The Na+/H+ antiporter SALT OVERLY SENSITIVE 1 regulates salt compensation of circadian rhythms by stabilizing GIGANTEA in Arabidopsis.

The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na+/H+ antiporter SOS1 specifically functions as a salt-specific circadian clock regulator via GIGANTEA (GI) in Arabidopsis thaliana. SOS1 directly interacts with GI in a salt-dependent manner and stabilizes this protein to sustain a proper clock period under salinity conditions. SOS1 function in circadian clock regulation requires the salt-mediated secondary messengers cytosolic free calcium and reactive oxygen species, pointing to a distinct regulatory role for SOS1 in addition to its function as a transporter to maintain Na+ homeostasis. Our results demonstrate that SOS1 maintains homeostasis of the salt response under high or daily fluctuating salt levels. These findings highlight the genetic capacity of the circadian clock to maintain timekeeping activity over a broad range of salinity levels.

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.

Nucleoredoxin 1 positively regulates heat stress tolerance by enhancing the transcription of antioxidants and heat-shock proteins in tomato.

Thioredoxins (TRXs) are small oxidoreductase proteins located in various subcellular compartments. Nucleoredoxin (NRX) is a nuclear-localized TRX and a key component for the integration of the antioxidant system with the immune response. Although NRX is well characterized in biotic stress responses, its functional role in abiotic stress responses is still elusive. To understand whether NRX contributes to heat stress response in tomato (Solanum lycopersicum), we generated CRISPR/Cas9-mediated mutations in SlNRX1 (slnrx1). Interestingly, the slnrx1 mutant was extremely sensitive to heat stress with higher electrolyte leakage, malondialdehyde contents, and H2O2 concentration compared to wild-type tomato plants, suggesting that SlNRX1 negatively regulates heat stress-induced oxidative damage. We also found that transcripts encoding antioxidant enzymes and Heat-Shock Proteins (HSPs) in slnrx1 were down-regulated either in the absence or presence of heat stress. These data suggest that NRX1 is a positive regulator for heat stress tolerance by elevating antioxidant capacity and inducing HSPs to protect cells against heat stress-induced oxidative damage and protein denaturation, respectively.

Transcriptome Changes Reveal the Molecular Mechanisms of Humic Acid-Induced Salt Stress Tolerance in Arabidopsis.

Humic acid (HA) is a principal component of humic substances, which make up the complex organic matter that broadly exists in soil environments. HA promotes plant development as well as stress tolerance, however the precise molecular mechanism for these is little known. Here we conducted transcriptome analysis to elucidate the molecular mechanisms by which HA enhances salt stress tolerance. Gene Ontology Enrichment Analysis pointed to the involvement of diverse abiotic stress-related genes encoding HEAT-SHOCK PROTEINs and redox proteins, which were up-regulated by HA regardless of salt stress. Genes related to biotic stress and secondary metabolic process were mainly down-regulated by HA. In addition, HA up-regulated genes encoding transcription factors (TFs) involved in plant development as well as abiotic stress tolerance, and down-regulated TF genes involved in secondary metabolic processes. Our transcriptome information provided here provides molecular evidences and improves our understanding of how HA confers tolerance to salinity stress in plants.

Arabidopsis GIGANTEA negatively regulates chloroplast biogenesis and resistance to herbicide butafenacil.

KEY MESSAGE: Arabidopsis GI negatively regulates chloroplast biogenesis and resistance to the herbicide butafenacil by enhanced activity and transcriptional levels of antioxidant enzymes Chloroplast biogenesis is blocked by retrograde signaling triggered by diverse internal and external cues, including sugar, reactive oxygen species (ROS), phytohormones, and abiotic stress. Efficient chloroplast biogenesis is essential for crop productivity due to its effect on photosynthetic efficiency, and is associated with agronomic traits such as insect/disease resistance, herbicide resistance, and abiotic stress tolerance. Here, we show that the circadian clock-controlled gene GIGANTEA (GI) regulates chloroplast biogenesis in Arabidopsis thaliana. The gi-2 mutant showed reduced sensitivity to the chloroplast biogenesis inhibitor lincomycin, maintaining high levels of photosynthetic proteins. By contrast, wild-type and GI-overexpressing plants were sensitive to lincomycin, with variegated leaves and reduced photosynthetic protein levels. GI is degraded by lincomycin, suggesting that GI is genetically linked to chloroplast biogenesis. The GI mutant alleles gi-1 and gi-2 were resistant to the herbicide butafenacil, which inhibits protoporphyrinogen IX oxidase activity and triggers ROS-mediated cell death via the accumulation of chlorophyll precursors. Butafenacil-mediated accumulation of superoxide anions and H2O2 was not detected in gi-1 or gi-2, as revealed by histochemical staining. The activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase were 1.2-1.4-fold higher in both gi mutants compared to the wild type. Finally, the expression levels of antioxidant enzyme genes were 1.5-2-fold higher in the mutants than in the wild type. These results suggest that GI negatively regulates chloroplast biogenesis and resistance to the herbicide butafenacil, providing evidence for a genetic link between GI and chloroplast biogenesis, which could facilitate the development of herbicide-resistant crops.

Ethylene enhances transcriptions of asparagine biosynthetic genes in soybean (Glycine max L. Merr) leaves.

Soybean, a vital protein-rich crop, offers bioactivity that can mitigate various chronic human diseases. Nonetheless, soybean breeding poses a challenge due to the negative correlation between enhanced protein levels and overall productivity. Our previous studies demonstrated that applying gaseous phytohormone, ethylene, to soybean leaves significantly boosts the accumulation of free amino acids, particularly asparagine (Asn). Current studies also revealed that ethylene application to soybeans significantly enhanced both essential and non-essential amino acid contents in leaves and stems. Asn plays a crucial role in ammonia detoxification and reducing fatigue. However, the molecular evidence supporting this phenomenon remains elusive. This study explores the molecular mechanisms behind enhanced Asn accumulation in ethylene-treated soybean leaves. Transcriptional analysis revealed that ethylene treatments to soybean leaves enhance the transcriptional levels of key genes involved in Asn biosynthesis, such as aspartate aminotransferase (AspAT) and Asn synthetase (ASN), which aligns with our previous observations of elevated Asn levels. These findings shed light on the role of ethylene in upregulating Asn biosynthetic genes, subsequently enhancing Asn concentrations. This molecular insight into amino acid metabolism regulation provides valuable knowledge for the metabolic farming of crops, especially in elevating nutraceutical ingredients with non-genetic modification (GM) approach for improved protein content.

HOS15 represses flowering by promoting GIGANTEA degradation in response to low temperature in Arabidopsis.

Flowering is the primary stage of the plant developmental transition and is tightly regulated by environmental factors such as light and temperature. However, the mechanisms by which temperature signals are integrated into the photoperiodic flowering pathway are still poorly understood. Here, we demonstrate that HOS15, which is known as a GI transcriptional repressor in the photoperiodic flowering pathway, controls flowering time in response to low ambient temperature. At 16°C, the hos15 mutant exhibits an early flowering phenotype, and HOS15 acts upstream of photoperiodic flowering genes (GI, CO, and FT). GI protein abundance is increased in the hos15 mutant and is insensitive to the proteasome inhibitor MG132. Furthermore, the hos15 mutant has a defect in low ambient temperature-mediated GI degradation, and HOS15 interacts with COP1, an E3 ubiquitin ligase for GI degradation. Phenotypic analyses of the hos15 cop1 double mutant revealed that repression of flowering by HOS15 is dependent on COP1 at 16°C. However, the HOS15-COP1 interaction was attenuated at 16°C, and GI protein abundance was additively increased in the hos15 cop1 double mutant, indicating that HOS15 acts independently of COP1 in GI turnover at low ambient temperature. This study proposes that HOS15 controls GI abundance through multiple modes as an E3 ubiquitin ligase and transcriptional repressor to coordinate appropriate flowering time in response to ambient environmental conditions such as temperature and day length.

Nucleoredoxin gene SINRX1 negatively regulates tomato immunity by activating SA signaling pathway.

The tomato (Solanum lycopersicum) is widely consumed globally and renowned for its health benefits, including the reduction of cardiovascular disease and prostate cancer risk. However, tomato production faces significant challenges, particularly due to various biotic stresses such as fungi, bacteria, and viruses. To address this challenges, we employed the CRISPR/Cas9 system to modify the tomato NUCLEOREDOXIN (SlNRX) genes (SlNRX1 and SlNRX2) belonging to the nucleocytoplasmic THIOREDOXIN subfamily. CRISPR/Cas9-mediated mutations in SlNRX1 (slnrx1) plants exhibited resistance against bacterial leaf pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326, as well as the fungal pathogen Alternaria brassicicola. However, the slnrx2 plants did not display resistance. Notably, the slnrx1 demonstrated elevated levels of endogenous salicylic acid (SA) and reduced levels of jasmonic acid after Psm infection, in comparison to both wild-type (WT) and slnrx2 plants. Furthermore, transcriptional analysis revealed that genes involved in SA biosynthesis, such as ISOCHORISMATE SYNTHASE 1 (SlICS1) and ENHANCED DISEASE SUSCEPTIBILITY 5 (SlEDS5), were upregulated in slnrx1 compared to WT plants. In addition, a key regulator of systemic acquired resistance, PATHOGENESIS-RELATED 1 (PR1), exhibited increased expression in slnrx1 compared to WT. These findings suggest that SlNRX1 acts as a negative regulator of plant immunity, facilitating infection by the Psm pathogen through interference with the phytohormone SA signaling pathway. Thus, targeted mutagenesis of SlNRX1 is a promising genetic means to enhance biotic stress resistance in crop breeding.

Phytochrome B Positively Regulates Red Light-Mediated ER Stress Response in Arabidopsis.

Light plays a crucial role in plant growth and development, and light signaling is integrated with various stress responses to adapt to different environmental changes. During this process, excessive protein synthesis overwhelms the protein-folding ability of the endoplasmic reticulum (ER), causing ER stress. Although crosstalk between light signaling and ER stress response has been reported in plants, the molecular mechanisms underlying this crosstalk are poorly understood. Here, we demonstrate that the photoreceptor phytochrome B (phyB) induces the expression of ER luminal protein chaperones as well as that of unfolded protein response (UPR) genes. The phyB-5 mutant was less sensitive to tunicamycin (TM)-induced ER stress than were the wild-type plants, whereas phyB-overexpressing plants displayed a more sensitive phenotype under white light conditions. ER stress response genes (BiP2 and BiP3), UPR-related bZIP transcription factors (bZIP17, bZIP28, and bZIP60), and programmed cell death (PCD)-associated genes (OXI1, NRP1, and MC8) were upregulated in phyB-overexpressing plants, but not in phyB-5, under ER stress conditions. The ER stress-sensitive phenotype of phyB-5 under red light conditions was eliminated with a reduction in photo-equilibrium by far-red light and darkness. The N-terminal domain of phyB is essential for signal transduction of the ER stress response in the nucleus, which is similar to light signaling. Taken together, our results suggest that phyB integrates light signaling with the UPR to relieve ER stress and maintain proper plant growth.

The thiol-reductase activity of YUCCA6 enhances nickel heavy metal stress tolerance in Arabidopsis.

Anthropogenic activities cause the leaching of heavy metals into groundwater and their accumulation in soil. Excess levels of heavy metals cause toxicity in plants, inducing the production of reactive oxygen species (ROS) and possible death caused by the resulting oxidative stress. Heavy metal stresses repress auxin biosynthesis and transport, inhibiting plant growth. Here, we investigated whether nickel (Ni) heavy metal toxicity is reduced by exogenous auxin application and whether Ni stress tolerance in Arabidopsis thaliana is mediated by the bifunctional enzyme YUCCA6 (YUC6), which functions as an auxin biosynthetic enzyme and a thiol-reductase (TR). We found that an application of up to 1 µM exogenous indole-3-acetic acid (IAA) reduces Ni stress toxicity. yuc6-1D, a dominant mutant of YUC6 with high auxin levels, was more tolerant of Ni stress than wild-type (WT) plants, despite absorbing significantly more Ni. Treatments of WT plants with YUCASIN, a specific inhibitor of YUC-mediated auxin biosynthesis, increased Ni toxicity; however yuc6-1D was not affected by YUCASIN and remained tolerant of Ni stress. This suggests that rather than the elevated IAA levels in yuc6-1D, the TR activity of YUC6 might be critical for Ni stress tolerance. The loss of TR activity in YUC6 caused by the point-mutation of Cys85 abolished the YUC6-mediated Ni stress tolerance. We also found that the Ni stress-induced ROS accumulation was inhibited in yuc6-1D plants, which consequently also showed reduced oxidative damage. An enzymatic assay and transcriptional analysis revealed that the peroxidase activity and transcription of PEROXIREDOXIN Q were enhanced by Ni stress to a greater level in yuc6-1D than in the WT. These findings imply that despite the need to maintain endogenous IAA levels for basal Ni stress tolerance, the TR activity of YUC6, not the elevated IAA levels, plays the predominant role inNi stress tolerance by lowering Ni-induced oxidative stress.

Retarding Ion Exchange between Conducting Polymers and Ionic Liquids for Printable Top Electrodes in Semitransparent Organic Solar Cells.

Semitransparent organic solar cells (ST-OSCs) are considered to be an influential tool for aesthetic and economic building-integrated photovoltaics, which can be fabricated by the printing technology. A poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) and ionic liquid (IL) composite has been considered as an electrode for ST-OSCs because of its high electrical conductivity, high transparency, and printability. However, we found that the introduction of IL into the PEDOT:PSS solution for enhancing its electrical conductivity results in (1) nonreliable printing of PEDOT:PSS/IL composite films because of gradual gelation of the mixture solution and (2) the production of chemically reactive ion pairs during ion exchange between PSS and IL, which induces the oxidation of the underlying organic semiconductors during printing. To solve these problems, we developed a sequential printing method using pristine PEDOT:PSS and IL solutions to retard ion exchange, thus preventing chemical doping of organic semiconductors by newly generated ion pairs. Finally, by using only solution processes, we demonstrate efficient ST-OSCs with a printed PEDOT:PSS/IL composite as the top electrode, exhibiting a power conversion efficiency of 6.32% at an average visible transmittance of 35.4%.

Humic acid enhances heat stress tolerance via transcriptional activation of Heat-Shock Proteins in Arabidopsis.

Humic acid (HA) is composed of a complex supramolecular association and is produced by humification of organic matters in soil environments. HA not only improves soil fertility, but also stimulates plant growth. Although numerous bioactivities of HA have been reported, the molecular evidences have not yet been elucidated. Here, we performed transcriptomic analysis to identify the HA-prompted molecular mechanisms in Arabidopsis. Gene ontology enrichment analysis revealed that HA up-regulates diverse genes involved in the response to stress, especially to heat. Heat stress causes dramatic induction in unique gene families such as Heat-Shock Protein (HSP) coding genes including HSP101, HSP81.1, HSP26.5, HSP23.6, and HSP17.6A. HSPs mainly function as molecular chaperones to protect against thermal denaturation of substrates and facilitate refolding of denatured substrates. Interestingly, wild-type plants grown in HA were heat-tolerant compared to those grown in the absence of HA, whereas Arabidopsis HSP101 null mutant (hot1) was insensitive to HA. We also validated that HA accelerates the transcriptional expression of HSPs. Overall, these results suggest that HSP101 is a molecular target of HA promoting heat-stress tolerance in Arabidopsis. Our transcriptome information contributes to understanding the acquired genetic and agronomic traits by HA conferring tolerance to environmental stresses in plants.

Expression of Arabidopsis thaliana Thioredoxin-h2 in Brassica napus enhances antioxidant defenses and improves salt tolerance.

Salt stress limits crop productivity worldwide, particularly in arid and heavily irrigated regions. Salt stress causes oxidative stress, in which plant cells accumulate harmful levels of reactive oxygen species (ROS). Thioredoxins (Trxs; EC 1.8.4.8) are antioxidant proteins encoded by a ubiquitous multigene family. Arabidopsis thaliana Trx h-type proteins localize in the cytoplasm and other subcellular organelles, and function in plant responses to abiotic stresses and pathogen attack. Here, we isolated the Arabidopsis genes encoding two cytosolic h-type Trx proteins, AtTrx-h2 and AtTrx-h3 and generated transgenic oilseed rape (Brassica napus) plants overexpressing AtTrx-h2 or AtTrx-h3. Heterologous expression of AtTrx-h2 in B. napus conferred salt tolerance with plants grown on 50 mM NaCl having higher fresh weight and chlorophyll contents compared with controls in hydroponic growth system. By contrast, expression of AtTrx-h3 or the empty vector control did not improve salt tolerance. In addition, AtTrx-h2-overexpressing transgenic plants exhibited lower levels of hydrogen peroxide and higher activities of antioxidant enzymes including peroxidase, catalase, and superoxide dismutase, compared with the plants expressing the empty vector control or AtTrx-h3. These results suggest that AtTrx-h2 is a promising candidate for engineering or breeding crops with enhanced salt stress tolerance.

Long-Term Stable Recombination Layer for Tandem Polymer Solar Cells Using Self-Doped Conducting Polymers.

Recently, the most efficient tandem polymer solar cells (PSCs) have used poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) ( PEDOT: PSS) as a p-type component of recombination layer (RL). However, its undesirable acidic nature, originating from insulating PSS, of PEDOT: PSS drastically reduces the lifetime of PSCs. Here, we demonstrate the efficient and stable tandem PSCs by introducing acid-free self-doped conducting polymer (SCP), combined with zinc oxide nanoparticles (ZnO NPs), as RL for PEDOT: PSS-free tandem PSCs. Moreover, we introduce an innovative and versatile nanocomposite system containing photoactive and p-type conjugated polyelectrolyte (p-CPE) into the tandem fabrication of an ideal self-organized recombination layer. In our new RL, highly conductive SCP facilitates charge transport and recombination process, and p-CPE helps to achieve nearly loss-free charge collection by increasing effective work function of indium tin oxide (ITO) and SCP. Because of the synergistic effect of extremely low electrical resistance, ohmic contact, and pH neutrality, tandem devices with our novel RL performed well, exhibiting a high power conversion efficiency of 10.2% and a prolonged lifetime. These findings provide a new insight for strategic design of RLs using SCPs to achieve efficient and stable tandem PSCs and enable us to review and extend the usefulness of SCPs in various electronics research fields.