Patent Application
20230416772
Plant seeds are specialized propagation vectors that can mature to a quiescent desiccated state, allowing them to remain viable in harsh conditions anywhere from a few years to millennia (1, 2). Water is essential for life but plant embryos can survive extreme desiccation by accumulating protective molecules and profoundly changing their cellular biophysical properties (3, 4). Upon the uptake of water, called imbibition, seeds rapidly undergo a cascade of biochemical events and the resumption of cellular activities (5). Seeds can endure multiple hydration-dehydration cycles while remaining viable and desiccation tolerant (6). But once committed to germination, they are no longer able to revert to their stress tolerant state (5). Thus, poor timing of germination can severely limit the chances of seedling survival (7), especially in times of drought. Despite the fundamental importance of germination control for plant biology and agriculture, the molecular underpinnings controlling this decision remain incompletely understood.
We identified an uncharacterized Arabidopsis prion-like protein. FLOE1, that phase separates upon hydration and allows the embryo to sense water stress. We demonstrated that the emergent properties of FLOE1 condensates are intimately linked to its biological function in vivo, where it functions as a negative regulator of seed germination in unfavorable environmental conditions. These findings provide evidence of a functional role of phase separation in a multicellular organism and have direct implications for plant ecology and agriculture, especially for generating drought resistant crops, in the face of climate change. Additionally provided herein are methods of modulating seed germination by modulating FLOE1 expression.
“Modulating” seed germination as used herein refers to modulating the percentage of FLOE1-modified seeds that germinate in a given time frame compared to control wildtype seeds maintained under the same conditions, e.g., drought. Similarly, “modulating” seed viability (“viability” may also be referred to herein as “longevity”) refers to modulating the percentage of FLOE-1 modified seeds that are viable after a period of time, e.g., 1, 2, 3, 4, or 5, or more years, compared to control wildtype seeds maintained under the same conditions. Viability and germination can he assessed using routine methods. In some embodiments, germination and viability are assessed using methodology as shown in the examples.
Modifications to FLOE1 that influence germination rates include modulating the levels of expression of wildtype and mutant FLOE1. For example, decreasing the level of endogenous FLOE1 results in increases in germination rates under certain environmental conditions, such as drought, whereas increasing the level of expression of a wildtype FLOE1 decreases germination rate under certain environmental conditions, such as drought. In some embodiments, seeds having decreased endogenous FLOE1 expression will germinate faster, compared to control, under normal growth conditions. In some embodiments, seeds having increased levels of a wildtype FLOE1 remain viable longer compared to control, wildtype seeds.
An illustrative FLOE 1 sequence is provided below:
Arabidopsis thaliana FLOE1 (including the starting methionine):
Domains include:
The DS-Rich Domain (DS Domain (Shown without the Start methionine)):
Domains were defined based on their disorder scores or previous annotations. There are three structured regions: the nucleation domain, coiled-coil and DUF1421. The other two regions are highly disordered and were named based on their amino acid profiles: the DS-rich domain is enriched in D and S amino acids and the QPS-rich is rich in Q, P and S amino acids. Domains of a native FLOE1 polypeptide of a plant can be identified as described herein. Illustrative domain sequences of FLOE1 homologs are shown in
In some embodiments, germination rates are modulated by mutating FLOE1, e.g., as described herein. In some embodiments, seeds are modified to remove all or a substantial portion of (e.g., removal of at least 60%, 70%, 80%, 90% or greater), of the QPS or DS domain, resulting in faster germination of seeds, e.g., under stress conditions such as drought.
In some embodiments, the levels of natural splice variants may be modified to modulate seed germination. For example, in some plants, a splice variant in which the DS domain is partly truncated can be up-regulated to enhance seed germination rates.
In some embodiments, seed gemination is modulated by introducing amino acid substitutions in FLOE1. For example, QPS has regularly spaced aromatic tyrosine residues along its sequence. In sonic embodiments, tyrosine residues in the QPS domain may be substituted with serine residues in multiple positions (see, e.g.,
Plants may be modified to introduce mutations and/or to increase or decrease FLOE1 expression using various techniques, including gene editing techniques. Exemplary genome editing proteins include targeted nucleases such as engineered zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs), and engineered meganucleases. In addition, systems which rely on an engineered guide RNA (a gRNA) to guide an endonuclease to a target cleavage site can be used. The most commonly used of these systems is the CRISPR/Cas system with an engineered guide RNA to guide the Cas-9 endonuclease to the target cleavage site. Alternatively, gene expression may be modified using interfering RNA, antisense or other methodology to reduce expression; or by overexpressing a gene to enhance expression.
Illustrative mutant FLOE1 sequences are provided below:
In some embodiments, homologs are defined based on whether they contain an annotated DUF1421 domain. FLOE1 homologs can also exhibit conserved variation in their disordered domains. Illustrative homolog sequences are provided below:
Arabidopsis thaliana FLOE1 (FIG. 4D)
Dunaliella salina FLOE2L (FIG. 12B)
Selaginella moellendorffii (Smo-FLOE2L)
Wollemia nobilis Wno-FLOE1L (FIG. 4H)
Theobroma cacao Tca-FLOE1L (FIG. 4H)
Marchantia polymorpha (Mpo-FLOE2L)
Chlamydomonas reinhardtii (Cre-FLOE2L)
Klebsormidium nitens (Kni-FLOE2L) (FIG. 4H)
Bathycoccus prasinos (Bpr-FLOE2L) (FIG. 4H)
Solanum lycopersicum 2 Sly-FLOE2L (FIG. 4H)
Coffea canephora FLOE1L (FIG. 12B)
Arabidopsis thaliana FLOE2 (FIG. 4D)
Arabidopsis thaliana FLOE3 (FIG. 4D)
Physcomitrella patens (Ppa-FLOE2L) (FIG. 4H)
Solanum tuberosum (Stu-FLOE1L) (FIG. 4H)
Solanum lycopersicum (Sly-FLOE1L) (FIG. 4H)
Sphagnum fallax FLOE2L (FIG. 12B)
Theobroma cacao 2 (Tca-FLOE2L) (FIG. 4H)
Ostreococcus tauri (Ota-FLOE2L) (FIG. 4H)
Wollemia nobilis 2 (Wno-FLOE2L) (FIG. 4H)
Because limited water availability dramatically alters protein solubility and plant seeds are known to undergo a cytoplasmic liquid-to-glass transition during maturation (3, 4), we investigated how plant seed proteins might have adapted to these extreme conditions (
FLOE1 accumulates during embryo development and its expression peaks in the mature desiccated state (
Numerous yeast proteins undergo oligomerization or phase separation upon stress-induced quiescence (15) but to our knowledge FLOE1 is the first example of a protein undergoing biotnolecular condensation upon release from the quiescent state. To define the mechanism by which FLOE1 undergoes this switch, we dissected the molecular grammar underlying this behavior. FLOE1 harbors a predicted short coiled-coil domain and a conserved plant-specific domain of unknown function (DUF1421) (
In line with their role in driving phase separation of other prion-like proteins, deletion of the QPS PrLD reduced condensate formation (
We next asked whether these various physical states of FLOE1 have a role in germination. Lines carrying the knockout allele floe1-1 did not show any obvious developmental defects, and floe1-1 seeds had the same size and weight as the wildtype (
If FLOE1 acts as a molecular tuning knob, we predict there should be natural variation in its phase separation behavior. FLOE1 has an annotated shorter splice isoform that lacks the majority of the DS domain (
Phase separation is emerging as a universal mechanism to explain how cells compartmentalize biomolecules. Recent work in yeast suggests that phase separation of prion-like and related proteins is important for their function (22, 23), but this picture is less clear for multicellular organisms, especially since aggregation of these proteins is implicated in human disease (24). There is evidence suggesting the functionality of prion-like condensates in plants (25-27) and flies (28), but strong in vivo evidence for a functional role of the emergent properties of phase separation remains lacking. While conformational switches between liquid and solid-like states of yeast prions can drive functional phenotypic variability via bet-hedging strategies (13, 23), we provide evidence that the same is true for a multicellular organism. Plant seed germination follows a bet-hedging strategy by spreading the risk of potential deleterious conditions (e.g., drought) across different phenotypes in a population (19-21). Our data show that altering both FLOE1 expression levels and its material properties can tune these strategies in different environments. While the exact molecular mode of action of this newly discovered protein is still unclear, RNAseq analysis suggests that its function is upstream of key germination pathways in a stress-dependent manner (
All references, including publications, accession numbers, patent applications, and patents, cited in the disclosure are hereby incorporated by reference for the purpose for which it is cited to the same extent as if each reference were individually and specifically indicated to be incorporated by reference.
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Arabidopsis thaliana genes were scored via the Expression Angler tool based on similarity to a “Developmental Map” expression pattern with “High Relative Expression” in “Dry Seed” and “Low Relative Expression” for all other tissues (http address bar.utoronto.ca/ExpressionAngler/) (I). The output were then normalized to Z-scores (data not shown) and genes were considered as seed-specifie if they had a Z score of 3 or higher. The MobiDB-lite disorder scores of each gene in the “Z>3” and “Z<3” groups were retrieved from the MobiDB (version 3.1) A. thailana dataset (http address mobidb.bionnipd.itldataset) (2), and their amino acid profiles were obtained using the protr package (3) in R. Genes in the “Z>3” group were then checked for the presence of a predicted prion-like domain (4). For FLOE1 disorder prediction we used PONDR VSL2 (web address pondr.com) (5) and for identifying its prion-like domain we used PLAAC (web address.wi.mit.edu/) (6).
Arabidopsis thaliana plants from which seeds were harvested for the experimental assays were grown in soil (PRO-MIX® HP Mycorrhizae) inside growth cabinets (Percival) held at 22° C. and 55% humidity with a 16/8 hour photoperiod (32-watt T8 light bulbs emitting 3000k white light). Seeds were stratified for 3 days at 4° C. in the darkness to break dormancy. Plants from each line were randomly distributed and rotated every day until bolting to minimize environmental variations. When siliques began to mature, humidity was decreased to 45% as recommended by the Arabidopsis Biological Resource Center (see, ftp://ftp.arabidopsis.org/ABRC/abrc_plant_growth.pdf). Harvested seeds were air-dried for a week before being stored in Eppendorf tubes at 4° C.
Arabidopsis thaliana plants that were used for line propagation were grown in soil (PRO-MIX® HP Mycorrhizae) inside chambers held at 22° C. with a 16/8 hour photoperiod. Seeds were stratified fbr 3 days at 4° C. in the darkness to break dormancy.
Nicotiana benthamiana plants were grown in soil (PRO-MIX® PDX) inside chambers held at 22° .C with a 16/8 hour photoperiod.
floe1-1 T-DNA mutant:
The mutant line floe1-1 (SALK_048257C) was obtained from the Arabidopsis Biological Resource Center (ARRC') and gcnotyped using primers priFLOE1cds-FWD/REV and the Salk genotyping primer LBb1.3 (sequences not shown). It was confirmed to be a knockout mutant by RT-qPCR (
Transgenic plants were generated by Agrobacterium-mediated (GV3101 strain) transformation (7) of floe1-1 with the constructs described in the Plant plasmid construction section, with the exception of the control transgenic line overexpressing YFP-FLAG used in
FLOE1 CRISPR lines were generate (using the Staphylococcus aureus CRISPR-Cas9 system (8) and by following the protocol described in (web address botanik.kit.edu/molbio/940.php). A region within the QPS-rich region was identified as having a NNGGGT protospacer adjacent motif (PAM) downstream of a protospacer sequence (5′TTACAGCCCCCAGACTGGC3′) that did not have any significant similarities to other genomic regions. The corresponding guide RNA was inserted in the Bbsil site of the pEn-Sa-Chimera vector through digestion-ligation following hybridization of the oligo duplex priCRISPR-FWD/REV. The resulting sgRNA coding vector was then transferred to pDe-Sa-CAS9 through LR recombination, The final binary destination vector was then used to transform Agrobacterium (GV3101 strain), which was used to transform Col-0 plants using the floral dip method (7). Seeds obtained from the T0 parental lines were sown on MS media (0.5X Murashige and Skoog basal salt mixture (MS) media (PhytoTechnologies Laboratories) (pH 5.7), supplemented with 0.8% agar (Difco) and 1% sucrose (Sigma-Aldrich)) supplemented with 30 mg/L Kanamycin (G-Biosciences) for selection of successfully transformed transgenics, Selected T1 seedlings were then transferred to soil to mature. Genomic DNA was extracted from mature rosette leaves of each of these T1 plants and the Cas9-recognition site within FLOE1 was amplified through PCR with Phusion DNA polymerase (Thermo Fisher Scientific) using primers prigenoCRISPR-FWD/REV. Sequencing (Sequetech Inc.) of the amplicons revealed that 12 plants demonstrated heterogenous sequences at the targeted region, which were subsequently selected for growing the T2 generation. For each selected T1 plant, 8 T2 progeny were grown, and PCR amplification followed by sequencing of the FLOE1 amplicon was again performed on genomic DNA extracted from mature rosette leaves. Four individuals from this T2 generation (floe1-2, floe1-3, floe1-4, floe1-5) presented different homozygous mutations in the FLOE1 amplicon, leading to frameshift mutations and pre-mature stop codons in the QPS region, and were selected for further assays.
Constructs were generated using the Gateway system Titrogen the pGWB601-661 collection (9) as follows:
Transgenes for Arabidopsis experiments: FLOE1's genomic region spanning its promoter, as predicted by AGRIS (10), to its last coding codon was amplified by PCR from Col-0 DNA (extracted with IDNeasy Plant Mini Kit (Qiagen)) using the prigFLOE1-FWD/REV primers. The amplicon was first cloned into pDONR221 (Thermo Fisher Scientific) using BP Clonase II (Thermo Fisher Scientific) and then subcloned into pGWB604, pGWB610 and pGWB633 using LR Clonase II (Thermo Fisher Scientific) to generate pFLOE1p:FLOE1-GFP, pFLOE 1p:FLOE1-FLAG and pFLOE1p:FLOE1-GUS respectively.
FLOE1p:FLOE1ΔDS-GFP, FLOE 1p:FLOE1ΔQPS-GFP, and FLOE1p:FLOE1ΔDUF-GFP were obtained by moditing pFLOE1p:FLOE1-GFP using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) with primers priDSdeletion-FWD/REV, priQPSdeletion-FWD/REV, and priQPSdeletion-FWD/REV, and priDUFdeletion-FWD/REV respectively.
An entry vector containing the YFP gene was donated by Dr. Zhiyong Wang (Carnegie Institution for Science, USA) and another one, G18395, containing FLOE1's coding sequence was obtained from ABRC. The two genes were then transferred from the entry vector into the binary vector pB7HFC3_0 (11) using Gateway cloning (Life Technologies), to create the vector p35S:YIT-FLAG and p35S:FLOE1-FLAG.
Transgenes for tobacco (Nicotiana benthamiana) experiments:
A. Arabidopsis genes: The coding sequences of FLOE1's isoforms, FLOE1.1 and FLOE1.2 were amplified by PCR from the entry vector G18395 using priFLOE1.1-FWD/REV and priFLOE1.2-FWD/REV and then BP recombined into pDONR221 (Thermo Fisher Scientific). These were then transferred by LR recombination into pGWB605 to generate p35S:FLOE1.1-GFP and p35S:FLOE1.2-GFP. Similarly, p35S:FLOE1.2-RFP was generated by subcloning FLOE1.2 into pGWB660. The N-terrninal version p35S:GFP-FLOE was generated by LR recombination of G18395 into pGWB606. To generate p35S:FLOE2-GFP and p35S:FLOE3-GFP, the coding sequences of FLOE2 and FLOE3 were obtained from 5-day old Col-0 seedlings cDNA by PCR amplification using Phusion DNA polymerase (Thermo Fisher Scientific) and the primers priFLOE2 -FWD/REV and priFLOE3-FWD/REV. Total cDNA was obtained by reverse transcription using M-MLV Reverse Transcriptase (Thermo Fisher Scientific) from total RNA extracted with the RNeasy Plant Mini Kit (Qiagen). The FLOE2 and FLOE3 amplicons were then BP recombined into pDONR221 before being transferred into pGWB605 by LR recombination.
B. Mutated FLOE1 versions: FLOE1 wt, FLOE1Δnucl, FLOE1ΔCC, FLOE1ΔQPS, and FLOE1-QPS-15×Y-S were amplified from the corresponding human expression vectors described in Human plasmid construction using prihFLOE1-FWD/REV and BP recombined into pDONR221 (Thermo Fisher Scientific) before being transferred by LR recombination into pGWB605 to generate p35S:wt.FLOE1-GFP, p35S:FLOE1Δnucl-GFP, p35S:FLOE1ΔCC-GFP, p35S:FLOE1ΔQPS-GFP, and p35S:FLOE1-QPS-15×Y-S-GFP. p35S:FLOE1ΔDS-GFP and p35S:FLOE1ΔDUF-GFP were obtained by the same process but with different primer pairs: prihFLOE1ΔDS-FWD/prihFLOE1-REV and prihFLOE1-FWD/prihFLOE1ΔDUF-REV, respectively.
C. Non-Arabidopsis FLOE1 homologs: Protein sequences for all FLOE1 homologs shown in
Phylogenetic tree construction: All Viridiplantae protein sequences containing the highly-conserved DUF1421 domain were retrieved from UniProt (12). After removal of duplicates due to re-annotations, the remaining 791 sequences were submitted to the phylogenetic analysis tool, NGPhylogeny,fr (14) with default settings. The FastIVIE Output Tree was then uploaded to iTOL (version 5) (15) for tree visualization.
QPS and DS domains lengths: All monocot and eudicot sequences from the FLOE1 and FLOE2/3 groups were aligned using the msa package (version 1.20,0) in R (16). The DS and QPS reions of the homologs were defined as aligning to the DS and QPS regions of FLOE1. The lengths of these regions were used for subsequent analysis.
Alignments: The figure showing the alignment and protein characteristic of select FLOE1 homologs was conducted using the msaPrettyPrint( )function of the msa package (16) in R and MacTex.
Agrobacterium cultures (GV3101 strain) carrying the relevant constructs were grown overnight at 28° C., in LB broth (Fisher BioReagents) containing 25 mg/L rifampicin (Fisher BioReagents), 50 mg/mL gentamicin (GoldBio) and 50 mg/L spectinomycin (GoldBio). Cultures were washed four times with infiltration buffer (10 mM MgCl2 (omniPur, EMD), 10 mM MES (pH 5.6) (J. T. Baker) and 100 uM acetosyringone (Sigma-Aldrich)) and diluted to reach an OD600 of 0.8. Fully expanded 3rd, 4th or 5th leaves from 6-week-old tobacco plants were infiltrated with these diluted Agrobacterium cultures using Monoject 1 mL Tuberculin Syringes (Covidien). For the FLOE1,1-GFP and FLOE1,2-RFP colocalization experiment, an equal amount of each culture was pre-mixed before infiltration. For each construct or combination of constructs, at least three individual tobacco plants were infiltrated.
Seeds were first sterilized by vortexing in 70% ethanol for 5 minutes after which the solution was removed and replaced with 100% ethanol. Seeds were then placed on pre-sterilized filter papers (Grade 410, VWR) and left to dr in a laminar flow hood. Sterilized seeds were then sown on square petri dishes (120×120 wide×15 mm high (VWR)) containing 40 mL of MS media (0.5X Murashige and Skoog basal salt mixture (MS) media (PhytoTechnologies Laboratories) (pH 5.7), supplemented with 0.8% agar (Difco) and 1% sucrose (Sigma-Aldrich)) supplemented with NaCl (Sigma-Aldrich) and mannitol (Sigma-Aldrich) at the concentrations indicated in the manuscript. Plates were then sealed with micropore surgical tape (3M) and covered in aluminum foil befbre being placed at 4° C. After exactly 120 h (5 days) of stratification to break seed dormancy, plates were transferred to a 24 h light (17-watt T8 light bulbs emitting 4100k white light), 22° C. growth cabinet (Percival). Germination (identified by radicle protrusion) was counted under a dissecting microscope the following day for the normal conditions and 15 days later for the stress conditions.
Germination experiments were performed on seeds from three independent batches of plants (A. B, and C) grown as described in the Plant growth conditions section.
Batch A (
Batch B (
Batch C (
Salt, inainiitol, sorbitol, cycloheximide and water assays: Seeds of the relevant GFP-tagged lines were submerged in either glycerin or in solutions of NaCl (Sigma-Aldrich). mannitol (Sigma-Aldrich), sorbitol (Sigma-Aldrich), cycloheximide (GoldBio) or double distilled water at concentrations indicated in the manuscript for 15-30 min (NaCl: 0, 0.2M, 0.4M, 0.6M, 0.8M, 1M, 1.2M, 1.4M, 1.6M, 1.8M, 2M; mannitol: 0, 950 mM; sorbitol: 0, 0.725M, 1.45M; cycloheximide: 1 g/L). They were then dissected to remove the seed coat and imaged by confocal microscopy (see Plant microscopy and image analysis). As controls, 35S: (11) and Col-0 seeds were dissected in water to verify that. GFP alone could not induce condensate formation and to indicate the level of autofluorescence of the protein storage vacuoles in the absence of GFP, respectively.
Condensate reversibility assays: Three different types of FLOE1 condensate reversibility assays were performed: 1) Embryos from dry seeds were first dissected in glycerin as described above, and after imaging, glycerin was washed off from the embryos with water and the same embryos were imaged in water; 2) Seeds were submerged in water for 1 hr before being transferred to 2M NaCl for 10 min and imaged and vice versa (1 h in 2M NaCl followed by 10 min in water); and 3) Seeds were submerged in water overnight and then left to dry for an additional day. Seeds were then either dissected in glycerin to obtain the condensate state of the dry seeds or in water to assess the ability to re-form condensates.
End of germination experiment analysis: At the end of the 230 mM NaCl germination experiment described in the Germination Experiments section (15 days in light following 5 days of stratification on MS media supplemented with 230 mM NaCl), seeds that did not germinate were either: 1) dissected directly in glycerin to maintain the hydration state of the seed; or 2) transferred first to normal MS media and dissected in glycerin two hours later. Dissected embryos were then imaged by confocal microscopy to obtain a snapshot of their final condensate state (see Plant microscopy and image analysis).
Developmental stages: FLOE1p:FLOE1-GFP and 35S:YFP-FLAG flower buds were self-crossed 11, 8, 6 and 4 days before dissection to obtain developing siliques carrying embryos at mature, torpedo, heart and globular stages respectively. Seeds from the various developmental stages were dissected either in glycerin or water and imaged by confocal microscopy (see Plant microscopy and image analysis).
FLOE1p:FLOE1-GUS seeds carrying embryos at different stages of maturation were incubated at 37° C. overnight in GUS staining solution (17)In the case of dry seeds, seed coats were first removed as they were impermeable to the staining solution and incubated at 37° C. for one hour in GUS staining solution. Following the incubation, samples were destained in 70% ethanol at room temperature for 24 hours and embryos were dissected out (in the case of developing siliques) before imaging. Pictures were taken with a compound microscope (Nikon) and dissecting scope (Leica MZ6 microscope).
Image acquisition: Embryos and tobacco leaves were imaged at room temperature on a LECIA TCS SP8 laser scanning confocal microscope in resonant scanning mode using the LASX software. All samples were imaged with a Hf PL APO CS2 63X/1.20 water objective with the exception of embryos submerged in glycerin that were imaged with a 63X/1.30 glycerin objective and of embryos of early developmental stages that were imaged with a HC PL APO CS2 20×/0.75 dry objective. GFP, RFP, and YFP fluorescence was detected by exciting with a white light laser at 488 nm, 561 nm and 514 nm, respectively, and by collecting emission from 500-500 nm, 591-637 nm and 524-574 nm, respectively, on a HyD SMD hybrid detector (Leica) with a lifetime gate filter of 1-6 ns to reduce background autofluorescence due to chlorophyll (tobacco) or protein storage vacuoles (embryos). Z-stacks were collected with a bidirectional 96-line averaging while single-frame images (tobacco images displayed in the publication) were collected with a bidirectional 1024-line averaging. For the colocalization experiments, samples were imaged sequentially between each line to ensure that the colocalization signals were not due to bleed-throughs. Images displayed in the publication were representative of at least three biological replicates for each construct (tobacco) or line (Arabidopsis). All samples that were compared in the publication were imaged with the same magnification and laser intensity.
Heterogeneity analysis: For each radicle and experimental condition, maximum projection images of their corresponding Z-stacks were obtained using the LASX software. ROIs were then manually drawn around each individual cell to obtain their standard deviation (RMS) and mean intensity levels. Heterogeneity scores were obtained by dividing the standard deviation by the mean. Between 363 and 461 cells were measured per embryo with a total of 3 embryos per condition. Cells were characterized as exhibiting FLOE1 condensates if their heterogeneity score was higher than the top 5 percentile of the 2M NaCl condition (heterogeneity cut-off=0.3 a.u.).
Granule size: Individual slices of a radicle Z-stack were analyzed using FIJI (18). Individual granules were identified using a threshold, followed by a watershed, and subsequently measured for their area. A total of 3-4 embryos per condition were analyzed.
Seed weight: Twelve and fourteen biological replicates of floe1-1 and Col-0 seeds, respectively, were used for the seed weight analysis. Seeds were weighed on a Sartorius M2P scale in batches of nine to twenty seeds and the process was replicated three times per biological replicate. The average weight per seed was calculated and used for subsequent statistical analysis.
Seed size and aspect ratio: Fourteen and sixteen biological replicates of floe1-1 and Col-0 seeds, respectively, were used for the seed size and aspect ratio analysis. Seed images were scanned using a Canon CanoScan LiDE 700 F (Canon Inc). All images were scanned at 600 dpi and, for ease of collection, the seeds were placed in transparent bags before scanning. The number of seeds per image varied, but ten seeds per sample were randomly selected and analyzed for area quantification and aspect ratio using ImageJ (version 2.0.0) (19). This process was replicated ten times per biological replicate to obtain a total of hundred seeds per biological replicate.
DNA-free total RNA was extracted from seeds and siliques (20). The extraction buffer utilized 0.5% β-mercaptoethanol. RNA quantity and purity from all samples were assessed using a NanoDrop Spectrophotometer (Thermo Fisher Scientific).
cDNA was synthesized from I pg of extracted RNA using M-MLV Reverse Transcriptase (Invitrogen), per manufacturer's protocol. qPCR was performed using the SensiFAST SYBR No-ROX Kit (Bioline). Primers used to quantify FLOE1 expression were priqPCRFLOE1set1-FWD-REV, with the exception of the qPCRs conducted on the CRISPR lines as well as on siliques and seeds from different developmental stages (
Experimental design: Six conditions were utilized in the RNA-seq analysis: 1) dry floe1-1 seeds; 2) dry Col-0 seeds; 3) imbibed floe1-1 seeds; 4) imbibed col-0 seeds; 5) salt-stressed imbibed floe1-1 seeds; and 6) salt-stressed imbibed Col-0 seeds. Three biological replicates corresponding to pooled seeds from 20 different plants were performed per condition, with 50 mg of mature seeds used per biological replicate. For conditions (1) and (2), RNA was extracted directly from dry seeds using the protocol described in the RNA extraction from seeds section. For conditions (3) and (4), and for each biological replicate, dry seeds were sown onto separate but identical agar plates of normal MS media conditions (0.5X Murashige and Skoog basal salt mixture (MS) media (PhytoTechnologies Laboratories) (pH 5.7), supplemented with 0.8% agar (Difco) and 1% sucrose (Sigma-Aldrich)) and cold-stratified for 5 days at 4° C. in the dark. All plates were subsequently transferred to and held in a growth cabinet (Percival) for exactly 4 hours under light and 22° C. After the 4-hour incubation, imbibed seeds were scraped from each plate and transferred to a clean mortar and pestle and ground in liquid nitrogen. Conditions (5) and (6) were conducted in parallel and using the exact same experimental setting with the only difference being that the MS media was supplemented with 220 mM NaCl.
For all biological replicates, 2 μL of extracted RNA was combined with 2 μL of DNase/RNase-free dH2O for a 1:2 dilution and sent to the Stanford University Protein and Nucleic Acid Facility for quantification and quality analysis using an Agilent 2100 Bioanalyzer. After analysis, 5 μL of extracted RNA was combined with 20 μL of DNase/RNase-free dH2O for a 1:5 dilution and sent to Novogene Corporation Inc. (Sacramento, CA) for RNA-seq library preparation (250-300 by insert cDNA library) and sequencing (2×150 by paired-end reads on an Illumina Platform).
Analysis: Reads were mapped with HISAT2 to the Arabidopsis thaliana TAIR10 reference genome using the Galaxy (Version 2.1.0+galaxy5) web platform (https usegalaxy.eu) (23). The resulting BAM files were then analyzed on R using the DESeq2 (24) and T×DB.Athaliana.BioMart.plantsmart28 (Bioconductor) packages. Genes with padj<0.05 were considered differentially expressed. Gene ontology and KEGG enrichment of the differentially expressed genes was obtained using g:Profiler (biit.cs.utee/gprofiler/gost) (25).
FLOE1 and derived mutant constructs for expression in human cells were optimized for human expression and generated through custom synthesis and subcloning into the pcDNA3.1+N-eGFP backbone by Genscript (Piscataway, USA).
U2OS cells (ATCC, HTB-96) were grown at 37° C. in a humidified atmosphere with 5% CO2 for 24 h in DMEM, high glucose, GlutaMAX+10% FBS and pen/strep (Thermo Scientific). Cells were transiently transfected using Lipofectamine 3000 (Invitrogen) according to manufacturer's instructions. Cells grown on cover slips were fixed 24 h after transfection in 4% formaldehyde in PBS. Slides were mounted using ProLong Gold antifade reagent (Life Technologies). Confocal images were obtained using a Zeiss LSM 710 confocal microscope. Images were processed using FIJI (18).
U2OS cells were cultured in glass bottom dishes (Ibidi) and transfected with GFP-FLOE1 constructs as described above. After 24 hr GFP-FLOE1 condensates were bleached and fluorescence recovery after bleaching was monitored using Zen software on a Zeiss LSM 710 confocal microscope with incubation chamber at 37° C. and 5% CO2. Data were analysed as described previously (28). In brief, raw data were background subtracted and normalized using Excel, and plotted using GraphPad Prism 8.4.1 software.
All data was analyzed using Graphpad Prism 8.4.1 and Excel. Statistical tests details are shown in the figure legends.
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This application claims benefit of U.S. provisional application No. 63/063,009, filed Aug. 7, 2020, which is herein incorporated by referenced for all purposes.
This invention was made with Government support under contract DE-SC0018277 awarded by the Department of Energy, under contract DE-SC0008769 awarded by the Department of Energy, under contract 617020 awarded by the National Science Foundation and under contract NS097263 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/045103 | 8/6/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63063009 | Aug 2020 | US |
