Effect of water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit,leaves and phloem exudate

Abscisic acid (ABA) was identified by combined gas liquid chromatography-mass spectrometry in sieve-tube exudate collected from the cut stylar ends of white lupin fruit. Water stress caused an increase in ABA levels in leaf, seed and pod tissues and phloem exudate. When compared with levels in extracts of these tissues, the concentration of ABA in sieve-tube sap was very high. It is suggested that ABA is actively transported out of mature leaves in the phloem and this finding is discussed in terms of the ABA balance of the plant.Abbreviations ABA abscisic acid - GLC gas liquid chromatography     

A simple system for determining the phloem mobility of compounds using excised pods of lupin (Lupinus albus L.)

Lupin pods were isolated from the plant and the pedicels placed in solutions of either radiolabeled sucrose, glucose, abscisic acid, or gibberellin A₉ (GA₉). Phloem exudate was collected from the cut stylar ends of the pods and both quantitative and qualitative data were obtained on the activity in the sap. Fed sucrose, abscisic acid, and GA₉ were found in phloem exudate, but labeled glucose was immobile as the monosaccharide and activity in exudate appeared in sucrose.Abbreviations GA gibberellic acid - ABA abscisic acid - TLC thin layer chromatography     

Determination of phytohormones in phloem exudate from tree species by radioimmunoassay

A sensitive and specific radioimmunoassay was used to determine quantitatively four of the most important phytohormones in the phloem exudate from 14 different tree species of 8 genera. For cytokinins and indole-3-acetic acid (IAA) we found higher concentrations than those reported previously for other species. The gibberellin values were of the same order of magnitude as in earlier analyses (with different methods) of tree phloem exudates, but lower than the ones reported for Ricinus. Free abscisic acid (ABA) was found in tree phloem exudates in similar concentrations as before in Yucca or palm phloem exudate, but at considerably lower ones than reported for Ricinus and in Lupinus phloem exudate.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA gibberellic acid     

Androgenic potential in coconut (Cocos nucifera L.)

Conditions for induction of androgenesis in coconut cv. Sri Lanka Tall were studied. Anthers collected from inflorescences at four maturity stages were given heat (38°C) or cold (4°C) pretreatments for 1, 3, 6 and 14 days, either prior to or post inoculation. Three different basal media and different anther densities were also tested. Androgenesis was observed only in anthers collected from inflorescences 3 weeks before splitting (WBS) and after a heat pretreatment at 38°C for 6 days. Modified Eeuwens Y₃ liquid medium supplemented with 100 μM 2,4-dichlorophenoxyacetic acid (2,4-d), 0.1% activated charcoal and 9% sucrose was effective in inducing an androgenic response. The lowest anther density tested, 10 per petri plate, was found to be the optimal density. When androgenic calli or embryos were subcultured to Y₃ medium containing 66 μM 2,4-d, followed by transfer to Y₃ medium without plant growth regulators and finally to Y₃ medium containing 5 μM 6-benzyladenine (BA) and 0.35 μM gibberellic acid (GA₃), plantlets regenerated at a frequency of 7%. Histological study indicated that the calli and embryos originated from the inner tissues of the anthers. Ploidy analysis of calli and embryos showed that they were haploid. This is the first report of successful androgenesis yielding haploid plants from coconut anthers.     

The isolation and some properties of a lectin (Haemagglutinin) from Cucurbita phloem exudate

The occurrence of high haemagglutinating (lectin) activity in phloem exudate from three cucurbit species is reported. The protein responsible for this lectin activity in Cucurbita maxima Duch. has been isolated by cation exchange chromatography on Sepharose and identified by gel electrophoresis. The lectin showed agglutinating activity at concentrations as low as 0.1 g/ml. No sugar, including those transported in the phloem of these species, interacted with agglutination. The lectin could not be extracted from cucurbit seed, but appeared in 5-day old seedlings. The possible role of a lectin in the sieve element is discussed.     

Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

Summary Abscisic acid (ABA) has been implicated as a regulatory factor in plant cold acclimation. In the present work, the cold-acclimation properties of an ABA-deficient mutant (aba) of Arabidopsis thaliana (L.) Heynh. were analyzed. The mutant had apparently lost its capability to cold acclimate: the freezing tolerance of the mutant was not increased by low temperature treatment but stayed at the level of the nonacclimated wild type. The mutational defect could be complemented by the addition of exogenous ABA to the growth medium, restoring freezing tolerance close to the wild-type level. This suggests that ABA might have a central regulatory function in the development of freezing tolerance in plants. Cold acclimation has been previously correlated to the induction of a specific set of proteins that have been suggested to have a role in freezing tolerance. However, these proteins were also induced in the aba mutant by low temperature treatment.     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.     

Dehydration improves cryopreservation of coconut (Cocos nucifera L.)

Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatisation and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut cultivars and is useful for the routine cryopreservation of coconut genetic resources.     

Studies on potassium-magnesium interaction in coconut (Cocos nucifera)

The widespread deficiency of magnesium in coconut acts as a limiting factor to increased production. Two ongoing experiments on potassium-magnesium (K-Mg) interaction in coconut conducted in lateritic gravels (ultisols), in the wet and intermediate agroclimatic zones of Sri Lanka showed significant yield responses (P=0.01) to differential K treatments, in the wet zone, but not in the intermediate zone. Differential Mg treatments, however, did not give rise to yield responses. Leaf and nut water analysis showed significant changes in the concentrations of Na, K and Cl (P=0.001), with a distinct inverse relationship between Na and K when K was applied. Differential Mg applications showed a significant effect only for leaf Mg (P=0.001), in the fourth year of the experiment, in the wet zone. Results indicate the usefulness of nut water analysis as an additional diagnostic tool, for Na, K and Cl.     

Abscisic acid in shoots and roots of Scots pine (Pinus sylvestris L.) seedlings grown in controlled long-day and short-day environments

Scots pine (Pinus sylvestris L.) seedlings grown in nutrient solution in controlled-environment chambers were used. The effects of a shortday (SD, early autumn) treatment on growth and the content of free and alkaline hydrolysable abscisic acid (ABA) in shoots and roots were investigated. The weekly relative growth rates of seedlings grown continuously under long-day (LD, summer) conditions were stable at approx. 0.08 g g^–1 d^–1 between weeks four and eight from germination. Weekly relative growth rates of seedlings transferred to SD conditions decreased rapidly to a then stable level of approx. 0.04 g g^–1 d⁰¹. Shoot elongation ceased within two weeks of SD treatment. The content of both free and alkaline hydrolysable ABA was approx. 40–50% higher in shoots of seedlings grown for five weeks in LD plus one week in SD than in shoots of seedlings grown for five or six weeks in LD. Two additional weeks of SD did not change the free ABA content. Three weeks in simulated late autumn (SD but decreased temperatures) and three weeks in simulated winter (lower light intensity and temperature) further increased the content of free ABA in the shoots. A transfer back to LD conditions reduced the ABA content to a level equal to the level found during the first LD period. The recovery of radioactive ABA at certain times after application ofr[³H] ABA was the same in shoots and roots of LD-grown and SD-treated seedlings.Abbreviations ABA abscisic acid - LD long day(s) - RGR₇ weekly relative growth rates - SD short day(s)     

Abscisic acid and apical dominance in Phaseolus coccineus L.

Abscisic acid (ABA) in lanolin, applied to the internode of decapitated runner bean plants enhances the outgrowth of lateral buds. The optimum concentration of the paste is 10^-5 M. The effect of ABA is counteracted by indoleacetic acid (IAA) but not by gibberellic acid (GA₃). There is no effect when ABA is applied to the apical bud or lateral buds of intact plants. However, 13.2 ng given to the lateral buds of decapitated plants stimulate their growth, whereas higher concentrations are inhibitory. Consequently, ABA enhances growth of lateral buds directly, but only when apical dominance is already weakened. The growth of the decapitated 2^nd internode was not affected by ABA. Radioactivity from [2-¹⁴C] ABA, applied to nonelongating 2^nd internode stumps of decapitated runner bean plants moves to the lateral buds, whereas [1-¹⁴C]IAA-and [³H]GA₁-translocation is much weaker. ABA transport is inhibited if IAA or [³H]GA₁ is applied simultaneously. In elongating internodes [¹⁴C]ABA is almost completely immobile. [¹⁴C]IAA-and [³H]GA₁-translocation is not affected by ABA. The amount of radioactivity from labelled ABA, translocated to the lateral buds, is highest during the early stages of bud outgrowth.Abbreviations ABA 2,4-cis, trans-(+)-abscisic acid - GA gibberellic acid - IAA indoleacetic acid - p.l. plain lanolin     

Linkage mapping and QTL analysis in coconut (Cocos nucifera L.)

Different DNA marker types were used to construct linkage maps in coconut (Cocos nucifera L.; 2n = 32) for the two parents of the cross Malayan Yellow Dwarf (MYD) × Laguna Tall (LAGT). A total of 382 markers was sufficient to generate 16 linkage groups for each parent. The total genome length corresponded to 2226 cM for the LAGT map and 1266 cM for the MYD map with 4–32 markers per linkage group. Common markers allowed the association of 9 linkage groups for the two parents MYD and LAGT. QTL analysis for the trait early germination identified six loci. These QTLs correlate with early flowering and yield, representing characters which are important in coconut breeding. The co-segregation of markers with these QTLs provides the first opportunity for marker-assisted selection in coconut breeding programmes. Received: 22 September 1999 / Accepted: 29 November 1999     

Calcium-binding protein in sieve tube exudate

A calcium-binding macromolecule, with an estimated molecular weight greater than 100,000, was detected in phloem exudate from Cucurbita maxima and related species. The macromolecule was a component of sieve tube sap, rather than a contaminant leached from cell walls or cut parenchyma cells during exudate collection. The protein nature of this macromolecule was deduced from its size, lability, susceptibility to proteolytic digestion, and by the dependence of calcium-binding activity on thiol-protecting agents. This protein is distinct from the major proteins of exudate and does not appear to be related to calmodulin.Abbreviations SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - CBP calcium-binding protein     

Endogenous isoprenoid and aromatic cytokinins in different plant parts of Cocos nucifera (L.)

The present study reports the analyses of both isoprenoid and aromatic cytokinins in the coconut palm by combined high performance liquid chromatography and group specific enzyme immunoassays (HPLC-ELISA). The results showed that the isoprenoid cytokinins were several fold more abundant than the aromatic cytokinins in each of the plant parts analysed: immature inflorescence, shoot apical meristem (SAM), spear leaf and embryo. Within the isoprenoid cytokinins, the most abundant ones by type were the zeatin- (Z-), the isopentenyladenine- (iP-) and the dihydrozeatin- (DHZ-) type in decreasing order for most plant parts studied, and individually, zeatin riboside (ZR) or zeatin riboside-5-monophosphate (ZR5P) depending on the part. In the case of the iP-type cytokinins, the results showed that its 9-glucoside was the most abundant one in most parts. The isoprenoid cytokinin profiles in coconut showed a predominant pattern of 9-conjugation as a major metabolism route for these cytokinins. Analyses also showed the occurrence of the aromatic cytokinin 6-benzylaminopurine (BAP) and its riboside (BAPR), 9-glucoside (BAP9G), and nucleotide (BAPR5P). Their presence in coconut palm was unequivocally identified after permethylation by gas chromatography-mass spectrometry. They were more concentrated in the embryo and in the immature inflorescence than in the other two parts studied, however their concentration in each part was several times lower than that of isoprenoid cytokinins. All four were detected in each of the parts studied. The most abundant ones were BAPR and BAP9G in immature inflorescence; and BAPR in all of the other parts. When all cytokinins analysed are considered, differences between the plant parts studied were found. The zygotic embryos showed the highest content, double that in immature inflorescence, and five times more that in spear leaf and SAM. These differences are even greater when individual cytokinins are compared.     

Wound phloem in transition to bundle phloem in primary roots ofPisum sativum L.

Summary 48 hours after interrupting the root stele ofPisum, wound phloem initiated (proximally or distally to the wound) to reconnect the vascular stumps was found to contain some nucleate wound-sieve elements. At the elongating end of an incomplete wound-sieve tube these elements exhibit a sequence of ultrastructural changes as known from protophloem-sieve tubes. Elongation occurs by the addition of newly divided (wound-) sieve-element/companion-cell complexes. In order to dedifferentiate and assume a new specialization formerly quiescent stelar or cortical cells require at least one (mostly more) preliminary division. Companion cells are consequently obligatory sister cells to wound-sieve elements.By reconstruction using serial sections it could be shown that wound-sieve tubes elongate bidirectionally, starting in an early activated procambial cell of the stele. The elongation is directed by the existence of plasmodesmata, preferably when lying in primary pit fields, and by the plane of preceding divisions. Thus, the developing wound-sieve tube can deviate from the damaged bundle and radiate into the cortex as soon as the plane of the preceding divisions is favourable. In the opposite direction, elongating wound-sieve tubes run parallel to pre-existing phloem traces, thus broading their base at the bundle for the deviating part of the wound-sieve tube. Frequently an individual wound-sieve tube is supplemented at the bundle by a further wound-sieve tube which is partly running parallel to it. Both sieve tubes are interlinked with sieve plates by three-poled sieve elements.Ultrastructurally, the developmental changes of nucleate wound-sieve elements follow the known pattern. In spite of its contrasting origin and odd shape a mature wound-sieve element eventually has the same contents as regular sieve elements: sieve-element plastids, mitochondria, stacked ER and small amounts of P-protein within an electronlucent cytoplasm.     

Sieve-tube exudate from Ricinus communis L. seedlings contains ubiquitin and chaperones

The cut hypocotyl of Ricinus communis L. seedlings exudes phloem sap which contains a characteristic set of proteins (Sakuth et al. 1993, Planta 191, 207–213). These sieve-tube exudate proteins were probed with antibodies to highly conserved proteins, namely ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), Rubisco subunit-binding protein, heat-shock protein (HSP 70), chaperonin GroEL and ubiquitin. Homologous proteins in the sieve-tube exudate were identified with antisera to HSP 70, Rubisco-subunit-binding protein and ubiquitin. Ribulose-1,5-bisphosphate carboxylase-oxygenase, which was present in the tissue, was not detected. Of all the cross-reactive proteins detected, ubiquitin was special because the ubiquitin-to-protein ratio in the sieve-tube exudate was higher than in both the surrounding hypocotyl and in the cotyledonary tissues. Therefore, ubiquitin features properties which favour its transfer into the sieve tubes and which might rely on efficient transport through plasmodesmata. It is assumed that chaperones and ubiquitin are needed for the maintenance of sieve-tube function, e.g. to ensure correct folding of proteins. Their possible involvement in protein translocation through plasmodesmata from companion cells to sieve tubes is discussed.Abbreviations HSP heat-shock protein - Rubisco ribulose1,5-bisphosphate carboxylase-oxygenase - RBP Rubisco-subunit-binding protein - STEP sieve-tube exudate protein This research was supported by a TEMPUS grant European Community, Brüssel to E.K., which enabled the stay of A.P. The authors thank Dr. A. Bachmair (Institut für Botanik, Universität Wien, Austria), Prof. D. Wolf and Dr. A. Finger (Institut für Biochemie, Universität Stuttgart, Germany), Dr. S. Jentsch (Friedrich-Miescher Laboratorium, Max-Planck Institut Tübingen, Germany), Prof. U. Kull (Biologisches Institut, Universität Stuttgart, Germany), and Dr. T. Gatenby (Dupont, Wilmington, Del., USA) for generous supply of antisera used in this study. Improvement of English style was due to D. Schobert-Wiese.     

Abscisic acid levels and metabolism in the leaf epidermal tissue of Tulipa gesneriana L. and Commelina communis L.

We have shown the presence of abscisic acid (ABA) in abaxial epidermal strips taken from Tulipa gesneriana and Commelina communis and that the ABA level rises in the epidermis when leaves are water stressed. ABA levels had risen 50% in the abaxial epidermis of C. communis 30 min after the leaves lost 10% of their fresh weight. Epidermis from both T. gesneriana and C. communis metabolize [¹⁴C]ABA to several products probably including phaseic acid (PA) and dihydrophaseic acid (DPA).Abbreviations ABA abscisic acid - RIA radioimmunoassay - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography     

Specific proteins in the sieve-tube exudate of Ricinus commuais L. seedlings: separation,characterization and in-vivo labelling

Ricinus communis L. seedlings exuded pure phloem sap from the cut hypocotyl for several hours. Throughout the entire exudation period proteins were present in the phloem exudate at a constant concentration ranging from 0.11 to 0.41 mg·ml^–1 depending on the culture conditions and the age of the seedlings. Manipulation of the nutrient supply at the cotyledons after removal of the endosperm did not change the protein concentration in the exudate. Comparison of sieve-tube exudate proteins (STEPs) with soluble proteins extracted from the hypocotyl and the cotyledons showed a unique abundance of small proteins in the exudate, with molecular weights ranging from 10 to 25 kDa. Bands at 18, 19 and 20 kDa were especially dominant. The proteins found transiently in the xylem exudate, which might represent proteins secreted at the wound surface, were different in pattern. Two-dimensional separation of STEPs revealed that more than 100 distinct polypeptides occurred in the sieve-tube exudate, most of them slightly acidic with isoelectric points ranging from 4 to 6 and a few basic ones around 8. [³⁵S]Methionine fed to the cotyledons led to labelling of STEPs, demonstrating their rapid synthesis. It is concluded that there is a continuous synthesis and translocation of specific sieve-tube proteins, whose function is unknown.Abbreviations IEF isoelectric focussing - pI isoelectric point - STEP sieve-tube exudate protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid We wish to thank Pia Großmann and Libuse Badewitz for technical help.     

Relation between seed exudate and host susceptibility in gram (Cicer arietinum L.) toRhizoctonia bataticola

Summary Pre-emergence damping-off of gram in Haryana was determined to be caused primarily byRhizoctonia bataticola. The quantity of soluble carbohydrate exuded from seed of the susceptible variety L-550 is more than five times from the resistant variety BG-203. A third variety G-130 was intermediate, both in amount of carbohydrate in its seed exudate and in the amount of pre-emergence damping-off. No qualitative differences were detected in the exudates from seed of resistant and susceptible varieties. A direct relationship between the amount of soluble carbohydrate exuded by a germinating seed and pre-emergence damping-off caused byR. bataticola was shown in these studies.     

Population structures of Brazilian tall coconut (Cocos nucifera L.) by microsatellite markers

Coconut palms of the Tall group were introduced to Brazil from the Cape Verde Islands in 1553. The present study sought to evaluate the genetic diversity among and within Brazilian Tall coconut populations. Samples were collected of 195 trees from 10 populations. Genetic diversity was accessed by investigating 13 simple sequence repeats (SSR) loci. This provided a total of 68 alleles, ranging from 2 to 13 alleles per locus, with an average of 5.23. The mean values of gene diversity (H_e ) and observed heterozygosity (H_o ) were 0.459 and 0.443, respectively. The genetic differentiation among populations was estimated at θ^P=0.1600and the estimated apparent outcrossing rate was t_a = 0.92. Estimates of genetic distances between the populations varied from 0.034 to 0.390. Genetic distance and the corresponding clustering analysis indicate the formation of two groups. The first consists of the Baía Formosa, Georgino Avelino, and São José do Mipibu populations and the second consists of the Japoatã, Pacatuba, and Praia do Forte populations. The correlation matrix between genetic and geographic distances was positive and significant at a 1% probability. Taken together, our results suggest a spatial structuring of the genetic variability among the populations. Geographically closer populations exhibited greater similarities.