A moso bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>) miniature inverted-repeat transposable element (MITE): the possible role of a suppressor

Transposable elements (transposons) are fragments of DNA sequences which can move within host genome. Miniature inverted-repeat transposable elements (MITEs) are widespread and high-copy transposable elements in eukaryotic genomes. Tourist-like MITEs are especially abundant in plant kingdom. Earlier genome-wide analysis has shown that MITEs are widely distributed in the moso bamboo genome and preferentially inserted into gene regions. In the present study, in order to examine the potential influence of MITEs on the moso bamboo gene expressions, a highly conserved Tourist-like MITE family, which distributed near genes, was selected as research focus and named PhTst-3 (Phyllostachys edulis Tourist-like element 3). The MITEs’ insertion sites were tested in moso bamboo half-sib seedlings by real-time fluorescence quantitative PCR. Amplification polymorphisms were found in a copy of PhTst-3 (PhTst-3-55) which was located in the intron of PH01002699G0010. This inserted PhTst-3-55 had a significant impact on the gene expression revealed by the real-time fluorescence quantitative PCR. The gene expression levels were four times higher in the absence of PhTst-3-55 than those in the presence of it. This finding suggests that the PhTst-3 located in the intron is involved in the regulation of the gene. In order to examine the impact of PhTst-3-55 on the near genes, the PhTst-3-55 was inserted into a promoter analysis vector, pxk7S2D, between the two promoter sequences. The Agrobacterium-mediated transient expression showed that PhTst-3-55 insertion decreases the expression level of upstream GUS gene and downstream GFP gene. So, PhTst-3-55 can have a silencing role by bidirectionally inhibiting gene expression.     

Isolation and Expression Analysis of <Emphasis Type="Italic">PeDWF1</Emphasis> in <Emphasis Type="Italic">Phyllostachys edulis</Emphasis>

Brassinosteroids (BRs) are steroidal hormones that play crucial roles in various processes of plant growth and development. DWF1 encodes a delta(24)-sterol reductase that participates in one of the early stage in the brassinosteroids’ biosynthetic pathway: the conversion of 24-methylenecholesterol to campesterol. Here we report the isolation and expression of one DWF1 homologous gene, PeDWF1, in moso bamboo (Phyllostachys edulis (Carrière) J. Houz.). Sequence analysis revealed that the open reading frame of PeDWF1 was 1686-bp encoding a protein composed of 561 amino acid residues with a calculated molecular weight of 65.1 kD and a theoretic isoelectric point of 8.32. Phylogenetic analysis indicated that PeDWF1 was very close to the cell elongation protein Dwarf1 in rice (Oryza sativa). Furthermore, transient expression of a PeDWF1::GFP fusion protein showed that PeDWF1 was an integral membrane protein most probably associated with the endoplasmic reticulum similar to Dwarf1. Tissue specific expression analysis showed that PeDWF1 was constitutively expressed in moso bamboo with the highest level in shoots and the lowest level in mature leaves. In the early growing stage of shoots, the expression level of PeDWF1 had a rising trend with the increasing height of shoots. These results indicated that PeDWF1 might be involved in the regulation of shoot development by participating in BRs biosynthesis. Moreover, PeDWF1 was heterologously expressed in Escherichia coli and the recombinant protein was about 65 kD, which facilitated further study on the gene function of PeDWF1 in bamboo.     

Genome-wide identification and characterization of aquaporin gene family in moso bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>)

Aquaporins (AQPs) are known to play a major role in maintaining water and hydraulic conductivity balance in the plant system. Numerous studies have showed AQPs execute multi-function throughout plant growth and development, including water transport, nitrogen, carbon, and micronutrient acquisition etc. However, little information on AQPs is known in bamboo. In this study, we present the first genome-wide identification and characterization of AQP genes in moso bamboo (Phyllostachys edulis) using bioinformatics. In total, 26 AQP genes were identified by homologous analysis, which were divided into four groups (PIPs, TIPs, NIPs, and SIPs) based on the phylogenetic analysis. All the genes were located on 26 different scaffolds respectively on basis of the gene mapped to bamboo genome. Evolutionary analysis indicated that Ph. edulis was more close to Oryza sativa than Zea mays in the genetic relationship. Besides, qRT-PCR was used to analyze gene expression profiles, which revealed that AQP genes were expressed constitutively in all the detected tissues, and were all responsive to the environmental cues such as drought, water, and NaCl stresses. This data suggested that AQPs may play fundamental roles in maintaining normal growth and development of bamboo, which would contribute to better understanding for the complex regulation mechanism involved in the fast-growing process of bamboo. Furthermore, the result could provide valuable information for further research on bamboo functional genomics.     

Genome-wide characterization and evolution analysis of long terminal repeat retroelements in moso bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>)

The availability of nearly complete moso bamboo genome sequences permits the detailed discovery and cross-species comparison of transposable elements (TEs) between Bambusoideae and other Poaceae species at the whole genome level. Long terminal repeat retroelements (LTR-retroelements) are the single largest components of most plant genomes and can substantially impact the genome in various ways. Through a combination of structure- and homology-based approaches, we initially investigated 982 LTR-retroelement families comprising 2,004,644 LTR-retroelement sequences, which accounted for more than 40% of the moso bamboo genome. Further analysis revealed that the ratio of solo LTRs to intact elements (S/I) in moso bamboo is significantly low (approximately 0.28:1), indicating that bamboo LTR-retroelements might have undergone relatively low frequencies of unequal recombination and illegitimate recombination. Phylogenetic analysis revealed four Ty1-copia and five Ty3-gypsy evolutionary lineages that were present before the divergence of eudicot and monocot species, but the scales and timeframes within which they proliferated significantly varied across families and lineages. Insertion time estimates showed that LTR-retroelements were amplified for approximately 0~3 million years and had longer periods of activity than those of rice and Arabidopsis. These findings suggest that the expansion of LTR-retroelements might be responsible for host large genome size during moso bamboo evolution.     

Two active bamboo <Emphasis Type="Italic">mariner</Emphasis>-like transposable elements (<Emphasis Type="Italic">Ppmar1</Emphasis> and <Emphasis Type="Italic">Ppmar2</Emphasis>) identified as the transposon-based genetic tools for mutagenesis

Bamboo is one of the most important non-timber forest species in the world, but their molecular breeding lags far behind in contrast to other economic plants. Regarding the difficulties of hybridization and gene modification, the transposon-based insertional mutagenesis might be an alternative, feasible way for molecular breeding of bamboo. A systematic search for potential active transposons identified two full-length mariner-like elements (MLEs) (Ppmar1 and Ppmar2) from moso bamboo in the previous study. Both MLEs contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase. Two transposases contain intact DNA-binding motifs and a DD39D catalytic domain which indicates that Ppmar1 and Ppmar2 are likely active. Here, we deployed a heterologous transposition system of Arabidopsis thaliana to study the transposition activity of Ppmar1 and Ppmar2. The results show that both MLEs could transpose in A. thaliana. Excisions of Ppmar1 and Ppmar2 are usually unperfect as they leave 1–4 bp in excision sites. The reinsertions of both Ppmar1 and Ppmar2 occur at TA dinucleotides and prefer to insert into the TA-rich regions. The insertion sites are dispersed and non-linked. Two active bamboo transposons identified here not only could be applied to construction of the bamboo mutant libraries but also would provide another choice for other plant transposon-based gene tagging.     

Root-favoured biomass allocation improves growth and yield of field-grown rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) plants only when the shoot sink is expandable

We tested the hypothesis that high root/shoot (R/S) in rice improves plant growth and yield when the shoot sink is expandable, and that in a genotype with exaggerated R/S ratio, the shoot growth is not limited by root resources. This study involved the three rice genotypes, Giza 178, PM12, and Moroberekan with a range of R/S ratios and shoot sink sizes. Root regrowth after trimming or high- and low-nitrogen treatments revealed that Moroberekan has consistently high root-favoured biomass partitioning than Giza 178 or PM12. Increasing the R/S ratios by detillering improved the culm growth in Giza 178 and PM12 (by 43.4 and 17.7% of control, respectively) but not Moroberekan, indicating that PM12 was closer to achieving its growth potential than Giza 178 but Moroberekan was operating at maximal shoot growth potential because of high R/S ratio and small sink size. Under drought, shoot growth, gas exchange, and grain yield correlated strongly with R/S ratio and root length density (RLD) in the droughted but not the well-watered plants. We further hypothesized that R/S ratio of Moroberekan was in excess of shoot requirement for optimum growth. Crossing Moroberekan to PM12 generated three F1 hybrids with intermediate R/S ratios but higher growth, gas exchange, and yield than either parent. We conclude that increasing the R/S ratio improved growth and yield in PM12 but not Moroberekan, because the shoot sink size was expandable in PM12. Moreover, lower R/S ratios than that of Moroberekan could support higher shoot growth if shoot sink is expandable.     

Genome-wide characterization and evolution analysis of miniature inverted-repeat transposable elements (MITEs) in moso bamboo (<Emphasis Type="Italic">Phyllostachys heterocycla</Emphasis>)

Main conclusion

Moso bamboo MITEs were genome-wide identified first time, and data shows that MITEs contribute to the genomic diversity and differentiation of bamboo. Miniature inverted-repeat transposable elements (MITEs) are widespread in animals and plants. There are a large number of transposable elements in moso bamboo (Phyllostachys heterocycla var. pubescens) genome, but the genome-wide information of moso bamboo MITEs is not known yet. Here we identified 362 MITE families with a total of 489,592 MITE-related sequences, accounting for 4.74 % of the moso bamboo genome. The 362 MITE families are clustered into six known and one unknown super-families. Our analysis indicated that moso bamboo MITEs preferred to reside in or near the genes that might be involved in regulation of host gene expression. Of the seven super-families, three might undergo major expansion event twice, respectively, during 8–11 million years ago (mya) ago and 22–28 mya ago; two might experience a long expansion period from 6 to 13 mya. Almost 1/3 small RNAs might be derived from the MITE sequences. Some MITE families generate small RNAs mainly from the terminals, while others predominantly from the central region. Given the high copy number of MITEs, many siRNAs and miRNAs derived from MITE sequences and the preferential insertion of MITE into gene regions, MITEs may contribute to the genomic diversity and differentiation of bamboo.

     

Prolonged culture of <Emphasis Type="Italic">Boesenbergia rotunda</Emphasis> cells reveals decreased growth and shoot regeneration capacity

We evaluated the ploidy levels and tissue culture responses of 16 Japanese Miscanthus accessions, which are registered and vegetatively maintained in the National Agriculture and Food Research Organization GeneBank, Japan, to screen suitable genotypes for the molecular breeding of Miscanthus species. A ploidy analysis showed that most M. sinensis and M. sinensis var. condensatus (var. condensatus) were putative diploids, but one accession identified as M. sinensis was unexpectedly a putative tetraploid. Additionally, M. sacchariflorus and its hybrid accessions were putative tetraploids. The deoxyribonucleic acid levels in var. condensatus were significantly higher than those in the diploid M. sinensis. Of the accessions, 10, including M. sinensis and var. condensatus, could induce plant regenerable embryogenic calli from apical meristems. We selected three of these M. sinensis accessions for further experiments because their calli growth rates were faster than those of the var. condensatus accessions. Tissue culture experiments with the selected accessions indicated that the frequencies of callus and green shoot formation strongly correlated with genotype. The broad-sense heritabilities of the embryogenic callus and green shoot formation frequencies in the selected accessions were 0.75 and 0.65, respectively, indicating that the cultures’ responses were mainly controlled by genetic factors. Thus, we further selected one accession that had the highest efficiencies in callus and green shoot formation, and we observed that light during callus culturing significantly inhibited calli growth, but promoted plant regeneration from calli in the selected accession.     

Involvement of reactive oxygen species and Ca<Superscript>2+</Superscript> in the differential responses to low-boron in rapeseed genotypes

Background and aims

Invasive weeds may exert negative impact on other plant species and soil processes. The observed negative impact of an invasive weed species may be driven by allelopathy or nutrient availability.

Methodology

Sorghum halepense is one of the worst invasive weeds in crop fields. We quantified the species richness in the S. halepense-invaded communities and communities not yet invaded by the weed. Sorghum soil and no-sorghum soil were analysed for total phenolics, microbial activity, available nitrogen (N) and organic carbon. Manipulative experiments were carried out to understand the interference potential of S. halepense. Soil was amended with root or shoot leachate of S. halepense, and its impact on plant growth and soil properties was studied.

Results

Out of four S. halepense-sites, lower plant species richness was observed in one site compared to uninvaded sites. S. halepense-invaded soil had higher levels of total phenolics and lower levels of available N. Higher inhibition in the root growth of Brassica juncea or Bidens pilosa was observed in root leachate-amended soil than shoot leachate-amended soil. Shoot leachate-amended soil had higher levels of total phenolics and available N than root leachate-amended soils. Significant reduction in the available N was observed in soil amended with root leachate. Significant decline in the total phenolics over a period of time was observed in soil amended with root leachate or shoot leachate of S. halepense. Higher CO₂ release was observed 24 h after amending soil with root leachate or shoot leachate of S. halepense.

Conclusions

Sorghum halepense interference potential in its soil is likely due to lower levels of available N. Greater reduction in root dry weight of assay species in root leachate amended soil compared to shoot leachate amended soil was likely due to lower levels of available N in root leachate-amended soil. Relative interference potential of both root and shoot leachates or extracts should be evaluated in allelopathy bioassays and further experiments should be designed to distinguish the role of allelochemicals and nutrient availability in plant growth inhibition.

     

Microsatellite markers revealed moderate genetic diversity and population differentiation of moso bamboo (<Emphasis Type="Italic">Phyllostachys edulis</Emphasis>)—a primarily asexual reproduction species in China

As an ecologically and economically important endemic bamboo species, moso (Phyllostachys edulis) has been widely distributed in Southern China. In the paper, 20 fluorescently labeled microsatellite markers were used to evaluate the genetic structure of Ph. edulis including 34 representative populations (803 individuals) covering the geographic range in China. Moderate genetic diversity (H?= 0.376) and differentiation (Gst?= 0.162) were detected at the species level, with the majority of genetic diversity occurring within populations (84.55%). Bayesian model-based structure analysis and sNMF/ANLS-AS method revealed the presence of two and three clusters. When K?=?2, majority of populations (except SX) were clustered together (C1). It implied that SX, known as an introduced and isolated founder population, significantly differed from other populations for distinct environmental selection and allele mutation with the proof of scarce outcrossing and relatively high frequency of private allele. While K?=?3, two subgroups (C1a of 18 populations and C1b of 14 populations) were detected within C1. The C1b displayed as a belt-shape region with an east-west direction. It coincided with the extremely high artificial selection in C1b (lower genetic diversity than that of C1a) due to the intensive plantation in the last four decades. Our results implied that the population protection and germplasm collection of moso bamboo should be not only from representative populations in Zhejiang, Jiangxi, Fujian, and other places with intensive cultivation in the east of China, but from populations with high level of gene and genotypic diversity in the west (e.g., HN5, GD1, GZ2, YN1, and SX).     

Micropropagation of Ajuga species: a mini review

The genus Ajuga L., belonging to Lamiaceae family, is widespread. The demand for Ajuga species has risen sharply because of their medicinal, ornamental, and pharmacological properties. These wide-ranging plants are being rapidly depleted due to over-collection for ornamental and medicinal purposes, as well as by habitat destruction and deforestation. Ajuga boninsimae, A. bracteosa, A. ciliate, A. genevensis, A. incisa, A. makinoi, A. multiflora, A. pyramidalis, A. shikotanensis, A. reptans, and A. vestita are categorized and protected as endangered plants. In vitro plant culture has therefore emerged for the conservation and mass clonal propagation of rare plants. This mini-review covers the current in vitro scenario in the propagation of Ajuga species. Adventitious or axillary shoots are initiated on the leaf, petiole and internodes, as well as roots, nodes, and shoot tip explants. Shoot induction is predominantly dependent on plant growth regulators added to the culture medium. Full- or half-strength Murashige and Skoog medium with or without auxin is used for in vitro rooting. Rooted shoots need to be acclimatized in the greenhouse with an estimated 82–100% survival rate.     

<Emphasis Type="Italic">Azospirillum</Emphasis> spp. from native forage grasses in Brazilian Pantanal floodplain: biodiversity and plant growth promotion potential

A sustainable alternative to improve yield and the nutritive value of forage is the use of plant growth-promoting bacteria (PGPB) that release nutrients, synthesize plant hormones and protect against phytopathogens (among other mechanisms). Azospirillum genus is considered an important PGPB, due to the beneficial effects observed when inoculated in several plants. The aim of this study was to evaluate the diversity of new Azospirillum isolates and select bacteria according to the plant growth promotion ability in three forage species from the Brazilian Pantanal floodplain: Axonopus purpusii, Hymenachne amplexicaulis and Mesosetum chaseae. The identification of bacterial isolates was performed using specific primers for Azospirillum in PCR reactions and partial sequencing of the 16S rRNA and nifH genes. The isolates were evaluated in vitro considering biological nitrogen fixation (BNF) and indole-3-acetic acid (IAA) production. Based on the results of BNF and IAA, selected isolates and two reference strains were tested by inoculation. At 31 days after planting the plant height, shoot dry matter, shoot protein content and root volume were evaluated. All isolates were able to fix nitrogen and produce IAA, with values ranging from 25.86 to 51.26 mg N mL^?1 and 107–1038 µmol L^?1, respectively. The inoculation of H. amplexicaulis and A. purpusii increased root volume and shoot dry matter. There were positive effects of Azospirillum inoculation on Mesosetum chaseae regarding plant height, shoot dry matter and root volume. Isolates MAY1, MAY3 and MAY12 were considered promising for subsequent inoculation studies in field conditions.     

Halophilic rhizobacteria from <Emphasis Type="Italic">Distichlis spicata</Emphasis> promote growth and improve salt tolerance in heterologous plant hosts

Rhizobacteria are central components of the plant microbiome and influence root development and function. Desciphering how rhizobacteria contribute to plant performance under adverse environments is a major research challenge. The aims of the present study were to isolate and characterize rhizobacteria from the halophilic grass Distichlis spicata and to test their possible growth promoting and salt protective properties in Arabidopsis thaliana, Cucumis sativus, and Citrullus lanatus. To determine their possible plant growth promoting properties, 38 rhizobacterial isolates were co-cultivated with Arabidopsis seedlings in vitro. Out of these, two halophilic bacteria, LBEndo1 and KBEcto4, were selected following their strong shoot and root biostimulation. 16S rRNA sequencing identified LBEndo1 as Bacillus sp. and KBEcto4 as Pseudomonas lini. Both strains improved growth under standard and saline conditions, which correlated with IAA and siderophore production, as well as phosphate solubilization. Additionally, the KBEcto4 strain expresses the ACC deaminase enzyme (acdS gene), and slightly increases auxin redistribution within Arabidopsis roots expressing an auxin-inducible gene construct. These data reveal the potential of saltgrass (Distichlis spicata) rhizobacteria to promote growth and confer salt tolerance to Arabidopsis and crop plants.     

Plant species-specificity and effects of bioinoculants and fertilization on plant performance for nickel phytomining

Aims

To investigate the effects of fertilization and bacterial inoculation on the growth, health and Ni phytoextraction capacity of three Ni-hyperaccumulators, Odontarrhena bracteata, O. inflata and O. serpyllifolia.

Methods

Plants were grown for three months in serpentine soil fertilized with inorganic NPK or amended with cow manure and inoculated with five rhizobacterial strains (previously isolated from O. serpyllifolia). Shoot and root dry weight (DW) yields, Ni accumulation and removal, nutritive status and stress indicators were determined.

Results

Plants grown in manure-amended soil showed significantly higher DW yields, improved nutritive status and higher total Ni phytoextracted. Some bacterial inoculants enhanced Ni removal due to the stimulation in growth and/or increase in shoot Ni concentration but this depended on the plant species, soil type and inoculant. Pseudoarthrobacter oxydans strain SBA82 enhanced shoot DW yield of all three Odontarrhena spp. in at least one soil type. Paenarthrobacter sp. strain LA44 and Stenotrophomonas sp. strain MA98 promoted growth of O. serpyllifolia and O. bracteata. Inoculated plants showing growth promotion presented lower activities of antioxidative enzymes, and concentrations of malondialdehyde (MDA) and H₂O₂, indicating a protective effect of these inoculants on the plants.

Conclusion

Rhizobacterial inoculants applied in combination with manure can improve plant growth and health, and Ni phytoextraction, in some hyperaccumulating Odontarrhena spp.