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.

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.

Stem Rot Disease of Juglans sigillata Dode Caused by Fusarium fujikuroi in China.

Juglans sigillata Dode is an endemic species in the southwest China, and is an important nut and woody oil tree. The shell of its fruit is hard and can be used to make various crafts. From 216 to 2019, typical stem rot symptoms of 8-year-old J. sigillata were observed in cultivated fields in a 600-ha orchard in Zigong, Sichuan province, China. At this orchard, approximately 35% of the trees have been seriously damaged over the past few years. The typical symptoms were water-soaking on the stem, rotting, wilting, and blighting, eventually leading to the death of the plant. In June, ten diseased tissues were collected and surface-sterilized by 3% NaClO and 75% alcohol. Morphological observations were made from the isolates grown on Potato dextrose agar (PDA) and incubated at 25°C for 3 to 9 days. Morphological characteristics were made on pure cultures grown on Synthetic low nutrient agar (SNA). Five isolates with similar morphology were isolated from single spores. Colonies on PDA reached 8.3 cm in diameter after 6 days at 25 °C, aerial mycelia were white to cream and wol-like, later turning violet and dark purple with age. The hyphae of the strain were colorless and septate. There were two types of conidia on SNA, microconidia and macroconidia. Microconidia (n = 50) were oval, elliptic or clavate, no septate, 2.2 to 3.8 × 7.6 to11.7 µm. Conidiophores were branched or unbranched, solitary or in groups, phialides cylindrical to flask-shaped, monophialidic and polyphialidic. Macroconidia (n = 50) were long slender with a curved apical cell and foot-like basal cell, 3 to 4 septate and 2.1 to 3.9 × 26.2 to 53.4 µm. For molecular identification, the internal transcribed spacer (ITS), ß-tubulin (TUB2), translation elongation factor (TEF1) and large subunit (LSU) were amplified with the corresponding primer pairs ITS1/ITS4 (White et al. 1990), BT2A/BT2B, EF1/EF2 (O'Donnell et al. 1997), and LROR/LR5 (Rehner and Samuels 1994), respectively. BLAST search results indicated that the ITS, TUB2, TEF1, LSU sequences (GenBank acc. nos. MT791384, MT786729, MN853324, and MT705246) showed 99 to100% identity with Fusarium fujikuroi sequences at NCBI (GenBank acc. nos. MG798789, MH398245, MK604519 and KJ954504). The results were confirmed by multilocus phylogenetic analysis. Based on the morphological characteristics and molecular analysis of the isolates, the fungus was identified as F. fujikuroi (Leslie and Summerell 2006). Koch's postulates were checked under controlled conditions. Fifteen 2-year-old healthy potted J. sigillata were inoculated by pricking the epidermis of stem with a needle and applying 150 µl of a microconidial suspension (1 × 106 spores/ml) to the wounded surface with a brush. Sterilizd distilled water was used as the control. The experiment was repeated three times. All the plants were incubated at 25 ± 2°C after inoculation for daily observation of disease development. After 12 days, the inoculated plants showed the same symptoms as observed in the original diseased plants, while the control plants were asymptomatic. The fungus was re-isolated from the symptomatic stems and was completely identical to the isolates used to inoculate the plants. Thus, we confirmed that F. fujikuroi caused the stem rot of J. sigillata. To our knowledge, this is the first report of this fungus causing stem rot in J. sigillata in China. Our results can help identify stem rot disease of J. sigillata and develop control measures for the disease.

Phenolic constituents from the fresh pericarps of Juglans sigillata.

Three new phenolic compounds, 2,3,4-trihydroxy-1-(4'-hydroxybenzyl)benzoic acid (1), 2,3,4-trihydroxy-1-(4'-hydroxy-3'-methoxybenzyl)benzoic acid (2), and 2-(4'-hydroxy-3'-methoxybenzyl)-3,5-dimethoxy-1,4-benzoquinone (3), were isolated from the fresh pericarps of Juglans sigillata Dode. Their structures were elucidated by integrated spectroscopic techniques. Bioactivity screening results showed that compounds 1-3 exhibited moderate neuroprotective effects against H2O2-induced or CoCl2-induced cellular damage in human neuroblastoma SH-SY5Y cell line.

Precocious genotypes and homozygous tendency generated by self-pollination in walnut.

BACKGROUND: Observations of precocious (early bearing) genotypes of walnut (Juglans regia L.) under natural conditions encouraged us to study the origin and genetic control of these fascinating traits. RESULTS: In this study, the self-fertility, progeny performance, and simple sequence repeat (SSR) locus variation of iron walnut (Juglans sigillata Dode), an ecotype of J. regia, were investigated. The average self-pollinated fruit set rate of J. sigillata cv. 'Dapao' (DP) was 7.0% annually from 1979 to 1982. The average germination rate of self-pollinated seeds was 45.2% during the 4-year period. Most progeny had inbreeding depression. Nine representative self-pollinated progeny (SP1-SP9), with special or typical traits of DP, were selected. SP1-SP4 were precocious because they initiated flowers as early as 2 years after germination, compared to the 7-10-yr period that is typical of DP. SP9 had not flowered since 1980. Twelve SSR markers were used to analyze the SP and DP. The genome of SP had a tendency toward high levels of homozygosity. The high levels of homozygosity reported in 18 additional precocious walnut genotypes complemented the results of this study. CONCLUSIONS: These results provide evidence of precocious phenotypes and genomes with high levels of homozygosity that might be generated from self-pollinating walnut. This suggests that self-pollination might facilitate the generation of unique homozygous parents for subsequent use in walnut-breeding programs. The results also indicate that more attention should be focused on adequate management of precocious walnut to avoid early depression in the production of nuts.

Study on chemical constituents from green husk of Juglans sigillata / 中草药

Objective: To investigate the chemical constituents in the green husk of Juglans sigillate. Methods: The chemical constituents were isolated by silica gel, RP-18, MCI column chromatographies and so on. The structures were identified on the basis of spectroscopic analysis (MS, 13C-NMR, 1H-NMR). Results: Eleven compounds were isolated from the extract of green husk of J. sigillate. And their structures were characterized as: sinapaldehyde (1), (Z)-10-eicosenoic acid (2), 5α, 8α-epidioxyergosta-6, 22E-diene-3β-ol (3), 5, 8-dihydroxy-4-methoxy-α-tetralone (4), regiolone (5), 4, 5-dihydroxy-α-tetralone (6), 4, 5, 8-trihydroxy-α-tetralone (7), 5-hydroxy-4-methoxy-α-tetralone (8), 5-hydroxy-2-methoxy-1, 4-naphthoquinone (9), naringenin (10), and β-sitosterol (11). Conclusion: Compounds 1-2 are isolated from the plants of Juglans Linn. for the first time.

Study on chemical constituents in shells of Juglans sigillata / 中草药

Objective: To investigate the chemical constituents in the shells of Juglans sigillata. Methods: The chemical constituents were isolated by silica gel, RP18, Sephadex LH-20, and MCI column chromatography and semi-preparative HPLC and so on. The structures were identified on the basis of spectroscopic analysis and chemical evidence. Results: Fifteen compounds were isolated and identified in the 70% ethanol extract from the shells of J. sigillata including seven phenolic glycosides: tachioside (1), mudanoside A (2), 4-O-β-D-glucopyranosylvanillc acid (3), breynioside A (4), 1-O-vanilloyl-β-D-glucose (5), 6'-O-vanilloyltachioside (6), and 6'-O-vanilloylisotachioside (7); three phenylpropanoide acid glycosides: 6-O-feruloyl-D-glucopyranose (8), methyl-4-O-coumaroylquinate (9), and 5-p-cis-coumaroylquinic acid (10); two tetralone glycosides: juglanin A (11) and juglanin E (12); one norsesquiterpenes glycoside: roseoside (13); one flavone: toxifolin (14); and one glucosylated abscisic acid derivate: (1'R, 3'R, 5'R, 8'S)-epi-dihydrophaseic acid β-D-glucoside (15). Conclusion: Except compound 14, the other compounds are isolated from the shells of J. sigillata for the first time. And compounds 1-4, 13, and 15 are reported for the first time from the plants in genus of Juglans L.