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The New Phytologist Aug 2024Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events...
Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed processing events in vivo. This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana.
Topics: Nicotiana; Proteome; Plant Proteins; Proteolysis; Proteomics; Carboxylic Ester Hydrolases; Lipase; Peptide Hydrolases; Glycoside Hydrolases
PubMed: 38853453
DOI: 10.1111/nph.19891 -
The New Phytologist Jun 2024Modern cultivated rice (Oryza sativa) typically experiences limited growth benefits from arbuscular mycorrhizal (AM) symbiosis. This could be due to the long-term...
Modern cultivated rice (Oryza sativa) typically experiences limited growth benefits from arbuscular mycorrhizal (AM) symbiosis. This could be due to the long-term domestication of rice under favorable phosphorus conditions. However, there is limited understanding of whether and how the rice domestication has modified AM properties. This study compared AM properties between a collection of wild (Oryza rufipogon) and domesticated rice genotypes and investigated the mechanisms underlying their differences by analyzing physiological, genomic, transcriptomic, and metabolomic traits critical for AM symbiosis. The results revealed significantly lower mycorrhizal growth responses and colonization intensity in domesticated rice compared to wild rice, and this change of AM properties may be associated with the domestication modifications of plant phosphorus utilization efficiency at physiological and genomic levels. Domestication also resulted in a decrease in the activity of the mycorrhizal phosphorus acquisition pathway, which may be attributed to reduced mycorrhizal compatibility of rice roots by enhancing defense responses like root lignification and reducing carbon supply to AM fungi. In conclusion, rice domestication may have changed its AM properties by modifying P nutrition-related traits and reducing symbiotic compatibility. This study offers new insights for improving AM properties in future rice breeding programs to enhance sustainable agricultural production.
PubMed: 38853449
DOI: 10.1111/nph.19901 -
The New Phytologist Aug 2024IRE1, BI-1, and bZIP60 monitor compatible plant-potexvirus interactions though recognition of the viral TGB3 protein. This study was undertaken to elucidate the roles of...
IRE1, BI-1, and bZIP60 monitor compatible plant-potexvirus interactions though recognition of the viral TGB3 protein. This study was undertaken to elucidate the roles of three IRE1 isoforms, the bZIP60U and bZIP60S, and BI-1 roles in genetic reprogramming of cells during potexvirus infection. Experiments were performed using Arabidopsis thaliana knockout lines and Plantago asiatica mosaic virus infectious clone tagged with the green fluorescent protein gene (PlAMV-GFP). There were more PlAMV-GFP infection foci in ire1a/b, ire1c, bzip60, and bi-1 knockout than wild-type (WT) plants. Cell-to-cell movement and systemic RNA levels were greater bzip60 and bi-1 than in WT plants. Overall, these data indicate an increased susceptibility to virus infection. Transgenic overexpression of AtIRE1b or StbZIP60 in ire1a/b or bzip60 mutant background reduced virus infection foci, while StbZIP60 expression influences virus movement. Transgenic overexpression of StbZIP60 also confers endoplasmic reticulum (ER) stress resistance following tunicamycin treatment. We also show bZIP60U and TGB3 interact at the ER. This is the first demonstration of a potato bZIP transcription factor complementing genetic defects in Arabidopsis. Evidence indicates that the three IRE1 isoforms regulate the initial stages of virus replication and gene expression, while bZIP60 and BI-1 contribute separately to virus cell-to-cell and systemic movement.
Topics: Arabidopsis; Basic-Leucine Zipper Transcription Factors; Plant Diseases; Arabidopsis Proteins; Potexvirus; Plants, Genetically Modified; Gene Expression Regulation, Plant; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Mutation; Tunicamycin; Membrane Proteins; Protein Kinases
PubMed: 38853429
DOI: 10.1111/nph.19882 -
The New Phytologist Aug 2024The 'assimilates inhibition hypothesis' posits that accumulation of nonstructural carbohydrates (NSCs) in leaves reduces leaf net photosynthetic rate, thus internally...
The 'assimilates inhibition hypothesis' posits that accumulation of nonstructural carbohydrates (NSCs) in leaves reduces leaf net photosynthetic rate, thus internally regulating photosynthesis. Experimental work provides equivocal support mostly under controlled conditions without identifying a particular NSC as involved in the regulation. We combined 3-yr in situ leaf gas exchange observations (natural dynamics) in the upper crown of mature Betula pendula simultaneously with measurements of concentrations of sucrose, hexoses (glucose and fructose), and starch, and similar measurements during several one-day shoot girdling (perturbation dynamics). Leaf water potential and water and nitrogen content were measured to account for their possible contribution to photosynthesis regulation. Leaf photosynthetic capacity (A/C) was temporally negatively correlated with NSC accumulation under both natural and perturbation states. For developed leaves, leaf hexose concentration explained A/C variation better than environmental variables (temperature history and daylength); the opposite was observed for developing leaves. The weaker correlations between NSCs and A/C in developing leaves may reflect their strong internal sink strength for carbohydrates. By contrast, the strong decline in photosynthetic capacity with NSCs accumulation in mature leaves, observed most clearly with hexose, and even more tightly with its constituents, provides support for the role of assimilates in regulating photosynthesis under natural conditions.
Topics: Photosynthesis; Plant Leaves; Betula; Seasons; Hexoses; Carbon Sequestration; Water; Nitrogen; Carbon; Starch
PubMed: 38853424
DOI: 10.1111/nph.19883 -
The New Phytologist Aug 2024Shifts in pollinator occurrence and their pollen transport effectiveness drive the evolution of mating systems in flowering plants. Understanding the genomic basis of...
Shifts in pollinator occurrence and their pollen transport effectiveness drive the evolution of mating systems in flowering plants. Understanding the genomic basis of these changes is essential for predicting the persistence of a species under environmental changes. We investigated the genomic changes in Brassica rapa over nine generations of pollination by hoverflies associated with rapid morphological evolution toward the selfing syndrome. We combined a genotyping-by-sequencing (GBS) approach with a genome-wide association study (GWAS) to identify candidate genes, and assessed their functional role in the observed morphological changes by studying mutations of orthologous genes in the model plant Arabidopsis thaliana. We found 31 candidate genes involved in a wide range of functions from DNA/RNA binding to transport. Our functional assessment of orthologous genes in A. thaliana revealed that two of the identified genes in B. rapa are involved in regulating the size of floral organs. We found a protein kinase superfamily protein involved in petal width, an important trait in plant attractiveness to pollinators. Moreover, we found a histone lysine methyltransferase (HKMT) associated with stamen length. Altogether, our study shows that hoverfly pollination leads to rapid evolution toward the selfing syndrome mediated by polygenic changes.
Topics: Pollination; Brassica rapa; Animals; Genes, Plant; Biological Evolution; Genome-Wide Association Study; Self-Fertilization; Flowers; Reproduction; Arabidopsis; Mutation; Diptera; Phenotype; Pollen
PubMed: 38853408
DOI: 10.1111/nph.19880 -
Life Sciences Aug 2024Long-term oral anticoagulation is the primary therapy for preventing ischemic stroke in patients with atrial fibrillation (AF). Different types of oral anticoagulant...
AIMS
Long-term oral anticoagulation is the primary therapy for preventing ischemic stroke in patients with atrial fibrillation (AF). Different types of oral anticoagulant drugs can have specific effects on the metabolism of patients. Here we characterize, for the first time, the serum metabolomic and lipoproteomic profiles of AF patients treated with anticoagulants: vitamin K antagonists (VKAs) or direct oral anticoagulants (DOACs).
MATERIALS AND METHODS
Serum samples of 167 AF patients (median age 78 years, 62 % males, 70 % on DOACs treatment) were analyzed via high resolution H nuclear magnetic resonance (NMR) spectroscopy. Data on 25 metabolites and 112 lipoprotein-related fractions were quantified and analyzed with multivariate and univariate statistical approaches.
KEY FINDINGS
Our data provide evidence that patients treated with VKAs and DOACs present significant differences in their profiles: lower levels of alanine and lactate (odds ratio: 1.72 and 1.84), free cholesterol VLDL-4 subfraction (OR: 1.75), triglycerides LDL-1 subfraction (OR: 1.80) and 4 IDL cholesterol fractions (ORs ∼ 1.80), as well as higher levels of HDL cholesterol (OR: 0.48), apolipoprotein A1 (OR: 0.42) and 7 HDL cholesterol fractions/subfractions (ORs: 0.40-0.51) are characteristic of serum profile of patients on DOACs' therapy.
SIGNIFICANCE
Our results support the usefulness of NMR-based metabolomics for the description of the effects of oral anticoagulants on AF patient circulating metabolites and lipoproteins. The higher serum levels of HDL cholesterol observed in patients on DOACs could contribute to explaining their reduced cardiovascular risk, suggesting the need of further studies in this direction to fully understand possible clinical implications.
Topics: Humans; Atrial Fibrillation; Male; Female; Aged; Vitamin K; Anticoagulants; Administration, Oral; Aged, 80 and over; Metabolomics; Metabolome; Middle Aged; Magnetic Resonance Spectroscopy
PubMed: 38852797
DOI: 10.1016/j.lfs.2024.122796 -
The New Phytologist Aug 2024Agroinfiltration of Nicotiana benthamiana is routinely used in plant science and molecular pharming to transiently express proteins of interest. Here, we discuss four... (Review)
Review
Agroinfiltration of Nicotiana benthamiana is routinely used in plant science and molecular pharming to transiently express proteins of interest. Here, we discuss four phenomena that should be avoided to improve transient expression. Immune responses can be avoided by depleting immune receptors and employing pathogen-derived effectors; transcript degradation by using silencing inhibitors or RNA interference machinery mutants; endoplasmic reticulum stress by co-expressing chaperones; and protein degradation can be avoided with subcellular targeting, protease mutants and co-expressing protease inhibitors. We summarise the reported increased yields for various recombinant proteins achieved with these approaches and highlight remaining challenges to further improve the efficiency of this versatile protein expression platform.
Topics: Nicotiana; Recombinant Proteins; Plant Proteins; Proteolysis; Gene Expression Regulation, Plant; Endoplasmic Reticulum Stress
PubMed: 38849321
DOI: 10.1111/nph.19894 -
The New Phytologist Jun 2024Flowering is a vital agronomic trait that determines the economic value of most ornamental plants. The flowering time of rose (Rosa spp.) is photoperiod insensitive and...
Flowering is a vital agronomic trait that determines the economic value of most ornamental plants. The flowering time of rose (Rosa spp.) is photoperiod insensitive and is thought to be tightly controlled by light intensity, although the detailed molecular mechanism remains unclear. Here, we showed that rose plants flower later under low-light (LL) intensity than under high-light (HL) intensity, which is mainly related to the stability of PHYTOCHROME-INTERACTING FACTORs (RcPIFs) mediated by OPEN STOMATA 1-Like (RcOST1L) under different light intensity regimes. We determined that HL conditions trigger the rapid phosphorylation of RcPIFs before their degradation. A yeast two-hybrid screen identified the kinase RcOST1L as interacting with RcPIF4. Moreover, RcOST1L positively regulated rose flowering and directly phosphorylated RcPIF4 on serine 198 to promote its degradation under HL conditions. Additionally, phytochrome B (RcphyB) enhanced RcOST1L-mediated phosphorylation of RcPIF4 via interacting with the active phyB-binding motif. RcphyB was activated upon HL and recruited RcOST1L to facilitate its nuclear accumulation, in turn leading to decreased stability of RcPIF4 and flowering acceleration. Our findings illustrate how RcPIF abundance safeguards proper rose flowering under different light intensities, thus uncovering the essential role of RcOST1L in the RcphyB-RcPIF4 module in flowering.
PubMed: 38849320
DOI: 10.1111/nph.19885 -
The New Phytologist Aug 2024Jasmonic acid (JA) and gibberellin (GA) coordinately regulate plant developmental programs and environmental cue responses. However, the fine regulatory network of the...
The MdNAC72-MdABI5 module acts as an interface integrating jasmonic acid and gibberellin signals and undergoes ubiquitination-dependent degradation regulated by MdSINA2 in apple.
Jasmonic acid (JA) and gibberellin (GA) coordinately regulate plant developmental programs and environmental cue responses. However, the fine regulatory network of the cross-interaction between JA and GA remains largely elusive. In this study, we demonstrate that MdNAC72 together with MdABI5 positively regulates anthocyanin biosynthesis through an exquisite MdNAC72-MdABI5-MdbHLH3 transcriptional cascade in apple. MdNAC72 interacts with MdABI5 to promote the transcriptional activation of MdABI5 on its target gene MdbHLH3 and directly activates the transcription of MdABI5. The MdNAC72-MdABI5 module regulates the integration of JA and GA signals in anthocyanin biosynthesis by combining with JA repressor MdJAZ2 and GA repressor MdRGL2a. MdJAZ2 disrupts the MdNAC72-MdABI5 interaction and attenuates the transcriptional activation of MdABI5 by MdNAC72. MdRGL2a sequesters MdJAZ2 from the MdJAZ2-MdNAC72 protein complex, leading to the release of MdNAC72. The E3 ubiquitin ligase MdSINA2 is responsive to JA and GA signals and promotes ubiquitination-dependent degradation of MdNAC72. The MdNAC72-MdABI5 interface fine-regulates the integration of JA and GA signals at the transcriptional and posttranslational levels by combining MdJAZ2, MdRGL2a, and MdSINA2. In summary, our findings elucidate the fine regulatory network connecting JA and GA signals with MdNAC72-MdABI5 as the core in apple.
Topics: Oxylipins; Malus; Cyclopentanes; Ubiquitination; Gene Expression Regulation, Plant; Plant Proteins; Signal Transduction; Gibberellins; Proteolysis; Anthocyanins; Protein Binding; Ubiquitin-Protein Ligases; Models, Biological
PubMed: 38849319
DOI: 10.1111/nph.19888 -
The New Phytologist Aug 2024The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that...
The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1-aminocyclopropane-1-carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl-ACC (MACC), glutamyl-ACC (GACC), and jasmonyl-ACC (JA-ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild-type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA-ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene-independent role of ACC.
Topics: Arabidopsis; Ethylenes; Tandem Mass Spectrometry; Chromatography, High Pressure Liquid; Amino Acids, Cyclic; Biosynthetic Pathways; Stress, Physiological; Reproducibility of Results; Mutation; Liquid Chromatography-Mass Spectrometry
PubMed: 38849316
DOI: 10.1111/nph.19878