Cunjin Zhang

SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana.

Significance The photoreceptor phytochrome-B (phyB) cycles between its active Pfr [far red light (FRL)-absorbing state λmax, 730 nm] and inactive Pr [red light (RL)-absorbing state λmax, 660 nm] forms and regulates as red/far red light-activated/inactivated molecular switch plant growth and development. Here we show that conjugation of small ubiquitin-like modifier to the photoreceptor inhibits interaction of phyB Pfr with its immediate signaling partner PHYTOCHROME INTERACTING FACTOR 5 (PIF5). The impaired interaction of these proteins negatively affects photomorphogenic responses; thus, SUMOylation similar to phosphorylation plays a role in desensitizing phyB-mediated signaling. OVERLY TOLERANT TO SALT 1 and 2 (OTS1 and OTS2) are involved in regulating phyB action as these SUMO proteases mediate deconjugation of SUMO from phyB. The red/far red light absorbing photoreceptor phytochrome-B (phyB) cycles between the biologically inactive (Pr, λmax, 660 nm) and active (Pfr; λmax, 730 nm) forms and functions as a light quality and quantity controlled switch to regulate photomorphogenesis in Arabidopsis. At the molecular level, phyB interacts in a conformation-dependent fashion with a battery of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR transcription factors, and by modulating their activity/abundance, it alters expression patterns of genes underlying photomorphogenesis. Here we report that the small ubiquitin-like modifier (SUMO) is conjugated (SUMOylation) to the C terminus of phyB; the accumulation of SUMOylated phyB is enhanced by red light and displays a diurnal pattern in plants grown under light/dark cycles. Our data demonstrate that (i) transgenic plants expressing the mutant phyBLys996Arg-YFP photoreceptor are hypersensitive to red light, (ii) light-induced SUMOylation of the mutant phyB is drastically decreased compared with phyB-YFP, and (iii) SUMOylation of phyB inhibits binding of PHYTOCHROME INTERACTING FACTOR 5 to phyB Pfr. In addition, we show that OVERLY TOLERANT TO SALT 1 (OTS1) de-SUMOylates phyB in vitro, it interacts with phyB in vivo, and the ots1/ots2 mutant is hyposensitive to red light. Taken together, we conclude that SUMOylation of phyB negatively regulates light signaling and it is mediated, at least partly, by the action of OTS SUMO proteases.

Deubiquitinating enzymes AtUBP12 and AtUBP13 and their tobacco homologue NtUBP12 are negative regulators of plant immunity

• Signalling by ubiquitination is implicated in diverse aspects of the plant lifecycle, and enzymes of ubiquitin metabolism are overrepresented in the Arabidopsis genome compared with other model eukaryotes. Despite the importance of ubiquitination in the regulation of signalling, little is known about deubiquitinating enzymes, which reverse the process of ubiquitination. • Transgenic RNA interference-based cosuppression and the isolation of Atubp12/13 double mutants collectively provides the first report that AtUBP12 and AtUBP13 are functionally redundant and are required for immunity against virulent Pseudomonas syringae pv tomato in Arabidopsis. The Solanaceous AtUBP12 orthologue NtUBP12 was identified. Viral-induced gene silencing and transient gain-of-function assays were employed to establish that the NtUBP12 protein functions as a negative regulator of the Cf-9-triggered hypersensitive response. • Here, we demonstrate that NtUBP12 and AtUBP12 are bona fide deubiquitinating enzymes capable of cleaving lysine-48-linked ubiquitin chains. AtUBP12 and NtUBP12 are functionally interchangeable and their deubiquitinating activity is required to suppress plant cell death. • Overall, our data implicate AtUBP12- and NtUBP12-dependent deubiquitination in the stabilization of common substrates across Solanaceae and Brassicaceae which regulate disease resistance.

Stability of small ubiquitin-like modifier (SUMO) proteases OVERLY TOLERANT TO SALT1 and -2 modulates salicylic acid signalling and SUMO1/2 conjugation in Arabidopsis thaliana

Small ubiquitin-like modifier proteases 1 and 2 (SUMO1/2) have been linked to the regulation of salicylic acid (SA)-mediated defence signalling in Arabidopsis thaliana. In order to define the role of the SUMO proteases OVERLY TOLERANT TO SALT1 and -2 (OTS1/2) in defence and to provide insight into SUMO1/2-mediated regulation of SA signalling, we examined the status of SA-mediated defences in ots1/2 mutants. The ots1 ots2 double mutant displayed enhanced resistance to virulent Pseudomonas syringae and higher levels of SA compared with wild-type (WT) plants. Furthermore, ots1 ots2 mutants exhibited upregulated expression of the SA biosynthesis gene ICS1 in addition to enhanced SA-responsive ICS1 expression beyond that of WT. SA stimulated OTS1/2 degradation and promoted accumulation of SUMO1/2 conjugates. These results indicate that OTS1 and -2 act in a feedback loop in SA signalling and that de novo OTS1/2 synthesis works antagonistically to SA-promoted degradation, adjusting the abundance of OTS1/2 to moderate SA signalling. Accumulation of SUMO1/2 conjugates coincides with SA-promoted OTS degradation and may play a positive role in SA-mediated signalling in addition to its repressive roles reported elsewhere.

A functional Small Ubiquitin-like Modifier (SUMO) interacting motif (SIM) in the gibberellin hormone receptor GID1 is conserved in cereal crops and disrupting this motif does not abolish hormone dependency of the DELLA-GID1 interaction

Plants survive adversity by modulating their growth in response to changing environmental signals. The phytohormone Gibberellic acid (GA) plays a central role in regulating these adaptive responses by stimulating the degradation of growth repressing DELLA proteins which accumulate during stress. The current model for GA signaling describes how this hormone binds to its receptor GID1 so promoting association of GID1 with DELLA, which then undergoes ubiquitin-mediated proteasomal degradation. Recent data revealed that conjugation of DELLAs to the Small Ubiquitin-like Modifier (SUMO) protein enables plants to modulate its abundance during environmental stress. This is achieved by SUMOylated DELLAs sequestering GID1 via its SUMO interacting motif (SIM) allowing non-SUMOylated DELLAs to accumulate leading to growth restraint under stress and potential yield loss. We demonstrate that GID1 proteins across the major cereal crops contain a functional SIM able to bind SUMO1. Site directed mutagenesis and yeast 2 hybrid experiments reveal that it is possible to disrupt the SIM-SUMO interaction motif without affecting the GA dependent DELLA–GID1 interaction and thereby uncoupling SUMO–mediated inhibition from DELLA degradation. Arabidopsis plants overexpressing a SIM mutant allele of GID1 perform better at relieving DELLA restraint than wild–type GID1. This evidence suggests that manipulating the SIM motif in the GA receptor may provide a possible route to developing stress tolerant crops plants.

BTB-BACK domain protein POB1 suppresses immune cell death by targeting ubiquitin E3 ligase PUB17 for degradation.

Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses.

Rice SUMO protease Overly Tolerant to Salt 1 targets the transcription factor, OsbZIP23 to promote drought tolerance in rice

Conjugation of SUMO (Small Ubiquitin-like Modifier) protein to cellular targets is emerging as a very influential protein modification system. Once covalently bound, SUMO conjugation can change the stability or functionality of its cognate target proteins. SUMO protease can rapidly reverse SUMO conjugation making this modification system highly dynamic. A major factor in the variation of SUMO-target function is the balance between the conjugated/de-conjugated forms. The mechanistic role of these regulatory SUMO proteases in mediating stress responses has not been defined in any crops. In this study, we reveal the role of the SUMO protease, OsOTS1 in mediating tolerance to drought in rice. OsOTS1 depleted transgenic plants accumulate more ABA and exhibit more productive agronomic traits during drought while OsOTS1 overexpressing lines are drought sensitive but ABA insensitive. Drought and ABA treatment stimulates the degradation of OsOTS1 protein indicating that SUMO conjugation is an important response to drought stress in rice achieved through down-regulation of OTS1/2 activity. We reveal that OsOTS1 SUMO protease directly targets the ABA and drought responsive transcription factor OsbZIP23 for de-SUMOylation affecting its stability. OsOTS-RNAi lines show increased abundance of OsbZIP23 and increased drought responsive gene expression while OsOTS1 overexpressing lines show reduced levels of OsbZIP23 leading to suppressed drought responsive gene expression. Our data reveal a mechanism in which rice plants govern ABA-dependant drought responsive gene expression by controlling the stability of OsbZIP23 by SUMO conjugation through manipulating specific SUMO protease levels.

The ubiquitin conjugating enzyme, TaU4 regulates wheat defence against the phytopathogen Zymoseptoria tritici

Mycosphaerella graminicola (Zymoseptoria tritici commonly known as Septoria), the causal agent of Septoria Leaf Blotch (STB), is considered one of the major threats to European wheat production. Previous studies have shown the importance of ubiquitination in plant defence against a multitude of pathogens. However the ubiquitination machinery in wheat is under studied, particularly E2 enzymes that have the ability to control the ubiquitination and thereby the fate of many different target proteins. In this study we identify an E2 enzyme, Triticum aestivum Ubiquitin conjugating enzyme 4 (TaU4) that functions in wheat defence against Septoria. We demonstrate TaU4 to be a bona fide E2 enzyme through an E2 charging assay. TaU4 localises in both the cytoplasm and nucleus, therefore potentially interacting with E3 ligases and substrate proteins in multiple compartments. Virus Induced Gene Silencing of TaU4 in wheat leaves resulted in delayed development of disease symptoms, reduced Septoria growth and reproduction. We conclude that TaU4 is a novel negative regulator of defence against Septoria.

Rice OVERLY TOLERANT TO SALT 1 (OTS1) SUMO protease is a positive regulator of seed germination and root development

ABSTRACT Salinity is one of the major environmental stresses affecting rice production worldwide. Improving rice salt tolerance is a critical step for sustainable food production. Posttranslational modifications of proteins greatly expand proteome diversity, increase functionality and allow quick responses to environmental stresses, all at low cost to the cell. SUMO mediated modification of substrate proteins is a highly dynamic process governed by the balance of activities of SUMO E3 ligases and deconjugating SUMO proteases. In recent years, SUMO (Small Ubiquitin like Modifier) conjugation of proteins has emerged as an influential regulator of stress signaling in the model plant Arabidopsis. However SUMOylation remain largely under studied in crop plants. We recently identified the SUMO protease gene family in rice and demonstrated a role for OsOTS1 SUMO proteases in salt stress. Interestingly, rice plants silencing OsOTS1 also show significantly reduced germination rate. Knockdown of OsOTS1 gene expression affects root growth by primarily reducing cell size rather than cell division.

Erratum: SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana (Proceedings of the National Academy of Sciences of the United States of America (2015) 112 (11108-11113))

PLANT BIOLOGY Correction for “SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana,” by Ari Sadanandom, Éva Ádám, Beatriz Orosa, András Viczián, Cornelia Klose, Cunjin Zhang, Eve-Marie Josse, László Kozma-Bognár, and Ferenc Nagy, which appeared in issue 35, September 1, 2015, of Proc Natl Acad Sci USA (112:11108–11113; first published August 17, 2015; 10.1073/ pnas.1415260112). The authors note that Fig. S2 and Fig. S8 appeared incorrectly. The SI has been corrected online.

SUMO Suppresses the Activity of the Jasmonic Acid Receptor CORONATINE INSENSITIVE 1

Small Ubiquitin-like Modifier protein, SUMO, regulates jasmonic acid (JA) signaling by suppressing the JA receptor CORONATINE INSENSITIVE1 activity independently of JA levels. Plants respond rapidly to sudden environmental cues, often responding prior to changes in the hormone levels that coordinate these responses. How this is achieved is not fully understood. The integrative role of the phytohormone jasmonic acid (JA) relies upon the plant’s ability to control the levels of JASMONATE ZIM (JAZ) domain-containing repressor proteins. Here, we demonstrate that regardless of intrinsic JA levels, Small Ubiquitin-like Modifier (SUMO)-conjugated JAZ proteins inhibit the JA receptor CORONATINE INSENSITIVE1 (COI1) from mediating non-SUMOylated JAZ degradation. The SUMO-deconjugating proteases OVERLY TOLERANT TO SALT1 (OTS1) and OTS2 regulate JAZ protein SUMOylation and stability. The ots1 ots2 double mutants accumulate SUMOylated and non-SUMOylated JAZ repressor proteins but show no change in endogenous JA levels compared with wild-type plants. SUMO1-conjugated JAZ proteins bind to COI1 independently of the JA mimic coronatine. SUMO inhibits JAZ binding to COI1. We identify the SUMO interacting motif in COI1 and demonstrate that this is vital to SUMO-dependent inhibition of COI1. Necrotroph infection of Arabidopsis thaliana promotes SUMO protease degradation, and this increases JAZ SUMOylation and abundance, which in turn inhibits JA signaling. This study reveals a mechanism for rapidly regulating JA responses, allowing plants to adapt to environmental changes.