A Decoy Library Uncovers U-Box E3 Ubiquitin Ligases That Regulate Flowering Time

Genetics: Early Online, published on May 20, 2020 as 10.1534/genetics.120.303199 1 1 A Decoy Library Uncovers U-box E3 Ubiquitin Ligases that Regulate Flowering Time 2 in Arabidopsis 3 4 Authors: Ann M. Feke*, Jing Hong*†, Wei Liu*, Joshua M. Gendron* 5 6 Affiliations: *Department of Molecular, Cellular, and Developmental Biology, Yale 7 University, New Haven, CT 06511, USA. 8 †School of Food Science and Engineering, South China University of Technology, 9 Guangzhou, China Copyright 2020. 2 10 Running Title: E3 Ubiquitin Ligases in Flowering Time 11 Key Words: Protein degradation, flowering time, dominant negative strategies, plant 12 biology 13 Corresponding Author: Joshua Gendron; YSB 424, 260 Whitney Avenue, New Haven, CT 14 06511; 203-432-7317; [email protected] 3 15 ABSTRACT 16 Targeted degradation of proteins is mediated by E3 ubiquitin ligases and is 17 important for the execution of many biological processes. Redundancy has prevented the 18 genetic characterization of many E3 ubiquitin ligases in plants. Here, we performed a 19 reverse genetic screen in Arabidopsis using a library of dominant negative U-box type E3 20 ubiquitin ligases in order to identify their roles in flowering time and reproductive 21 development. We identified five U-box decoy transgenic populations that have defects in 22 flowering time or the floral development program. We used additional genetic and 23 biochemical studies to validate PLANT U-BOX 14 (PUB14), MOS4-ASSOCIATED COMPLEX 3A 24 (MAC3A), and MAC3B as bona fide regulators of flowering time. This work demonstrates the 25 widespread importance of E3 ubiquitin ligases in floral reproductive development. 26 Furthermore, it reinforces the necessity of dominant negative strategies for uncovering 27 previously unidentified regulators of developmental transitions in an organism with 28 widespread genetic redundancy, and provides a basis on which to model other similar 29 studies. 4 30 INTRODUCTION 31 Flowering is the first committed step in the plant reproductive process, leading to 32 the production of reproductive organs and eventually offspring. Plants use highly complex 33 gene networks that integrate a wide array of internal and external signals to regulate 34 flowering. In the model plant Arabidopsis thaliana, six pathways have been identified to 35 control flowering time. Four of these six pathways regulate the production of the florigen, 36 the protein FT, while the remaining two bypass FT to promote flowering in a more direct 37 manner (Srikanth and Schmid 2011). In addition, abiotic and biotic stress can modulate 38 flowering time by altering the function of one or multiple flowering pathways (Park et al. 39 2016; Takeno 2016). 40 In Arabidopsis, the transition to flowering is an irreversible decision the plant 41 makes in response to external and internal signals. In order for the response to occur at the 42 appropriate time, plants need to promote the activity of floral activators and repress the 43 activity of floral repressors. One way that plants accomplish this is by leveraging the 44 ubiquitin proteasome system to accurately degrade floral regulator proteins (Nelson et al. 45 2000; McGinnis et al. 2003; Imaizumi et al. 2005; Sawa et al. 2007; Hu et al. 2014). E3 46 ubiquitin ligases provide substrate specificity for the ubiquitin proteasome system and 47 mediate ubiquitylation of target proteins (Vierstra 2009). E3 ubiquitin ligases play central 48 roles in the regulation of the photoperiodic, vernalization, and GA flowering time pathways 49 (Nelson et al. 2000; McGinnis et al. 2003; Imaizumi et al. 2005; Sawa et al. 2007; Jang et al. 50 2008; Lazaro et al. 2012; Hu et al. 2014) demonstrating their important functions in this 51 critical developmental decision. 5 52 Despite these well-characterized roles, it is likely that E3 ubiquitin ligases that 53 regulate flowering have not been identified due to the numerous genome duplications that 54 have resulted in widespread gene redundancy (Risseeuw et al. 2003; Yee and Goring 2009; 55 Navarro-Quezada et al. 2013). To overcome these issues, we previously created and 56 validated a library of transgenic plants expressing E3 ubiquitin ligase decoys (Feke et al. 57 2019). E3 ubiquitin ligase decoys are forms of E3 ubiquitin ligases that lack the protein 58 domain that coordinates substrate ubiquitylation but retain other protein domains. Thus, 59 E3 ubiquitin ligase decoys can still interact with substrate proteins but can no longer 60 promote ubiquitylation and proteasomal degradation (Lee and Feke et al., 2018). In the 61 case of the U-box family of E3 ubiquitin ligases, the U-box domain coordinates substrate 62 ubiquitylation (Azevedo et al. 2001; Finn et al. 2016). A U-box decoy protein lacks the U- 63 box domain but retains the N-terminal and/or C-terminal protein regions that typically 64 contain protein-protein interaction domains (Feke et al. 2019). 65 Here, we employ the decoy library to identify U-box-type E3 ubiquitin ligases that 66 control flowering time and reproductive development. We focus on four metrics of 67 reproductive development: the number of rosette leaves, the age of the plant in days when 68 1 cm bolting occurs, the first occurrence of anthesis, and the rate of stem elongation. Using 69 these metrics, we uncover six U-box proteins which regulate 1 cm bolting, six U-box 70 proteins which regulate rosette leaf number, four U-box proteins that control stem 71 elongation, and one U-box protein that controls anthesis. 72 We perform focused genetic studies on three U-box genes, PLANT U-BOX 14 73 (PUB14), MAC3A, and MAC3B. We confirm their roles in flowering time regulation by 74 observing delayed flowering phenotypes in three T-DNA insertion mutants, pub14-1 6 75 (SALK_118095C), mac3a, and mac3b mutants (Monaghan et al. 2009). We also perform 76 immunoprecipitation mass spectrometry with the PUB14 decoy, similar to what we did 77 previously for MAC3B (Feke et al. 2019), and find a list of proteins involved in the 78 regulation of flowering time. These findings demonstrate the widespread importance of E3 79 ubiquitin ligases in the regulation of reproductive development and illustrate the strength 80 of the decoy technique to quickly identify novel E3 ubiquitin ligases in diverse biological 81 processes. 7 82 MATERIALS AND METHODS 83 Phenotypic Screening: The construction of the decoy library, the MAC3B-OX, and the 84 MAC3B-WD was described previously (Feke et al. 2019). Control pCCA1 Luciferase and 85 decoy seeds were surface sterilized in 70% ethanol and 0.01% Triton X∷-100 for 20 minutes 86 prior to being sown on ½ MS plates (2.15 g/L Murashige and Skoog medium, pH 5.7, 87 Cassion Laboratories, cat#MSP01 and 0.8% bacteriological agar, AmericanBio cat# 88 89 (SantaAB01185) Cruz with Biotechnology, or without appropriatecat# 77182- antibiotics82-2) for vectors (15 μg/mL pB7- ammoniumHFN and pB7 glufosinate-HFC, or 50 90 -HFN). Seeds were stratified for two days 91 atμg/mL 4°, transferred kanamycin to sulfate 12 hours (AmericanBio) light/12 hours for darkpK7 conditions for seven days, then to 92 constant light conditions for 7 days in order to do screening for circadian clock studies 93 shown in Feke et al. 2019. Seedlings were then transferred to soil (Fafard II) and grown at 94 22° in inductive 16 hours light/8 hours dark conditions with a light fluence rate of 135 95 mol m-2 s-1. Plants were monitored daily for flowering status, recording the dates upon 96 whichμ each individual reached 1 cm inflorescence height, 10 cm inflorescence height, and 97 the first occurrence of anthesis. Additionally, leaf number at 1 cm inflorescence height was 98 recorded. 99 Homozygous pub14-1, mac3a, mac3b, and mac3a/mac3b mutant seeds were surface 100 sterilized, sown on ½ MS plates without antibiotics as described above. Seeds were 101 stratified for 3 days at 4°, then transferred to 12 hours light/ 12 hours dark conditions for 102 two weeks prior to transfer to soil and growth under inductive conditions as described 103 above. Plants were monitored daily for flowering status as described above. 8 104 Data Normalization and Statistical Analysis: As the age at anthesis depends on the 105 initiation of flowering, we used anthesis delay as a measurement of anthesis. Anthesis 106 delay was calculated by taking the age at anthesis and subtracting the age at 1 cm 107 inflorescence height. Similarly, the age at 10 cm inflorescence height depends on the 108 initiation of flowering. Thus we calculated the stem elongation period by subtracting the 109 age at 1 cm inflorescence height from the age at 10 cm inflorescence height. These modified 110 metrics were used for all analyses. 111 To allow for comparison across independent experiments, data was normalized to 112 the individual wildtype control performed concurrently. The average value of the wildtype 113 control plants was calculated for every experiment, then this average was subtracted from 114 the value of each individual T1 insertion or control wildtype plant done concurrently. This 115 normalized value was used for statistical analyses, and are presented in Table S1. 116 Welch’s t-test was used to compare each normalized T1 insertion plant population 117 or subpopulation to the population of all normalized control plants. In order to decrease 118 the number of false positives caused by multiple testing, we utilized a Bonferroni corrected 119 - 120 throughout.α as the p value threshold. The α applied differs between experiments, and is noted 121 Measurement of Gene Expression in U-box mutants: Homozygous mac3a/mac3b 122 mutant plants in the Col-0 background were generated previously (Monaghan et al. 2009). 123 Col-0, pub14-1, mac3a, mac3b, and mac3a/mac3b seeds were stratified on ½ MS plates at 124 4° for two days prior to growth in 16 hour light/8 hour dark conditions at a fluence rate of 125 2 s 1 at 22°.

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