp maidenii.     

Adventitious bud induction in Eucalyptus globulus Labill

Summary Adventitious buds and shoots of Eucalyptus globulus Labill. (Tasmanian Bluegum) have been regenerated from cotyledons and hypocotyls from mature embryos and seedlings. Adventitious buds, were induced at high frequency with 0.05 μM thidiazuron in combination with 0.2 μM 2,4-dichlorophenoxyacetic acid or 5 μM α-naphthaleneacetic acid. Culture of explants in the dark inhibited bud induction, but up to 86% of cotyledons, longitudinally split just prior to culture, produced adventitious buds, in the light. Development of buds into shoots occurred only at low frequency, after transfer to media containing N⁶-benzylaminopurine.     

Endophytic fungi from flowers, capsules and seeds of Eucalyptus globulus

The main goal of this work was to detect whether Cytospora chrysosperma and Fusicoccum eucalypti are present as endophytes of symptomless hypocotyls, cotyledons, flowers, capsules, peduncles of flowers in order to interpret an earlier finding of their presence in seeds, seedling stems and twigs of E. globulus. Segments from these organs as well as from bark and the xylem from flower peduncles were surface-sterilized and plated on 2% malt-agar. All plates were incubated at 24 degrees C for six weeks or more depending on the growth rate of fungi. C. chrysosperma was asymptomatically present in flowers, capsules, hypocotyls, cotyledons and peduncles. F. eucalypti was isolated from asymptomatic flowers and capsules. It is probable that C. chrysosperma spreads during seed germination colonizing seedling stems through hypocotyl and cotyledon.     

Reduction of generation time in Eucalyptus globulus

The plant growth retardant paclobutrazol was applied by foliar spray to seedlings of Eucalyptus globulus (Labill.) to determine its effects on reproductive activity. It was shown to induce flowering in vegetatively juvenile seedlings and increase reproductive activity in vegetatively adult material. The former case represented a 50% (three year) reduction in generation time for commercial seedlings. Paclobutrazol treatment did not appear to have a negative effect on any aspect of reproductive development, germination of new seed, or early seedling growth, while having a distinct positive effect on reproductive output. This suggests that paclobutrazol may be an effective tool enabling tree breeders to reduce generation time and increase reproductive output in E. globulus. Foliar spraying during autumn was shown to be an effective means and timing of paclobutrazol application, with results evident in the following flowering season. Considerable persistence of paclobutrazol effects was noted in the second season after application. Pot size and growth temperatures also play roles in determining the extent of new reproductive activity each season in E. globulus seedlings.     

Maternal inheritance of mitochondria in Eucalyptus globulus

It is important to verify mitochondrial inheritance in plant species in which mitochondrial DNA (mtDNA) will be used as a source of molecular markers. We used a polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) approach to amplify mitochondrial introns from subunits 1, 4, 5, and 7 of NADH dehydrogenase (nad) and cytochrome oxidase subunit II (cox2) in Eucalyptus globulus. PCR fragments were then either sequenced or cut with restriction enzymes to reveal polymorphism. Sequencing cox2 showed that eucalypts lack the intron between exons 1 and 2. One polymorphism was found in intron 2-3 of nad7 following restriction digests with HphI. Fifty-four F1 progeny from seven families with parents distinguishable in their mitochondrial nad7 were screened to show that mitochondria were maternally inherited in E. globulus. These results constitute the first report of mitochondrial inheritance in the family Myrtaceae.     

Acclimatization of secondary somatic embryos derived plants of Eucalyptus globulus Labill.: an ultrastructural approach

This paper reports the complete process from secondary emblings (SE-derived plants) regeneration to acclimatization of Eucalyptus globulus and describes histocytological changes that occur in leaves from in vitro to ex vitro acclimatization for a 3-month period. After elongation, plants were transferred to pots with sterilized peat:perlite and acclimatized in a phytotron, with progressive reduction of RH and increase of light intensity. Histocytological analyses were performed in fixed material using light microscopy and ultrastructural changes followed by electron microscopy (SEM and TEM). The protocol used allowed the successful acclimatization of the emblings. Plants looked morphologically normal and FCM screening revealed no ploidy or DNA content abnormalities. Histocytological analyses showed significant changes along time, mostly in stomata shape and aperture, starch reserves, chloroplast morphology and mesophyll differentiation. This is the first report concerning emblings acclimatization to ex vitro conditions in Eucalyptus. It was clearly demonstrated that during acclimatization emblings suffered profound changes in leaf morphology in order to successfully adapt to ex vitro conditions.