Mechanical properties of pea starch films associated with xanthan gum and glycerol

The aim of this study was to evaluate the effect of the addition of xanthan gum and glycerol to the starch of green pea with high content of AM (cv. Utrillo) in the preparation of films and their physical characteristics. Filmogenic solution (FS) with different levels of pea starch (3, 4, and 5%), xanthan gum (0, 0.05, and 0.1%), and glycerol (glycerol-starch ratio of 1: 5 w/w) were studied. The FS was obtained by boiling (5 min), followed by autoclaving for 1 h at 120 degrees C. The films were prepared by casting. Films prepared only with pea starch were mechanically resistant when compared to other films, prepared with corn, cassava, rice, and even other pea cultivars (yellow, commercial). The tensile strength of these films is comparable to synthetic films prepared with high-density polyethylene and linear low-density polyethylene. However, they are films of low elasticity when compared to other films, such as rice starch films, and especially when compared to polyethylene films. The increased concentration of starch in the solution increased the puncture force. The increased concentration of glycerol slightly decreased the film crystallinity and interfered in the mechanical properties of the films, causing reduction of the maximum values of tensile strength, strain at break, and puncture force. The plasticizer also caused an increase of elongation at break. Xanthan gum was important to formation of films; however, it did not affect their mechanical properties.

Growth-active gibberellins overcome the very slow shoot growth of Hancornia speciosa, an important fruit tree from the Brazilian ""Cerrado""

Shoot elongation of Hancornia speciosa, an endangered tree from the Brazilian savannah ""Cerrado"", is very slow, thus limiting nursery production of plants. Gibberellins (GAs) A(1), A(3), and A(5), and two inhibitors of GA biosynthesis, trinexapac-ethyl and ancymidol were applied to shoots of Hancornia seedlings. GA(1) and GA(3) significantly stimulated shoot elongation, while GA(5) had no significant effect. Trinexapac-ethyl and ancymidol, both at 100 A mu g per seedling, inhibited shoot elongation up to 45 days after treatment, though the effect was statistically significant only for ancymidol. Somewhat surprisingly, exogenous GA(3) more effectively stimulated shoot elongation in SD-grown plants, than in LD-grown plants. The results from exogenous application of GAs and inhibitors of GA biosynthesis imply that Hancornia shoot growth is controlled by GAs, and that level of endogenous growth-active GAs is likely to be the limiting factor for shoot elongation in Hancornia. Application of GAs thus offer a practical method for nursery production of Hancornia seedlings for outplanting into the field.

SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees.

Glycine metabolism by plant roots and its occurrence in Australian plant communities

Soluble organic nitrogen, including protein and amino acids, was found to be a ubiquitous form of soil N in diverse Australian environments. Fine roots of species representative of these environments were found to be active in the metabolism of glycine. The ability to incorporate [N-15]glycine was widespread among plant species from subantarctic to tropical communities. In species from subantarctic herbfield, subtropical coral cay, subtropical rainforest and wet heathland, [N-15]glycine incorporation ranged from 26 to 45% of (NH4+)-N-15 incorporation and was 2- to 3-fold greater than (NO3-)-N-15 incorporation. Most semiarid mulga and tropical savanna woodland species incorporated [N-15]glycine and (NO3-)-N-15 in similar amounts, 18-26% of (NH4+)-N-15 incorporation. We conclude that the potential to utilise amino acids as N sources is of widespread occurrence in plant communities and is not restricted to those from low temperature regimes or where N mineralisation is limited. Seedlings of Hakea (Proteaceae) were shown to metabolise glycine, with a rapid transfer of N-15 from glycine to serine and other amino compounds. The ability to take up and metabolise glycine was unaffected by the presence of equimolar concentrations of NO3- and NH4+. Isonicotinic acid hydrazide (INH) did not inhibit the transfer of N-15-label from glycine to serine indicating that serine hydroxymethyltransferase was not active in glycine catabolism. In contrast aminooxyacetate (AOA) strongly inhibited transfer of N-15 from glycine to serine and labelling of other amino compounds, suggesting that glycine is metabolised in roots and cluster roots of Hakea via an aminotransferase.

Thylakoid membrane architecture

Confocal scanning laser microscopic observations were made on live chloroplasts in intact cells and on mechanically isolated, intact chloroplasts. Chlorophyll fluorescence was imaged to observe thylakoid membrane architecture. C-3 plant species studied included Spinacia oleracea L., Spathiphyllum sp. Schott, cv. 'Mauna Loa', and Pisum sativum L. C-4 plants were also investigated: Saccharum officinarum L., Sorghum bicolor L. Moench, Zea mays L. and Panicum miliaceum L. Some Spinacia chloroplasts were treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to enhance or sodium dithionite (SD) to reduce the photosystem II fluorescence signal. Confocal microscopy images of C-3 chloroplasts differed from electron microscopy pictures because they showed discrete spots of bright fluorescence with black regions between them. There was no evidence of fluorescence from stroma thylakoids. The thylakoid membrane system at times appeared to be string-like, with brightly fluorescing grana lined up like beads. C-4 bundle sheath chloroplasts were imaged from three different types of C-4 plants. Saccharum and Sorghum bundle sheath chloroplasts showed homogeneous fluorescence and were much dimmer than mesophyll chloroplasts. Zea had rudimentary grana, and dim, homogeneous intergrana fluorescence was visualised. Panicum contained thylakoids similar in appearance and string-like arrangement to mesophyll chloroplasts. Isolated Pisum chloroplasts, treated with a drop of 5 mM MgCl2 showed a thylakoid membrane system which appeared to be unravelling. Spongy mesophyll chloroplasts of Spinacia treated with 5 mM sodium dithionite showed a granal thylakoid system with distinct regions of no fluorescence. A time-series experiment provided evidence of dynamic membrane rearrangements over a period of half an hour.

Long-distance signalling and a mutational analysis of branching in pea

Four ramosus mutants with increased branching at basal and aerial nodes have been used to investigate the genetic regulation of bud outgrowth in Pisum sativum L. (garden pea). Studies of long-distance signalling, xylem sap cytokinin concentrations, shoot auxin level, auxin transport and auxin response are discussed. A model of branching control is presented that encompasses two graft-transmissible signals in addition to auxin and cytokinin. Mutants rms1 through rms4 are not deficient in indole-3-acetic acid (IAA) or in the basipetal transport of this hormone. Three of the four mutants, rms1, rms3 and rms4, have very reduced cytokinin concentrations in xylem sap from roots. This reduction in xylem sap cytokinin concentration appears to be caused by a property of the shoot and may be part of a feedback mechanism induced by an aspect of bud outgrowth. The shoot-to-root feedback signal is unlikely to be auxin itself, as auxin levels and transport are not correlated with xylem sap cytokinin concentrations in various intact and grafted mutant and wild-type plants. Rms1 and Rms2 act in shoot and rootstock to regulate the level or transport of graft-transmissible signals. Various grafting studies and double mutant analyses have associated Rms2 with the regulation of the shoot-to-root feedback signal. Rms1 is associated with a second unknown graft-transmissible signal that is postulated to move in the direction of root-to-shoot. Exogenous auxin appears to interact with both of the signals regulated by Rms1 and Rms2 in the inhibition of branching after decapitation. The action of Rms3 and Rms4 is less apparent at this stage, although both appear to act largely in the shoot.

The shoot controls zeatin riboside export from pea roots. Evidence from the branching mutant rms4

The rms4 mutant of pea (Pisum sativum L.) was used in grafting studies and cytokinin analyses of the root xylem sap to provide evidence that, at least for pea, the shoot can modify the import of cytokinins from the root. The rms4 mutation, which confers a phenotype with increased branching in the shoot, causes a very substantial decrease (down to 40-fold less) in the concentration of zeatin riboside (ZR) in the xylem sap of the roots. Results from grafts between wild-type (WT) and rms4 plants indicate that the concentration of cytokinins in the xylem sap of the roots is determined almost entirely by the genotype of the shoot. WT scions normalize the cytokinin concentration in the sap of rms4 mutant roots, whereas mutant scions cause WT roots to behave like those of self-grafted mutant plants. The mechanism whereby rms4 shoots of pea cause a down-regulation in the export of cytokinins from the roots is unknown at this time. However, our data provide evidence that the shoot transmits a signal to the roots and thereby controls processes involved in the regulation of cytokinin biosynthesis in the root.

Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance.

The rms1 mutant of pea has elevated indole-3-acetic acid levels and reduced root-sap zeatin riboside content but increased branching controlled by graft-transmissible signal(s)

Rms1 is one of the series of five ramosus loci in pea (Pisum sativum L.) in which recessive mutant alleles confer increased branching at basal and aerial vegetative nodes. Shoots of the nonallelic rms1 and rms2 mutants are phenotypically similar in most respects. However, we found an up to 40-fold difference in root-sap zeatin riboside ([9R]Z) concentration between rms1 and rms2 plants. Compared with wild-type (WT) plants, the concentration of [9R]Z in rms1 root sap was very low and the concentration in rms2 root sap was slightly elevated. To our knowledge, the rms1 mutant is therefore the second ramosus mutant (rms4 being the first) to be characterized with low root-sap [9R]Z content. Like rms2, the apical bud and upper nodes of rms1 plants contain elevated indole-3-acetic acid levels compared with WT shoots. Therefore, the rms1 mutant demonstrates that high shoot auxin levels and low root-sap cytokinin levels are not necessarily correlated with increased apical dominance in pea. A graft-transmissible basis of action has been demonstrated for both mutants from reciprocal grafts between mutant and WT plants. Branching was also largely inhibited in rms1 shoots when grafted to rms2 rootstocks, but was not inhibited in rms2 shoots grafted to rms1 rootstocks. These grafting results are discussed, along with the conclusion that hormone-like signals other than auxin and cytokinin are also involved.

Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm

Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the ""Simple"" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the ""Simple"" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the ""Simple"" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the ""Simple"" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species. (c) 2008 Elsevier Inc. All rights reserved.

Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin

The process of cryopreservation impairs sperm cell function, potentially leading to a reduction in fertility. The objectives of the present study were to evaluate the effects that cryopreservation using two different extenders has on sperm motility and mitochondrial function, as well as on the integrity of plasma membranes, acrosomal membranes and chromatin, using practical and objective techniques. The focus of the present study was to identify correlations between alterations in sperm membranes and sperm motility in cryopreserved bovine spermatozoa. Seven ejaculates were collected from eight Simmental bulls (n = 56). After collection, semen volume and concentration were assessed for purposes of dilution. Sperm motility was evaluated subjectively and by computer-assisted semen analysis, morphological characteristics were evaluated by differential interference microscopy, the integrity of plasma and acrosomal membranes, as well as mitochondrial function, were determined using a combination of fluorescent probes containing fluorescein isothiocyanate-Pisum sativum agglutinin, propidium iodide or 5,5`,6,6`-tetrachloro-1,1`,3,3`-tetraethylbenzimidazolearbocyanine iodide. Chromatin integrity was evaluated using the acridine orange technique. The semen was subsequently divided into two aliquots and diluted with one of two extenders (Bioxcell(R) or Botu-Bov(R)), after which both were packaged in 0.5 mL straws and frozen using an automated system. Two straws of semen from each treatment were thawed, and the semen parameters were evaluated as described above. Cryopreservation of sperm reduced motility, damaging plasma and acrosomal membranes, as well as decreasing mitochondrial function. The Botu-Bov(R) extender was more effective in preserving sperm motility and membrane integrity than was the Bioxcell(R) extender. (C) 2007 Elsevier B.V. All rights reserved.

Hormone physiology of pea mutants prevented from flowering by mutations gi or veg1

The veg1 (vegetative) mutant in pea (Pisum sativum L.) does not flower under any circumstances and gi (gigas) mutants remain vegetative under certain conditions. gi plants are deficient in production of floral stimulus, whereas veg1 plants lack a response to floral stimulus. During long days in particular, these non-flowering mutant plants eventually enter a stable compact phase characterised by a large reduction in internode length, small leaves and growth of lateral shoots from the upper-stem (aerial) nodes. The first-order laterals in turn produce second-order laterals and so on in a reiterative pattern. The apical bud is reduced in size but continues active growth. Endogenous hormone measurements and gibberellin application studies with gi-1, gi-2 and veg1 plants indicate that a reduction in gibberellin and perhaps indole-3-acetic acid level may account, at least partially, for the compact aerial shoot phenotype. In the gi-1 mutant, the compact phenotype is rescued by transfer from a 24- to an 8-h photoperiod. We propose that in plants where flowering is prevented by a lack of floral stimulus or an inability to respond, the large reduction in photoperiod gene activity during long days may lead to a reduction in apical sink strength that is manifest in an altered hormone profile and weak apical dominance.

Long-distance signaling and the control of branching in the rms1 mutant of peal

The ramosus (rms) mutation (rms1) of pea (Pisum sativum) causes increased branching through modification of graft-transmissible signal(s) produced in rootstock and shoot. Additional grafting techniques have led us to propose that the novel signal regulated by Rms1 moves acropetally in shoots and acts as a branching inhibitor. Epicotyl interstock grafts showed that wild-type (WT) epicotyls grafted between rms1 scions and rootstocks can revert mutant scions to a WT non-branching phenotype. Mutant scions grafted together with mutant and WT rootstocks did not branch despite a contiguous mutant root-shoot system. The primary action of Rms1 is, therefore, unlikely to be to block transport of a branching stimulus from root to shoot. Rather, Rms1 may influence a long-distance signal that functions, directly or indirectly, as a branching inhibitor. It can be deduced that this signal moves acropetally in shoots because WT rootstocks inhibit branching in rms1 shoots, and although WT scions do not branch when grafted to mutant rootstocks, they do not inhibit branching in rms1 cotyledonary shoots growing from the same rootstocks. The acropetal direction of transport of the Rms1 signal supports previous evidence that the rms1 lesion is not in an auxin biosynthesis or transport pathway. The different branching phenotypes of WT and rms1 shoots growing from the same rms1 rootstock provides further evidence that the shoot has a major role in the regulation of branching and, moreover, that root-exported cytokinin is not the only graft-transmissible signal regulating branching in intact pea plants.

Mutational analysis of branching in pea. Evidence that Rms1 and Rms5 regulate the same novel signal

The fifth increased branching ramosus (rms) mutant, rms5, from pea (Pisum sativum), is described here for phenotype and grafting responses with four other rms mutants. Xylem sap zeatin riboside concentration and shoot auxin levels in rms5 plants have also been compared with rms1 and wild type (WT). Rms1 and Rms5 appear to act closely at the biochemical or cellular level to control branching, because branching was inhibited in reciprocal epicotyl grafts between rms5 or rms1 and WT plants, but not inhibited in reciprocal grafts between rms5 and rmsl seedlings. The weakly transgressive or slightly additive phenotype of the rmsl rms5 double mutant provides further evidence for this interaction. Like rms1, rms5 rootstocks have reduced xylem sap cytokinin concentrations, and rms5 shoots do not appear deficient in indole-3-acetic acid or 4-chloroindole-3-acetic acid. Rms1 and Rms5 are similar in their interaction with other Rms genes. Reciprocal grafting studies with rmsl, rms2, and rms5, together with the fact that root xylem sap cytokinin concentrations are reduced in rms1 and rms5 and elevated in rms2 plants, indicates that Rms1 and Rms5 may control a different pathway than that controlled by Rms2. Our studies indicate that Rms1 and Rms5 may regulate a novel graft-transmissible signal involved in the control of branching.