Morphological studies in the tribe Colletieae (Rhamnaceae): analysis of architecture and inflorescences.

A morphological study of vegetative and reproductive structures was carried out on the tribe Colletieae (Rhamnaceae) composed of six genera and 20 species. The plant habit variation, from trees (up to 8 m high) to prostrate shrubs and tangled bushes, suggests structural differences within the group. All the species have an architecture represented by a sequence of monopodial shoots with limited growth, linked sympodially and with acrotonic dichasial ramification. Foliage leaves are decussate and an axillary complex with two serial meristems is associated with each leaf axil. Ancillary shoots (thorn-tipped branches, short shoots, secondary branches and reproductive shoots) are present at the nodes. A single structural plan was observed throughout the tribe. Most of the variability may be ascribed to different strategies for adaptation to environmental conditions, specially xeromorphy. Reproductive structures, in terms of inflorescences, also show conspicuous variation throughout the tribe. Three different models are described; in the first two models individual flowers or 3–7 flowered cymes constitute the paracladia of either deciduous monotelic synflorescences, or of synflorescences proliferating into vegetative long shoots; in the third, uniflorous paracladia arise from perennial short-shoots. Nevertheless, a link within them can be established through steps of truncation, proliferation, reduction and acquisition of rhythmic growth.     

Cyclic peptide alkaloids from the bark of Discaria americana

The isolation and structure determination of cyclic peptide alkaloids, discarine-M and discarine-N, along with seven known cyclic peptide alkaloids, adouetine-Y', franganine, frangulanine, discarines-A, -B, -C, and -D from the root bark of Discaria americana are described. Structures were determined spectroscopically, especially using 2D NMR spectroscopic analysis. The crude methanol extract, the basic ether extract, and the alkaloids 6 and 7 also weakly inhibited growth of gram-negative and gram-positive bacteria.     

Ammonium assimilation in root nodules of actinorhizal Discaria trinervis. Regulation of enzyme activities and protein levels by the availability of macronutrients (N,P and C)

Asparagine was found to be the main N compound exported from Discaria trinervis nodules. Aspartate (Asp), glutamate (Glu), alanine (Ala) and serine (Ser) were also detected in root xylem sap, but at lower concentrations. A comparable picture is found in nodulated alfalfa. We hypothesized that a similar set of enzymes for Asn synthesis was present in D. trinervis nodules. We demonstrate the expression of most of the enzymes involved in the synthesis of Asn from NH⁺ ₄ and oxoacids, in nodules – but not in roots – of fully symbiotic D. trinervis. By complementation of enzyme assays (^A) and immunodetection (^I) we detected glutamane-synthetase (GS^{A, I}), Asp-aminotransferase (AAT^A), malate-dehydrogenase (MDH^{A, I}, at least two isoforms), Glu-dehydrogenase (GDH^A), Glu-synthase (GOGAT^I) and Asn-synthetase (AS^I). PEP-carboxylase (PEPC) activity was not detected. We previously shown that N acts as a negative regulator of nodulation and nodule growth, while P is a strong stimulator for nodule growth. We present data on the regulation of nodule N metabolism by altering, during 4 weeks, the availability of N, P and light in symbiotic D. trinervis. NH₄NO₃ (2 mM) induced inactivation and degradation of nodule GS, MDH and AS, but activation of GDH and AAT; the amount of nitrogenase components was not affected. A 10-fold increase in P supply did not greatly affect activity and amount of enzymes, suggesting that N metabolism is not P-limited in nodules. On the other hand, suppression of P supply induced an important reduction of nodule GS, GOGAT, MDH and AS protein levels, although nitrogenase was not affected. GDH was the only measured activity that was stimulated by limiting P supply. Shading plants did result in complete degradation of nitrogenase and partial degradation of GS, AS and nodule-specific MDH isoform, but GDH and AAT were activated. These results are discussed in connection with the regulation of nodulation and nodule growth in D. trinervis.     

Isolation of an effective strain of Frankia from nodules of Discaria trinervis (Rhamnaceae)

Frankia strain BCU110501 was isolated from root nodules of the native Patagonian actinorhizal plant Discaria trinervis. The strain was grown on BAP medium with sodium propionate or glucose as carbon sources. Colonies grown in nitrogen-free medium showed branched hyphae bearing polymorphic sporangia and vesicles, which were capable of nitrogen fixation. Old cultures produced a red pigment. The infectivity and effectivity of a Frankia strain isolated from Discaria on its own host, D. trinervis and also in D. chacaye, is reported for the first time. Frankia BCU110501 has physiological properties that are intermediate between categories proposed by Lechevalier et al. (1983) to classify Frankia.     

The regulation of nodulation,nitrogen fixation and ammonium assimilation under a carbohydrate shortage stress in the Discaria trinervis-Frankia symbiosis

N₂-fixation is sensitive to limitation in the availability of newly synthesised carbohydrates for the nodules. We decided to explore the response of the D. trinervis - Frankia symbiosis to a transient decrease in carbohydrate supply to nodules. Feedback inhibition of nodulation as well as nodule growth was not released by a 6-day dark stress in D. trinervis nodulated plants. However, nitrogen fixation and assimilation were affected by the imposed stress. Nitrogenase activity was totally inhibited after 4 days of darkness although high levels of nitrogenase components were still detected at this time. Degradation of FeMo and Fe nitrogenase subunits – both at similar rates – was observed after 6 days of dark stress, revealing the need for inactivation to precede enhancement of protein turnover. Glutamine synthetase (GS), malate dehydrogenase (MDH) and asparagine synthetase (AS) polypeptides were also degraded during the dark stress, although at a lower rate than nitrogenase. ARA and nitrogenase were totally recovered 8 days after resuming normal illumination. It seems that current nitrogenase activity and ammonium assimilation are not, or are only weakly linked with the feedback control of nodulation in D. trinervis. These observations give support to the persistence of an autoregulatory signal in mature nodules that is not sensitive to transient shortages of carbon supply and sustains the inhibition of nodulation in the transient absence of N₂ fixation.     

Effects of Zn<Superscript>2+</Superscript> on nodulation and growth of a South American actinorhizal plant, <Emphasis Type="Italic">Discaria americana</Emphasis> (Rhamnaceae)

Discaria americana is a xerophytic shrub which lives in symbiosis with an actinomycete of the genus Frankia. The objective of this paper was to investigate the effects of high soil Zn²⁺ concentrations on growth and nodulation on the association Discaria americana–Frankia with the aim of determining if this association is suitable for improving contaminated soils. Two experiments were performed in 1 dm³ pots containing soil and different Zn additions, from 0 to 2,000 mg Zn²⁺ kg⁻¹ dry soil, with or without N fertilization. Zn additions strongly delayed shoot and root growth, but once growth was initiated, the biomass production of the plants supplied with moderate Zn amounts did not differ from the control plants. Zn reduced the final nodule number, but not the total nodule biomass. At the end of the experiment only the highest Zn treatments showed a lower nodule weight than the control plants, while N addition completely inhibited nodulation. It is concluded than Zn reduces the number of Frankia infections, but once the actinomycete is inside the roots, nodules can continue growing according to plant demand for N, compensating the reduced nodule number with more biomass. On the other hand, there is a toxic effect of Zn itself on plants when present in very high concentrations.     

Phylogeny of the tribe Colletieae, Rhamnaceae

A cladistic analysis based on 63 morphological characters was carried out on the tribe Colletieae including two presumed closely related genera, Geanothus and Noltea as outgroupS. In addition to a parsimony analysis of the equally weighted characters, analyses investigating the effects of character weighting, removal of a presumed hybrid species as well as the impact of uncertainly scored characters were undertaken. In all analyses Noltea was placed as sister group to a well supported monophyletic Colletieae. Nineteen different in group topologies were found, with the additional analyses mainly supporting two of them. Within the Colletieae a basal dichotomy divides Trevoa and Retanilla from the remainder of the tribe. While the Trevoa-Retanilla clade is fully resolved, the second lacks detailed resolution. Within this clade the Colletia species from a well supported monophyletic group, while monophyly of the disjunct genus Discaria could not be confirmed.     

Grazing and community structure as determinants of invasion success by Scotch broom in a New Zealand montane shrubland

Abstract. Scotch broom ( Cytisus scoparius (L.) Link; Fabaceae) is a problematic invasive plant in many countries, and while attention has been paid to traits that make it a successful invader, there has been less focus on the properties of ecosystems that it invades. We conducted an experiment in a New Zealand montane shrubland with tussock grasses that has been invaded by Scotch broom to determine features that rendered it susceptible to invasion. We planted broom seedlings into the shrubland (control) and into three treatments: (1) resident shrubs removed, (2) tussocks removed and (3) shrubs and tussocks removed. We measured broom seedling mortality and growth over two growing seasons. The site was grazed by sheep in the first season, and scarcely grazed in the second, wetter season. Survivorship across all treatments after 19 months was 42%, and was lowest where shrubs were retained but tussocks removed. Broom seedlings grew taller and had greater leaf areas in treatments that retained shrubs. Neighbouring (within 49 cm) shrubs had no effects on survivorship or growth of broom seedlings. Neighbouring tussocks increased survivorship of broom seedlings but depressed their growth. Grazing by sheep was the most important determinant of survivorship and growth of broom seedlings, and effects were uniform regardless of experimental treatments. Initial high mortality of seedlings (48% in the first 3 months) was due to grazing, and height growth was often negative during periods of grazing. In the second growing season when the site was less grazed and there was greater rainfall, there was a rapid increase in height across all treatments. Continued grazing of the site by sheep is likely to be the chief means of retarding the invasion.     

Time course of nodule development in the Discaria trinervis (Rhamnaceae) –Frankia symbiosis

The time course of initiation and development of root nodules was investigated in the South American actinorhizal shrub Discaria trinervis (Rhamnaceae). A local strain of Frankia (BCU110501) which was isolated from D. trinervis nodules, was used as inoculum. Inoculated seedlings were periodically studied under the light microscope after clearing with aqueous NaClO. In parallel, semithin and ultrathin sections were analysed by light and electron microscopy. Infection by Frankia BCU110501 involved intercellular penetration among epidermal and cortical root cells. Nodule primordia were detected from 6 d after inoculation, while bacteria were progressing through intercellular spaces of the outer layers of cortical cells. Invasion of host cells by the symbiont occurred 7–9 d after inoculation, and hypertrophy of the primordium cells was associated with Frankia penetration. Root hairs were not deformed during the early events of nodule formation. From 13 to 16 d after inoculation, the proximal cellular zone of the primordia behaved differently from the other tissues after NaClO treatment and remained darkly pigmented. At the same time, differentiation of Frankia vesicles started to occur inside already infected cells. By 16 d after inoculation, spherical vesicles of BCU110501 were homogeneously distributed in the host cells. These vesicles were septate and surrounded by void space. Frankia spores or sporangia were not observed in the nodule tissue. This study has clarified the mode of Frankia penetration in D. trinervis , one of the Rhamnaceae which also includes Ceanothus . The events involved in infection, nodule induction, host-cell infection and vesicle differentiation have been characterized and identified as time-segregated developmental processes in the ontogeny of D. trinervis root nodules.