Shutang Zhao

Expression Analysis of Two NAC Transcription Factors PtNAC068 and PtNAC154 from Poplar

NAM/ATAF/CUC (NAC) family genes comprise one of the largest families of transcription factors in plant genomes and are widely expressed in developing woody tissues. In the present study, we constructed plant transformation vectors using the β-glucuronidase (GUS) reporter gene system and detected the promoter expression patterns derived from the PtNAC068 and PtNAC154 genes of Chinese white poplar (Populus tomentosa Carr.) in transgenic poplars (Populus alba × Populus glandulosa). The results showed that the GUS expression driven by PtNAC068 and PtNAC154 promoters may be more complex in poplar than they are in Arabidopsis. Histochemical GUS assays showed that GUS activity driven by PtNAC068 promoter was mainly in vascular tissues of stems, leaves, petioles, and roots, while that driven by PtNAC154 promoter was confined to the developing secondary xylem of stems and veins of leaves. The transcript level of both PtNAC068 and PtNAC154 in successive internodes below the apex was found to be much higher in IN5-10 compared to that in IN2-4 as measured by real-time RT-PCR, suggesting that PtNAC068 and PtNAC154 upregulation is related to secondary growth in poplar. GUS expression in internodes 3–8 of ProNAC068::GUS transgenic plants was 30-fold higher than that in ProNAC154::GUS transgenic plants. The differences in the expression pattern and transcript level of mRNA accumulation indicate that PtNAC068 and PtNAC154 may be involved in two distinct aspects of vascular tissue development.

Bacillus thuringiensis Cry3Aa fused to a cellulase-binding peptide shows increased toxicity against the longhorned beetle.

Cry3 class toxins are used extensively for biological control of coleopteran larvae. We previously identified a peptide (PCx) from a phage display library that specifically binds Cx-cellulase from the midgut of Anoplophora glabripennis Motschulsky (Asian longhorn beetle) larvae. Here, we added a DNA fragment that encodes the peptide onto either end of the cry3Aa gene and tested the expressed PCx-Cry3Aa and Cry3Aa-PCx proteins for insecticidal activity in the longhorned beetle. An insect bioassay revealed that, compared with native Cry3Aa, the two modified Cry3Aa proteins had significantly higher lethality, with PCx-Cry3Aa exhibiting a mortality rate almost three times that of Cry3Aa. We also proposed that the increased lethality in larvae fed with PCx-Cry3Aa or Cry3Aa-PCx would be attributable to the binding of the toxin with Cx-cellulase, thereby increasing toxin retention in the midgut. The significantly enhanced insecticidal activity of Cry3Aa fused with the Cx-cellulase binding peptide provides a new strategy for increasing toxin efficacy against the longhorned beetle. These uniquely modified Cry3Aa proteins have potential use for pest control.

A Populus TIR1 gene family survey reveals differential expression patterns and responses to 1-naphthaleneacetic acid and stress treatments.

The plant hormone auxin is a central regulator of plant growth. TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) is a component of the E3 ubiquitin ligase complex SCFTIR1/AFB and acts as an auxin co-receptor for nuclear auxin signaling. The SCFTIR1/AFB-proteasome machinery plays a central regulatory role in development-related gene transcription. Populus trichocarpa, as a model tree, has a unique fast-growth trait to which auxin signaling may contribute. However, no systematic analyses of the genome organization, gene structure, and expression of TIR1-like genes have been undertaken in this woody model plant. In this study, we identified a total of eight TIR1 genes in the Populus genome that are phylogenetically clustered into four subgroups, PtrFBL1/PtrFBL2, PtrFBL3/PtrFBL4, PtrFBL5/PtrFBL6, and PtrFBL7/PtrFBL8, representing four paralogous pairs. In addition, the gene structure and motif composition were relatively conserved in each paralogous pair and all of the PtrFBL members were localized in the nucleus. Different sets of PtrFBLs were strongly expressed in the leaves, stems, roots, cambial zones, and immature xylem of Populus. Interestingly, PtrFBL1 and 7 were expressed mainly in vascular and cambial tissues, respectively, indicating their potential but different roles in wood formation. Furthermore, Populus FBLs responded differentially upon exposure to various stresses. Finally, over-expression studies indicated a role of FBL1 in poplar stem growth and response to drought stress. Collectively, these observations lay the foundation for further investigations into the potential roles of PtrFBL genes in tree growth and development.

Identification of microRNAs Involved in Regeneration of the Secondary Vascular System in Populus tomentosa Carr

Wood formation is a complex developmental process primarily controlled by a regulatory transcription network. MicroRNAs (miRNAs) can modulate the expression of target genes involved in plant growth and development by inducing mRNA degradation and translational repression. In this study, we used a model of secondary vascular system regeneration established in Populus tomentosa to harvest differentiating xylem tissues over time for high-throughput sequencing of small RNAs. Analysis of the sequencing data identified 209 known and 187 novel miRNAs during this regeneration process. Degradome sequencing analysis was then performed, revealing 157 and 75 genes targeted by 21 known and 30 novel miRNA families, respectively. Gene ontology enrichment of these target genes revealed that the targets of 15 miRNAs were enriched in the auxin signaling pathway, cell differentiation, meristem development, and pattern specification process. The major biological events during regeneration of the secondary vascular system included the sequential stages of vascular cambium initiation, formation, and differentiation stages in sequence. This study provides the basis for further analysis of these miRNAs to gain greater insight into their regulatory roles in wood development in trees.

Transcriptional and temporal response of Populus stems to gravi-stimulation: Gravitropism time-series

Plants modify development in response to external stimuli, to produce new growth that is appropriate for environmental conditions. For example, gravi-stimulation of leaning branches in angiosperm trees results in modifications of wood development, to produce tension wood that pulls leaning stems upright. Here, we use gravi-stimulation and tension wood response to dissect the temporal changes in gene expression underlying wood formation in Populus stems. Using time-series analysis of seven time points over a 14-d experiment, we identified 8,919 genes that were differentially expressed between tension wood (upper) and opposite wood (lower) sides of leaning stems. Clustering of differentially expressed genes showed four major transcriptional responses, including gene clusters whose transcript levels were associated with two types of tissue-specific impulse responses that peaked at about 24-48 h, and gene clusters with sustained changes in transcript levels that persisted until the end of the 14-d experiment. Functional enrichment analysis of those clusters suggests they reflect temporal changes in pathways associated with hormone regulation, protein localization, cell wall biosynthesis and epigenetic processes. Time-series analysis of gene expression is an underutilized approach for dissecting complex developmental responses in plants, and can reveal gene clusters and mechanisms influencing development.

An Empirical Assessment of Transgene Flow from a Bt Transgenic Poplar Plantation.

To assess the possible impact of transgenic poplar plantations on the ecosystem, we analyzed the frequency and distance of gene flow from a mature male transgenic Populus nigra plantation carrying the Bacillus thuringiensis toxin gene (Bt poplar) and the survival of Bt poplar seeds. The resultant Bt poplar seeds occurred at a frequency of ~0.15% at 0 m to ~0.02% at 500 m from the Bt poplar plantation. The germination of Bt poplar seeds diminished within three weeks in the field (germination rate from 68% to 0%) compared to 48% after three weeks of storage at 4°C. The survival rate of seedlings in the field was 0% without any treatment but increased to 1.7% under the addition of four treatments (cleaning and trimming, watering, weeding, and covering with plastic film to maintain moisture) after being seeded in the field for eight weeks. The results of this study indicate that gene flow originating from the Bt poplar plantation occurred at an extremely low level through pollen or seeds under natural conditions. This study provides first-hand field data on the extent of transgene flow in poplar plantations and offers guidance for the risk assessment of transgenic poplar plantations.

Preliminary study of Cell Wall Structure and its Mechanical Properties of C3H and HCT RNAi Transgenic Poplar Sapling

This research focused on the cell wall structure and its mechanical properties of down-regulated Coumaroyl shikimate 3-hydroxylase (C3H) transgenic poplar and down-regulated hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) transgenic poplar (Populus alba × P. glandulosa cv ‘84 k’). The wood samples with respect to microstructure, the longitudinal elastic modulus (MOE) and hardness of wood fiber secondary cell wall were investigated. The results show that the lignin contents in the two transgenic poplar woods were lower than non-modified wood. The C3H transgenic poplar and HCT transgenic poplar have more than 18.5% and 16.1% cellulose crystalline regions than non-modified poplar respectively. The diameter of the fiber cell and the vessel element of transgenic poplars are smaller. Double radial vessel cell wall thicknesses of both transgenic poplars were smaller than non-modified poplar. Cell wall ratios for the transgenic poplar were higher than non-modified poplar and cell wall density was significantly lower in both C3H and HCT transgenic poplar. The cell wall MOEs of C3H and HCT transgenic poplar was 5.8% and 7.0% higher than non-modified poplar. HCT can be more effective than C3H to modify the trees by considerably increasing mechanical properties of the cell wall.

AtBET5 is essential for exine pattern formation and apical meristem organization in Arabidopsis

BET5 is a component of trafficking protein particle (TRAPP) which has been studied extensively in non-plant organisms where they are involved in membrane trafficking within Golgi and between Golgi and early endosomes. Recent analysis of TRAPP in different classes of organisms indicates that TRAPP function might exhibit differences among organisms. A single copy of the BET5 gene named AtBET5 was found in the Arabidopsis genome based on sequence similarity. Developmental phenotype and the underlying mechanisms have been characterized upon transcriptional knock-down lines generated by both T-DNA insertion and RNAi. Pollen grains of the T-DNA insertional line present reduced fertility and pilate exine instead of tectate exine. Perturbation of the AtBET5 expression by RNAi leads to apical meristematic organization defects and reduced fertility as well. The reduced fertility was due to the pollination barrier caused by an altered composition and structure of pollen walls. Auxin response in root tip cells is altered and there is a severe disruption in polar localization of PIN1-GFP, but to a less extent of PIN2-GFP in the root tips, which causes the apical meristematic organization defects and might also be responsible for the secretion of sporopollenin precursor or polar targeting of sporopollenin precursor transporters.

The auxin receptor TIR1 homolog (PagFBL1) regulates adventitious rooting through interactions with Aux/IAA28 in Populus

Summary Adventitious roots occur naturally in many species and can also be induced from explants of some tree species including Populus, providing an important means of clonal propagation. Auxin has been identified as playing a crucial role in adventitious root formation, but the associated molecular regulatory mechanisms need to be elucidated. In this study, we examined the role of PagFBL1, the hybrid poplar (Populus alba × P. glandulosa clone 84K) homolog of Arabidopsis auxin receptor TIR1, in adventitious root formation in poplar. Similar to the distribution pattern of auxin during initiation of adventitious roots, PagFBL1 expression was concentrated in the cambium and secondary phloem in stems during adventitious root induction and initiation phases, but decreased in emerging adventitious root primordia. Overexpressing PagFBL1 stimulated adventitious root formation and increased root biomass, while knock‐down of PagFBL1 transcript levels delayed adventitious root formation and decreased root biomass. Transcriptome analyses of PagFBL1 overexpressing lines indicated that an extensive remodelling of gene expression was stimulated by auxin signalling pathway during early adventitious root formation. In addition, PagIAA28 was identified as downstream targets of PagFBL1. We propose that the PagFBL1‐PagIAA28 module promotes adventitious rooting and could be targeted to improve Populus propagation by cuttings.

Increased Bacillus thuringiensis δ-endotoxin Cry3Aa toxicity against longhorned beetle by fusing to peptide specifically binding to beetle Cx-cellulase

Background Bacillus thuringiensis (Bt) Cry toxins have specific toxicity to susceptible insects. They are being used in transgenic plants or spray to control insect pests in agriculture [1, 2, 3]. Cry3A toxins are used extensively for biological control of coleopteran larvae [4, 5]. A Bt886-Cry3Aa gene that exhibited a high activity against Coleoptera insects isolated Our laboratory. Insect bioassay performed on Anoplophora glabripennis Motsch and Apriona germari Hope showed that the mortality of larvae fed with the product of this gene was over 60% [6]. However, both transgenic poplar with native Cry3Aa and withmodified-Cry3Aa by using poplar-prefered codons did little effects on longhorned beetles probably due to its low expression level in poplar.A peptide (LPPNPTK) named PCx that specifically bind to cellulase from midgut of longhorned beetle larvae was screened out from a phage display library previously in our laboratory[7].