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Takehiro A. Ozawa

@tkozawa.bsky.social

Great preprint by Zhang, Aizezi et al. (2025) on how #brassinosteroids promote phosphoenolpyruvate carboxykinase (PCK) activity for sugar synthesis in C3 and C4 plants, by inhibiting BIN2-mediated PCK phosphorylation at two conserved residues (Ser-62 and Thr-66). 🌱🌽

www.biorxiv.org/content/10.1...

Fig. 8: Summary of the roles of BIN2-PCK1 regulatory module in Arabidopsis and sorghum/maize.

a) Brassinolide activates PCK1, by inhibiting BIN2 phosphorylation, to promote the conversion of lipids into sugar for Arabidopsis seedling establishment.

b) BR and light regulate PCK1 in bundle sheath cells (BSC) for photosynthesis in sorghum and maize. CO2 is fixed by PEP carboxylase (PEPC) to make OAA in mesophyll cells (MC). OAA is then converted to Asp and transported to BSC, where OAA is decarboxylated by PCK1, thus releasing CO2 for photosynthesis. PEP returns to the MC for the next round of C4 cycle.

October 27, 2025 at 8:40 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by O’Leary et al. (2025) on how Gln-driven #PlantTOR signaling occurs during the early development of pea embryos (i.e. cell division phase), but is unexpectedly inactive during #legume seed storage protein biosynthesis. #Pisum #glutamine

🔗 nph.onlinelibrary.wiley.com/doi/10.1111/...

Fig. 8 Hypothetical model showing the reciprocal regulation of target of rapamycin (TOR) and abscisic acid (ABA) signaling during the transition from the cell division to the cell expansion phase of legume seed development.
The activators of TOR observed in this paper are shown, but further molecular and hormonal influences on the developmental transition are omitted for simplicity.
The bottom panel extends a basic model by Weber et al. (2005) to show the approximate timing of relative changes in three established signaling metabolites: Glc, glucose; Suc, sucrose; and Gln, glutamine. Sucrose levels are known to be correlated with the signaling metabolite T6P.

Fig. 5. Glutamine (Gln) activates target of rapamycin (TOR) in developing pea cotyledons.
(a) The in vivo RPS6-Ser240 phosphorylation status in pea embryos throughout development. Replicate immunoblots are shown in Supporting Information Fig. S5.
(b) The relative RPS6-Ser240 phosphorylation signal is plotted, and the fresh weight of the embryos is plotted against days after pollination (DAP). Error bars represent the standard error of the mean.
(c–e) RPS6-Ser240 phosphorylation was assessed in 90–130 mg pea embryos incubated for 2 or 4 h with or without Gln (62.5 mM) and sucrose (142 mM) (c), Gln (62.5 mM) and glucose (100 mM) (d) or individual amino acids (62.5 mM) and NH4NO3 (20 mM) (e).
Two biological replicates are shown. Replicate immunoblots are shown in Fig. S6.

October 16, 2025 at 9:24 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great preprint by Upadhyaya et al. (2025) on using a #multiomics approach to study #PlantTOR signaling in the unicellular green alga #Chromochloris zofingiensis, particularly showing the upregulation of #AminoAcid biosynthesis pathways during TOR inhibition with AZD8055.
#Algae #Phosphoproteomics 🔗⬇️

Fig. 8. Summary of the mechanism of TOR inhibition-dependent accumulation of amino acids in Chromochloris zofingiensis.

TOR inhibition by AZD8055 induces the expression of transcription factors G2- like, HSF, and bHLH, which bind to amino acid biosynthesis and nitrogen metabolism genes to potentially regulate their transcription.
This process, which is independent of glucose-TOR signaling, leads to an accumulation of amino acids during TOR inhibition. Glucose addition via hexokinase1 (HXK1) also upregulates amino acid biosynthesis.

Fig. 1. TOR inhibition prevents glucose-driven growth and suppresses photosynthesis in the green alga Chromochloris zofingiensis.

(A) Representative C. zofingiensis (WT) cultures over the 96 h experiment with the following treatments: control, TOR inhibition by 1 µM AZD8055, 100 mM glucose addition, and 100mM glucose + 1 µM AZD treatment.

(B) Cell density and (C) maximum PSII photosynthetic efficiency (Fv/Fm). For (B) and (C), datapoints represent biological replicates with lines representing means (n = 3).

(D) PCA of WT 1 h and 24 h transcriptome, proteome, and metabolome with treatments: control, 1 µM AZD, 15 mM glucose, 15 mM glucose + 1 µM AZD, and 15 mM sorbitol as an osmotic control.
For the transcriptome, normalized log2-transformed counts of 500 genes with highest variance were considered.
For the proteome and the metabolome, the raw intensity values were log2-transformed, and proteins or metabolites with highest variance were used to calculate and plot the PCA.

Modified Fig. 2. Phosphoproteomics of TOR-inhibited Chromochloris zofingiensis identifies novel TOR players.

(B) The log2 fold changes of TOR-dependent phosphosites between TOR inhibition by 1 µM AZD, 15 mM glucose addition, 15 mM glucose + 1 µM AZD, and 15 mM sorbitol-treated C. zofingiensis (WT) cultures at 1 h and 24 h from cluster 4 were labelled according to their protein symbols and phosphorylated Ser/Thr residue on the left and by their ortholog name identified on the right.
The phosphopeptides were labelled if they are known conserved TOR targets as “known targets” and if they are unknown as “novel targets”. RbcL, MAPK, PP2C, PP2C, and SnRK2 were identified as putative novel conserved TOR targets. White boxes in the heatmap indicate that the phosphopeptides were not detected. Data represent means of fold changes log2 intensities of phosphopeptides (n = 3-4 biological replicates).

October 2, 2025 at 4:27 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Wang et al. (2025) on how #PlantTOR dynamically regulates the translation of a chromatin-associated complex for growth (CACG) in #Arabidopsis, which represses the expression of stress-responsive genes to coordinate plant growth and stress tolerance 🌱⚖️🏜️.
🔗 www.nature.com/articles/s41...

Fig. 6 | Working model for the role of the nutrient–TOR–CACG axis in balancing plant growth and stress tolerance.

In the presence of adequate nutrients, TOR is active and promotes the translation of CACG mRNAs. The pyrimidine-rich motifs present in the 5′ UTRs of CACG mRNAs facilitate target selection by TOR. The CACG complex directly binds to numerous stress-responsive genes marked by histone acetylation, thereby repressing their transcription, which contributes to plant growth and development.

Conversely, under nutrient-deficient conditions, TOR becomes inactive, leading to decreased translation of CACG mRNAs. This results in the alleviation of the repression of stress-response genes, causing reduced growth but increased stress tolerance. The nutrient–TOR–CACG axis is crucial for balancing plant growth with stress tolerance, promoting survival and reproductive success in variable environmental conditions. conditions.

Image partially created in BioRender. He, X.

Fig. 1 | Identification of a multi-subunit protein complex involved in nutrient-responsive growth.

a) Protein–protein interaction networks were generated from mass spectrometry data using Cytoscape. The edges show the interactions identified by AP–MS. CACG subunits are shown in pink, while COMPASS subunits and the INO80 complex (INO80-C) are shown in blue.

b) Interactions among CACG subunits as determined by Y2H and pull-down assays. Interactions revealed by Y2H assays are shown in yellow, those shown by pull-down assays in red and those confirmed by both methods in orange. Protein names in bold denote those analysed through pull-down assays.

c) Detection of a high-molecular-weight complex by gel filtration.
Proteins extracted from Flag-tagged GTE1, DP1, EMB1967 and CP2 transgenic plants were separated in a Superose 6 column (10/300 GL; GE Healthcare Life Sciences) and were detected by immunoblotting.

d) Immunoblot analysis of protein levels in the indicated Flag-tagged transgenic plants under different nutrient conditions. Twelve-day-old plants grown in solid MS medium were transferred to liquid MS, H2O or reduced concentrations of MS for 4 days. The actin protein level is shown as a loading control.

e) Fresh weight of wild-type plants irrigated with water or liquid MS medium (n = 24). The values are means ± s.d.

f) Immunoblot analysis of protein levels in the indicated Flag-tagged transgenic plants irrigated with water or liquid MS medium. The actin protein level is shown as a loading control.

g) Morphological phenotypes of 20-day-old wild-type and mutant plants (top) and 12-day-old wild-type and mutant plants (bottom). Scale bars, 1 cm.

The experiments in c, d and f were repeated independently twice and showed similar results.

Modified Fig. 4 | The CACG complex suppresses the expression of stress-responsive genes to coordinate plant growth and stress tolerance.

g) Morphological phenotypes of wild-type plants and CACG mutants under control, drought stress and rehydration conditions. Twelve-day-old plants grown in solid MS medium were transferred to soil and subsequently subjected to drought treatment until the Col-0 plants began to show mortality. The phenotypes are shown before and three days after rehydration with MS. Plants watered with MS serve as the control.

h) The survival rate of wild-type plants and CACG mutants under drought stress conditions (n = 24). Each bar shows the mean ± s.d. from three independent biological replicates. P values were determined using two-tailed Student’s t-tests.

i) Immunoblot analysis of the protein levels of CACG components under the indicated conditions. TOR activity was monitored by the phosphorylation state of S6K. The actin protein level is shown as a loading control. The experiments were independently repeated twice and showed similar results.

j) Statistical analysis of pS6K protein levels. Actin was used as a loading control. Each black dot represents an independent biological replicate.

September 26, 2025 at 8:26 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Interesting work by Lu et al. (2025) on introducing a synthetic #CO2 fixing cycle, malyl-CoA-glycerate (McG), into #Arabidopsis chloroplast by overexpressing six heterologous enzymes.
The McG plants showed increased biomass, leaf number, seed yield and #lipid production.
🔗 doi.org/10.1126/scie...

Fig. 1. The CBB-McG dual-cycle carbon fixation.
The chloroplast-localized synthetic McG cycle consists of six heterologously expressed enzymes (Mc, pink-shaded ovals; G, purple ovals) and four endogenous enzymes (black).
The Mc part consists of phosphoenolpyruvate carboxylase (PPC), malate thiokinase (MTK), and malyl-CoAlyase (MCL).
The G part consists of glyoxylatecarboligase (GCL), tartonic semialdehyde reductase (TSR), and glycolate dehydrogenase (GDH).
Endogenous enzymes are malate dehydrogenase (MDH), plastidial glyceratekinase (PGK), phosphoglycerate mutase (PGAM), and enolase (ENO).

The McG cycle can use both the RuBisCO carboxylation product 3-phosphoglycerate (3PG) and oxygenation product glycolate to produce acetyl-CoA through bicarbonate fixation catalyzed by PPC. The CBB and McG cycles are interconnected by a reversible path between 2PG and 3PG and an irreversible path through glycerate and 3PG, so that the two cycles can balance each other.

Modified Fig. 2. Increased plant biomass and seed yield in McG plants.
(E) The rosette leaf number of the 6-week- old plants (n = 4; mean ± SEM). The phenotypes are shown in figs. S5 and S7.
(F) Representative images of individual rosette leaves from 6-week- old WT and McG plants.
(G) The silique number from the primary inflorescence of the WT and McG plants at 14 days after fertilization (DAF) (n = 4; mean ± SEM).
(H) Representative images of siliques from the primary inflorescence of the WT and McG plants.

Modified Fig. 5. Images of lipid droplets and TAG/DAG production in the
McG plants.
(D) The levels of different lipid classes in the leaves of the WT and the McG plants. Rosette leaves of the 5-week- old plants were harvested for the measurements of triacylglycerol (TAG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and fatty acid (FA). One-way Welch’s ANOVA test was used, followed by Dunnett’s T3 multiple comparison test against WT for significance difference (P < 0.05). The adjusted P values are indicated. Cases with no significant difference are indicated with “ns.”
(F) The levels of TAG and its precursors diacylglycerol (DAG) and phosphatidylcholine (PC) and FA in the dried matured seeds from the WT and the McG lines.

September 13, 2025 at 3:26 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great preprint by Lihanova et al. (2025) on the development of an activation tagging system in the green alga #Chlamydomonas, called enhancer-driven random gene overexpression (ERGO), allowing the discovery of a new gene (putative F-box protein) involved in the regulation of #carotenoid metabolism⬇️.

Figure 1: ERGO screen in C. reinhardtii.

(A) ERGO workflow. C. reinhardtii strain CC-3840 with a yellowin-the-dark phenotype was transformed by electroporation with a DNA cassette containing the enhancer Ehist cons with four copies of a conserved 8 bp-motif (four green triangles), and the APHVIII selection marker (Supplementary Fig. S1, S2). Transformants were screened for a visible color change on agar plates containing 15 µg ml-1 paromomycin in the dark. Mutants with a pronounced and reproducible phenotype were chosen for mapping of the Ehist cons insertion sites and further characterization.

(B) Classification of orange-colored mutants (OCMs) based on the insertion type. The Ehist cons insertion sites were mapped in the nuclear genome of 30 OCMs by mapping-by-sequencing (MBS). In one OCM with a complex insertion, an exogenous 396 bp fragment of chloroplast DNA, co-inserted with the transformation cassette, is shown in blue. Enhancer insertions were sometimes accompanied by short indels or duplications of genomic DNA at the insertion site

September 8, 2025 at 3:29 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Ma et al. (2025) on how NRT1.1B, a traditionally recognized #nitrate receptor, can directly perceive extracellular #AbscisicAcid (ABA) in a nitrate-antagonistic manner, thus enabling 🌾plants to integrate nutrient and abiotic stress cues (N level & drought)
www.cell.com/cell/fulltex...

Figure 7. NRT1.1B integrates compound environmental signals in plants.

(M) A proposed model of nitrate-ABA coordinated responses mediated by NRT1.1B.
Under low-nitrate conditions without ABA, NLP4 interacts with SPX4 and is sequestered in the cytoplasm. ABA perception enhances the NRT1.1B-SPX4 interaction, thereby promoting NLP4 release and translocation into the nucleus, and ultimately activates downstream ABA responses.
Under high-nitrate conditions, nitrate competes with ABA for NRT1.1B binding, and NRT1.1B shifts to sense the nitrate signal.

August 12, 2025 at 7:24 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Interesting work by Maharjan et al. (2025) on the 3D #bioprinting of plant- and animal cell-based hybrid foods, especially noodles that are made of 30–40% edible #microalgae ( #Chlamydomonas or Chlorella) and 60–70% muscle cells (C2C12 or chicken #myoblasts) 🍜🍗🧐.
🔗 www.nature.com/articles/s41...

Fig. 1. Automated continuous chaotic bioprinting.

a) Schematic illustration showing automated chaotic bioprinting of noodles with internally aligned lamellar microstructures, using the three-element KSM (Kenics static mixer) printhead, with highlighted lateral and transverse views.
b) Schematics of various three-dimensional (3D) hybrid food items, produced by simultaneous chaotic bioprinting of microalgae and muscle cells.
c) Schematic illustration showing bioink designs for microalga and muscle cell-bioprinting.

Fig. 6. 3D-bioprinted hybrid food.

a) Photographs showing 3D hybrid drumsticks on day 0 and day 16, bioprinted with C2C12 cell- and Chlamydomonas cell-bioinks.
b) Viability values of bioprinted C2C12 cells and Chlamydomonas cells within the hybrid drumsticks, assessed over the 15-day culture period at 37 °C. Data are presented as themean values ± SEMs (n = 3 independent samples, each obtained from a separate bioprinting run).
c) Fluorescence confocal micrographs showing the expression of skeletal muscle myosin (green) by C2C12 cells and Chlamydomonas cells with red autofluorescence of chlorophyll on day 16, within bioprinted hybrid drumsticks.
d) Photographs of bioprinted hybrid cuboids at day 0 and day 14, using C2C12 cell- and Chlamydomonas cell-bioinks.
e) Photographs of cut pieces of bioprinted hybrid cuboids on day 21, used for cooking studies.
f) Comparison of mechanical properties of hybrid food before and after cooking on day 21. Data are presented as the mean values ± SEMs (n = 4 independent samples, each obtained from a separate bioprinting run) and a two-tailed paired t-test was applied to measure p values.

Fig. 7. Chaotic-bioprinted chicken-microalga hybrid noodle.

a) Photograph of hybrid chicken-microalga noodles on day 21, bioprinted with chicken cell- and Chlamydomonas cell-laden bioinks.
b) Fluorescence micrograph showing live chicken cells (green) and Chlamydomonas cells with red autofluorescence of chlorophyll, within the bioprinted hybrid chicken-microalga noodles on day 21.
c) Viability values of chicken cells and Chlamydomonas cells within bioprinted hybrid chicken-microalga noodles, assessed over the 21-day culture period at 37 °C. Data are presented as the mean values ± SEMs (n = 3 independent samples, each obtained from a separate bioprinting run).
d) Fluorescence confocal micrographs showing the expressions of F-actin, MYH2, and skeletal muscle myosin (green) by chicken cells and Chlamydomonas cells with red autofluorescence of chlorophyll, within bioprinted chicken-microalga hybrid noodles on day 21.

July 31, 2025 at 3:30 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Chen et al. (2025) on introducing the tartronyl-CoA (TaCo) pathway into rice chloroplasts as a synthetic, carbon-positive photorespiratory bypass to enhance CO2 fixation efficiency and increase biomass, grain yield and grain size in rice 🌾. #PlantBiotechnology
🔗 doi.org/10.1111/pbi....

Figure 1. The tartronyl-coenzyme A (TaCo) pathway as a photorespiratory bypass in plant chloroplast.
(a) A schematic representation of plant photorespiration pathway (black) and GS2/Fd-GOGAT Cycle (light blue) and the TaCo bypass (orange).

Figure S1. Schematic diagram depicting the multigene expression vectors TaCo-pYL1300H designed for TaCo bypass implementation.

Each enzyme was fused at the N-terminus with RC2, a highly efficient chloroplast-targeting peptide, and at the C-terminus with specific tags: 3 × FLAG for glycolyl-CoA synthase (GCS), 3 × HA for glycolyl-CoA carboxylase (GCCM5), and Myc for tartronyl-CoA reductase (TCR).
Each cassette was driven by different promoters and terminators: the enhanced CaMV 35S promoter (proE35S) and OCS terminator for RC2-GCS-3×FLAG, the rice Actin promoter (proACT) and pea (Pisum sativum) 3A terminator for RC2-GCC-3×HA, and the maize (Zea mays) Ubiquitin promoter (proUbi) and NOS terminator for RC2-TCR-Myc.

Figure 1. The tartronyl-coenzyme A (TaCo) pathway as a photorespiratory bypass in rice chloroplast.

(j) Biomass per plant (n = 6).
(k) Grain yield per plant (n = 6).
(l) Grain number per panicle (n = 6).
(m) Mature paddy rice grains. Scale bar, 5 mm.
(n) Grain length (n = 20).
(o) Grain width (n = 20).
(p) The 1000-grain weight (n = 6).

Bars (j) to (k) and (n) to (p) represent means ± SDs. Significant differences (P < 0.05) are indicated by letters using ANOVA with Tukey's multiple-comparison test (Data S4).

July 22, 2025 at 3:13 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Jacobebbinghaus et al. (2025) on transcriptional gene fusions via #CRISPR/Cas9-mediated targeted integration at safe harbors (e.g. LHCBM1 locus, 50 bp homology arms) to enhance #transgene expression in #Chlamydomonas compared with randomly-inserted transgenes.
🔗 doi.org/10.1111/nph....

Fig. 2 Effect of transgene fusion on native expression.
(a) Selection of natively high expressed genes (Schmollinger et al., 2014).

(b) Schematic of a highly expressed gene locus. Repair template integrated in native coding sequence and is composed of 50-homology arm, the FMDV 2A peptide, mVenus and aadA fusion CDS and 30-homology arm.

(c) Relative mRNA fold change of respective gene for biological triplicates compared with UVM4. Error bars (SD) were calculated from technical triplicates. PCR products were separated in agarose gels (Fig. S5).

(d) Western blot with immunodetection (anti-GFP for YFP-aadA fusion protein) and Coomassie Brilliant Blue (CBB) as loading control.

(e) Single-cell microscopy of Chlamydomonas reinhardtii showing yellow fluorescent protein (YFP) emission, Chl emission, a combined overlay as well as a differential interference contrast (DIC) picture for UVM4, cytosolic and chloroplast control (PSAD), representative RPL10, RBCS2 and Light harvesting Chl a/b binding protein of LHCII (LHCBM1) transformants.

Fig. 4 Biotechnological approach.
(a) Reaction from Farnesylpyrophosphate (FPP; originating from methylerythritol phosphate pathway) to valencene through the Callitropsis nootkatensis Valencene synthase.

(b) Schematic constructs for random insertion and scar-less targeted integration of the CnVs in between native coding sequence and 30-UTR.

(c) Valencene production by Chlamydomonas reinhardtii is presented for best random integrated (n = 20) and scar-less integrated CnVs after Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) (n = 3) and Light harvesting Chl a/b binding protein of LHCII (LHCBM1) native loci (n = 6).
For random integrated CnVs, best 20 expressing transformants were chosen based on yellow fluorescent protein (YFP) fluorescence out of 288 random isolated transformants. displays mean production of each group and error bars represent the SD. Asterisks (*) indicate the significance level of an unpaired, two-tail Students t-test for mean production assuming unequal variances (**, P < 0.01).

July 10, 2025 at 4:00 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Li et al. (2025) on identifying the regulatory loop between #PlantTOR complex (TORC) via its RAPTOR1B subunit (Ser897) and the CBL4/CBL10–CIPK24 module in #Arabidopsis, which regulates the balance between plant growth and #SaltStress response ⚖️.
#PlantScience

🔗 doi.org/10.1093/plce...

Figure 1. Target of rapamycin complex (TORC) activity is rapidly repressed by salt stress.

A) Dose dependance of salt-mediated TOR repression. 35S:S6K1-HA seedlings were treated with different NaCl concentrations for 4 h. Band intensity in the immunoblots indicates the level of total protein (anti-HA in lower panel) or T449-phosphorylated form of S6K1 (anti-p-T449 in upper panel). B) Quantification of relative p-T449 intensity in A). The data represent the ratio of intensity of p-T449 over S6K1-HA, and the value for the sample “0 mM NaCl” was set as 1.

C) Time course of salt-mediated TOR repression. 35S:S6K1-HA seedlings were treated with 200 mM NaCl for indicated times. D) Quantification of relative p-T449 intensity in C). The value at 0 min was set as 1.

E) TORC activity was restored following salt recovery treatment. 35S:S6K1-HA seedlings were treated with 200 mM NaCl for 15 min, and then transferred to the recovery medium for indicated times. Rec, recovery. F) Quantification of relative p-T449 intensity in E).

In B), D), and F), the data represent the ratio of intensity of p-T449 over S6K1-HA, data are means±SEM, n=3 (biologically independent experiments). Different letters in B) and D) represent significant difference between samples by one-way ANOVA (P<0.05, Supplementary Data Set 1). In F), *P<0.05 (one-way ANOVA, Supplementary Data Set 1)

Figure 6.The salt-tolerant phenotype shown in raptor1b mutant is abolished in the absence of SOS2/Calcineurin B-Like (CBL)-Interacting Protein Kinase 24 (CIPK24).

A) Representative images of Col-0, cipk24, raptor1-2, and raptor1-2cipk24 mutant under normal or salt conditions in post-germination assays. Scale bar: 1 cm.

B) Chlorophyll content per 4 seedlings measured at the end of the assay shown in A). Quantitative data are mean±SD of three biological repeats. Statistical analyses were performed by one-way ANOVA. (***P<0.001, ****P<0.0001, n.s., not significant).

C) Working model for target of rapamycin complex (TORC), abscisic acid (ABA), and CBL–CIPK signaling pathways to orchestrate growth and salt stress response under changing salinity status. In response to salt stress, ABA pathway and CBL–CIPK pathway dominate, suppressing TORC by phosphorylating RAPTOR1B and slowing down the growth to facilitate stress adaptation. Upon the removal of salt stress, TORC rapidly activates to suppress the ABA- and CBL–CIPK-mediated salt stress response network and promotes plant growth.

July 2, 2025 at 4:15 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great #preprint by Chen et al. (2025) on revealing the role of #SalicylicAcid (SA) in controlling central metabolic regulators #SnRK1 and #PlantTOR to coordinate plant immunity and growth by differential phosphorylation of #Arabidopsis NPR1, a key SA receptor (Ser-557 and Ser-55/59, respectively)🤯.

Figure 7H. Proposed model in which central metabolic regulators SNF1-RELATED KINASE 1 (SnRK1) and TARGET OF RAPAMYCIN (TOR) govern plant immunity through differential phosphorylation of NONEXPRESSER OF PR GENES 1 (NPR1), the central plant immune regulator of salicylic acid (SA)-mediated defense responses, uncovering a mechanistic link to SA signaling and NPR1 activation in coordinating defense and growth.

Under normal growth conditions (left), NPR1 is phosphorylated at Ser-55 and Ser-59 (Ser-55/59) by active TOR (bright green) and sequestered in the cytoplasm via oligomerization, while SnRK1 (dim red) is inhibited by nutrient availability.

During immune responses (right), SA activates SnRK1 (bright red), leading to TOR inhibition and Ser-55/59 dephosphorylation. SA-triggered redox changes also facilitate nuclear translocation of NPR1. SnRK1 then phosphorylates NPR1 at Ser-557, partially activating it and priming it for subsequent PTMs. Ub, ubiquitination.

June 19, 2025 at 3:53 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Interesting work by Einhaus et al. (2025) on how CRISPR/Cas9-mediated combinatorial disruption of #epigenetic factors in the green alga #Chlamydomonas (e.g. ΔA51 strain = ΔSRTA, ΔDMC5 + ΔVIG1) improved transgene expression strength and stability after 3 months.
www.sciencedirect.com/science/arti...

Graphical abstract
Einhaus, A., Krieger, A., Köhne, L., Rautengarten, B., Jacobebbinghaus, N., Saudhof, M., Baier, T., & Kruse, O. (2025). Genome editing of epigenetic transgene silencing in Chlamydomonas reinhardtii. Trends in Biotechnology.
Created in BioRender. Kruse, O. (2025) https://BioRender.com/ucys7zl

Green microalgal biotechnology is limited by strong transgene silencing.
CRISPR-mediated combinatorial disruption of epigenetic factors in Chlamydomonas reinhardtii identified transgene silencing factors and improved transgene expression strength and stability.
In addition, an intein-based split selectable marker system was established for dual-targeted CRISPR, reducing the need for selectable markers.

Figure 4. Engineered terpenoid production from improved mutant strains.

(A) Top expressing transformants from each mutant strain were used for the production of patchoulol. The top-ten expressing transformants of 288 isolated transformants per biological triplicate for each knockout (KO) mutant strain (based on the initial evaluation, see Figure 2A in the main text) were combined into the respective boxplots. Volumetric production in mg l–1 is depicted in box and whiskers plots and was compared with the CC4350 wild-type (WT) strain by two-sided Student’s t-test for mean production assuming non-homogenous variances; ***P <0.001, *P <0.05, n.s. P >0.05.

(B) Initial transgene expression capacity of the ΔA51 and control strains depicted as fluorescence/OD750 normalized to the mean expression of the WT (left). The top-20 expressing transformants of 288 isolated transformants per biological triplicate of ΔA51 were combined into the respective boxplots. UVM4 and ΔA51 were compared with the WT by two-sided Student’s t-test for mean expression assuming non-homogenous variances; ***P <0.001, *P <0.05, n.s. P >0.05. Long-term stability of transgene expression (right) was assessed by repeated quantification of mVenus fluorescence after 3 months and compared with the initial quantification by two-sided Student’s t-test for mean expression assuming non-6homogenous variances; ***P <0.001, *P <0.05, n.s. P >0.05. The percentage change in mean expression of respective strains is detailed.

(C) Engineered terpenoid production from high cell-density cultivation of the nitrate-complemented UVM4 and ΔA51 strains. The top-ten expressing transformants of 288 isolated transformants each strain (initial evaluation) were analyzed. Volumetric terpenoid production from the ΔA51 strain was compared to UVM4 by two-sided Student’s t-test for mean production assuming non-homogenous variances; ***P <0.001, *P <0.05, n.s. P >0.05.

Abbreviations: DMC5= 5 HLM4= 4 MUT9= 9 SRTA= A VIG1= 1

June 2, 2025 at 7:22 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Mallén-Ponce et al. (2025) on how they identified #dihydroxyacetone phosphate (DHAP) as a key metabolite regulating the activation of #PlantTOR in the green alga #Chlamydomonas reinhardtii in response to #CO2 availability and light signals 🌅.
@ibvf-sevilla.bsky.social #PlantScience ⬇️

Fig 3E. Proposed model for the regulation of target of rapamycin (TOR) activity by dihydroxyacetone phosphate (DHAP) in the model green alga Chlamydomonas reinhardtii.

Photosynthesis provides reducing power [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)] and energy [ATP (adenosine 5′-triphosphate)] for CO2 fixation and the biosynthesis of organic carbon compounds. DHAP is synthesized in the chloroplast and exported to the cytoplasm through the TPT3 transporter. The abundance of cytoplasmic DHAP is transmitted to TOR to modulate its activity.

Figure S3. Schematic diagram of central carbon metabolism in the model green alga Chlamydomonas reinhardtii.

The Calvin Benson Bassham (CBB), tricarboxylic acid (TCA) and glutamine synthetase-glutamate synthase (GS-GOGAT) cycles, gluconeogenesis, glycolysis and lipid synthesis are shown.

The effect of dihydroxyacetone (DHA), dimethyl-alpha-ketoglutarate (DMKG), L-methionine sulfoximine (MSX) or cerulenin on central metabolism is also shown.
The fatty acid synthase (FAS) inhibitor cerulenin and the GS inhibitor MSX are indicated with red lines whereas the metabolic input of DHA and DMKG as precursors of DHAP and AKG, respectively, are indicated with blue arrows. The DHAP chloroplast-to-cytosol transporter triose-phosphate-translocator 3 (TPT3) is shown in pink.

Enzyme abbreviations are as follows: PRK, phosphoribulokinase; RuBisCo, ribulose 1,5-bisphosphate carboxylase oxygenase; PGK, phosphoglucokinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TK, triokinase; TPI, triosephosphate isomerase; FAS: fatty acid synthase; GS, glutamine synthetase; GOGAT, glutamate synthase; TPT3, triose-phosphatetranslocator 3; PDH, pyruvate dehydrogenase; CS, citrate synthase; ACO, aconitase; IL, isocitrate lyase; IDH, isocitrate dehydrogenase; ADH, alphaketoglutarate dehydrogenase; SCS, succinyl-CoA-synthase; SDH, succinicdehydrogenase; Fum, fumarase; MDH, malate dehydrogenase; DAK1, dihydroxyacetone kinase 1. Metabolite abbreviations: DHAP, dihydroxyacetone phosphate; Ru5P, ribulose 5-phosphate; RuBP, ribulose 1,5-bisphosphate; 3- PGA, 3-phosphoglycerate; GAP, glyceraldehyde 3-phosphate; Glc, glucose; Glc1P, glucose 1-phosphate; Glc6P, glucose 6-phosphate; AKG, alphaketoglutarate.

Fig. 2. Effect of dihydroxyacetone (DHA), cerulenin, dimethyl-α-ketoglutarate (DMKG), or L-methionine sulfoximine (MSX) on TOR activity.

(A) Immunoblot (upper panel) of P-RPS6/ RPS6 in Chlamydomonas cells treated with 2 mM DHA (0, 15, 30, and 60 min) over the night part of the diurnal cycle. Relative abundance (lower panel) of DHAP in cells treated with 2 mM DHA (0, 15, 30, and 60 min).

(B) Immunoblot (upper panel) of P-RPS6/ RPS6 in cells treated with 20 μM cerulenin (0, 15, 30, and 60 min) over the day part of the diurnal cycle. Relative abundance (lower panel) of DHAP in cells treated with 20 μM cerulenin (0, 15, 30, and 60 min).

(C) Immunoblot (upper panel) of P-RPS6/ RPS6 in cells treated with 2 mM DMKG (0, 15, 30, and 60 min) over the night part of the diurnal cycle. Relative abundance (lower panel) of DHAP in cells under the same conditions. (D) Immunoblot (upper panel) of P-RPS6/ RPS6 and relative abundance (lower panel) of Gln in cells treated or not with 5 mM MSX for 2 hours over the night part of the diurnal cycle and then reilluminated for 30 min.

(E) Immunoblot (upper panel) of P-RPS6/ RPS6 and relative abundance (lower panel) of Gln and DHAP in cells treated or not with 5 mM MSX for 2 hours and then 2 mM DHA for 30 min over the day part of the diurnal cycle.

Coomassie brilliant blue–stained gels were used as a loading control. Chlamydomonas cells were grown in TP medium. Error bars correspond to SD from at least three biological replicates. Asterisks represent significant differences according to one-way ANOVA: ***P < 0.001; **P < 0.01; *P < 0.05; not significant, ≥0.05. The statistical analyses described apply to all statistical analyses in this figure (detailed in table S8). White and black bars indicate light and dark phases, respectively.

April 22, 2025 at 3:02 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Very interesting review by Liu, Hu et al. (2025) on the signaling landscape of #PlantTOR pathway (i.e. upstream signals and downstream effectors) and the physiological roles of #PlantTOR signaling in plant growth, development, and stress responses 🌱. www.annualreviews.org/content/jour...

Figure 2. Representative TOR-specific chemical inhibitors in the green plant lineage.

The structures of ATP and TOR-specific chemical inhibitors docking to plant TOR complexes were predicted by using AlphaFold 3.

For more detail, please refer to Supplemental Table 2. Abbreviations: Ath, Arabidopsis thaliana; FKBP12, FK506-binding protein 12; FRB, FKBP-rapamycin binding; LST8, lethal with SEC13 protein 8; TOR, target of rapamycin.

Figure 3. Upstream signals and regulators of plant TOR signaling.

The plant TORC integrates a variety of upstream signals to gate its own activity and function. Activation of TORC only occurs when glucose/energy, mineral nutrients, growth-related hormones, and favorable environmental conditions are all readily available.

Conversely, low energy, nutrient scarcity, and stresses lead to TORC inhibition. Transporters depicted include AKT, Arabidopsis potassium (KC) transporter (65); AMT, ammonium (NH4 C) transporter (74); IRT, iron (Fe2C) transporter (24); NRT, nitrate (NO3 -) transporter (74); PHT, phosphate (PO4 3-) transporter (23); and SULTR, sulfate (SO4 2-) transporter (141). Positive regulators of TORC are depicted in orange; negative regulators are in light gray blue.

Figure 4. Downstream targets and molecular functions of plant TOR signaling.

Plant TORC orchestrates a myriad of cellular, molecular, and metabolic changes to balance anabolic and catabolic programs by directly or indirectly regulating key components.

(a–e, left) Summary of plant TORC downstream substrates, regulators, and their related molecular functions. (Right) Illustration of the central dogma in the plant cell.
( f ) Summary of plant TORC-regulated hormone signaling pathways.

Positive regulators are depicted in light green; negative
regulators are in light gray-blue. Phosphorylation regulation is depicted by the phosphate group shown in pink, and the biological
processes are shown in light cyan. Solid lines with triangle arrows (positive) and with vertical bars (negative) represent confirmed
signaling regulation. The dashed line indicates that this regulation needs further experimental validation.

Figure 5. Physiological roles of TOR signaling in plant growth, development, and stress adaptation.

(a, i–vii) By integrating upstream signals and regulating multiple cellular processes, plant TOR signaling ultimately regulates every stage of an individual life cycle, from embryo development to senescence, and (b) participates in diverse stress adaptation and growth–defense trade-offs. The key upstream signals and regulators are shown. External and internal signals involved in TOR regulating plant growth, development, and stress adaptation processes are labeled in brown (activation signals) and blue (inhibition signals).

Positive and negative regulators are labeled in orange and black, respectively. Black lines with triangle arrows (positive) and with vertical bars (negative) represent signaling regulation.

April 9, 2025 at 3:02 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Zheng et al. (2025) on how employing pupylation-based proximity labeling (PUP-IT) unravels a comprehensive #interactome of #Arabidopsis TOR complex.
Newly identified #PlantTOR interactors, like PANK2, were also supported by #AlphaFold2.
advanced.onlinelibrary.wiley.com/doi/10.1002/...

Figure 1. Combining different treatments and baits determines different components of the TORC interactome.

A) Scheme illustrating PUP-IT in the context of TOR signaling. PafA fused to the baits LST8-1, RAPTOR1, and ScFKBP labels individual and TOR complex associated protein interactors, while a GFP fusion acts as a control for unspecific interactions.

B–E) Volcano plots showing proteins significantly enriched in LST8-1 B,D) and RAPTOR1 C,E) baits using constitutive B,C) or inducible D,E) FLAG::PUP(E) expression.

F–G) Treatment-dependency of identified interactors using LST8-1 G) or RAPTOR1 H) baits and inducible FLAG::PUP(E) expression. Treatment-specific interactors are those only identified in one of the two treatments.

H) Rapamycin response of WT and ScFKBP-producing seedlings (n = 10 seedlings). Bar height indicates group median.

I) Volcano plot showing proteins significantly enriched in the ScFKBP bait using constitutive FLAG::PUP(E) expression.

In all volcano plots, fold changes are calculated from n = 3 replicates using MsqRob2, p-values are corrected for multiple comparisons using the Benjamini–Hochberg FDR method. Different letters in panel (H) indicate statistically significant differences between groups based on a one-way ANOVA followed by a Tukey-HSD test (𝛼 = 0.05).

Figure 2. Identification of phosphorylated direct interactors of the TOR complex and in silico corroboration using AlphaFold2.

A,B) Phosphorylated proteins enriched in LST8-1 and RAPTOR1 against the GFP control after 4 h A) and 24 h B) of sucrose treatment and FLAG::PUP(E) induction. Phosphorylation sites previously associated with TOR are indicated in blue, new sites in proteins previously reported to be phosphorylated by TOR in turquoise. Fold changes are calculated from n = 3 replicates using MsqRob2, p-values are corrected for multiple comparisons using the Benjamini–Hochberg FDR method.

C,D) The two significantly enriched motifs among the identified phosphorylation sites. The “SP” motif C) mirrors previous reports from TOR substrates, while the “RxxS” motif D) has previously been associated with TOR downstream interactor S6K1.

E) Interactions of 20 proteins from the four shown groups with the TORC components TOR, LST8-1, and RAPTOR1 were predicted using AlphaFold2 multimer. Vertical lines indicate the median of the local interaction score (LIS) distribution by group.

F) Predicted structures of the top-scoring interactions for each group: newly identified TORC interactor PANK2, TORC subunit RAPTOR1, senescence regulator S40-7, and KIN10 paralog KIN11.
Models are colored by predicted local distance difference test (pLDDT), with values above 70 indicating high confidence predictions.

March 24, 2025 at 8:15 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Le, Choi, Kim et al. (2025) on how the functional fusion of #Agrobacterium VirD2-derived nuclear localization signal (NLS) to #Cas9 improved its nuclear import, thereby doubling the gene-editing frequency in #algae, #Chlamydomonas and Chlorella Sp. HS2.
🔗 www.pnas.org/doi/10.1073/...

Fig. 4. Visual confirmation of localization using NLS-bearing mCherry reporters and Cas9 variants, and in vitro cleavage assay of NLS-fused Cas9 variants.

(E) Box and whisker plots show the fluorescence ratio (as a localization measure) derived from four biological replicates and quantified using ImageJ software. The median (the center line) ± whiskers (1.5 × the interquartile range of the lower and upper quartiles) are shown. Student’s t test was performed. The fold-changes are presented with the statistical significance. The region of interest for the quantification is indicated in the inset.

(F) Expected cut site and lengths of the resulting cleavage fragments when PCR-amplified MAA7 (Cre03.g161400) gene of Chlamydomonas reinhardtii is subjected to in vitro cleavage.
cCas9 indicates a commercial Cas9. cPAM: complementary protospacer adjacent motif; U.D.: uncleaved DNA (i.e., intact PCR product); and C.F.: cleavage fragment.

Fig. 5. In vivo application of Cas9 variants for algal mutagenesis and investigation of the genetic variance as a result of NHEJ.

(A) Representative plate images obtained from the mutagenesis of Chlamydomonas reinhardtii by each Cas9 variant.

(B) Simplified description of a strain selection strategy based on tryptophan auxotrophy, when the MAA7 gene is disrupted. The synthesis of the proteinogenic amino acid, L-Trp, from intracellularly synthesized indole is impaired by ΔMAA7. It also deprives cells of the ability to synthesize the cytotoxic, nonproteinogenic amino acid, 5-Fluoro- L- Trp, which is produced when 5-fluoroindole is supplied extracellularly.

(C) Mutagenesis frequencies from two scoring methods, namely the number of colony formations and short-read deep sequencing (SR deep seq.), are compared. The absence and presence of L-Trp and 5-FI in each assay are indicated below two vertical axes. The mutation frequencies from the SR deep seq. were corrected with the error rate from the WT sample (SI Appendix, Table S2) and are presented as bars. The individual colony count data are shown as dots with biological triplicates. Floating bars represent the mean. Student’s t test was performed on the frequencies from the colony counts. The fold-changes are presented together with the statistical significance. The mutagenesis frequency is further calibrated with the mutagenesis efficiency calculated from the Sanger sequencing results (Table 2).

Fig. 6. Validation of cross-species versatility using industrial microalga, Chlorella Sp. HS2.

(A) The three-dimensional structure of the putative Impα (Hscell_00006266) in Chlorella Sp. HS2 was predicted using AlphaFold. The structural homology provided a rationale for applying the NLS-mediated delivery enhancement strategy. Armadillo repeat motifs (Arms) in the predicted structure are presented. N and C termini are indicated. A microscopic image of Chlorella Sp. HS2 is shown at the Upper Right. (Scale bar, 3 μm.)

(B) Representative plate images (from replicate 1 in SI Appendix, Table S3) obtained from the mutagenesis of Chlorella Sp. HS2 targeting the MAA7 gene by each Cas9 variant.

(C) Mutagenesis frequencies from the targeted mutagenesis frequency derived from colony formation followed by Sanger sequencing and SR deep seq. are compared. The absence and presence of L-Trp and 5-FI in each assay are indicated below two vertical axes. The mutation frequencies from the SR deep seq. were corrected with the error rate from the WT sample (SI Appendix, Table S4) and are presented as bars. The individual targeted mutagenesis frequency data are shown as dots with technical triplicates. Floating bars represent the mean. Student’s t test was performed on the targeted mutagenesis frequencies. The fold-changes are presented together with the statistical significance.

March 4, 2025 at 5:01 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Betterle, Gasparotto et al. (2025) on the engineering of the fast-growing cyanobacterium #Synechococcus sp. PCC 11901 to synthesize #astaxanthin 🔴 by heterologous expression of bKT and CtrZ genes, reaching an Asta productivity of ~ 9.6 mg/L/day under photoautotrophic growth conditions.

Fig.7 Cultivation of Synechococcus sp. PCC 11901 strain that is expressing β-carotene ketolase (bKT) and β-carotene hydroxylase (CtrZ) recombinant enzymes to produce astaxanthin (called BC) in an industrial photobioreactor (PBR).

a) BC strain was photoautotrophically grown in batch mode in a 330L-PBR with limited sterility. The numbers on top of each picture indicate the day of growth.

b) Biomass accumulation curve of BC strain, as measured from the optical density of the cultures at 720 nm. Cultures were inoculated to an initial cell concentration corresponding to OD720 0.037 ± 0.003. Error bars represent standard deviation (n = 4)

March 3, 2025 at 5:47 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Chaudhary et al. (2025) on how #FERONIA (FER), a cell wall integrity sensor, is essential for limiting #PlantCellWall damage and preventing cell rupture during #brassinosteroid (BR)-induced cell elongation in #Arabidopsis.
BR signaling promotes FER accumulation at the plasma membrane.

Figure 6. The BR/BIN2-FER signaling circuit ensures integrity during brassinosteroid (BR)-induced cell expansion in Arabidopsis.

When the BR level is low (-BR), the GSK3-like kinase BIN2 inhibits cell expansion by phosphorylating and inactivating BZR1 family transcription factors and cellulose synthase (CesA). BIN2 phosphorylates the wall-sensing receptor kinase FERONIA (FER) to maintain a low level of FER on the plasma membrane (PM) when the cell is not expanding.

When the BR level is high (+BR), BR activates the receptor kinase BRI1, leading to BIN2 inactivation, which results in BZR1 accumulation, expression of growth-promoting genes, activation of cellulose synthesis, and accumulation of FER at the PM.
BRI1 also phosphorylates the PM H+-ATPase (AHA) to acidify the cell wall and polarize the PM, which loosens the cell wall and increases the turgor pressure through water uptake by aquaporin (PIP). When cell expansion causes initial wall damage, cell wall pectin fragments and RALF peptide together activate FER, which inhibits AHA and PIP, leading to PM depolarization, cell wall alkalinization, and reduced water uptake and turgor pressure. FER signaling also increases calcium and ROS levels.

Together, the FER-induced cellular responses rapidly reduce turgor pressure, slow down cell expansion, and strengthen cell wall to prevent cell rupture. Thin and thick lines show inactivated and activated functions, whereas arrows and bars show activation and inhibition, respectively.

February 7, 2025 at 7:05 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Urrea-Castellanos et al. (2025) on how RAPTOR1B, a subunit of #PlantTOR complex (TORC), post-transcriptionally regulates CONSTANS (CO), a component of the #photoperiod pathway to promote flowering, via #circadian clock-associated protein GIGANTEA (GI) 🌻.
🔗 www.pnas.org/doi/10.1073/...

Fig. 5. RAPTOR1B functions upstream of GIGANTEA (GI) to contribute to CONSTANS (CO) stability at dusk and thus promote the floral transition in Arabidopsis.

(F) Proposed model for the role of RAPTOR during floral transition in long days (LD).

The length of the light period is primarily sensed in leaves by the combined action of light signaling (mediated by PHYA and CRYs) and the circadian clock (driving gene expression of effector proteins, such GI and FKF1). This accounts for protein accumulation of CONSTANS at dusk, which in turn, upregulates the florigen, FT. FT protein travels from the leaves into the shoot apex via the vasculature to induce the expression of floral integrator genes (such SOC1) and flower promoting genes (such SPL3/4/5). GI, similar to CO, accumulates over the day and is degraded at night. Independent, or together with FKF1 in response to blue light-mediated by CRYs, GI helps to stabilize CONSTANS at dusk in LD.

RAPTOR, which is part of the TORC, contributes to CO stability at dusk by promoting GI levels posttranscriptionally. TORC is known to be activated by light and sugars, suggesting that RAPTOR conveys nutrient status, in this case carbon availability, to regulate GI accumulation and thus fine-tune flowering via the photoperiod pathway. Remarkably, TOR is essential for sugar-mediated regulation of the circadian period, while GI integrates signals from the circadian clock, thus strengthening the potential crosstalk between these proteins.

February 4, 2025 at 6:19 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great #preprint by Rawat and Laxmi (2025) on how salicylic acid (SA)-induced primary root growth inhibition in #Arabidopsis seedlings is regulated by the glucose-auxin-TOR-E2Fa module. #PlantTOR
🔗 www.biorxiv.org/content/10.1...

Figure 8. The interplay of glucose, auxin and TOR-E2Fa in mediating root growth repression by the defense hormone, salicylic acid (SA).

A testable model for glucose and SA crosstalk. Glucose promotes root growth by stimulating auxin biosynthesis, signaling and transport. Both glucose and auxin activate TOR kinase, which, in turn, activate RPS6 and E2Fa, key players in cell cycle regulation. E2Fa binds to S-phase cell cycle gene promoters, upregulating their expression and promoting cell growth and proliferation. Conversely, SA appears to inhibit root growth by antagonizing auxin signaling and transport. Additionally, SA reduces the phosphorylation levels of both RPS6 and E2Fa, negatively impacting cell growth and proliferation processes, ultimately leading to root growth inhibition.

February 3, 2025 at 4:08 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by Li, Kim et al. (2025) on how knocking out the carboxyltransferase interactor 1 (CTI1) in #Chlamydomonas boosted #triacylglycerol (TAG) content by 5-fold compared to wild-type without compromising #microalgae growth under mixotrophic conditions.
🔗 onlinelibrary.wiley.com/doi/full/10....

Figure 3. Chlamydomonas reinhardtii carboxyltransferase interactor 1 (CrCTI1) mutants made more oil during mixotrophic growth without compromising photosynthesis yield and growth.

(a) Total fatty acid content measured by GC–MS. FAME, fatty acid methyl ester.
(b) TAG content was measured by thin-layer chromatography (TLC).
(c) Confocal fluorescence microscopy images of Chlamydomonas cells. Green, Bodipy fluorescence; red, chlorophyll autofluorescence. Images were present in high and low magnification. Scale bars = 10 µm.
(d) Chlamydomonas cell growth curve.
(e) PSII yield (Y(II)) measurement.

Data are means of three biological replicates with standard deviation shown. *P < 0.05 and ***P < 0.001 (Student’s t-test).

January 31, 2025 at 3:28 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great #preprint by @wsmagghe.bsky.social (2024) on applying this #ASP-SPARK sensor in combination with tissue-specific knockout (CRISPR-TSKO) of #SnRK1 to dissect its roles at cellular resolution during #Arabidopsis plant growth and development. #PlantScience
🔗 www.researchsquare.com/article/rs-5...

Figure 4. CRISPR-based tissue-specific knockout (CRISPR-TSKO) of the key energy sensor in plants, Sucrose non-fermenting-1 related kinase 1 (SnRK1), during Arabidopsis root development.

a. Scheme illustrating the CRISPR-TSKO strategy that was used to target the two SnRK1 catalytic α-subunits in different hotspots of SnRK1 activity during growth and development. Cas9 is only expressed in the tissue of interest, allowing the assessment of SnRK1 function in that tissue, omitting pleiotropic effects of SnRK1 knock-out elsewhere.

b. Overview of the different tissue-specific promotors that were used.

c. Representative confocal images of the SPARK signal and Cas9-BFPnls expression in the different CRISPR-TSKO mutants harboring the pSMB-Cas9 constructs. In the SnRK11/2 double mutant, the SPARK signal is lost in the root cap cells (indicated with white arrowheads). Scale bars = 50 μm.

d. Widefield microscopic images of roots from the SnRK11/2 TSKO double mutant stained with Ruthenium red for pectin content.

e. Bar plot showing the mean relative pectin content from the ASP-SPARK WT, and pSMB #4 and pSMB #5 mutants (T2 seedlings from two independent T1 plants). Error bars represent the standard deviation, N>5, One-way ANOVA, followed by a Fisher’s LSD test, compared to the WT. **** = p<0.001.

f. Bar plot with the mean relative expression levels of genes related to root cap cell release in WT ASP-SPARK and pSMB #4 and pSMB #5 CRISPR-TSKO ASP-SPARK seedlings. Error bars represent the standard deviation, N=4, One-way ANOVA, followed by a Fisher’s LSD test, compared to the WT. ns = p>0.05, **** = p<0.001.

December 19, 2024 at 7:35 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Great work by He et al. (2024) on boosting transcriptional activities in #TranscriptionFactors (TFs) by employing tandemly repeated activation domains, especially two R2R3-MYB TFs from #Arabidopsis and #Epimedium that are involved in #anthocyanin biosynthesis 🌺. #PlantSci
🔗 doi.org/10.1093/plce...

Figure 9. Schematic diagram showing a proposed strategy for boosting the transcriptional activities of transcription factors (TFs).

A) Natural TFs usually have a DNA-binding domain (DBD) and a transcriptional activation domain (AD), and these natural TFs can regulate the expression of multiple genes. TFs recruited co-activator and other TFs to establish a transcription initiation complex by stabilization of binding and in part via cofactor interactions.

B) Synthetic TFs, with tandemly repeated DBDs leading to protein misfolding, fail to bind to the promoter of target genes, resulting in the loss of transcriptional activation of downstream genes.

C) Synthetic TFs with tandemly repeated ADs show increased transcriptional activation activity, possibility because synthetic TFs recruit more coactivators into the transcription complexes.

Modified Figure. Functional analysis of synthetic transcription factors (TFs) containing tandemly repeated DNA-binding domains (DBDs) or tandemly repeated transcriptional activation domains (ADs), which are derived from the R2R3-MYB TF, AtPAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1 from Arabidopsis thaliana).

Fig. 4A and 6A) Schematic diagram of AtPAP1, which contains one DBD and one AD, and synthetic TFs containing tandemly repeated DBDs or ADs. P2R contains two tandemly repeated DBDs containing R2R3-MYB motifs. P2C and P4C have two and four tandemly repeated ADs from native AtPAP1, respectively.

Modified Fig. 4E and 6E) Phenotypes of the transgenic tobacco flowers overexpressing P2R, P2C or P4C. Bars=10 mm.

Fig. 4G and 6G) Anthocyanin content in the transgenic tobacco flowers overexpressing P2R, P2C or P4C.

December 19, 2024 at 4:38 PM

Takehiro A. Ozawa

@tkozawa.bsky.social

Amazing resource by @mjonikas.bsky.social Lab (2024), providing a next-generation #Chlamydomonas mutant library (CLiP2) with greatly-increased genome coverage.
CLiP2 contains 71,700 strains that have different background, mating type and antibiotic marker (hygromycin) than the original collection ⬇️.

Chlamydomonas mutants with insertions in genes of interest can be searched in CLiP website (https://www.chlamylibrary.org/).
In this case, new PSR1 mutants were found in the CLiP2 library.

These mutants can also be ordered online in the Chlamydomonas Resource Center website (https://www.chlamycollection.org/).

December 18, 2024 at 4:40 AM

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