The ability to detect and respond to stress is central to a plant's survival in a host of unfavorable environments and a key determinant of agricultural productivity under sub-optimal field conditions. Although a number of pathways have been described that confer specific protection to various abiotic and biotic challenges, a recent discovery of a potentially universal protective mechanism involving the Small Ubiquitin-related MOdifier (SUMO) may transform the current appreciation of stress biology. Specifically, it has been shown that the Arabidopsis SUMO polypeptide becomes covalently attached to numerous nuclear proteins and that the levels of these conjugates rise rapidly and reversibly after exposing plants to various abiotic stresses. Using novel quantitative proteomic approaches, it has been discovered that many of the SUMOylation targets are known critical regulators with their collective functions implying that SUMO addition engages a protective response that broadly alters chromatin accessibility, transcription, and mRNA processing/export. Taken together, these results suggest that SUMO might offer unique opportunities to globally manipulate the stress response for agricultural benefit. Unfortunately, the organization and functions of the SUMO system are largely unknown in other plant species, including all important agricultural crops, thus precluding rational redesign to improve crop plant productivity. Moreover, preliminary genome analyses of maize and rice revealed that the organization of the SUMO system in cereals might differ significantly from that in observed in Arabidopsis. This EAGER project proposes to define how SUMOylation works during stress in crops using maize (Zea mays) as the model. The specific aims are to: (i) delineate the SUMOylation system in maize using bioinformatic and biochemical methods and define kinetically how the system responds to stress; (ii) generate a library of maize mutants and transgenic lines affecting key components required for SUMO addition and release; (iii) define the "SUMOylome" of maize, quantify how the SUMOylation status of individual targets changes during stress and after recovery; and (iv) analyze SUMO pathway mutants phenotypically to determine how stress-induced SUMOylation may help maize survive adverse environments. Collectively, this project will generate much-needed tools and germplasm that can be exploited to understand how SUMO might reorganize maize chromatin and its transcriptome during stress, and identify key points in plant stress responses involving SUMOylation that can be manipulated for improved yield.
The current understanding of SUMOylation in plants is still rudimentary and almost nonexistent in crop species where its manipulation may have substantial agricultural impact. This project will provide interdisciplinary training of the next generation of plant scientists working on crops. This research will collectively incorporate postdocs, graduate students, and undergraduates as well as high school students sponsored by the Wisconsin Youth Apprenticeship Program (YAP) in Biotechnology. During the course of this project, reagents, techniques, mutants, and transgenic lines will be generated that will provide a much needed foundation to investigate SUMOylation in maize, and hopefully offer new strategies to rationally alter the SUMO system for agricultural and medicinal benefit. Plant resources will be available through the Maize Genetics Cooperative Stock Center (http://maizecoop.cropsci.uiuc.edu). Raw and processed experimental data will be deposited into NCBI's Gene Expression Omnibus (GEO) and at Maize GDB (www.maizegdb.org/).
The ability of plants to cope with environmental challenges involves the rapid modification of intracellular proteins with SUMO. Our NSF EAGER project aims to define of the SUMO pathway in maize by: (i) characterizing the pathway genetically, (ii) establishing the kinetics of SUMOylation, and (iii) developing germplasm to dissect the function of SUMO and identify the suite of SUMOylated maize proteins. Defining the Maize SUMO System by Bioinformatics. Using Arabidopsis SUMO pathway as a model, we identified the corresponding maize components. While our preliminary analysis accurately predicted the number of SUMO and E1 genes, we refined the maize pathway to include an additional SCE1 E2 isoform (ZmSce1g), two additional relatives of the SIZ1 E3 (ZmSiz1b and ZmSiz1c), a novel SUMO E3 ligase (ZmSms1), and 3 more deSUMOylating proteases (DSPs) related to ESD4/ELS1 (Figure 1). Two SUMO proteins are identical at the amino acid level (Sumo1a/b) while a third, more divergent, SUMO isoform (Sumo2) is also present that appears common among land plants (Figure 2-1). Maize encodes seven SCE1 isoforms that subdivide into two classes: Class I SCE1 common among land plants and a cereal-specific Class II – invoking the possibility of novel enzymatic function(s) (Figure 2-2). Previously, we identified a single maize Siz1 protein bearing the signature SAP, PHD, and MIZ/SP-RING domains. We identified two additional SAP domain-containing proteins with downstream PHD domains. Manually scanning 3’ to the predicted stop codon revealed additional ORFs for ZmSIZ1b and ZmSIZ1c ~3- and ~20-kbp downstream, respectively, that were confirmed to be transcriptionally connected to the SAP/PHD domains. We also discovered a potentially novel ligase designated SUMO MIZ/SP-RING ligase-1 (Sms1) (Figure 1). ZmSms1 shares homology and intron/exon structures with genes present in many other land plants, suggestive of functional conservation. We also expanded the ESD4/ELS1-type SUMO protease family to a total of 4 genes in maize (Figure 1). We validated the SUMO pathway gene models shown in Figure 1 by analysis of the full-length coding sequences PCR amplified from B73 cDNA. For the ZmSae2 gene, we detected a splice variant that introduces a premature stop codon (Figure 3B). This splicing variant is also in rice, suggesting that it has physiological significance. We are now attempt to express these clones into Escherichia coli for eventual SUMOylation assays in vitro. Gene Expression Analysis A comprehensive RNA-Seq analysis of 60 maize tissues over a developmental time series revealed nearly constitutive expression for most SUMO pathway genes with highest expression generally prevalent in whole seed and endosperm tissues (Figure 4) . ZmSumo2, ZmDSUL, Class II ZmSce1 transcripts (ZmSce1e-g), and ZmEsd4d were either not detectable or had tissue-specific expression patterns. The Class II Sce1 genes have a restricted expression profile implying that they have subfunctionalized from the more constitutive Class I genes. Stress-Induced SUMOylation Response By testing the SUMOylation levels at varying temperatures we showed that the rapid and robust conjugation of SUMO to other proteins requires temperatures above 37ºC (Figure 5-1A). This SUMOylation occurs within minutes of stress induction, and is reversible upon return to the normal growth temperature (Figure 5-1B). After initial heat stress-induced SUMOylation, maize requires a refractory period of ~6-8 hours to again fully recover its ability to undergo robust heat stress-induced SUMOylation (Figure 5-1C). Development of Germplasm Affecting SUMOylation. We have collected a suite of Mu transposon insertion lines predicted to impact the SUMO pathway (diagram see Figure 1). We are backcrossing many of these to the W22 parent and are actively screening for homozygous lines following a self cross. One particularly exciting mutant sae1-3 disrupts correct splicing of the ZmSae1 transcript (Figure 6-1). Protein gel blot analysis of wild-type and homozygous mutant sae1-3 segregants challenged with heat stress reveals that sae1-3 plants have a severely dampened stress-induced SUMOylation response (Figure 6-1C) despite the increased steady-state of free SUMO protein levels. Interestingly, sce1-3 plants show constitutive up-regulation of the free SUMO, suggesting that they can sense and adjust to reduced SUMOylation capacity. We also found Mu lines bearing insertions within the first intron of ZmSumo-v (sumo-v-1) or within the coding region of ZmSms1 (sms1-1) (Figures 1 and 6-1A). Both Mu insertions significantly reduce the steady-state transcript levels as compared with wild-type progenitor or segregant controls (Figure 6-2B). The identification of SUMOylated targets is key to understanding the role of stress-induced SUMOylation in maize. To accomplish this goal, we created transgenic maize lines expressing 6His-Sumo1a-H89R and 6His-Sumo1a-(K0)-H89R transgenes designed for affinity-purification and proteomic footprinting of the SUMO moiety using mass spectrometric methods. In addition, we synthesized a concatenated ubiquitin (Ub) transgene that will express high levels of a Ub variant tagged with 6His-Ub [hexa(6His-Ub)] to aid in the purification and identification of ubiquitylated maize targets. Broader Impacts During the past year a high school student Noah Cotter and an UW undergraduate Cole McBee assisted with this project under the direction of Dr. Robert Augustine.
