Comparative study on fruit development and oil synthesis in two cultivars of Camellia oleifera.

BACKGROUND: The oil-tea tree (Camellia oleifera Abel.) is a woody tree species that produces edible oil in the seed. C. oleifera oil has high nutritional value and is also an important raw material for medicine and cosmetics. In China, due to the uncertainty on maturity period and oil synthesis mechanism of many C. oleifera cultivars, growers may harvest fruits prematurely, which could not maximize fruit and oil yields. In this study, our objective was to explore the mechanism and differences of oil synthesis between two Camellia oleifera cultivars for a precise definition of the fruit ripening period and the selection of appropriate cultivars. RESULTS: The results showed that 'Huashuo' had smaller fruits and seeds, lower dry seed weight and lower expression levels of fatty acid biosynthesis genes in July. We could not detect the presence of oil and oil bodies in 'Huashuo' seeds until August, and oil and oil bodies were detected in 'Huajin' seeds in July. Moreover, 'Huashuo' seeds were not completely blackened in October with up to 60.38% of water and approximately 37.98% of oil in seed kernels whose oil content was much lower than normal mature seed kernels. The oil bodies in seed endosperm cells of 'Huajin' were always higher than those of 'Huashuo' from July to October. CONCLUSION: Our results confirmed that C. oleifera 'Huashuo' fruits matured at a lower rate compared to 'Huajin' fruits and that 'Huajin' seeds entered the oil synthesis period earlier than 'Huashuo' seeds. Moreover, 'Huashuo' fruits did not mature during the Frost's Descent period (October 23-24 each year).

Effects of Funneliformis mosseae on the utilization of organic phosphorus in Camellia oleifera Abel.

Arbuscular mycorrhizal (AM) fungi play an important role in the acquisition of phosphorus (P) by plants. The external hyphae of AM fungi function as an extension of plant roots and may downregulate related functions in the roots. It is not clear whether the ability of AM fungi to mineralize organic P affects root phosphatase activities. A pot experiment was conducted to investigate the effect of Funneliformis mosseae on soil organic P mineralization under phytate application and to explore root phosphatase activities, P uptake, and growth in Camellia oleifera Abel. The plants and their growth substrates were harvested 4 and 8 months after planting. The results showed that organic P application had no effect on the total dry mass of nonmycorrhizal plants, but differences in dry mass under P application were observed in mycorrhizal plants in both harvests. Inoculation with F. mosseae increased soil acid phosphatase, phytase, and alkaline phosphatase activities and reduced the soil organic P content. Mycorrhizal plants had higher root activity, shoot and root P contents and root acid phosphatase and phytase activities than nonmycorrhizal plants irrespective of organic P application. In conclusion, AM fungi enhanced the mineralization of soil organic P and positively affect root phosphatase activities.

Identification of miRNA-mRNA Regulatory Modules Involved in Lipid Metabolism and Seed Development in a Woody Oil Tree (Camellia oleifera).

Tea oil camellia (Camellia oleifera), an important woody oil tree, is a source of seed oil of high nutritional and medicinal value that is widely planted in southern China. However, there is no report on the identification of the miRNAs involved in lipid metabolism and seed development in the high- and low-oil cultivars of tea oil camellia. Thus, we explored the roles of miRNAs in the key periods of oil formation and accumulation in the seeds of tea oil camellia and identified miRNA-mRNA regulatory modules involved in lipid metabolism and seed development. Sixteen small RNA libraries for four development stages of seed oil biosynthesis in high- and low-oil cultivars were constructed. A total of 196 miRNAs, including 156 known miRNAs from 35 families, and 40 novel miRNAs were identified, and 55 significantly differentially expressed miRNAs were found, which included 34 upregulated miRNAs, and 21 downregulated miRNAs. An integrated analysis of the miRNA and mRNA transcriptome sequence data revealed that 10 miRNA-mRNA regulatory modules were related to lipid metabolism; for example, the regulatory modules of ath-miR858b-MYB82/MYB3/MYB44 repressed seed oil biosynthesis, and a regulation module of csi-miR166e-5p-S-ACP-DES6 was involved in the formation and accumulation of oleic acid. A total of 23 miRNA-mRNA regulatory modules were involved in the regulation of the seed size, such as the regulatory module of hpe-miR162a_L-2-ARF19, involved in early seed development. A total of 12 miRNA-mRNA regulatory modules regulating growth and development were identified, such as the regulatory modules of han-miR156a_L+1-SPL4/SBP2, promoting early seed development. The expression changes of six miRNAs and their target genes were validated using quantitative real-time PCR, and the targeting relationship of the cpa-miR393_R-1-AFB2 regulatory module was verified by luciferase assays. These data provide important theoretical values and a scientific basis for the genetic improvement of new cultivars of tea oil camellia in the future.

New perspective for evaluating the main Camellia oleifera cultivars in China.

To assess the adaptability of Camellia oleifera for introduction in new growth locations, this study evaluated 10 representative C. oleifera cultivars from the main areas in China where this oil-producing evergreen crop is grown. Cluster analysis, correlation analysis, and membership function analysis were used to evaluate various indices of the selected C. oleifera cultivars, including flowering phenology, cold tolerance, leaf structure, pollen characteristics, and pollen viability. The correlation analysis identified the full blossoming time, leaf palisade and spongy tissue thickness, pollen deformity rate, and pollen activity as key indices for determining the adaptability of the cultivars to new areas. The membership function analysis of the 10 C. oleifera cultivars revealed the following order of adaptability: 'XLC25' > 'Changlin4hao' > 'Ganzhouyou8hao' > 'Ganzhouyou6hao' > 'Tiechengyihao' > 'Eyou465' > 'XLC10' > 'Changlin3hao' > 'Changlin18hao' > 'QY235.' When introducing C. oleifera cultivars to new regions, the higher-ranked cultivars are more likely to be successful. The results of this study may provide a new direction for the comprehensive assessment of plant introduction and domestication potential, i.e., the assessment of the vegetative and reproductive growth, adversity resistance, and blossoming time of plants.

Soil C-N-P pools and stoichiometry as affected by intensive management of camellia oleifera plantations.

Intensive management of C. oleifera has produced many pure C. oleifera plantations. The transmission of C. oleifera plantation will potentially affect soil C, N, and P pools as well as their stoichiometric characteristics both in top soil layer and vertical soil profile due to the intensive management. To understand changes in vertical pools and stoichiometric characteristics of soil C, N, and P as affected by intensive management of C. oleifera plantations, both mixed and pure C. oleifera plantations were studied. We conducted studies in five locations in Jiangxi, China with both pure and mixed C. oleifera plantations, to compare changes in vertical pools and stoichiometry of C, N, and P. Both C and N pools were significantly different between mixed and pure plantation types of C. oleifera. However, the ratio of C:N, C:P, and N:P was consistently higher in mixed plantations with C:P and N:P altered but C:N ratio did not change with soil depth. The intensive management significantly impact both C and N pools and the stoichiometry of C, N, and P. Intensive management of C. oleifera plantations decreased both C and N pools, especially at the depth of 30-50 cm soil layer. C. oleifera plantation alteration from mixed to pure should be considered in future forest management practice considering the substantial effects on soil element cycling and distribution along vertical soil profile.

Manganese accumulation and plant physiology behavior of Camellia oleifera in response to different levels of nitrogen fertilization.

Manganese (Mn) pollution in soil, especially around the mining areas, is a severe problem in China. Seeking for effective remediation methods for Mn-contaminated soil is therefore urgent and necessary. Camellia oleifera (C. oleifera) is one of the world's four major woody oil plants, which is widely cultivated in subtropical acidic soils for oil production and has become an important economic and ecological resource in Guangxi Province. Nitrogen (N) is one of the most common limiting factors for plant growth and development in soils. We carried out this study to evaluate the effects of different N fertilization levels (0, 100, 300 and 500 mg kg-1) on the morphological and physiological characteristics of C. oleifera in two soils with different Mn-contamination degrees. The results indicate that N fertilization affected the plant growth and the content of photosynthetic pigments, while C. oleifera accumulated great amounts of Mn in both soils. However, the plant biomass reduced significantly at the high-level N fertilization (≥300 mg kg-1), and the oxidative stress was stimulated under Mn contamination. As a comparison, the plant biomass remained unaffected at the low-level N fertilization (100 mg kg-1), and the ascorbate peroxidase (APX) activity in C. oleifera leaves were enhanced to alleviate the oxidative stress and therefore protecting the plant from Mn contamination. Meanwhile, plants supplemented with a low-level of N fertilizer (100 mg kg-1) had appropriate antioxidant enzyme and nonenzymatic antioxidant activities, which indicates that this was favorable growth conditions for C. oleifera. Thus, the recommended N fertilization level for maintaining plant biomass and increasing Mn accumulation in plant is 100 mg kg-1 N; at which level the efficiency of Mn phytoremediation by C. oleifera can be further enhanced.

Changes of pollen viability of ornamental plants after long-term preservation in a cryopreservation pollen bank.

This study determined the changes in pollen viability of 102 species/cultivars of ornamental plants (affiliated to 32 genera of 14 families) following long-term liquid nitrogen storage in a cryopreservation pollen bank. The goal was to provide information on the safety and stability of pollen cryopreservation technology. Fresh pollen at the time of storage was used as the control, and the study examined the pollen viability of ornamental plants cryopreserved for 8, 9, or 10 years. The results show that pollen of the 102 species/cultivars in the cryopreservation pollen bank retained viability ranging from 1% to 58%, After long-term storage there were changes in viability: 11.76% (12 species/cultivars) had increased viability, 16.67% (17 species/cultivars) had stable viability, and the viability of 71.57% (73 species/cultivars) showed a decreasing trend.

Comparative transcriptomic analysis identifies genes responsible for fruit count and oil yield in the oil tea plant Camellia chekiangoleosa.

Fruit yield is an important trait for horticultural crops. However, the limited fruit yield of Camellia chekiangoleosa, a novel promising oil tree, restricts the production of oil. The breeding improvement is little due to its long generation time and lack of available genomic sequence. We identified distinct fruit count phenotypes, high-yield (HY) and low-yield (LY). To understand the underlying genetic basis, we focused on global gene discovery and expression levels in floral buds, which affect fruit count. A total of 140,299 de novo assembled unigenes were obtained using RNA-seq technology, and more genes were expressed in HY than in LY. 2395 differentially expressed genes (DEGs) were identified and enriched in membrane, energy metabolism, secondary metabolism, fatty acid biosynthesis and metabolism, and 18 other metabolic pathways. Of the DEGs, twelve identified transcription factors, including AP2, mostly involve in inflorescence and flower development and in hormone networks. Key DEGs in fatty acid biosynthesis (Fab) FabB, FabF, FabZ, and AccD were highly expressed in floral buds and associated with high oil yield in fruits. We hypothesized that a potential link exists between fruit count and its oil yield. These findings help to elucidate the molecular cues affecting fruit count and oil yield.

Influence of Electric Fields and Conductivity on Pollen Tube Growth assessed via Electrical Lab-on-Chip.

Pollen tubes are polarly growing plant cells that are able to rapidly respond to a combination of chemical, mechanical, and electrical cues. This behavioural feature allows them to invade the flower pistil and deliver the sperm cells in highly targeted manner to receptive ovules in order to accomplish fertilization. How signals are perceived and processed in the pollen tube is still poorly understood. Evidence for electrical guidance in particular is vague and highly contradictory. To generate reproducible experimental conditions for the investigation of the effect of electric fields on pollen tube growth we developed an Electrical Lab-on-Chip (ELoC). Pollen from the species Camellia displayed differential sensitivity to electric fields depending on whether the entire cell or only its growing tip was exposed. The response to DC fields was dramatically higher than that to AC fields of the same strength. However, AC fields were found to restore and even promote pollen growth. Surprisingly, the pollen tube response correlated with the conductivity of the growth medium under different AC frequencies--consistent with the notion that the effect of the field on pollen tube growth may be mediated via its effect on the motion of ions.

A cytological study of anther and pollen development in Camellia oleifera.

The plant species Camellia oleifera is an important producer of edible oil in China. However, it suffers from a low fruit-setting rate. This study used high resolution scanning electron microscopy of semi-thin tissue sections to investigate anther development and pollen formation, and to determine whether problems in forming functional pollen cause low seed setting rates. During anther development, cell sizes within the epidermis and end othecium gradually increase, and at the beginning of the microsporocyte phase, the cells become highly vacuolated. The cell walls of the inner wall cells thicken in a ribbon pattern during the later part of the two-cell pollen stage. Middle layer cells become flattened during the later part of the secondary sporogenous cell stage. A tapetum also forms at this stage; the secondary sporogenous cells begin to degrade at the early microspore stage and completely disappear by the time the pollen is mature, thus forming a glandular tapetum. In microsporocyte, cytokinesis occurs simultaneously with microsporocyte meiosis, and a tetrahedral arrangement is present in tetrads. Reproductive cells begin to form at the later stage of microsporogenesis; the nuclei of the reproductive cells are enlarged as the reproductive cells begin to move away from the pollen wall. Two-cell pollen is first formed after reproductive cells have left the pollen wall, and the number of inclusions in the pollen subsequently increases. The pollen matures for 5-8 days before flowering occurs. No pollen abortion phenomena were observed, suggesting that anther development in C. oleifera is normal and that functional pollen are formed.

Low caffeine content in novel grafted tea with Camellia sinensis as scions and Camellia oleifera as stocks.

Caffeine, a purine alkaloid, is a major secondary metabolite in tea leaves. The demand for low caffeine tea is increasing in recent years, especially for health reasons. We report a novel grafted tea material with low caffeine content. The grafted tea plant had Camellia sinensis as scions and C. oleifera as stocks. The content of purine alkaloids was determined in the leaves of one-year-old grafted tea plants by HPLC. We also characterized caffeine synthase (CS), a key enzyme involved in caffeine biosynthesis in tea plants, at the expression level. The expression patterns of CS were examined in grafted and control leaves by Western blot, using a self-prepared polyclonal antibody with high specificity and sensitivity. The expression of related genes (TCS1, tea caffeine synthase gene, GenBank accession No. AB031280; sAMS, SAM synthetase gene, AJ277206; TIDH, IMP dehydrogenase gene, EU106658) in the caffeine biosynthetic pathway was investigated by qRT-PCR. HPLC showed that the caffeine content was only 38% as compared with the non-grafted tea leaves. Immunoblotting analysis showed that CS protein decreased by half in the leaves of grafted tea plants. qRT-PCR revealed no significant changes in the expression of two genes in the upstream pathway (sAMS and TIDH), while the expression of TCS1 was greatly decreased (50%). Taken together, these data revealed that the low caffeine content in the grafted tea leaves is due to low TCS1 expression and CS protein accumulation.

Hypotriglyceridemic potential of fermented mixed tea made with third-crop green tea leaves and camellia (Camellia japonica) leaves in Sprague-Dawley rats.

Fermented mixed tea made with third-crop green tea leaves and camellia leaves by a tea-rolling process has been developed. The objective of this study was to investigate hypotriglyceridemic potential of the mixed tea in rats. The mixed tea contained theasinensins and theaflavins. Rats fed the mixed tea extract at the level of 1% exerted significantly lower body weight and adipose tissue weight compared to animals fed third-crop green tea or camellia tea extract alone for 4 weeks. Serum and hepatic triglyceride was significantly and dose-dependently decreased by the mixed tea. This decrease was associated with lowered lipogenic enzyme activities in the liver. Furthermore, an oral administration of 4 or 8% of the mixed tea extract followed by fat emulsion suppressed the increment of serum triglyceride level. These results suggest that the mixed tea has hypotriglyceridemic action, partially via delaying triglyceride absorption in the small intestine and repressing hepatic lipogenic enzymes.

Fermentation characteristics of some assamica clones and process optimization of black tea manufacturing.

Changes in the specific activities of polyphenol oxidase (PPO), peroxidase (POD), and protease and in the relative amounts of flavan-3-ols for eight genetically derived cultivated teas at various stages of leaf maturity and in four succescive seasons were examined. A series of investigations were carried out to study the cross-reactivity of complex polyphenols and PPO-generated orange-yellow theaflavins, as well as of POD oxidized substrates, producing brown so-called thearubigins during fermented tea processing. From the estimation of five major catechins, PPO activities in young shoots, and theaflavin and thearubigin contents of crushed, torn, and curled (CTC) black teas, the superior variety and flavorful flush characteristics were refined. Notable protein hydrolysis by endogenous protease as measured from free amino acids and formation of tannin-protein complex (browning products) was obtained for cultivar character and product quality. Results showed that process optimization with respect to time, temperature, moisture, and pH maximizes PPO-catalyzed desirable theaflavin pigments, whereas POD-mediated chemical reaction produces dull color.