Phloem transport of amino acids in two Brassica napus L. genotypes and one B. carinata genotype in relation to their seed protein content

 In order to investigate the relationship between the amino acid concentration in the phloem sap of leaves and the protein content in seeds, two Brassica napus genotypes and one B. carinata genotype with low, medium and high seed protein contents were analyzed. Phloem sap was collected from the B. napus winter rapeseed breeding line DSV15 with 19% protein of dry weight in the seeds, the spring cultivar ‘Duplo’ with 25% protein in the seeds and from the B. carinata line BRA1151/90 with 39% protein in the seeds by using the aphid-stylet technique. The total amino acid contents measured in the phloem varied considerably among the three genotypes analysed, and correlated positively with their respective seed protein contents. The total amino acid-to-sucrose ratio was lowest in B. napus line DSV15 which had the lowest seed protein content and highest in the B. carinata line BRA1151/90 which had the highest seed protein content. The amino-N translocation in the phloem during the light period was about 2-fold higher in the B. carinata line BRA1151/90 than in the B. napus lines Dulpo and DSV15. Predominant amino acids in the phloem were glutamine and glutamate, followed by serine, aspartate, and threonine. The amino acid patterns in the leaves resembled those in the phloem, although their absolute concentrations were higher in the phloem than in the cytosol of mesophyll tissue. Furthermore, the concentration gradient of amino acids between the cytosol of mesophyll cells and the phloem was higher in the B. carinata line BRA1151/90 than in the B. napus lines Duplo and DSV15. These results lead to the conclusion that the phloem translocation of amino-N and the phloem loading process of amino acids are decisive factors for the protein content in the seeds of Brassica species. Received: 28 November 1999 / Accepted: 10 April 2000     

Expression of GFP and Bt transgenes in Brassica napus and hybridization with Brassica rapa

It is possible to monitor the movement of transgenes by tagging them with green fluorescent protein (GFP). In order to develop a model to study transgene flow, canola (Brassica napus cv Westar) was transformed with two GFP constructs, mGFP5er (GFP only) and pSAM 12 [GFP linked to a synthetic Bacillus thuringiensis (Bt) cry1Ac endotoxin gene]. Transformed callus sectors that fluoresced green were preferentially selected in the tissue culture process. Four independent GFP canola events and 12 events of GFP/Bt canola were regenerated through tissue culture. GFP fluorescence was macroscopically detectable throughout the entire life cycle of canola. The GFP/Bt events were insecticidal to neonate corn earworm (Helicoverpa zea) larvae and prevented herbivory damage. Fluorescence intensity at 508 nm varied between the independent transformation events, and ranged from 7.6×10⁵ to 13.8×10⁵ (counts per second) in contrast with the wild-type at 5.3×10⁵ cps. Nine GFP/Bt and three GFP events were hybridized with three wild accessions of B. rapa. The resultant hybrids fluoresced green and were insecticidal to neonate corn earworm larvae to the same degree as the transgenic canola parents. However, fluorescence intensities of the hemizygous F₁ hybrid lines were lower than the respective original homozygous canola parents. Each F₁ hybrid line was backcrossed by hand onto the B. rapa parent, and transgenic backcrosses were produced at rates ranging from 15% to 34%. These data suggest that GFP can be used as a tool to monitor transgene flow from crop species to wild relatives. Received: 11 September 2000 / Accepted: 1 February 2001     

Morphogenesis in cucumber seedlings is negatively controlled by gravity

 Seedlings of most cucurbitaceous plants develop a peg (protuberance caused by cell outgrowth) on the transition zone between the hypocotyl and root. The peg is necessary for removing the seed coat after germination. In our spaceflight experiments on the STS-95 space shuttle, Discovery, we found that cucumber (Cucumis sativus L.) seedlings grown under microgravity conditions developed two pegs symmetrically at the transition zone. Thus, cucumber seedlings potentially develop two pegs and do not require gravity for peg formation itself, but on the ground the development of one peg is suppressed in response to gravity. This may be considered as negative control of morphogenesis by gravity. Received: 17 August 1999 / Accepted: 4 October 1999     

Physical distances between S-locus genes in various S haplotypes of Brassica rapa and B. oleracea

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B. oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes. Received: 20 September 1999 / Accepted: 8 October 1999     

Arginine degradation by arginase in mitochondria of soybean seedling cotyledons

 Arginase (EC 3.5.3.1) localization was studied in soybean (Glycine max L.) seedling cotyledons. Subcellular fractionation in a discontinuous Percoll gradient showed that arginase was localized in the mitochondrion. Arginine (Arg) uptake by mitochondria was demonstrated by co-sedimentation of [³H]Arg-derived label and the mitochondrial marker enzyme cytochrome c oxidase. Arginine uptake was complete in about 10 min. Since detergent but not NaCl released most label, we conclude that Arg was taken up and not bound to the organellar surface. Arginine transport was not saturable, at least up to 20 mM. Basic amino acids were the best inhibitors of Arg uptake. The uncoupler 2,4-dinitrophenol did not inhibit Arg uptake. At least 30% of l-[guanido-¹⁴C]Arg taken up by mitochondria was degraded by arginase in seedling cotyledons, while little or no degradation was detected in mitochondria from developing embryos, even though the Arg uptake level was similar in both mitochondrial preparations. These results are consistent with our previously reported pattern of arginase expression and urea accumulation during embryo development and seed germination (A. Goldraij and J.C. Polacco, 1999, Plant Physiol. 119: 297–303). The lack of Arg degradation allows developing embryos to conserve Arg, the main N-reserve amino acid utilized by germinating soybean. Received: 7 July 1999 / Accepted: 21 September 1999     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

Control of gravimorphogenesis by auxin: accumulation pattern of CS-IAA1 mRNA in cucumber seedlings grown in space and on the ground

Cucumber (Cucumis sativus L.) seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave side of the gravitropically bending transition zone. The morphological features of the space-grown seedlings were similar to those of seedlings grown in a vertical position on the ground with their radicles pointing down: both became two-pegged seedlings. Morphogenesis of cucumber seedlings is thus inhibited by gravity. Analysis by in-situ hybridization of an auxin-inducible gene, CS-IAA1, showed that its mRNA accumulated to a much greater extent on the lower side of the transition zone in the horizontally placed seedlings on the ground just prior to and during the initiation period of peg formation. On the other hand, when seedlings were grown in microgravity or in a vertical position on the ground, accumulation of CS-IAA1 mRNA occurred all around the transition zone. Accumulation of CS-IAA1 mRNA in horizontally grown seedlings appreciably decreased on the upper side of the transition zone and increased on the lower side upon gravistimulation, compared with the two-pegged seedlings. Application of IAA to seedlings in a horizontal position caused the development of a peg on each side of the transition zone, or a collar-like protuberance, depending on the concentration used. These results suggest that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold value necessary for peg formation. Space-grown seedlings of cucumber might develop two pegs symmetrically because the auxin level in the entire transition zone is maintained above the threshold. This spaceflight experiment verified for the first time that auxin does not redistribute in microgravity. Received: 10 February 2000 / Accepted: 15 March 2000     

Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins

The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel β-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa     

Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives

 The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. × P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours. Received: 12 June 1999 / Accepted: 20 October 1999     

The Lupinus albus class-III chitinase gene, IF3, is constitutively expressed in vegetative organs and developing seeds

 A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants). Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related (PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against fungal infection, though our results do not exclude other functions for this protein. Received: 15 March 1999 / Accepted: 12 July 1999     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

Crossability of Brassica tournefortii and B. rapa, and morphology and cytology of their F1 hybrids

The crossability between Brassica tournefortii (TT, 2n = 20) and Brassica rapa (AA, 2n = 20) and the cytomorphology of their F₁ hybrids were studied. Hybrids between these two species were obtained only when B. tournefortii was involved as a female parent. The hybrid plants were intermediate for most of the morphological attributes and were found to be free from white rust under field conditions. The F₁ plants showed poor pollen fertility, although occasional seed set was achieved from open pollination. Self-pollination or backcrosses did not yield any seeds in these plants. The occurrence of chromosome association ranging from bivalents (0–7), trivalents (0–2) to a rare quadrivalent (0–1) in the dihaploid hybrids indicates pairing between the T and A genomes. The homoeologous pairing coupled with seed set in the F₁ plants offer an opportunity for interspecific gene transfers from B. tournefortii to B. rapa and vice-versa through interspecific hybridization. Received: 3 July 2000 / Accepted: 22 September 2000     

Analytical electron microscopical investigations on the apoplastic pathways of lanthanum transport in barley roots

 In transmission electron microscopy studies, lanthanum ions have been used as electron-opaque tracers to delineate the apoplastic pathways for ion transport in barley (Hordeum vulgare L.) roots. To localize La³⁺ on the subcellular level, e.g. in cell walls and on the surface of membranes, electron-energy-loss spectroscopy and electron-spectroscopic imaging were used. Seminal and nodal roots were exposed for 30 min to 1 mM LaCl₃ and 10 mM LaCl₃, respectively. In seminal roots, possessing no exodermis, La³⁺ diffusion through the apoplast was stopped by the Casparian bands of the endodermis. In nodal roots with an exodermis, however, La³⁺ diffusion through the cortical apoplast had already stopped at the tight junctions of the exodermal cell walls resembling the Casparian bands of the endodermis. Therefore, we conclude that in some specialized roots such as the nodal roots of barley, the physiological role of the endodermis is largely performed by the exodermis. Received: 28 July 1999 / Accepted: 24 February 2000     

Fast activation of a time-dependent outward current in protoplasts derived from coats of developing Phaseolus vulgaris seeds

An outward current that appeared to activate instantaneously in response to depolarising voltage pulses at low sampling frequencies predominated in the plasma membrane of ground-parenchyma protoplasts derived from coats of developing Phaseolus vulgaris L. (cv. Redland Pioneer) seeds. However, the outward current showed time-dependent activation when higher sampling frequencies were used to measure the current. Activation of the current was best described as a double-exponential time course with the fast and slow time constants being 1 and 20 ms, respectively. The current also exhibited a rapid deactivation that followed a double-exponential time course with time constants of approximately 2 and 30 ms, respectively. “Tail-current” analysis allowed us to show that this current exhibited a low selectivity between K⁺ and Cl⁻ (P _K:Cl=1.8). Such a fast-activating current may account for some of the reports of time-independent, instantaneous currents that have been observed in plasma membranes of plant cells digitised at low sampling frequencies. Therefore, when “instantaneous” currents appear it is advisable to characterise these currents using higher sampling frequencies with correspondingly higher filtering frequency cut-offs. Received: 12 May 2000 / Accepted: 26 June 2000     

Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study

In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalancho? species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.