A proposed role for selective autophagy in regulating auxin-dependent lateral root development under phosphate starvation in Arabidopsis

Abstract

Plants respond to limited soil nutrient availability by inducing more lateral roots (LR) to increase the root surface area. At the cellular level, nutrient starvation triggers the process of autophagy through which bulk degradation of cellular materials is achieved to facilitate nutrient mobilization. Whether there is any link between the cellular autophagy and induction of LR had remained unknown. We recently showed that the S-Domain receptor Kinase (ARK2) and U Box/Armadillo Repeat-Containing E3 ligase (PUB9) module is required for lateral root formation under phosphate starvation in Arabidopsis thaliana.¹ We also showed that PUB9 localized to autophagic bodies following either activation by ARK2 or under phosphate starvation and ark2–1/pub9–1 plants displayed lateral root defects with inability to accumulate auxin in the root tips under phosphate starvation.¹ Supplementing exogenous auxin was sufficient to rescue the LR defects in ark2–1/pub9–1 mutant. Blocking of autophagic responses in wild-type Arabidopsis also resulted in inhibition of both lateral roots and auxin accumulation in the root tips indicating the importance of autophagy in mediating auxin accumulation under phosphate starved conditions.¹ Here, we propose a model for ARK2/AtPUB9 module in regulation of lateral root development via selective autophagy.

Plants respond to low phosphate availability by reducing their primary root length and increasing the lateral root density.² It has been proposed that enhanced root branching seen in Pi starvation response is an auxin-dependent process while primary root growth inhibition is independent of polar auxin transport.³ Auxin is known to be responsible for establishment of founder cells committed to LR formation^4,-6 and auxin responses are mediated by auxin response factors (ARFs) and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) proteins.⁷ ARFs function by acting as transcriptional activators/repressors of auxin response genes, while AUX/IAA proteins bind to ARFs to repress their transcriptional activity.^8,9 In presence of auxin, AUX/IAA repressors are degraded via the proteasome through the ubiquitin protein ligase complex SCF^TIR1, which allows the ARFs to regulate the expression of auxin responsive genes involved in growth and development.^10,11 Interestingly, SCF^TIR1 complex has also been proposed to regulate lateral root development during low Pi availability.¹¹

One of the common responses of plants to nutrient starvation is autophagy, required for nutrient mobilization and recycling.^12,14,15 Pi starvation has been shown to induce the ubiquitin-like autophagy protein ATG8 in root tips.¹³ Despite this, a clear mechanism of how autophagy could regulate Pi starvation responses has been lacking. In our recently published work we showed that the Arabidopsis module comprising of S-domain receptor kinase1–6 or Arabidopsis Receptor Kinase2 (ARK2) and the E3 ligase plant U-box/armadillo repeat protein9 (AtPUB9) E3 ligase mediates lateral root development under Pi starvation. The S-domain receptor kinases act as upstream activators of PUBs and can effectively phosphorylate them.¹⁴ We demonstrated that lack of ARK2 and PUB9 resulted in abrogation of auxin accumulation in the LR tips following Pi starvation and supplementing exogenous auxin was sufficient to restore LR formation. Interestingly, the single mutants did not display this phenotype indicating functional redundancy across gene families that mediate LR formation under Pi starvation. Such genetic interactions across gene families have not been explored in detail in plant systems.

Genetic interactions are usually identified when a combination of 2 mutations leads to an unexpected phenotype that cannot be observed in the single mutants. In plants, large gene families and vast number of genes make it a difficult task to set up screens to identify genetic interactions at a genome-wide scale.  In contrast, in yeast, synthetic genetic interaction screens called ‘Synthetic Genetic Array’ analyses have been used exhaustively to identify genes whose functions overlap. In these screens, double mutants are created at a high-throughput rate by crossing a mutant line defective in the chosen gene of interest with viable deletion mutants of all the genes in the genome.¹⁵ This provides a global analysis of genetic interactions and reveals gene products that function in the same essential pathway. The extensive duplication and rearrangement of Arabidopsis genome¹⁶ makes it extremely difficult to reveal functional redundancies across gene families via conventional crossing and epistatic analysis. Since these ARK2 and PUB9 interacted in a yeast 2-hybrid screen and PUB9 was re-located to autophagosome following phosphorylation, we created the double mutants and tested it under various conditions that induced autophagy. This led to the identification of the genetic interaction between these 2 diverse genes. When close relatives of ARK2 and PUB9 are examined,^17-20 these genes are also expressed in the root tissue and a subset is modulated by Pi starvation (Fig. 1). This suggests that multiple combination of SD-1 kinase and PUBs can exist that can be involved to different extents in LR formation under Pi limited conditions with a major contribution from ARK2/PUB9 module.

Figure 1.

Expression analysis of SDK and PUB gene family members in Arabidopsis roots (A). Graphs representing the absolute gene expression values from Whole Genome Tiling Array 1.0 of SDK gene family members (left) and plant U Box genes closely related to PUB9 (right). Values generated using the Arabidopsis eFP Browser at bar.utoronto.ca (B). Heat maps representing the absolute expression values of available SDK (left) and PUB9 gene family members (right) in Arabidopsis roots under Pi rich and Pi deficient conditions (6 h and 24 h post-transfer). Values represent the average of 3 replicates generated from Co-expression Networks of Phosphate Deficiency genes in BAR Expression Browser (bar.utoronto.ca).

Expression of IAA28, an AUX/IAA repressor which was previously reported to be a suppressor of lateral root development²¹ was up-regulated in Pi starved ark2–1/pub9–1 plants compared to wild type Col-0 and auxin transport genes PINFORMED1 (PIN1), PIN3, PIN5, PIN6 and PIN7 were significantly down-regulated.¹ It has been proposed that auxin regulates PIN gene expression through AUX/IAA pathway²² and AUX/IAA-ARF mediate PIN polarity and the redirection of auxin flow, resulting in establishment of a new primordium axis during lateral root formation.²³ Although there are at least 29 AUX/IAA and 23 ARF gene family members in Arabidopsis thaliana²⁴ with a staggering number of possible combinations, the biological roles of most of these are as yet unidentified. Auxin is known to control lateral root development through multiple auxin-signaling modules.^24-27 Typically AUX/IAAs have a conserved 13 amino acid core sequence in domain II that is known to mediate auxin dependent TIR1 binding and degradation.^25,26 There are AUX/IAAs in Arabidopsis that lack this domain II (IAA20, IAA30 and IAA32–34) and IAA 31 has partial domain sequence.²⁷ It has been suggested that there could be degrons outside domain II, which could also influence proteolysis^27-29 and function as a convergence point for other signals. Thus, existence of more than one pathway and alternative mechanisms in regulation of these proteins cannot be precluded.

Based on our observations with ark2–1/pub9–1 phenotypes and our current knowledge about auxin signaling, we propose a mechanism of selective autophagy that operates to regulate auxin accumulation and lateral root development under Pi starvation. As per our model, Pi starvation triggers the phosphorylation-mediated activation of PUB9 by ARK2 and subsequently the activated PUB9 selectively targets the AUX/IAA or other repressors of auxin accumulation to the autophagosomes for degradation. This in turn allows either AUXIN RESPONSE FACTORS (ARFs) or alternate mechanisms to cause auxin accumulation and lateral root development (Fig. 2). The nature of the targets of PUB9 that regulate auxin accumulation is not known. This pathway could be operating simultaneously with the previously proposed SCF^TIR1-dependent pathway that also functions during Pi starvation, although ARK2/PUB9 likely operates upstream regulating auxin accumulation in the LR tips.

Figure 2.

Model for ARK2/PUB9 controlling lateral root growth under Pi starvation In presence of Pi, ARK2/PUB9 is likely not active. Under sufficient Pi levels either AUX/IAA or other repressors prevent accumulation of auxin in later root (LR) initiation sites, allowing normal root development (left). Activation of ARK2, under Pi limiting conditions leads to PUB9 phosphorylation. Phosphorylated PUB9 is likely involved in selective targeting of either AUX/IAA or other repressors of auxin accumulation to the autophagosome. This releases the suppression on ARF/other signals, which results auxin accumulation in LR initiation sites leading to enhanced lateral root growth (right).

There is emerging evidence in animal systems that support a role for ubiquitin in autophagy and cross talks between the ubiquitin-proteasome system (UPS) and autophagy.^30,31,32 Despite being a new theme, the concept of selective autophagy has been shown in plants, with the discovery of ATG8 protein and its interacting partner NBR1, the homolog of mammalian autophagic adaptor P62.^33,34 Identification of the interactors of phosphorylated PUB9 would reveal the identity of the candidates involved in mediating auxin accumulation in LR tips under Pi starvation. Understanding the genetic and physiological signals behind root growth and development in Arabidopsis will allow translation of this technology to generate crops that can sustain nutrient retrieval and growth under nutrient deprived conditions.

Acknowledgments

This work was supported by Natural Sciences and Engineering Research Council of Canada and funding from University of Calgary to M.A.S. S.S is supported by an Eyes High International Doctoral Scholarship and Global Open Doctoral Scholarship from the University of Calgary.