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1. The localization of PHRAGMOPLAST ORIENTING KINESIN1 at the division site depends on the microtubule-binding proteins TANGLED1 and AUXIN-INDUCED IN ROOT CULTURES9 in Arabidopsis

   https://doi.org/10.1093/plcell/koac266  

   Mills, Alison M. ; Morris, Victoria H. ; Rasmussen, Carolyn G. ( August 2022 , The Plant Cell)

   Proper plant growth and development require spatial coordination of cell divisions. Two unrelated microtubule-binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), are together required for normal growth and division plane orientation in Arabidopsis (Arabidopsis thaliana). The tan1 air9 double mutant has synthetic growth and division plane orientation defects, while single mutants lack obvious defects. Here we show that the division site-localized protein, PHRAGMOPLAST ORIENTING KINESIN1 (POK1), was aberrantly lost from the division site during metaphase and telophase in the tan1 air9 mutant. Since TAN1 and POK1 interact via the first 132 amino acids of TAN1 (TAN11–132), we assessed the localization and function of TAN11–132 in the tan1 air9 double mutant. TAN11–132 rescued tan1 air9 mutant phenotypes and localized to the division site during telophase. However, replacing six amino-acid residues within TAN11–132, which disrupted the POK1–TAN1 interaction in the yeast-two-hybrid system, caused loss of both rescue and division site localization of TAN11–132 in the tan1 air9 mutant. Full-length TAN1 with the same alanine substitutions had defects in phragmoplast guidance and reduced TAN1 and POK1 localization at the division site but rescued most tan1 air9 mutant phenotypes. Together, these data suggest that TAN1 and AIR9 are required for POK1 localization, and yet unknown proteins may stabilize TAN1–POK1 interactions.

    

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2. A phosphorylation‐deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana , rescuing mutant defects from global translation and gene expression to photosynthesis and growth

   https://doi.org/10.1002/pld3.566  

   Dasgupta, Anwesha ; Urquidi Camacho, Ricardo A. ; Enganti, Ramya ; Cho, Sung Ki ; Tucker, Lindsey L. ; Torreverde, John S. ; Abraham, Paul E. ; von Arnim, Albrecht G. ( January 2024 , Plant Direct)

   The eukaryote‐specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data fromArabidopsis thaliana, which indicate that plants expressing only a phospho‐deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in bothrps6aandrps6bgenes when expressed at approximately twice the wild‐type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl‐terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes ofrps6that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho‐enabled version of eS6z, the phospho‐deficient seedlings retained a mild pointed‐leaf phenotype, root growth was reduced, and certain cell cycle‐related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho‐deficient seedlings could be understood as an incomplete rescue of therps6mutant defects. There was little or no evidence for gain‐of‐function defects. As previously published, the phospho‐deficient eS6z also rescued therps6aandrps6bsingle mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho‐deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

    

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3. Genetic and physical interactions between the organellar mechanosensitive ion channel homologs MSL 1, MSL 2, and MSL 3 reveal a role for inter‐organellar communication in plant development

   https://doi.org/10.1002/pld3.124  

   Lee, Josephine S. ; Wilson, Margaret E. ; Richardson, Ryan A. ; Haswell, Elizabeth S. ( March 2019 , Plant Direct)

   Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their responses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical interactions between threeMechanosensitive channel ofSmall conductance‐Like (MSL) proteins fromArabidopsis thaliana.MSLproteins areArabidopsishomologs of the bacterialMechanosensitivechannel ofSmall conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock.MSL1 localizes to the inner mitochondrial membrane, whileMSL2 andMSL3 localize to the inner plastid membrane and are required to maintain plastid osmotic homeostasis during normal growth and development. In this study, we characterized the phenotypic effect of a genetic lesion inMSL1, both in wild type and inmsl2 msl3mutant backgrounds.msl1single mutants appear wild type for all phenotypes examined. The characteristic leaf rumpling inmsl2 msl3double mutants was exacerbated in themsl1 msl2 msl3triple mutant. However, the introduction of themsl1lesion into themsl2 msl3mutant background suppressed othermsl2 msl3mutant phenotypes, including ectopic callus formation, accumulation of superoxide and hydrogen peroxide in the shoot apical meristem, decreased root length, and reduced number of lateral roots. All these phenotypes could be recovered by molecular complementation with a transgene containing a wild type version ofMSL1. In yeast‐based interaction studies,MSL1 interacted with itself, but not withMSL2 orMSL3. These results establish that the abnormalities observed inmsl2 msl3double mutants is partially dependent on the presence of functionalMSL1 and suggest a possible role for communication between plastid and mitochondria in seedling development.

    

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4. Interactions between a mechanosensitive channel and cell wall integrity signaling influence pollen germination in Arabidopsis thaliana

   https://doi.org/10.1093/jxb/erab525  

   Wang, Yanbing ; Coomey, Joshua ; Miller, Kari ; Jensen, Gregory S. ; Haswell, Elizabeth S. ; Dobritsa, ed., Anna ( November 2021 , Journal of Experimental Botany)

   Cells employ multiple systems to maintain cellular integrity, including mechanosensitive ion channels and the cell wall integrity (CWI) pathway. Here, we use pollen as a model system to ask how these different mechanisms are interconnected at the cellular level. MscS-Like 8 (MSL8) is a mechanosensitive channel required to protect Arabidopsis thaliana pollen from osmotic challenges during in vitro rehydration, germination, and tube growth. New CRISPR/Cas9 and artificial miRNA-generated msl8 alleles produced unexpected pollen phenotypes, including the ability to germinate a tube after bursting, dramatic defects in cell wall structure, and disorganized callose deposition at the germination site. We document complex genetic interactions between MSL8 and two previously established components of the CWI pathway, MARIS and ANXUR1/2. Overexpression of MARISR240C-FP suppressed the bursting, germination, and callose deposition phenotypes of msl8 mutant pollen. Null msl8 alleles suppressed the internalized callose structures observed in MARISR240C-FP lines. Similarly, MSL8-YFP overexpression suppressed bursting in the anxur1/2 mutant background, while anxur1/2 alleles reduced the strong rings of callose around ungerminated pollen grains in MSL8-YFP overexpressors. These data show that mechanosensitive ion channels modulate callose deposition in pollen and provide evidence that cell wall and membrane surveillance systems coordinate in a complex manner to maintain cell integrity.

    

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5. Molecular analyses of zebrafish V0v spinal interneurons and identification of transcriptional regulators downstream of Evx1 and Evx2 in these cells

   https://doi.org/10.1186/s13064-023-00176-w  

   England, Samantha J. ; Rusnock, Amber K. ; Mujcic, Amra ; Kowalchuk, Angelica ; de Jager, Sarah ; Hilinski, William C. ; Juárez-Morales, José L. ; Smith, Matthew E. ; Grieb, Ginny ; Banerjee, Santanu ; et al ( November 2023 , Neural Development)

   V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells.

   To identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type andevx1;evx2double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes inevx1;evx2double mutants and wild-type siblings.

   Our data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development,evx1;evx2double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes arehmx2andhmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression ofskor1aandnefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulateskor1aandnefmaexpression in V0v interneurons by repressing Hmx2/3a expression.

   This study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

    

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