Developmental Physiology of Cluster-Root Carboxylate Synthesis and Exudation in Harsh Hakea. Expression of Phosphoenolpyruvate Carboxylase and the Alternative Oxidase

Abstract

Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrientimpoverished 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 O 2 consumption, CO 2 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 O 2 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.In some plant species, a shortage of phosphorus induces the development of dense clusters of determinate branch roots (rootlets) that arise, en masse, from a localized region of the parent root axis. These short-lived structures have been termed proteoid roots because they were first described for Proteaceae (Purnell, 1960) but have since been found in a wide range of other species and families and are now often referred to as cluster roots . Most of our advances in cluster-root biology have been derived from studies of the crop species Lupinus albus (Fabaceae family) (Gardner et al

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea

“…Consistent with earlier results (Shane et al, 2004a), (1) anti-PEPC immunoblots of stage I root extracts revealed comparable amounts of immunoreactive p110 and p107 that comigrated with the purified, partially monoubiquitinated PEPC heterotetramer from germinating COS, whereas (2) a progressive loss of immunoreactive p110 in stages II to III was paralleled by increased levels of p107 such that the total amount of immunoreactive PEPC polypeptides remained relatively constant (Fig. 2B).…”

“…Similarly, a 2-fold increase in SUS activity occurred during maturation of white lupin proteoid roots (Massonneau et al, 2001). The peak in SUS and PEPC activities at stage III of hakea proteoid root development correlates with their maximal rates of citrate and malate exudation (Shane et al, 2004a). By contrast, citrate synthase (CS) and malate dehydrogenase (MDH) activities remained relatively constant throughout development (except in senescing stage IV roots, where they showed a pronounced decline; Supplemental Fig.…”

“…Immunoblotting indicated that PEPC abundance remained relatively constant during proteoid root development, except in senescing 3-week-old roots, where it showed a marked decline. The PEPC immunoblots also revealed approximately 110-and 100-kD immunoreactive polypeptides that were of equal intensity in young proteoid roots, whereas mature proteoid roots showed a marked reduction in the p110 (Shane et al, 2004a). The possible contribution of PTMs such as phosphorylation to the in vivo activation of proteoid root PEPCs is currently unclear (e.g.…”

“…A crucial adaptation of harsh hakea is its proteoid roots, which excrete copious quantities of citrate and malate to mediate Pi solubilization and acquisition from the soil's mineralbound Pi (Supplemental Figs. S1 and S2; Shane et al, 2003Shane et al, , 2004aShane et al, , 2004bShane and Lambers, 2005). Shane and coworkers (2004a) correlated proteoid root development in -Pi harsh hakea with marked increases in respiration, internal carboxylate concentrations, and rates of carboxylate exudation.…”

“…S1), a common abiotic stress that frequently limits plant growth in natural ecosystems. The marked induction of Class-1 PEPCs during Pi stress has been linked to the synthesis and excretion of large amounts of organic acid anions by roots of Pi-starved (-Pi) plants (O'Leary et al, 2011a;Uhde-Stone et al, 2003;Vance et al, 2003;Shane et al, 2004a). The excreted organic acids chelate metal cations such as Al 3+ and Ca 2+ that immobilize Pi in the soil, thus increasing soluble Pi concentrations by up to 1,000-fold .…”