Nitrogen supply enhances the physiological resistance of Chinese fir plantlets under polyethylene glycol (PEG)-induced drought stress.

Water and nitrogen stresses are major constraints for agricultural and forest productivity. Although the effects of water scarcity or nitrogen stress on plant growth, physiology, and yield have been widely studied, few studies have assessed the combined effects of both stresses. In the present study, we investigated the effects of different nitrogen forms (NO3-N, NH4+-N, and a combination of NO3-N + NH4+-N) on antioxidant enzyme activity, osmotic regulatory substances, and nitrogen assimilation in Chinese fir (Cunninghamia lanceolata) plantlets under drought stress (induced by 10% polyethylene glycol). We found that different N ionic forms had different effects on drought-stressed plantlets. Nitrogen supply greatly increased the activities of superoxide dismutase (SOD), peroxidase (POD) and polyphenol oxidase (PPO) when plantlets were exposed to water stress. The malondialdehyde (MDA) contents significantly decreased under the NH4+ + water stress treatment. The proline (Pr) contents significantly increased in both the NO3-N and NH4+-N + water stress treatment. The nitrate reductase (NR) increased by 7.1% in the NO3- + water stress treatment, and the glutamine synthetase (GS), and the glutamate synthase (GOGAT) activity increased in all the nitrogen + water stress treatments. These results suggested that nitrogen supply could alleviate the adverse effects of drought stress on plants by enhancing antioxidant defense and improving nitrogen assimilation, while the effects on plant tolerance to drought stress varied with nitrogen ionic forms.

Transcriptomic Revelation of Phenolic Compounds Involved in Aluminum Toxicity Responses in Roots of Cunninghamia lanceolata (Lamb.) Hook.

: Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) is one of the most important coniferous evergreen tree species in South China due to its desirable attributes of fast growth and production of strong and hardy wood. However, the yield of Chinese fir is often inhibited by aluminum (Al) toxicity in acidic soils of South China. Understanding the molecular mechanisms of Chinese fir root responses to Al toxicity might help to further increase its productivity. Here we used the Illumina Hiseq4000 platform to carry out transcriptome analysis of Chinese fir roots subjected to Al toxicity conditions. A total of 88.88 Gb of clean data was generated from 12 samples and assembled into 105,732 distinct unigenes. The average length and N50 length of these unigenes were 839 bp and 1411 bp, respectively. Among them, 58362 unigenes were annotated through searches of five public databases (Nr: NCBI non-redundant protein sequences, Swiss-Prot: A manually annotated and reviewed protein sequence database, GO: Gene Ontology, KOG/COG: Clusters of Orthologous Groups of proteins, and KEGG: the Kyoto Encyclopedia of Genes and Genomes database), which led to association of unigenes with 44 GO terms. Plus, 1615 transcription factors (TFs) were functionally classified. Then, differentially expressed genes (DEGs, |log2(fold change)| ≥ 1 and FDR ≤ 0.05) were identified in comparisons labelled TC1 (CK-72 h/CK-1 h) and TC2 (Al-72 h/Al-1 h). A large number of TC2 DEGs group were identified, with most being down-regulated under Al stress, while TC1 DEGs were primarily up-regulated. Combining GO, KEGG, and MapMan pathway analysis indicated that many DEGs are involved in primary metabolism, including cell wall metabolism and lipid metabolism, while other DEGs are associated with signaling pathways and secondary metabolism, including flavonoids and phenylpropanoids metabolism. Furthermore, TFs identified in TC1 and TC2 DEGs represented 21 and 40 transcription factor families, respectively. Among them, expression of bHLH, C2H2, ERF, bZIP, GRAS, and MYB TFs changed considerably under Al stress, which suggests that these TFs might play crucial roles in Chinese fir root responses to Al toxicity. These differentially expressed TFs might act in concert with flavonoid and phenylpropanoid pathway genes in fulfilling of key roles in Chinese fir roots responding to Al toxicity.

Effects of LED Light on Acacia Melanoxylon Bud Proliferation in Vitro and Root Growth ex Vitro.

This study examines the effects of light emitting diodes (LEDs) on tissue culture proliferation of Acacia melanoxylon plantlets among five different clones (FM1, FM2, FM4, FM5, and FM10). Shoot bud apex cuttings were transplanted onto Murashige and Skoog basal medium containing 0.1 mg L-1 6-benzyladenine and 0.5 mg L-1 naphthalene acetic acid and cultured in vitro for 40 days. Root growth was studied under different light intensities and photoperiods ex vitro. The bud proliferation coefficient was greatest under a light intensity of 45 µmol m-2 s-1 photosynthetic photon flux and photoperiod of 16 h light, but decreased as the light intensity increased. However, the greatest light intensity was beneficial for the growth of robust plantlets. Plantlets exposed to red and blue LED combinations grew tall and green, with a small number of roots. Plantlets also grew taller and some roots expanded under the longer photoperiod. Increased light intensity had positive effects on root number and rooting rate, and prolonged light greatly increased root number. Therefore, lower light intensity and a short photoperiod were beneficial for bud proliferation, while red/blue LED combinations, increased light intensity, and longer light illumination were beneficial for plantlet growth and root growth of Acacia melanoxylon.

Complete chloroplast genome of Isoetes sinensis, an endemic fern in China.

Isoetes sinensis is the only aquatic pteridophyte in China with high research value of phylogeny. It is in endangered status. A conservation strategy is therefore imperative for this endangered pteridophyte. In the study, the complete chloroplast (cp) genome of Isoetes sinensis (Isoetaceae) was assembled and annotated. It is the full length of 145,492 bp, include large single-copy (LSC) region of 91,865 bp, small single-copy (SSC) region of 13,207 bp, and a pair of invert repeats (IR) regions of 27,213 bp. Plastid genome contains 135 genes, 71 protein-coding genes, 36 tRNA genes, and eight rRNA genes. Phylogenetic analysis suggested I. sinensis was most closely related to the clade of I. melanospora, I. mattaponica, I. graniticola, I. engelmannii, I. flaccida, I. valida, and I. butleri, with strong support (bootstrap = 100%). The cp genome will contribute to further research and conservation of I. sinensis.

Metabolomic Analysis of Pollen Grains with Different Germination Abilities from Two Clones of Chinese Fir (Cunninghamia lanceolata (Lamb) Hook).

Pollen grains produce certain metabolites, which can improve or inhibit germination and tube growth. Metabolomic analysis of germinating and growing Chinese fir pollen has not been reported. Therefore, this study aimed to analyse metabolites changes, content and expression in the germinating pollen of Chinese fir. To understand the metabolic differences, two clones from Chinese fir were selected. Metabolomics analyses were performed on three stages (1-, 24- and 48-h) during in vitro pollen germination. The metabolites profiles at different time points were analyzed by using liquid chromatography-mass spectrometry. The results showed that 171 peaks were screened; the corresponding differential metabolites of 121 peaks were classified into nine types of substances. The expression of metabolites showed significant differences across and between clones, and the variation was evident at all germination stages. The expression was obvious at the early stage of germination, which differed clearly from that of the late stage after pollen tube growth. Moreover, the metabolites were mainly enriched in 14 metabolic pathways. Pollen germination and tube growth and metabolites expressions changed per incubation time. Since this work is preliminary, we suggest further investigations to understand the relationship between the differential metabolites and pollen development, and factors affecting pollen germination process.

Optimization of hormone combinations for root growth and bud germination in Chinese fir (Cunninghamia lanceolata) clone leaf cuttings.

In order to ascertain the optimal hormone combination for Chinese fir (Cunninghamia lanceolata) leaf cuttings, an L16(4)4 orthogonal test of 6-benzylaminopurine (6-BA), 1-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA) (0, 10, 30, or 50 mg · L-1 of each exogenous hormone) immersion for 5, 10, 15, or 20 min was conducted. Callus initiation rate and rooting promotion rate were mainly affected by treatment time, root length increase by 6-BA concentration, and bud germination rate and plantlet formation rate by NAA concentration. The expected optimal combination for callus initiation rate was 50 mg · L-1 6-BA + 0 mg · L-1 NAA + 30 mg · L-1 IBA + 10 min; for rooting promotion rate, it was 0-10 mg · L-1 6-BA + 10 mg · L-1 NAA + 30 mg · L-1 IBA + 20 min; for bud germination rate, it was 50 mg · L-1 6-BA + 0-10 mg · L-1 NAA + 0-30 mg · L-1 IBA + 20 min; and for seedling formation rate for No. 4, it was 10 mg · L-1 6-BA + 10 mg · L-1 NAA + 0 mg · L-1 IBA + 20 min. Light microscopy image analysis revealed that a cluster of primordial cells was produced 40 days after cutting, and mastoid cells developed into peninsula cells in calli that were cultured for 50 days.

Genetic diversity and variation of Chinese fir from Fujian province and Taiwan, China, based on ISSR markers.

Genetic diversity and variation among 11 populations of Chinese fir from Fujian province and Taiwan were assessed using inter-simple sequence repeat (ISSR) markers to reveal the evolutionary relationship in their distribution range in this report. Analysis of genetic parameters of the different populations showed that populations in Fujian province exhibited a greater level of genetic diversity than did the populations in Taiwan. Compared to Taiwan populations, significant limited gene flow were observed among Fujian populations. An UPGMA cluster analysis showed that the most individuals of Taiwan populations formed a single cluster, whereas 6 discrete clusters were formed by each population from Fujian. All populations were divided into 3 main groups and that all 5 populations from Taiwan were gathered into a subgroup combined with 2 populations, Dehua and Liancheng, formed one of the 3 main groups, which indicated relative stronger relatedness. It is supported by a genetic structure analysis. All those results are suggesting different levels of genetic diversity and variation of Chinese fir between Fujian and Taiwan, and indicating different patterns of evolutionary process and local environmental adaption.

Ion Flux in Roots of Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook) under Aluminum Stress.

Chinese fir is a tall, fast-growing species that is unique to southern China. In Chinese fir plantations, successive plantings have led to a decline in soil fertility, and aluminum toxicity is thought to be one of the main reasons for this decline. In this study, Non-invasive Micro-test Technology was used to study the effect of aluminum stress on the absorption of 4 different ions in the roots of the Chinese fir clone FS01. The results are as follows: with increased aluminum concentration and longer periods of aluminum stress, the H+ ion flow gradually changed from influx into efflux; there was a large variation in the K+ efflux, which gradually decreased with increasing duration of aluminum stress; and 1 h of aluminum stress uniformly resulted in Ca2+ influx, but it changed from influx to efflux after a longer period of aluminum stress. Changes in the different concentrations of aluminum had the largest influence on Mg2+.

Isolation of High-Quality Total RNA from Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook).

RNA isolation with RNA in a high quantity is a basic analytical method in plant genetics, molecular biology and related physiological investigations. To understand the genetic and molecular biology of Chinese fir, sufficient high-quality total RNA must be obtained for cDNA library construction and other downstream molecular applications. However, extracting RNA from Chinese fir is difficult and often requires the modification of existing protocols. Chinese fir tissues containing large amounts of polysaccharides and polyphenol compounds and are one of the most difficult plant tissues for RNA isolation. Therefore, we developed a simple method for extracting high-quality RNA from Chinese fir tissues. RNA isolations were performed within two hours, RNA quality was measured for yield and purity. Total RNA obtained from this procedure was successfully used for cDNA library construction, RT-PCR and transcriptome sequencing. It was proven that extracted RNA was intact and suitable for downstream molecular applications, including RT-PCR and qPCR, and other downstream molecular applications. Thus, this protocol represents a simple, efficient, and low-cost method.