article addendum
article addendum
Plant Signaling & Behavior 5:5, 561-563; May 2010; © 2010 Landes Bioscience
Non-proteolytic protein ubiquitination is crucial for iron deficiency signaling Wenfeng Li and Wolfgang Schmidt* Institute of Plant and Microbial Biology; Academia Sinica; Taipei, Taiwan
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Key words: root hairs, iron deficiency, Lys 63-linked polyubiquitylation, ubiquitin-conjugase, post-transcriptional regulation Submitted: 02/05/10 Accepted: 02/05/10 Previously published online: www.landesbioscience.com/journals/psb/ article/11424 *Correspondence to: Wolfgang Schmidt; Email:
[email protected] Addendum to: Li W, Schmidt W. A lysine 63-linked ubiquitin chain forming conjugase, UBC13, promotes the developmental responses to iron deficiency in Arabidopsis roots. Plant J 2010; In press; PMID: 20113438; DOI: 10.1111/j.1365313X.2010.04150.x.
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biquitination generally targets proteins for recognition and degradation via the 26S proteasome. Activated ubiquitin (E1) is transferred to an ubiquitin conjugase (UBC, E2) which associates with a ubiquitin ligase (E3), and multiple ubiquitin molecules are attached via linkage of Lys 48. By contrast, Lys 63-linked ubiquitin chains modify proteins in a non-proteolytic manner. We recently reported that UBC13, the only known ubiquitin conjugase capable of catalyzing Lys 63-linked polyubiqitination, is responsive to the iron (Fe) regime at the post-transcriptional level and may play a crucial role for the morphological alterations triggered by Fe deficiency in cucumber and Arabidopsis roots. It is assumed that UBC13 participates, most likely via the non-proteolytic polyubiquitination of proteins, in the signal transduction cascade associated with the acclimation of plants to the prevailing availability of Fe. In this Addendum, we present a possible scenario that occurs downstream of UBC13, which ultimately leads to Fe deficiency-specific changes in postembryonic development of Arabidopsis roots. Iron (Fe) is an indispensible mineral nutrient for all organisms. Plants respond to Fe deficiency by altering mRNA availability and protein activity in order to improve Fe uptake from pools with limited solubility and to adjust the inter and intracellular distribution of the mineral. In addition, alterations in post-embryonic development, generally aimed at increasing the
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absorptive surface area of the root are induced by Fe deficiency. Root morphological changes, such as the formation of extra root hairs, are widespread among plants; however, in contrast to the major physiological responses to Fe deficiency, the processes that mediate these developmental alterations have not yet been explored. UBC13 is Responsive to the Iron Regime In a paper appearing in the 2010 edition of the Plant Journal, we report on a protein isolated in a screen for proteins that are post-transcriptionally responsive to Fe deficiency from cucumber root tips.1 We subsequently identified a protein that displayed high amino acid sequence similarity to the ubiquitin conjugase enzyme 13 of Arabidopsis (AtUBC13A). UBC13 is highly conserved among eukaryotes and has been demonstrated to exert a function in promoting the assembly of non-canonical ubiquitin chains that are linked at lysine 63.2,3 In contrast to classical Lys 48-linked polyubiquitin which targets protein into 26S proteasome degradation pathway, Lys 63 polyubiquitin chains instead are thought to alter protein activity in a post-translational manner, similar to other protein modifications such as phosphorylation. Ectopic expression of the cucumber gene in Arabidopsis promoted the formation of bifurcated root hairs, a striking feature characteristic of Fe-deficient Arabidopsis plants. By contrast, the formation of branched root hairs was repressed in Fe-deficient Atubc13A mutant plants.
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Figure 1. Computational predictions of protein-protein interactions for UBC13A. Potential and experimentally confirmed partners of UBC13A are indicated in bold letters. The figure has been generated by The Bio-Array Resource for Plant Functional Genomics (http://bar.utoronto.ca/), and has been modified to highlight putative interactions important for the processes discussed here.
The Level of UBC13 Protein Controls Downstream Reactions In mammals, yeast and plants, the formation of Lys 63-linked polyubiquitin chains is catalyzed by UBC13 and requires an ubiquitin conjugating enzyme variant (named Uev1A in mammals and Mms2 in budding yeast) and an associated E3 ligase. In Arabidopsis, four Mms2 homologs (UEV1s) have been identified.4 All four UEV1 genes can form stable complexes with UBC13 and can promote the formation of Lys 63-linked polyubiquitin chains. Computational predictions of protein-protein interactions suggest two putative E3 ligase partners for UBC13A (Fig. 1). The RING domain Ligase2 (RGLG2) protein has been shown to interact with UBC13A in yeast-two hybrid assays and to be capable of forming Lys 63-linked ubiquitin chains in vitro.5 Astonishingly, rglg1 rglg2 double mutants showed a constitutive formation of root hair branching, independent on the Fe supply. This was an unexpected finding since disruption of the E2-E3 cascade by knockout of UBC13A compromised the response. A possible
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explanation is that UBC13A interacts with different E3 ligases. Database search revealed strong interactions between UBC13 and At5g43530, which encodes a SNF2 domain containing protein with strong homology to the yeast ScRAD5 gene, a key component of the error-free branch of post-replication repair in yeast.6 In Arabidopsis, sequence comparison revealed two close homologs to ScRAD5 named AtRAD5A (At5g22750) and AtRAD5B (At5g43530).7 RAD5 proteins possess a RING domain, have E3 ligase activity (at least in yeast) and are putatively involved in Lys 63-linked chain formation.7,8 While Atrad5A mutants were found to be hypersensitive to DNAdamaging agents, no such phenotype was observed for Atrad5B mutants.7 This finding suggests that AtRAD5B could possess an alternative or an yet unspecified function. The phenotype of rglg1 rglg2 mutants could be explained if we consider the possibility that different E3 ligases compete for a limited amount of UBC13A protein (Fig. 2). Under control conditions, this competition allows only a small percentage of branched root hairs to be formed. Under Fe-deficient
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conditions, the level of UBC13A protein is increased post-transcriptionally, causing a high percentage of branched root hairs. Knockout of RGLG2 provides more UBC13A protein for the Fe-specific pathway, resulting in a constitutively high number of bifurcated root hairs. As reported previously, this phenomenon is highly specific to Fe-deficient Arabidopsis plants and is observed in extremely low occurrence under control or phosphatedeficient conditions.9 In all genotypes under investigation, root hair branching was repressed under phosphate-deficient conditions. Interestingly, the rglg1 rglg2 mutants, even though exhibiting constitutive formation of bi- or trifurcated root hairs, do not form branched hairs under phosphate-deficient conditions. Also this phenotype can be explained if it is assumed that UBC13A is the decisive factor for root hair branching. It is well known that phosphate deficiency causes an overload of Fe and a subsequent downregulation of Fe-regulated genes.10,11 Therefore, under phosphate-deficient conditions, the level of UBC13A can be assumed to be severely reduced, effectively compromising the response.
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Figure 2. Hypothetical model for the role of UBC13A in the regulation of the morphological Fe deficiency responses and other processes in Arabidopsis. The level of UBC13A is regulated post-transcriptionally by the availability of Fe. Depending on the availability of UBC13A protein, downstream processes are induced or repressed. Ubiquitin ligase activity has only been proven for RGLG2.
A Role for Auxin?
Final Remarks
While it seems clear that UBC13A, probably act via the formation of Lys 63-linked polyubiquitin chain, is crucial for the morphological responses to Fe deficiency, it remains to be elucidated what happens downstream of the E2-E3 cascade. The (constitutive) formation of branched root hairs has been observed in a number of mutant backgrounds. For example, the axr2 mutant, which harbors a defect in a gene encoding a member of the Aux/IAA protein family, IAA7, is auxin-related.12,13 The rglg1 rglg2 double mutants were shown to lack apical dominance due to a defect in auxin transport.5 It is thus tempting to speculate that the Fe deficiency-induced root hair branching and the phenotype of plants overexpressing UBC13 is the result of a compromise in auxin transport. From the fact that auxin can induce morphological alterations resembling developmental responses to Fe deficiency,14 and the instance that the formation of branched root hairs in circumvented under phosphate-deficient conditions under which root hair formation was shown to be independent of auxin, a direct or indirect interaction of auxin-related processes with Lys 63-polyubiquinated substrates appears to be a plausible explanation for the case of branched hairs induced under Fe deficiency.
Responses to Fe deficiency in Arabidopsis and other species have been mainly deciphered at the transcriptional level. UBC13 is the first key player in the Fe deficiency syndrome that is regulated chiefly or entirely at the post-transcriptional level. It remains to be shown if UBC13 is the exception in a transcriptionally ruled world or rather represents the tip of the iceberg of an additional layer of regulation in the complex interplay to acclimate plants to changing Fe availability.
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References 1. Li W, Schmidt W. A Lysine 63-Linked Ubiquitin Chain Forming Conjugase, UBC13, Promotes the Developmental Responses to Iron Deficiency in Arabidopsis Roots. Plant Journal 2010; in press. 2. Hofmann RM, Pickart CM. Noncanonical MMS2encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 1999; 96:645-53. 3. Pickart CM, Fushman D. Polyubiquitin chains: polymeric protein signals. Curr Opin Chem Biol 2004; 8:610-6. 4. Wen R, Torres-Acosta JA, Pastushok L, Lai X, Pelzer L, Wang H, Xiao W. Arabidopsis UEV1D promotes Lysine-63-linked polyubiquitination and is involved in DNA damage response. Plant Cell 2008; 20:21327. 5. Yin XJ, Volk S, Ljung K, Mehlmer N, Dolezal K, Ditengou F, et al. Ubiquitin lysine 63 chain forming ligases regulate apical dominance in Arabidopsis. Plant Cell 2007; 19:1898-911.
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6. Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 2002; 419:135-41. 7. Chen IP, Mannuss A, Orel N, Heitzeberg F, Puchta H. A homolog of ScRAD5 is involved in DNA repair and homologous recombination in Arabidopsis. Plant Physiol 2008; 146:1786-96. 8. Chang DJ, Cimprich KA. DNA damage tolerance: when it’s OK to make mistakes. Nat Chem Biol 2009; 5:82-90. 9. Müller M, Schmidt W. Environmentally induced plasticity of root hair development in Arabidopsis. Plant Physiol 2004; 134:409-19. 10. Misson J, Raghothama KG, Jain A, Jouhet J, Block MA, Bligny R, et al. A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc Natl Acad Sci USA 2005; 102:11934-9. 11. Ward JT, Lahner B, Yakubova E, Salt DE, Raghothama KG. The effect of iron on the primary root elongation of Arabidopsis during phosphate deficiency. Plant Physiol 2008; 147:1181-91. 12. Wilson AK, Pickett FB, Turner JC, Estelle M. A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid. Mol Gen Genet 1990; 222:377-83. 13. Nagpal P, Walker LM, Young JC, Sonawala A, Timpte C, Estelle M, Reed JW. AXR2 encodes a member of the Aux/IAA protein family. Plant Physiol 2000; 123:563-74. 14. Schmidt W, Michalke W, Schikora A. Proton pumping by tomato roots. Effect of Fe deficiency and hormones on the activity and distribution of plasma membrane H + -ATPase in rhizodermal cells. Plant Cell & Environment 2003; 26:361-70.
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