Xin-Qiang He

The regulation of cambial activity in Chinese fir (Cunninghamia lanceolata) involves extensive transcriptome remodeling

Chinese fir (Cunninghamia lanceolata), a commercially important tree for the timber and pulp industry, is widely distributed in southern China and northern Vietnam, but its large and complex genome has hindered the development of genomic resources. Few efforts have focused on analysis of the modulation of transcriptional networks in vascular cambium during the transition from active growth to dormancy in conifers. Here, we used Illumina sequencing to analyze the global transcriptome alterations at the different stages of vascular cambium development in Chinese fir. By analyzing dynamic changes in the transcriptome of vascular cambium based on our RNA sequencing (RNA-Seq) data at the dormant, reactivating and active stages, many potentially interesting genes were identified that encoded putative regulators of cambial activity, cell division, cell expansion and cell wall biosynthesis and modification. In particular, the genes involved in transcriptional regulation and hormone signaling were highlighted to reveal their biological importance in the cambium development and wood formation. Our results reveal the dynamics of transcriptional networks and identify potential key components in the regulation of vascular cambium development in Chinese fir, which will contribute to the in-depth study of cambial differentiation and wood-forming candidate genes in conifers.

Phloem transdifferentiation from immature xylem cells during bark regeneration after girdling in Eucommia ulmoides Oliv

Eucommia ulmoides Oliv. (Eucommiaceae), a traditional Chinese medicinal plant, was used to study phloem cell differentiation during bark regeneration after girdling on a large scale. Here it is shown that new sieve elements (SEs) appeared in the regenerated tissues before the formation of wound cambium during bark regeneration after girdling, and they could originate from the transdifferentiation of immature/differentiating axial xylem cells left on the trunk. Assays of water-cultured twigs revealed that girdling blocked sucrose transport until the formation of new SEs, and the regeneration of the functional SEs was not dependent on the substance provided by the axis system outside the girdled areas, while exogenous indole acetic acid (IAA) applied on the wound surface accelerated SE differentiation. The experiments suggest that the immature xylem cells can transdifferentiate into phloem cells under certain conditions, which means xylem and phloem cells might share some identical features at the beginning of their differentiation pathway. This study also showed that the bark regeneration system could provide a novel method for studying xylem and phloem cell differentiation.

Molecular features of secondary vascular tissue regeneration after bark girdling in Populus

Regeneration is a common strategy for plants to repair damage to their tissue after attacks from other organisms or physical assaults. However, how differentiating cells acquire regenerative competence and rebuild the pattern of new tissues remains largely unknown. Using anatomical observation and microarray analysis, we investigated the morphological process and molecular features of secondary vascular tissue regeneration after bark girdling in trees. After bark girdling, new phloem and cambium regenerate from differentiating xylem cells and rebuild secondary vascular tissue pattern within 1 month. Differentiating xylem cells acquire regenerative competence through epigenetic regulation and cell cycle re-entry. The xylem developmental program was blocked, whereas the phloem or cambium program was activated, resulting in the secondary vascular tissue pattern re-establishment. Phytohormones play important roles in vascular tissue regeneration. We propose a model describing the molecular features of secondary vascular tissue regeneration after bark girdling in trees. It provides information for understanding mechanisms of tissue regeneration and pattern formation of the secondary vascular tissues in plants.

Induction of PtoCDKB and PtoCYCB transcription by temperature during cambium reactivation in Populus tomentosa Carr.

Cell cycle progression requires interaction between cyclin-dependent kinase B (CDKB) and cyclin B (CYCB). The seasonal expression patterns of the CDKB and CYCB homologues from Populus tomentosa Carr. were investigated, and effects of temperature and exogenous indole-3-acetic acid (IAA) on their expression were further studied in water culture experiments. Based on the differential responses of dormant cambium cells to exogenous IAA, four stages of cambium dormancy were confirmed for P. tomentosa: quiescence 1 (Q1), rest, quiescence 2-1 (Q2-1), and quiescence 2-2 (Q2-2). PtoCDKB and PtoCYCB transcripts were strongly expressed in the active phases, weakly in Q1, and almost undetectable from rest until late Q2-2. Climatic data analysis showed a correlation between daily air temperature and PtoCDKB and PtoCYCB expression patterns. Water culture experiments with temperature treatment further showed that a low temperature (4 °C) kept PtoCDKB and PtoCYCB transcripts at undetectable levels, while a warm temperature (25 °C) induced their expression in the cambium region. Meanwhile, water culture experiments with exogenous IAA treatment showed that induction of PtoCDKB and PtoCYCB transcription was independent of exogenous IAA. The results suggest that, in deciduous hardwood P. tomentosa growing in a temperate zone, the temperature in early spring is a vital environmental factor for cambium reactivation. The increasing temperature in early spring may induce CDKB and CYCB homologue transcription in the cambium region, which is necessary for cambium cell division.

Deep sequencing on a genome-wide scale reveals diverse stage-specific microRNAs in cambium during dormancy-release induced by chilling in poplar

BackgroundTrees in temperate zones show periodicity by alternating active and dormant states to adapt to environmental conditions. Although phytohormones and transcriptional regulation were found to be involved in growth cessation and dormancy transition, little is known about the mechanisms of the dormancy-active growth transition, especially dormancy maintenance and release. Small RNAs are a group of short non-coding RNAs regulating gene expressions at the post-transcriptional level during plant development and the responses to environmental stress. No report on the expression profiling of small RNAs in the cambial meristem during the dormancy-active growth transition has been reported to date.ResultsThree small RNA libraries from the cambium of poplar, representing endodormancy induced by short day conditions, ecodormancy induced by chilling and active growth induced by long day conditions, respectively, were generated and sequenced by Illumina high-throughput sequencing technology. This yielded 123 known microRNAs (miRNAs) with significant expression changes, which included developmental-, phytohormone- and stress-related miRNAs. Interestingly, miR156 and miR172 showed opposite expression patterns in the cambial dormancy-active growth transition. Additionally, miR160, which is involved in the auxin signaling pathway, was expressed specifically during endodormancy release by chilling. Furthermore, 275 novel miRNAs expressed in the cambial zone were identified, and 34 of them had high detection frequencies and unique expression patterns. Finally, the target genes of these novel miRNAs were predicted and some were validated experimentally by 5′RACE.ConclusionsOur results provided a comprehensive analysis of small RNAs in the cambial meristem during dormancy-release at the genome-wide level and novel evidence of miRNAs involved in the regulation of this biological process.

An improved CTAB-ammonium acetate method for total RNA isolation from cotton.

INTRODUCTION Cotton is an important economic crop. Genetic, developmental and molecular studies of cotton require high-quality total RNA from different tissues. Due to the richness in polyphenols and polysaccharides, the Trizol-based methods and other commercial kits are unsuitable for RNA isolation from cotton. Available methods are generally laborious and time-consuming. OBJECTIVE To develop an easy, simple and rapid cetyltrimethylammonium bromide (CTAB)-ammonium acetate protocol that takes less time and obtains high yield and quality of RNA from polysaccharide- and polyphenol-rich cotton tissues. METHODOLOGY Based on the original CTAB protocol, we used phenol-chloroform and chloroform-isoamyl alcohol to remove proteins, polysaccharides and polyphenols, and ammonium acetate to precipitate RNA, reducing the incubation time prior to RNA precipitation. After adding ammonium acetate to precipitate RNA, all centrifugation steps (14000 × g) were carried out at 4°C to avoid degradation. RESULTS The procedure took only 1.5 h and was suitable for different cotton tissues. The A(260) : A(280) ratios ranged from 1.80 to 1.85 with clear 28 s and 18 s ribosomal RNA bands in 1.2% agarose gel. The isolated RNA was usable for downstream molecular studies, such as reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR. CONCLUSION The CTAB-ammonium acetate method is easy, rapid, low-cost and effective for high-quality RNA isolation from polysaccharide- and polyphenol-rich cotton tissues.

The Ca2+ -dependent DNases are involved in secondary xylem development in Eucommia ulmoides.

Secondary xylem development has long been recognized as a typical case of programmed cell death (PCD) in plants. During PCD, the degradation of genomic DNA is catalyzed by endonucleases. However, to date, no endonuclease has been shown to participate in secondary xylem development. Two novel Ca(2+) -dependent DNase genes, EuCaN1 and EuCaN2, were identified from the differentiating secondary xylem of the tree Eucommia ulmoides Oliv., their functions were studied by DNase activity assay, in situ hybridization, protein immunolocalization and virus-induced gene silencing experiments. Full-length cDNAs of EuCaN1 and EuCaN2 contained an open reading frame of 987 bp, encoding two proteins of 328 amino acids with SNase-like functional domains. The genomic DNA sequence for EuCaN1 had no introns, while EuCaN2 had 8 introns. EuCaN1 and EuCaN2 digested ssDNA and dsDNA with Ca(2+) -dependence at neutral pH. Their expression was confined to differentiating secondary xylem cells and the proteins were localized in the nucleus. Their activity dynamics was closely correlated with secondary xylem development. Secondary xylem cell differentiation is influenced by RNAi of endonuclease genes. The results provide evidence that the Ca(2+) -dependent DNases are involved in secondary xylem development.

Preplasma effects on the generation of high-energy protons in ultraintense laser interaction with foil targets

It is shown that the intense quasistatic electric and magnetic fields self-generated near the axis of the laser-driven channel in an appropriately profiled preplasma during ultraintense laser interaction with a thin target can create dense relativistic electron bunches. The latter easily penetrate through the target and can greatly enhance the sheath field at the rear, resulting in significant increase in the laser-to-ion energy conversion efficiency and the maximum energy of the target normal sheath accelerated ions. Particle-in-cell simulations show that with a hydrogen targets a proton beam of peak energy ∼38 MeV and energy conversion efficiency ≥6.5% can be produced by a linearly polarized 5 × 1019 W/cm2 laser. An analytical model is also proposed and its results agree well with those of the simulations.

Tissue regeneration after bark girdling: an ideal research tool to investigate plant vascular development and regeneration

Regeneration is a common strategy for plants to survive the intrinsic and extrinsic challenges they face through their life cycle, and it may occur upon wounding. Bark girdling is applied to improve fruit production or harvest bark as medicinal material. When tree bark is removed, the cambium and phloem will be peeled off. After a small strip of bark is removed from trees, newly formed periderm and wound cambium develop from the callus on the surface of the trunk, and new phloem is subsequently derived from the wound cambium. However, after large-scale girdling, the newly formed sieve elements (SEs) appear earlier than the regenerated cambium, and both of them derive from differentiating xylem cells rather than from callus. This secondary vascular tissue regeneration mainly involves three key stages: callus formation and xylem cell dedifferentiation; SEs appearance and wound cambium formation. The new bark is formed within 1 month in poplar, Eucommia; thus, it provides high temporal resolution of regenerated tissues at different stages. In this review, we will illustrate the morphology, gene expression and phytohormone regulation of vascular tissue regeneration after large-scale girdling in trees, and also discuss the potential utilization of the bark girdling system in studies of plant vascular development and tissue regeneration.

Proteomic analysis provides insights into changes in the central metabolism of the cambium during dormancy release in poplar.

Seasonal cycling of growth and dormancy is an important feature for the woody plants growing in temperate zone, and dormancy is an effective strategy for surviving the winter stress. But the mechanisms of dormancy maintenance and its release are still not clear, especially little information is available with regard to the changes of proteome during the process. A better understanding in the function of proteins and their related metabolic pathways would expand our knowledge of the mechanisms of dormancy maintenance and its release in trees. In this study, we employed the isobaric tags for relative and absolute quantification (iTRAQ) approach with LC-MS/MS analysis to investigate the protein profile changes during dormancy release in poplar. In addition, the change of lipid, total insoluble carbohydrates and starch granules in the cambium was investigated by histochemical methods. A total of 3789 proteins were identified in poplar cambial tissues, 1996 of them were significantly altered during the dormancy release. Most of the altered proteins involved in signaling, phytohormone, energy metabolism, stress and secondary metabolism by functional analysis. Our data shows that the lipid metabolism proteins changed significantly both in the release stage of eco- and endodormancy, while the changes of carbohydrate metabolism proteins were mainly in endo-dormancy release stage. Moreover, histochemical results were consistent with the proteomic data. Our results reveal diverse stage-specific metabolism changes during the dormancy-release process induced by chilling in poplar, which provided new information regarding the regulation mechanisms of dormancy maintenance and its release in trees.