Evolutionary Variation in MADS-box Dimerization Affects Floral Development and Protein Abundance in Maize

25 Interactions between MADS-box transcription factors are critical in the regulation of floral development, 26 and shifting MADS-box protein-protein interactions are predicted to have influenced floral evolution. 27 However, precisely how evolutionary variation in protein-protein interactions affects MADS-box protein 28 function remains unknown. To assess the impact of changing MADS-box protein-protein interactions on 29 transcription factor function, we turned to the grasses, where interactions between B-class MADS-box 30 proteins vary. We tested the functional consequences of this evolutionary variability using maize (Zea 31 mays) as an experimental system. We found that differential B-class dimerization was associated with 32 subtle, quantitative differences in stamen shape. In contrast, differential dimerization resulted in large33 scale changes to downstream gene expression. Differential dimerization also affected B-class complex 34 composition and abundance, independent of transcript levels. This indicates that differential B-class 35 dimerization affects protein degradation, revealing an important consequence for evolutionary variability 36 in MADS-box interactions. Our results highlight complexity in the evolution of developmental gene 37 networks changing protein-protein interactions could affect not only the composition of transcription 38 factor complexes, but also their degradation and persistence in developing flowers. Our results also show 39 how coding change in a pleiotropic master regulator could have small, quantitative effects on 40 development. 41 42 INTRODUCTION 43 Floral organ morphology is diverse, but the master regulators controlling floral organ development are 44 conserved. Many of the master regulators controlling floral organ development are transcription factors 45 encoded by the ABC(DE) genes. The ABC(DE) model proposes how these transcription factors act 46 together to regulate the development of floral organs. All but one of the original ABC(DE) genes encode 47


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Floral organ morphology is diverse, but the master regulators controlling floral organ development are  Table 1).

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In contrast, adult flowers in si1 mutants carrying STS1-HOM were indistinguishable from non-transgenic 117 si1 mutants (Fig. 1C, 1F). This indicates that STS1-HOM can rescue sts1 mutants and is functional, but 118 cannot compensate for the loss of SI1 function.

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To explore differences between STS1-HET and STS1-HOM in more detail, we analyzed the development 121 of complemented sts1 mutants. We examined protein localization of both STS1-HET and STS1-HOM 122 over the course of development using confocal microscopy. We found that STS1-HET was restricted to 123 lodicule and stamen primordia, as expected ( Fig. 1G-H) (Bartlett et al., 2015). In contrast, protein 124 localization was relaxed in STS1-HOM lines, appearing in gynoecia in addition to lodicule and stamen 125 primordia (Fig. 1J). However, this gynoecial localization was not evident in our immunolocalizations 126 using an anti-STS1 antibody (Supplemental Fig. 1). In contrast to the proteins, STS1-HET and STS1-HOM 127 RNAs showed similar localization patterns (Supplemental Fig. 1). This suggests that the subtle 128 localization differences we detected were regulated at the protein level.

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In addition to protein localization differences, morphology differed quantitatively between STS1-HOM 131 and STS1-HET flowers. This included differences in anther aspect ratio, such that anthers of STS1-HOM 132 flowers were wider and shorter than those of STS1-HET flowers while they were still developing (t-test p-133 value = 6.8e-10, Fig. 1K-L, Supplemental Dataset 1). We assayed anther shape at anthesis using eFourier 134 analysis, as implemented in the R package MOMOCS (Bonhomme et al., 2014). This analysis identified a 135 small, but significant difference in shape between anthers from sts1 mutants complemented with either 136 STS1-HET or STS1-HOM (MANOVA, p-value = 0.006). Anthers from STS1-HET flowers occupied a 137 larger morphospace, and tended to be wider than STS1-HOM anthers (Supplemental Fig. 2). Indeed, 138 aspect ratio measurements of mature anthers showed that anthers from STS1-HOM flowers were narrower 139 than those from STS1-HET flowers (t-test, p-value = 0.003, Fig. 1M-N, Supplemental Dataset 2).

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Differences in anther aspect ratio, both during development and at anthesis, were despite similarities in 141 anther surface area, which we used as a proxy for size (developing anthers: t-test p-value = 0.259, Fig 1K; 142 anthesis: t-test p-value = 0.367, Fig. 1M). Taken together, our results suggest that B-class dimerization 143 may affect anther shape, potentially by affecting anther growth dynamics over the course of development.

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Differential dimerization of maize B-class proteins affects downstream gene regulation

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The morphological differences between STS1-HET and STS1-HOM flowers were subtle, indicating small 147 phenotypic consequences of differential B-class dimerization. We were curious if downstream 148 transcription was similarly conserved between STS1-HET and STS1-HOM. To understand the effect of 149 STS1 dimerization on gene expression, we performed RNA-seq in sts1 or si1 mutants complemented with 150 either STS1-HET or STS1-HOM. We harvested inflorescence tissue shortly after stamen primordium 151 emergence, to capture gene expression just after initiation of STS1 expression (Bartlett et al., 2015).

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Because of the high genetic diversity in maize, we compared expression profiles within genetic 153 backgrounds to control for potential differences due to incomplete introgression of the STS1 transgenes 154 (Buckler et al., 2006). To this end, we measured differential expression by comparing expression in each 155 line (STS1-HOM or STS1-HET) against expression in their mutant siblings.

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These analyses revealed more differentially expressed genes in STS1-HOM than in STS1-HET, as 158 compared to mutant siblings. At a 5% false discovery rate (FDR), there were 501 differentially expressed 159 genes in inflorescences expressing STS1-HET, as compared to sts1 mutant siblings ( Fig

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To determine whether the general functions of genes regulated by STS1-HET and STS1-HOM were 174 qualitatively similar, we performed gene ontology (GO)-enrichment analyses with our differentially

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HET and STS1-HOM constructs were so similar, and since our morphological data suggested subtle 178 functional differences between STS1-HET and STS1-HOM (Fig. 1), we reasoned that STS1-HET and 179 STS1-HOM were regulating similar processes. Therefore, we made the threshold for calling a GO-term 180 unique to either dataset very stringent; only GO-terms with an enrichment p-value of less than 0.01 in one 181 dataset, and an enrichment p-value of more than 0.25 in the other dataset were called 'unique' (Fig. 2E, 182 sectors i and v). Using these comparisons, we found many GO-terms related to development shared 183 between STS1-HET and STS1-HOM (Fig. 2E). Indeed, 29 of the 65 enriched GO-terms shared between 184 STS1-HET and STS1-HOM were related to development (p-value in both datasets <0.01, Fig. 1E, sector 185 iii). GO-terms related to signaling and metabolism were also enriched in both datasets, although more of 186 these terms were specifically enriched in STS1-HOM. However, the most highly enriched GO-terms in 187 STS1-HOM were not significantly enriched in STS1-HET (p-value > 0.25). These GO-terms, specific to 188 STS1-HOM, were almost all related to chromatin assembly and protein modification (Fig. 2E, sector i).

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Thus, core floral developmental programs were activated in inflorescences expressing STS1-HOM, but B-190 class dimerization also affected the expression of unique sets of genes, particularly genes involved in 191 chromatin assembly and remodeling.

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Differential dimerization may affect the composition of protein complexes, which is crucial for MADS-194 box function (Theissen and Saedler, 2001;Theissen et al., 2016). To determine how differential B-class 195 dimerization might impact the composition of MADS-box complexes, we performed 196 immunoprecipitations (IPs) of STS1-HET and STS1-HOM in an sts1 mutant background, using a specific 197 antibody against GFP (ChromoTek). We analyzed precipitated complexes using quantitative mass 198 spectrometry (MS). After confirming the presence of STS1 in the IP complex through immunoblotting, 199 we performed trypsin digestion and LC-MS/MS followed by label-free protein quantification (Sinitcyn et 200 al., 2018). We used protein from sts1 mutant siblings as a negative control, to detect non-specific 201 proteins. We compared abundances of identified proteins using the iBAQ (intensity Based Absolute 202 Quantification) method (He et al., 2019;Krey et al., 2014). Protein identification was based on at least 5 203 exclusive peptides and two replicates were performed for each sample; protein abundances were similar 204 between replicates (Supplemental Datasets 9 and 10). We found 1,278 total proteins in complex with 205 STS1-HET; 453 of these were either specific to STS1-HET, or were at least 2 fold higher than in mutant 206 siblings (Supplemental Dataset 9). In contrast, we found 1,597 proteins in complex with STS1-HOM; 486 207 of these proteins were either specific to STS1-HOM, or were at least 2 fold higher than in mutant siblings 208 (Supplemental Dataset 10). We found 125 proteins in common to both the STS1-HET and STS1-HOM 209 immunoprecipitations (either absent in sts1 siblings, or ≥ 2 fold change). This result indicates that, while 210 many proteins are common to STS1-HET and STS1-HOM complexes, each STS1 variant is associated 211 with a distinct set of proteins.

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The first set of proteins in our immunoprecipitations that we explored further were the MADS-box 213 proteins. Ancestral protein resurrection and in vitro surveys of protein-protein interactions predict that 214 complexes of B-, C-, and E-class MADS-box proteins are conserved across flowering plants (Zhang et al., 215 2018;Theissen et al., 2016;Veron et al., 2007). However, this prediction remains largely untested in 216 planta, particularly in monocots. Therefore, we specifically searched for other MADS-box proteins in 217 our IP-MS data. In the STS1-HET IP-MS results, we found both STS1 and SI1 peptides, as well as 218 peptides for three E-class MADS-box proteins (ZMM27, ZMM7, ZMM6), and two C-class proteins 219 (AGAMOUS (AG) co-orthologs, ZAG1 and ZMM23 (Münster et al., 2002;Zahn et al., 2005) 220 (Supplemental Table 2). ZMM6, ZMM7, and ZMM27 are all in a single clade of E-class proteins, co-221 orthologous to the A. thaliana protein SEPALLATA3 (Zahn et al., 2005). In STS1-HOM, we found the 222 same three E-class proteins and one C-class protein, ZAG1, but did not find ZMM23. ZMM23 was  The MADS-box proteins we identified in both IP datasets were at much higher levels in STS1-HOM than 231 in STS1-HET (SI1 was the only exception). STS1-HOM was far more abundant than STS1-HET:

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absolute iBAQ values for STS1-HOM were 4.0 times higher than for STS1-HET, 3.58 times higher 233 normalized to SI1 (Fig. 3). The higher abundance of STS1-HOM in our immunoprecipitations could have 234 been due to complex stoichiometry, where STS1-HOM homodimerization caused double the number of 235 STS1-HOM proteins in MADS-box complexes. Alternatively, STS1-HOM protein levels could also have 236 been generally higher in developing flowers. We suspected that STS1-HOM levels were generally higher; 237 our immunolocalizations suggested a higher abundance of STS1-HOM vs. STS1-HET, despite the same 238 experimental conditions (Supplemental Fig. 1). However, these immunolocalizations were not 239 quantitative. To directly measure protein levels, we carried out immunoblots using a polyclonal antibody 240 against STS1. The same amount of protein for each sample was loaded. After immunoblotting (described 241 in methods), we carried out densitometry analysis using Image J (1.4 NIH software, Schneider et al.,  Table 3). Together with our IP-MS results, these data indicate 245 that STS1-HOM accumulated to a higher abundance than STS1-HET in inflorescence tissue.

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STS1-HOM protein could have been more abundant than STS1-HET protein because the STS1-HOM 247 transgene was transcribed to higher levels than the STS1-HET transgene. To test for this, we carried out 248 RT-qPCR using specific primers for STS1 and SI1 in sts1 mutants complemented with either STS1-HET 249 or STS1-HOM. We found that SI1 was expressed to the same level in both lines; however, the expression 250 of STS1 in STS1-HET was 3.7 fold higher than in STS1-HOM (relative to Actin, Fig. 3C). Similarly, in our 251 RNA-Seq results, normalized STS1-HET expression was consistently double normalized STS1-HOM 252 expression; approximately 15,000 counts vs. 8,600 counts respectively. Thus, despite relatively low STS1-253 HOM expression, STS1-HOM protein accumulated to higher levels than STS1-HET in floral tissue. This

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indicates that STS1 homodimerization led to increased protein accumulation independent of RNA levels.

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Therefore the higher abundance of STS1-HOM is likely regulated post-transcriptionally.

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The other MADS-box proteins we detected in our immunoprecipitations were also more abundant in 257 STS1-HOM than in STS1-HET. ZAG1, ZMM6 and ZMM7 increased in abundance 2-to 9-fold in STS1-

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HOM, as compared to STS1-HET (Fig. 3A, Supplemental Table 2). This higher abundance of ZAG1, 259 ZMM6, ZMM7, and ZMM27 could also have been because of differences in transcription, or because of 260 differences post-transcriptionally. To distinguish between these possibilities, we looked for these genes in 261 our RNA-Seq data, and determined that they were not differentially expressed between STS1-HOM and 262 their sts1 mutant siblings (Supplemental Dataset 4). Therefore, the higher abundance of these C-and E-

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We also found a class of proteins related to signaling in our immunoprecipitation datasets, specifically 292 kinases. Ten kinases were immunoprecipitated with STS1-HET, and eleven with STS1-HOM; only two 293 of these were in both samples (Supplemental Datasets 9 and 10, Supplemental Table 4). The kinases 294 found in both STS1-HET and STS1-HOM were a non-specific serine/threonine protein kinase 295 (Zm00001d028733) and Calcium dependent protein kinase 11 (Zm00001d004812). In A. thaliana, 296 calcium dependent protein kinases are involved in signal transduction pathways that involve calcium as a 297 second messenger and regulate the calcium-mediated abscisic acid signaling pathway (Zhu et al., 2007).

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In general, the kinases found only with STS1-HET were related to basal metabolism, for example a 299 phosphoglycerate kinase and a pyruvate kinase, likely involved in the synthesis and degradation of 300 carbohydrates (Rosa-Téllez et al., 2018;Lu and Hunter, 2018). In contrast, the kinases that 301 immunoprecipitated with STS1-HOM were related to signaling and membrane receptors; for example 302 brassinosteroid (BR)-signaling kinase 2 and BR-LRR receptor kinase (Tang et al., 2008;Xu et al., 2014).

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analyses are necessary to confirm interactions between STS1 and the kinases we found.

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One new set of GO-terms that emerged in our analysis was related to protein modification and 307 ubiquitination. These GO-terms were more enriched in STS1-HOM, but were still present in STS1-HET 308 ( Fig. 3D). When we explored which proteins might be represented by these enriched GO-terms 309 (Supplemental Table 5

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We found that differential B-class dimerization did not result in qualitatively different organ identities in 343 maize. Instead, the impacts of differential B-class dimerization on maize floral development were subtle 344 and quantitative; anthers from STS1-HOM flowers were narrower than those from STS1-HET flowers at 345 anthesis ( Fig. 1). At a molecular level, we found STS1 in complex with C-class and E-class proteins,

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We found that STS1-HOM protein was more abundant than STS1-HET, despite lower levels of RNA 360 (Fig. 3). This means that STS1-HOM abundance was likely regulated post-transcriptionally; leaving 361 either (1) impaired degradation of the STS1-HOM protein, or (2)   376 data lead us to favor differential protein degradation as an explanation for higher STS1-HOM abundance.

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It seems likely that STS1-HOM was more abundant than STS1-HET because of differential degradation.

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However, we are left wondering what mechanism may underlie this difference. Both differential

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Similarly, strong DNA-binding of an Epstein-Barr virus protein inhibits its degradation in the proteasome 392 (Coppotelli et al., 2011). STS1 homodimers and STS1/SI1 heterodimers may differ in their DNA-binding 393 profiles or affinity, which could, in turn, affect protein degradation dynamics.

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We discovered that STS1 likely interacts with kinases, and is phosphorylated (Fig. 3)

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We found that variation in B-class MADS-box dimerization affected one aspect of anther shape in maize:

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The small phenotypic change that we found indicates that floral development is largely robust to

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In all of our experiments, we focused on a single maize PI-like protein, STS1. However, there are three PI

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The transcriptional tuning mediated by differential B-class dimerization that we discovered has important

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RNAseq tissue collection and sequencing. Plants were grown in the greenhouse as described above.

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Shortly after stamen primordium emergence (4-5 weeks after planting), plants were harvested and 507 inflorescence meristems were flash frozen in liquid nitrogen. Samples were harvested at the same time of 508 day, beginning at 3pm. Three plants per genotype were pooled to generate one biological replicate, with 509 three biological replicates per genotype (genotyping primers in Supplemental Table 6

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Antibody production and immunolocalization. Anti-STS1 antibody was developed from the full-length 592 coding sequence of STS1 cloned into pDEST17 at the Bartlett Lab, using the protocol described in (

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RT-qPCR. Total RNA was isolated from 1.8 cm tassels using Trizol reagent according to the 642 manufacturer's instructions. cDNA was synthesized using 1 μg of RNA, Oligo dT (20)                        aspect ratio (AR) (anther width/anther length) was higher in STS1-HOM anthers than in STS1-HET anthers (p-value = p-value = 6.8e-10, left), while anther area (anther width X anther length) was not significantly different (p-value = 0.2592, right). (L) Confocal images of developing anthers measured in (K). (M) At anthesis, anther aspect ratio is lower in STS1-HOM anthers than in STS1-HET anthers (p-value = 0.003, left), while anther area is not significantly different (p-value = 0.367, right). p-values calculated N) 25 randomly selected anthers from the first (bottom) and fourth (top) quartiles of anthers measured in M, colored according to STS1 transgene genotype.  Degraded by the proteosome

A B
CHR126b, CHR12 FRL4a, BRM1 CHR126b (A) STS1-HET/SI1 heterodimers, in complex with other MADS-box proteins, recruits chromatin remodelers like the SWI/SNF ATP-ase CHR126b, ultimately resulting in the upregulation of target genes. This transcription is likely halted by the proteasome-mediated degradation of MADS-box complexes. (B) STS1-HOM homodimers and their MADS-box partners also recruit chromatin remodelers and scaffolding proteins, resulting in upregulated transcription. However, STS1 homodimerization disrupts degradation, resulting in higher or more sustained transcription of target genes.

IN A NUTSHELL
Background. The regulatory network that controls floral organ formation is complex and still not completely understood. This network has also been changing over the course of evolution, leading to immense floral diversity. MADS-box transcription factors are important regulators of floral organ development, and changing interactions between MADS-box proteins are predicted to have influenced floral evolution. However, the functional consequences of evolutionary change to MADS-box protein-protein interactions were unknown. Grasses are an excellent family to explore these consequences due to high genetic diversity, evolutionary variation in MADS-box protein-protein interactions, and the numerous genetic model systems in the family. We used the genetic and genomic resources available in maize to determine the functional consequences of evolutionary variation in dimerization of a key regulator of floral development, STERILE TASSEL SILKY EAR1 (STS1).
Question. STS1 dimerization varies across the grasses. In this work, we set out to discover the effect of some of this evolutionary variation on floral development, downstream gene expression and protein complex assembly in maize.

Findings.
We found that evolutionary variants of STS1 differentially affect downstream gene expression and protein complex assembly in maize. Surprisingly, we found that STS1 dimerization changed protein degradation dynamics, which had not been described. This discovery adds a new layer of complexity to the regulation of flower development, and how that regulation changes over deep time. We also found that a coding change to STS1, a master regulator of development, had subtle effects on floral development. Our results indicate that small changes to MADS-box genes may have influenced the gradual evolution of floral form. This dissection of the effects of a single base-pair change, using an allelic series provided by evolution, is critical in this era of genome editing.
Next steps. Next projects are focused on explore mechanisms by which protein modifications and interactions affect floral organ formation and evolution. In the future, our findings might help to identify additional factors influencing floral development and evolution.