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1.
Plant Physiol ; 193(2): 1395-1415, 2023 09 22.
Article in English | MEDLINE | ID: mdl-37335933

ABSTRACT

The selective turnover of macromolecules by autophagy provides a critical homeostatic mechanism for recycling cellular constituents and for removing superfluous and damaged organelles, membranes, and proteins. To better understand how autophagy impacts seed maturation and nutrient storage, we studied maize (Zea mays) endosperm in its early and middle developmental stages via an integrated multiomic approach using mutants impacting the core macroautophagy factor AUTOPHAGY (ATG)-12 required for autophagosome assembly. Surprisingly, the mutant endosperm in these developmental windows accumulated normal amounts of starch and Zein storage proteins. However, the tissue acquired a substantially altered metabolome, especially for compounds related to oxidative stress and sulfur metabolism, including increases in cystine, dehydroascorbate, cys-glutathione disulfide, glucarate, and galactarate, and decreases in peroxide and the antioxidant glutathione. While changes in the associated transcriptome were mild, the proteome was strongly altered in the atg12 endosperm, especially for increased levels of mitochondrial proteins without a concomitant increase in mRNA abundances. Although fewer mitochondria were seen cytologically, a heightened number appeared dysfunctional based on the accumulation of dilated cristae, consistent with attenuated mitophagy. Collectively, our results confirm that macroautophagy plays a minor role in the accumulation of starch and storage proteins during maize endosperm development but likely helps protect against oxidative stress and clears unneeded/dysfunctional mitochondria during tissue maturation.


Subject(s)
Endosperm , Zea mays , Endosperm/metabolism , Zea mays/metabolism , Starch/metabolism , Autophagy/genetics , Mitochondria/metabolism , Oxidative Stress
2.
PLoS Genet ; 17(10): e1009830, 2021 10.
Article in English | MEDLINE | ID: mdl-34695110

ABSTRACT

The post-translational addition of SUMO plays essential roles in numerous eukaryotic processes including cell division, transcription, chromatin organization, DNA repair, and stress defense through its selective conjugation to numerous targets. One prominent plant SUMO ligase is METHYL METHANESULFONATE-SENSITIVE (MMS)-21/HIGH-PLOIDY (HPY)-2/NON-SMC-ELEMENT (NSE)-2, which has been connected genetically to development and endoreduplication. Here, we describe the potential functions of MMS21 through a collection of UniformMu and CRISPR/Cas9 mutants in maize (Zea mays) that display either seed lethality or substantially compromised pollen germination and seed/vegetative development. RNA-seq analyses of leaves, embryos, and endosperm from mms21 plants revealed a substantial dysregulation of the maize transcriptome, including the ectopic expression of seed storage protein mRNAs in leaves and altered accumulation of mRNAs associated with DNA repair and chromatin dynamics. Interaction studies demonstrated that MMS21 associates in the nucleus with the NSE4 and STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC)-5 components of the chromatin organizer SMC5/6 complex, with in vitro assays confirming that MMS21 will SUMOylate SMC5. Comet assays measuring genome integrity, sensitivity to DNA-damaging agents, and protein versus mRNA abundance comparisons implicated MMS21 in chromatin stability and transcriptional controls on proteome balance. Taken together, we propose that MMS21-directed SUMOylation of the SMC5/6 complex and other targets enables proper gene expression by influencing chromatin structure.


Subject(s)
Arabidopsis Proteins/genetics , Genome, Plant/genetics , Genomic Instability/genetics , Ligases/genetics , SUMO-1 Protein/genetics , Sumoylation/genetics , Zea mays/genetics , Chromatin/genetics , Chromosomes, Plant/genetics , Proteome/genetics , Transcription, Genetic/genetics , Ubiquitin-Protein Ligases/genetics
3.
Plant Cell ; 33(4): 1093-1094, 2021 05 31.
Article in English | MEDLINE | ID: mdl-35234968
4.
Plant Cell ; 32(9): 2699-2724, 2020 09.
Article in English | MEDLINE | ID: mdl-32616663

ABSTRACT

Autophagic recycling of proteins, lipids, nucleic acids, carbohydrates, and organelles is essential for cellular homeostasis and optimal health, especially under nutrient-limiting conditions. To better understand how this turnover affects plant growth, development, and survival upon nutrient stress, we applied an integrated multiomics approach to study maize (Zea mays) autophagy mutants subjected to fixed-carbon starvation induced by darkness. Broad metabolic alterations were evident in leaves missing the core autophagy component ATG12 under normal growth conditions (e.g., lipids and secondary metabolism), while changes in amino acid-, carbohydrate-, and nucleotide-related metabolites selectively emerged during fixed-carbon starvation. Through combined proteomic and transcriptomic analyses, we identified numerous autophagy-responsive proteins, which revealed processes underpinning the various metabolic changes seen during carbon stress as well as potential autophagic cargo. Strikingly, a strong upregulation of various catabolic processes was observed in the absence of autophagy, including increases in simple carbohydrate levels with a commensurate drop in starch levels, elevated free amino acid levels with a corresponding reduction in intact protein levels, and a strong increase in the abundance of several nitrogen-rich nucleotide catabolites. Altogether, this analysis showed that fixed-carbon starvation in the absence of autophagy adjusts the choice of respiratory substrates, promotes the transition of peroxisomes to glyoxysomes, and enhances the retention of assimilated nitrogen.


Subject(s)
Amino Acids/metabolism , Autophagy/physiology , Carbon/metabolism , Zea mays/cytology , Zea mays/metabolism , Carbohydrate Metabolism/genetics , Carbohydrate Metabolism/physiology , Darkness , Gene Expression Regulation, Plant , Lipid Metabolism/genetics , Lipid Metabolism/physiology , Mutation , Plant Leaves/metabolism , Plant Proteins/genetics , Zea mays/genetics
6.
Plant Cell ; 32(8): 2446-2448, 2020 08.
Article in English | MEDLINE | ID: mdl-32554623
7.
Plant Cell ; 31(12): 2821-2822, 2019 12.
Article in English | MEDLINE | ID: mdl-31628164
8.
Plant Cell ; 31(11): 2558, 2019 11.
Article in English | MEDLINE | ID: mdl-31548255
9.
Nat Plants ; 4(12): 1056-1070, 2018 12.
Article in English | MEDLINE | ID: mdl-30478358

ABSTRACT

The turnover of cytoplasmic material by autophagic encapsulation and delivery to vacuoles is essential for recycling cellular constituents, especially under nutrient-limiting conditions. To determine how cells/tissues rely on autophagy, we applied in-depth multi-omic analyses to study maize (Zea mays) autophagy mutants grown under nitrogen-replete and -starvation conditions. Broad alterations in the leaf metabolome were evident in plants missing the core autophagy component ATG12, even in the absence of stress, particularly affecting products of lipid turnover and secondary metabolites, which were underpinned by substantial changes in the transcriptome and/or proteome. Cross-comparison of messenger RNA and protein abundances allowed for the identification of organelles, protein complexes and individual proteins targeted for selective autophagic clearance, and revealed several processes controlled by this catabolism. Collectively, we describe a facile multi-omic strategy to survey autophagic substrates, and show that autophagy has a remarkable influence in sculpting eukaryotic proteomes and membranes both before and during nutrient stress.


Subject(s)
Autophagy-Related Protein 12/metabolism , Autophagy , Metabolome , Nitrogen/deficiency , Proteome , Transcriptome , Zea mays/metabolism , Autophagy-Related Protein 12/genetics , Lipid Metabolism , Mutation , Plant Proteins/genetics , Plant Proteins/metabolism , Secondary Metabolism , Stress, Physiological , Vacuoles/metabolism , Zea mays/genetics
10.
Curr Opin Plant Biol ; 45(Pt A): 143-154, 2018 10.
Article in English | MEDLINE | ID: mdl-30014889

ABSTRACT

Conjugation of small ubiquitin-related modifier (SUMO) to intracellular proteins provides a dynamic regulatory mechanism that enables plants to rapidly defend against environmental challenges. SUMOylation of mostly nuclear proteins is among the fastest stress responses observed but precisely how this post-translational modification provides stress resilience remains unclear. Here, we describe the plant SUMO system and its expanding target catalog, which implicates this modification in DNA repair, chromatin modification/remodeling, transcriptional activation/repression, epigenetics, and RNA metabolism, with a likely outcome being extensive nuclear re-wiring to withstand stress. In parallel, studies have linked SUMO to developmental programs such as gametogenesis and gene silencing. The accumulating data support the notion that SUMOylation substantially influences the transcriptional and epigenetic landscapes to promote stress tolerance and developmental progression.


Subject(s)
Cell Nucleus/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Nucleus/genetics , DNA Repair/genetics , DNA Repair/physiology , Protein Processing, Post-Translational , Sumoylation/genetics , Sumoylation/physiology
13.
Plant Cell ; 30(5): 1077-1099, 2018 05.
Article in English | MEDLINE | ID: mdl-29588388

ABSTRACT

The posttranslational addition of small ubiquitin-like modifier (SUMO) is an essential protein modification in plants that provides protection against numerous environmental challenges. Ligation is accomplished by a small set of SUMO ligases, with the SAP-MIZ domain-containing SIZ1 and METHYL METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in Arabidopsis thaliana, we used siz1 and mms21 mutants for proteomic analyses of SUMOylated proteins enriched via an engineered SUMO1 isoform suitable for mass spectrometric studies. Through multiple data sets from seedlings grown at normal temperatures or exposed to heat stress, we identified over 1000 SUMO targets, most of which are nuclear localized. Whereas no targets could be assigned to MMS21, suggesting that it modifies only a few low abundance proteins, numerous targets could be assigned to SIZ1, including major transcription factors, coactivators/repressors, and chromatin modifiers connected to abiotic and biotic stress defense, some of which associate into multisubunit regulatory complexes. SIZ1 itself is also a target, but studies with mutants protected from SUMOylation failed to uncover a regulatory role. The catalog of SIZ1 substrates indicates that SUMOylation by this ligase provides stress protection by modifying a large array of key nuclear regulators.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Heat-Shock Response/genetics , Heat-Shock Response/physiology , Proteomics/methods , Seedlings/genetics , Seedlings/metabolism , Sumoylation/genetics , Sumoylation/physiology , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
14.
Plant Physiol ; 171(3): 2191-210, 2016 07.
Article in English | MEDLINE | ID: mdl-27208252

ABSTRACT

In response to abiotic and biotic challenges, plants rapidly attach small ubiquitin-related modifier (SUMO) to a large collection of nuclear proteins, with studies in Arabidopsis (Arabidopsis thaliana) linking SUMOylation to stress tolerance via its modification of factors involved in chromatin and RNA dynamics. Despite this importance, little is known about SUMOylation in crop species. Here, we describe the plant SUMO system at the phylogenetic, biochemical, and transcriptional levels with a focus on maize (Zea mays). In addition to canonical SUMOs, land plants encode a loosely constrained noncanonical isoform and a variant containing a long extension upstream of the signature ß-grasp fold, with cereals also expressing a novel diSUMO polypeptide bearing two SUMO ß-grasp domains in tandem. Maize and other cereals also synthesize a unique SUMO-conjugating enzyme variant with more restricted expression patterns that is enzymatically active despite a distinct electrostatic surface. Maize SUMOylation primarily impacts nuclear substrates, is strongly induced by high temperatures, and displays a memory that suppresses subsequent conjugation. Both in-depth transcript and conjugate profiles in various maize organs point to tissue/cell-specific functions for SUMOylation, with potentially significant roles during embryo and endosperm maturation. Collectively, these studies define the organization of the maize SUMO system and imply important functions during seed development and stress defense.


Subject(s)
Endosperm/growth & development , Plant Proteins/metabolism , Sumoylation , Zea mays/metabolism , Endosperm/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Plant , Oxidative Stress , Peptide Hydrolases/metabolism , Phylogeny , Plant Proteins/genetics , Stress, Physiological , Up-Regulation , Zea mays/genetics , Zea mays/growth & development
15.
Plant Cell ; 23(10): 3696-710, 2011 Oct.
Article in English | MEDLINE | ID: mdl-22003077

ABSTRACT

The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics.


Subject(s)
Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors/metabolism , Actins/metabolism , Bryopsida/metabolism , Microfilament Proteins/metabolism , Actin Cytoskeleton/ultrastructure , Actin Depolymerizing Factors/genetics , Base Sequence , Bryopsida/genetics , Bryopsida/growth & development , Bryopsida/ultrastructure , Cytosol/metabolism , DNA, Plant/chemistry , DNA, Plant/genetics , Gene Knockout Techniques , Genetic Complementation Test , Microfilament Proteins/genetics , Molecular Sequence Data , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/metabolism , Plant Roots/ultrastructure , Plants, Genetically Modified , RNA, Plant/genetics , Reverse Genetics , Sequence Analysis, DNA , Time Factors
16.
Plant Cell ; 22(6): 1868-82, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20525854

ABSTRACT

Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5' untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells.


Subject(s)
5' Untranslated Regions , Bryopsida/genetics , Myosins/metabolism , Plant Proteins/metabolism , Plant Shoots/growth & development , Actins/metabolism , Bryopsida/growth & development , DNA, Complementary/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genes, Plant , Genetic Complementation Test , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Myosins/genetics , Plant Proteins/genetics , Plant Shoots/genetics , RNA Interference , RNA, Plant/genetics
17.
Plant Physiol ; 151(2): 506-14, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19666707

ABSTRACT

We developed a simple and fast method to identify temperature-sensitive alleles of essential plant genes. We used primary and tertiary structure information to identify residues in the core of the protein of interest. These residues were mutated and tested for temperature sensitivity, taking advantage of the exceptionally rapid 1-week complementation assay in the moss Physcomitrella patens. As test molecules, we selected the actin-binding proteins profilin and actin-depolymerizing factor, because they are essential and their loss-of-function phenotype can be fully rescued. Screening a small number of candidate mutants, we successfully identified temperature-sensitive alleles of both profilin and actin-depolymerizing factor. Plants harboring these alleles grew well at the permissive temperature of 20 degrees C to 25 degrees C but showed a complete loss of function at the restrictive temperature of 32 degrees C. Notably, the profilin mutation identified in the moss gene can be transferred to profilins from other plant species, also rendering them temperature sensitive. The ability to routinely generate temperature-sensitive alleles of essential plant proteins provides a powerful tool for the study of gene function in plants.


Subject(s)
Alleles , Genetic Techniques , Plants/genetics , Temperature , Amino Acid Sequence , Arabidopsis/genetics , Bryopsida/genetics , Lilium/genetics , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation/genetics , Plant Proteins/chemistry , Plant Proteins/genetics , Profilins/genetics , Protein Structure, Secondary , Saccharomyces cerevisiae/genetics
18.
Proc Natl Acad Sci U S A ; 106(32): 13341-6, 2009 Aug 11.
Article in English | MEDLINE | ID: mdl-19633191

ABSTRACT

Formins are present in all eukaryotes and are essential for the creation of actin-based structures responsible for diverse cellular processes. Because multicellular organisms contain large formin gene families, establishing the physiological functions of formin isoforms has been difficult. Using RNAi, we analyzed the function of all 9 formin genes within the moss Physcomitrella patens. We show that plants lacking class II formins (For2) are severely stunted and composed of spherical cells with disrupted actin organization. In contrast, silencing of all other formins results in normal elongated cell morphology and actin organization. Consistent with a role in polarized growth, For2 are apically localized in growing cells. We show that an N-terminal phosphatase tensin (PTEN)-like domain mediates apical localization. The PTEN-like domain is followed by a conserved formin homology (FH)1-FH2 domain, known to promote actin polymerization. To determine whether apical localization of any FH1-FH2 domain mediates polarized growth, we performed domain swapping. We found that only the class II FH1-FH2, in combination with the PTEN-like domain, rescues polarized growth, because it cannot be replaced with a similar domain from a For1. We used in vitro polymerization assays to dissect the functional differences between these FH1-FH2 domains. We found that both the FH1 and the FH2 domains from For2 are required to mediate exceptionally rapid rates of actin filament elongation, much faster than any other known formin. Thus, our data demonstrate that rapid rates of actin elongation are critical for driving the formation of apical filamentous actin necessary for polarized growth.


Subject(s)
Actin Cytoskeleton/metabolism , Bryopsida/cytology , Bryopsida/growth & development , Nerve Tissue Proteins/metabolism , Actins/metabolism , Bryopsida/anatomy & histology , Cell Polarity , Fetal Proteins/chemistry , Fetal Proteins/metabolism , Formins , Gene Silencing , Genetic Complementation Test , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , PTEN Phosphohydrolase/chemistry , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Tertiary , Protein Transport
19.
Plant J ; 54(5): 863-75, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18298672

ABSTRACT

Actin depolymerizing factor (ADF)/cofilin is important for regulating actin dynamics, and in plants is thought to be required for tip growth. However, the degree to which ADF is necessary has been elusive because of the presence of multiple ADF isoforms in many plant species. In the moss Physcomitrella patens, ADF is encoded by a single, intronless gene. We used RNA interference to demonstrate that ADF is essential for plant viability. Loss of ADF dramatically alters the organization of the F-actin cytoskeleton, and leads to an inhibition of tip growth. We show that ADF is subject to phosphorylation in vivo, and using complementation studies we show that mutations of the predicted phosphorylation site partially rescue plant viability, but with differential affects on tip growth. Specifically, the unphosphorylatable ADF S6A mutant generates small polarized plants with normal F-actin organization, whereas the phosphomimetic S6D mutant generates small, unpolarized plants with a disorganized F-actin cytoskeleton. These data indicate that phosphoregulation at serine 6 is required for full ADF function in vivo, and, in particular, that the interaction between ADF and actin is important for tip growth.


Subject(s)
Bryophyta/metabolism , Destrin/metabolism , Plant Roots/growth & development , Bryophyta/genetics , Bryophyta/growth & development , Destrin/genetics , Genes, Plant , Phosphorylation
20.
Plant Cell ; 19(11): 3705-22, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17981997

ABSTRACT

The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 3' untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-l-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-l-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-l-proline ligands are required to properly organize F-actin.


Subject(s)
Bryopsida/growth & development , Profilins/metabolism , Actins/metabolism , Amino Acid Sequence , Binding Sites , Bryopsida/cytology , Bryopsida/genetics , Bryopsida/ultrastructure , Cell Nucleus/metabolism , Cell Proliferation , Fluorescent Antibody Technique , Gene Expression Regulation, Plant , Genes, Plant , Genetic Complementation Test , Molecular Sequence Data , Mutation/genetics , Peptides/metabolism , Phenotype , Profilins/chemistry , Profilins/genetics , RNA Interference , Sequence Homology, Amino Acid
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