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• W3048672132 abstract "•HTB encodes a SUMO protease required for fruit shape in Capsella•Anisotropic cell growth is suppressed in the fruit valves of the htb mutant•HTB stabilizes CrIND through de-SUMOylation to facilitate local auxin biosynthesis Morphological variation is the basis of natural diversity and adaptation. For example, angiosperms (flowering plants) evolved during the Cretaceous period more than 100 mya and quickly colonized terrestrial habitats [1Soltis P.S. Folk R.A. Soltis D.E. Darwin review: angiosperm phylogeny and evolutionary radiations.Proc. Royal Soc. B: Biol. Sci. 2019; 286: 20190099Crossref Scopus (31) Google Scholar]. A major reason for their astonishing success was the formation of fruits, which exist in a myriad of different shapes and sizes [2Seymour G.B. Østergaard L. Chapman N.H. Knapp S. Martin C. Fruit development and ripening.Annu. Rev. Plant Biol. 2013; 64: 219-241Crossref PubMed Scopus (350) Google Scholar]. Evolution of organ shape is fueled by variation in expression patterns of regulatory genes causing changes in anisotropic cell expansion and division patterns [3Carroll S.B. Endless forms: the evolution of gene regulation and morphological diversity.Cell. 2000; 101: 577-580Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 4Carroll S.B. Evolution at two levels: on genes and form.PLoS Biol. 2005; 3: e245Crossref PubMed Scopus (611) Google Scholar, 5Wray G.A. The evolutionary significance of cis-regulatory mutations.Nat. Rev. Genet. 2007; 8: 206-216Crossref PubMed Scopus (990) Google Scholar]. However, the molecular mechanisms that alter the polarity of growth to generate novel shapes are largely unknown. The heart-shaped fruits produced by members of the Capsella genus comprise an anatomical novelty, making it particularly well suited for studies on morphological diversification [6Eldridge T. Łangowski Ł. Stacey N. Jantzen F. Moubayidin L. Sicard A. Southam P. Kennaway R. Lenhard M. Coen E.S. Østergaard L. Fruit shape diversity in the Brassicaceae is generated by varying patterns of anisotropy.Development. 2016; 143: 3394-3406Crossref PubMed Scopus (28) Google Scholar, 7Łangowski Ł. Stacey N. Østergaard L. Diversification of fruit shape in the Brassicaceae family.Plant Reprod. 2016; 29: 149-163Crossref PubMed Scopus (21) Google Scholar, 8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. Here, we show that post-translational modification of regulatory proteins provides a critical step in organ-shape formation. Our data reveal that the SUMO protease, HEARTBREAK (HTB), from Capsella rubella controls the activity of the key regulator of fruit development, INDEHISCENT (CrIND in C. rubella), via de-SUMOylation. This post-translational modification initiates a transduction pathway required to ensure precisely localized auxin biosynthesis, thereby facilitating anisotropic cell expansion to ultimately form the heart-shaped Capsella fruit. Therefore, although variation in the expression of key regulatory genes is known to be a primary driver in morphological evolution, our work demonstrates how other processes—such as post-translational modification of one such regulator—affects organ morphology. Morphological variation is the basis of natural diversity and adaptation. For example, angiosperms (flowering plants) evolved during the Cretaceous period more than 100 mya and quickly colonized terrestrial habitats [1Soltis P.S. Folk R.A. Soltis D.E. Darwin review: angiosperm phylogeny and evolutionary radiations.Proc. Royal Soc. B: Biol. Sci. 2019; 286: 20190099Crossref Scopus (31) Google Scholar]. A major reason for their astonishing success was the formation of fruits, which exist in a myriad of different shapes and sizes [2Seymour G.B. Østergaard L. Chapman N.H. Knapp S. Martin C. Fruit development and ripening.Annu. Rev. Plant Biol. 2013; 64: 219-241Crossref PubMed Scopus (350) Google Scholar]. Evolution of organ shape is fueled by variation in expression patterns of regulatory genes causing changes in anisotropic cell expansion and division patterns [3Carroll S.B. Endless forms: the evolution of gene regulation and morphological diversity.Cell. 2000; 101: 577-580Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 4Carroll S.B. Evolution at two levels: on genes and form.PLoS Biol. 2005; 3: e245Crossref PubMed Scopus (611) Google Scholar, 5Wray G.A. The evolutionary significance of cis-regulatory mutations.Nat. Rev. Genet. 2007; 8: 206-216Crossref PubMed Scopus (990) Google Scholar]. However, the molecular mechanisms that alter the polarity of growth to generate novel shapes are largely unknown. The heart-shaped fruits produced by members of the Capsella genus comprise an anatomical novelty, making it particularly well suited for studies on morphological diversification [6Eldridge T. Łangowski Ł. Stacey N. Jantzen F. Moubayidin L. Sicard A. Southam P. Kennaway R. Lenhard M. Coen E.S. Østergaard L. Fruit shape diversity in the Brassicaceae is generated by varying patterns of anisotropy.Development. 2016; 143: 3394-3406Crossref PubMed Scopus (28) Google Scholar, 7Łangowski Ł. Stacey N. Østergaard L. Diversification of fruit shape in the Brassicaceae family.Plant Reprod. 2016; 29: 149-163Crossref PubMed Scopus (21) Google Scholar, 8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. Here, we show that post-translational modification of regulatory proteins provides a critical step in organ-shape formation. Our data reveal that the SUMO protease, HEARTBREAK (HTB), from Capsella rubella controls the activity of the key regulator of fruit development, INDEHISCENT (CrIND in C. rubella), via de-SUMOylation. This post-translational modification initiates a transduction pathway required to ensure precisely localized auxin biosynthesis, thereby facilitating anisotropic cell expansion to ultimately form the heart-shaped Capsella fruit. Therefore, although variation in the expression of key regulatory genes is known to be a primary driver in morphological evolution, our work demonstrates how other processes—such as post-translational modification of one such regulator—affects organ morphology. Organs in multicellular organisms have evolved into specific shapes that are critical for their function. Accordingly, little diversity is observed in organ morphology between individuals of the same species, with organs consistently and robustly developing into specific shapes [9Moyroud E. Glover B.J. The evolution of diverse floral morphologies.Curr. Biol. 2017; 27: R941-R951Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar]. By contrast, major variation in organ shape can exist between closely related species, as observed for fruits, leaves, insect wings, or the outer ears of mammals [7Łangowski Ł. Stacey N. Østergaard L. Diversification of fruit shape in the Brassicaceae family.Plant Reprod. 2016; 29: 149-163Crossref PubMed Scopus (21) Google Scholar, 10Runions A. Tsiantis M. Prusinkiewicz P. A common developmental program can produce diverse leaf shapes.New Phytol. 2017; 216: 401-418Crossref PubMed Scopus (59) Google Scholar, 11Salcedo M.K. Hoffmann J. Donoughe S. Mahadevan L. Computational analysis of size, shape and structure of insect wings.Biol. Open. 2019; 8: bio040774Crossref PubMed Scopus (11) Google Scholar, 12Coleman M.N. Ross C.F. Primate auditory diversity and its influence on hearing performance.Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 2004; 281: 1123-1137Crossref PubMed Scopus (46) Google Scholar]. Changes in the expression pattern of key regulatory genes is a major driver of such morphological diversity, ultimately giving rise to changes in cell division patterns and cell expansion [13Huu C.N. Kappel C. Keller B. Sicard A. Takebayashi Y. Breuninger H. Nowak M.D. Bäurle I. Himmelbach A. Burkart M. et al.Presence versus absence of CYP734A50 underlies the style-length dimorphism in primroses.eLife. 2016; 5: e17956Crossref PubMed Scopus (52) Google Scholar, 14Sicard A. Kappel C. Lee Y.W. Woźniak N.J. Marona C. Stinchcombe J.R. Wright S.I. Lenhard M. Standing genetic variation in a tissue-specific enhancer underlies selfing-syndrome evolution in Capsella.Proc. Natl. Acad. Sci. USA. 2016; 113: 13911-13916Crossref PubMed Scopus (35) Google Scholar]. We have shown that sequence variation in regulatory domains of the fruit-tissue identity gene, INDEHISCENT (IND) (CrIND in Capsella), is responsible for augmentation of its expression domain in the heart-shaped fruits from Capsella rubella. In turn, CrIND induces expression of auxin biosynthesis genes required for growth of the shoulders of the heart [8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. To identify genetic factors controlling this process and required for the formation of heart-shaped fruits in Capsella, we carried out a forward genetic screen of an ethyl methanesulfonate (EMS)-induced Capsella rubella (Cr22.5) mutant population. One mutant, heartbreak (htb), was isolated because of its strong defects in fruit development with compromised outgrowth of the fruit shoulders (Figures 1A, 1B, and 1D ). Moreover, compared with wild type (WT), the htb mutant exhibits defects throughout both vegetative and reproductive development (Figures S1A–S1J). This demonstrates that the HTB gene regulates multiple developmental processes. In WT Capsella, the heart-shaped fruit develops from a disc-formed (ovate spheroid) gynoecium soon after pollination [6Eldridge T. Łangowski Ł. Stacey N. Jantzen F. Moubayidin L. Sicard A. Southam P. Kennaway R. Lenhard M. Coen E.S. Østergaard L. Fruit shape diversity in the Brassicaceae is generated by varying patterns of anisotropy.Development. 2016; 143: 3394-3406Crossref PubMed Scopus (28) Google Scholar] (Figure 1E). From stage 13 onward, directional outgrowths of the apical parts of the valves found formation of the heart shape by stage 14 (Figures 1F and 1G; developmental stages defined in [8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]). Comparative ontogenetic analysis revealed no defects between WT and htb during early gynoecium development (Figures 1E and 1H). In contrast to WT, however, the outgrowth of the htb valve apex is significantly suppressed from stage 13 (Figures 1F, 1G, 1I, and 1J). During postfertilization development, anisotropic cell expansion drives fruit growth toward the final size and shape [7Łangowski Ł. Stacey N. Østergaard L. Diversification of fruit shape in the Brassicaceae family.Plant Reprod. 2016; 29: 149-163Crossref PubMed Scopus (21) Google Scholar, 15Ripoll J.J. Zhu M. Brocke S. Hon C.T. Yanofsky M.F. Boudaoud A. Roeder A.H.K. Growth dynamics of the Arabidopsis fruit is mediated by cell expansion.Proc. Natl. Acad. Sci. USA. 2019; 116: 25333-25342Crossref PubMed Scopus (11) Google Scholar]. To assess the cellular basis underlying the htb phenotype, we traced the cell growth dynamics by time-lapse imaging of developing fruits [16Barbier de Reuille P. Routier-Kierzkowska A.-L. Kierzkowski D. Bassel G.W. Schüpbach T. Tauriello G. Bajpai N. Strauss S. Weber A. Kiss A. et al.MorphoGraphX: a platform for quantifying morphogenesis in 4D.eLife. 2015; 4: 05864Crossref PubMed Scopus (247) Google Scholar]. We chose three specific stages: stage 12 (immediately preceding the initiation of shoulder outgrowth); stage 13 (outgrowth begins); and stage 14 (shoulders are clearly formed; Figures 1E–1G). In stage-13 WT fruits, cells in the apical part of the valve grew anisotropically along the medio-lateral axis (Figures 1K and S1K). At stage 14, most of the cells in the apical part of the WT fruit had become highly anisotropic, growing toward the developing fruit shoulders, although cells in the basal part of the fruit remained largely isotropic from stages 12 to 14 (Figure 1K). In WT, the overall cell expansion rate was similar between apical and basal parts of the fruit (Figures 1M and S1L). In contrast to WT, cells in the valves of the htb mutant grew isotropically throughout all the stages studied here, leading to reduced growth rate in the shoulders (Figures 1L and S1K). Also, in comparison to WT, the htb mutant displayed a decreased overall cell expansion rate in the apical part of the fruit (Figures 1N and S1L). These data demonstrate that the HTB locus functions to promote anisotropic cell growth in the fruit valves. The htb mutation segregates as a single-locus recessive trait (Figure S2B). By whole-genome sequencing and associative mapping, we identified two candidate mutations in the predicted genes, Carubv10012951 and Carubv10008238. A synonymous mutation in the first exon of Carubv10012951 precluded it for further consideration. Instead, a potential causal mutation in the acceptor site of the first intron of a predicted gene, Carubv10008238, was investigated further. This gene encodes a putative small ubiquitin modifer (SUMO) protease, a member of the ULP2 subfamily of cysteine proteases, and is orthologous to the Arabidopsis SPF1/ASP1 gene (Figure S2A) [17Kong X. Luo X. Qu G.P. Liu P. Jin J.B. Arabidopsis SUMO protease ASP1 positively regulates flowering time partially through regulating FLC stability.J. Integr. Plant Biol. 2017; 59: 15-29Crossref PubMed Scopus (30) Google Scholar, 18Castro P.H. Santos M.Â. Freitas S. Cana-Quijada P. Lourenço T. Rodrigues M.A.A. Fonseca F. Ruiz-Albert J. Azevedo J.E. Tavares R.M. et al.Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development.J. Exp. Bot. 2018; 69: 4633-4649Crossref PubMed Scopus (16) Google Scholar]. The mutation disrupts the splicing of the first intron, which instead occurs after an alternative site 7 bp into the second exon, resulting in a frameshift and premature stop codon (Figures 2A and S2C). We will refer to this mutant allele as htb-1. Verification of the causality of this mutation on fruit shape was confirmed as follows: (1) expression of Carubv10008238 driven by its native promoter fully complemented the htb-1 mutant (Figures 1C and 1D); (2) a knockout line of Carubv10008238 using CRISPR-Cas9, leading to a single-base deletion in the second exon (htb-2ge), phenocopied the htb-1 fruit character alongside other developmental defects (Figures 2A and S2D–S2F); and (3) F1 plants of htb-1 crossed with the htb-2ge mutant show the same phenotype as htb-1 (Figure S2G). Together, these experiments show that the developmental defects observed in the htb-1 mutant are caused by loss of the Carubv10008238 gene, which we henceforth refer to as HEARTBREAK (HTB), encoding a putative SUMO protease. In agreement with the wide range of developmental defects of the htb-1 mutant, we found a pHTB:GUS reporter line to be expressed throughout plant development, including vascular tissue of cotyledons and roots and in root tips of seedlings (Figures S2H and S2I). pHTB:GUS signal seemed uniformly distributed in the inflorescences and young gynoecia (Figures 2B–2D). Notably, in the developing fruit, stronger HTB promoter activity is detected in the shoulders from stage 13 to stage 14, when the heart shape starts to develop, although at stage 15, only residual HTB expression is detected (Figures 2E–2G). HTB expression therefore correlates spatially and temporally with fruit growth in agreement with its role in promoting anisotropic cell growth in the valves. The SPF1/ASP1 protein is located in the nucleus of Arabidopsis cells [18Castro P.H. Santos M.Â. Freitas S. Cana-Quijada P. Lourenço T. Rodrigues M.A.A. Fonseca F. Ruiz-Albert J. Azevedo J.E. Tavares R.M. et al.Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development.J. Exp. Bot. 2018; 69: 4633-4649Crossref PubMed Scopus (16) Google Scholar, 19Liu L. Jiang Y. Zhang X. Wang X. Wang Y. Han Y. Coupland G. Jin J.B. Searle I. Fu Y.-F. Chen F. Two SUMO proteases SUMO PROTEASE RELATED TO FERTILITY1 and 2 are required for fertility in Arabidopsis.Plant Physiol. 2017; 175: 1703-1719Crossref PubMed Scopus (23) Google Scholar]. To test the subcellular localization of HTB, we used a pHTB:HTB:GFP reporter line, which fully complements the htb-1 mutant (Figures 1C and 1D). Strong GFP signal was seen specifically within the nucleus but excluded from the nucleolus in root cells (Figures 2H and 2I). A similar nuclear localization pattern was observed using transient overexpression of an HTB-GFP fusion protein in WT leaf protoplasts (Figure S2J). These data suggest that HTB exerts its function on fruit-shape formation by affecting the activity of nuclear proteins. The SUMOylation of proteins is a dynamic process with reversibility in conjugation and deconjugation [20Mukhopadhyay D. Dasso M. Modification in reverse: the SUMO proteases.Trends Biochem. Sci. 2007; 32: 286-295Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar]. SUMO proteases falling into the class of ubiquitin-like proteases (ULPs) belong to the cysteine protease family and are able to mediate SUMO maturation as well as SUMO deconjugation from protein targets through their endopeptidase and isopeptidase activity, respectively [21Yates G. Srivastava A.K. Sadanandom A. SUMO proteases: uncovering the roles of deSUMOylation in plants.J. Exp. Bot. 2016; 67: 2541-2548Crossref PubMed Scopus (46) Google Scholar]. In order to determine whether HTB affects SUMO-conjugation levels, we compared the SUMOylation profiles between WT and htb-1 by western blotting using specific anti-SUMO1 antibodies. Compared with WT, high-molecular-weight SUMO conjugates constitutively accumulated in total-protein extracts from the htb-1 mutant. This was particularly evident in inflorescence tissue and stage-13 fruits (Figure 2J), suggesting that the developmental defects observed in the htb-1 mutant (Figures S1A–S1J) is due to over-SUMOylation of proteins that are targets of the HTB SUMO protease. SUMO proteases have been reported to control SUMOylation levels of transcription factors, chromatin remodeling factors, and/or transcriptional co-repressors [18Castro P.H. Santos M.Â. Freitas S. Cana-Quijada P. Lourenço T. Rodrigues M.A.A. Fonseca F. Ruiz-Albert J. Azevedo J.E. Tavares R.M. et al.Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development.J. Exp. Bot. 2018; 69: 4633-4649Crossref PubMed Scopus (16) Google Scholar, 22Gill G. Something about SUMO inhibits transcription.Curr. Opin. Genet. Dev. 2005; 15: 536-541Crossref PubMed Scopus (412) Google Scholar, 23Orosa-Puente B. Leftley N. von Wangenheim D. Banda J. Srivastava A.K. Hill K. Truskina J. Bhosale R. Morris E. Srivastava M. et al.Root branching toward water involves posttranslational modification of transcription factor ARF7.Science. 2018; 362: 1407-1410Crossref PubMed Scopus (87) Google Scholar, 24Srivastava M. Srivastava A.K. Orosa-Puente B. Campanaro A. Zhang C. Sadanandom A. SUMO conjugation to BZR1 enables Brassinosteroid signaling to integrate environmental cues to shape plant growth.Curr. Biol. 2020; 30: 1410-1423.e3Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. In order to understand the relationship between the transcriptional profile and HTB function in fruit development, we performed a comparative transcriptomic analysis of stage-13 fruits between WT and htb-1, when the developmental difference started to emerge (Figures 1F and 1I). The RNA-profiling analysis generated a total of 605 significant differentially expressed genes (DEGs) between WT and htb-1. Among them, 190 were upregulated and 415 were downregulated (Data S1A and S1B). Gene Ontology (GO) and pathway-enrichment analyses identified enrichment of DEGs in processes such as oxidation-reduction, protein phosphorylation, responses to light stimulus, and cell wall organization and modification (Figures S3A and S3C; Data S2A and S2B). Intriguingly, genes involved in hormone response were over-represented in the DEGs, especially among the downregulated genes (Figures S3B and S3D; Data S2A and S2B). Among the 26 downregulated DEGs associated with hormone response, 11 were associated with auxin response, pinpointing a possible role of HTB in regulating auxin dynamics during fruit-shape determination (Figures S3D and S3E; Data S2B). We recently reported that the development of the heart-shaped Capsella fruit requires an auxin maximum in the fruit shoulders ensured by local expression of auxin biosynthesis genes, CrTAA1 and CrYUC9 [8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. Hence, we analyzed whether auxin dynamics was disrupted in the htb-1 fruits compared to WT. To visualize the auxin signaling pattern in the fruit valves, we used the pDR5v2:GUS reporter whose expression marks and precedes shoulder growth and introduced it into htb-1. In stage-14 WT fruit, a gradient of auxin signaling was observed in the valves with a maximum in the fruit shoulders (Figure 3A). In contrast, in the htb-1 mutant, the auxin maxima in the shoulders were lost, signifying a reduction of auxin response in the htb-1 fruits (Figure 3B). We next asked whether the lack of auxin maxima in the htb-1 fruit shoulders was due to low auxin levels. Direct measurements of both the predominant natural auxin, indole-3-acetic acid (IAA), and its precursor, indole-3-pyruvate (IPA), showed a significant reduction in the shoulders of htb-1 compared to WT (Figures 3C and 3D). In correlation with reduced IPA and IAA levels, we found that expression of CrTAA1 and CrYUC9 was lower in the htb-1 fruit shoulders compared to WT (Figures 3E–3J). These data suggest that the decrease in auxin response observed in htb-1 fruits can be attributed to low levels of auxin biosynthesis, resulting from reduced CrTAA1 and CrYUC9 expression. Rescue of the htb-1 phenotype by exogenous application of IAA and valve-shoulder-specific expression of a bacterial auxin biosynthesis gene (pCrIND:iaaM) provided further evidence that auxin biosynthesis is a downstream output of HTB activity required for fruit-shape formation (Figures 3K and 3O). In Capsella, shoulder-specific expression of CrTAA1 and CrYUC9 is regulated by the basic-helix-loop-helix (bHLH) transcription factor, CrIND [8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. In the crind-1ge mutant, the fruit shoulders fail to fully expand due to depletion of auxin in the fruits compared to WT [8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. The htb-1 mutant exhibits a similar phenotype as crind-1ge, and lack of an obvious exacerbation of the single mutant phenotypes in the htb-1 crind-1ge double mutant suggests that HTB and CrIND function in the same pathway (Figure 4A). To explore this possibility further, we crossed htb-1 with the crful-1 mutant previously shown to be partially rescued by mutations in the CrIND gene [8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar] (Figures 3P–3R, 3T–3V, and S3F). The htb-1 mutant also partially rescues the strong growth defect of crful-1, although to a lesser extent than crind-1ge (Figures 3S, 3W, and S3F). It is therefore possible that the absence of HTB partially overturns the effect of ectopic CrIND expression previously reported to occur in crful-1 [8Dong Y. Jantzen F. Stacey N. Łangowski Ł. Moubayidin L. Šimura J. Ljung K. Østergaard L. Regulatory diversification of INDEHISCENT in the Capsella genus directs variation in fruit morphology.Curr. Biol. 2019; 29: 1038-1046.e4Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar]. Interestingly, expression of CrIND was unchanged in htb-1 compared to WT (Figure 3X). This led us to test whether CrIND function is regulated post-translationally by HTB through SUMOylation. In plant cells, SUMOylation occurs through an isopeptide-bond formation between the di-glycine at the C-terminal of the SUMO peptide and the accessible lysyl ε-amino group within the targets [25Miura K. Hasegawa P.M. Sumoylation and other ubiquitin-like post-translational modifications in plants.Trends Cell Biol. 2010; 20: 223-232Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar]. Cumulative SUMO target datasets suggest a consensus ψ-K-X-D/E canonical SUMOylation motif (ψ, hydrophobic amino acid; X, any amino acid) [26Rytz T.C. Miller M.J. McLoughlin F. Augustine R.C. Marshall R.S. Juan Y.T. Charng Y.Y. Scalf M. Smith L.M. Vierstra R.D. SUMOylome profiling reveals a diverse array of nuclear targets modified by the SUMO ligase SIZ1 during heat stress.Plant Cell. 2018; 30: 1077-1099Crossref PubMed Scopus (72) Google Scholar]. Searching the CrIND sequence identified a consensus SUMO motif in amino acid positions 123–126 (AKMD) with lysine in position 124 (K124) as a potential SUMO-conjugation residue. To investigate the functional relevance of K124 in CrIND with HTB, we produced a mutant variant of CrIND, in which K124 is mutated to the related but unSUMOylatable amino acid, arginine (R), and compared the function of CrIND and CrINDK124R in htb-1 background. The K124R mutation did not change the protein function, as both pCrIND:CrIND:GFP and pCrIND:CrINDK124R:GFP fully rescued the crind mutant (Figures S4A–S4D). In the htb-1 background, however, we observed a different behavior of these two proteins. Although pCrIND:CrIND:GFP failed to complement the htb-1 mutant, pCrIND:CrINDK124R:GFP effectively rescued the fruit defects of htb-1, developing fully heart-shaped fruits (Figures 4B–4D). This implies that post-translational modification of the K124 residue in CrIND is the primary cause of the defect in htb-1 fruit-shoulder growth and suggests that HTB functions to de-SUMOylate CrIND on this residue. We then tested whether CrIND SUMOylation status depends on HTB. To this end, overexpression of FLAG-tagged versions of CrIND and CrINDK124R in WT and the htb-1 mutant was achieved using a two-component dexamethasone (DEX)-inducible system (Figure S4E). A pull-down experiment of FLAG-tagged CrIND/CrINDK124R detected a high-molecular-weight version of SUMOylated CrIND only in the htb-1 mutant background (Figure 4E). Moreover, western blotting with FLAG antibody revealed low abundance of CrIND in htb-1 compared to WT, whereas no reduction was observed with the CrINDK124R version in htb-1 (Figure 4E). These data demonstrate that HTB positively controls CrIND levels through de-SUMOylation, suggesting that SUMOylation on K124 of CrIND leads to its destabilization. In agreement with reduced stability of CrIND in the htb-1 mutant, chromatin immunoprecipitation (ChIP) assays in htb-1 revealed that promoter regions of CrTAA1 and CrYUC9 were less enriched with CrIND-GFP compared to CrINDK124R-GFP (Figure 4F). On the other hand, the binding affinities to CrTAA1 and CrYUC9 promoters were not significantly different between CrIND-GFP and CrINDK124R-GFP when ChIP assays were carried out in the crind-1ge background (Figure S4F). Furthermore, CrTAA1 and CrYUC9 expression in the fruit shoulders is restored in htb-1 carrying the pCrIND:CrINDK124R:GFP transgene (Figures 3I and 3J). Together, these biochemical and genetic data demonstrate that HTB acts directly on CrIND, leading to local expression of auxin biosynthesis genes. Although the effect of SUMOylation can vary widely between proteins, our results align with observations in both plants and animals that SUMOylation of transcription factors affects their stability and activity toward target genes [22Gill G. Something about SUMO inhibits transcription.Curr. Opin. Genet. Dev. 2005; 15: 536-541Crossref PubMed Scopus (412) Google Scholar, 23Orosa-Puente B. Leftley N. von Wangenheim D. Banda J. Srivastava A.K. Hill K. Truskina J. Bhosale R. Morris E. Srivastava M. et al.Root branching toward water involves posttranslational modification of transcription factor ARF7.Science. 2018; 362: 1407-1410Crossref PubMed Scopus (87) Google Scholar, 24Srivastava M. Srivastava A.K. Orosa-Puente B. Campanaro A. Zhang C. Sadanandom A. SUMO conjugation to BZR1 enables Brassinosteroid signaling to integrate environmental cues to shape plant growth.Curr. Biol. 2020; 30: 1410-" @default.
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