1 An atypical aspartic protease modulates lateral root development in Arabidopsis 2 thaliana 3 4 André Soares1,2,3,4#, Stefan Niedermaier5, Rosário Faro3, Andreas Loos6§, Bruno 5 Manadas3, Carlos Faro3, Pitter F. Huesgen5*, Alice Y. Cheung4*, Isaura Simões2,3*, 6 1 7 PhD Programme in Experimental Biology and Biomedicine, Center for Neuroscience 8 and Cell Biology, University of Coimbra, Portugal; 2 9 Institute for Interdisciplinary Research, University of Coimbra, Portugal; 3 10 CNC-Center for Neuroscience and Cell Biology, University of Coimbra; 4 11 Department of Biochemistry and Molecular Biology, University of Massachusetts, 12 Amherst, MA 01003, USA; 5 13 Central Institute for Engineering, Electronics and Analytics, ZEA-3, 14 Forschungszentrum Jülich, Jülich, Germany; 15 6Department of Applied Genetics and Cell Biology, University of Natural Resources 16 and Life Sciences, Vienna, Austria; 17 18 #Current address: FairJourney Biologics, Porto, Portugal 19 §Current address: Aridis Pharmaceuticals, 5941 Optical Court, CA 95138, US. 20 *Corresponding authors 21 22 E-mail addresses: AS ([email protected]); SN ([email protected]); 23 RF ([email protected]); AL ([email protected]); BM 24 ([email protected]); CF ([email protected]); PFH ([email protected]); 25 AYC ([email protected]); IS ([email protected]) Phone: +351 231 26 249196 27 28 This is a pre-copyedited, author-produced version of the article accepted for publication 29 in the Journal of Experimental Botany following peer review. The version of record is 30 available online at: https://doi.org/10.1093/jxb/erz059 31 32 1 33 Running title: 34 An atypical AP in lateral root development 35 36 Highlight 37 Aspartic protease (AP) ASPR1 displays atypical enzymatic properties, including fungal- 38 like specificity preferences. A role in lateral root development is anticipated. This work 39 further strengthens a sophisticated specialization among atypical APs. 40 41 Abstract 42 Few atypical aspartic proteases (APs) present in plants have been functionally studied to 43 date despite having been implicated in developmental processes and stress responses, 44 and diverse biochemical properties were reported for these enzymes. Here we 45 characterize a novel atypical AP that we name ATYPICAL ASPARTIC PROTEASE IN 46 ROOTS 1 (ASPR1), denoting its expression in Arabidopsis roots. Recombinant ASPR1 47 produced by transient expression in N. benthamiana was active and displayed atypical 48 properties, combining optimum acidic pH, partial sensitivity to pepstatin and 49 pronounced sensitivity to redox agents, and unique specificity preferences resembling 50 those of fungal APs. ASPR1 overexpression suppressed primary root growth and lateral 51 root development, implicating a previously unknown biological role for an AP. 52 Quantitative comparison of WT and aspr1 root proteomes revealed deregulation of 53 proteins associated with both ROS and auxin homeostasis in the mutant. Together, our 54 findings on ASPR1 reinforce the varied pattern of enzymatic properties and biological 55 roles of atypical APs and raise exciting questions on how these distinctive features 56 impact functional specialization among these proteases. 57 58 Keywords 59 Arabidopsis thaliana; ASPR1; Atypical aspartic protease; auxin; lateral root; 60 magnICON expression system; root development; ROS 61 62 Abbreviations: AP, aspartic protease; LR, lateral root 2 63 Introduction 64 Aspartic proteases (APs) represent the second-largest class of plant proteases 65 (van der Hoorn, 2008). Members of the pepsin-like family (A1) are widely distributed 66 in a variety of plants (Simões and Faro, 2004), with a large representation of genes 67 encoding putative A1 members found in the genomes of Arabidopsis thaliana, Oryza 68 sativa and Vitis vinifera L. (Chen et al., 2009; Faro and Gal, 2005; Guo et al., 2013). 69 Depending on their domain organizations and active site sequence motifs, plant A1 70 family members are generally grouped into three classes – typical, atypical and 71 nucellin-like (Faro and Gal, 2005). Typical APs have been extensively studied and are 72 characterized by the presence of a signal peptide, a prosegment and an extra protein 73 domain known as the plant-specific insert, which is absent in the mature form of the 74 enzymes. These proteases are active at acidic pH, are specifically inhibited by pepstatin 75 A and are mostly localized to the vacuole (Simões and Faro, 2004). Strikingly, the vast 76 majority of putative plant APs are grouped as atypical and nucellin-like, exhibiting 77 distinct features when compared with typical APs (Faro and Gal, 2005). These features 78 include the absence of the plant-specific insert; an unusually high number of cysteine 79 residues; the nature of the amino acids preceding the first catalytic aspartate; and 80 unexpected localizations such as the chloroplast, mitochondria or endoplasmic 81 reticulum/extracellular (Gao et al., 2017; Paparelli et al., 2012; Phan et al., 2011). 82 Although studies on atypical APs are still nascent, unusual enzymatic properties have 83 already been documented for several of these enzymes, including more diverse optimal 84 pHs (less-acidic), very broad sensitivity to pepstatin A, proteolytic activity without 85 prosegment removal, as well as distinct specificities (Olivares et al., 2011; Paparelli et 86 al., 2012; Prasad et al., 2010; Simões et al., 2007; Yao et al., 2012). Therefore, the large 87 number of atypical and nucellin-like APs in plants is suggestive of greater 88 diversification of protein functions and a more regulatory role for these APs, as 89 compared to the housekeeping function generally attributed to typical plant APs 90 (Simões and Faro, 2004). Indeed, some functions are starting to be uncovered for these 91 atypical APs, with proposed roles in important and highly regulated processes. For 92 instance, several APs have been demonstrated to be important for responses to biotic 93 (Alam et al., 2014; Breitenbach et al., 2014; Li et al., 2016; Prasad et al., 2009; Xia et 94 al., 2004) and abiotic stresses (Yao et al., 2012). APs are also involved in a range of 95 other functions, including chloroplast metabolism (Nakano et al., 1997; Nakano et al., 96 2003; Paparelli et al., 2012), hybrid sterility (Chen et al., 2008; Ji et al., 2012), and 3 97 reproductive development (Chen and Foolad, 1997; Gao et al., 2017; Ge et al., 2005; 98 Huang et al., 2013; Niu et al., 2013; Phan et al., 2011). These findings implicate 99 functional specialization of plant APs and suggest tight activity regulation. 100 Root developmental processes depend on the complex interplay between various 101 transcriptional regulators, multiple hormones as well as other signaling molecules such 102 as reactive oxygen species (ROS). Auxin plays a central role in almost every aspect, 103 including the integration of developmental cues with changes in environmental 104 conditions (Du and Scheres, 2018; Kazan, 2013; Lavenus et al., 2013; Malekpoor 105 Mansoorkhani et al., 2014; Manzano et al., 2014; Peret et al., 2009; Petricka et al., 106 2012; Tognetti et al., 2012; Tsukagoshi, 2016). Proteolysis is an important post- 107 translational modification process known to modulate different plant developmental and 108 environmental cues (Schwechheimer and Schwager, 2004; van der Hoorn, 2008). For 109 example, during root development, the ubiquitin-26S proteasome system (UPS) 110 regulates auxin signaling through the degradation of Aux/IAA transcriptional repressors 111 that inhibit the activity of the transcription factors AUXIN RESPONSIVE FACTORS 112 (ARFs) (Rogg and Bartel, 2001; Schwechheimer and Schwager, 2004; Wang and 113 Estelle, 2014). Plant proteases are also critical regulators of different biological 114 processes. Although their role in root development remains largely unclear (van der 115 Hoorn, 2008), there are several examples of proteases that have been implicated in this 116 process, such as the putative zinc carboxypeptidase SOL1 (Casamitjana-Martinez et al., 117 2003), subtilase SBT6.1 (Ghorbani et al., 2016), the caspase family member separase 118 (Wu et al., 2010), and the membrane-associated aminopeptidase M1 (APM1) (Peer et 119 al., 2009) from Arabidopsis, and the rice sumo protease OTS1 (Srivastava et al., 2016). 120 Other proteases described in roots are linked to nutrient acquisition in response to 121 abiotic stress conditions or symbiotic or pathogenic relationships (Kohli et al., 2012). 122 From the atypical APs implicated in reproduction, the Arabidopsis PCS1 (Ge et 123 al., 2005), UNDEAD (Phan et al., 2011), A36 and A39 (Gao et al., 2017), and rice 124 OsAP25 and OsAP37 (Niu et al., 2013), and OsAP65 (Huang et al., 2013) have been 125 shown to modulate programmed cell death (PCD) events that determine the 126 development of male gametophyte. When evaluating the function of the transcription 127 factor MALE STERILITY 1, whose absence resulted in a severe male sterility phenotype, 128 Ito and co-workers identified a gene encoding another atypical AP significantly 129 repressed in ms1 plants (At2g03200), suggesting its possible function during male 130 reproductive development (Ito et al., 2007). 4 131 In this work, we explore the biochemical properties and functional role of this 132 novel putative atypical AP (herein named ASPR1, for Atypical Aspartic Protease in 133 Roots 1).We demonstrate that ASPR1 is indeed an active AP displaying a set of atypical 134 enzymatic properties. Moreover, overexpression