Application of Metabolomics Tools to Determine Possible Biomarker Metabolites Linked to Leaf Blackening in Protea.

The postharvesting disorder leaf blackening is the main cause of product rejection in Protea during export. In this study, we report an investigation into metabolites associated with leaf blackening in Protea species. Methanol extracts of leaf and involucral bract tissue were analyzed by liquid chromatography hyphenated to photodiode array and high-resolution mass spectrometry (LC-PDA-HRMS), where 116 features were annotated. Analytical data obtained from 37 Protea species, selections, and hybrids were investigated using metabolomics tools, which showed that stems susceptible to leaf blackening cluster together and contained features identified as benzenetriol- and/or hydroquinone-derived metabolites. On the other hand, species, selections, and cultivars not prone to blackening were linked to metabolites with known protective properties against biotic and abiotic stressors. During the browning process, susceptible cultivars also produce these protective metabolites, yet at innately low levels, which may render these species and cultivars more vulnerable to blackening. Metabolites that were found to be correlated to the instigation of the browning process, all comprising benzenetriol- and hydroquinone-glycoside derivatives, are highlighted to provide preliminary insights to guide the development of new Protea cultivars not susceptible to leaf blackening.

High nutrient-use efficiency during early seedling growth in diverse Grevillea species (Proteaceae).

Several hypotheses have been proposed to explain the rich floristic diversity in regions characterised by nutrient-impoverished soils; however, none of these hypotheses have been able to explain the rapid diversification over a relatively short evolutionary time period of Grevillea, an Australian plant genus with 452 recognised species/subspecies and only 11 million years of evolutionary history. Here, we hypothesise that the apparent evolutionary success of Grevillea might have been triggered by the highly efficient use of key nutrients. The nutrient content in the seeds and nutrient-use efficiency during early seedling growth of 12 species of Grevillea were compared with those of 24 species of Hakea, a closely related genus. Compared with Hakea, the Grevillea species achieved similar growth rates (root and shoot length) during the early stages of seedling growth but contained only approximately half of the seed nutrient content. We conclude that the high nutrient-use efficiency observed in Grevillea might have provided a selective advantage in nutrient-poor ecosystems during evolution and that this property likely contributed to the evolutionary success in Grevillea.

Lipid biosynthesis and protein concentration respond uniquely to phosphate supply during leaf development in highly phosphorus-efficient Hakea prostrata.

Hakea prostrata (Proteaceae) is adapted to severely phosphorus-impoverished soils and extensively replaces phospholipids during leaf development. We investigated how polar lipid profiles change during leaf development and in response to external phosphate supply. Leaf size was unaffected by a moderate increase in phosphate supply. However, leaf protein concentration increased by more than 2-fold in young and mature leaves, indicating that phosphate stimulates protein synthesis. Orthologs of known lipid-remodeling genes in Arabidopsis (Arabidopsis thaliana) were identified in the H. prostrata transcriptome. Their transcript profiles in young and mature leaves were analyzed in response to phosphate supply alongside changes in polar lipid fractions. In young leaves of phosphate-limited plants, phosphatidylcholine/phosphatidylethanolamine and associated transcript levels were higher, while phosphatidylglycerol and sulfolipid levels were lower than in mature leaves, consistent with low photosynthetic rates and delayed chloroplast development. Phosphate reduced galactolipid and increased phospholipid concentrations in mature leaves, with concomitant changes in the expression of only four H. prostrata genes, GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE1, N-METHYLTRANSFERASE2, NONSPECIFIC PHOSPHOLIPASE C4, and MONOGALACTOSYLDIACYLGLYCEROL3. Remarkably, phosphatidylglycerol levels decreased with increasing phosphate supply and were associated with lower photosynthetic rates. Levels of polar lipids with highly unsaturated 32:x (x = number of double bonds in hydrocarbon chain) and 34:x acyl chains increased. We conclude that a regulatory network with a small number of central hubs underpins extensive phospholipid replacement during leaf development in H. prostrata. This hard-wired regulatory framework allows increased photosynthetic phosphorus use efficiency and growth in a low-phosphate environment. This may have rendered H. prostrata lipid metabolism unable to adjust to higher internal phosphate concentrations.

Does cluster-root activity benefit nutrient uptake and growth of co-existing species?

Species that inhabit phosphorus- (P) and micronutrient-impoverished soils typically have adaptations to enhance the acquisition of these nutrients, for example cluster roots in Proteaceae. However, there are several species co-occurring in the same environment that do not produce similar specialised roots. This study aims to investigate whether one of these species (Scholtzia involucrata) can benefit from the mobilisation of P or micronutrients by the cluster roots of co-occurring Banksia attenuata, and also to examine the response of B. attenuata to the presence of S. involucrata. We conducted a greenhouse experiment, using a replacement series design, where B. attenuata and S. involucrata shared a pot at proportions of 2:0, 1:2 and 0:4. S. involucrata plants grew more in length, were heavier and had higher manganese (Mn) concentrations in their young leaves when grown next to one individual of B. attenuata and one individual of S. involucrata than when grown with three conspecifics. All S. involucrata individuals were colonised by arbuscular mycorrhizal fungi, and possibly Rhizoctonia. Additionally, P concentration was higher in the young leaves of B. attenuata when grown with another B. attenuata than when grown with two individuals of S. involucrata, despite the smaller size of the S. involucrata individuals. Our results demonstrate that intraspecific competition was stronger than interspecific competition for S. involucrata, but not for B. attenuata. We conclude that cluster roots of B. attenuata facilitate the acquisition of nutrients by neighbouring shrubs by making P and Mn more available for their neighbours.

Soil nitrogen, and not phosphorus, promotes cluster-root formation in a South American Proteaceae, Embothrium coccineum.

PREMISE OF THE STUDY: Cluster roots are a characteristic root adaptation of Proteaceae species. In South African and Australian species, cluster roots promote phosphorus (P) acquisition from poor soils. In a South American Proteaceae species, where cluster roots have been scarcely studied and their function is unknown, we tested whether cluster-root formation is stimulated by low soil nutrition, in particular low P-availability. METHODS: Small and large seedlings (< 6- and > 6-months old, respectively) of Embothrium coccineum and soil were collected across four different sites in Patagonia (Chile). We determined cluster-root number and relative mass, and leaf Pi concentration per mass (Pimass) and per area (Piarea) for each seedling, and tested relationships with Olsen-P (OP), sorbed-P (sP) and total nitrogen (N) using generalized linear mixed-effects models and model selection to assess the relative strength of soil and plant drivers. KEY RESULTS: Best-fit models showed a negative logarithmic relationship between cluster-root number and soil nitrogen (N), and between cluster-root relative mass and both leaf Piarea and soil N, and a positive logarithmic relationship between cluster-root number and leaf Piarea. Cluster-root relative mass was higher in small than in large seedlings. CONCLUSIONS: Contrary to that found in South African and Australian Proteaceae, cluster roots of E. coccineum do not appear to be driven by soil P, but rather by soil N and leaf Piarea. We suggest that cluster roots are a constitutive and functional trait that allows plants to prevail in poor N soils.

Downregulation of net phosphorus-uptake capacity is inversely related to leaf phosphorus-resorption proficiency in four species from a phosphorus-impoverished environment.

BACKGROUND AND AIMS: Previous research has suggested a trade-off between the capacity of plants to downregulate their phosphorus (P) uptake capacity and their efficiency of P resorption from senescent leaves in species from P-impoverished environments. METHODS: To investigate this further, four Australian native species (Banksia attenuata, B. menziesii, Acacia truncata and A. xanthina) were grown in a greenhouse in nutrient solutions at a range of P concentrations [P]. Acacia plants received between 0 and 500 µm P; Banksia plants received between 0 and 10 µm P, to avoid major P-toxicity symptoms in these highly P-sensitive species. KEY RESULTS: For both Acacia species, the net P-uptake rates measured at 10 µm P decreased steadily with increasing P supply during growth. In contrast, in B. attenuata, the net rate of P uptake from a solution with 10 µm P increased linearly with increasing P supply during growth. The P-uptake rate of B. menziesii showed no significant response to P supply in the growing medium. Leaf [P] of the four species supported this finding, with A. truncata and A. xanthina showing an increase up to a saturation value of 19 and 21 mg P g(-1) leaf dry mass, respectively (at 500 µm P), whereas B. attenuata and B. menziesii both exhibited a linear increase in leaf [P], reaching 10 and 13 mg P g(-1) leaf dry mass, respectively, without approaching a saturation point. The Banksia plants grown at 10 µm P showed mild symptoms of P toxicity, i.e. yellow spots on some leaves and drying and curling of the tips of the leaves. Leaf P-resorption efficiency was 69 % (B. attenuata), 73 % (B. menziesii), 34 % (A. truncata) and 36 % (A. xanthina). The P-resorption proficiency values were 0·08 mg P g(-1) leaf dry mass (B. attenuata and B. menziesii), 0·32 mg P g(-1) leaf dry mass (A. truncata) and 0·36 mg P g(-1) leaf dry mass (A. xanthina). Combining the present results with additional information on P-remobilization efficiency and the capacity to downregulate P-uptake capacity for two other Australian woody species, we found a strong negative correlation between these traits. CONCLUSIONS: It is concluded that species that are adapted to extremely P-impoverished soils, such as many south-western Australian Proteaceae species, have developed extremely high P-resorption efficiencies, but lost their capacity to downregulate their P-uptake mechanisms. The results support the hypothesis that the ability to resorb P from senescing leaves is inversely related to the capacity to downregulate net P uptake, possibly because constitutive synthesis of P transporters is a prerequisite for proficient P remobilization from senescing tissues.

Fire-adapted Gondwanan Angiosperm floras evolved in the Cretaceous.

BACKGROUND: Fires have been widespread over the last 250 million years, peaking 60-125 million years ago (Ma), and might therefore have played a key role in the evolution of Angiosperms. Yet it is commonly believed that fireprone communities existed only after the global climate became more arid and seasonal 15 Ma. Recent molecular-based studies point to much earlier origins of fireprone Angiosperm floras in Australia and South Africa (to 60 Ma, Paleocene) but even these were constrained by the ages of the clades examined. RESULTS: Using a molecular-dated phylogeny for the great Gondwanan family Proteaceae, with a 113-million-year evolutionary history, we show that the ancestors of many of its characteristic sclerophyll genera, such as Protea, Conospermum, Leucadendron, Petrophile, Adenanthos and Leucospermum (all subfamily Proteoideae), occurred in fireprone habitats from 88 Ma (83-94, 95% HPD, Mid-Upper Cretaceous). This coincided with the highest atmospheric oxygen (combustibility) levels experienced over the past 150 million years. Migration from non-fireprone (essentially rainforest-climate-type) environments was accompanied by the evolution of highly speciose clades with a range of seed storage traits and fire-cued seed release or germination mechanisms that was diagnostic for each clade by 71 Ma, though the ant-dispersed lineage (as a soil seed-storage subclade) was delayed until 45 Ma. CONCLUSIONS: Focusing on the widespread 113-million-year-old family Proteaceae, fireproneness among Gondwanan Angiosperm floras can now be traced back almost 90 million years into the fiery Cretaceous. The associated evolution of on-plant (serotiny) and soil seed storage, and later ant dispersal, affirms them as ancient adaptations to fire among flowering plants.

Phosphorus-mobilization ecosystem engineering: the roles of cluster roots and carboxylate exudation in young P-limited ecosystems.

BACKGROUND: Carboxylate-releasing cluster roots of Proteaceae play a key role in acquiring phosphorus (P) from ancient nutrient-impoverished soils in Australia. However, cluster roots are also found in Proteaceae on young, P-rich soils in Chile where they allow P acquisition from soils that strongly sorb P. SCOPE: Unlike Proteaceae in Australia that tend to proficiently remobilize P from senescent leaves, Chilean Proteaceae produce leaf litter rich in P. Consequently, they may act as ecosystem engineers, providing P for plants without specialized roots to access sorbed P. We propose a similar ecosystem-engineering role for species that release large amounts of carboxylates in other relatively young, strongly P-sorbing substrates, e.g. young acidic volcanic deposits and calcareous dunes. Many of these species also fix atmospheric nitrogen and release nutrient-rich litter, but their role as ecosystem engineers is commonly ascribed only to their diazotrophic nature. CONCLUSIONS: We propose that the P-mobilizing capacity of Proteaceae on young soils, which contain an abundance of P, but where P is poorly available, in combination with inefficient nutrient remobilization from senescing leaves allows these species to function as ecosystem engineers. We suggest that diazotrophic species that colonize young soils with strong P-sorption potential should be considered for their positive effect on P availability, as well as their widely accepted role in nitrogen fixation. Their P-mobilizing activity possibly also enhances their nitrogen-fixing capacity. These diazotrophic species may therefore facilitate the establishment and growth of species with less-efficient P-uptake strategies on more-developed soils with low P availability through similar mechanisms. We argue that the significance of cluster roots and high carboxylate exudation in the development of young ecosystems is probably far more important than has been envisaged thus far.

Root of edaphically controlled Proteaceae turnover on the Agulhas Plain, South Africa: phosphate uptake regulation and growth.

The influence of phosphorus (P) availability on growth and P uptake was investigated in South African Proteaceae: (1) Protea compacta R.Br., endemic on severely nutrient-impoverished colluvial sands; (2) Protea obtusifolia Bueck ex Meissner; and (3) Leucadendron meridianum I. J. Williams, the latter both endemic on comparatively fertile limestone-derived soils. Plants were grown hydroponically in 1000 L tanks at 0.01, 0.1 or 1.0 microm P for 14 weeks. Biomass accumulation was influenced by P availability, doubling as [P] increased from 0.1 to 1.0 microm. Total biomass was greatest for P. compacta, but L. meridianum and P. obtusifolia had two to four times greater relative biomass accumulation at 0.1 and 1.0 microm [P]. Proteoid root clusters developed at both 0.01 and 0.1 microm[P], but were suppressed at 1.0 microm [P]; this was a 10-fold lower [P] than previously reported to inhibit cluster root formation. Rates of net P uptake at 5 microm P decreased in response to increased P availability from 0.01 to 1.0 microm P. Significant between-species differences in rates of P uptake and capacity to down-regulate P uptake were observed: P. compacta < P. obtusifolia < L. meridianum. The species responses are discussed in terms of adaptation to mosaics in soil P availability and the high beta diversity in the natural habitat.

Banksia species (Proteaceae) from severely phosphorus-impoverished soils exhibit extreme efficiency in the use and re-mobilization of phosphorus.

Banksia species (Proteaceae) occur on some of the most phosphorus (P)-impoverished soils in the world. We hypothesized that Banksia spp. maximize P-use efficiency through high photosynthetic P-use efficiency, long leaf lifespan (P residence time), effective P re-mobilization from senescing leaves, and maximizing seed P concentration. Field and glasshouse experiments were conducted to quantify P-use efficiency in nine Banksia species. Leaf P concentrations for all species were extremely low (0.14-0.32 mg P g(-1) DM) compared with leaf P in other species reported and low relative to other plant nutrients in Banksia spp.; however, moderately high rates of photosynthesis (13.8-21.7 micromol CO2 m(-2) s(-1)), were measured. Some of the Banksia spp. had greater P proficiency (i.e. final P concentration in senesced leaves after re-mobilization; range: 27-196 microg P g(-1) DM) than values reported for any other species in the literature. Seeds exhibited significantly higher P concentrations (6.6-12.2 mg P g(-1 )DM) than leaves, and species that sprout after fire ('re-sprouters') had significantly greater seed mass and P content than species that are killed by fire and regenerate from seed ('seeders'). Seeds contained only small amounts of polyphosphate (between 1.3 and 6 microg g(-1) DM), and this was not correlated with P concentration or fire response. Based on the evidence in the present study, we conclude that Banksia species are highly efficient in their use of P, explaining, in part, their success on P-impoverished soils, with little variation between species.

Increased ecological amplitude through heterosis following wide outcrossing in Banksia ilicifolia R.Br. (Proteaceae).

To assess whether wide outcrossing (over 30 km) in the naturally fragmented Banksia ilicifolia R.Br. increases the ecological amplitude of offspring, we performed a comparative greenhouse growth study involving seedlings of three hand-pollinated progeny classes (self, local outcross, wide outcross) and a range of substrates and stress conditions. Outcrossed seedlings outperformed selfed seedlings, with the magnitude of inbreeding depression as high as 62% for seed germination and 37% for leaf area. Wide outcrossed seedlings outperformed local outcrossed seedlings, especially in non-native soils, facilitated in part by an improved capacity to overcome soil constraints through greater root carboxylate exudation. Soil type significantly affected seedling growth, and waterlogging and water deficit decreased growth, production of cluster roots, root exudation and total plant P uptake. Our results suggest that the interaction of narrow ecological amplitude and the genetic consequences of small fragmented populations may in part explain the narrow range of local endemics, but that wide outcrossing may provide opportunities for increased genetic variation, increased ecological amplitude and range expansion.

Systemic suppression of cluster-root formation and net P-uptake rates in Grevillea crithmifolia at elevated P supply: a proteacean with resistance for developing symptoms of 'P toxicity'.

Grevillea crithmifolia R. Br. is a species of Proteaceae that is resistant to developing P-toxicity symptoms at phosphorus supplies in the root environment that induce P-toxicity symptoms in the closely related Hakea prostrata (Proteaceae). It was discovered previously that development of P-toxicity symptoms in H. prostrata is related to its low capacity to down-regulate net P-uptake rates (i.e. its low plasticity). The plasticity of net P-uptake rates and whole-plant growth responses in G. crithmifolia has now been assessed in two separate experiments: (i) a range of P, from 0 to 200 micromol P d-1, was supplied to whole root systems; (ii) using a split-root design, one root half was supplied with 0, 3, 75, or 225 micromol P d-1, while the other root half invariably received 3 micromol P d-1. Fresh mass was significantly greater in G. crithmifolia plants that had received a greater daily P supply during the pretreatments, but symptoms of P toxicity were never observed. Cluster-root growth decreased from about half the total root fresh mass when the leaf [P] was lowest (c. 0.1 mg P g-1 DM) to complete suppression of cluster-root growth when leaf [P] was 1-2 mg P g-1 DM. Split-root studies revealed that cluster-root initiation and growth, and net P-uptake rates by roots were regulated systemically, possibly by shoot P concentration. It is concluded that, in response to higher P supply, G. crithmifolia does not develop symptoms of P toxicity because of (i) greater plasticity of its net P-uptake capacity, and (ii) its greater plasticity for allocating P to growth and P storage in roots. This ecologically important difference in plasticity is most probably related to a slightly higher nutrient availability in the natural habitat of G. crithmifolia when compared with that of H. prostrata.

Developmental physiology of cluster-root carboxylate synthesis and exudation in harsh hakea. Expression of phosphoenolpyruvate carboxylase and the alternative oxidase.

Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrient-impoverished soils in southwest Australia. When phosphorus is limiting, harsh hakea develops proteoid or cluster roots that release carboxylates that mobilize sparingly soluble phosphate in the rhizosphere. To investigate the physiology underlying the synthesis and exudation of carboxylates from cluster roots in Proteaceae, we measured O2 consumption, CO2 release, internal carboxylate concentrations and carboxylate exudation, and the abundance of the enzymes phosphoenolpyruvate carboxylase and alternative oxidase (AOX) over a 3-week time course of cluster-root development. Peak rates of citrate and malate exudation were observed from 12- to 13-d-old cluster roots, preceded by a reduction in cluster-root total protein levels and a reduced rate of O2 consumption. In harsh hakea, phosphoenolpyruvate carboxylase expression was relatively constant in cluster roots, regardless of developmental stage. During cluster-root maturation, however, the expression of AOX protein increased prior to the time when citrate and malate exudation peaked. This increase in AOX protein levels is presumably needed to allow a greater flow of electrons through the mitochondrial electron transport chain in the absence of rapid ATP turnover. Citrate and isocitrate synthesis and accumulation contributed in a major way to the subsequent burst of citrate and malate exudation. Phosphorus accumulated by harsh hakea cluster roots was remobilized during senescence as part of their efficient P cycling strategy for growth on nutrient impoverished soils.

Tissue and cellular phosphorus storage during development of phosphorus toxicity in Hakea prostrata (Proteaceae).

Storage of phosphorus (P) in stem tissue is important in Mediterranean Proteaceae, because proteoid root growth and P uptake is greatest during winter, whereas shoot growth occurs mostly in summer. This has prompted the present investigation of the P distribution amongst roots, stems, and leaves of Hakea prostrata R.Br. (Proteaceae) when grown in nutrient solutions at ten P-supply rates. Glasshouse experiments were carried out during both winter and summer months. For plants grown in the low-P range (0, 0.3, 1.2, 3.0, or 6.0 micromol d(-1)) the root [P] was > stem and leaf [P]. In contrast, leaf [P] > stem and root [P] for plants grown in the high-P range (6.0, 30, 60, 150, or 300 micromol P d(-1)). At the highest P-supply rates, the capacity for P storage in stems and roots appears to have been exceeded, and leaf [P] thereafter increased dramatically to approximately 10 mg P g(-1) dry mass. This high leaf [P] was coincident with foliar symptoms of P toxicity which were similar to those described for many other species, including non-Proteaceae. The published values (tissue [P]) at which P toxicity occurs in a range of species are summarized. X-ray microanalysis of frozen, full-hydrated leaves revealed that the [P] in vacuoles of epidermal, palisade and bundle-sheath cells were in the mM range when plants were grown at low P-supply, even though very low leaf [P] was measured in bulk leaf samples. At higher P-supply rates, P accumulated in vacuoles of palisade cells which were associated with decreased photosynthetic rates.

Fast pollen tube growth in Conospermum species.

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.