Interplay between NIN-LIKE PROTEINs 6 and 7 in nitrate signaling.

NLP7 (NIN-LIKE-PROTEIN 7) is the major transcriptional factor responsible for the primary nitrate response (PNR), but the role of its homolog, NLP6, in nitrogen signaling and the interplay between NLP6 and NLP7 remain to be elucidated. In this study, we show that, like NLP7, nuclear localization of NLP6 via a nuclear retention mechanism is nitrate dependent, but nucleocytosolic shuttling of both NLP6 and NLP7 is independent of each other. Compared with single mutants, the nlp6nlp7 double mutant displays a synergistic growth retardation phenotype in response to nitrate. The transcriptome analysis of the PNR showed that NLP6 and NLP7 govern ∼50% of nitrate-induced genes, with cluster analysis highlighting 2 distinct patterns. In the A1 cluster, NLP7 plays the major role, whereas in the A2 cluster, NLP6 and NLP7 are partially functionally redundant. Interestingly, comparing the growth phenotype and PNR under high- and low-nitrate conditions demonstrated that NLP6 and NLP7 exert a more dominant role in the response to high nitrate. Apart from nitrate signaling, NLP6 and NLP7 also participated in high ammonium conditions. Growth phenotypes and transcriptome data revealed that NLP6 and NLP7 are completely functionally redundant and may act as repressors in response to ammonium. Other NLP family members also participated in the PNR, with NLP2 and NLP7 acting as broader regulators and NLP4, -5, -6, and -8 regulating PNR in a gene-dependent manner. Thus, our findings indicate that multiple modes of interplay exist between NLP6 and NLP7 that differ depending on nitrogen sources and gene clusters.

Mitigating the allergenicity of peanut allergen Ara h 1 by cold atmospheric pressure argon plasma jet.

BACKGROUND: Peanut allergy is recognized as a major food allergy that triggers severe and even fatal symptoms. Avoidance of peanuts in the diet is the main option for current safety management. Processing techniques reducing peanut allergenicity are required to develop other options. Cold plasma is currently considered as a novel non-thermal approach to alter protein structure and has the potential to alleviate immunoreactivity of protein allergen. RESULTS: The application of a cold argon plasma jet to peanut protein extract could reduce the amount of a 64 kDa protein band corresponding to a major peanut allergen Ara h 1 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but the overall protein size distribution did not change significantly. A decrease in peanut protein solubility was a possible cause that led to the loss of protein content in the soluble fraction. Immunoblotting and enzyme-linked immunosorbent assay elucidated that the immunoreactivity of Ara h 1 was significantly decreased with the time treated with plasma. Ara h 1 antigenicity reduced by 38% after five scans (approximately 3 min) of cold argon plasma jet treatment, and the reduction was up to 66% after approximately 15 min of treatment. CONCLUSION: The results indicate that cold argon plasma jet treatment could be a suitable platform for alleviating the immunoreactivity of peanut protein. This work demonstrates an efficient, compact, and rapid platform for mitigating the allergenicity of peanuts, and shows great potential for the plasma platform as a non-thermal technique in the food industry. © 2023 Society of Chemical Industry.

Producing high quality mung bean sprout using atmospheric cold plasma treatment: better physical appearance and higher γ-aminobutyric acid (GABA) content.

BACKGROUND: Germination of mung beans increases the content of dietary fiber, vitamin C, antioxidants, and γ-aminobutyric acid (GABA). Atmospheric cold plasma is a recently developed technology that can rapidly modify the surface properties of an object. In this work, atmospheric cold plasma was utilized to promote higher moisture absorption of mung bean seeds and, thus, enhance the germination ratio and GABA level. The selected healthy seeds that were exposed to plasma generated at different ionizing powers. RESULT: According to the experimental results, atmospheric cold plasma treatments on mung bean seeds could induce significantly more water absorption and lead to a higher rate of germination. The physical appearance of the sprout developed after plasma treatment was noticeably modified to a more desirable form, which has a short radicle and longer hypocotyls with a larger diameter. The content of the bioactive component GABA in plasma-treated beans was approximately three times higher than the untreated group due to the response of seed to the environmental stress created by the plasma treatment. CONCLUSION: The result from this work will serve as a good reference for future investigation that is searching for a solution to enhance bioactive compound production in natural products. © 2021 Society of Chemical Industry.

Restoring Fertility for Novel Interspecific Hybrids between Kalanchoe garambiensis and K. nyikae Using Colchicine Treatment.

Interspecific hybridization is an effective strategy in Kalanchoe breeding programs for the introduction of new traits. Wild species within the Kalanchoe genus are valuable genetic resources for providing new horticulture traits and to improve environmental adaptations. However, reproductive barriers associated with fertilization and hybrid sterility must be overcome to produce fertile hybrid progenies. To approach the breeding objectives for Kalanchoe cut flower cultivars with long stem traits and adaptation to tropical/subtropical regions, a tropical species endemic to Taiwan, Kalanchoe garambiensis Kudo, was used as a parent to cross with other long stem Kalanchoe species. Reciprocal crossing was effective in overcoming interspecific unilateral incompatibility in our crossed pairs. One superior hybrid, '103-1', produced capsules without seeds by selfing and backcrossing with pollens from either parent. Other than the seedless trait, failure of pollen releasing from anther, pollen aggregation and no pollen germination in '103-1' suggested its F1 sterility. Colchicine treatments on apical buds of '103-1' successfully overcame potential meiotic abnormalities by doubling ploidy. For the first time, fertile interspecific hybrids of K. garambiensis and K. nyikae Engler were generated. The fertile hybrid has further produced progeny populations by crossing with K. nyikae or K. blossfeldiana von Poelln, 'Ida'.

Bioactive compounds with anti-oxidative and anti-inflammatory activities of hop extracts.

The aim of this study is to simultaneously evaluate anti-oxidative and anti-inflammatory activities of the hop extracts by different solvents. Hop water extract (HWE) and hop ethanol extracts (HEEs) were prepared by extracting hop pellets with hot water at 90 °C and ethanol solutions (55%, 75%, and 95%), respectively. Bioactive compound such as α-acid, ß-acid, total phenolic, and total flavonoid contents were determined. All the HEEs showed higher anti-oxidative activities than the HWEs. The HEEs showing the highest anti-oxidative activities are different in the experiments with different free radicals. For anti-inflammatory activities, both the HWE and HEEs decreased NO productions. HWE decreased TNF-α and IL-6 secretion but showed no effect on IL-1ß, while HEEs decreased IL-1ß and IL-6 secretion but increased TNF-α secretion. Except for TNF-α secretion, the HEEs showed higher anti-inflammatory activities than the HWE. Future work is to explore the possible mechanism to improve the ethanol extraction procedure.

A High Resolution Melting Analysis-Based Genotyping Toolkit for the Peach (Prunus persica) Chilling Requirement.

The chilling requirement (CR) is the main factor controlling the peach floral bud break and subsequent reproductive growth. To date, several peach CR quantitative trait loci (QTLs) have been identified. To improve the accessibility and convenience of this genetic information for peach breeders, the aim of this study was to establish an easy-to-use genotype screening system using peach CR molecular markers as a toolkit for marker-assisted selection. Here, we integrated 22 CR-associated markers from three published QTLs and positioned them on the Prunus persica physical map. Then, we built a PCR-based genotyping platform by using high-resolution melting (HRM) analysis with specific primers and trained this platform with 27 peach cultivars. Due to ambiguous variant calls from a commercial HRM software, we developed an R-based pipeline using principal component analysis (PCA) to accurately differentiate genotypes. Based on the PCA results, this toolkit was able to determine the genotypes at the CR-related single nucleotide polymorphisms (SNPs) in all tested peach cultivars. In this study, we showed that this HRM-PCA pipeline served as a low-cost, high-throughput, and non-gel genotyping solution. This system has great potential to accelerate CR-focused peach breeding.

Characterization of an insecticidal toxin and pathogenicity of Pseudomonas taiwanensis against insects.

Pseudomonas taiwanensis is a broad-host-range entomopathogenic bacterium that exhibits insecticidal activity toward agricultural pests Plutella xylostella, Spodoptera exigua, Spodoptera litura, Trichoplusia ni and Drosophila melanogaster. Oral infection with different concentrations (OD = 0.5 to 2) of wild-type P. taiwanensis resulted in insect mortality rates that were not significantly different (92.7%, 96.4% and 94.5%). The TccC protein, a component of the toxin complex (Tc), plays an essential role in the insecticidal activity of P. taiwanensis. The ΔtccC mutant strain of P. taiwanensis, which has a knockout mutation in the tccC gene, only induced 42.2% mortality in P. xylostella, even at a high bacterial dose (OD = 2.0). TccC protein was cleaved into two fragments, an N-terminal fragment containing an Rhs-like domain and a C-terminal fragment containing a Glt symporter domain and a TraT domain, which might contribute to antioxidative stress activity and defense against macrophagosis, respectively. Interestingly, the primary structure of the C-terminal region of TccC in P. taiwanensis is unique among pathogens. Membrane localization of the C-terminal fragment of TccC was proven by flow cytometry. Sonicated pellets of P. taiwanensis ΔtccC strain had lower toxicity against the Sf9 insect cell line and P. xylostella larvae than the wild type. We also found that infection of Sf9 and LD652Y-5d cell lines with P. taiwanensis induced apoptotic cell death. Further, natural oral infection by P. taiwanensis triggered expression of host programmed cell death-related genes JNK-2 and caspase-3.

Plant defense after flooding.

Since the first study of hypoxic response in plants with cDNA microarray in 2002, the number of hypoxia-responsive genes has grown to more than 2000. However, to date, only small numbers of hypoxia-responsive genes are known to confer hypoxic resistance. Most investigations in this area have focused on identifying which genes are responsive and then characterized how these genes are induced during hypoxia, but the roles of numerous genes in hypoxic response are still unknown. In our recent study, we demonstrated that a group of genes are induced by submergence to trigger plant immunity, which is a response to protect plants against a higher probability of pathogen infection during or after flooding. This work offered a brand new perspective, i.e., that hypoxia-responsive genes can be induced for reasons other than conferring hypoxic resistance. Possible reasons why these responses were triggered are discussed herein.

Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis.

Transcriptional control plays an important role in regulating submergence responses in plants. Although numerous genes are highly induced during hypoxia, their individual roles in hypoxic responses are still poorly understood. Here, we found that expression of genes that encode members of the WRKY transcription factor family was rapidly and strongly induced upon submergence in Arabidopsis thaliana, and this induction correlated with induction of a large portion of innate immunity marker genes. Furthermore, prior submergence treatment conferred higher resistance to the bacterial pathogen Pseudomonas syringae in Arabidopsis. Among the WRKY genes tested, WRKY22 had the highest level of induction during the early stages of submergence. Compared with the wild type, WRKY22 T-DNA insertion mutants wrky22-1 and wrky22-2 had lower disease resistance and lower induction of innate immunity markers, such as FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1) and WRKY53, after submergence. Furthermore, transcriptomic analyses of wrky22-2 and chromatin immunoprecipitation identified several potential targets of WRKY22, which included genes encoding a TIR domain-containing protein, a plant peptide hormone, and many OLIGO PEPTIDE TRANSPORTER genes, all of which may lead to induction of innate immunity. In conclusion, we propose that submergence triggers innate immunity in Arabidopsis via WRKY22, a response that may protect against a higher probability of pathogen infection either during or after flooding.

Insights into hypoxic systemic responses based on analyses of transcriptional regulation in Arabidopsis.

We have adopted a hypoxic treatment system in which only roots were under hypoxic conditions. Through analyzing global transcriptional changes in both shoots and roots, we found that systemic signals may be transduced from roots to trigger responses in tissues not directly subjected to hypoxia. The molecular mechanisms of such systemic responses under flooding are currently largely unknown. Using ontological categorization for regulated genes, a systemic managing program of carbohydrate metabolism was observed, providing an example of how systemic responses might facilitate the survival of plants under flooding. Moreover, a proportion of gene expressions that regulated in shoots by flooding was affected in an ethylene signaling mutation, ein2-5. Many systemic-responsive genes involved in the systemic carbohydrate managing program, hormone responses and metabolism, ubiquitin-dependent protein degradation were also affected in ein2-5. These results suggested an important role of ethylene in mediation of hypoxic systemic responses. Genes associated with abscisic acid (ABA) biosynthesis are upregulated in shoots and down regulated in roots. An ABA signaling mutation, abi4-1, affects expression of several systemic responsive genes. These results suggested that regulation of ABA biosynthesis could be required for systemic responses. The implications of these results for the systemic responses of root-flooded Arabidopsis are discussed.

The AP2/ERF transcription factor AtERF73/HRE1 modulates ethylene responses during hypoxia in Arabidopsis.

A number of APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) genes have been shown to function in abiotic and biotic stress responses, and these genes are often induced by multiple stresses. We report here the characterization of an AP2/ERF gene in Arabidopsis (Arabidopsis thaliana) that is specifically induced during hypoxia. We show that under normoxic conditions, the expression of AtERF73/HRE1 can be induced by exogenous addition of 1-aminocyclopropane-1-carboxylic acid and that a combination of hypoxia and 1-aminocyclopropane-1-carboxylic acid results in hyperinduction of AtERF73/HRE1 expression. In addition, hypoxic induction of AtERF73/HRE1 is reduced but not completely abolished in ethylene-insensitive mutants and in the presence of inhibitors of ethylene biosynthesis and responses. These results suggest that, in addition to ethylene, an ethylene-independent signal is also required to mediate hypoxic induction of AtERF73/HRE1. To assess the role of AtERF73/HRE1, we generated three independent RNA interference (RNAi) knockdown lines of AtERF73/HRE1. Under normoxic conditions, the AtERF73/HRE1-RNAi seedlings displayed increased ethylene sensitivity and exaggerated triple responses, indicating that AtERF73/HRE1 might play a negative regulatory role in modulating ethylene responses. Gas chromatography analyses showed that the production of ethylene was similar between wild-type and RNAi lines under hypoxia. Quantitative reverse transcription-polymerase chain reaction analyses showed that hypoxia-inducible genes could be affected by AtERF73/HRE1-RNAi lines in two different ways: hypoxic induction of glycolytic and fermentative genes was reduced, whereas induction of a number of peroxidase and cytochrome P450 genes was increased. Taken together, our results show that AtERF73/HRE1 is involved in modulating ethylene responses under both normoxia and hypoxia.

Arabidopsis Hsa32, a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation.

Plants and animals share similar mechanisms in the heat shock (HS) response, such as synthesis of the conserved HS proteins (Hsps). However, because plants are confined to a growing environment, in general they require unique features to cope with heat stress. Here, we report on the analysis of the function of a novel Hsp, heat-stress-associated 32-kD protein (Hsa32), which is highly conserved in land plants but absent in most other organisms. The gene responds to HS at the transcriptional level in moss (Physcomitrella patens), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). Like other Hsps, Hsa32 protein accumulates greatly in Arabidopsis seedlings after HS treatment. Disruption of Hsa32 by T-DNA insertion does not affect growth and development under normal conditions. However, the acquired thermotolerance in the knockout line was compromised following a long recovery period (>24 h) after acclimation HS treatment, when a severe HS challenge killed the mutant but not the wild-type plants, but no significant difference was observed if they were challenged within a short recovery period. Quantitative hypocotyl elongation assay also revealed that thermotolerance decayed faster in the absence of Hsa32 after a long recovery. Similar results were obtained in Arabidopsis transgenic plants with Hsa32 expression suppressed by RNA interference. Microarray analysis of the knockout mutant indicates that only the expression of Hsa32 was significantly altered in HS response. Taken together, our results suggest that Hsa32 is required not for induction but rather maintenance of acquired thermotolerance, a feature that could be important to plants.