Identification of ARF genes in Juglans Sigillata Dode and analysis of their expression patterns under drought stress.

BACKGROUND: Auxin response factor (ARF), a transcription factors that controls the expression of genes responsive to auxin, plays a key role in the regulation of plant growth and development. Analyses aimed at identifying ARF family genes and characterizing their functions in Juglans sigillata Dode are lacking. METHODS AND RESULTS: We used bioinformatic approaches to identify members of the J. sigillata ARF gene family and analyze their evolutionary relationships, collinearity, cis-acting elements, and tissue-specific expression patterns. The expression patterns of ARF gene family members under natural drought conditions were also analyzed. The J. sigillata ARF gene family contained 31 members, which were unevenly distributed across 16 chromosomes. We constructed a phylogenetic tree of JsARF genes and other plant ARF genes. Cis-acting elements in the promoters of JsARF were predicted. JsARF28 showed higher expressions in both the roots and leaves. A heat map of the transcriptome data of the cluster analysis under drought stress indicated that JsARF3/9/11/17/20/26 are responsive to drought. The expression of the 11 ARF genes varied under PEG treatment and JsARF18 and JsARF20 were significantly up-regulated. CONCLUSIONS: The interactions between abiotic stresses and plant hormones are supported by our cumulative data, which also offers a theoretical groundwork for comprehending the ARF mechanism and drought resistance in J. sigillata.

Establishment of virus-induced gene-silencing system in Juglans sigillata Dode and functional analysis of JsFLS2 and JsFLS4.

Juglans sigillata Dode is one of the important tree species in southwest China, and it has significant economic and ecological value. However, there is still a lack of effective methods to identify the functional genes of J. sigillata. By verifying the model plant tobacco, the pTRV2::JsPDS vector was able to cause photobleaching. This study showed that photobleaching occurred 24 and 30 d after the silencing vector was infected with aseptic seedlings and fruits of J. sigillata, respectively. When the OD600 was 0.6, and the injection dose was 500 µL, the gene silencing efficiency of aseptic seedlings was the highest at 16.7 %, significantly better than other treatments. Moreover, when the OD600 was 0.8, and the injection dose was 500 µL, the gene silencing efficiency in the walnut fruit was the highest (20 %). In addition, the VIGS system was successfully used to silence JsFLS2 and JsFLS4 genes in J. sigillata. This study also showed that the flavonol content and gene expression in the treatment group were decreased compared to the control group. In addition, the proteins transcribed and translated from the JsFLS4 gene may have higher catalytic activity for dihydroquercetin. The above results indicate that the TRV-mediated VIGS system can be an ideal tool for studying J. sigillata gene function.

Overexpression of JsFLS5 in calli improves salinity tolerance by maintaining active oxygen balance and reducing Na+ toxicity in Juglans sigillata.

The escalating global climate change significantly threatens plant growth, development, and production through salinity stress. Flavonoids, a crucial category of secondary metabolites, have been extensively studied for their role in modulating plant growth and development mechanisms in the face of biological and abiotic stress. The flavonol synthetase (FLS) gene plays a key role in the biosynthesis and accumulation of flavonoids. To investigate the correlation between salt tolerance and flavonol synthesis, JsFLS5 was overexpressed in the callus of Juglans sigillata cv. "Qianhe-7." This study shows that the upregulation of JsFLS5 significantly elevates the overall flavonoid content by modulating the expression of genes associated with flavonoid synthesis under salinity stress conditions. Additionally, the overexpressing callus exhibited enhanced resistance to salt stress compared to the wild-type callus, as evidenced by reduced levels of reactive oxygen species accumulation, electrolyte leakage, and malondialdehyde content in the overexpressing callus relative to the wild type (WT). Moreover, the overexpressing callus showed higher antioxidant enzyme activity and a more efficient ascorbic acid-glutathione cycle. Furthermore, the concentration of Na+ in the overexpressing callus was lower than WT, resulting in a decreased Na+ /K+ ratio. These findings suggest that JsFLS5 overexpression in calli effectively mitigates the oxidative damage induced by osmotic stress and reduces Na+ toxicity by enhancing flavonoid synthesis under salt stress conditions. Consequently, this study offers a novel perspective for comprehending the role of JsFLS5 in the response to abiotic stress in J. sigillata.

What government interventions are effective in regulating the use and recycling of high-standard mulch film in China?

The widespread use of ultra-thin mulch films has been considered the root cause of China's difficulties in recycling waste mulch films and their severe residue and pollution. Therefore, environmental regulation to guide farmers to use and recycle high-standard mulch has become a key measure to prevent and control mulch pollution at the source. In this paper, we review environmental regulation measures for mulch pollution and their implementation characteristics in the northwest dry farming restoration region as a case study, and explore their impact on the use and recycling of high-standard mulch. ​The results show that China's environmental regulations on mulch use and recycling can be classified into three types: guidance, incentives, and restrictions. However, the measures implemented so far primarily consist of guidance, while the restrictive and incentive regulations are inadequate. The percentage of farmers using and recycling high-standard mulch under environmental regulations is relatively high (88.18% and 79.83%, respectively), but there is still room for improvement. The study also reveals that while environmental regulations promote the use of high-standard plastic film and its recycling, not all measures are equally effective. Specifically, the implementation of measures such as benefits publicity, price subsidies, technical training, behavior supervision, and punishment mechanisms can be effective in improving farmers' adoption of high-standard plastic mulch; publicizing the hazards of mulch residue, training on recycling techniques, establishing recycling sites, regulating recycling practices, and penalizing violations can effectively help increase the recycling rate of used mulch film. Finally, this study provides a valuable reference for improving regulatory policies for mulch pollution control.

Chromosome-scale genome assembly provides insights into flower coloration mechanisms of Canna indica.

Canna indica, which produce conspicuous and colorful flowers, are widely appreciated as ornamental plants. We used Pacific Biosciences sequencing (PacBio) and chromosome conformation capture (Hi-C) genome scaffolding to build a high-quality chromosome-scale genome assembly of C. indica and the genome assembly was 821Mb with a contig N50 of 48Mb assembled into nine chromosomes. The genome of C. indica was predicted to contain 31,130 genes and 30,816 genes were functionally annotated. Genome annotation identified 522 Mb (63.59 %) as repetitive sequences. Genome evolution analysis showed that whole-genome duplication occurred 53.4 million years ago. Transcriptome analysis revealed that petal coloration was linked with the expression of genes encoding enzymes involved in anthocyanin biosynthesis, carotenoid biosynthesis, and the methylerythritol phosphate (MEP) pathway. Furthermore, modules of co-expressed genes and hub genes were identified via weighted gene co-expression network analysis. These results suggested that, in Canna indica, deep red petal coloration was regulated by CHS2 and yellow petal coloration was associated with expression of ARF6 and NAC14. Considered together, the current study revealed a high-quality reference genome which may provide new insights into the molecular basis of flower coloration in Canna indica and help enhance the conservation and breeding of ornamental plants in general.

Establishment of Agrobacterium-mediated transformation system to Juglans sigillata Dode 'Qianhe-7'.

An efficient genetic transformation system is of great significance for verifying gene function and improving plant breeding efficiency by gene engineering. In this study, a stable Agrobacterium mediated genetic transformation system of Juglans sigillata Dode 'Qianhe-7' was investigated using callus and negative pressure-assisted and ultrasonic-assisted transformation selection. The results showed that the axillary shoot leaves were suitable to induce callus and the callus proliferation rate could reach 516.27% when induction calli were cultured on DKW medium containing 0.5 mg L-1 indole-3-butyric acid, 1.2 mg L-1 2,4-dichlorophenoxyacetic acid and 0.5 mg L-1 kinetin for 18 d. In addition, negative pressure infection was the optimal infection method with the lowest browning rate (0.00%), high GFP conversion rate (16.67%), and better growth status. To further prove the feasibility of this genetic transformation system, the flavonol synthetase (JsFLS5) gene was successfully transformed into the into leaf-derived callus of 'Qianhe-7'. JsFLS5 expression and the content of total flavonoids in transformed callus were improved significantly compared with the untransformed callus, which proved that we had an efficient and reliable genetic transformation system using leaf-derived callus of Juglans sigillata.

One-Step Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Canna Yellow Streak Virus.

Canna yellow streak virus (CaYSV) is a potyvirus that causes severe damage to the ornamental plant canna in the United Kingdom and Brazil. Here, we identified CaYSV in China by isolating total RNA from an infected plant, amplifying the virus genome segments, and cloning and sequencing the amplicons. After assembly, the full-length genome of the virus was obtained and uploaded to the NCBI database. Phylogenetic analysis results showed that the Guizhou isolate (OL546222) was most closely related to the KS isolate (MG545919.1). Virus detection is essential for virus disease control but the subclinical infection of CaYSV on canna in its early development increases the difficulty of CaYSV diagnosis. The goal of this study was to develop an efficient method for detection of CaYSV. We designed the primers, optimized the reaction conditions, and finally established a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method. The product of RT-LAMP can be analyzed by both agarose gel electrophoresis and visible color change. The established one-step RT-LAMP assay showed high specificity and sensitivity in detecting CaYSV. This RT-LAMP method was also applied in analysis of 61 field samples collected from Guizhou and Jiangsu Provinces. The results showed that the infection rates of CaYSV on canna samples from these two provinces were very high (63 and 96% respectively).

Effect of Melatonin on Fruit Quality via Decay Inhibition and Enhancement of Antioxidative Enzyme Activities and Genes Expression of Two Mango Cultivars during Cold Storage.

The postharvest deterioration of mango fruits is a critical issue limiting mango storage and preservation due to its climacteric nature. This study evaluated the storage behavior of two mango cultivars and their response to exogenous melatonin (MT, 1000 µmol L-1) treatment in attenuating fruit decay and enhancing fruits' physiological and metabolic processes and gene relative expression subjected to cold storage. MT treatment in both mango cultivars significantly delayed weight loss, firmness, respiration rate, and decay incidence. However, MT did not influence the TSS, TA, and TSS:TA ratio regardless of the cultivar. Moreover, MT inhibited the decrease in total phenol and flavonoid content and AsA content while delaying the increase in the MDA content of mango during storage in both cultivars. In addition, MT dramatically inhibited the enzyme activity of PPO. In contrast, an increase in the activities of antioxidant enzymes (SOD and APX) and PAL and their genes' relative expression was noticed in MT-treated fruits versus control in both cultivars. However, MT treatment was cultivar dependent in most parameters under study. These results demonstrated that MT treatment could be an essential postharvest treatment in minimizing decay, maintaining fruit quality, and extending mango fruits' postharvest shelf life by enhancing the physiological and metabolic processes during cold storage.

Enhancing the Nutritional and Functional Properties of Auricularia auricula through the Exploitation of Walnut Branch Waste.

As the third most edible fungus in the world, Auricularia auricular needs a lot of sawdust for cultivation; thus, it is a win-win method to develop waste wood sawdust suitable for black agaric cultivation. This study evaluated the growth, agronomic characters and nutritional quality of A. auricula cultured on different ratios of miscellaneous sawdust and walnut waste wood sawdust, and comprehensively analyzed the feasibility of cultivating black agaric with walnut sawdust using principal component method (PCA). The results showed that the macro mineral elements and phenolic substances in walnut sawdust were significantly higher than those of miscellaneous sawdust by 18.32-89.00%. The overall activity of extracellular enzymes reached the highest when the ratio of the substrate was 0:4 (miscellaneous sawdust: walnut sawdust). The mycelia of 1:3 substrates grew well and fast. In addition, the growth cycle for A. auricula was significantly lower for 0:4 (116 d) than for 4:0 (126 d). Then, the single bag yield and biological efficiency (BE) were highest at 1:3. Moreover, the nutrients and mineral elements of A. auricula cultivated in walnut sawdust were significantly higher than that of miscellaneous sawdust, expect for total sugar and protein, and the highest overall value was found at 1:3. Finally, the results of comprehensive evaluation by PCA showed that the D value was the highest when the substrate was 1:3 and the lowest when the substrate was 4:0. Therefore, the substrate ratio of 1:3 was the most suitable for the growth of A. auricula. In this study, the high yield and quality of A. auricula were cultivated by waste walnut sawdust, which provided a new way to utilize walnut sawdust.

Effect of storage temperature and time on the nutritional quality of walnut male inflorescences.

The objective of this study was to investigate the effect of storage temperature and time on nutrients, bioactive compounds, and antioxidant activities of walnut male inflorescences. The results showed that the moisture, saccharides, fat, protein, amino acids, ascorbic acid, phenolic and flavonoid compound contents, and antioxidant activities of walnut male inflorescences were markedly influenced by storage temperature, and different degrees of decrease in these parameters were observed during the entire storage period. Moreover, higher storage temperature had a more significant effect on the nutrients, bioactive compounds, and antioxidant activities of walnut male flowers, and the loss rate of these components at 25°C was higher than that determined at 4°C. However, the results also presented that the ash and mineral contents did not appear to be influenced significantly by the storage temperature, and slightly significant changes were observed in crude fiber throughout storage, which indicated that the influence of storage on the individual mineral and crude fiber content was minimal. Based on the findings in this study, in order to maximize nutrients concentration, walnut male inflorescences should be kept at 4°C for <6 days and be consumed as fresh as possible.

[Drought-resistance evaluation of marigold cultivars based on multiple statistics analysis].

By the methods of principal component analysis, subordinate function, and cluster analysis, a comprehensive evaluation was conducted on the drought-resistance of 16 physiological indices of nine marigold cultivars. Under drought stress, the 16 physiological indices had different responses, among which, free proline (Pro), H2O2, and ascorbate peroxidase (APX) showed the greatest sensitivity. There existed significant correlations in the drought-resistance coefficients of parts of the physiological indices. Four principal factors presented 88.6% of the information of the 16 indices, and the cultivars 'Janie' and 'Gate gold', 'Bonanzn', 'Janie', and 'Chokdee' had the strongest drought-resistance for the 4 principal factors, respectively. The comprehensive evaluation value of the drought-resistance of the 9 cultivars was in the order of 'Janie' > 'Gate gold' > 'Bonanza' > 'Chokdee' > 'Giant' > 'Great hero' > 'Little hero' > 'Durango' > 'Discovery'. The 9 cultivars could be clustered into 3 groups, and 'Gate gold', 'Janie', 'Bonanza' and 'Chokdee' belonged to the drought-resistance group.