Search Results (9)

Cool-season creeping bentgrass (Agrostis stolonifera L., As) is extensively used on golf courses worldwide and is negatively affected by several fungal diseases and abiotic stresses including drought and salinity. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) gene editing technology was employed in this project to knock out the AsDREBL (dehydration responsive element binding-like factor) gene, a potential negative regulator in stress tolerance. With our established single guide RNA (sgRNA)-based CRISPR-editing vector and optimized creeping bentgrass tissue culture system using mature seed-derived embryogenic calli of cv. Crenshaw as explant, more than 20 transgenic plants were produced by gene gun bombardment. Fifteen confirmed AsDREBL mutant plants were tested for drought and salinity tolerance by withholding water and applying salt spray in greenhouse settings. Some of the mutants were shown to be more tolerant of drought and salinity stress compared to the non-edited, wild type Crenshaw plants. Our results demonstrate that CRISPR-gene editing technology can be successfully applied to improve the agronomical traits of turfgrass. Full article

The chloroplast signal recognition particle (cpSRP) components cpSRP43 and cpSRP54 not only form a complex with light-harvesting chlorophyll (Chl)-binding proteins to direct them to the thylakoid membrane, but also serve other functions. cpSRP43 independently acts as a chaperone for some tetrapyrrole biosynthesis (TBS) enzymes, while cpSRP54 participates in the co-translational targeting of plastid-encoded proteins. However, it remains unclear to what extent the two cpSRP components are coregulated—despite their distinct functions—and whether both participate in genomes-uncoupled (GUN)-mediated retrograde signaling. Here, we demonstrate that cpSRP43 and cpSRP54 accumulation is strongly interdependently controlled: overexpression of one protein increases the level of the other, while a deficiency in one of the two proteins leads to a simultaneous decrease in the other component. Disruption of this balance, e.g., by combining the overexpression of one component with a knockout of the other, results in severe chlorosis, stunted growth, and reduced levels of Chl and tetrapyrrole intermediates. Moreover, cpSRP43 deficiency exacerbates the pale-green phenotype of gun4 and gun5 mutants, highlighting a synergistic impact on TBS; however, cpSRP43 overexpression fails to rescue these defects. Remarkably, loss of cpSRP43 does not affect the expression of nuclear-encoded photosynthetic genes under intrinsic plastid stress, clearly demonstrating that cpSRP43 is not involved in plastid-to-nucleus retrograde signaling. Overall, our findings underscore that the fine-tuned expression of cpSRP43 and cpSRP54 is crucial for proper chloroplast function and pigment biosynthesis, while cpSRP43 alone does not participate in the retrograde signaling pathway. Full article

Albinism is a unique problem encountered in tissue culture experiments, but the underlying mechanism remains unclear in most bamboo species. In this study, we identified the putative regulatory genes in an albino mutant of Bambusa oldhamii using comparative physiology and transcriptome analysis. The degeneration of chloroplasts, low chlorophyll (Chl) content and reduced photosynthetic capacity were observed in albinotic B. oldhamii compared to normal lines. A total of 6191 unigenes were identified that were clearly differentially expressed between albino and normal lines by transcriptome sequencing. Most genes related to chloroplast development (such as Psa, Psb) and pigment biosynthesis (such as LHC, GUN4, ZEP) were downregulated significantly in albinotic lines, which might be responsible for the albino phenotype. Moreover, some transcription factors (TFs) such as PIF and GLK1 were identified to be involved in chloroplast development and Chl synthesis, indicating the involvement of putative regulatory pathways PIF-LHC and GLK1-LHC/Psa/Psb in albinotic B. oldhamii. Finally, the downregulation of some stress responsive TFs (like ICE1 and EREB1) suggested a reduction in stress resistance of albinotic B. oldhamii. The above findings provided new insights into the molecular mechanism of albinism in bamboo. Full article

The chloroplast is a key organelle for photosynthesis and perceiving environmental information. GENOME UNCOUPLED 4 (GUN4) has been shown to be required for the regulation of both chlorophyll synthesis, reactive oxygen species (ROS) homeostasis and plastid retrograde signaling. In this study, we found that growth of the gun4 mutant was significantly improved under medium strong light (200 μmol photons m⁻²s⁻¹) compared to normal light (100 μmol photons m⁻²s⁻¹), in marked contrast to wild-type (WT). Further analysis revealed that GUN4 interacts with SIGNAL RECOGNITION PARTICLE 54 KDA SUBUNIT (SRP43) and SRP54. RNA-seq analysis indicated that the expression of genes for light signaling and the circadian clock is altered in gun4 compared with (WT). qPCR analysis confirmed that the expression of the clock genes CLOCK-RELATED 1 (CCA1), LATE ELONGATION HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO RESPONSE REGULATOR 7 (PRR7) is significantly changed in the gun4 and srp54 mutants under normal and medium strong light conditions. These results suggest that GUN4 may coordinate the adaptation of plants to changing light conditions by regulating the biological clock, although it is not clear whether the effect is direct or indirect. Full article

Chloroplasts are semi-autonomous organelles governed by the precise coordination between the genomes of their own and the nucleus for functioning correctly in response to developmental and environmental cues. Under stressed conditions, various plastid-to-nucleus retrograde signals are generated to regulate the expression of a large number of nuclear genes for acclimation. Among these retrograde signaling pathways, the chloroplast protein GENOMES UNCOUPLED 1 (GUN1) is the first component identified. However, in addition to integrating aberrant physiological signals when chloroplasts are challenged by stresses such as photooxidative damage or the inhibition of plastid gene expression, GUN1 was also found to regulate other developmental processes such as flowering. Several partner proteins have been found to interact with GUN1 and facilitate its different regulatory functions. In this study, we report 15 possible interacting proteins identified through yeast two-hybrid (Y2H) screening, among which 11 showed positive interactions by pair-wise Y2H assay. Through the bimolecular fluorescence complementation assay in Arabidopsis protoplasts, two candidate proteins with chloroplast localization, DJC31 and HCF145, were confirmed to interact with GUN1 in planta. Genes for these GUN1-interacting proteins showed different fluctuations in the WT and gun1 mutant under norflurazon and lincomycin treatments. Our results provide novel clues for a better understanding of molecular mechanisms underlying GUN1-mediated regulations. Full article

During a plant’s life cycle, plastids undergo several modifications, from undifferentiated pro-plastids to either photosynthetically-active chloroplasts, ezioplasts, chromoplasts or storage organelles, such as amyloplasts, elaioplasts and proteinoplasts. Plastid proteome rearrangements and protein homeostasis, together with intracellular communication pathways, are key factors for correct plastid differentiation and functioning. When plastid development is affected, aberrant organelles are degraded and recycled in a process that involves plastid protein ubiquitination. In this study, we have analysed the Arabidopsis gun1-102 ftsh5-3 double mutant, lacking both the plastid-located protein GUN1 (Genomes Uncoupled 1), involved in plastid-to-nucleus communication, and the chloroplast-located FTSH5 (Filamentous temperature-sensitive H5), a metalloprotease with a role in photosystem repair and chloroplast biogenesis. gun1-102 ftsh5-3 seedlings show variegated cotyledons and true leaves that we attempted to suppress by introgressing second-site mutations in genes involved in: (i) plastid translation, (ii) plastid folding/import and (iii) cytosolic protein ubiquitination. Different phenotypic effects, ranging from seedling-lethality to partial or complete suppression of the variegated phenotype, were observed in the corresponding triple mutants. Our findings indicate that Plant U-Box 4 (PUB4) E3 ubiquitin ligase plays a major role in the target degradation of damaged chloroplasts and is the main contributor to the variegated phenotype observed in gun1-102 ftsh5-3 seedlings. Full article

Chloroplast biogenesis requires the coordinated expression of the chloroplast and nuclear genomes, which is achieved by communication between the developing chloroplasts and the nucleus. Signals emitted from the plastids, so-called retrograde signals, control nuclear gene expression depending on plastid development and functionality. Genetic analysis of this pathway identified a set of mutants defective in retrograde signaling and designated genomes uncoupled (gun) mutants. Subsequent research has pointed to a significant role of tetrapyrrole biosynthesis in retrograde signaling. Meanwhile, the molecular functions of GUN1, the proposed integrator of multiple retrograde signals, have not been identified yet. However, based on the interactions of GUN1, some working hypotheses have been proposed. Interestingly, GUN1 contributes to important biological processes, including plastid protein homeostasis, through transcription, translation, and protein import. Furthermore, the interactions of GUN1 with tetrapyrroles and their biosynthetic enzymes have been revealed. This review focuses on our current understanding of the function of tetrapyrrole retrograde signaling on chloroplast biogenesis. Full article

The SA-4-1BBL, an oligomeric novel form of the natural ligand for the 4-1BB co-stimulatory receptor of the tumor necrosis factor (TNF) superfamily, as a recombinant protein has potent pleiotropic effects on cells of innate, adaptive, and regulatory immunity with demonstrated therapeutic efficacy in several tumor models. However, the production of soluble form of SA-4-1BBL protein and quality control is time and resource intensive and face various issues pertinent to clinical development of biologics. The present study sought to take advantage of the simplicity and translatability of DNA-based vaccines for the production and delivery of SA-4-1BBL for cancer immune prevention and therapy. A chimeric HPV-16 E7 DNA vaccine (SP-SA-E7-4-1BBL) was constructed that contains the signal peptide (SP) of calreticulin (CRT), streptavidin (SA) domain of SA-4-1BBL, HPV-16 E7 double mutant gene, and the extracellular domain of mouse 4-1BBL. Immunization by gene gun with SP-SA-E7-4-1BBL induced greater prophylactic as well as therapeutic effects in C57BL/6 mice against TC-1 tumor model compared with immunization with E7wt, SP-SA-4-1BBL or reference-positive control CRT-E7wt. The therapeutic efficacy of the DNA vaccine was associated with increased frequency of E7-specific T cells producing interferon (IFN)-γ. Overall, our data suggest that this DNA-based vaccine strategy might represent a translational approach because it provides a simpler and versatile alternative to a subunit vaccine based on SA-4-1BBL and E7 proteins. Full article

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis. Full article