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Very few of the >650 Proteaceae species in south-western Australia cope with the high calcium (Ca) levels in young, calcareous soils (soil indifferent); most are Ca sensitive and occur on nutrient-impoverished, acidic soils (calcifuge). We assessed possible control points for Ca transport across roots of two soil-indifferent (Hakea prostrata and Banksia prionotes) and two calcifuge (H. incrassata and B. menziesii) Proteaceae. Using quantitative X-ray microanalysis, we investigated cell-specific elemental Ca concentrations at two positions behind the apex in relation to development of apoplastic barriers in roots of plants grown in nutrient solution with low or high Ca supply. In H. prostrata, Ca accumulated in outer cortical cells at 20 mm behind the apex, but [Ca] was low in other cell types. In H. incrassata, [Ca] was low in all cells. Accumulation of Ca in roots of H. prostrata corresponded to development of apoplastic barriers in the endodermis. We found similar [Ca] profiles in roots and similar [Ca] in leaves of two contrasting Banksia species. Soil-indifferent Hakea and Banksia species show different strategies to inhabit calcareous soils: H. prostrata intercepts Ca in roots, reducing transport to shoots, whereas B. prionotes allocates Ca to specific leaf cells.

Endemism and rarity have long intrigued scientists. We focused on a rare endemic and critically-endangered species in a global biodiversity hotspot, Grevillea thelemanniana (Proteaceae). We carried out plant and soil analyses of four Proteaceae, including G. thelemanniana, and combined these with glasshouse studies. The analyses related to hydrology and plant water relations as well as soil nutrient concentrations and plant nutrition, with an emphasis on sodium (Na) and calcium (Ca). The local hydrology and matching plant traits related to water relations partially accounted for the distribution of the four Proteaceae. What determined the rarity of G. thelemanniana, however, was its accumulation of Ca. Despite much higher total Ca concentrations in the leaves of the rare G. thelemanniana than in the common Proteaceae, very few Ca crystals were detected in epidermal or mesophyll cells. Instead of crystals, G. thelemanniana epidermal cell vacuoles contained exceptionally high concentrations of noncrystalline Ca. Calcium ameliorated the negative effects of Na on the very salt-sensitive G. thelemanniana. Most importantly, G. thelemanniana required high concentrations of Ca to balance a massively accumulated feeding-deterrent carboxylate, trans-aconitate. This is the first example of a calcicole species accumulating and using Ca to balance accumulation of an antimetabolite.

We introduce the AusTraits database - a compilation of values of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 448 traits across 28,640 taxa from field campaigns, published literature, taxonomic monographs, and individual taxon descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological attributes (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual- and species-level measurements coupled to, where available, contextual information on site properties and experimental conditions. This article provides information on version 3.0.2 of AusTraits which contains data for 997,808 trait-by-taxon combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data, which also provides a template for other national or regional initiatives globally to fill persistent gaps in trait knowledge., Measurement(s)plant traitTechnology Type(s)digital curationSample Characteristic - OrganismViridiplantaeSample Characteristic - LocationAustralia Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.14545755

Maintenance of photosynthesis and thus salinity tolerance in chickpea is related to the capacity to ‘exclude’ Na from leaflets and the differential distribution of Na between epidermis and photosynthetically active mesophyll cells., Salinity tolerance is associated with Na ‘exclusion’ from, or ‘tissue tolerance’ in, leaves. We investigated whether two contrasting chickpea genotypes, salt-tolerant Genesis836 and salt-sensitive Rupali, differ in leaf tissue tolerance to NaCl. We used X-ray microanalysis to evaluate cellular Na, Cl, and K concentrations in various cell types within leaflets and also in secretory trichomes of the two chickpea genotypes in relation to photosynthesis in control and saline conditions. TEM was used to assess the effects of salinity on the ultrastructure of chloroplasts. Genesis836 maintained net photosynthetic rates (A) for the 21 d of salinity treatment (60 mM NaCl), whereas A in Rupali substantially decreased after 11 d. Leaflet tissue [Na] was low in Genesis836 but had increased markedly in Rupali. In Genesis836, Na was accumulated in epidermal cells but was low in mesophyll cells, whereas in Rupali cellular [Na] was high in both cell types. The excessive accumulation of Na in mesophyll cells of Rupali corresponded to structural damage to the chloroplasts. Maintenance of photosynthesis and thus salinity tolerance in Genesis836 was associated with an ability to ‘exclude’ Na from leaflets and in particular from the photosynthetically active mesophyll cells, and to compartmentalize Na in epidermal cells.

Ca increased the allocation of P to palisade mesophyll cells in Proteaceae, supporting our model of Ca-enhanced P toxicity and demonstrating its role in the distribution of Proteaceae., Over 650 Proteaceae occur in south-western Australia, contributing to the region’s exceptionally high biodiversity. Most Proteaceae occur exclusively on severely nutrient-impoverished, acidic soils (calcifuge), whilst only few also occur on young, calcareous soils (soil-indifferent), higher in calcium (Ca) and phosphorus (P). The calcifuge habit of Proteaceae is explained by Ca-enhanced P toxicity, putatively linked to the leaf cell-specific allocation of Ca and P. Separation of these elements is essential to avoid the deleterious precipitation of Ca-phosphate. We used quantitative X-ray microanalysis to determine leaf cell-specific nutrient concentrations of two calcifuge and two soil-indifferent Proteaceae grown in hydroponics at a range of Ca and P concentrations. Calcium enhanced the preferential allocation of P to palisade mesophyll (PM) cells under high P conditions, without a significant change in whole leaf [P]. Calcifuges showed a greater PM [P] compared with soil-indifferent species, corresponding to their greater sensitivity. This study advances our mechanistic understanding of Ca-enhanced P toxicity, supporting the proposed model, and demonstrating its role in the calcifuge distribution of Proteaceae. This furthers our understanding of nutrient interactions at the cellular level and highlights its importance to plant functioning.

The Jurien Bay dune chronosequence in south‐western Australia’s biodiversity hotspot comprises sites differing in nutrient availability, with phosphorus (P) availability declining strongly with increasing soil age. We have explored the exceptionally high photosynthetic P‐use efficiency (PPUE) of Proteaceae in this region, triggering the question what the PPUE of co‐occurring species in other families might be along the Jurien Bay chronosequence. We explored how traits associated with PPUE, photosynthetic nitrogen (N)‐use efficiency (PNUE) and leaf respiration might converge along the chronosequence, and whether Proteaceae and non‐Proteaceae species differ in leaf traits associated with nutrient use. Seven to 10 species were sampled at three sites differing in nutrient availability (ranging from N‐ to P‐limited). Measurements of leaf light‐saturated photosynthesis and dark respiration were integrated with measurements of total N and P concentration in both mature and senesced leaves, and leaf mass per unit area (LMA). Contrary to what is known for other chronosequences, rates of photosynthesis and respiration did not decrease with increasing soil age and LMA along the Jurien Bay chronosequence. However, they increased when expressed per unit leaf P. Both N and P were used much more efficiently for photosynthesis on nutrient‐poor sites, in both Proteaceae and non‐Proteaceae species. Proteaceae had the fastest rate of photosynthesis per unit leaf P, followed by species that preferentially allocate P to mesophyll cells, rather than epidermal cells. Synthesis. Our results show that with declining soil P availability, photosynthetic P‐use efficiency of all investigated species from different families increased. Plants growing on the oldest, most nutrient‐impoverished soils exhibited similar rates of CO₂ exchange as plants growing on more nutrient‐rich younger soils, and extraordinarily high photosynthetic P‐use efficiency. This indicates convergence in leaf traits related to photosynthetic nutrient use on severely P‐impoverished sites.

Crops with improved uptake of fertilizer phosphorus (P) would reduce P losses and confer environmental benefits. We examined how P-sufficient 6-week-old soil-grown Trifolium subterraneum plants, and 2-week-old seedlings in solution culture, accumulated P in roots after inorganic P (Pi) addition. In contrast to our expectation that vacuoles would accumulate excess P, after 7 days, X-ray microanalysis showed that vacuolar [P] remained low (3,000 mmol kg −1 ), potassium, magnesium, and sodium. Similar structures were evident in seedlings, both before and after P addition, with their [P] increasing threefold after P addition. Nuclear magnetic resonance (NMR) spectroscopy showed seedling roots accumulated Pi following P addition, and transmission electron microscopy (TEM) revealed large plastids. For seedlings, we demonstrated that roots differentially expressed genes after P addition using RNAseq mapped to the T. subterraneum reference genome assembly and transcriptome profiles. Among the most up-regulated genes after 4 hr was TSub_g9430.t1, which is similar to plastid envelope Pi transporters (PHT4;1, PHT4;4): expression of vacuolar Pi-transporter homologs did not change. We suggest that subcellular P accumulation in globular structures, which may include plastids, aids cytosolic Pi homeostasis under high-P availability.

The calcifuge habit of plants is commonly explained in terms of high soil pH and its effects on nutrient availability, particularly that of phosphorus (P). However, most Proteaceae that occur on nutrient-impoverished soils in south-western Australia are calcifuge, despite their ability to produce cluster-roots, which effectively mobilize soil P and micronutrients. We hypothesize that the mechanism explaining the calcifuge habit in Proteaceae is their sensitivity to P and calcium (Ca), and that soil-indifferent species are less sensitive to the interaction of these nutrients. In this study, we analyzed growth, gas-exchange rate, and chlorophyll fluorescence of two soil-indifferent and four calcifuge Hakea and Banksia (Proteaceae) species from south-western Australia, across a range of P and Ca concentrations in hydroponic solution. We observed Ca-enhanced P toxicity in all analyzed species, but to different extents depending on distribution type and genus. Increasing P supply enhanced plant growth, leaf biomass, and photosynthetic rates of soil-indifferent species in a pattern largely independent of Ca supply. In contrast, positive physiological responses to increasing [P] in calcifuges were either absent or limited to low Ca supply, indicating that calcifuges were far more sensitive to Ca-enhanced P toxicity. In calcifuge Hakeas, we attributed this to higher leaf [P], and in calcifuge Banksias to lower leaf zinc concentration. These differences help to explain these species' contrasting sensitivity to Ca-enhanced P toxicity and account for the exclusion of most Proteaceae from calcareous habitats. We surmise that Ca-enhanced P toxicity is a major factor explaining the calcifuge habit of Proteaceae, and, possibly, other P-sensitive plants.

Background Sponges are increasingly recognised as key ecosystem engineers in many aquatic habitats. They play an important role in nutrient cycling due to their unrivalled capacity for processing both dissolved and particulate organic matter (DOM and POM) and the exceptional metabolic repertoire of their diverse and abundant microbial communities. Functional studies determining the role of host and microbiome in organic nutrient uptake and exchange, however, are limited. Therefore, we coupled pulse-chase isotopic tracer techniques with nanoscale secondary ion mass spectrometry (NanoSIMS) to visualise the uptake and translocation of 13C- and 15N-labelled dissolved and particulate organic food at subcellular level in the high microbial abundance sponge Plakortis angulospiculatus and the low microbial abundance sponge Halisarca caerulea. Results The two sponge species showed significant enrichment of DOM- and POM-derived 13C and 15N into their tissue over time. Microbial symbionts were actively involved in the assimilation of DOM, but host filtering cells (choanocytes) appeared to be the primary site of DOM and POM uptake in both sponge species overall, via pinocytosis and phagocytosis, respectively. Translocation of carbon and nitrogen from choanocytes to microbial symbionts occurred over time, irrespective of microbial abundance, reflecting recycling of host waste products by the microbiome. Conclusions Here, we provide empirical evidence indicating that the prokaryotic communities of a high and a low microbial abundance sponge obtain nutritional benefits from their host-associated lifestyle. The metabolic interaction between the highly efficient filter-feeding host and its microbial symbionts likely provides a competitive advantage to the sponge holobiont in the oligotrophic environments in which they thrive, by retaining and recycling limiting nutrients. Sponges present a unique model to link nutritional symbiotic interactions to holobiont function, and, via cascading effects, ecosystem functioning, in one of the earliest metazoan–microbe symbioses.

Transcriptomes from sponges are important resources for studying the stress responses of these ecologically important filter feeders, the interactions between sponges and their symbionts, and the evolutionary history of metazoans. Here, we generated reference transcriptomes for two common and cosmopolitan Indo-Pacific sponge species: Carteriospongia foliascens and Cliona orientalis. We also created a reference transcriptome for the primary symbiont of C. orientalis – Gerakladium endoclionum. To ensure a full repertoire of transcripts were included, clones of each sponge species were exposed to a range of individual stressors: decreased salinity, elevated temperature, elevated suspended sediment concentrations, sediment deposition and light attenuation. RNA extracted from all treatments was pooled for each species, using equal concentrations from each clone. Sequencing of pooled RNA yielded 409 and 418 million raw reads for C. foliascens and C. orientalis holobionts (host and symbionts), respectively. Reads underwent quality trimming before assembly with Trinity. Assemblies were filtered into sponge-specific or, for G. endoclionum, symbiont-specific assemblies. Assemblies for C. foliascens, C. orientalis, and G. endoclionum contained 67,304, 82,895, and 28,670 contigs, respectively. Contigs represented 15,248-37,344 isogroups (∼genes) per assembly, and N50s ranged 1,672-4,355 bp. Gene ortholog analysis verified a high level of completeness and quality for sponge-specific transcriptomes, with an average 93% of core EuKaryotic Orthologous Groups (KOGs) and 98% of single-copy metazoan core gene orthologs identified. The G. endoclionum assembly was partial with only 56% of core KOGs and 32% of single-copy eukaryotic core gene orthologs identified. These reference transcriptomes are a valuable resource for future molecular research aimed at assessing sponge stress responses.

Lack of O2 and high concentrations of iron (Fe) and manganese (Mn) commonly occur in waterlogged soils. The development of a barrier to impede radial O2 loss (ROL) is a key trait improving internal O2 transport and waterlogging tolerance in plants. We evaluated the ability of the barrier to ROL to impede the entry of excess Fe into the roots of the waterlogging-tolerant grass Urochloa humidicola. Plants were grown in aerated or stagnant deoxygenated nutrient solution with 5 µM or 900 µM Fe. Quantitative X-ray microanalysis was used to determine cell-specific Fe concentrations at two positions behind the root apex in relation to ROL and the formation of apoplastic barriers. At a mature zone of the root, Fe was ‘excluded’ at the exodermis where a suberized lamella was evident, a feature also associated with a strong barrier to ROL. In contrast, the potassium (K) concentration was similar in all root cells, indicating that K uptake was not affected by apoplastic barriers. The hypothesis that the formation of a tight barrier to ROL impedes the apoplastic entry of toxic concentrations of Fe into the mature zones of roots was supported by the significantly higher accumulation of Fe on the outer side of the exodermis.

Kapsulotaenia tidswelli is a proteocephalidean cestode that utilizes varanid lizards as definitive hosts. Fresh specimens of this cestode were observed with endogenous red pigmentation in the neck region that disappeared rapidly if specimens were not preserved in glutaraldehyde. The ultrastructural characteristics of the red pigment, which are described, suggest it is a carotenoid. Phylogenetic analysis confirmed a close relationship between K. tidswelli and other species of Kapsulotaenia for which sequence information is available. There is thus no reason to consider that the red pigmentation is because K. tidswelli is atypical, and it is proposed the carotenoids are likely to be associated with the diet of its varanid host.

Elevated suspended sediment concentrations (SSCs) often impact coral fertilisation success, but sediment composition can influence effect thresholds, which is problematic for accurately predicting risk. Here, we derived concentration–response thresholds and cause-effect pathways for SSCs comprising a range of realistic mineral and organic compositions on coral fertilisation success. Effect concentration thresholds (EC10: 10% fertilisation inhibition) varied markedly, with fertilisation highly sensitive to inshore organic-clay rich sediments and bentonite clay at 40 mg L−1). The effect thresholds for relevant sediment types were combined with in situ turbidity data from locations near dredging operations to assess the risks posed by dredging to coral fertilisation at these locations.

In contrast to the situation in plants inhabiting most of the world’s ecosystems, mycorrhizal fungi are usually absent from roots of the only two native vascular plant species of maritime Antarctica, Deschampsia antarctica and Colobanthus quitensis. Instead, a range of ascomycete fungi, termed dark septate endophytes (DSEs), frequently colonise the roots of these plant species. We demonstrate that colonisation of Antarctic vascular plants by DSEs facilitates not only the acquisition of organic nitrogen as early protein breakdown products, but also as non‐proteinaceous d‐amino acids and their short peptides, accumulated in slowly‐decomposing organic matter, such as moss peat. Our findings suggest that, in a warming maritime Antarctic, this symbiosis has a key role in accelerating the replacement of formerly dominant moss communities by vascular plants, and in increasing the rate at which ancient carbon stores laid down as moss peat over centuries or millennia are returned to the atmosphere as CO2., In contrast to the situation in plants inhabiting most of the world's ecosystems, mycorrhizal fungi are usually absent from roots of the only two native vascular plant species of maritime Antarctica, Deschampsia antarctica and Colobanthus quitensis. Instead, a range of ascomycete fungi, termed dark septate endophytes (DSEs), frequently colonise the roots of these plant species. We demonstrate that colonisation of Antarctic vascular plants by DSEs facilitates not only the acquisition of organic nitrogen as early protein breakdown products, but also as non‐proteinaceous D‐amino acids and their short peptides, accumulated in slowly‐decomposing organic matter, such as moss peat.

Previous studies have shown that pH, rather than calcium (Ca), is the main reason why some Lupinus species are sensitive to nutrient solutions mimicking calcareous soils; however, a hydroponic system is quite different from soil systems, and plants may respond differently to these two growing conditions. Thus, studies with Lupinus species grown in calcareous soils are needed. Two calcicole and two calcifuge species were grown in river sand with different Ca forms and amounts, pH levels, and [bicarbonate (HCO3−)] (HCO3− concentration, which is produced by calcium carbonate (CaCO3)). Leaf symptoms, leaf area, gas exchange, biomass, and root morphology were recorded; whole leaf and root nutrient concentrations were analysed. We observed leaf chlorosis of the youngest leaves under high pH (adjusted by KOH) and high pH + high Ca (representing high [HCO3−], high pH and high Ca) treatments for all Lupinus species. However, after 2 weeks, leaf chlorosis of all Lupinus species under high pH started to disappear, with calcicole species fully, and calcifuge species only partly recovering. Leaf chlorosis symptoms of calcicole species under high pH + high Ca partly disappeared as well, while those of calcifuge species did not disappear at all. High pH (resulting from either KOH or HCO3−) inhibited root growth, and subsequently uptake of some nutrients and shoot growth of Lupinus species. However, the strong buffering capacity of HCO3− is the key factor determining if Lupinus species can survive in calcareous soils. Among all studied Lupinus species, L. pilosus was the most tolerant to high [HCO3−] and/or high pH, followed by L. cosentinii and L. angustifolius, while L. hispanicus was the most sensitive.

Turbidity associated with elevated suspended sediment concentrations can significantly reduce underwater light availability. Understanding the consequences for sensitive organisms such as corals and crustose coralline algae (CCA), requires an understanding of tolerance levels and the time course of effects. Adult colonies of Acropora millepora and Pocillopora acuta, juvenile P. acuta, and the CCA Porolithon onkodes were exposed to six light treatments of ~0, 0.02, 0.1, 0.4, 1.1 and 4.3 mol photons m−2 d−1, and their physiological responses were monitored over 30 d. Exposure to very low light (-2 d-1) caused tissue discoloration (bleaching) in the corals, and discolouration (and partial mortality) of the CCA, yielding 30 d EI10 thresholds (irradiance which results in a 10% change in colour) of 1.2–1.9 mol photons m−2 d−1. Recent monitoring studies during dredging campaigns on a shallow tropical reef, have shown that underwater light levels very close (~500 m away) from a working dredge routinely fall below this value over 30 d periods, but rarely during the pre-dredging baseline phase. Light reduction alone, therefore, constitutes a clear risk to coral reefs from dredging, although at such close proximity other cause-effect pathways, such as sediment deposition and smothering, are likely to also co-occur.

The interaction between local, anthropogenic stressors, and larger scale regional/global stressors, is often used to explain the current poor condition of many corals reefs. This form of cumulative pressure is clearly manifested by situations where dredging projects happen to coincide with marine heatwaves that have caused coral bleaching. A key pressure associated with dredging is elevated sedimentation. In this study, 3 coral species (Acropora millepora, Porites spp. and Turbinaria reniformis), representing three common morphologies (branching, massive and foliose respectively), were experimentally induced to bleach by exposure to a temperature of 31 °C for 21 d. The corals were then subjected to a range of sedimentation rates (0, 11, 22 and 40 mg cm−2 d−1), and their sediment-rejection ability quantified after 1 and 7 successive sediment deposition events. Bleached corals were less capable of removing sediments from their surfaces, and sediment accumulated 3 to 4-fold more than on normally-pigmented corals. Repeated deposition resulted in a ~3-fold increase in the amount of sediment remaining on the corals, regardless of bleaching status. These results suggest that adaptive management practices need to be developed to reduce the impacts of future dredging projects that follow or coincide with elevated sea surface temperatures and coral bleaching events.

Zinc (as an essential phytonutrient) and cadmium (as a toxic but readily bioavailable nonessential metal for plants) share similar routes for crossing plant biomembranes, although with a substantially different potential for translocation into above-ground tissues. The in situ distribution of these metals in plant cells and tissues (particularly intensively-dividing and fast-growing areas) is poorly understood. In this study, 17-day-old radish (Raphanus sativus L.) plants grown in nutrient solution were subjected to short-term (24 h) equimolar contamination (2.2 &micro, M of each 70Zn and Cd) to investigate their accumulation and distribution in the shoot apex (leaf primordia) and edible fleshy hypocotyl tissues. After 24-h exposure, radish hypocotyl had similar concentration (in &micro, g/g dry weight) of 70Zn (12.1 &plusmn, 1.1) and total Cd (12.9 &plusmn, 0.8), with relatively limited translocation of both metals to shoots (concentrations lower by 2.5-fold for 70Zn and 4.8-fold for Cd) as determined by inductively-coupled plasma mass spectrometry (ICP-MS). The in situ Zn/Cd distribution maps created by high-resolution secondary ion mass spectrometry (NanoSIMS, Cameca, Gennevilliers, France) imaging corresponded well with the ICP-MS data, confirming a similar pattern and uniform distribution of 70Zn and Cd across the examined areas. Both applied techniques can be powerful tools for quantification (ICP-MS) and localisation and visualisation (NanoSIMS) of some ultra-trace isotopes in the intensively-dividing cells and fast-growing tissues of non-metalophytes even after short-term metal exposure. The results emphasise the importance of the quality of (agro)ecosystem resources (growing media, metal-contaminated soils/waters) in the public health risk, given that, even under low contamination and short-term exposure, some of the most toxic metallic ions (e.g., Cd) can relatively rapidly enter the human food chain.

Systemic hypersensitive resistance (SHR) caused by Turnip mosaic virus (TuMV) was studied by light microscopy and histochemical analysis in stem cross sections of Brassica juncea (Indian mustard) plants. Ten TuMV isolates were inoculated to leaves of susceptible line JM 06006, cv. Oasis CI, which carries TuMV systemic hypersensitivity gene TuRBJU 01, and F3 progeny plants obtained from a cross between them. Systemic mosaic (SM) symptoms were induced by all 10 isolates in plants of JM 06006, and by resistance-breaking isolate NSW-3 in all cv. Oasis CI and F3 plants. With the other nine isolates, cv. Oasis CI plants developed SHR while F3 progeny plants segregated for both phenotypes; mock-inoculated control plants never became infected. Presence of SHR did not delay systemic invasion as this commenced within 2 hours after inoculation (hai) and was almost complete by 72 hai regardless of whether plants subsequently developed SHR or SM. When stem cross sections sampled 9 to 12 days after inoculation were examined for the plant defense responses, phloem necrosis, hydrogen peroxide accumulation, and additional lignin deposition, sections from plants with SHR demonstrated all of these characteristics, but sections from plants with SM or mock-inoculation did not. Based on consolidated data from all isolates except NSW-3, stems developing SHR had significantly more occluded xylem vessels (P < 0.001) compared with stems from plants developing SM or mock-inoculated plants. Both light microscopy and histochemical tests with phloroglucinol-HCl and toluidine blue O indicated that the xylem occlusions could be gels. Thus, phloem necrosis, xylem occlusion, lignification, and hydrogen peroxide accumulation were all associated with the SHR in B. juncea plants carrying TuMV hypersensitivity gene TuRBJU 01. In addition, virus inclusion bodies were fewer in sections from plants with SHR. Phloem necrosis was apparently acting as the primary cause of SHR and xylem occlusion as an important secondary cause.

White leaf spot disease (Pseudocercosporella capsellae) is widespread across oilseed, vegetable and forage brassicas. Light (LM) and scanning electron (SEM) microscope studies were undertaken to investigate host–pathogen interactions on cotyledons of resistant and susceptible Brassica carinata, B. juncea and B. napus. Under LM, unique brown structures were present, particularly on susceptible genotypes, in two morphologically distinct forms: first, as thread-like structures within cortical tissue by 24 h post-inoculation (hpi) and secondly, as brown ropy strand structures either within cortical tissues (internal ropy strands), or extruded out through stomatal pores (ropy strand extrusions). Under LM, these brown structures were most prevalent in highly susceptible B. juncea ‘Vardan’ that had both a high incidence within cortical tissue (70%) and of ropy strand extrusions (73%), as did susceptible B. napus ‘Trilogy’ within cortical tissue (60%). Under SEM, both these genotypes showed thread-like structures smaller than hyphae forming highly branched networks and ropy strand-like structures. While there were fewer brown structures in susceptible B. carinata UWA #012 (35%), fine, thread-like structures forming networks were again prominent (SEM). In contrast, for resistant genotypes, brown structures (LM) were of very low frequency or absent; only 5% in resistant B. juncea ‘Dune’ and none in resistant B. napus ‘Hyola 42’ or highly resistant B. carinata ATC94129P. Under SEM, fine, thread-like structures were present in the resistant B. juncea ‘Dune’ and B. napus ‘Hyola 42’. Liquid chromatographic analyses of brown structures revealed that both internal ropy strands within cortical tissues and ropy strand extrusions contained the mycotoxin cercosporin.

Programmed cell death (PCD) pathways caused by Turnip mosaic virus (TuMV) infection before symptom appearance were studied by light microscopy and electrolyte leakage following sap inoculation of Brassica carinata (Ethiopian mustard) TZ-SMN-44-6 plants. Leaf responses to inoculation with avirulent (TuMV-avir) and virulent (TuMV-vir) isolates, and mock-inoculation, were compared at 2, 20 and 52 h after inoculation (hai). The phenotypes induced were localized resistance (TuMV-avir) and systemic susceptibility (TuMV-vir). No visible TuMV symptoms were recorded in any inoculated plants during the 2–52 hai sampling period, but appeared as chlorotic spots in inoculated leaves at 5 days after inoculation. With TuMV-vir alone, they were followed by systemic infection (mosaic). Dead cell number, deformation, percentage area and percentage integrated intensity, and conductivity of electrolyte leakage data, were analysed to examine their possible roles in stimulating cell death pathways. At 2 hai, dead cell number and percentage area were significantly greater for TuMV-avir than TuMV-vir infection or mock-inoculation. Overall, isolate TuMV-vir caused significantly greater cell deformation than TuMV-avir, whereas wounding by mock-inoculation had negligible effects. By 52 hai, isolate TuMV-avir caused significantly greater electrolyte leakage than isolate TuMV-vir or mock-inoculation. This suggests both isolates triggered morphological changes consistent with apoptotic-like PCD and necrosis-like PCD that depended upon isolate virulence and stage of infection, respectively. These findings highlight how quantification of dead cell deformation and electrolyte leakage offer a new understanding of compatible and incompatible plant responses to early virus infection in plants.

Studies were undertaken to compare susceptible and resistant host responses to Pseudocercosporella capsellae in cotyledons of Brassica carinata, B. juncea and B. napus in order to define the mechanisms of resistance in these three species. On both resistant and susceptible hosts, hyphal penetration was always through stomatal openings and without infection pegs or appressoria. On resistant B. carinata ATC94129P, up to 72% of spores disintegrated and, generally, germination (

Many Proteaceae are highly phosphorus (P)-sensitive and occur exclusively on old nutrient-impoverished acidic soils (calcifuge), whilst a few also occur on young calcareous soils (soil-indifferent) that are higher in available calcium (Ca) and P. Calcium increases the severity of P-toxicity symptoms, but its underlying mechanisms are unknown. We propose that Ca-enhanced P toxicity explains the calcifuge habit of most Proteaceae. Four calcifuge and four soil-indifferent Proteaceae from South-Western Australia were grown in hydroponics, at a range of P and Ca concentrations. Calcium increased the severity of P-toxicity symptoms in all species. Calcifuge Proteaceae were more sensitive to Ca-enhanced P toxicity than soil-indifferent ones. Calcifuges shared these traits: low leaf zinc concentration ([Zn]), low Zn allocation to leaves, low leaf [Zn]:[P], low root : shoot ratio, and high seed P content, compared with soil-indifferent species. This is the first demonstration of Ca-enhanced P toxicity across multiple species. Calcium-enhanced P toxicity provides an explanation for the calcifuge habit of most Proteaceae and is critical for the management of this iconic Australian family. This study represents a major advance towards an understanding of the physiological mechanisms of P toxicity and its role in the distribution of Proteaceae.

Phosphorus (P)-deficiency symptoms are known for Lupinus species grown in calcareous soil, but we do not know if this is due to a high calcium (Ca) availability or a low P availability in the soil. To address this problem, we explored both the effects of Ca and its interactions with P on nutrient status and growth of three Lupinus species. Two Ca-sensitive genotypes (L. angustifolius L. P26723 and L. cosentinii Guss. P27225) and two Ca-tolerant genotypes (L. angustifolius L. cv Mandelup and L. pilosus Murr. P27440) were grown hydroponically at two P (0.1 and 10 μM) and three Ca (0.1, 0.6 and 6 mM) levels. Leaf symptoms and biomass were recorded, whole leaf and root nutrient concentrations were analysed, and leaf cellular P and Ca concentrations were determined. Phosphorus-deficiency symptoms were only observed in the Ca-sensitive genotypes. Among all the genotypes in this study, the Ca-tolerant L. pilosus showed an ability to maintain stable leaf Ca and P concentrations whereas the Ca-tolerant L. angustifolius cv Mandelup did not maintain a stable whole leaf Ca concentration, but maintained a low cytosolic Ca2+ concentration through effective Ca compartmentation. However, the two Ca-sensitive genotypes, L. angustifolius P26723 and L. cosentinii, did not exhibit an ability to maintain a stable whole leaf Ca concentration or effectively compartmentalise Ca. Therefore, having the capacity to maintain a stable whole leaf Ca concentration or effectively compartmentalising Ca in leaves are likely critical for Lupinus species to be Ca tolerant.

Plants allocate nutrients to specific leaf cell types, with commelinoid monocots preferentially allocating phosphorus (P) to the mesophyll and calcium (Ca) to the epidermis, whereas the opposite is thought to occur in eudicots. However, Proteaceae from severely P-impoverished habitats present the same P-allocation pattern as monocots. This raises the question of whether preferential P allocation to mesophyll cells is a phylogenetically conserved trait, exclusive to commelinoid monocots and a few Proteaceae, or a trait that has evolved multiple times to allow plants to cope with very low soil P availability. We analysed the P-allocation patterns of 16 species from 10 genera, eight families and six orders within three major clades of eudicots across different P-impoverished environments in Australia and Brazil, using elemental X-ray mapping to quantitatively determine leaf cell-specific nutrient concentrations. Many of the analysed species showed P-allocation patterns that differed substantially from that expected for eudicots. Instead, P-allocation patterns were strongly associated with the P availability in the natural habitat of the species, suggesting a convergent evolution of P-allocation patterns at the cellular level, with P limitation as selective pressure and without a consistent P-allocation pattern within eudicots. Here, we show that most eudicots from severely P-impoverished environments preferentially allocated P to their mesophyll. We surmise that this preferential P allocation to photosynthetically active cells might contribute to the very high photosynthetic P-use efficiency of species adapted to P-impoverished habitats.

To study mechanism underpinning the calcifuge habit of some Lupinus species, especially under low-phosphorus (P) conditions, Lupinus species that were likely to respond differently to calcium (Ca) availability were assembled, and the sensitivity to Ca under a low-P supply was assessed. Seven Lupinus species (9 genotypes, L. albus L. cv Kiev, L. albus L. P26766, L. angustifolius L. cv Mandelup, L. angustifolius L. P26723, L. luteus L. cv Pootalong, L. hispanicus ssp. bicolor Boiss. and Reut. P22999, L. pilosus Murr. P27440, L. cosentinii Guss. P27225, and L. atlanticus Gladst. P27219) were grown hydroponically at 10 or 6000 μM Ca. Leaf symptoms, gas exchange and biomass were recorded; leaf and root nutrient concentrations were analysed, and the leaf cell types in which Ca and P accumulated were determined using elemental X-ray microanalyses. Calcium toxicity was demonstrated for L. angustifolius P26723, L. hispanicus ssp. bicolor. P22999, and L. cosentinii P27225, whereas the other species were tolerant of a high Ca supply under low-P conditions. In addition, genotypic differences in Ca toxicity were found within L. angustifolius. Most Ca accumulated in the mesophyll cells in all species, whereas most P was located in epidermal cells.

Plants allocate nutrients to specific leaf cell types; eudicots are thought to predominantly allocate phosphorus (P) to epidermal/bundle sheath cells. However, three Proteaceae species have been shown to preferentially allocate P to mesophyll cells instead. These Proteaceae species are highly adapted to P-impoverished habitats, with exceptionally high photosynthetic P-use efficiencies (PPUE). We hypothesized that preferential allocation of P to photosynthetic mesophyll cells is an important trait in species adapted to extremely P-impoverished habitats, contributing to their high PPUE. We used elemental X-ray mapping to determine leaf cell-specific nutrient concentrations for 12 Proteaceae species, from habitats of strongly contrasting soil P concentrations, in Australia, Brazil, and Chile. We found that only species from extremely P-impoverished habitats preferentially allocated P to photosynthetic mesophyll cells, suggesting it has evolved as an adaptation to their extremely P-impoverished habitat and that it is not a family-wide trait. Our results highlight the possible role of soil P in driving the evolution of ecologically relevant nutrient allocation patterns and that these patterns cannot be generalized across families. Furthermore, preferential allocation of P to photosynthetic cells may provide new and exciting strategies to improve PPUE in crop species.

Summary � The ability of plants to compete effectively for nitrogen (N) resources is critical to plant survival. However, controversy surrounds the importance of organic and inorganic sources of N in plant nutrition because of our poor ability to visualize and understand processes happening at the root–microbial–soil interface. � Using high-resolution nano-scale secondary ion mass spectrometry stable isotope imaging (NanoSIMS-SII), we quantified the fate of 15 N over both space and time within the rhizosphere. We pulse-labelled the soil surrounding wheat (Triticum aestivum) roots with either 15 NH þ 4 or 15 N-glutamate and traced the movement of 15 N over 24 h. � Imaging revealed that glutamate was rapidly depleted from the rhizosphere and that most 15 N was captured by rhizobacteria, leading to very high 15 N microbial enrichment. After microbial capture, approximately half of the 15 N-glutamate was rapidly mineralized, leading to the excretion of NH þ , which became available for plant capture. Roots proved to be poor competitors for 15 N-glutamate and took up N mainly as 15 NH þ . Spatial mapping of 15 N revealed differential patterns of 15 N uptake within bacteria and the rapid uptake and redistribution of 15 N within roots. � In conclusion, we demonstrate the rapid cycling and transformation of N at the soil–root interface and that wheat capture of organic N is low in comparison to inorganic N under the conditions tested.

As part of an investigation of the effects of water quality from dredging/natural resuspension on reefs, the effects of suspended sediment concentrations (SSCs) (0, 30, 100mgL-1) and light (~0, 1.1, 8.6molphotonsm-2d-1) were examined alone and in combination, on the corals Acropora millepora, Montipora capricornis and Porites spp. over an extended (28d) period. No effects were observed at any sediment concentrations when applied alone. All corals in the lowest light treatments lost chlorophyll a and discoloured (bleached) after a week. Coral mortality only occurred in the two lowest light treatments and was higher when simultaneously exposed to elevated SSCs. Compared to water quality data collected during large dredging programs and natural resuspension events (and in the absence of sediment deposition as a cause-effect pathway) these data suggest the light reduction associated with turbidity poses a proportionally greater risk than effects of elevated SSCs alone.

3D imaging exposes deformities and shows how ocean acidification can change the way juvenile corals build their skeletons., Rising atmospheric CO2 is causing the oceans to both warm and acidify, which could reduce the calcification rates of corals globally. Successful coral recruitment and high rates of juvenile calcification are critical to the replenishment and ultimate viability of coral reef ecosystems. Although elevated Pco2 (partial pressure of CO2) has been shown to reduce the skeletal weight of coral recruits, the structural changes caused by acidification during initial skeletal deposition are unknown. We show, using high-resolution three-dimensional x-ray microscopy, that ocean acidification (Pco2 ~900 μatm, pH ~7.7) not only causes reduced overall mineral deposition but also a deformed and porous skeletal structure in newly settled coral recruits. In contrast, elevated temperature (+3°C) had little effect on skeletal formation except to partially mitigate the effects of elevated Pco2. The striking structural deformities we observed show that new recruits are at significant risk, being unable to effectively build their skeletons in the Pco2 conditions predicted to occur for open ocean surface waters under a “business-as-usual” emissions scenario [RCP (representative concentration pathway) 8.5] by the year 2100.

Suspended sediments produced from dredging activities, or added to the sediment budget via river runoff, are a concern for marine resource managers. Understanding the impact of suspended sediments on critical life history stages of keystone species like corals is fundamental to effective management of coastlines and reefs. Coral embryos (Acropora tenuis and A. millepora) and larvae (A. tenuis, A. millepora and Pocillopora acuta) were subjected to a range of suspended sediment concentrations of different sediment types (siliciclastic and carbonate) to assess concentration-response relationships on ecologically relevant endpoints, including survivorship and ability to metamorphose. Embryos were subjected to short (12 h) suspended sediment exposures from ages of 3-12 hours old or a long (30 h) exposure at 6 hours old. Neither the survivorship nor metamorphosis function of embryos were significantly affected by realistic sediment exposures to ~1000 mg L-1. However, some embryos exhibited a previously undescribed response to dynamically suspended sediments, which saw 10% of the embryos form negatively buoyant cocoons at siliciclastic suspended sediment concentrations ≥35 mg L-1. Scanning electron and optical microscopy confirmed the presence of a coating on these embryos, possibly mucus with incorporated sediment particles. Cocoon formation was common in embryos but not in larvae, and occurred more often after exposure to siliciclastic rather than carbonate sediments. Once transferred into sediment-free seawater, functional ~36-h-old embryos began emerging from the cocoons, coinciding with cilia development. Ciliated (> 36-h-old) larvae exposed to suspended sediments for 60 h were also observed to secrete mucus and were similarly unaffected by suspended sediment concentrations to ~800 mg L-1. This study provides evidence that mucous secretion and cilia beating effectively protect coral embryos and larvae from suspended sediment and that these mechanisms may enhance their chances of successful recruitment.

Suspended sediment from dredging activities and natural resuspension events represent a risk to the reproductive processes of coral and therefore the ongoing maintenance of reefal populations. To investigate the underlying mechanisms that could reduce the fertilisation success in turbid water, we conducted several experiments exposing gametes of the corals Acropora tenuis and A. millepora to two sediment types. Sperm limitation was identified in the presence of siliciclastic sediment (230 and ~700 mg L−1), with 2–37 fold more sperm required to achieve maximum fertilisation rates, when compared with sediment-free treatments. This effect was more pronounced at sub-optimum sperm concentrations. Considerable (>45%) decreases in sperm concentration at the water’s surface was recorded in the presence of siliciclastic sediment and a >20% decrease for carbonate sediment. Electron microscopy then confirmed sediment entangled sperm and we propose entrapment and sinking is the primary mechanism reducing sperm available to the egg. Longer exposure to suspended sediments and gamete aging further decreased fertilisation success when compared with a shorter exposure. Collectively, these findings demonstrate that high concentrations of suspended sediments effectively remove sperm from the water’s surface during coral spawning events, reducing the window for fertilisation with potential subsequent flow-on effects for recruitment.

Background and aims Sclerotinia stem rot (SSR, Sclerotinia sclerotiorum) is a damaging disease of oilseed brassicas world-wide. Host resistance is urgently needed to achieve control, yet the factors that contribute to stem resistance are not well understood. This study investigated the mechanisms of resistance to SSR. Methods Stems of 5-week-old Brassica carinata, B. juncea and B. napus of known resistance were infected via filter paper discs impregnated with S. sclerotiorum mycelium under controlled conditions. Transverse sections of the stem and portions of the stem surface were examined using optical and scanning electron microscopy. The association of anatomical features with the severity of disease (measured by mean lesion length) was determined. Key results Several distinct resistance mechanisms were recorded for the first time in these Brassica-pathogen interactions, including hypersensitive reactions and lignification within the stem cortex, endodermis and in tissues surrounding the lesions. Genotypes showing a strong lignification response 72 h post-infection (hpi) tended to have smaller lesions. Extensive vascular invasion by S. sclerotiorum was observed only in susceptible genotypes, especially in the vascular fibres and xylem. Mean lesion length was negatively correlated with the number of cell layers in the cortex, suggesting progress of S. sclerotiorum is impeded by more cell layers. Hyphae in the centre of lesions became highly vacuolate 72 hpi, reflecting an ageing process in S. sclerotiorum hyphal networks that was independent of host resistance. The infection process of S. sclerotiorum was analogous in B. carinata and B. napus. Infection cushions of the highly virulent isolate of S. sclerotiorum MBRS-1 were grouped together in dense parallel bundles, while hyphae in the infection cushions of a less aggressive isolate WW-3 were more diffuse, and this was unaffected by host genotype. Conclusions A variety of mechanisms contribute to host resistance against S. sclerotiorum across the three Brassica species. These complex interactions between pathogen and host help to explain variable expressions of resistance often observed in the field.