The E3 ubiquitin ligase COP1 regulates salt tolerance via GIGANTEA degradation in roots.

Excess soil salinity significantly impairs plant growth and development. Our previous reports demonstrated that the core circadian clock oscillator GIGANTEA (GI) negatively regulates salt stress tolerance by sequestering the SALT OVERLY SENSITIVE (SOS) 2 kinase, an essential component of the SOS pathway. Salt stress induces calcium-dependent cytoplasmic GI degradation, resulting in activation of the SOS pathway; however, the precise molecular mechanism governing GI degradation during salt stress remains enigmatic. Here, we demonstrate that salt-induced calcium signals promote the cytoplasmic partitioning of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), leading to the 26S proteasome-dependent degradation of GI exclusively in the roots. Salt stress-induced calcium signals accelerate the cytoplasmic localization of COP1 in the root cells, which targets GI for 26S proteasomal degradation. Align with this, the interaction between COP1 and GI is only observed in the roots, not the shoots, under salt-stress conditions. Notably, the gi-201 cop1-4 double mutant shows an enhanced tolerance to salt stress similar to gi-201, indicating that GI is epistatic to COP1 under salt-stress conditions. Taken together, our study provides critical insights into the molecular mechanisms governing the COP1-mediated proteasomal degradation of GI for salt stress tolerance, raising new possibilities for developing salt-tolerant crops.

Generation of parthenocarpic tomato plants in multiple elite cultivars using the CRISPR/Cas9 system.

Tomato (Solanum lycopersicum L.) is one of the most important crops in the world for its fruit production. Advances in cutting-edge techniques have enabled the development of numerous critical traits related to the quality and quantity of tomatoes. Genetic engineering techniques, such as gene transformation and gene editing, have emerged as powerful tools for generating new plant varieties with superior traits. In this study, we induced parthenocarpic traits in a population of elite tomato (ET) lines. At first, the adaptability of ET lines to genetic transformation was evaluated to identify the best-performing lines by transforming the SlANT1 gene overexpression cassette and then later used to produce the SlIAA9 knockout lines using the CRISPR/Cas9 system. ET5 and ET8 emerged as excellent materials for these techniques and showed higher efficiency. Typical phenotypes of knockout sliaa9 were clearly visible in G0 and G1 plants, in which simple leaves and parthenocarpic fruits were observed. The high efficiency of the CRISPR/Cas9 system in developing new tomato varieties with desired traits in a short period was demonstrated by generating T-DNA-free homozygous sliaa9 knockout plants in the G1 generation. Additionally, a simple artificial fertilization method was successfully applied to recover seed production from parthenocarpic plants, securing the use of these varieties as breeding materials. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01452-1.

Single-strand annealing: Molecular mechanisms and potential applications in CRISPR-Cas-based precision genome editing.

BACKGROUND: Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival. Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown. Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated. PURPOSE AND SCOPE: Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing. SUMMARY AND CONCLUSION: The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5'-3' resection to reveal homologous repeats on the 3' ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.

Overexpression of oligouridylate binding protein 1b results in ABA hypersensitivity.

Oligouridylate binding protein 1b (UBP1b), a marker protein of plant stress granules (SGs), plays a role in heat stress tolerance in plants. A previous microarray analysis revealed that the expression of several ABA signaling-related genes is higher in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox) subjected to both non-stressed and heat stress conditions. Root elongation and seed germination assays demonstrated that UBP1b-ox exhibited hypersensitivity to ABA. RT-qPCR analysis confirmed that mitogen-activated protein kinase (MAPK) cascade genes, such as MPK3, MKK4, and MKK9 were upregulated in UBP1b-ox plants. ABA receptor genes, including PYL5 and PYL6, were also upregulated in UBP1b-ox plants. mRNA of WRKY33 - a downstream gene of MPK3 and an upstream gene of ethylene biosynthesis, exhibited high levels of accumulation, although the level of endogenous ABA was not significantly different between UBP1b-ox and control plants. In addition, RNA decay analysis revealed that WRKY33 was more stable in UBP1b-ox plants, indicating that the mRNA of WRKY33 was protected within UBP1b SGs. Collectively, these data demonstrate that UBP1b plays an important role in plant response to ABA.

Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance.

Stress granules (SGs), which are formed in the plant cytoplasm under stress conditions, are transient dynamic sites (particles) for mRNA storage. SGs are actively involved in protecting mRNAs from degradation. Oligouridylate binding protein 1b (UBP1b) is a component of SGs. The formation of microscopically visible cytoplasmic foci, referred to as UBP1b SG, was induced by heat treatment in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox). A detailed understanding of the function of UBP1b, however, is still not clear. UBP1b-ox plants displayed increased heat tolerance, relative to control plants, while ubp1b mutants were more sensitive to heat stress than control plants. Microarray analysis identified 117 genes whose expression was heat-inducible and higher in the UBP1b-ox plants. RNA decay analysis was performed using cordycepin, a transcriptional inhibitor. In order to determine if those genes serve as targets of UBP1b, the rate of RNA degradation of a DnaJ heat shock protein and a stress-associated protein (AtSAP3) in UBP1b-ox plants was slower than in control plants; indicating that the mRNAs of these genes were protected within the UBP1b SG granule. Collectively, these data demonstrate that UBP1b plays an integral role in heat stress tolerance in plants.