PLANT PHYSIOLOGY

Nguyen, Trinh-Don

doi: 10.1093/plphys/kiac457pmid: 36149328

Accepted manuscripts Accepted manuscripts are PDF versions of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout. Article PDF first page preview Close This content is only available as a PDF. © American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: [email protected]

Galindo-Trigo, Sergio

doi: 10.1093/plphys/kiac436pmid: 36135829

The epidermis in plants is the outermost cell layer and as such is crucial to mediate the plant’s interaction with its environment. In aerial tissues like leaves and stems, the epidermis protects the plant from the threats of pathogens, herbivores, and unfavorable environmental conditions. Meanwhile, the root epidermis actively increases the roots’ surface to favor water and nutrient intake, as well as to establish symbiotic relationships with beneficial fungi. To carry out such a diverse number of functions, epidermal cells can differentiate into aerial hairs or trichomes, stomata, or root hairs, among others. In some plants, trichomes act as a direct defence mechanism as they accumulate herbivore-deterrent chemicals, while in Arabidopsis (Arabidopsis thaliana) trichomes act as mechanosensors, initiating Ca2+ waves that reach and alert distant cells of the presence of a threat (Matsumura et al., 2022). However, not all epidermal cells differentiate into trichomes or root hairs. Specific genes regulate epidermal cell fate and therefore the ratio of epidermal cell types (Zuch et al., 2022). For example, GLABRA2 (GL2) encodes a transcription factor (TF) and is one of the best characterized regulators of epidermal cell fate in Arabidopsis, controlling trichome differentiation, root hair-less cell file determination, as well as mucilage synthesis in seeds (for review, see Ariel et al., 2007). TFs bind specific DNA sequences (or target sites) and execute changes in the expression of genes, usually by recruiting additional TFs and components of the basal transcriptional machinery. TFs are modular molecules comprising distinct protein domains, each of which typically contributes to the TF activity in a different manner: the DNA binding domain determines the DNA sequence to which the TF will bind, whereas other protein domains may influence the TF’s protein interactome, intracellular transport dynamics, and protein stability. Distinguishing the contributions of individual protein domains in a TF is crucial to harness their full potential to fine-tune transcription for better yields or to efficiently design synthetic transcriptional networks (Khalil et al., 2012). GL2 belongs to the homeodomain-leucine zipper (HD-ZIP) class IV TF family that has a plant-specific domain arrangement consisting of an HD-ZIP DNA-binding domain tandem near the N-terminus, followed by a steroidogenic acute regulatory protein-related lipid-transfer (START) domain, and a START-associated domain (STAD) near the protein C-terminus (Schena and Davis, 1992). In animals and the liverwort Marchantia polymorpha, the START domain binds to lipids and influences their transport (Clark, 2020; Hirashima et al., 2021). Nevertheless, the functional contribution of the START domain to GL2 transcriptional activity has remained speculative until now. In the current issue of Plant Physiology, Mukherjee et al. (2022) molecularly dissect and characterize the roles of the START domain in GL2 function as well as its interactions with the better-characterized HD-ZIP domains. The approach taken by Mukherjee et al. (2022) involved designing a comprehensive set of GL2 mutant variants that could potentially impair the HD and START domain activities. Knockout gl2 plants were transformed with the set of GL2 variants fused to a fluorescent protein, and their individual capacity to rescue the well-characterized gl2 developmental defects was scored. Confirming the predictive power of homology-based structural models, all mutant variants of GL2 failed to rescue gl2 trichome development, root hair density, and seed mucilage accumulation, indicating that the START domain, similarly to HD, is indispensable for GL2 transcriptional function. Similar phenotypic defects were quantified in domain-specific START and HD mutants, indicating that both domains are equally necessary for GL2 activity. HDs allow DNA binding and dictate the target site of TFs. Consequently, and based on genetic complementation assays, the START domain must govern another equally indispensable molecular mechanism for GL2 activity. The authors first analyzed the subcellular localization of the mutant GL2 variants and concluded that neither the HD nor START domains impair GL2 nuclear localization. Next, the importance of the START domain for the ability of HD-ZIP(IV) proteins to bind DNA was tested in electrophoretic mobility shift assays (EMSAs). Due to difficulties expressing recombinant GL2, a close relative, PROTODERMAL FACTOR2 (PDF2), and PDF2 mutant variants equivalent to those designed for GL2 were used in these experiments. EMSAs and western blots indicated that the START domain of PDF2 does not influence the TF DNA binding in vitro and that the START domain is necessary for homodimerization. A ChIP-seq experiment using several GL2 mutant variants revealed that the presence or functionality of the START domain does not substantially alter GL2 DNA binding specificity in vivo. Yeast-two hybrid and split-fluorescent protein assays using a comprehensive set of full-length and domain deletion GL2 variants confirmed the requirement of the START domain for effective GL2 homodimerization in yeast and in planta. Finally, Mukherjee and colleagues investigated whether the START domain could influence protein stability by applying protein synthesis inhibitors to seedlings expressing different GL2 mutant variants and quantifying the corresponding GL2 protein in a time-course experiment. The latter experiment clearly indicated that GL2 variants lacking a functional START domain were degraded more rapidly than wild-type GL2. Using a proteasome degradation inhibitor extended the lifetime of START-domain mutant GL2 variants several hours, suggesting that the proteasome degradation pathway could be controlling GL2 protein stability. The authors therefore concluded the START domain is vital for HD-ZIP(IV) TF activity as it (i) enables homodimerization, therefore likely allowing a more effective recruitment of the basal transcriptional machinery; and (ii) increases protein stability, increasing the active window for each HD-ZIP(IV) molecule to conduct its cellular activity before protein turnover. Moving forward it would be very informative to address the lipid-binding capacity of the HD-ZIP(IV) START domains by means of biochemical and evo-devo approaches, that is, testing their capacity to directly bind collections of lipids, and investigating whether they can genetically complement the previously identified START domain of the M. polymorpha STAR2 protein. Pinpointing the type of lipid and the conditions under which it is bound by HD-ZIP(IV) TFs would provide us with a clearer picture of how these TFs are mechanistically regulated and perhaps reveal how these TFs may perceive and respond to readouts of cellular metabolism and/or important small signaling lipids. Conflict of interest statement. None declared. References Ariel FD , Manavella PA, Dezar CA, Chan RL ( 2007 ) The true story of the HD-Zip family . Trends Plant Sci 12 : 419 – 426 Google Scholar Crossref Search ADS PubMed WorldCat Clark BJ ( 2020 ) The START-domain proteins in intracellular lipid transport and beyond . Mol Cell Endocrinol 504 : 110704 Google Scholar Crossref Search ADS PubMed WorldCat Hirashima T , Jimbo H, Kobayashi K, Wada H ( 2021 ) A START domain-containing protein is involved in the incorporation of ER-derived fatty acids into chloroplast glycolipids in Marchantia polymorpha . Biochem Biophys Res Commun 534 : 436 – 441 Google Scholar Crossref Search ADS PubMed WorldCat Khalil AS , Lu TK, Bashor CJ, Ramirez CL, Pyenson NC, Joung JK, Collins JJ ( 2012 ) A synthetic biology framework for programming eukaryotic transcription functions . Cell 150 : 647 – 658 Google Scholar Crossref Search ADS PubMed WorldCat Matsumura M , Nomoto M, Itaya T, Aratani Y, Iwamoto M, Matsuura T, Hayashi Y, Mori T, Skelly MJ, Yamamoto YY, et al. ( 2022 ) Mechanosensory trichome cells evoke a mechanical stimuli-induced immune response in Arabidopsis thaliana . Nat Commun 13 : 1216 Google Scholar Crossref Search ADS PubMed WorldCat Mukherjee T , Subedi B, Khosla A, Begler EM, Stephens PM, Warner AL, Lerma-Reyes R, Thompson KA, Gunewardena S, Schrick K ( 2022 ) The START domain mediates Arabidopsis GLABRA2 transcription factor dimerization and turnover independently of homeodomain DNA binding . Plant Physiol https://doi.org/10.1093/plphys/kiac383 Google Scholar OpenURL Placeholder Text WorldCat Schena M , Davis RW ( 1992 ) HD-Zip proteins: members of an Arabidopsis homeodomain protein superfamily . Proc Natl Acad Sci USA 89 : 3894 – 3898 Google Scholar Crossref Search ADS PubMed WorldCat Zuch DT , Doyle SM, Majda M, Smith RS, Robert S, Torii KU ( 2022 ) Cell biology of the leaf epidermis: fate specification, morphogenesis, and coordination . Plant Cell 34 : 209 – 227 Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.

Lanctot, Amy

doi: 10.1093/plphys/kiac453pmid: 36161497

Accepted manuscripts Accepted manuscripts are PDF versions of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout. Article PDF first page preview Close This content is only available as a PDF. © The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.

Kerwin, Rachel E; Dubois, Marieke

doi: 10.1093/plphys/kiac417pmid: 36086956

Accepted manuscripts Accepted manuscripts are PDF versions of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout. Article PDF first page preview Close This content is only available as a PDF. © The Author(s) (2022) . Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) (2022) . Published by Oxford University Press on behalf of American Society of Plant Biologists.

Kazachkova, Yana

doi: 10.1093/plphys/kiac438pmid: 36124986

Accepted manuscripts Accepted manuscripts are PDF versions of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout. Article PDF first page preview Close This content is only available as a PDF. © The Author(s) (2022). Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) (2022). Published by Oxford University Press on behalf of American Society of Plant Biologists.

Mishra, Divya

doi: 10.1093/plphys/kiac416pmid: 36063464

Accepted manuscripts Accepted manuscripts are PDF versions of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout. Article PDF first page preview Close This content is only available as a PDF. © American Society of Plant Biologists 2022. All rights reserved. [br]For permissions, please email: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © American Society of Plant Biologists 2022. All rights reserved. [br]For permissions, please email: [email protected]

Wege, Stefanie

doi: 10.1093/plphys/kiac429pmid: 36124988

Accepted manuscripts Accepted manuscripts are PDF versions of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout. Article PDF first page preview Close This content is only available as a PDF. © The Author(s) (2022). Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) (2022). Published by Oxford University Press on behalf of American Society of Plant Biologists.

Courbier, Sarah

doi: 10.1093/plphys/kiac443pmid: 36165704

Accepted manuscripts Accepted manuscripts are PDF versions of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout. Article PDF first page preview Close This content is only available as a PDF. © American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: [email protected]

Fernández-Milmanda, Guadalupe L

doi: 10.1093/plphys/kiac454pmid: 36169164

Gibberelins (GAs) function in a myriad of plant developmental processes, such as germination, tissue elongation, and the transition from vegetative growth to reproduction. The action of GA relies on transcriptional regulation through DELLA proteins, which interact with and modify the activity of several transcription factors. In the presence of GA, the receptor GA-INSENSITIVE DWARF1 (GID1) interacts with DELLA, which allows the recognition of DELLA by the F-box protein SLEEPY1 (SLY1), part of an ubiquitin E3 ligase, targeting DELLA for destruction (Blázquez et al., 2020). Although the GA–GID1–DELLA link represents the canonical GA core signaling, some observations suggest the involvement of additional pathways within GA responses. sly1 knockout lines, which cannot degrade DELLA, display the typical phenotype of GA insensitivity, such as increased seed dormancy, dwarfism, and delayed flowering (Steber et al., 1998; McGinnis et al., 2003). Overexpression of GID1 can partially rescue sly1 phenotypes (Ariizumi et al., 2008; Ariizumi et al., 2013) without inducing DELLA degradation, suggesting that some aspects of GA signaling can still occur independently of DELLA destruction, possibly through the interaction of GID1 with other proteins (Ariizumi et al., 2013). In this issue of The Plant Physiology, Hauvermale et al. (2022) found that the PLANT UBX DOMAIN-CONTAINING PROTEIN 1 (PUX1), which has been previously implicated in growth regulation (Rancour et al., 2004), functions in GA signaling. First, the authors performed a screen for GID1 interactors, and PUX1 emerged as one of the strongest candidates. PUX1 was able to bind to the three GID1 homologs in Arabidopsis (Arabidopsis thaliana), and the interaction required the UBX domain of PUX1 and was independent of GA. UBX proteins associate with CELL DIVISION CYCLE 48 (CDC48), a segregase that unfolds and extracts polyubiquitinated proteins from membranes and complexes, acting as a major component of the ubiquitin-dependent protein degradation pathway (Bègue et al., 2019). CDC48 acts in a hexameric complex, and binding of PUX1 induces disassembly of the complex, thus modulating CDC48 functions (Rancour et al., 2004). To investigate if the ability of PUX1 to associate with CDC48 could affect GID1 functions, the authors performed a velocity sedimentation analysis with extracts of suspension-cultured cells. Velocity sedimentation measures the movement of molecules through a solution (in this case a 15%–40% glycerol gradient) under a centrifugal force. Bigger and more voluminous particles sediment faster and are assigned a higher Sverberg (S) value. After centrifugation, fractions of the solution were analyzed through immunoblot to determine the presence of the different proteins. CDC48 was found in fractions of around 17S, which corresponds with its conformation as part of a hexamer. In extracts from cells overexpressing PUX1, CDC48 also appeared in fractions related to proteins of about 5–8S, which supports a role of PUX1 in promoting the disassembly of CDC48 complexes. In the extracts from control cells, GID1 co-fractionated with PUX1 but not with hexameric CDC48, whereas in the extracts of PUX1 overexpressors, a shift in GID1 migration was detected, co-fractioning with PUX1 and CDC48 subunits at 5–8S. Altogether, these results suggest the possibility of a GID1–PUX1–CDC48 complex acting in vivo. Finally, to assess the physiological role of PUX1 in GA responses, the authors characterized the phenotype of pux1 knockout lines. In normal conditions, pux1 plants had longer stems and roots, as previously reported in Rancour et al. (2004), and flowered earlier than the wild-type (Figure 1), a phenotype consistent with increased GA signaling. Furthermore, the pux1 plants were less sensitive to the GA inhibitor, paclobutrazol (PAC), as the PAC effects on repressing germination and root elongation were less severe in the pux1 lines than in the wild-type. Altogether, these results suggest that PUX1 acts as a negative regulator of GA signaling. Figure 1 Open in new tabDownload slide pux1 knockout lines display an early flowering phenotype. A, Both pux1-1 and pux1-2 begin to bolt 20-day after germination (DAG), ∼8–12 days earlier than the Wassilewskija (Ws) wild-type. B, Comparison between Ws and the pux1 plants at 32 DAG. Adapted from Hauvermale et al. (2022). In summary, the work of Hauvermale et al. pinpoints PUX1 as a molecular player in GA signaling, possibly through its ability to interact with the GA receptor GID1 and CDC48, a key member of the protein degradation pathway. Future research may focus on the mechanism downstream of the GID1–PUX–CDC48 interaction and the possible role of PUX1 or CDC48 in regulating GID1 activity or stability. Conflict of interest statement. None declared. References Ariizumi T , Hauvermale AL, Nelson SK, Hanada A, Yamaguchi S, Steber CM ( 2013 ) Lifting DELLA repression of Arabidopsis seed germination by nonproteolytic gibberellin signaling . Plant Physiol 162 : 2125 – 2139 Google Scholar Crossref Search ADS PubMed WorldCat Ariizumi T , Murase K, Sun T, Steber CM ( 2008 ) Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1 . Plant Cell 20 : 2447 – 2459 Google Scholar Crossref Search ADS PubMed WorldCat Bègue H , Mounier A, Rosnoblet C, Wendehenne D ( 2019 ) Toward the understanding of the role of CDC48, a major component of the protein quality control, in plant immunity . Plant Sci 279 : 34 – 44 Google Scholar Crossref Search ADS PubMed WorldCat Blázquez MA , Nelson DC, Weijers D ( 2020 ) Evolution of plant hormone response pathways . Ann Rev Plant Biol 71 : 327 – 353 Google Scholar Crossref Search ADS WorldCat Hauvermale AL , Cárdenas JJ, Bednarek SY, Steber CM ( 2022 ) GA signaling expands: the plant UBX domain-containing protein 1 is a binding partner for the GA receptor . Plant Physiol , https://doi.org/10.1093/plphys/kiac406 Google Scholar OpenURL Placeholder Text WorldCat McGinnis KM , Thomas SG, Soule JD, Strader LC, Zale JM, Sun T, Steber CM ( 2003 ) The Arabidopsis SLEEPY1 gene encodes a putative F-Box subunit of an SCF E3 ubiquitin ligase[W] . Plant Cell 15 : 1120 – 1130 Google Scholar Crossref Search ADS PubMed WorldCat Rancour DM , Park S, Knight SD, Bednarek SY ( 2004 ) Plant UBX domain-containing protein 1, PUX1, regulates the oligomeric structure and activity of Arabidopsis CDC48 . J Biol Chem 279 : 54264 – 54274 Google Scholar Crossref Search ADS PubMed WorldCat Steber CM , Cooney SE, McCourt P ( 1998 ) Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1 in Arabidopsis thaliana . Genetics 149 : 509 – 521 Google Scholar Crossref Search ADS PubMed WorldCat © American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: [email protected] This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) © American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: [email protected]

Dubois, Marieke

doi: 10.1093/plphys/kiac413pmid: 36063030

Accepted manuscripts Accepted manuscripts are PDF versions of the author’s final manuscript, as accepted for publication by the journal but prior to copyediting or typesetting. They can be cited using the author(s), article title, journal title, year of online publication, and DOI. They will be replaced by the final typeset articles, which may therefore contain changes. The DOI will remain the same throughout. Article PDF first page preview Close This content is only available as a PDF. © The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.