Light-dependent activation of phosphoenolpyruvate carboxylase by reversible phosphorylation in cluster roots of white lupin plants: diurnal control in response to photosynthate supply.

Background and Aims Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated enzyme that controls carbohydrate partitioning to organic acid anions (malate, citrate) excreted in copious amounts by cluster roots of inorganic phosphate (Pi)-deprived white lupin plants. Excreted malate and citrate solubilize otherwise inaccessible sources of mineralized soil Pi for plant uptake. The aim of this study was to test the hypotheses that (1) PEPC is post-translationally activated by reversible phosphorylation in cluster roots of illuminated white lupin plants, and (2) light-dependent phosphorylation of cluster root PEPC is associated with elevated intracellular levels of sucrose and its signalling metabolite, trehalose-6-phosphate. Methods White lupin plants were cultivated hydroponically at low Pi levels (≤1 µm) and subjected to various light/dark pretreatments. Cluster root PEPC activity and in vivo phosphorylation status were analysed to assess the enzyme's diurnal, post-translational control in response to light and dark. Levels of various metabolites, including sucrose and trehalose-6-phosphate, were also quantified in cluster root extracts using enzymatic and spectrometric methods. Key Results During the daytime the cluster root PEPC was activated by phosphorylation at its conserved N-terminal seryl residue. Darkness triggered a progressive reduction in PEPC phosphorylation to undetectable levels, and this was correlated with 75-80 % decreases in concentrations of sucrose and trehalose-6- phosphate. Conclusions Reversible, light-dependent regulatory PEPC phosphorylation occurs in cluster roots of Pi-deprived white lupin plants. This likely facilitates the well-documented light- and sucrose-dependent exudation of Pi-solubilizing organic acid anions by the cluster roots. PEPC's in vivo phosphorylation status appears to be modulated by sucrose translocated from CO2-fixing leaves into the non-photosynthetic cluster roots.

Nutritional regulation of root development.

Mineral nutrients such as nitrogen (N), phosphorus (P), and iron (Fe) are essential for plant growth, development, and reproduction. Adequate provision of nutrients via the root system impacts greatly on shoot biomass and plant productivity and is therefore of crucial importance for agriculture. Nutrients are taken up at the root surface in ionic form, which is mediated by specific transport proteins. Noteworthy, root tips are able to sense the local and internal concentrations of nutrients to adjust growth and developmental processes, and ultimately, to increase or decrease the exploratory capacity of the root system. Recently, important progress has been achieved in identifying the mechanisms of nutrient sensing in wild- and cultivated species, including Arabidopsis, bean, maize, rice, lupin as well as in members of the Proteaceae and Cyperaceae families, which develop highly sophisticated root clusters as adaptations to survive in soils with very low fertility. Major findings include identification of transporter proteins and transcription factors regulating nutrient sensing, miRNAs as mobile signals and peptides as repressors of lateral root development under heterogeneous nutrient supply. Understanding the roles played by N, P, and Fe in gene expression and biochemical characterization of proteins involved in root developmental responses to homogeneous or heterogeneous N and P sources has gained additional interest due to its potential for improving fertilizer acquisition efficiency in crops.

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

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

Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient harsh hakea.

Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al(3+), Fe(3+), and Ca(2+) phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme's activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced Km [PEP] coupled with elevated I50 [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots.

Opportunities for improving phosphorus-use efficiency in crop plants.

Limitation of grain crop productivity by phosphorus (P) is widespread and will probably increase in the future. Enhanced P efficiency can be achieved by improved uptake of phosphate from soil (P-acquisition efficiency) and by improved productivity per unit P taken up (P-use efficiency). This review focuses on improved P-use efficiency, which can be achieved by plants that have overall lower P concentrations, and by optimal distribution and redistribution of P in the plant allowing maximum growth and biomass allocation to harvestable plant parts. Significant decreases in plant P pools may be possible, for example, through reductions of superfluous ribosomal RNA and replacement of phospholipids by sulfolipids and galactolipids. Improvements in P distribution within the plant may be possible by increased remobilization from tissues that no longer need it (e.g. senescing leaves) and reduced partitioning of P to developing grains. Such changes would prolong and enhance the productive use of P in photosynthesis and have nutritional and environmental benefits. Research considering physiological, metabolic, molecular biological, genetic and phylogenetic aspects of P-use efficiency is urgently needed to allow significant progress to be made in our understanding of this complex trait.

Development and persistence of sandsheaths of Lyginia barbata (Restionaceae): relation to root structural development and longevity.

BACKGROUND AND AIMS: Strongly coherent sandsheaths that envelop perennial roots of many monocotyledonous species of arid environments have been described for over a century. This study, for the first time, details the roles played by the structural development of the subtending roots in the formation and persistence of the sheaths. METHODS: The structural development of root tissues associated with persistent sandsheaths was studied in Lyginia barbata, native to the Western Australian sand plains. Cryo-scanning electron microscopy CSEM, optical microscopy and specific staining methods were applied to fresh, field material. The role of root hairs was clarified by monitoring sheath development in roots separated from the sand profile by fine mesh. KEY RESULTS AND CONCLUSIONS: The formation of the sheaths depends entirely on the numerous living root hairs which extend into the sand and track closely around individual grains enmeshing, by approx. 12 cm from the root tip, a volume of sand more than 14 times that of the subtending root. The longevity of the perennial sheaths depends on the subsequent development of the root hairs and of the epidermis and cortex. Before dying, the root hairs develop cellulosic walls approx. 3 µm thick, incrusted with ferulic acid and lignin, which persist for the life of the sheath. The dead hairs remain in place fused to a persistent platform of sclerified epidermis and outer cortex. The mature cortex comprises this platform, a wide, sclerified inner rim and a lysigenous central region - all dead tissue. We propose that the sandsheath/root hair/epidermis/cortex complex is a structural unit facilitating water and nutrient uptake while the tissues are alive, recycling scarce phosphorus during senescence, and forming, when dead, a persistent essential structure for maintenance of a functional stele in the perennial Lyginia roots.

Seasonal water relations of Lyginia barbata (Southern rush) in relation to root xylem development and summer dormancy of root apices.

*Periods of dormancy in shallow roots allow perennial monocotyledons to establish deep root systems, but we know little about patterns of xylem maturation, water-transport capacities and associated economies in water use of growing and dormant roots. *Xylem development, anatomy, conductance and in situ cellular [K] and [Cl] were investigated in roots of field-grown Lyginia barbata (Restionaceae) in Mediterranean southwestern Australia. Parallel studies of gas exchange, culm relative water loss and soil water content were conducted. *Stomatal conductance and photosynthesis decreased during summer drought as soil profiles dried, but rates recovered when dormant roots became active with the onset of wetter conditions. Anatomical studies identified sites of close juxtaposition of phloem and xylem in dormant and growing roots. Ion data and dye tracing showed mature late metaxylem of growing roots was located >or= 100 mm from the tip, but at only

Summer dormancy and winter growth: root survival strategy in a perennial monocotyledon.

Here, we tested the alternation of root summer dormancy and winter growth as a critical survival strategy for a long-lived monocotyledon (Restionaceae) adapted to harsh seasonal extremes of Mediterranean southwest Western Australia. Measurements of growth and the results of comparative studies of the physiology, water content, metabolites, osmotic adjustments, and proteomics of the dormant and growing perennial roots of Lyginia barbata (Restionaceae) were assessed in field-grown plants. Formation of dormant roots occurred before the onset of summer extremes. They resumed growth (average 2.3 mm d(-1)) the following winter to eventually reach depths of 2-4 m. Compared with winter-growing roots, summer dormant roots had decreased respiration and protein concentration and c. 70% water content, sustained by sand-sheaths, osmotic adjustment and presumably hydraulic redistribution. Concentrations of compatible solutes (e.g. sucrose and proline) were significantly greater during dormancy, presumably mitigating the effects of heat and drought. Fifteen root proteins showed differential abundance and were correlated with either winter growth or summer dormancy. None matched currently available libraries. The specific features of the root dormancy strategy of L. barbata revealed in this study are likely to be important to understanding similar behaviour in roots of many long-lived monocotyledons, including overwintering and oversummering crop species.

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.

Specialized 'dauciform' roots of Cyperaceae are structurally distinct, but functionally analogous with 'cluster' roots.

When grown in nutrient solutions of extremely low [P] (

Root structure and functioning for efficient acquisition of phosphorus: Matching morphological and physiological traits.

BACKGROUND: Global phosphorus (P) reserves are being depleted, with half-depletion predicted to occur between 2040 and 2060. Most of the P applied in fertilizers may be sorbed by soil, and not be available for plants lacking specific adaptations. On the severely P-impoverished soils of south-western Australia and the Cape region in South Africa, non-mycorrhizal species exhibit highly effective adaptations to acquire P. A wide range of these non-mycorrhizal species, belonging to two monocotyledonous and eight dicotyledonous families, produce root clusters. Non-mycorrhizal species with root clusters appear to be particularly effective at accessing P when its availability is extremely low. SCOPE: There is a need to develop crops that are highly effective at acquiring inorganic P (Pi) from P-sorbing soils. Traits such as those found in non-mycorrhizal root-cluster-bearing species in Australia, South Africa and other P-impoverished environments are highly desirable for future crops. Root clusters combine a specialized structure with a specialized metabolism. Native species with such traits could be domesticated or crossed with existing crop species. An alternative approach would be to develop future crops with root clusters based on knowledge of the genes involved in development and functioning of root clusters. CONCLUSIONS: Root clusters offer enormous potential for future research of both a fundamental and a strategic nature. New discoveries of the development and functioning of root clusters in both monocotyledonous and dicotyledonous families are essential to produce new crops with superior P-acquisition traits.

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.

The occurrence of dauciform roots amongst Western Australian reeds, rushes and sedges, and the impact of phosphorus supply on dauciform-root development in Schoenus unispiculatus (Cyperaceae).

* The incidence of species that develop specialised 'dauciform' lateral roots, which are hypothesised to be important for phosphorus (P) acquisition, is uncertain. We investigated their occurrence in Australian reed, rush and sedge species, grown at low P concentration in nutrient solution, and studied the response of Schoenus unispiculatus (Cyperaceae) to a range of P concentrations. * We assessed the fraction of root biomass invested in dauciform roots, their respiration and net P-uptake rate, and the P status of roots and leaves. * Dauciform-root development occurred only in particular genera of Cyperaceae when grown at low P supply. Increased P supply was associated with increased growth of S. unispiculatus and increased leaf [P]. Dauciform-root growth was reduced by increased P supply, and reduced P uptake co-occurred with the complete suppression of dauciform roots. * The P-induced suppression of dauciform roots in Cyperaceae is similar to that observed for proteoid roots in members of Proteaceae and Lupinus albus. The response of dauciform roots to altered P supply and their absence from root systems of some sedge species are discussed in terms of managed and natural systems.

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.