Transcriptomic and Metabolic Profiling Reveal the Mechanism of Ovule Development in Castanea mollissima.

Ovule abortion, which is the main cause of empty burs in the Chinese chestnut, affects the formation of embryos and further reduces yield; therefore, it is important to study the mechanism of ovule abortion. In this study, we analyzed the transcriptomic and metabolomic data of ovules at critical developmental stages to explore the key regulatory networks affecting ovule development. The metabolites were enriched mainly in pathways involved in phytohormone signaling, energy metabolism, and amino acid synthesis in the endoplasmic reticulum. Analysis of the differentially expressed genes (DEGs) revealed that the HSP genes were significantly down-regulated during fertilization, indicating that this process is extremely sensitive to temperature. The hormone and sucrose contents of ovules before and after fertilization and of fertile and abortive ovules at different developmental stages showed significant differences, and it is hypothesized that that abnormal temperature may disrupt hormone synthesis, affecting the synthesis and catabolism of sucrose and ultimately resulting in the abortive development of Chinese chestnut ovules. At the pollination and fertilization stage of chestnuts, spraying with ethylene, ACC, and AIB significantly increased the number of developing fruit in each prickly pod compared to CK (water) treatment. These results indicated that both ethylene and ACC increased the rate of ovule development. This study provides an important theoretical molecular basis for the subsequent regulation of ovule development and nut yield in the Chinese chestnut.

Genome-Wide Characterization and Functional Validation of the ACS Gene Family in the Chestnut Reveals Its Regulatory Role in Ovule Development.

Ovule abortion significantly contributes to a reduction in chestnut yield. Therefore, an examination of the mechanisms underlying ovule abortion is crucial for increasing chestnut yield. In our previous study, we conducted a comprehensive multiomic analysis of fertile and abortive ovules and found that ACS genes in chestnuts (CmACS) play a crucial role in ovule development. Therefore, to further study the function of ACS genes, a total of seven CmACS members were identified, their gene structures, conserved structural domains, evolutionary trees, chromosomal localization, and promoter cis-acting elements were analyzed, and their subcellular localization was predicted and verified. The spatiotemporal specificity of the expression of the seven CmACS genes was confirmed via qRT-PCR analysis. Notably, CmACS7 was exclusively expressed in the floral organs, and its expression peaked during fertilization and decreased after fertilization. The ACC levels remained consistently greater in fertile ovules than in abortive ovules. The ACSase activity of CmACS7 was identified using the genetic transformation of chestnut healing tissue. Micro Solanum lycopersicum plants overexpressing CmACS7 had a significantly greater rate of seed failure than did wild-type plants. Our results suggest that ovule fertilization activates CmACS7 and increases ACC levels, whereas an overexpression of CmACS7 leads to an increase in ACC content in the ovule prior to fertilization, which can lead to abortion. In conclusion, the present study demonstrated that chestnut ovule abortion is caused by poor fertilization and not by nutritional competition. Optimization of the pollination and fertilization of female flowers is essential for increasing chestnut yield and reducing ovule abortion.

Molecular characterization of SPL gene family during flower morphogenesis and regulation in blueberry.

The SPL gene is a plant-specific transcription factor involved in the regulation of plant growth and development, which have been identified in woody plants. The process of floral bud differentiation affects the timing of flowering and fruit set and regulates plant growth, however, the mechanism of regulation of flower development by SPL genes is less studied. In this study, 56 VcSPL genes were identified in the tetraploid blueberry. The VcSPL gene family was classified into six subfamilies, and analysis of cis-elements showed that VcSPL genes were regulated by light, phytohormones (abscisic acid, MeJA), and low temperature. In the evolutionary analysis, segmental replication may play an important role in VcSPL gene amplification. Interestingly, we also studied diploid blueberry (Bilberry), in which 24 SPL genes were identified, and 36 homologous pairs were found, suggesting a high degree of convergence in the syntenic relationship between blueberry (Vaccinium corymbosum L) and bilberry (Vaccinium darrowii). Based on the expression profile, VcSPL genes were expressed at high levels in flowers, shoots, and roots, indicating a diversity of gene functions. Then we selected 20 differentially-expressed SPL genes to further investigate the role of VcSPL in floral induction and initiation. It showed that the genes VcSPL40, VcSPL35, VcSPL45, and VcSPL53 may play a crucial role in the blueberry floral transition phase (from vegetative growth to flower initiation). These results provided important information for understanding and exploring the role of VcSPLs in flower morphogenesis and plant growth.

The sex expression and sex determining mechanism in Pistacia species.

Generally, Pistacia species are dioecious, but monoecious strains in several populations have been found, providing excellent models for studying sex differentiation and sex determination mechanisms. Although the mechanisms of sex determination and sex evolution have been extensively studied, related research on heterozygous woody plants is limited. Here, we discuss the expressions of various sex types, which showed broad diversity and complex instability. We have also reviewed the sex determination systems in the plant kingdom and the morphological, cytological, physiological, and molecular aspects of the sex-linked markers in Pistacia trees. Moreover, hypotheses to explain the origin of monoecy are discussed, which is more likely to be the interaction between sex-related genes and environment factors in female plants. Besides, further prospects for the utilization of monoecious resources and the research directions of sex determination mechanism are proposed. This study provides important information on sex expression and provides more insights into sex differentiation and determination.

Effects of different nitrogen application rates and picking batches on the nutritional components of Lycium barbarum L. fruits.

Lycium barbarum L., commonly known as wolfberry, is not only a traditional Chinese medicine but also a highly nutritious food. Its main nutrients include L. barbarum polysaccharide, flavonoid polyphenols, carotenoids, alkaloids, and other compounds, demonstrating its wide application value. This study investigated the effects of nitrogen application on the accumulation of the main nutrients and metabolites in wolfberry fruits under three different nitrogen application rates, namely, N1 (20% nitrogen (N) reduction, 540 kg·ha-2), N2 (medium N, 675 kg·ha-2), and N3 (20% nitrogen increase, 810 kg·ha-2,which is a local conventional nitrogen application amount.). Additionally, due to continuous branching, blossoming, and fruiting of wolfberry plants during the annual growth period, this research also explored the variation in nutritional composition among different harvesting batches. The contents of total sugar and polysaccharide in wolfberry fruit were determined by Fehling reagent method and phenol-sulfuric acid method, respectively;The content of betaine in fruit was determined by high-performance liquid chromatography,and the flavonoids and carotene in the wolfberry fruits were determined by spectrophotometry. Analysis of data over three consecutive years revealed that as nitrogen application increased, the total sugar content in wolfberry fruits initially decreased and then increased. The levels of L. barbarum polysaccharides, total flavonoids, and total carotenoids initially increased and then decreased, while the betaine content consistently increased. Different picking batches significantly impacted the nutrient content of wolfberry fruits. Generally, the first batch of summer wolfberry fruits had greater amounts of total sugar and flavonoids, whereas other nutrients peaked in the third batch. By employing a broadly targeted metabolomics approach, 926 different metabolites were identified. The top 20 differentially abundant metabolites were selected for heatmap generation, revealing that the contents of L-citrulline, 2-methylglutaric acid, and adipic acid increased proportionally to the nitrogen gradient. Conversely, the dibutyl phthalate and 2, 4-dihydroxyquinoline contents significantly decreased under high-nitrogen conditions. The remaining 15 differentially abundant metabolites, kaempferol-3-O-sophorosid-7-O-rhamnoside, trigonelline, and isorhamnosid-3-O-sophoroside, initially increased and then decreased with increasing nitrogen levels. Isofraxidin, a common differentially abundant metabolite across all treatments, is a coumarin that may serve as a potential biomarker for wolfberry fruit response to nitrogen. Differentially abundant metabolites were analyzed for GO pathway involvement, revealing significant enrichment in metabolic pathways and biosynthesis of secondary metabolites under different nitrogen treatments. In conclusion, a nitrogen application of 675 kg·ha-2, 20% less than the local farmers' actual application, was most beneficial for the quality of four-year-old Ningqi 7 wolfberry fruits. Consumers who purchase wolfberry-dried fruit for health benefits should not consider only the first batch of summer wolfberry fruits. These results offer a broader perspective for enhancing the quality and efficiency of the wolfberry industry.

Integrated transcriptome, metabolome and phytohormone analysis reveals developmental differences between the first and secondary flowering in Castanea mollissima.

Introduction: Chestnut (Castanea mollissima BL.) is an important woody grain, and its flower formation has a significant impact on fruit yield and quality. Some chestnut species in northern China re-flower in the late summer. On the one hand, the second flowering consumes a lot of nutrients in the tree, weakening the tree and thus affecting flowering in the following year. On the other hand, the number of female flowers on a single bearing branch during the second flowering is significantly higher than that of the first flowering, which can bear fruit in bunches. Therefore, these can be used to study the sex differentiation of chestnut. Methods: In this study, the transcriptomes, metabolomes, and phytohormones of male and female chestnut flowers were determined during spring and late summer. We aimed to understand the developmental differences between the first and secondary flowering stages in chestnuts. We analysed the reasons why the number of female flowers is higher in the secondary flowering than in the first flowering and found ways to increase the number of female flowers or decrease the number of male flowers in chestnuts. Results: Transcriptome analysis of male and female flowers in different developmental seasons revealed that EREBP-like mainly affected the development of secondary female flowers and HSP20 mainly affected the development of secondary male flowers. KEGG enrichment analysis showed that 147 common differentially-regulated genes were mainly enriched from circadian rhythm-plant, carotenoid biosynthesis, phenylpropanoid biosynthesis, and plant hormone signal transduction pathways. Metabolome analysis showed that the main differentially accumulated metabolites in female flowers were flavonoids and phenolic acids, whereas the main differentially accumulated metabolites in male flowers were lipids, flavonoids, and phenolic acids. These genes and their metabolites are positively correlated with secondary flower formation. Phytohormone analysis showed that abscisic and salicylic acids were negatively correlated with secondary flower formation. MYB305, a candidate gene for sex differentiation in chestnuts, promoted the synthesis of flavonoid substances and thus increased the number of female flowers. Discussion: We constructed a regulatory network for secondary flower development in chestnuts, which provides a theoretical basis for the reproductive development mechanism of chestnuts. This study has important practical implications for improving chestnut yield and quality.

Relationship of Soil Microbiota to Seed Kernel Metabolism in Camellia oleifera Under Mulched.

An experiment was conducted from 2016 to 2017 to assess the effect of kernel metabolism in development stages after organic mulching compared to control. Organic mulching significantly increased crop yields (higher 128% in 2016, higher 60% in 2017), oil content (the highest oil content was 27.6% higher than that of the control), and improved soil properties (SOC, SAN, AP, and AK). In this study, soil pH, SOC, AN, AP, and AK in 0-30 cm soil depth were measured. Results showed that the effect of mulching on soil pH was not significant at the harvesting stage. The greatest metabolic differences occurred during the period of high oil conversion (S2-S4), primarily involving 11 relevant metabolic pathways. This further verified that Camellia oleifera oil yield was improved after mulching. A total of 1,106 OTUs were detected by using 16S rRNA, and Venn diagram showed that there were 106 unique OTUs in control and 103 OTUs in the treatment, respectively. Correlation analysis showed that soil pH and soil temperature were two indicators with the most correlations with soil microbiota. The yield was significantly positively correlated with soil microbial Proteobacteria, Bacteroidetes, and soil nutrition indexes. Organic mulching improved the physicochemical properties of soils, caused differences in the relative abundance of dominant bacteria in soil bacteria, and improved the soil microbiological environment to promote plant growth, indicating that organic mulching is an effective measure to alleviate seasonal drought.

Maturity Grading and Identification of Camellia oleifera Fruit Based on Unsupervised Image Clustering.

Maturity grading and identification of Camellia oleifera are prerequisites to determining proper harvest maturity windows and safeguarding the yield and quality of Camellia oil. One problem in Camellia oleifera production and research is the worldwide confusion regarding the grading and identification of Camellia oleifera fruit maturity. To solve this problem, a Camellia oleifera fruit maturity grading and identification model based on the unsupervised image clustering model DeepCluster has been developed in the current study. The proposed model includes the following two branches: a maturity grading branch and a maturity identification branch. The proposed model jointly learns the parameters of the maturity grading branch and maturity identification branch and used the maturity clustering assigned from the maturity grading branch as pseudo-labels to update the parameters of the maturity identification branch. The maturity grading experiment was conducted using a training set consisting of 160 Camellia oleifera fruit samples and 2628 Camellia oleifera fruit digital images collected using a smartphone. The proposed model for grading Camellia oleifera fruit samples and images in training set into the following three maturity levels: unripe (47 samples and 883 images), ripe (62 samples and 1005 images), and overripe (51 samples and 740 images). Results suggest that there was a significant difference among the maturity stages graded by the proposed method with respect to seed oil content, seed soluble protein content, seed soluble sugar content, seed starch content, dry seed weight, and moisture content. The maturity identification experiment was conducted using a testing set consisting of 160 Camellia oleifera fruit digital images (50 unripe, 60 ripe, and 50 overripe) collected using a smartphone. According to the results, the overall accuracy of maturity identification for Camellia oleifera fruit was 91.25%. Moreover, a Gradient-weighted Class Activation Mapping (Grad-CAM) visualization analysis reveals that the peel regions, crack regions, and seed regions were the critical regions for Camellia oleifera fruit maturity identification. Our results corroborate a maturity grading and identification application of unsupervised image clustering techniques and are supported by additional physical and quality properties of maturity. The current findings may facilitate the harvesting process of Camellia oleifera fruits, which is especially critical for the improvement of Camellia oil production and quality.

Beta-Amylase and Phosphatidic Acid Involved in Recalcitrant Seed Germination of Chinese Chestnut.

Chinese chestnut (Castanea mollissima), a species with recalcitrant seeds, is an important source of nuts and forest ecosystem services. The germination rate of recalcitrant seeds is low in natural habitats and decreases under conditions of desiccation and low temperature. The germination rate of cultivated Chinese chestnut seeds is significantly higher than that of wild seeds. To explore the reasons for the higher germination rate of cultivated seeds in Chinese chestnut, 113,524 structural variants (SVs) between the wild and cultivated Chinese chestnut genomes were detected through genome comparison. Genotyping these SVs in 60 Chinese chestnut accessions identified allele frequency changes during Chinese chestnut domestication, and some SVs are overlapping genes for controlling seed germination. Transcriptome analysis revealed downregulation of the abscisic acid synthesis genes and upregulation of the beta-amylase synthesis genes in strongly selected genes of cultivated seeds. On the other hand, hormone and enzyme activity assays indicated a decrease in endogenous ABA level and an increase in beta-amylase activity in cultivated seeds. These results shed light on the higher germination rate of cultivated seeds. Moreover, phosphatidic acid synthesis genes are highly expressed in seed germination stages of wild Chinese chestnut and may play a role in recalcitrant seed germination. These findings provide new insight into the regulation of wild seed germination and promote natural regeneration and succession in forest ecosystems.

Lilac (Syringa oblata) genome provides insights into its evolution and molecular mechanism of petal color change.

Color change during flower opening is common; however, little is understood on the biochemical and molecular basis related. Lilac (Syringa oblata), a well-known woody ornamental plant with obvious petal color changes, is an ideal model. Here, we presented chromosome-scale genome assembly for lilac, resolved the flavonoids metabolism, and identified key genes and potential regulatory networks related to petal color change. The genome assembly is 1.05 Gb anchored onto 23 chromosomes, with a BUSCO score of 96.6%. Whole-genome duplication (WGD) event shared within Oleaceae was revealed. Metabolome quantification identified delphinidin-3-O-rutinoside (Dp3Ru) and cyanidin-3-O-rutinoside (Cy3Ru) as the major pigments; gene co-expression networks indicated WRKY an essential regulation factor at the early flowering stage, ERF more important in the color transition period (from violet to light nearly white), while the MBW complex participated in the entire process. Our results provide a foundation for functional study and molecular breeding in lilac.

Response of metabolic and lipid synthesis gene expression changes in Camellia oleifera to mulched ecological mat under drought conditions.

Plants respond to adverse conditions by activating defense mechanisms that alter metabolism and impact agricultural crop yield. Organic mulching of Camellia oleifera leads to increased oil yield compared to control. In this study, multi-platform untargeted metabolomics and qRT-PCR were used to measure the effects of organic mulching on seed kernel metabolism. Metabolomics analysis revealed that tyrosine, tryptophan, and several flavonoids and polyphenol metabolites were significantly lower in the mulched treatment compared to the control, indicating lower stress levels with mulching. The qRT-PCR analysis showed that EAR, SAD, and CoHCD were up-regulated by mulching, while CT, FAD7, FAD8, CoATS1, SQS, SQE, FATB, and ß-AS were down-regulated. Correlation network analysis was used to integrate data from this multi-omics investigation to analyze the relationships between differentially expressed genes, metabolites, and fruit and soil indicators concerning mulch treatment of C. oleifera.

Direct and Indirect Somatic Embryogenesis Induction in Camellia oleifera Abel.

Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.

Molecular cloning and functional characterization of CoFT1, a homolog of FLOWERING LOCUS T (FT) from Camellia oleifera.

FLOWERING LOCUS T (FT) is an important integrator of flowering genetic pathways and plays crucial roles in flowering transition. The tea-oil tree (Camellia oleifera Abel.) is a valuable woody oil crop, but the molecular mechanisms controlling flowering are still unclear. In this study, a FT-like gene, CoFT1, was isolated and characterized from C. oleifera. The CoFT1 protein was localized in the nucleus and cytoplasm of Arabidopsis protoplasts, and exhibited no transactivation activity in yeast cells. CoFT1 was highly expressed in mature leaves and reproductive organs, such as flower buds, flowers, flower organs, and young fruits. Its expression showed diurnal rhythms under both long-day and short-day conditions, and was photoperiod-dependent. Seasonal expression analysis revealed that the CoFT1 transcript in leaves increased during the floral induction period. Overexpression of CoFT1 in wild-type Arabidopsis resulted in precocious flowering and elevated the transcription levels of flowering related genes, such as SOC1, AP1, and LFY. Furthermore, the yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that CoFT1 was able to interact with both Arabidopsis FD and C. oleifera CoFD proteins. The sequence analysis revealed that the CoFT1 promoter contained a number of light-responsive elements, several hormonal- and stress-responsive motifs, and flowering related transcriptional factor binding sites, including CORE, CCAATBOX1, and CArG motifs. Our results suggested that CoFT1 might function as a flowering promoter in C. oleifera.

Combined effects of cotyledon excision and nursery fertilization on root growth, nutrient status and outplanting performance of Quercus variabilis container seedlings.

Artificial excision of the distal part of acorns in order to promote germination is well researched in oak seedling cultivation studies. However, studies of combined effects of cotyledon excision and nursery fertilization on container seedlings are lacking, especially for seedling root growth and outplanting performance. This study aimed to explore the main effects of cotyledon excision on Quercus variabilis seedling emergence characteristics and demonstrated the combined effects of cotyledon excision and nursery fertilization on seedling quality to improve Quercus variabilis seedling outplanting performance. Four cotyledon excision treatments and two classes of nursery fertilization were implemented. Seedling emergence was noted every week after sowing. Seedling dry mass, morphology, and nutrient status were assessed at the end of the nursery season. After the first outplanting season, the aforementioned measurements along with seedling survival were determined once again. The results showed that cotyledon excision generally induced greater and more rapid seedling emergence, but did not affect shoot emergence synchronicity. The highest total emergence and emergence rate occurred with Intermediate excision (1/2 of the distal end of acorn was excised). Effects of nutrient loss due to cotyledon excision on seedling quality and outplanting performance were somewhat compensated by nursery fertilization. Nursery fertilization promoted dry mass increment (the net increment from T0 to T2 for dry mass) for excised seedlings after outplanting, resulting in better performance for Slight (1/3 of the distal end of acorn was excised) and Intermediate excision treatments in the field. Thus we conclude Intermediate excision combined with reasonable nursery fertilization can be recommended for production of nursery grown seedlings for afforestation.