Genome sequencing provides insights into the evolution and antioxidant activity of Chinese bayberry.

BACKGROUND: Chinese bayberry (Myrica rubra Sieb. & Zucc.) is an economically important fruit tree characterized by its juicy fruits rich in antioxidant compounds. Elucidating the genetic basis of the biosynthesis of active antioxidant compounds in bayberry is fundamental for genetic improvement of bayberry and industrial applications of the fruit's antioxidant components. Here, we report the genome sequence of a multiple disease-resistant bayberry variety, 'Zaojia', in China, and the transcriptome dynamics in the course of fruit development. RESULTS: A 289.92 Mb draft genome was assembled, and 26,325 protein-encoding genes were predicted. Most of the M. rubra genes in the antioxidant signaling pathways had multiple copies, likely originating from tandem duplication events. Further, many of the genes found here present structural variations or amino acid changes in the conserved functional residues across species. The expression levels of antioxidant genes were generally higher in the early stages of fruit development, and were correlated with the higher levels of total flavonoids and antioxidant capacity, in comparison with the mature fruit stages. Based on both gene expression and biochemical analyses, five genes, namely, caffeoyl-CoA O-methyltransferase, anthocyanidin 3-O-glucosyltransferase, (+)-neomenthol dehydrogenase, gibberellin 2-oxidase, and squalene monooxygenase, were suggested to regulate the flavonoid, anthocyanin, monoterpenoid, diterpenoid, and sesquiterpenoid/triterpenoid levels, respectively, during fruit development. CONCLUSIONS: This study describes both the complete genome and transcriptome of M. rubra. The results provide an important basis for future research on the genetic improvement of M. rubra and contribute to the understanding of its genetic evolution. The genome sequences corresponding to representative antioxidant signaling pathways can help revealing useful traits and functional genes.

The red bayberry genome and genetic basis of sex determination.

Morella rubra, red bayberry, is an economically important fruit tree in south China. Here, we assembled the first high-quality genome for both a female and a male individual of red bayberry. The genome size was 313-Mb, and 90% sequences were assembled into eight pseudo chromosome molecules, with 32 493 predicted genes. By whole-genome comparison between the female and male and association analysis with sequences of bulked and individual DNA samples from female and male, a 59-Kb region determining female was identified and located on distal end of pseudochromosome 8, which contains abundant transposable element and seven putative genes, four of them are related to sex floral development. This 59-Kb female-specific region was likely to be derived from duplication and rearrangement of paralogous genes and retained non-recombinant in the female-specific region. Sex-specific molecular markers developed from candidate genes co-segregated with sex in a genetically diverse female and male germplasm. We propose sex determination follow the ZW model of female heterogamety. The genome sequence of red bayberry provides a valuable resource for plant sex chromosome evolution and also provides important insights for molecular biology, genetics and modern breeding in Myricaceae family.

A Novel Pathway for Triacylglycerol Biosynthesis Is Responsible for the Accumulation of Massive Quantities of Glycerolipids in the Surface Wax of Bayberry (Myrica pensylvanica) Fruit.

Bayberry (Myrica pensylvanica) fruits synthesize an extremely thick and unusual layer of crystalline surface wax that accumulates to 32% of fruit dry weight, the highest reported surface lipid accumulation in plants. The composition is also striking, consisting of completely saturated triacylglycerol, diacylglycerol, and monoacylglycerol with palmitate and myristate acyl chains. To gain insight into the unique properties of Bayberry wax synthesis, we examined the chemical and morphological development of the wax layer, monitored wax biosynthesis through [(14)C]-radiolabeling, and sequenced the transcriptome. Radiolabeling identified sn-2 monoacylglycerol as an initial glycerolipid intermediate. The kinetics of [(14)C]-DAG and [(14)C]-TAG accumulation and the regiospecificity of their [(14)C]-acyl chains indicated distinct pools of acyl donors and that final TAG assembly occurs outside of cells. The most highly expressed lipid-related genes were associated with production of cutin, whereas transcripts for conventional TAG synthesis were >50-fold less abundant. The biochemical and expression data together indicate that Bayberry surface glycerolipids are synthesized by a pathway for TAG synthesis that is related to cutin biosynthesis. The combination of a unique surface wax and massive accumulation may aid understanding of how plants produce and secrete non-membrane glycerolipids and also how to engineer alternative pathways for lipid production in non-seeds.

Relationship between sucrose metabolism and anthocyanin biosynthesis during ripening in Chinese bayberry fruit.

Two cultivars of Chinese bayberry fruit cvs 'Dongkui' and 'Biqi' with five different ripening stages were used to investigate the relationship between anthocyanin biosynthesis and sugar metabolism during fruit development. The results showed that anthocynin accumulated with the increased ripening stage in both of the two cultivars of bayberries. As compared to 'Biqi' fruit, a higher level of anthocyanin content was observed in 'Dongkui' fruit due to the increased expression of anthocyanin biosynthetic and regulatory genes especially MrCHI, MrDFR1, MrANS, and MrMYB1. Meanwhile, 'Dongkui' fruit also experienced higher levels of soluble sugars including sucrose, glucose, and fructose and expression of genes such as MrSPS1, MrSPS2, MrSPS3, MrINV1, MrINV2, and MrINV3 involved in sugar metabolism. Correlation analysis showed anthocyanin content had a significant relationship with all the three soluble sugars in bayberry fruit. Therefore, our results suggested that the higher anthocyanin content in 'Dongkui' fruit might be associated with its increased levels of soluble sugars.

Impact of soil water chemistry on the apparent sex ratio of the flowering ramets of the dioecious plant Myrica gale var. tomentosa.

We determined whether the apparent (M/Fl) sex ratio (male ramets/flowering ramets) and apparent reproductive ramet ratio (Fl/Li ratio; flowering ramets/living ramets) in 15 Myrica gale var. tomentosa populations varied with dissolved total nitrogen, dissolved total phosphorus, potassium, magnesium, calcium, or pH in the soil water. Our aim was to define the environmental factors affecting the M/Fl sex ratio and Fl/Li ratio of the populations. We also examined the habitat conditions of these populations by analyzing soil water chemistry and water dynamics. In 2007, 3 of the 15 populations had no females. The remaining 12 had significantly male-biased (M/Fl sex ratio = 0.59-0.97). Although we could not explain the absence of females by the current potassium levels alone, as potassium increased, so did the M/Fl sex ratio. As nitrogen increased and potassium decreased, Fl/Li ratio decreased. Our soil water chemistry analyses suggested that the potassium supply by soil surface erosion from flooding and the inflow of anthropogenic nitrogen were the important factors influencing the M/Fl sex ratio and Fl/Li ratio. Nitrogen management would be important in one of the endangered populations where inflow of nitrogen was the highest among 15 habitats.

Transcriptomic analysis of Chinese bayberry (Myrica rubra) fruit development and ripening using RNA-Seq.

BACKGROUND: Chinese bayberry (Myrica rubra Sieb. and Zucc.) is an important subtropical fruit crop and an ideal species for fruit quality research due to the rapid and substantial changes that occur during development and ripening, including changes in fruit color and taste. However, research at the molecular level is limited by a lack of sequence data. The present study was designed to obtain transcript sequence data and examine gene expression in bayberry developing fruit based on RNA-Seq and bioinformatic analysis, to provide a foundation for understanding the molecular mechanisms controlling fruit quality changes during ripening. RESULTS: RNA-Seq generated 1.92 G raw data, which was then de novo assembled into 41,239 UniGenes with a mean length of 531 bp. Approximately 80% of the UniGenes (32,805) were annotated against public protein databases, and coding sequences (CDS) of 31,665 UniGenes were determined. Over 3,600 UniGenes were differentially expressed during fruit ripening, with 826 up-regulated and 1,407 down-regulated. GO comparisons between the UniGenes of these two types and interactive pathways (Ipath) analysis found that energy-related metabolism was enhanced, and catalytic activity was increased. All genes involved in anthocyanin biosynthesis were up-regulated during the fruit ripening processes, concurrent with color change. Important changes in carbohydrate and acid metabolism in the ripening fruit are likely associated with expression of sucrose phosphate synthase (SPS) and glutamate decarboxylase (GAD). CONCLUSIONS: Mass sequence data of Chinese bayberry was obtained and the expression profiles were examined during fruit ripening. The UniGenes were annotated, providing a platform for functional genomic research with this species. Using pathway mapping and expression profiles, the molecular mechanisms for changes in fruit color and taste during ripening were examined. This provides a reference for the study of complicated metabolism in non-model perennial species.

Interactions between bacterial carbon monoxide and hydrogen consumption and plant development on recent volcanic deposits.

Patterns of microbial colonization and interactions between microbial processes and vascular plants on volcanic deposits have received little attention. Previous reports have shown that atmospheric CO and hydrogen contribute significantly to microbial metabolism on Kilauea volcano (Hawaii) deposits with varied ages and successional development. Relationships between CO oxidation and plant communities were not clear, however, since deposit age and vegetation status covaried. To determine plant-microbe interactions in deposits of uniform ages, CO and hydrogen dynamics have been assayed for unvegetated tephra on a 1959 deposit at Pu'u Puai (PP-bare), at the edge of tree 'islands' within the PP deposit (PP-edge) and within PP tree islands (PP-canopy). Similar assays have been conducted for vegetated and unvegetated sites on a 1969 Mauna Ulu (MU) lava flow. Net in situ atmospheric CO uptake was highest at PP-edge and PP-bare sites (2.2+/-0.5 and 1.3+/-0.1 mg CO m(-2) day(-1), respectively), and least for PP-canopy (-3.2+/-0.9 mg CO m(-2) day(-1), net emission). Respiration rates, microbial biomass and maximum CO uptake potential showed an opposing pattern. Comparisons of atmospheric CO uptake and CO(2) production rates indicate that CO contributes significantly to microbial metabolism in PP-bare and MU-unvegetated sites, but negligibly where vegetation is well developed. Nonetheless, maximum potential CO uptake rates indicate that CO oxidizer populations increase with increasing plant biomass and consume CO actively. Some of these CO oxidizers may contribute to elevated nitrogen fixation rates (acetylene reduction) measured within tree islands, and thus, support plant successional development.

[Carbohydrate metabolism during fruit development of bayberry (Myrica rubra Sieb. et Zucc.)].

The dynamics of dry and fresh weight, the glucose, fructose, sucrose, titratable acid contents, and activities of sucrose-metabolizing and hexose-metabolizing enzymes were examined in developing fruits of bayberry (Myrica rubra Sieb. et Zucc. cvs. 'Wuzi' and 'Biqi'). The results showed the dry and fresh weight of bayberry fruit increased with fruit development and maturation (Fig. 1), with the highest increase rate of dry matters and water occurring during later stage of fruit development (about 10 d before maturation). The change in titratable acid followed a course of "low-high-low" in developing bayberry fruits (Fig. 3). The titratable acid content reached its peak at about 18 d before fruit maturation, and then decreased rapidly. The sugar compositions in fruits of bayberry cv. 'Wuzi' were different from those in fruits of bayberry cv. 'Biqi'. The main sugar accumulated in fruits of bayberry cv. 'Wuzi' was sucrose, accounting for 2/3 of total sugars but the sucrose content in fruits of bayberry cv. 'Biqi' was below 50% of total sugars. The fructose content in fruits of bayberry cv. 'Wuzi' was 4% higher, but that in fruits of bayberry cv. 'Biqi' was 12% lower than glucose content (Fig. 2). The activities of sucrose cleavage enzymes (invertase and cleavage activity of SS) in the fruit of bayberry cv. 'Biqi' increased with fruit development and maturation, but those activities in fruit bayberry cv. 'Wuzi' were almost stable during fruit development with lower levels of enzyme activities in fruit of cv. 'Wuzi' than in cv. 'Biqi' throughout fruit development (Fig. 4 and Fig. 5A). The SPS activity increased during fruit development (Fig. 6), however, the activity peak of synthetic activity of SS occurred at the middle stage of fruit development (Fig. 5B). The FRK activity in fruit of bayberry cv. 'Wuzi' was higher than that of HXK, but the reverse was in fruit of bayberry cv. 'Biqi' (Fig. 7). These results suggested that the 2-3 weeks before fruit maturation was a key phase for the bayberry development and the formation of fruit quality. There was a correlation between water transport and dry matter accumulation. The different sucrose constitutions between two varieties may be attributed to the differences in the activity levels of the sucrose cleavage enzymes while the difference in the ratio of glucose content to fructose content may be caused by the different activity levels of the hexose-metabolizing enzymes.

Mode of pollen-tube growth in Pistils of Myrica rubra (Myricaceae): a comparison with related families.

BACKGROUND AND AIMS: It is generally known that fertilization is delayed for more than a few weeks after pollination in Fagales. Recent studies showed that, during that period, pollen tubes grew in pistils in close association with the development of the ovule in a five-step process in Casuarina (Casuarinaceae) and a four-step process in Alnus (Betulaceae). The number of pollen tubes was reduced from many to one, a fact suggesting that delayed fertilization plays a role for gametophyte selection. Myrica (Myricaceae) also shows delayed fertilization for >2 weeks after pollination, but nothing is known of how pollen tubes grow in the pistil during that period. METHODS: Pollen-tube growth and the development of the ovule in pistils was investigated by fluorescent and scanning electron microscopy and analysis of microtome sections of the pistils. KEY RESULTS: Developmental study of the pollen-tube growth in the pistil of M. rubra showed that the tip of the pollen tube was branched or lay in a zigzag pattern in the upper space of the ovarian locule or near the tip of the integument, and subsequently was swollen on the nucellar surface. Such morphological changes indicate that the pollen-tube growth was temporarily arrested before fertilization. The pollen-tube growth in M. rubra can therefore be summarized as occurring in three steps: (1) from the stigma to the ovarian locule; (2) from the ovarian locule to the nucellar surface; and (3) from the nucellar surface to the embryo sac. CONCLUSION: Myrica differs from other families in that the pollen tubes arrest their growth on the nucellar surface, probably digesting nutrient from nucellar cells. There is little information on five other families of Fagales. An extensive study is needed to better understand the diversity and function of the mode of pollen-tube growth within the order.

Possible input of nitrogen of visitors' origin on a protected peatland.

The Ozegahara peatland, in the Nikko-Oze National Park in Japan, is ecologically significant because of its oligotrophic environment; it is one of the most strictly preserved areas in the country. The isotope ratio of nitrogen (15N/14N) and carbon (13C/12C) and C/N ratio of peat moss (Sphagnum spp.) and sundew (Drosera rotundifolia) in the peatland were analyzed. The correlation of the isotope ratio with some parameters (sundew population density, number of trapped insects, water level, bog myrtle coverage, and visitor density) was investigated. The nitrogen isotope ratio of sundew showed the most significant covariation with visitor density, where sundew from lunch areas or along busy walkways showed a higher nitrogen isotope ratio. The nitrogen isotope ratio of peat moss covaried, not only with route traffic frequency but also with water level, bog myrtle coverage, and number of trapped insects by sundew, indicating that factors other than the visitor level influence the local nitrogen cycle. This study suggests that the nitrogen imported into the peatland by visitors is a principal factor to be monitored for the maintenance of the natural environment.