Differences in foliar phosphorus fractions, rather than in cell-specific phosphorus allocation, underlie contrasting photosynthetic phosphorus use efficiency among chickpea genotypes.

Although significant intraspecific variation in photosynthetic phosphorus (P) use efficiency (PPUE) has been shown in numerous species, we still know little about the biochemical basis for differences in PPUE among genotypes within a species. Here, we grew two high PPUE and two low PPUE chickpea (Cicer arietinum) genotypes with low P supply in a glasshouse to compare their photosynthesis-related traits, total foliar P concentration ([P]) and chemical P fractions (i.e. inorganic P (Pi), metabolite P, lipid P, nucleic acid P, and residual P). Foliar cell-specific nutrient concentrations including P were characterized using elemental X-ray microanalysis. Genotypes with high PPUE showed lower total foliar [P] without slower photosynthetic rates. No consistent differences in cellular [P] between the epidermis and mesophyll cells occurred across the four genotypes. In contrast, high PPUE was associated with lower allocation to Pi and metabolite P, with PPUE being negatively correlated with the percentage of these two fractions. Furthermore, a lower allocation to Pi and metabolite P was correlated with a greater allocation to nucleic acid P, but not to lipid P. Collectively, our results suggest that a different allocation to foliar P fractions, rather than preferential P allocation to specific leaf tissues, underlies the contrasting PPUE among chickpea genotypes.

Phosphorus toxicity, not deficiency, explains the calcifuge habit of phosphorus-efficient Proteaceae.

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.

Accumulation of phosphorus and calcium in different cells protects the phosphorus-hyperaccumulator Ptilotus exaltatus from phosphorus toxicity in high-phosphorus soils.

Ptilotus exaltatus accumulates phosphorus (P) to > 40 mg g-1 without toxicity symptoms, while Kennedia prostrata is intolerant of increased P supply. What physiological mechanisms underlie this difference and protect P. exaltatus from P toxicity? Ptilotus exaltatus and K. prostrata were grown in a sandy soil with low-P, high-P and P-pulse treatments. Both species hyperaccumulated P (>20 mg g-1) under high-P and P-pulse treatments; shoot dry weight was unchanged for P. exaltatus, but decreased by >50% for K. prostrata. Under high-P, in young fully-expanded leaves, both species accumulated P predominantly as inorganic P. However, P. exaltatus preferentially allocated P to mesophyll cells and stored calcium (Ca) as occasional crystals in specific lower mesophyll cells, separate from P, while K. prostrata preferentially allocated P to epidermal and spongy mesophyll cells, but co-located P and Ca in palisade mesophyll cells where granules with high [P] and [Ca] were evident. Mesophyll cellular [P] correlated positively with [potassium] for both species, and negatively with [sulfur] for P. exaltatus. Thus, P. exaltatus tolerated a very high leaf [inorganic P] (17 mg g-1), associated with P and Ca allocation to different cell types and formation of Ca crystals, thereby avoiding deleterious precipitation of Ca3(PO4)2. It also showed enhanced [potassium] and decreased [sulfur] to balance high cellular [P]. Phosphorus toxicity in K. prostrata arose from co-location of Ca and P in palisade mesophyll cells. This study advances understanding of leaf physiological mechanisms for high P tolerance in a P-hyperaccumulator and indicates P. exaltatus as a promising candidate for P-phytoextraction.

Role of roots in adaptation of soil-indifferent Proteaceae to calcareous soils in south-western Australia.

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.

Calcium modulates leaf cell-specific phosphorus allocation in Proteaceae from south-western Australia.

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.

Globular structures in roots accumulate phosphorus to extremely high concentrations following phosphorus addition.

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 (<12 mmol kg-1 ). However, in the plants after P addition, some cortex cells contained globular structures extraordinarily rich in P (often >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.

Calcium-enhanced phosphorus toxicity in calcifuge and soil-indifferent Proteaceae along the Jurien Bay chronosequence.

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.

Eudicots from severely phosphorus-impoverished environments preferentially allocate phosphorus to their mesophyll.

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.

Sensitivity of different Lupinus species to calcium under a low phosphorus supply.

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.

Proteaceae from phosphorus-impoverished habitats preferentially allocate phosphorus to photosynthetic cells: An adaptation improving phosphorus-use efficiency.

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.

The chiton stylus canal: An element delivery pathway for tooth cusp biomineralization.

A detailed investigation of the stylus canal situated within the iron mineralized major lateral teeth of the chiton Acanthopleura hirtosa was undertaken in conjunction with a row-by-row examination of cusp mineralization. The canal is shown to contain columnar epithelial tissue similar to that surrounding the mineralized cusps, including the presence of iron rich particles characteristic of the iron storage protein ferritin. Within the tooth core, a previously undescribed internal pathway or plume is evident above the stylus canal, between the junction zone and mineralizing posterior face of the cusp. Plume formation coincides with the appearance of iron in the superior epithelium and the onset of mineralization at tooth row 13. The plume persists during the delivery of phosphorous and calcium into the tooth core, and is the final region of the cusp to become mineralized. The presence of the stylus canal was confirmed in a further 18 chiton species, revealing that the canal is common to polyplacophoran molluscs. These new data strongly support the growing body of evidence highlighting the importance of the junction zone for tooth mineralization in chiton teeth, and indicate that the chemical and structural environment within the tooth cusp is under far greater biological control than previously considered.

A novel method for the study of the biophysical interface in soils using nano-scale secondary ion mass spectrometry.

The spatial location of microorganisms and their activity within the soil matrix have major impacts on biological processes such as nutrient cycling. However, characterizing the biophysical interface in soils is hampered by a lack of techniques at relevant scales. A novel method for studying the distribution of microorganisms that have incorporated isotopically labelled substrate ('active' microorganisms) in relation to the soil microbial habitat is provided by nano-scale secondary ion mass spectrometry (NanoSIMS). Pseudomonas fluorescens are ubiquitous in soil and were therefore used as a model for 'active' microorganisms in soil. Batch cultures (NCTC 10038) were grown in a minimal salt medium containing 15N-ammonium sulphate (15/14N ratio of 1.174), added to quartz-based white sand or soil (coarse textured sand), embedded in Araldite 502 resin and sectioned for NanoSIMS analysis. The 15N-enriched P. fluorescens could be identified within the soil structure, demonstrating that the NanoSIMS technique enables the study of spatial location of microbial activity in relation to the heterogeneous soil matrix. This technique is complementary to the existing techniques of digital imaging analysis of soil thin sections and scanning electron microscopy. Together with advanced computer-aided tomography of soils and mathematical modelling of soil heterogeneity, NanoSIMS may be a powerful tool for studying physical and biological interactions, thereby furthering our understanding of the biophysical interface in soils.