Arabidopsis Phytochrome B Promotes SPA1 Nuclear Accumulation to Repress Photomorphogenesis under Far-Red Light

Phytochrome A (phyA) is the primary photoreceptor mediating deetiolation under far-red (FR) light, whereas phyB predominantly regulates light responses in red light. SUPPRESSOR OF PHYA-105 (SPA1) forms an E3 ubiquitin ligase complex with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), which is responsible for the degradation of various photomorphogenesis-promoting factors, resulting in desensitization to light signaling. However, the role of phyB in FR light signaling and the regulatory pathway from light-activated phytochromes to the COP1-SPA1 complex are largely unknown. Here, we confirm that PHYB overexpression causes an etiolation response with reduced ELONGATED HYPOCOTYL5 (HY5) accumulation under FR light. Notably, phyB exerts its nuclear activities and promotes seedling etiolation in both the presence and absence of phyA in response to FR light. PhyB acts upstream of SPA1 and is functionally dependent on it in FR light signaling. PhyB interacts and forms a protein complex with SPA1, enhancing its nuclear accumulation under FR light. During the dark-to-FR transition, phyB is rapidly imported into the nucleus and facilitates nuclear SPA1 accumulation. These findings support the notion that phyB plays a role in repressing FR light signaling. Activity modulation of the COP1-SPA E3 complex by light-activated phytochromes is an effective and pivotal regulatory step in light signaling.     

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.     

Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants

Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs.     

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

Aminoalcoholphosphotransferase (AAPT) catalyzes the synthesis of phosphatidylcholine (PC) and phosphotidylethanolamine (PE), which are the most prevalent membrane phospholipids in all eukaryotic cells. Here, we show that suppression of AAPTs results in extensive membrane phospholipid remodeling in Arabidopsis thaliana. Double knockout (KO) mutants that are hemizygous for either aapt1 or aapt2 display impaired pollen and seed development, leading to embryotic lethality of the double KO plants, whereas aapt1 or aapt2 single KO plants show no overt phenotypic alterations. The growth rate and seed yield of AAPT RNA interference (RNAi) plants are greatly reduced. Lipid profiling shows decreased total galactolipid and phospholipid content in aapt1-containing mutants, including aapt1, aapt1/aapt1 aapt2/AAPT2, aapt1/AAPT1 aapt2/aapt2, and AAPT RNAi plants. The level of PC in leaves was unchanged, whereas that of PE was reduced in all AAPT-deficient plants, except aapt2 KO. However, the acyl species of PC was altered, with increased levels of C34 species and decreased C36 species. Conversely, the levels of PE and phosphatidylinositol were decreased in C34 species. In seeds, all AAPT-deficient plants, including aapt2 KO, displayed a decrease in PE. The data show that AAPT1 and AAPT2 are essential to plant vegetative growth and reproduction and have overlapping functions but that AAPT1 contributes more than AAPT2 to PC production in vegetative tissues. The opposite changes in molecular species between PC and PE and unchanged PC level indicate the existence of additional pathways that maintain homeostatic levels of PC, which are crucial for the survival and proper development of plants.     

Pipecolic Acid,an Endogenous Mediator of Defense Amplification and Priming,Is a Critical Regulator of Inducible Plant Immunity

Metabolic signals orchestrate plant defenses against microbial pathogen invasion. Here, we report the identification of the non-protein amino acid pipecolic acid (Pip), a common Lys catabolite in plants and animals, as a critical regulator of inducible plant immunity. Following pathogen recognition, Pip accumulates in inoculated Arabidopsis thaliana leaves, in leaves distal from the site of inoculation, and, most specifically, in petiole exudates from inoculated leaves. Defects of mutants in AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) in systemic acquired resistance (SAR) and in basal, specific, and β-aminobutyric acid–induced resistance to bacterial infection are associated with a lack of Pip production. Exogenous Pip complements these resistance defects and increases pathogen resistance of wild-type plants. We conclude that Pip accumulation is critical for SAR and local resistance to bacterial pathogens. Our data indicate that biologically induced SAR conditions plants to more effectively synthesize the phytoalexin camalexin, Pip, and salicylic acid and primes plants for early defense gene expression. Biological priming is absent in the pipecolate-deficient ald1 mutants. Exogenous pipecolate induces SAR-related defense priming and partly restores priming responses in ald1. We conclude that Pip orchestrates defense amplification, positive regulation of salicylic acid biosynthesis, and priming to guarantee effective local resistance induction and the establishment of SAR.     

Cytoplasmic Nucleation and Atypical Branching Nucleation Generate Endoplasmic Microtubules in Physcomitrella patens

The mechanism underlying microtubule (MT) generation in plants has been primarily studied using the cortical MT array, in which fixed-angled branching nucleation and katanin-dependent MT severing predominate. However, little is known about MT generation in the endoplasm. Here, we explored the mechanism of endoplasmic MT generation in protonemal cells of Physcomitrella patens. We developed an assay that utilizes flow cell and oblique illumination fluorescence microscopy, which allowed visualization and quantification of individual MT dynamics. MT severing was infrequently observed, and disruption of katanin did not severely affect MT generation. Branching nucleation was observed, but it showed markedly variable branch angles and was occasionally accompanied by the transport of nucleated MTs. Cytoplasmic nucleation at seemingly random locations was most frequently observed and predominated when depolymerized MTs were regrown. The MT nucleator γ-tubulin was detected at the majority of the nucleation sites, at which a single MT was generated in random directions. When γ-tubulin was knocked down, MT generation was significantly delayed in the regrowth assay. However, nucleation occurred at a normal frequency in steady state, suggesting the presence of a γ-tubulin-independent backup mechanism. Thus, endoplasmic MTs in this cell type are generated in a less ordered manner, showing a broader spectrum of nucleation mechanisms in plants.