The function of chloroplast ferredoxin-NADP+ oxidoreductase positively regulates the accumulation of bamboo mosaic virus in Nicotiana benthamiana

A gene down-regulated in Nicotiana benthamiana after bamboo mosaic virus (BaMV) infection had high identity to the nuclear-encoded chloroplast ferredoxin NADP⁺ oxidoreductase gene (NbFNR). NbFNR is a flavoenzyme involved in the photosynthesis electron transport chain, catalysing the conversion of NADP⁺ into NADPH. To investigate whether NbFNR is involved in BaMV infection, we used virus-induced gene silencing to reduce the expression of NbFNR in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and RNA was significantly reduced. The transient expression of NbFNR fused with orange fluorescent protein (OFP) localized in the chloroplasts and elevated the level of BaMV coat protein. These results suggest that NbFNR could play a positive role in regulating BaMV accumulation. Expressing a mutant that failed to translocate to the chloroplast did not assist in BaMV accumulation. Another mutant with a catalytic site mutation could support BaMV accumulation to some extent, but accumulation was significantly lower than that of the wild type. In an in vitro replication assay, the replicase complex with FNR inhibitor, heparin, the RdRp activity was reduced. Furthermore, BaMV replicase was revealed to interact with NbFNR in yeast two-hybrid and co-immunoprecipitation experiments. Overall, these results suggest that NbFNR localized in the chloroplast with functional activity could efficiently assist BaMV accumulation.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Isolation of amaranthin synthetase from Chenopodium quinoa and construction of an amaranthin production system using suspension‐cultured tobacco BY‐2 cells

Betalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti‐inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow‐2 (BY‐2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 μm (mean ± SEM) amaranthin and 26.60 ± 1.53 μm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV‐1 protease activity, whereas betanin did not.     

Identification of ESTs differentially expressed in green and albino mutant bamboo (Bambusa edulis) by suppressive subtractive hybridization (SSH) and microarray analysis

To gain a better understanding of gene expression in bamboo (Bambusa edulis Murno), we have used a combination of suppressive subtractive hybridization (SSH), microarray hybridization analysis, sequencing, and bioinformatics to identify bamboo genes differentially expressed in a bamboo albino mutant. Ten expressed sequence tags (ESTs) were found to be differentially expressed; these were isolated and sequenced. RT-PCR analysis of these ESTs supported the results of the microarray analysis. Six ESTs that were nucleus-encoded exhibited differential expression patterns in the green wild-type bamboo relative to the albino mutant. These genes (exception being the Rubisco small subunit) were non-photosynthesis-related genes. The development of a specific SSH cDNA library in which most of the chloroplast-encoded or photosynthesis-related genes had been subtracted proved to be useful for studying the function of non-photosynthesis-related genes in the albino bamboo mutants with aberrant chloroplast genome. The combined use of this SSH library with microarray analysis will provide a powerful analytical tool for future studies of the bamboo genome.     

NbRABG3f,a member of Rab GTPase,is involved in Bamboo mosaic virus infection in Nicotiana benthamiana

The screening of differentially expressed genes in plants after pathogen infection can uncover the potential host factors required for the pathogens. In this study, an up‐regulated gene was identified and cloned from Nicotiana benthamiana plants after Bamboo mosaic virus (BaMV) inoculation. The up‐regulated gene was identified as a member of the Rab small guanosine triphosphatase (GTPase) family, and was designated as NbRABG3f according to its in silico translated product with high identity to that of RABG3f of tomato. Knocking down the expression of NbRABG3f using a virus‐induced gene silencing technique in a protoplast inoculation assay significantly reduced the accumulation of BaMV. A transiently expressed NbRABG3f protein in N. benthamiana plants followed by BaMV inoculation enhanced the accumulation of BaMV to approximately 150%. Mutants that had the catalytic site mutation (NbRABG3f/T22N) or had lost their membrane‐targeting capability (NbRABG3f/ΔC3) failed to facilitate the accumulation of BaMV in plants. Because the Rab GTPase is responsible for vesicle trafficking between organelles, a mutant with a fixed guanosine diphosphate form was used to identify the donor compartment. The use of green fluorescent protein (GFP) fusion revealed that GFP‐NbRABG3f/T22N clearly co‐localized with the Golgi marker. In conclusion, BaMV may use NbRABG3f to form vesicles derived from the Golgi membrane for intracellular trafficking to deliver unidentified factors to its replication site; thus, both GTPase activity and membrane‐targeting ability are crucial for BaMV accumulation at the cell level.     

A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus

An up‐regulated gene derived from Bamboo mosaic virus (BaMV)‐infected Nicotiana benthamiana plants was cloned and characterized in this study. BaMV is a single‐stranded, positive‐sense RNA virus. This gene product, designated as NbTRXh2, was matched with sequences of thioredoxin h proteins, a group of small proteins with a conserved active‐site motif WCXPC conferring disulfide reductase activity. To examine how NbTRXh2 is involved in the infection cycle of BaMV, we used the virus‐induced gene silencing technique to knock down NbTRXh2 expression in N. benthamiana and inoculated the plants with BaMV. We observed that, compared with control plants, BaMV coat protein accumulation increased in knockdown plants at 5 days post‐inoculation (dpi). Furthermore, BaMV coat protein accumulation did not differ significantly between NbTRXh2‐knockdown and control protoplasts at 24 hpi. The BaMV infection foci in NbTRXh2‐knockdown plants were larger than those in control plants. In addition, BaMV coat protein accumulation decreased when NbTRXh2 was transiently expressed in plants. These results suggest that NbTRXh2 plays a role in restricting BaMV accumulation. Moreover, confocal microscopy results showed that NbTRXh2‐OFP (NbTRXh2 fused with orange fluorescent protein) localized at the plasma membrane, similar to AtTRXh9, a homologue in Arabidopsis. The expression of the mutant that did not target the substrates failed to reduce BaMV accumulation. Co‐immunoprecipitation experiments revealed that the viral movement protein TGBp2 could be the target of NbTRXh2. Overall, the functional role of NbTRXh2 in reducing the disulfide bonds of targeting factors, encoded either by the host or virus (TGBp2), is crucial in restricting BaMV movement.     

The association of hormone signalling genes,transcription and changes in shoot anatomy during moso bamboo growth

Moso bamboo is a large, woody bamboo with the highest ecological, economic and cultural value of all the bamboo types and accounts for up to 70% of the total area of bamboo grown. However, the spatiotemporal variation role of moso bamboo shoot during growth period is still unclear. We found that the bamboo shoot growth can be divided into three distinct periods, including winter growth, early growth and late growth based on gene expression and anatomy. In the early growth period, lateral buds germinated from the top of the bamboo joint in the shoot tip. Intercalary meristems grew vigorously during the winter growth period and early growth period, but in the late growth period, mitosis in the intercalary meristems decreased. The expression of cell cycle‐associated genes and the quantity of differentially expressed genes were higher in early growth than those in late growth, appearing to be influenced by hormonal concentrations. Gene expression analysis indicates that hormone signalling genes play key roles in shoot growth, while auxin signalling genes play a central role. In situ hybridization analyses illustrate how auxin signalling genes regulate apical dominance, meristem maintenance and lateral bud development. Our study provides a vivid picture of the dynamic changes in anatomy and gene expression during shoot growth in moso bamboo, and how hormone signalling‐associated genes participate in moso bamboo shoot growth.     

An experimental study of bamboo ants in western Amazonia

Numerous ant taxa naturally inhabit stems of live and dead Guadua bamboo (Bambusoidea, Poaceae) in western Amazonia. In an experiment at the onset of the wet season in Peru’s Manu National Park, we augmented potential nest sites in stems of live bamboo, dead bamboo and dead ca?a brava (Gynerium sagittatum, another woody grass) at five stations within each of ten bamboo patches and ten control areas outside those patches. Each experimental stem possessed three vacant and available internodes, pre-drilled with, respectively, large, small and linear holes, mimicking the range of forms of surveyed natural entrances. After 24 days, approximately 13% of 798 available internodes had been colonized, the majority by fragments of existing colonies. Ignoring entrance type, which did not affect colonization for any species or species group, and censoring non-independent internodes of the same stem, we used individual stems as independent sample units in other tests. One specialist in live bamboo (Camponotus longipilis), and a likely specialist in dead bamboo (Camponotus depressus), were identified based on overrepresentation in bamboo habitat and disproportionate occurrence in live or dead bamboo stems. A third species, Camponotus (Pseudocolobopsis sp.) was more abundant in bamboo areas but colonized both dead bamboo and dead ca?a. Relatively high abundance of standing dead stems in Guadua forests may account for the presence of a dead stem specialist. The experiment missed detecting specialization in one live culm specialist (Camponotus mirabilis), likely due to its failure to simulate conditions required for the species’ unique modes of colony establishment and spread into new culms. Most opportunistic stem nesters colonized dead bamboo at significantly greater rates than dead ca?a, but were either equally well represented in bamboo and control areas, or underrepresented in bamboo habitat. Given low colonization rates overall, underrepresentation in bamboo cannot be attributed to competition from bamboo specialists for nesting space. Rather, it may be due to combined effects of seasonal flooding of bamboo habitat, and greater importance of food limitation, relative to nest site limitation, in that habitat. Received 9 May 2005; revised 25 August 2005; accepted 29 August 2005.     

Consumption of cyanogenic bamboo by a newly discovered species of bamboo lemur

Three species of bamboo-eating lemurs were found to be sympatric in the southeastern rain forests of Madagascar. Sympatric species generally differ in habitat utilization or diet, but these three closely related bamboo lemurs lived in the same habitat and all ate bamboo. Behavioral observation revealed that they did select different parts of the bamboo, and chemical analyses confirmed that there was a difference in the secondary compound content present in those selections. The growing tips of Cephalostachyum ef uiguieri selected by the golden bamboo lemur (Hapalemuraureus) contained 15 mg of cyanide per 100 g fresh weight bamboo while the leaves of C. perrieri selected by the gentle bamboo lemur (H. griseus)and the mature culms of C. cf uiguieri selected by the greater bamboolemur (H. simus) did not contain cyanide. Since each individual golden bamboo lemur ate about 500 g of bamboo per day, they daily ingestedabout 12 times the lethal dose of cyanide. The mechanism by which this small primate avoids the acute and chronic symptoms of cyanide poisioning is unknown.     

Identification of Single Nucleotide Polymorphisms and Analysis of Linkage Disequilibrium in Different Bamboo Species Using the Candidate Gene Approach

Bamboos are one of the most beautiful and useful plants on Earth. The genetic background and population structure of bamboos are well known, which helps accelerate the process of artificial domestication of bamboo. Partial sequences of six genes involved in nitrogen use efficiency in 32 different bamboo species were analyzed for occurrence of single nucleotide polymorphisms (SNPs). The nucleotide diversity θw and total nucleotide polymorphisms π_T of the sequenced DNA regions was 0.05137 and 0.03332, respectively. Both πnonsyn /πsyn and Ka/Ks values were <1. The nucleotide sequences of these six genes were inferred to be relatively conserved, and the haplotype diversity was relatively high. The results of evolutionary neutrality tests showed that the six genes were in line with neutral evolution, and that the NRT2.1 and AMT2.1 gene sequences may have experienced negative selection. An inter-SNP recombination event at the NRT2.1 gene in the all pooled sample, of all 32 bamboo species was the lowest at 0.0645, whereas the AMT gene recombination events were all >0.1. Estimation and analysis of linkage disequilibrium of five genes revealed that with the increase in nucleotide sequence length, the degree of SNP linkage disequilibrium decreased rapidly. We inferred the population genetic structure of 32 bamboo species based on the SNP loci of six genes with frequencies >18%. 32 bamboo species were divided into five categories, which indicated that the combined population of all bamboo species had obvious multivariate characteristics and was heterogeneous; red (Group 1) and green (Group 2) were the main groups.     

Ser/Thr Kinase-Like Protein of Nicotiana benthamiana Is Involved in the Cell-to-Cell Movement of Bamboo mosaic virus

To investigate the plant genes affected by Bamboo mosaic virus (BaMV) infection, we applied a cDNA-amplified fragment length polymorphism technique to screen genes with differential expression. A serine/threonine kinase-like (NbSTKL) gene of Nicotiana benthamiana is upregulated after BaMV infection. NbSTKL contains the homologous domain of Ser/Thr kinase. Knocking down the expression of NbSTKL by virus-induced gene silencing reduced the accumulation of BaMV in the inoculated leaves but not in the protoplasts. The spread of GFP-expressing BaMV in the inoculated leaves is also impeded by a reduced expression of NbSTKL. These data imply that NbSTKL facilitates the cell-to-cell movement of BaMV. The subcellular localization of NbSTKL is mainly on the cell membrane, which has been confirmed by mutagenesis and fractionation experiments. Combined with the results showing that active site mutation of NbSTKL does not change its subcellular localization but significantly affects BaMV accumulation, we conclude that NbSTKL may regulate BaMV movement on the cell membrane by its kinase-like activity. Moreover, the transient expression of NbSTKL does not significantly affect the accumulation of Cucumber mosaic virus (CMV) and Potato virus X (PVX); thus, NbSTKL might be a specific protein facilitating BaMV movement.     

Species delimitation in the Chinese bamboo partridge Bambusicola thoracica (Phasianidae; Aves)

Although tropical and subtropical Asia harbour a high level of species diversity, their species richness can be underestimated because species which are in fact distinct have not been separately identified. In this study, we delimit Bambusicola thoracica into two full species, the Chinese bamboo partridge (B. thoracica) in continental Asia and the Taiwanese bamboo partridge (B. sonorivox) on the island of Taiwan, using coalescent‐based multilocus division and diagnosable vocalization patterns. Isolation‐with‐migration analysis indicated that the two bamboo partridges diverged approximately 1.8 million years ago, with gene flow present most probably during the early stages of their divergence. This conclusion supports the hypothesis that diverging lowland lineages spread across the Asian mainland, and continental islands have more opportunities for secondary contact than highland ones when the sea level was low. Our results imply that conservation of biodiversity in tropical and subtropical Asia could be hindered by overlooking numerous ‘hidden’ species and highlight the importance of re‐examining the taxonomic statuses of species in this region traditionally defined as polytypic.     

四个竹秆变异毛竹变型的全基因组序列分析

毛竹是我国重要的经济竹种,在长期栽培适应过程中产生了丰富的变异。为揭示毛竹竹秆变异变型的全基因组突变类型,以黄皮毛竹、金丝毛竹、绿皮花毛竹和花毛竹4个毛竹变型为实验材料,采用高通量重测序技术获得全基因组序列,进行单核苷多态性(SNP)、小片段插入缺失(InDel)和结构变异(SV)检测和注释,并将变异基因进行功能注释。结果表明:花毛竹基因组检测得到的基因变异数最多,为12 555个; 金丝毛竹样品变异位点数最少,为11 923个; 4个样品都有7 000多个变异基因得到功能注释。GO注释分类包括细胞组件、分子功能和生物过程三个基因功能分类体系的56个功能组。在细胞组件方面,叶绿素合成相关基因有2 431个; 在生物过程方面,参与类胡萝卜素合成过程的基因有75个,参与花青素合成过程中的调控以及紫外光下组织中花青素积累的相关基因有80个。COG分类表明参与复制、重组和修复的基因数为369个,信号转导机制的基因数为291个,转录的相关基因为222个。通过KEGG数据库系统地分析变异基因参与的黄酮类、类胡萝卜素等物质代谢合成途径。深入研究这些差异基因的调控途径,从DNA水平上解释竹秆的变异机制,可为深入研究毛竹种内丰富的多态性和遗传变异提供数据支持,阐析不同变异类型的基因家族、功能基因等遗传基础。