ARTICLE Received 11 Feb 2011 | Accepted 21 Jun 2011 | Published 19 July 2011 DOI: 10.1038/ncomms1408 Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1 Bong Soo Park1,2, Jong Tae Song3 & Hak Soo Seo1,2,4 Small ubiquitin-related modifier SUMO( ) is a small polypeptide that modulates protein activity and regulates hormone signalling, abiotic and biotic responses in plants. Here we show that AtSIZ regulates nitrogen assimilation in Arabidopsis through its E3 SUMO ligase function. Dwarf plants of siz1-2 flower early, show abnormal seed development and have high salicylic acid content and enhanced resistance to bacterial pathogens. These mutant phenotypes are reverted to wild-type phenotypes by exogenous ammonium but not by nitrate, phosphate or potassium. Decreased nitrate reductase activity in siz1-2 plants resulted in low nitrogen concentrations, low nitric oxide production and high nitrate content in comparison with wild-type plants. The nitrate reductases, NIA1 and NIA2, are sumoylated by AtSIZ1, which dramatically increases their activity. Both sumoylated and non-sumoylated NIA1 and NIA2 can form dimers. Our results indicate that AtSIZ1 positively controls nitrogen assimilation by promoting sumoylation of NRs in Arabidopsis. 1 Department of Plant Science, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. 2 Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-921, Korea. 3 School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Korea. 4 Bio-MAX Institute, Seoul National University, Seoul 151-818, Korea. Correspondence and requests for materials should be addressed to H.S.S. (email: [email protected]). NatURE COMMUNicatiONS | 2:400 | DOI: 10.1038/ncomms1408 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLE NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms1408 itrogen is one of the most important inorganic nutrients 10 d 20 d 10 d 20 d and a major component of nucleic acids, proteins, various WT siz1-2 WT siz1-2 WT siz1-2 WT siz1-2 cofactors and secondary metabolites. Plants have evolved N None None to utilize nitrate as a source of environmental nitrogen, and nitrate serves as a potent signal that regulates nitrogen and carbon metabo- 1–4 KNO lism as well as organ growth and development . Nitrate from the NaCI 3 soil is transported actively into cells by a nitrate transporter and is reduced sequentially to ammonia, which enters the amino-acid NaNO NaH2PO4 3 pool primarily via the action of glutamine synthetase. Homodimers of plant nitrate reductase (NR) catalyse the NAD(P)H-dependent (NH ) SO reduction of nitrate to nitrite5,6, which is reduced further to ammo- KCI 4 2 4 nium by NR in the second step of the nitrate assimilation pathway7. The Arabidopsis genome contains two NR genes, NIA1 and NIA2. NH4NO3 The nia1/nia2 double mutant flowers early 8. Small ubiquitin-related modifier (SUMO), a key regulator of biological processes, is covalently conjugated to a lysine residue in Figure 1 | Growth patterns of WT and siz1-2 plants and the effect of a substrate protein via a reversible posttranslational modification nitrogen sources on their growth. (a) After germination on MS media, process that is facilitated by E3 SUMO ligases9. As in other eukaryo- seedlings were transferred to soil and treated with 5 mM phosphate and tes, SUMO modification in plants has been implicated in numerous potassium. (b) Seedlings were also treated with 5 mM of each nitrogen basic cellular processes, such as stress and defence responses and source for 10 and 20 days. The growth of siz1-2 plants was recovered by 10–18 ammonium sources such as (NH ) SO and NH NO , but not by nitrate the regulation of flowering . 4 2 4 4 3 sources (such as KNO or NaNO ), phosphate or potassium. Scale bar, AtSIZ1 is an SP-RING-finger protein that contains a SAP domain 3 3 and a zinc-finger Miz domain. AtSIZ1 is a key regulator of signalling 5 cm. pathways that mediate responses to nutrient deficiency and environ- mental stresses14–17,19–22. The observation that mutations in AtSIZ1 result in dwarf plants with smaller leaves indicates that AtSIZ1 also mutant reproductive phenotypes are caused by impairment of the has a role in vegetative growth and development16,17,23. nitrate reduction pathway because of the AtSIZ1 mutation. Despite the involvement of sumoylation in a wide range of NR is the only enzyme to catalyse the conversion of nitrite to physiological processes, only a few Arabidopsis proteins, includ- nitric oxide (NO) in several higher plants29–32, although the pri- ing the SUMO machinery proteins AtSIZ1 and AtSCE1, have been mary function of NR is nitrogen assimilation through the reduc- experimentally demonstrated to be sumoylated24. Very recently, tion of nitrate to nitrite. Low levels of NO in the NR double mutant two groups have identified massive SUMO conjugates by proteom- nia1/nia2 have been reported to cause early flowering8,33, similar ics methods25 and yeast two-hybrid screening26 in Arabidopsis to siz1-2 plants21. Interestingly, ammonium-treated siz1-2 plants under non-stress and stress conditions. The results indicated that showed restored flowering time (Supplementary Fig. S1), implying sumoylation can regulate diverse biological processes, although the that nitrate reduction is impaired in siz1-2. functional consequences of this modification were not fully characterized. Decreased nitrogen content and NR activity in siz1-2 plants. Nitrogen assimilation is a fundamental biological process We assessed NR activity in siz1-2 plants using chlorate, the that has a marked effect on plant productivity, biomass and crop chlorine analogue of nitrate, which is reduced to the toxic chlo- yield. NR is a key enzyme in a plant’s nitrogen assimilation path- rite by NR. Phenotypic analysis revealed a less severe inhibition of way. However, few components that influence NR activity have been growth of chlorate-treated siz1-2 plants compared with WT plants identified27,28, and NR regulation at the posttranslational level has (Supplementary Fig. S3). This result indicates that the siz1-2 plants not been well characterized. have low NR activity. Further analysis showed that NR activity was Here, we investigated the contribution of AtSIZ1-mediated decreased in both the leaves and roots of the siz1-2 plants compared nitrate reduction to nitrogen assimilation in Arabidopsis. siz1 with WT plants (Fig. 2a,b). mutants were recovered as to the wild-type (WT) phenotype when On the basis of the low NR activity of siz1-2 plants, nitrate supplemented with exogenous ammonium and NR activity of NIA1 reduction and nitrogen assimilation were assessed by measuring and NIA2 was increased by sumoylation through E3 ligase acti­ the nitrogen and nitrate concentrations and NO production in the vity of AtSIZ1. Our results provide the first biochemical evidence mutant plants. As expected, nitrogen content was low (Fig. 2c,d; that sumoylation is a critical protein modification required for the Supplementary Table S2) and nitrate content was high in siz1-2 regulation of NR activity. plants (Fig. 2e,f). In addition, NO production was decreased in siz1-2 plants (Fig. 2g,h), which supports the previous finding that Results NO accumulation is mediated by an NR-associated pathway34. Ammonium recovers the siz1-2 mutant phenotype. The Arabi­ dopsis siz1-2 mutants display a dwarf phenotype, early flowering E3 SUMO ligase activity of AtSIZ1 on NIA1 and NIA2. Our results and abnormal seed development. We examined the capacity imply that AtSIZ1 may modulate the activity or stability of NIA1 of Arabidopsis siz1-2 mutants to recover growth and normal and NIA2 by acting as an E3 SUMO ligase. The deduced amino- development in response to the provision of a nitrogen source and/ acid sequences of the NRs, NIA1 and NIA2, revealed that both pro- or supplementation with phosphate or potassium, as these factors teins have putative sumoylation sites (ψKXE), which were located are essential for plant growth and development. During vegetative at lysine 356 (K356) of NIA1 and lysine 353 (K353) of NIA2 (Sup- growth, the WT phenotype was restored with the use of ammo­ plementary Fig. S4). Therefore, to evaluate the possible interaction nium, but not nitrate, as a nitrogen source (Fig. 1; Supplementary between AtSIZ1 and NIA1 or NIA2, glutathione sulphur transferase Fig. S1). During the reproductive phase, siz1-2 mutant phenotypes (GST)-tagged AtSIZ1 and histidine-tagged NIA1 or NIA2 were such as short silique length, lower seed number (about 50% of WT) overexpressed in Escherichia coli and purified with glutathione 2 + and sterility were restored to WT phenotypes with ammonium and Ni -NTA resins, respectively. In vitro pull-down of His6-NIA1 (Supplementary Fig. S2 and Table S1). This result indicates that the or His6-NIA2 with GST and GST-AtSIZ1 revealed that AtSIZ1 NatUre cOMMUNicatiONS | 2:400 | DOI: 10.1038/ncomms1408 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms1408 ARTICLE 0.6 0.14 14 70 0.5 0.12 12 60 10 50 0.4 0.10 ) 0.08 8 –1 40 0.3 g 0.06 6 30 0.2 (mg 0.04 (mg/plants) 4 20 0.1 0.02 2 10 Nitrogen concentration 0.0 0.00 0 Nitrogen concentration 0 Nitrate reductase activity Nitrate reductase activity (Abs at 540 nm/100 plants) (Abs at 540 nm/100 plants) WT siz1-2 WT siz1-2 WT siz1-2 WT siz1-2 0.5 20 t 0.4 15 WT WT ) –1 0.3 g 10 0.2 (mg 5 Nitrate conten 0.1 siz1-2 siz1-2 0.0 0 WT siz1-2 Nitrate content (mg/plant) WT siz1-2 Figure 2 | NR activity is decreased in siz1-2 plants.