E and M SARS-CoV-2 membrane protein expression and enrichment with plant lipid droplets.

Plants are gaining traction as a cost-effective and scalable platform for producing recombinant proteins. However, expressing integral membrane proteins in plants is challenging due to their hydrophobic nature. In our study, we used transient and stable expression systems in Nicotiana benthamiana and Camelina sativa respectively to express SARS-CoV-2 E and M integral proteins, and target them to lipid droplets (LDs).

ABSCISIC ACID-DEFICIENT4 Has an Essential Function in Both cis-Violaxanthin and cis-Neoxanthin Synthesis.

Abscisic acid (ABA), a plant hormone synthesized from carotenoids, functions in seed germination and abiotic stress responses. ABA is derived from the cleavage of 9-cis-isomers of violaxanthin and neoxanthin, which are oxygenated carotenoids, also called xanthophylls. Although genes encoding enzymes responsible for most steps of the ABA biosynthesis pathway have been identified, enzymatic reactions leading to the production of these cis-isomers from trans-violaxanthin remain poorly understood.

Correction to "Dynamic Contrast for Plant Phenotyping".

[This corrects the article DOI: 10.1021/acsomega.0c00957.

Dynamic Contrast for Plant Phenotyping.

Noninvasiveness, minimal handling, and immediate response are favorable features of fluorescence readout for high-throughput phenotyping of labeled plants.Yet, remote fluorescence imaging may suffer from an autofluorescent background and artificial or natural ambient light. In this work, the latter limitations are overcome by adopting reversibly photoswitchable fluorescent proteins (RSFPs) as labels and Speed OPIOM (out-of-phase imaging after optical modulation), a fluorescence imaging protocol exploiting dynamic contrast.

Synchrotron FTIR and Raman spectroscopy provide unique spectral fingerprints for Arabidopsis floral stem vascular tissues.

Cell walls are highly complex structures that are modified during plant growth and development. For example, the development of phloem and xylem vascular cells, which participate in the transport of sugars and water as well as providing support, can be influenced by cell-specific wall composition. Here, we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) and Raman spectroscopy to analyse the cell wall composition of floral stem vascular tissues of wild-type Arabidopsis and the double-mutant sweet11-1 sweet12-1, which has impaired sugar transport.

Author Correction: Resonant out-of-phase fluorescence microscopy and remote imaging overcome spectral limitations.

The Peer Review File associated with this Article was updated shortly after publication to redact from the authors' point-by-point response a description of unpublished work describing how Speed OPIOM may in future be used to facilitate discrimination between FRET and direct excitation.

Resonant out-of-phase fluorescence microscopy and remote imaging overcome spectral limitations.

We present speed out-of-phase imaging after optical modulation (OPIOM), which exploits reversible photoswitchable fluorophores as fluorescent labels and combines optimized periodic illumination with phase-sensitive detection to specifically retrieve the label signal. Speed OPIOM can extract the fluorescence emission from a targeted label in the presence of spectrally interfering fluorophores and autofluorescence. Up to four fluorescent proteins exhibiting a similar green fluorescence have been distinguished in cells either sequentially or in parallel.

Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid Camelina sativa.

In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants.

Dual Fatty Acid Elongase Complex Interactions in Arabidopsis.

Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation.

OCTOPUS Negatively Regulates BIN2 to Control Phloem Differentiation in Arabidopsis thaliana.

The phloem is a vascular strand that conducts photoassimilates and systemic signals throughout the plant to coordinate growth. To date, few molecular genetic determinants have been identified to control both specification and differentiation of this tissue [1-3]. Among them, OCTOPUS (OPS) protein was previously identified as a polarly localized plasma membrane-associated protein of unknown biochemical function whose broad provascular expression becomes restricted to the phloem upon differentiation [2].

The VASCULATURE COMPLEXITY AND CONNECTIVITY gene encodes a plant-specific protein required for embryo provasculature development.

The molecular mechanisms by which vascular tissues acquire their identities are largely unknown. Here, we report on the identification and characterization of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC), a member of a 15-member, plant-specific gene family in Arabidopsis (Arabidopsis thaliana) that encodes proteins of unknown function with four predicted transmembrane domains. Homozygous vcc mutants displayed cotyledon vein networks of reduced complexity and disconnected veins.

Inhibition of very long acyl chain sphingolipid synthesis modifies membrane dynamics during plant cytokinesis.

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure, the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the formation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosyl-ceramides with long (16 carbons) or very long (24 carbons) acyl chains.

Elevated levels of MYB30 in the phloem accelerate flowering in Arabidopsis through the regulation of FLOWERING LOCUS T.

In Arabidopsis thaliana, the R2R3 MYB-like transcription factor MYB30 is a positive regulator of the pathogen-induced hypersensitive response and of brassinosteroid and abscisic acid signaling. Here, we show that MYB30 expressed under the control of the strong phloem-specific SUC2 promoter accelerates flowering both in long and short days. Early flowering is mediated by elevated expression of flowering locus T (FT), which can be observed in the absence and presence of CONSTANS (CO), the main activator of FT.

Increased expression of a phloem membrane protein encoded by NHL26 alters phloem export and sugar partitioning in Arabidopsis.

The complex process of phloem sugar transport involves symplasmic and apoplasmic events. We characterized Arabidopsis thaliana lines ectopically expressing a phloem-specific gene encoding NDR1/HIN1-like26 (NHL26), a putative membrane protein. NHL26 overexpressor plants grew more slowly than wild-type plants, accumulated high levels of carbohydrates in mature leaves, and had a higher shoot biomass, contrasting with slower root growth and a lower seed yield.

Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria.

In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins.

Very-long-chain fatty acids are required for cell plate formation during cytokinesis in Arabidopsis thaliana.

Acyl chain length is thought to be crucial for biophysical properties of the membrane, in particular during cell division, when active vesicular fusion is necessary. In higher plants, the process of cytokinesis is unique, because the separation of the two daughter cells is carried out by de novo vesicular fusion to generate a laterally expanding cell plate. In Arabidopsis thaliana, very-long-chain fatty acid (VLCFA) depletion caused by a mutation in the microsomal elongase gene PASTICCINO2 (PAS2) or by application of the selective elongase inhibitor flufenacet altered cytokinesis.

Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis.

Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development.

The simultaneous repression of CCR and CAD, two enzymes of the lignin biosynthetic pathway, results in sterility and dwarfism in Arabidopsis thaliana.

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the last steps of monolignol biosynthesis. In Arabidopsis, one CCR gene (CCR1, At1g15950) and two CAD genes (CAD C At3g19450 and CAD D At4g34230) are involved in this pathway. A triple cad c cad d ccr1 mutant, named ccc, was obtained.

Very-long-chain fatty acids are involved in polar auxin transport and developmental patterning in Arabidopsis.

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis.

Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response.

Flowering of Arabidopsis is induced by long summer days (LDs). The transcriptional regulator CONSTANS (CO) promotes flowering, and its transcription is increased under LDs. We systematically misexpressed transcription factors in companion cells and identified several DOF proteins that delay flowering by repressing CO transcription.

A neomorphic sgs3 allele stabilizing miRNA cleavage products reveals that SGS3 acts as a homodimer.

The putative RNA-binding protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) protects RNA from degradation before transformation into dsRNA by the RNA-dependent RNA polymerase RDR6 during plant post-transcriptional gene silencing and trans-acting small interfering (siRNA) pathways. In this study, we show that SGS3 acts as a homodimer, and that the point mutation sgs3-3 impairs post-transcriptional gene silencing in a dominant-negative manner through the formation of SGS3/sgs3-3 heterodimers. Unlike complete-loss-of-function sgs3 mutants, which are impaired in the accumulation of both micro RNA-directed TAS cleavage products and mature trans-acting siRNAs, the sgs3-3 mutant overaccumulates TAS cleavage products and exhibits slightly reduced trans-acting siRNA accumulation.

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development.

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation.

Beta-subunits of the SnRK1 complexes share a common ancestral function together with expression and function specificities; physical interaction with nitrate reductase specifically occurs via AKINbeta1-subunit.

The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one alpha-type catalytic subunit and two gamma- and beta-type regulatory subunits. In yeast it has been proposed that the beta-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets.