Invasive Plants: Turning Enemies into Value.

In this review, a brief description of the invasive phenomena associated with plants and its consequences to the ecosystem is presented. Five worldwide invasive plants that are a threat to Portugal were selected as an example, and a brief description of each is presented. A full description of their secondary metabolites and biological activity is given, and a resume of the biological activity of extracts is also included. The chemical and pharmaceutical potential of invasive species sensu lato is thus acknowledged. With this paper, we hope to demonstrate that invasive species have potential positive attributes even though at the same time they might need to be controlled or eradicated. Positive attributes include chemical and pharmaceutical properties and developing these could help mitigate the costs of management and eradication.

Tight control of sulfur assimilation: an adaptive mechanism for a plant from a severely phosphorus-impoverished habitat.

Hakea prostrata (Proteaceae) has evolved in extremely phosphorus (P)-impoverished habitats. Unlike species that evolved in P-richer environments, it tightly controls its nitrogen (N) acquisition, matching its low protein concentration, and thus limiting its P requirement for ribosomal RNA (rRNA). Protein is a major sink for sulfur (S), but the link between low protein concentrations and S metabolism in H. prostrata is unknown, although this is pivotal for understanding this species' supreme adaptation to P-impoverished soils. Plants were grown at different sulfate supplies for 5 wk and used for nutrient and metabolite analyses. Total S content in H. prostrata was unchanged with increasing S supply, in sharp contrast with species that typically evolved in environments where P is not a major limiting nutrient. Unlike H. prostrata, other plants typically store excess available sulfate in vacuoles. Like other species, S-starved H. prostrata accumulated arginine, lysine and O-acetylserine, indicating S deficiency. Hakea prostrata tightly controls its S acquisition to match its low protein concentration and low demand for rRNA, and thus P, the largest organic P pool in leaves. We conclude that the tight control of S acquisition, like that of N, helps H. prostrata to survive in P-impoverished environments.

Tight control of nitrate acquisition in a plant species that evolved in an extremely phosphorus-impoverished environment.

Hakea prostrata (Proteaceae) has evolved in an extremely phosphorus (P)-limited environment. This species exhibits an exceptionally low ribosomal RNA (rRNA) and low protein and nitrogen (N) concentration in its leaves. Little is known about the N requirement of this species and its link to P metabolism, despite this being the key to understanding how it functions with a minimal P budget. H. prostrata plants were grown with various N supplies. Metabolite and elemental analyses were performed to determine its N requirement. H. prostrata maintained its organ N content and concentration at a set point, independent of a 25-fold difference nitrate supplies. This is in sharp contrast to plants that are typically studied, which take up and store excess nitrate. Plants grown without nitrate had lower leaf chlorophyll and carotenoid concentrations, indicating N deficiency. However, H. prostrata plants at low or high nitrate availability had the same photosynthetic pigment levels and hence were not physiologically compromised by the treatments. The tight control of nitrate acquisition in H. prostrata retains protein at a very low level, which results in a low demand for rRNA and P. We surmise that the constrained nitrate acquisition is an adaptation to severely P-impoverished soils.

Soil bacteria hold the key to root cluster formation.

Root clusters are bunches of hairy rootlets that enhance nutrient uptake among many plants. Since first being reported in 1974, the involvement of rhizobacteria in their formation has received conflicting support. Attempts to identify specific causative organisms have failed and their role has remained speculative. We set up a gnotobiotic experiment using two root-clustered species, Viminaria juncea (Fabaceae) and Hakea laurina (Proteaceae), and inoculated them with two plant-growth-promoting rhizobacteria (PGPR), Bradyrhizobium elkanii and Bacillus mageratium, that produce indole-3-acetic-acid (IAA). Plants were suspended in water culture with four combinations of nitrogen and phosphorus. Clusters only developed in the presence of PGPR in two treatments, were greatly enhanced in another four, suppressed in five, and unaffected in five. Nitrogen amendment was associated with a higher density of clusters. Bradyrhizobium promoted cluster formation in Hakea, whereas Bacillus promoted cluster formation in Viminaria and suppressed it in Hakea. Greater root cluster numbers were due either to a larger root system induced by PGPR (indirect resource effect) and/or to more clusters per unit length of parent root (direct morphogenetic effect). The results are interpreted in terms of greater IAA production by Bradyrhizobium than Bacillus and greater sensitivity of Viminaria to IAA than Hakea.

Paraphyly changes understanding of timing and tempo of diversification in subtribe Hakeinae (Proteaceae), a giant Australian plant radiation.

PREMISE OF THE STUDY: Subtribe Hakeinae (526 spp.) represents a large Australian plant radiation central to our understanding of that flora's evolution and ecology. It contains Grevillea-the third largest plant genus in Australia and a group inferred to have among the highest diversification rates in the angiosperms. However, we lack a robust phylogenetic framework for understanding subtribe Hakeinae and recognize that Grevillea lacks an unambiguous synapomorphy supporting its monophyly. METHODS: We used four plastid and one nuclear DNA region from a taxonomically even sampling of a third of the species to infer a time-calibrated phylogeny of Hakeinae and absolute diversification rates of major clades. We developed the R package addTaxa to add unsampled taxa to the tree for diversification rate inference. KEY RESULTS: Grevillea is paraphyletic with respect to Hakea and Finschia. Under most parameter combinations, Hakea contains the major clade with the highest diversification rate in Hakeinae, rather than Grevillea. The crown age of the Grevillea+Hakea+Finschia crown group is about double that of prior estimates. CONCLUSIONS: We demonstrate that the paraphyly of Grevillea considerably enlarges the number of Australian descendants from its most recent common ancestor but has also misled investigators who considered a single operational taxonomic unit as adequate to represent the genus for inferences of diversification rate and timing. Our time-calibrated phylogeny can form the basis of future evolutionary, comparative ecology, and biogeography studies involving this large Australian plant radiation, as well as nomenclatural changes.

Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae).

Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native 'extremophile' plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors' knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested.

Stereo Camera Setup for 360° Digital Image Correlation to Reveal Smart Structures of Hakea Fruits.

About forty years after its first application, digital image correlation (DIC) has become an established method for measuring surface displacements and deformations of objects under stress. To date, DIC has been used in a variety of in vitro and in vivo studies to biomechanically characterise biological samples in order to reveal biomimetic principles. However, when surfaces of samples strongly deform or twist, they cannot be thoroughly traced. To overcome this challenge, different DIC setups have been developed to provide additional sensor perspectives and, thus, capture larger parts of an object's surface. Herein, we discuss current solutions for this multi-perspective DIC, and we present our own approach to a 360° DIC system based on a single stereo-camera setup. Using this setup, we are able to characterise the desiccation-driven opening mechanism of two woody Hakea fruits over their entire surfaces. Both the breaking mechanism and the actuation of the two valves in predominantly dead plant material are models for smart materials. Based on these results, an evaluation of the setup for 360° DIC regarding its use in deducing biomimetic principles is given. Furthermore, we propose a way to improve and apply the method for future measurements.

Capturing Genetic Diversity in Seed Collections: An Empirical Study of Two Congeners with Contrasting Mating Systems.

Plant mating systems shape patterns of genetic diversity and impact the long-term success of populations. As such, they are relevant to the design of seed collections aiming to maximise genetic diversity (e.g., germplasm conservation, ecological restoration). However, for most species, little is known empirically about how variation in mating systems and genetic diversity is distributed. We investigated the relationship between genetic diversity and mating systems in two functionally similar, co-occurring species of Hakea (Proteaceae), and evaluated the extent to which genetic diversity was captured in seeds. We genotyped hundreds of seedlings and mother plants via DArTseq, and developed novel implementations of two approaches to inferring the mating system from SNP data. A striking contrast in patterns of genetic diversity between H. sericea and H. teretifolia was revealed, consistent with a contrast in their mating systems. While both species had mixed mating systems, H. sericea was found to be habitually selfing, while H. teretifolia more evenly employed both selfing and outcrossing. In both species, seed collection schemes maximised genetic diversity by increasing the number of maternal lines and sites sampled, but twice as many sites were needed for the selfing species to capture equivalent levels of genetic variation at a regional scale.

Differential tolerance of the woody invasive Hakea sericea to drought and terminal heat stress.

Drought and extreme temperatures are likely to be more common and intense in the Mediterranean region as a consequence of climate change. Both stresses usually arise together in the field, but our understanding of their joint influence on the performance of invasive alien species (IAS) is limited. Thus, the main objective of the present study is to fill this gap by analyzing the individual and combined effects of drought and terminal heat stress on the leaf physiology, biochemistry and growth of Hakea sericea Schrader, one of the most problematic IAS in the Mediterranean-type ecosystems. In this study, 1-year-old plants of H. sericea were exposed to four treatments under controlled conditions: control (CT), drought (DS), terminal heat stress (Ht), and combined Ht and DS (DHt). The DS treatment alone caused a marked reduction in shoot biomass, net photosynthetic (A) rate and stomatal conductance, while increasing the proline content, as compared with CT plants. In turn, the Ht treatment promoted the accumulation of malondialdehyde but hastened the decline in all gas exchange parameters, and also decreased leaf photosynthetic pigments, carotenoids, proline and relative water contents Exposure of H. sericea plants to the combined DHt exacerbated the impacts of Ht, which was accompanied by significant decreases in net photosynthetic and transpiration rates, and intrinsic water-use efficiency. Principal component analysis clearly separated the DHt from the other treatments and revealed similarities between DS and CT treatment. These findings suggest that xerothermic weather conditions might modify the fitness, competitive ability, resilience and spread of this IAS, thereby providing opportunities for its control.

Seasonal Variation in Selected Biochemical Traits in the Leaves of Co-Occurring Invasive and Native Plant Species under Mediterranean Conditions.

The success of invasive alien species (IAS) is often linked to differences in functional traits in relation to other, either native or non-invasive, species. Two of the most problematic IAS in the Mediterranean area belong to Hakea and Acacia genera that often invade pine plantations. Therefore, the aim of this study was to assess the seasonal variations in photosynthetic pigments, total phenolics, and non-structural carbohydrates (NSC), including total soluble sugars (SS) and starch (St), and lipid peroxidation, in terms of malondialdehyde (MDA) in the leaves of evergreen species, two IAS (Hakea sericea and Acacia melanoxylon) and one native (Pinus pinaster), throughout 2019. All parameters showed a pronounced seasonal variability while also differing across species. Generally, the lowest contents of photosynthetic pigments, phenolics and SS were noted in early spring, along with the highest St and NSC values. On the other hand, higher photosynthetic pigment and lower NSC contents were measured in early autumn and early winter. When these parameters were compared across the three species, the IAS had significantly higher content of photosynthetic pigments, mainly chlorophyll b and total chlorophyll, and lower total phenolics and MDA concentrations in their leaves than Pinus pinaster. Differences in seasonal patterns were also observed. Hakea sericea and Acacia melanoxylon had considerably higher chlorophyll, SS and NSC contents in the early autumn, while Pinus pinaster had higher St and MDA contents in early summer. Overall, the biochemical characteristics of leaves of the studied IAS can explain their success in the Mediterranean area, in terms of tolerance to stressful environmental conditions.

Aspects of the reproductive ecology of four australian Hakea species (Proteaceae) in South Africa.

Four shrub species of the Australian Proteaceae (Hakea sericea, H. gibbosa, H. suaveolens and H. salicifolia) were introduced to South African fynbos shrublands between 1840 and 1860. H. sericea is highly invasive, H. gibbosa and H. suaveolens are moderately invasive and H. salicifolia is not invasive. The allocation of reproductive energy, germinability, the ability to survive fires and to germinate in burnt and unburnt areas, and the nutrient content of seeds were assessed for the four species. The information was used to investigate whether the success of H. sericea relative to the other three species could be explained by the superior expression of any trait. The most important trait which separates H. sericea from the other species is its ability to produce a large seed bank in its adopted environment in the absence of seed predators. Seed production in H. sericea shrubs with an above-ground dry mass of 8 kg is four times greater than H. gibbosa and more than 16 times that of H. suaveolens. Although H. salicifolia also produces a large seed bank, its seeds are unable to survive fires due to inadequate insulation by the small follicles. The results are compared to dispersal and seed bank data for indigenous South African Proteaceae, which have low dispersal and suffer high pre-dispersal seed predation. We suggest that potential invasives in the fynbos can be identified as species that have: (i) a potentially high seed production that is limited by specialized predators; (ii) an ability to disperse over long distances; and (iii) are pre-adapted to frequent fires and low soil nutrients. The data also support the current strategy of combatting H. sericea using specialized insect seed predators.

Post-fire mortality and water relations of three congeneric shrub species under extreme water stress - a trade-off with fecundity?

Mortality and water relations of three cooccurringHakea species were studied over the first year following a wildfire in scrub-health in southwestern Australia.Hakea ruscifolia regenerated from both resprouting adults and seedlings, whereasH. smilacifolia andH. polyanthema regenerated only from seedlings. We monitored seedling and resprout survival relative to pre-fire numbers and water relations (shoot water potential, transpiration, pressure-volume parameters) in order to determine the relationship between plant mortality and water relations over the critical first summer drought. Seedlings ofH. polyanthema (few initial seedlings per parent) showed little mortality (8%), achieved through both drought avoidance (low transpiration, high predawn water potential and relative water content (RWC), substantial osmotic adjustment) and drought tolerance later in the season. Seedlings ofH. smilacifolia andH. ruscifolia (high seedling/parent ratios) showed little drought avoidance and high mortality (54-68%). The remaining seedlings spent 3-4 months in a wilted condition (up to 3.6 MPa and 45% RWC below the turgor loss point inH. ruscifolia) indicating marked drought tolerance of the survivors. In contrast to its seedlings,H. ruscifolia resprouts were successful drought avoiders and experienced no mortality. The high level of survival and drought resistance ofH. polyanthema was consistent with its large seedlings (via large seeds) and low initial fecundity. The study highlights the importance of the interaction between ecophysiology-morphology and demography in determining the recruitment strategies of plants.

Antimicrobial, antibiofilm and cytotoxic activities of Hakea sericea Schrader extracts.

BACKGROUND: Hakea sericea Schrader is an invasive shrub in Portuguese forests. OBJECTIVE: The goal of this work was to evaluate the antimicrobial activity of H. sericea extracts against several strains of microorganisms, including the ability to inhibit the formation of biofilms. Additionally the cytotoxic properties of these extracts, against human cells, were assessed. MATERIALS AND METHODS: The antimicrobial activity of the methanolic extracts of H. sericea was assessed by disk diffusion assay and Minimum Inhibitory Concentration (MIC) value determination. The antibiofilm activity was determined by quantification of total biofilm biomass with crystal violet. Cytotoxicity was evaluated by hemolysis assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. RESULTS: For Gram-positive bacteria, MIC values of H. sericea methanolic extracts ranged between 0.040 and 0.625 mg/mL, whereas the fruits extract yielded the lowest MIC for several strains of microorganisms, namely, S. aureus, B. cereus, L. monocytogenes and clinical methicillin-resistant S. aureus (MRSA). Stems and fruits extract at 2.5 mg/mL effectively eradicated the biofilm of S. aureus ATCC 25923, SA 01/10 and MRSA 12/10. Regarding leaves extract, hemolysis was not observed, and in the case of stems and fruits, hemolysis was verified only for higher concentrations, suggesting its low toxicity. Fruits extract presented no toxic effect to normal human dermal fibroblasts (NHDF) cells however for concentrations of 0.017 and 0.008 mg/mL this extract was able to decrease human breast adenocarcinoma cells (MCF-7) viability in about 60%, as MTT test results had confirmed. This is a clearly demonstrator of the cytotoxicity of this extract against MCF-7 cells.