Hailong Shen

Influence of nitrogen and phosphorous on the growth and root morphology of Acer mono

Nitrogen and phosphorous are critical determinants of plant growth and productivity, and both plant growth and root morphology are important parameters for evaluating the effects of supplied nutrients. Previous work has shown that the growth of Acer mono seedlings is retarded under nursery conditions; we applied different levels of N (0, 5, 10, and 15 g plant-1) and P (0, 4, 6 and 8 g plant-1) fertilizer to investigate the effects of fertilization on the growth and root morphology of four-year-old seedlings in the field. Our results indicated that both N and P application significantly affected plant height, root collar diameter, chlorophyll content, and root morphology. Among the nutrient levels, 10 g N and 8 g P were found to yield maximum growth, and the maximum values of plant height, root collar diameter, chlorophyll content, and root morphology were obtained when 10 g N and 8 g P were used together. Therefore, the present study demonstrates that optimum levels of N and P can be used to improve seedling health and growth during the nursery period.

Effects of KNO3 pretreatment and temperature on seed germination of Sorbus pohuashanensis

We characterized the effects of KNO3 pretreatment and germination temperature on dormancy breaking and germination of mature mountain ash seeds. Seeds treated with KNO3 and germinated at 25 °C followed by 5 °C had significantly higher germination percentages and germination potentials (51% and 49%, respectively), compared with controls. These treated seeds also exhibited reduced germination initiation times (minimum of 48 days), and elevated germination rate indices (up to 97). The germination of seeds subjected to long-term cold storage (2 years at 0–5 °C) was also significantly improved by 3 days of 4% KNO3 pretreatment before germinating under a variable temperature regimen (5 °C followed by 25 °C, and followed by 5 °C). Germination percentages and germination potentials for these cold-stored seeds reached 67% and 54%, respectively, and the germination rate index increased to 126.99. Pretreatment of mountain ash seeds with KNO3 represents a practical, effective, and pollution-free method for improving germination, and can be implemented easily within a variety of nursery settings.

Proteomic Analysis of Immature Fraxinus mandshurica Cotyledon Tissues during Somatic Embryogenesis: Effects of Explant Browning on Somatic Embryogenesis

Manchurian ash (Fraxinus mandshurica Rupr.) is a valuable hardwood species in Northeast China. In cultures of F. mandshurica, somatic embryos were produced mainly on browned explants. Therefore, we studied the mechanism of explant browning and its relationship with somatic embryogenesis (SE). We used explants derived from F. mandshurica immature zygotic embryo cotyledons as materials. Proteins were extracted from browned embryogenic explants, browned non-embryogenic explants, and non-brown explants, and then separated by 2-dimensional electrophoresis. Differentially and specifically expressed proteins were analyzed by mass spectrometry to identify proteins involved in the browning of explants and SE. Some stress response and defense proteins such as chitinases, peroxidases, aspartic proteinases, and an osmotin-like protein played important roles during SE of F. mandshurica. Our results indicated that explant browning might not be caused by the accumulation and oxidation of polyphenols only, but also by some stress-related processes, which were involved in programmed cell death (PCD), and then induced SE.

Proteome Analysis of Dormancy-Released Seeds of Fraxinus mandshurica Rupr. in Response to Re-Dehydration under Different Conditions

Desiccation tolerance is the ability of orthodox seeds to achieve equilibrium with atmospheric relative humidity and to survive in this state. Understanding how orthodox seeds respond to dehydration is important for improving quality and long-term storage of seeds under low temperature and drought stress conditions. Long-term storage of seeds is an artificial situation, because in most natural situations a seed that has been shed may not remain in a desiccated state for very long, and if dormant it may undergo repeated cycles of hydration. Different types of seeds are differentially sensitive to desiccation and this directly affects long-term storage. For these reasons, many researchers are investigating loss of desiccation tolerance during orthodox seed development to understand how it is acquired. In this study, the orthodox seed proteome response of Fraxinus mandshurica Rupr. to dehydration (to a relative water content of 10%, which mimics seed dehydration) was investigated under four different conditions viz. 20 °C; 20 °C with silica gel; 1 °C; and 1 °C after pretreatment with Ca2+. Proteins from seeds dehydrated under different conditions were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2919 protein spots were detected, and high-resolution 2D-DIGE indicated there were 27 differentially expressed. Seven of these were identified using MALDI TOF/TOF mass spectrometry. Inferences from bioinformatics annotations of these proteins established the possible involvement of detoxifying enzymes, transport proteins, and nucleotide metabolism enzymes in response to dehydration. Of the seven differentially abundant proteins, the amounts of six were down-regulated and one was up-regulated. Also, a putative acyl-coenzyme A oxidase of the glyoxylate cycle increased in abundance. In particular, the presence of kinesin-1, a protein important for regulation and cargo interaction, was up-regulated in seeds exposed to low temperature dehydration. Kinesin-1 is present in all major lineages, but it is rarely detected in seed desiccation tolerance of woody species. These observations provide new insight into the proteome of seeds in deep dormancy under different desiccation conditions.

Genetic Variation in Quercus acutissima Carruth., in Traditional Japanese Rural Forests and Agricultural Landscapes, Revealed by Chloroplast Microsatellite Markers

Quercus acutissima Carruth. is an economically important species that has long been cultivated in Japan, so is a valuable subject for investigating the impact of human activities on genetic variation in trees. In total, 2152 samples from 18 naturally regenerated populations and 28 planted populations in Japan and 13 populations from the northeastern part of Eurasia, near Japan, were analyzed using six maternally inherited chloroplast (cpDNA) simple sequence repeat (SSR) markers. Although 23 haplotypes were detected in total, both the Japanese natural and artificial populations exhibited much lower genetic diversity than the continental populations. The level of genetic differentiation among natural populations in Japan was also much lower (G’ST = 0.261) than that on the continent (G’ST = 0.856). These results suggest that human activities, such as historical seed transfer, have reduced genetic diversity within and among populations and resulted in a homogeneous genetic structure in Japan. The genetic characteristics of natural and artificial populations of Quercus acutissima in Japan are almost the same and it is likely that most of the natural populations are thought to have originated from individuals that escaped from plantations.

Genetic relationships among sympatric varieties of Acer mono in the Chichibu Mountains and Central Hokkaido, Japan

Acer mono Maxim. is one of the major components of cool temperate forests in Japan. Some of its many varieties are distributed sympatrically. Because of its great variability, the intraspecific taxonomy and nomenclature of the species are controversial. To understand the genetic relationships among these varieties and whether hybridization or introgression occurred among the sympatric varieties, we studied the genetic relationships among sympatric varieties of A. mono in the Chichibu Mountains (A. mono var. ambiguum, A. mono var. connivens, A. mono var. marmoratum) and Central Hokkaido (A. mono var. mayrii and A. mono var. glabrum) in Japan. Our results showed that varieties in Chichibu are genetically close, suggesting that hybridization or introgression might occur between these varieties, which could explain the higher genetic diversity of varieties in Chichibu than in Hokkaido. In contrast to the close relationships between the varieties in Chichibu, varieties in Hokkaido seemed relatively separated from each other; indeed, there may be reproductive isolation between the two varieties. The results provide new insight for the taxonomy of the varieties of A. mono, especially the sympatric varieties, in Japan.

Comparison of methods for extracting high-throughput sequencing RNA from Korean pine seeds

Korean pine (Pinus koraiensis Sieb. et Zucc.) is an ecologically and economically important tree species in East Asia. Molecular studies of seed development of this species are limited due to the lack of effective RNA extraction protocols. This study aimed to obtain an effective method to extract high-quality RNA from Korean pine seeds. The TRIzol kit and CTAB methods were used to extract the total RNA from Korean pine seeds at different developmental stages. The bands of RNA extracted by CTAB were not clear, whereas the bands of RNA extracted by the TRIzol kit were brighter and clearer, indicating higher quality and integrity of the RNA products extracted by the TRIzol kit. The 28S rRNA band was approximately 1.5- to 2-fold brighter than the 18S rRNA band on the agarose gel electrophoresis. The absorbance value A 260/280 was 1.8–2.0, and the absorbance value A 260/230 was >1.9. The Bioanalyzer RNA integrity results showed that the RNA integrity number of the RNA extracted using the TRIzol kit was acceptable for high-throughput sequencing. Therefore, the total RNA extracted using the TRIzol kit method can be used for high-throughput sequencing and other molecular biology experiments.