White lupin cluster root acclimation to phosphorus deficiency and root hair development involve unique glycerophosphodiester phosphodiesterases

Abstract

White lupin (Lupinus albus) is a legume that is very efficient in accessing unavailable phosphorus (Pi). It develops short, densely clustered tertiary lateral roots (cluster/proteoid roots) in response to Pi limitation. In this report, we characterize two glycerophosphodiester phosphodiesterase (GPX-PDE) genes (GPX-PDE1 and GPX-PDE2) from white lupin and propose a role for these two GPX-PDEs in root hair growth and development and in a Pi stress-induced phospholipid degradation pathway in cluster roots. Both GPX-PDE1 and GPX-PDE2 are highly expressed in Pi-deficient cluster roots, particularly in root hairs, epidermal cells, and vascular bundles. Expression of both genes is a function of both Pi availability and photosynthate. GPX-PDE1 Pi deficiency-induced expression is attenuated as photosynthate is deprived, while that of GPX-PDE2 is strikingly enhanced. Yeast complementation assays and in vitro enzyme assays revealed that GPX-PDE1 shows catalytic activity with glycerophosphocholine while GPX-PDE2 shows highest activity with glycerophosphoinositol. Cell-free protein extracts from Pi-deficient cluster roots display GPX-PDE enzyme activity for both glycerophosphocholine and glycerophosphoinositol. Knockdown of expression of GPX-PDE through RNA interference resulted in impaired root hair development and density. We propose that white lupin GPX-PDE1 and GPX-PDE2 are involved in the acclimation to Pi limitation by enhancing glycerophosphodiester degradation and mediating root hair development.

Figure 1.

Phylogenetic tree of white lupin GPX-PDEs with related GPX-PDE proteins from various species. The phylogenetic tree of white lupin GPX-PDEs with proteins containing GPX-PDE domains was generated using MEGA4 from a ClustalX alignment (sequence alignment is available as Supplemental Data Set S1). Numbers represent bootstrap values obtained from 1,000 trials. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances are displayed in units of the number of amino acid substitutions per site. Abbreviations for species are as follows: At, Arabidopsis thaliana; Ec, Escherichia coli; Gg, Gallus gallus; Gm, Glycine max; Hs, Homo sapiens; La, Lupinus albus; Mm, Mus musculus; Os, Oryza sativa; Sb, Sorghum bicolor; Sc, Saccharomyces cerevisiae; Stc, Streptomyces coelicolor.

Figure 2.

Cluster root development and qRT-PCR analysis of GPX-PDE expression in cluster roots of white lupin grown under Pi-sufficient and Pi-deficient conditions. A, Lupin cluster roots from Pi-deficient and Pi-sufficient plants can be separated into five zones (Zn): zone 1, root tip; zone 2, early meristems; zone 3, unemerged rootlets; zone 4, newly emerged and juvenile rootlets; zone 5, mature cluster roots covered with abundant root hairs (Neumann et al., 1999). Note that cluster roots can form on Pi-sufficient plants but constitute 10% or less of the root mass. However, on Pi-deficient plants, they constitute greater than 60% of the root mass (Johnson et al., 1994, 1996). B and C, Relative expression of white lupin GPX-PDE1 and GPX-PDE2 in the five developmental zones of Pi-deficient and Pi-sufficient cluster roots. qRT-PCR is shown for first-strand cDNA generated from Pi-sufficient (+P) and Pi-deficient (−P) lupin cluster root zones and normal roots (NR) using specific primer pairs for GPX-PDE1 and GPX-PDE2. Data are expressed as relative values based on the GPX-PDE1 or GPX-PDE2 expression level in +P normal roots and referenced as 1.0. Lowercase letters denote significant differences between zones (P ≤ 0.05). Asterisks denote significant differences between –P and +P (P ≤ 0.05). Statistical significance was based on three biological replicates.

Figure 3.

The effect of Pi or Phi resupply on transcript abundance of Pi deficiency-induced GPX-PDE1 and GPX-PDE2. White lupin plants were grown to 14 d after emergence without Pi and then resupplied with 1 mm Pi. Control plants were grown for 14 d after emergence with +Pi (1 mm), −Pi (0 mm), or +Phi (1 mm). Cluster roots from control and treated Pi-resupplied plants excised at 1, 4, 6, and 24 h were used for RNA isolation and cDNA synthesis. A and B, GPX-PDE1 (A) and GPX-PDE2 (B) expression analyzed by qRT-PCR. C, Total P concentration in control and treated plants. Data are expressed as relative values based on the GPX-PDE1 or GPX-PDE2 expression level in +P normal roots and referenced as 1.0. Lowercase letters denote significant differences between treatments (P ≤ 0.05). Statistical significance was based on three biological replicates. DW, Dry weight.

Figure 4.

Effect of photosynthate deprivation on transcript abundance of Pi deficiency-induced GPX-PDE1 and GPX-PDE2. White lupin plants were grown to 14 d after emergence either with 1 mm Pi (+Pi) or without Pi (−Pi) under a 16/8-h photoperiod (D₀). Plants were then placed in total darkness for 24 h (D₂₄). After a 24-h dark treatment, plants were returned to continuous light for 24 h (D₂₄L₂₄) and 48 h (D₂₄L₄₈). Cluster roots and leaf tissue from the various treatments were harvested and used for RNA isolation and cDNA synthesis. qRT-PCR was used to assess transcript abundance. A and B, GPX-PDE1 transcript abundance in cluster roots (A) and leaves (B) in response to dark treatment and upon return to light. C and D, GPX-PDE2 transcript abundance in cluster roots (C) and leaves (D) in response to dark treatment and upon return to light. Data are expressed as relative values based on the GPX-PDE1 or GPX-PDE2 expression level in +P (D₀) and referenced as 1.0. Lowercase letters denote significant differences between treatments (P ≤ 0.05). Statistical significance was based on three biological replicates.

Figure 5.

GPX-PDE1 and GPX-PDE2 reporter gene activity in Pi-deficient and Pi-sufficient cluster roots of white lupin. The 1,681- and 1,743-bp 5′-upstream promoter regions of GPX-PDE1 and GPX-PDE2, respectively, were fused to the GUS coding sequence and transformed into white lupin roots through A. rhizogenes-mediated hairy root transformation. Transgenic roots from Pi-sufficient and Pi-deficient roots were harvested 5 weeks after transformation and stained for GUS activity. Six roots per plant from a total of 12 transgenic plants were evaluated. Transgenic root tissue was incubated in GUS solution for 2 h at 37°C. Tissue from zone 5 and pooled zone 2 and 3 tissue was used for MUG enzyme assays. Lupin cluster roots are separated into five zones as described in Figure 2. A, GUS staining occurs throughout the entire root of both GPX-PDE1 and GPX-PDE2, with strong staining in the mature cluster root zone of –Pi plants; cluster roots of +Pi transgenic plants showed very low GUS staining. B and C, Root hairs (rh) from rootlets of the mature cluster root zone 5 of GPX-PDE1 (B) and GPX-PDE2 (C) also showed strong GUS staining. Bars = 0.05 mm. D and E, Cross sections of GPX-PDE1 (D) and GPX-PDE2 (E) rootlets from zone 5 displayed strong GUS staining in the epidermal cells (ep) and the vascular bundle (vb) in Pi-deficient transgenic roots. Bars = 0.5 mm. F and G, MUG assays confirmed the high promoter:reporter activity response to –Pi for GPX-PDE1 (F) and GPX-PDE2 (G). Z2+3, Cluster root zone 2 and 3 tissue pooled; Z5, cluster root zone 5 tissue. Lowercase letters denote significant differences between zones (P ≤ 0.05). Statistical significance was based on four biological replicates.

Figure 6.

Subcellular localization of GPX-PDE1 protein in root hairs. A to C, Confocal laser scanning micrographs of root hair cells of transgenic M. truncatula roots expressing a fusion of GPX-PDE1, including the transit sequence, and EGFP driven by the GPX-PDE1 promoter (GPD-PDE1:GFP) stained with ER-Tracker Dye. A, GPX-PDE1:GFP fluorescence. B, ER-Tracker Red fluorescence. C, Merged images of A and B showing colocalization of GPX-PDE1 and ER-Tracker Red as orange-yellow color. Bar = 0.5 mm. D to F, Confocal laser scanning micrographs of N. benthamiana leaf epidermal cells infiltrated with A. tumefaciens carrying both GPX-PDE1:GFP and HDEL:RFP. D, GPX-PDE1:GFP fluorescence. E, HDEL:RFP fluorescence. F, Merged images of D and E. Images show colocalization of GPX-PDE1:GFP and HDEL:RFP as orange-yellow color. Bar = 25 μm.

Figure 7.

Effect of GPX-PDE1 or GPX-PDE2 RNAi silencing on root hair development and expression of Pi starvation-induced genes. Reduced expression of GPX-PDE1 and GPX-PDE2 was achieved by subcloning a 631-bp mRNA sequence of GPX-PDE1 and a 510-bp mRNA sequence of GPX-PDE2 into an RNAi vector containing DsRED as a reporter and subsequent transformation to white lupin roots using A. rhizogenes. The human myosin gene was used as a control. Transgenic plants were grown under Pi-deficient conditions and harvested after 5 weeks. The DsRED reporter was used to screen transformed roots. A total of 48, 54, and 30 RNAi plants were evaluated for GPX-PDE1, GPX-PDE2, and myosin, respectively. A to C, Control RNAi (A), GPX-PDE1 RNAi (B), and GPX-PDE2 RNAi (C) showing root hair density and length from a zone 5 mature rootlet. D and E, Root hair length (D) and root hair density (E) of GPX-PDE1 and GPX-PDE2 RNAi zone 5 mature cluster rootlets show a significant reduction relative to the control myosin RNAi root hair length and density grown under Pi-deficient conditions. No significant differences were evident when grown under Pi-sufficient conditions. Lowercase letters denote significant differences between genes (P ≤ 0.05). Statistical significance was based on 11, 10, and eight biological replicates for myosin, GPX-PDE1, and GPX-PDE2 RNAi transgenic plants, respectively. F and G, qRT-PCR confirmed the reduction of the GPX-PDE1 (F) and GPX-PDE2 (G) transcript in the corresponding RNAi roots (patterned columns) relative to the control (solid columns). The transcript abundance of two other Pi deficiency-induced genes (SAP1 and PT1) was also reduced in GPX-PDE1 and GPX-PDE2 RNAi roots. Asterisks denote significant differences in expression levels relative to the control myosin RNAi gene (P ≤ 0.05).

Figure 8.

A novel pathway for the degradation of phospholipids and Pi recycling via a GPX-PDE-mediated pathway during phosphate limitation. Under Pi limitation, plant phospholipids (PC and PI) decrease. The degradation of PC mediated by either nonspecific phospholipase C (NPC4 and NPC5) or phospholipase D (PLDζ1 and PLDζ2) with the subsequent release of Pi is well documented (pathway in box). We propose an alternative phospholipid degradation and Pi recycling pathway that catabolizes PC and PI. PC and PI are deacylated into GPC and GPI by Pi deficiency-induced phospholipase A₂ (PLA2) and lysophospholipase. GPC and GPI are further hydrolyzed by Pi deficiency-induced GPX-PDE1 and GPX-PDE2 to glycerol-3-phosphate (G3P) and choline (Chol) or inositol (Ins). G3P is subsequently catalyzed by Pi deficiency-induced acid phosphatase (AP) to release Pi and glycerol. DGDG, Digalactosyldiacylglycerol; DGD, DGDG synthase; FA, fatty acid; LysoPtdC, lysophosphatidylcholine; LysoPtdIns, lysophosphatidylinositol; MGDG, monogalactosyldiacylglycerol; MGD, MGDG synthase; PA, phosphatidic acid; PChol, phosphocholine.