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. 2014 Jun 25;165(4):1647–1656. doi: 10.1104/pp.114.244376

The S-Domain Receptor Kinase Arabidopsis Receptor Kinase2 and the U Box/Armadillo Repeat-Containing E3 Ubiquitin Ligase9 Module Mediates Lateral Root Development under Phosphate Starvation in Arabidopsis1,[C],[W],[OPEN]

Srijani Deb 1,2, Subramanian Sankaranarayanan 1,2, Gayathri Wewala 1, Ellen Widdup 1, Marcus A Samuel 1,*
PMCID: PMC4119045  PMID: 24965176

Autophagy plays an important role during lateral root development under phosphate starvation.

Abstract

When plants encounter nutrient-limiting conditions in the soil, the root architecture is redesigned to generate numerous lateral roots (LRs) that increase the surface area of roots, promoting efficient uptake of these deficient nutrients. Of the many essential nutrients, reduced availability of inorganic phosphate has a major impact on plant growth because of the requirement of inorganic phosphate for synthesis of organic molecules, such as nucleic acids, ATP, and phospholipids, that function in various crucial metabolic activities. In our screens to identify a potential role for the S-domain receptor kinase1-6 and its interacting downstream signaling partner, the Arabidopsis (Arabidopsis thaliana) plant U box/armadillo repeat-containing E3 ligase9 (AtPUB9), we identified a role for this module in regulating LR development under phosphate-starved conditions. Our results show that Arabidopsis double mutant plants lacking AtPUB9 and Arabidopsis Receptor Kinase2 (AtARK2; ark2-1/pub9-1) display severely reduced LRs when grown under phosphate-starved conditions. Under these starvation conditions, these plants accumulated very low to no auxin in their primary root and LR tips as observed through expression of the auxin reporter DR5::uidA transgene. Exogenous auxin was sufficient to rescue the LR developmental defects in the ark2-1/pub9-1 lines, indicating a requirement of auxin accumulation for this process. Our subcellular localization studies with tobacco (Nicotiana tabacum) suspension-cultured cells indicate that interaction between ARK2 and AtPUB9 results in accumulation of AtPUB9 in the autophagosomes. Inhibition of autophagy in wild-type plants resulted in reduction of LR development and auxin accumulation under phosphate-starved conditions, suggesting a role for autophagy in regulating LR development. Thus, our study has uncovered a previously unknown signaling module (ARK2-PUB9) that is required for auxin-mediated LR development under phosphate-starved conditions.

Roots are one of the most important adaptations of land plants, because they provide anchorage, facilitate absorption of water and minerals, and also, aid in specialty functions, such as storage of food and water and vegetative reproduction in some plant species. Plants have the unique ability to alter their root morphology depending on resource availability in the soil. Under nutrient-starved conditions, this allows them to modify the roots to efficiently explore the heterogeneous soil environment for nutrients. Phosphorous in its inorganic form is often present at low concentrations and hence, heavily supplemented through fertilizers in agriculture. Inorganic phosphate (Pi) is the main form of plant-assimilated phosphorous present in the soil. Pi starvation is an increasing global problem in agriculture, and even in fertile soils, Pi concentration rarely exceeds 10 µm (Bieleski, 1973). To cope with these chronically low Pi levels, plants have developed highly specialized physiological and biochemical mechanisms to efficiently acquire and use Pi from the environment. This involves changes in the root architecture, increase in root-shoot ratio, and root hair development (Williamson et al., 2001; López-Bucio et al., 2002; Al-Ghazi et al., 2003). When plants perceive Pi starvation, the most common response is the induction of numerous lateral roots (LRs), inhibition of primary roots (PRs), and formation of denser root hairs, thus restructuring the roots for exhaustive exploration of the topsoil for Pi (Raghothama, 1999; Lynch and Brown, 2001). During Pi deficiency, plant root hairs account for 90% of the total Pi uptake by the plants (Raghothama, 1999). Phosphate is acquired by high-affinity Pi transporters and loaded into the xylem in roots. Pi moves freely through both xylem and phloem. When Pi is not limiting, Pi efflux by anion channels ensures Pi homeostasis. The high-affinity and low-affinity Pi transporters also mediate phosphate transport across the plasma membrane and tonoplast, which is powered by the membrane proton ATPase (Raghothama, 2000).

Multiple signals and molecular mechanisms are initiated when plants are exposed to Pi starvation. They include transcriptional regulation of gene expression, gene silencing by microRNAs, and posttranslational modification, like sumoylation and ubiquitination (for review, see Rojas-Triana et al., 2013). Although the complete signaling network through which plants cope with Pi limitation has not been deciphered, there is precedence for the role of hormones in altering the root system architecture during Pi starvation. Studies suggest both auxin-dependent and independent mechanisms during adaptive response to Pi limitation (Williamson et al., 2001; López-Bucio et al., 2002; Al-Ghazi et al., 2003; Nacry et al., 2005). However, cytokinins are known to negatively regulate this response (Martín et al., 2000; Wang et al., 2006). In addition, cross talk between hormone signaling and sugar signaling pathways during response to Pi starvation has also been reported (Hammond and White, 2011).

Several ubiquitin (Ub)-conjugating and -deconjugating enzymes are known to function in controlling adaptive response to Pi starvation. F-Box Protein2 (FBX2) proteins, which contain both WD40 and F-box motifs, negatively regulate responses, like root hair formation, expression of phosphoenolpyruvate carboxylase kinase (PPCK; PPCK1 and PPCK2), and anthocyanin accumulation during Pi starvation (Chen et al., 2008). FBX2 also interacts with basic helix-loop-helix32 in vitro, another negative regulator of Pi starvation responses (Chen et al., 2008). Recently, a rice (Oryza sativa) U box containing E3 Ub ligase, OsUPS, that is induced by Pi starvation has also been isolated (Hur et al., 2012).

One common response of plant cells to nutrient starvation is the induction of autophagy or self-eating, in which autophagosomes are used for bulk degradation of cellular organelles to maintain the cell at a low metabolic state (Bassham, 2007). Depending on the size of the cytoplasmic material engulfed for degradation, plant autophagy can be classified as either microautophagy or macroautophagy (Bassham et al., 2006). Autophagy-specific gene 18a (AtATG18a) in Arabidopsis (Arabidopsis thaliana) is required for autophagic response during Suc/nitrogen starvation and senescence (Xiong et al., 2005). RNA interference lines with reduced expression of AtATG18a are hypersensitive to Suc and nitrogen starvation (Xiong et al., 2005). Autophagy is also known to play a role in nutrient remobilization. Autophagy mutants of Arabidopsis are reduced in their nitrogen remobilization efficiency, leading to lower biomass and yield (Guiboileau et al., 2012). Similarly, disruption of Arabidopsis ATG5 prevents formation of autophagosomes and causes sensitivity to nitrogen starvation (Thompson et al., 2005). Under Pi starvation, Ub-like protein ATG8 has been shown to be up-regulated in root tips, and disruption of ATG5 leads to early consumption of root meristem (Sakhonwasee and Abel, 2009). Despite all this evidence for the requirement of autophagy for maintaining root architecture, the mechanisms by which autophagy regulates this process during Pi starvation remain unclear.

Arabidopsis plant U box/armadillo repeat protein9 (AtPUB9) belongs to the armadillo repeat-containing proteins with a U box that is commonly present in many E3 ligases (Mudgil et al., 2004). Although the biological functions of most of these proteins are still unknown, the roles of other PUB family proteins range from self-incompatibility to hormone responses to defense and abiotic stress responses (for review, see Yee and Goring, 2009). S-domain receptor kinases are known to function as upstream activators of PUBs (Samuel et al., 2008). Previously, it has been shown that one of the S-domain receptor kinase1-6 Arabidopsis Receptor Kinase2 (ARK2) could efficiently phosphorylate AtPUB9 in vitro (Samuel et al., 2008), and when coexpressed in tobacco (Nicotiana tabacum) suspension-cultured cells, this interaction led to localization of AtPUB9 in punctate structures (Samuel et al., 2008). In this report, we show that these punctate structures are lytic compartments or autophagosomes. Because autophagosomes are induced under nutrient-starved conditions, we investigated pub9-1 and ark2-1 mutant phenotypes under nutrient-limiting conditions. We found out that, under Pi starvation, ark2-1/pub9-1 double mutants were deficient in their ability to develop LRs. Our additional investigation revealed that this defect is likely caused by lack of auxin accumulation at the LR initiation sites. The LR defects in the double mutants could be rescued through complementation with either ARK2 or PUB9 as well as application of exogenous auxin.

RESULTS

PUB9 Localizes to Punctate Structures and Colocalizes with the Autophagosomal Marker ATG8 in the Presence of ARK2

AtPUBs have been shown to interact with the Arabidopsis S-domain receptor kinases (Samuel et al., 2008) and likely function as downstream signaling molecules to these kinases. This interaction is known to lead to both phosphorylation of the AtPUBs and alteration in their cellular localization (Samuel et al., 2008). In tobacco Bright Yellow2 (BY2) cells, PUB9 localization could be influenced by its interaction with the cytosolic kinase domains of ARK1 and ARK2; whereas ARK1 redistributes PUB9 from the nucleus to the plasma membrane, ARK2 interaction leads to localization of PUB9 in punctate structures in 40% of the cells (Samuel et al., 2008). To identify these subcellular structures, we performed colocalization studies in the same system with fluorescent marker proteins or dyes that localize on intracellular punctate structures. When PUB9 and ARK2 were cotransformed with red fluorescent protein (RFP)-tagged oleosin (the major structural protein associated with oil bodies), they largely localized independent of each other (Fig. 1A). When PUB9 and ARK2 were coexpressed with either lysotracker Red, an acidotropic fluorescent dye that labels acidic compartments like vacuoles, or RFP-Syntaxin of Plants21 (SYP21), which is predominantly located on the membranes of prevacuolar compartments, colocalization could be observed in the punctate structures (Fig. 1, B and C). This suggested to us that these compartments could be lytic vacuoles that are destined to the central vacuole. When PUB9 and ARK2 were coexpressed with ATG8, an autophagosome marker, complete colocalization of PUB9 and ATG8 could be observed on the punctate subcellular compartments (Fig. 1D).

Figure 1.

Figure 1.

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Subcellular localization of transiently coexpressed glutathione S-transferase::ARK2 (kinase domain) and GFP::PUB9 with oil body marker RFP::Oleosin (A), vacuolar marker Lysotracker Red (B), prevacuolar marker RFP::SYP21 (C), and autophagy marker RFP-ATG8 (D); 5 to 10 µg of plasmids were biolistically transformed into tobacco BY2 cells, and images were captured through epifluorescence microscopy. To determine the colocalization pattern, the green and red channels were merged.

Plants Lacking Both AtPUB9 and ARK2 Are Defective in LR Formation under Phosphate Starvation

The localization pattern of AtPUB9 in the presence of ARK2 to autophagic compartments prompted us to investigate the in vivo role of these proteins under conditions that induced autophagosomes. For this, homozygous transfer DNA (T-DNA) insertional lines of PUB9 (pub9-1) and ARK2 (ark2-1) and the homozygous double mutant, ark2-1/pub9-1, carrying both the mutations were used (Fig. 2, A and B). When root tissue was examined for expression of these genes, the double mutant line ark2-1/pub9-1 lacked PUB9 or ARK2 expression compared with the wild type (ecotype Columbia-0 of Arabidopsis [Col-0]) as revealed by reverse transcription (RT)-PCR (Fig. 2C). When 3-d-old seedlings of the single as well as the double mutants were transferred to nutrient-rich (+Pi/+Suc) medium and allowed to grow vertically for 7 d, there was no observable difference in LR density (number of LRs per centimeter of PR length; Fig. 2D). In contrast, when 3-d-old seedlings were grown in medium lacking Pi and Suc (−Pi/−Suc), ark2-1/pub9-1 had severely reduced numbers of LRs compared with pub9-1, ark2-1, or Col-0. The PR length was also inhibited in the double mutant relative to Col-0 and the single mutants (Fig. 2E). To verify that the observed LR developmental defect in the double mutant is caused by a lack of these respective genes, complementation of ark2-1/pub9-1 with either PUB9 or ARK2 was performed. Overexpression (35S::PUB9) of PUB9 in ark2-1/pub9-1 led to the rescue of the LR defect phenotype observed under −Pi/−Suc conditions (Supplemental Fig. S1). However, the PR length was reduced as a result of PUB9 overexpression in ark2-1/pub9-1 independent of the nutrient status (Supplemental Fig. S1). Expression of ARK2 similarly rescued the LR defect observed in ark2-1/pub9-1 under −Pi/−Suc condition. (Supplemental Fig. S1). These results indicated that both ARK2 and PUB9 are required for induction of LR under Pi starvation. These two diverse interacting partners likely play a redundant role in regulating LR development under phosphate-starved conditions.

Figure 2.

Figure 2.

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Absence of ARK2-PUB9 module leads to LR defect under Pi/Suc-deficient conditions. Schematic representation of the location of T-DNA inserts in pub9-1 and ark2-1 mutant lines (A). Homozygous double mutant ark2-1/pub9-1 line obtained by crossing pub9-1 and ark2-1 was confirmed by PCR genotyping (B). RT-PCR analysis of AtPUB9 and AtARK2 expression in ark2-1/pub9-1 roots relative to Col-0 (C). The LR density (calculated as the number of LRs per centimeter of length of PR) of Arabidopsis seedlings under nutrient-rich (+Pi/+Suc; D) and Pi/Suc-starved (−Pi/−Suc; E) conditions of the various genotypes; 3-d-old seedlings were allowed to grow vertically in either +Pi/+Suc or −Pi/−Suc media for 7 d before being photographed. The values represent mean ± se (n = 15). Bars = 1 cm.

Phosphate Starvation Leads to PUB9 Localization on Punctate Subcellular Structures in Planta

To determine if PUB9 has a role in autophagy, the roots of pub9-1 constitutively expressing GFP-tagged PUB9 (pub9-1/GFP-PUB9) were analyzed by confocal microscopy. Under normal nutrient-rich conditions (+Pi/+Suc), GFP-PUB9 is distributed throughout the cytosol in root epidermal cells (Fig. 3A). When GFP-PUB9 was observed under Pi/Suc starvation (−Pi/−Suc), a few punctate structures could be observed on the edge of the large central vacuole (Fig. 3B). To observe the autophagic compartments, the transgenic seedlings were treated with concanamycin A (ConcA), a specific inhibitor of vacuolar ATPase that blocks vacuolar degradation and has been previously used to reveal autophagosomes (Thompson et al., 2005; Chung et al., 2010). After ConcA treatment, PUB9 could be observed to localize on punctate structures under nutrient-rich conditions (Fig. 3C). Interestingly, the number of these punctate structures dramatically increased in ConcA-treated and Pi/Suc-starved root cells (Fig. 3D; Supplemental Fig. S2). The punctate structures observed were 0.5 to 1.5 µm in diameter and appeared to be mobile and in the vacuolar lumen. The increase in the number of these structures after phosphate starvation likely indicates the occurrence of some degree of basal autophagy under normal conditions, which gets elevated on starvation.

Figure 3.

Figure 3.

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Subcellular localization of GFP::PUB9 in stably transformed Arabidopsis pub9-1 root epidermal cells grown under different nutrient conditions. Localization of GFP::PUB9 under nutrient-rich condition without ConcA (A), Pi/Suc-deficient condition without ConcA (B), 2 µm ConcA-supplemented nutrient-rich condition (C), and 2 µm ConcA-supplemented Pi/Suc-deficient condition (D). Effect of 3-MA on the LR formation of wild-type Col-0 under nutrient-rich (+Pi/+ Suc) and nutrient-deficient (−Pi/−Suc) conditions (E).

Inhibiting Autophagy Leads to Defective LR Formation under Phosphate Starvation in Wild-Type Plants

To determine if autophagy has a role in regulating LR development under phosphate starvation, we transferred 3-d-old seedlings of wild-type Col-0 to either a nutrient-rich (+Pi/+Suc) or nutrient-starved medium (−Pi/−Suc) supplemented with 1 mm 3-methyladenine (3-MA), a known autophagy inhibitor, and allowed them to grow for 7 d. Interestingly, only the seedlings grown on −Pi/−Suc medium with 3-MA showed severely reduced numbers of LRs phenocopying the ark2-1/pub9-1 double mutants (Figs. 2E and 3E) The seedlings grown in nutrient-rich media did not display any observable LR defects (Fig. 3E). These results indicate that autophagy is required for LR formation under phosphate starvation and that the LR defect in ark2-1/pub9-1 double mutants could be a result of a defective autophagy process in this mutant.

ark2-1/pub9-1 Plants Lack DR5 Promoter Activity in Root Tips under Starvation

Because auxin accumulation is tightly associated with root development, we aimed at investigating any possible role of auxin in ARK2-PUB9-mediated regulation of LR development. For this, pub9-1, ark2-1, and ark2-1/pub9-1 lines expressing GUS under the control of the auxin-responsive DR5 promoter (DR5::uidA) were generated. When 10-d-old seedlings grown in either rich medium or −Pi/−Suc medium were subjected to GUS assays, similar GUS staining could be observed in the PR tip across all genotypes under nutrient-rich conditions (Fig. 4A, top). This indicated that auxin accumulation response was similar in all these lines under unchallenged conditions. Under Pi/Suc-starved condition, the ark2-1/pub9-1 PRs did not exhibit any GUS staining, whereas Col-0 and other single mutants displayed GUS staining (Fig. 4A, bottom). This indicates severely reduced levels of auxin in ark2-1/pub9-1 PR tips under nutrient-starved conditions (Fig. 4A, bottom). A similar pattern was observed for LR tips, with severely reduced GUS staining in ark2-1/pub9-1 lines in the LR tips analyzed (Fig. 4B).

Figure 4.

Figure 4.

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DR5::uidA expression in PR (A) and LR tips (B). GUS staining was performed to detect the level of auxin accumulation in the PR and LR tips of seedlings of the various genotypes grown on medium with or without phosphate and Suc. Bars = 16 μm.

Autophagy Is Required for Sustained Auxin Accumulation in Root Tips under Starvation

To investigate whether autophagy has a role in controlling auxin accumulation under phosphate starvation, we carried out GUS assays with 10-d-old DR5::uidA-expressing Col-0 seedlings grown in either rich medium (+Pi/+Suc) with 1 mm 3-MA or −Pi/−Suc medium with 1 mm 3-MA. We observed that GUS staining was reduced in the primary and LR tips of plants grown in the presence of 3-MA under −Pi/−Suc-starved conditions (Supplemental Fig. S3) compared with plants grown in the presence of 3-MA under nutrient-rich conditions (Supplemental Fig. S3). These results indicate that auxin accumulation is controlled by autophagy under Pi starvation, and reduced GUS staining observed in ark2-1/pub9-1 lines could be a result of the defective autophagy.

Exogenous Auxin Rescues Phosphate Starvation-Induced LR Defect in ark2-1/pub9-1

Based on the lack of auxin accumulation in root tips of ark2-1/pub9-1, after phosphate starvation, we hypothesized that the interaction between these two proteins could mediate auxin accumulation in the root tips. If a defect in auxin accumulation is the sole reason for the observed LR developmental phenotypes in these lines, then supplementation of auxin should rescue this phenotype. To test this, 3-d-old seedlings of Col-0 and ark2-1/pub9-1 initially germinated on 0.5× Murashige and Skoog (MS) medium with 1% (w/v) Suc were transferred to either +Pi/+Suc or −Pi/−Suc medium with or without the addition of various concentrations of naphthylacetic acid (NAA; 0.001–1 µm; Fig. 5, A and B). Addition of 0.001 µm NAA was sufficient to rescue the ark2-1/pub9-1 LR defect in −Pi/−Suc medium (Fig. 5C). Both 0.001 and 0.01 µm NAA were the optimal concentrations for the rescue of LR phenotype in ark2-1/pub9-1 lines without much effect on the PR length in both NAA concentrations in Col-0 (Fig. 5D). Treatment with 0.1 and 1 µm NAA resulted in a significant increase in the number of LRs in both nutrient-rich and nutrient-starved conditions, and both Col-0 and ark2-1/pub9-1 produced a similar number of LRs under these conditions but had an inhibitory effect on the PR elongation (Fig. 5, C and D; Supplemental Fig. S4). The rescue of LR defect in ark2-1/pub9-1 by auxin supplementation suggests that auxin is required for LR development under nutrient-starved conditions and that these two proteins control the accumulation of auxin under phosphate-starved conditions.

Figure 5.

Figure 5.

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Rescue of the LR defect phenotype by exogenous auxin supplementation. Three-day-old Col-0 (A) and ark2-1/pub9-1 (B) seedlings were allowed to grow vertically for 7 d in either +Pi/+Suc or −Pi/−Suc media with or without 0.1 μm NAA. Graphical representation of the LR density (C) and PR length (D) for Col-0 and ark2-1/pub9-1 grown on −Pi/−Suc media in the presence of various concentrations of NAA. The values represent mean ± se (n = 6). [See online article for color version of this figure.]

Gene Expression Analysis in LRs Suggests an Auxin-Dependent Mechanism

To further confirm our hypothesis that the ARK2-PUB9 module regulates auxin accumulation, quantitative RT-PCR was performed using key genes involved in auxin signaling and transport. For this, 3-d-old seedlings were transferred to either +Pi/+Suc or −Pi/−Suc condition and allowed to grow vertically for 4 d. The LR initiation zone, 2 to 2.5 cm away from the PR region, was used as the tissue for expression analysis. The expression of indole-3-acetic acid28 (IAA28), an AUXIN (AUX) /IAA repressor of auxin-responsive genes, was found to be up-regulated in Pi/Suc-starved ark2-1/pub9-1 compared with Col-0 (Fig. 6). However, the auxin transport genes PIN-FORMED1 (PIN1), PIN3, PIN5, PIN6, and PIN7 were significantly down regulated several fold in Pi/Suc-starved ark2-1/pub9-1 compared with Col-0 (Fig. 6), with PIN5 showing the lowest level of expression, being reduced by 3.03-fold, and PIN2 displaying a moderately enhanced expression (Fig. 6). Under nutrient-rich conditions, the expression of these genes in ark2-1/pub9-1 was similar to that of Col-0 (Supplemental Fig. S5). These observations are indicative of the requirement of the ARK2-PUB9 module in maintaining the expression of the PIN transport proteins and the IAA28 repressor under nutrient-starved conditions.

Figure 6.

Figure 6.

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Altered expression pattern of auxin signaling and transport genes under Pi/Suc-deficient condition. Quantitative RT-PCR analysis was performed with RNA isolated from 7-d-old Arabidopsis seedlings that were subjected to Pi and Suc starvation for 4 d. Relative expression levels were calculated by the Δ-ΔCt method, with Pi/Suc-starved Col-0 as the calibrator, and all quantifications were normalized using Ubq10 mRNA as an internal control. Data represent mean ± se (n = 4).

DISCUSSION

Despite the identification of 417 receptor kinases with an extracellular domain in Arabidopsis, functions of most of these kinases and their downstream interacting partners remain unknown (Shiu and Bleecker 2001a, 2001b). Previously, it was shown that the Arabidopsis S domain1 receptor kinase subfamily could potentiate signaling through the PUBs (Samuel et al., 2008). In particular, PUB9 is a short E3 Ub ligase containing a U-box domain and armadillo repeats and is expressed in nearly all tissue types, except leaves (Mudgil et al., 2004). ARK1, ARK2, and M-locus protein kinase are capable of phosphorylating PUB9 based on in vitro phosphorylation assays (Samuel et al., 2008). Phosphorylation of AtPUB9 by ARK2 led to its relocalization from the nucleus to the cytosol, and 40% of the cells formed punctate structures (Samuel et al., 2008). In this study, we have identified these compartments as autophagic bodies based on its colocalization with markers for lytic compartments and autophagosome marker ATG8 (Fig. 1). AtPUB9 in root epidermal cells of transgenic pub9-1::GFP-PUB9 plants localized to punctate structures in the vacuole under starvation and on ConcA treatment (Fig. 3D). This suggests that PUB9 localizes to autophagic bodies after phosphate starvation. Previous studies have shown that ATG8 localizes to similar compartments on ConcA treatment (Thompson et al., 2005; Chung et al., 2010). ConcA is a specific inhibitor of vacuolar type H+ATPase (Dröse et al., 2001) that increases the pH in vacuoles, preventing the activity of vacuolar proteases and thus, enabling the visualization of GFP in the vacuole (Tamura et al., 2003).

When examining a role for the ARK2-PUB9 module in mediating stress responses under nutrient-starved conditions, we identified that the LR development phenotype manifested only during phosphate-starved conditions in the ark2-1/pub9-1 double mutants (Fig. 2). Our results with 3-MA using Col-0 seedlings also confirmed the need for autophagy to mediate LR development under phosphate-starved conditions (Fig. 3E). The developmental plasticity of the root system allows it to respond and adapt to the external environment by modifying itself through a cascade of hormone-mediated events. We observed that the lack of LR formation is because of the inability of ark2-1/pub9-1 plants to accumulate auxin in the root tips under phosphate starvation (Fig. 4). The phytohormone auxin and the associated polar auxin transport are the principal stimulators of LR initiation, primordium development, and emergence (for review, see Lavenus et al., 2013). Previous studies have shown that exogenous application of auxin promotes the production of numerous LRs (Malamy and Ryan, 2001; Himanen et al., 2002). Likewise, mutation or overexpression of genes involved in auxin biosynthesis, metabolism, transport, and signaling led to an altered number of LRs (Celenza et al., 1995; Hobbie and Estelle, 1995; Rogg et al., 2001; Fukaki et al., 2002; Swarup et al., 2008; Mashiguchi et al., 2011). Interestingly, IAA28, a repressor of auxin-responsive genes, was up-regulated in ark2-1/pub9-1 compared with the wild type under Pi starvation. IAA28 functions as a repressor of LR development, because gain-of-function mutant iaa28-1 is defective in LR development, and GATA TRANSCRIPTION FACTOR23 expression can rescue this phenotype (Rogg et al., 2001; De Rybel et al., 2010). Although the repressor IAA28 can regulate auxin signaling, auxin gradients facilitated by PIN efflux carrier proteins using both root- and shoot-derived auxin are crucial for LR development (Benková et al., 2003). During Pi starvation, expression levels of PIN1, PIN3, PIN5, PIN6, and PIN7 were found to be down-regulated in ark2-1/pub9-1 plants, providing additional evidence that both auxin efflux carriers and the signaling proteins are affected, which could lead to lack of auxin accumulation in the root tips of ark2-1/pub9-1 mutants. Recent studies have also unraveled a link between E3 ligases and the Ub proteasome system during LR development and are known to function by modulating auxin accumulation and signaling under limited phosphate availability. Pi starvation increases the expression of the auxin receptor TRANSPORT INHIBITOR RESPONSE1, which triggers ubiquitination and subsequent degradation of AUX/IAA repressors, thus relieving the AUXIN RESPONSE FACTORs and regulating the expression of genes involved in LR formation (Pérez-Torres et al., 2008).

The lack of auxin accumulation in the root tips of ark2-1/pub9-1 seedlings under phosphate starvation and the rescue of the LR phenotype by exogenous auxin (Fig. 5) are indicative of the ARK2-PUB9 module regulating auxin accumulation in the root tips during phosphate starvation. In accordance with this, 3-MA treatment of Col-0 plants showed a reduction in auxin accumulation in LRs after phosphate starvation (Supplemental Fig. S3), suggesting a role for autophagy in controlling auxin accumulation. Our results suggest that the lack of these two proteins could block autophagy under phosphate-starved conditions and possible degradation of repressors of auxin accumulation. Alternatively, activation of PUB9 by ARK2 could lead to ubiquitination of repressors of auxin accumulation, which are subsequently targeted to the autophagosomes through a selective autophagy process. In this scenario, ark2-1/pub9-1 would have normal formation of autophagosomes, except that selective targeting of proteins through the ARK2-PUB9 module would be absent. Identifying potential interactors of PUB9 would provide clues to possible downstream targets of this module that could negatively regulate auxin accumulation.

Whether these two proteins play a redundant role in parallel pathways or whether they form one of the many S domain1 kinase-PUB modules that are necessary for this process is not known. At the kinase level, ARK2 has the ability to interact with and phosphorylate multiple PUBs (Samuel et al., 2008). Kinase activity of ARK1 has been shown to be essential for ARK1-mediated relocalization of PUB9 from the nucleus to the plasma membrane, whereas in our study, ARK2 interaction with PUB9 results in localization of PUB9 to autophagosomes (Fig. 1). ARK1 and PUB9 function in a linear pathway, negatively regulating abscisic acid responses, because both the single and double ark1/pub9-1 mutants exhibited similar hypersensitivity to abscisic acid during seed germination (Samuel et al., 2008). In this study, only lack of both ARK2 and PUB9 resulted in the LR phenotypes, suggesting that genetic interaction between these two protein products plays a functionally redundant role during phosphate starvation. Thus, it is likely that PUB9 can be used by multiple upstream kinases to mediate unique tissue-specific responses. Nevertheless, our study has uncovered a previously unknown, redundant role for these highly diverse interacting partners and provides additional evidence for PUB proteins functioning as potential downstream signaling targets for S-domain receptor kinases.

MATERIALS AND METHODS

Plant Materials and Genetic Analysis

Arabidopsis (Arabidopsis thaliana) Col-0 was used as the wild type for all experiments. T-DNA insertion lines for AtPUB9 (SALK_020751) and AtARK2 (SAIL_594_A06) were obtained from the Arabidopsis Biological Resource Center. Homozygous lines were generated by PCR genotyping using primers listed in Supplemental Table S1. Double homozygotes (ark2-1/pub9-1) were created by crossing these single mutant lines.

Transient Expression Using BY2 Cells

The full-length AtPUB9 (At3g07360) and the kinase domain of AtARK2 (At1g65800) were cloned into plant expression vector pRTL2 as GFP-tagged and glutathione S-transferase-tagged constructs under the control of Cauliflower mosaic virus 35S promoter, respectively, as described in Samuel et al., 2008. RFP-Olesoin and SYP21 (At5g16830) were provided by Robert Mullen (University of Guelph, Guelph, Canada). Lysotracker Red (Invitrogen) staining was performed as per the manufacturer’s instructions. Cultured tobacco (Nicotiana tabacum) BY2 cells were used to perform biolistic bombardment as described previously (Stone et al., 2003). Cells were fixed in 4% (w/v) paraformaldehyde and visualized directly through a Leica DMR epifluorescence microscope for detecting GFP and RFP (Stone et al., 2003).

Cloning in Binary Vectors and Plant Transformation

AtPUB9 with GFP fused to its N terminus and ARK2 with yellow fluorescent protein fused to its C terminus were cloned into pCAMBIA binary vectors pCAM-ter and pCAMBIA1301, respectively. The absence of a single nucleotide in ARK2 (RAFL09-14-P09; RIKEN) was corrected by site-directed mutagenesis using the primers listed in Supplemental Table S1. These constructs were mobilized into Agrobacterium tumefaciens strain GV3101 separately and used for transforming ark2-1/pub9-1 plants by the floral dip method (Clough and Bent, 1998). The transgenic plants were obtained by selecting the harvested seeds on MS medium (Murashige and Skoog, 1962) with kanamycin for PUB9 and hygromycin for ARK2.

Phosphate Starvation and Root Growth Assay

Vapor-phase sterilization of seeds was carried out by adding 3 mL of concentrated HCl to 75 mL of commercial bleach and allowing it to sit for 4 h in a desiccator jar. Seeds were then plated on 0.5× MS medium supplemented with 1% (w/v) Suc and 0.75% (w/v) agar. After stratifying for 3 d in the dark, the plates were oriented vertically and allowed to germinate under light. After 3 d, the seedlings were transferred to either nutrient-rich (+Pi/+Suc) or phosphate starvation (−Pi/−Suc) medium.

The +Pi/+Suc was composed of 0.5× MS medium supplemented with 1.5% (w/v) Suc and 0.75% (w/v) agar, whereas the −Pi/−Suc medium contained 3 µm CaCl2, 3 µm MgSO40.7H2O, 5 µm KCl, 2 µm MES hydrate, 3 µm KNO3 1× micronutrient salt (Sigma), and 1× vitamins (Sigma) supplemented with 0.75% (w/v) agar. The plates were placed vertically to allow root growth on the agar surface at 22°C in a plant growth chamber with a photoperiod of 16 h of light and 8 h of dark. The seedlings were photographed after 7 d; the number of LRs was counted, and the PR lengths were measured using ImageJ software (Abramoff et al., 2004). For root growth assay with autophagy inhibitor 3-MA, seedlings were grown in similar media composition as described above (+Pi/+Suc and −Pi/−Suc) with the addition of 1 mm 3-MA (Sigma) in the respective media.

ConcA Treatment of Arabidopsis Seedlings and Confocal Microscopy

Two different lines of Arabidopsis pub9-1 seedlings constitutively expressing GFP-tagged PUB9 under the control of Cauliflower mosaic virus 35S promoter were grown vertically for 3 d in 0.5× MS medium with 1% (w/v) Suc and 0.75% (w/v) agar. The seedlings were then transferred to either +Pi/+Suc or −Pi/−Suc liquid media and incubated for 24 h at 22°C in light. Ten to twelve seedlings were then transferred to 24-well ELISA plates with +Pi/+Suc or −Pi/−Suc liquid media either with or without 2 µm ConcA (Santa Cruz Biotechnology; 1 mm stock in dimethyl sulfoxide). In treatments without ConcA, the same volume of dimethyl sulfoxide was added to the liquid media. The seedlings were then incubated for 12 to 16 h in the dark at room temperature, mounted in an aqueous environment, and visualized using a confocal microscopy. A Leica TCS SP5 confocal microscope system was used in this study. Quantification was carried out by counting the number of punctate structures (0.5–1.5 µm) in three different 100-µm2 areas of at least two different root epidermal cells for each treatment.

Histochemical GUS Assay

The mutant lines pub9-1, ark2-1, and ark2-1/pub9-1 containing the transgene DR5::uidA and wild-type Col-0 expressing DR5::uidA were grown under +Pi/+Suc or −Pi/−Suc as described above and incubated overnight in GUS reaction buffer (0.5 mg mL−1 of 5-bromo-4-chloro-3-indolyl-β-d-glucuronide, 0.1 mm K4[Fe(CN)6], 0.1 mm K3[Fe(CN)6], and Triton X-100 in 100 mm sodium phosphate buffer, pH 7.2). The seedlings were then cleared in 70% (v/v) ethanol, mounted on 50% (v/v) glycerol, and visualized under Leica DMR epifluorescence microscope bright-field optics. For GUS assay in the presence of autophagy inhibitor 3-MA, a similar protocol was followed using wild-type plants (Col-0) expressing the transgene DR5::uidA and grown under +Pi/+Suc or −Pi/−Suc with 1 mm 3-MA in the media.

Auxin Treatment of Arabidopsis Seedlings

For hormonal complementation of ark2-1/pub9-1 LR defect, 3-d-old seedlings were transferred to +Pi/+Suc or −Pi/−Suc medium supplemented with 0.001 to 1 µm NAA. After 7 d, the seedlings were photographed and analyzed by ImageJ software.

Quantitative RT-PCR

RNA was isolated from the LR forming zone of Col-0 and ark2-1/pub9-1 seedlings grown under either +Pi/+Suc or −Pi/−Suc condition for 4 d using TRIzol (Invitrogen). First-strand complementary DNA was synthesized from 800 ng of DNase-treated total RNA using oligo(dT)12–18 primer and SuperScript II Reverse Transcriptase (Invitrogen) following the manufacturer’s instructions. The quantitative PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). Primer pairs are listed in Supplemental Table S1. Each PCR reaction contained 1× Fast SYBR Green Master Mix (Applied Biosystems), 200 nm each primer, and 0.5 μL of complementary DNA in a final volume of 20 μL. PCR amplification was performed for 40 cycles at 95°C for 3 s and 60°C for 30 s with a preceding initial enzyme activation of 20 s at 95°C. Relative expression levels were calculated by the Δ-ΔCt method, and all quantifications were normalized using Ubq10 mRNA as an internal control. For each target gene, the reactions were carried out in duplicate for two biological replicates.

Supplemental Data

The following materials are available in the online version of this article.

Supplementary Material

Supplemental Data
supp_165_4_1647__index.html (1.5KB, html)

Acknowledgments

We thank Dr. Robert Mullen (University of Guelph) for providing the constructs for colocalization experiments.

Glossary

Col-0

ecotype Columbia-0 of Arabidopsis

ConcA

concanamycin A

IAA

indole-3-acetic acid

LR

lateral root

MS

Murashige and Skoog

NAA

naphthylacetic acid

Pi

inorganic phosphate

RFP

red fluorescent protein

RT

reverse transcription

Ub

ubiquitin

3-MA

3-methyladenine

T-DNA

transfer DNA

PR

primary root

Footnotes

1

This work was supported by the Natural Sciences and Engineering Research Council of Canada (funding to M.A.S.), and the University Research Grants Committee from the University of Calgary (grants to M.A.S.).

[C]

Some figures in this article are displayed in color online but in black and white in the print edition.

[W]

The online version of this article contains Web-only data.

[OPEN]

Articles can be viewed online without a subscription.

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