Immature Xylem Research Articles

Comparative proteomic analysis provides insights into wood formation in immature xylem at different ages in Eucalyptus urophylla × Eucalyptus grandis.

Wood formation is a crucial developmental stage in the life cycle of a woody plant; this process has substantial scientific research implications and practical applications. However, the mechanisms underlying woody plant development, especially the process of wood formation, remain poorly understood. As eucalyptus is one of the fastest growing tree species in the world, understanding the mechanism of wood formation in eucalyptus will greatly promote the development of molecular breeding technology for forest trees. In this study, we investigated the proteomic profile of immature xylem at four different ages of Eucalyptus urophylla × Eucalyptus grandis (E. urograndis) using iTARQ technology. We identified 5236 proteins and 492 differentially abundant proteins (DAPs). The expression profiles of the DAPs corresponding to coding genes associated with wood formation were assessed using qRT-PCR. From the different expression profiles, it is inferred that the genes encoding kinesin, CDKD3, EXPA13, EXPA2, XTH27, EGases, UGT76E2, LAC, CCoAMT, CesA3, PAL, and CAD may undergo posttranscriptional regulation (PTR). Additionally, the genes encoding EIN2, ETR, MC4-like, and XCP may undergo posttranslational modifications (PTMs). We investigated changes in wood formation-related proteins at the protein abundance level in the immature xylem of E. urograndis, thereby elucidating potential regulatory mechanisms of key proteins involved in eucalyptus wood formation. This study may provide theoretical guidance for further research on molecular breeding techniques and genetic improvement related to the cultivation of rapidly growing and high-quality trees.

Development of the Vascular Cambium of Taxodium ascendens and Its Seasonal Activities in Subtropical China

The vascular cambium is an extensive and permanent secondary meristem with wood cells products of periclinal divisions commonly contributed to two directions and arranged in radial files of trees. Cambium activity is the origin of timber production. Taxodium ascendens Brongn is an exotic species in China, and its apical meristem and cambial activity are still elusive, resulting in a lack of understanding about its wood formation and improvement. We thus addressed this knowledge gap by studying Cambium activity. For studying, twigs from five 30-year-old healthy trees were collected between February-2017 and March-2018. Anatomy deciphered its apical meristem with a Cryptomeria–Abies type. The procambium appeared after leaf primordium and initially presented five lobes as observed transversely from a one-year-old shoot. The procambium under the apical differentiated into protophloem first and then protoxylem toward the inside. It means that protoxylem differentiated later than protophloem did. After dormancy, the vascular cambium began to be active, starting in early April 2017, which was later than shoot differentiation. On 25 July 2017, the cambial zone had 9–10 immature xylem cell layers. Both initiation and cessation of the xylem preceded that of the phloem. Until 10 October 2017, few immature elements were found, indicating the translation of cells from activity to dormancy. On 15 November 2017, the cambium contained 3–4 cells in radial rows, which demonstrated the dormancy of the cambium until next spring. Furthermore, immature xylem elements increased as cell layers in the cambium zone and cell fission increased. The growth pattern of T. ascendens revealed that cambial activity is highly seasonal and dependent on changes in abiotic conditions. Thus, the wood formation in the species will be significantly altered in a changing climatic pattern. These enhance our understanding of tree growth science, wood formation, wood structure, wood properties variation and wood improvement in tree breeding.

Expression Patterns and Regulation of Non-Coding RNAs during Synthesis of Cellulose in Eucalyptus grandis Hill

Cellulose, an essential structural component in the plant cell wall and a renewable biomass resource, plays a significant role in nature. Eucalyptus’s excellent timber tree species (including Eucalyptus grandis Hill) provide many raw materials for the paper and wood industries. The synthesis of cellulose is a very complex process involving multiple genes and regulated by various biological networks. However, research on regulating associated genes and non-coding RNAs during cellulose synthesis in E. grandis remains lacking. In this study, the wood anatomical characteristics and chemical indexes of E. grandis were analyzed by taking three different parts (diameter at breast height (DBH), middle and upper part of the trunk) from the main stem of E. grandis as raw materials. The role of non-coding RNAs (Long non-coding RNA, lncRNA; Micro RNA, miRNA; Circle RNA, circRNA) on regulating candidate genes was presented, and the network map of ceRNA (Competing endogenous RNA) regulation during wood cellulose biosynthesis of E. grandis was constructed. The transcriptome sequencing of nine samples obtained from the trunk of the immature xylem in E. grandis at DBH, middle and upper parts had a 95.81 G clean reading, 57,480 transcripts, 7365 lncRNAs, and 5180 circRNAs. Each sample had 172–306 known miRNAs and 1644–3508 new miRNAs. A total of 190 DE-lncRNAs (Differentially expressed long non-coding RNAs), 174 DE-miRNAs (Differentially expressed micro RNAs), and 270 DE-circRNAs (Differentially expressed circle RNAs) were obtained by comparing transcript expression levels. Four lncRNAs and nine miRNAs were screened out, and the ceRNA regulatory network was constructed. LncRNA1 and lncRNA4 regulated the genes responsible for cellulose synthesis in E. grandis, which were overexpressed in 84K (Populus Alba × Populus glandulosa) poplar. The cellulose and lignin content in lncRNA4-oe were significantly higher than wild type 84K poplar and lncRNA1-oe. The average plant height, middle and basal part of the stem diameter in lncRNA4-oe were significantly higher than the wild type. However, there was no significant difference between the growth of lncRNA1-oe and the wild type. Further studies are warranted to explore the molecular regulatory mechanism of cellulose biosynthesis in Eucalyptus species.

The Osmotin-Like Protein Gene PdOLP1 Is Involved in Secondary Cell Wall Biosynthesis during Wood Formation in Poplar.

Osmotin-like proteins (OLPs) mediate defenses against abiotic and biotic stresses and fungal pathogens in plants. However, no OLPs have been functionally elucidated in poplar. Here, we report an osmotin-like protein designated PdOLP1 from Populus deltoides (Marsh.). Expression analysis showed that PdOLP1 transcripts were mainly present in immature xylem and immature phloem during vascular tissue development in P. deltoides. We conducted phenotypic, anatomical, and molecular analyses of PdOLP1-overexpressing lines and the PdOLP1-downregulated hybrid poplar 84K (Populus alba × Populus glandulosa) (Hybrid poplar 84K PagOLP1, PagOLP2, PagOLP3 and PagOLP4 are highly homologous to PdOLP1, and are downregulated in PdOLP1-downregulated hybrid poplar 84K). The overexpression of PdOLP1 led to a reduction in the radial width and cell layer number in the xylem and phloem zones, in expression of genes involved in lignin biosynthesis, and in the fibers and vessels of xylem cell walls in the overexpressing lines. Additionally, the xylem vessels and fibers of PdOLP1-downregulated poplar exhibited increased secondary cell wall thickness. Elevated expression of secondary wall biosynthetic genes was accompanied by increases in lignin content, dry weight biomass, and carbon storage in PdOLP1-downregulated lines. A PdOLP1 coexpression network was constructed and showed that PdOLP1 was coexpressed with a large number of genes involved in secondary cell wall biosynthesis and wood development in poplar. Moreover, based on transcriptional activation assays, PtobZIP5 and PtobHLH7 activated the PdOLP1 promoter, whereas PtoBLH8 and PtoWRKY40 repressed it. A yeast one-hybrid (Y1H) assay confirmed interaction of PtoBLH8, PtoMYB3, and PtoWRKY40 with the PdOLP1 promoter in vivo. Together, our results suggest that PdOLP1 is a negative regulator of secondary wall biosynthesis and may be valuable for manipulating secondary cell wall deposition to improve carbon fixation efficiency in tree species.

Identification and functional evaluation of accessible chromatin associated with wood formation in Eucalyptus grandis.

Accessible chromatin changes dynamically during development and harbours functional regulatory regions which are poorly understood in the context of wood development. We explored the importance of accessible chromatin in Eucalyptus grandis in immature xylem generally, and MYB transcription factor-mediated transcriptional programmes specifically. We identified biologically reproducible DNase I Hypersensitive Sites (DHSs) and assessed their functional significance in immature xylem through their associations with gene expression, epigenomic data and DNA sequence conservation. We identified in vitro DNA binding sites for six secondary cell wall-associated Eucalyptus MYB (EgrMYB) transcription factors using DAP-seq, reconstructed protein-DNA networks of predicted targets based on binding sites within or outside DHSs and assessed biological enrichment of these networks with published datasets. 25319 identified immature xylem DHSs were associated with increased transcription and significantly enriched for various epigenetic signatures (H3K4me3, H3K27me3, RNA pol II), conserved noncoding sequences and depleted single nucleotide variants. Predicted networks built from EgrMYB binding sites located in accessible chromatin were significantly enriched for systems biology datasets relevant to wood formation, whereas those occurring in inaccessible chromatin were not. Our study demonstrates that DHSs in E.grandis immature xylem, most of which are intergenic, are of functional significance to gene regulation in this tissue.

Proline-rich protein gene PdPRP regulates secondary wall formation in poplar

Proline-rich protein (PRP) is a plant cell wall associated protein. Its distinct patterns of regulation and localization studied in a number of plants indicate that it may play important roles in growth and development. However, the mechanism of how these genes control secondary cell wall development in tree species is largely unknown. Here, we report that a Populus deltoides (Marsh.) proline-rich protein gene PdPRP was preferentially expressed in immature/mature phloem and immature xylem in P. deltoides. PdPRP overexpression increased poplar plant height and diameter as well as the radial width of the phloem and xylem regions, facilitated secondary wall deposition, and induced expression of genes related to microfibril angle (MFA) and secondary wall biosynthesis. Downregulation of PdPRP retarded poplar growth, decreased the radial width of the secondary phloem and secondary xylem regions, reduced secondary wall thickening in fibers and vessels, and decreased the expression of genes related to MFA and secondary wall biosynthesis. These results suggest that PdPRP might positively regulate secondary cell wall formation by promoting secondary wall thickening and expansion in poplar. PdPRP-overexpressing poplar had a lower MFA, indicating that PdPRP may be useful for improving wood stiffness and properties in plants. Together, our results demonstrate that PdPRP is a proline-rich protein associated with cell wall development, playing a critical role in regulating secondary cell wall formation in poplar.

The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N.benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N.benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N.benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.

The structure and development of interxylary and external phloem in Aquilaria sinensis

The structure and development of interxylary phloem (IP) and external phloem in Aquilaria sinensis were investigated using light and scanning electron microscopy. Complete IP strands were isolated, measuring 14 ± 4 mm in length and 417 ± 124 μm in width. The outer margin of IP was composed of two to three layers of fusiform parenchyma cells. The development of IP can be divided into five stages: 1) Locally IP starts its differentiation within a small segment of a broad cambial zone, at the cost of xylem differentiation. 2) Inward growth of IP advances, and fibres and sieve tubes differentiate. 3) IP is constricted by the encroachment of immature xylem cells between cambium and immature IP. 4) IP is isolated from the cambium and surrounded by immature, non-lignified xylem tissue. 5) IP is surrounded by lignified xylem tissue, and the fibres within IP become lignified.In all the phloem islands in a ten-year-old stem, sieve elements showed positive staining of callose with aniline blue. However, no staining of callose was observed in the external secondary phloem of agarwood trees collected from two different sites. No sieve tubes or sieve pores were detected by SEM observation of numerous serial cross and radial sections of the external phloem. We therefore conclude that sieve tubes are absent from the external phloem or extremely rare and that the transport of photosynthetic products in the stem of A. sinensis takes place in the interxylary phloem.

Isolation, expression and single nucleotide polymorphisms (SNPs) analysis of LACCASE gene (LkLAC8) from Japanese larch (Larix kaempferi)

Nucleotide diversity (π) and linkage disequilibrium (LD) analysis based on SNP marker could provide a sound basis for choosing an association analysis method. Japanese larch (Larix kaempferi) is an important timber coniferous tree species for pulping and papermaking, but its high lignin content has significantly restricted it application potential. In this study, the LACCASE gene, that plays an important regulatory role for lignin biosynthesis, was selected as research target. The full-length cDNA and genomic sequences of the encoding LkLAC8 gene were isolated from the LACCASE expressed sequence tags of the Japanese larch transcriptome database using the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA was determined to be 1940 bp, with an open reading frame (ORF, 1734 bp) that encoded a protein of 577 AA. This protein contains four highly specific Cu2+ binding sites and 11 glycosylation sites, thus belonging to the LACCASE family. The deduced protein sequence shared an 89% identity with the PtaLAC from Pinus taeda. A real-time PCR analysis showed that the LkLAC8 transcript was expressed predominantly in mature xylem, with moderate levels in the immature xylem, cambium and mature leaves, the lowest in the roots. Lastly, the genomic sequences of LkLAC8 in 40 individuals from six naturally distributed populations of Japanese larch were amplified, and a total of 201 SNPs (103 and 98 mutation types of transition and transversion, respectively) were detected; the frequency of the SNPs was 1/19 bp. Nucleotide diversity among the six populations ranged from 0.0034 to 0.0053, which suggested that there were no significant differences among the populations. The LD analysis showed that the LD level decayed rapidly within the increasing length of the LkLAC8 gene. These results implied that LD mapping and association analysis based on candidate gene may be feasible for the marker-assisted breeding of new germplasms with low lignin in Japanese larch.

A cDNA microarray analysis of the molecular control of poplar wood properties

Molecular biological research into wood development and formation has been the focus in recent years, but the pace of discovery of related genes and their functions in the control of wood properties has been slow. The microarray technique—with its advantages of high throughput capacity, sensitivity, and reliability over other tools developed for investigating genes expression patterns—is capable of rapidly assaying thousands of genes. In this study, a cDNA microarray prepared from two cDNA libraries of developing poplar xylem tissues was used to assay gene expression patterns in immature xylem tissues at different heights from the main stem of Populus deltoides (15 years old), which was confirmed to have distinct wood properties (microfibrillar angle, woody density) by X-ray. Two hundred seventy-four transcripts with differential expression profiles between the chips were screened out, and the individual clones were subjected to 5′ sequencing. Using bioinformatic analysis, we identified candidate genes that may influence poplar wood properties, many of which belong to various regulatory and signal transduction gene families, such as zinc finger protein transcription factor, DNA-binding transcription factor, ethylene response factors, and so on. The results suggest that these genes may regulate enzymes involved in wood formation. Further work will be performed to clone these genes and determine how they influence poplar wood properties.

MISSING AND DARK RINGS ASSOCIATED WITH DROUGHT IN PINUS HALEPENSIS

The responses of the vascular cambium and tracheid differentiation to extreme drought in Aleppo pine (Pinus halepensis Mill.) were investigated. The research focused on the drought year of 2005, in the primary study area at Maigmo (MAI) in southeastern Spain, with comparisons in Jarafuel (JAL) and Guardamar (GUA). The climate in this region is typically warm and dry with hot summers. Wood formation throughout the 2005 growing season was studied in transverse microtome sections and integrated with a retrospective dendrochronological analysis of crossdated increment cores collected in 2009. For most anatomical sections collected throughout the growing season at MAI, the vascular cambium appeared to be dormant as indicated by the low number of cells per radial file. Occasionally, immature xylem derivatives were observed during the growing season but without production of an annual ring. In increment cores collected at MAI, the 2005 position in the annual ring series contained either a narrow ring of both earlywood and latewood (47% of samples), a narrow ring of apparent latewood with no earlywood (13%), or a missing ring (50%). We introduce the term “dark ring” to refer to those annual rings of apparent latewood with no earlywood. For trees at JAL, the 2005 ring had below-average width and contained both earlywood and latewood. At GUA, the trees produced the widest 2005 ring of all three sites and mainly contained an intra-annual density fluctuation (IADF). The IADF was formed after cambial reactivation in the autumn. Although dark rings, IADFs, and especially missing rings complicate dendrochronological analysis, these anatomical features may provide an additional proxy record from which to infer climate variability and change in the past.

Small GTP-binding protein PdRanBP regulates vascular tissue development in poplar.

BackgroundPrevious research has demonstrated that ectopic expression of Ran-binding protein (RanBP) in Arabidopsis results in more axillary buds and reduced apical dominance compared to WT plants. However, the function of RanBP in poplar, which has very typical secondary growth, remains unclear. Here, the Populus deltoides (Marsh.) RanBP gene (PdRanBP) was isolated and functionally characterized by ectopic expression in a hybrid poplar (P. davidiana Dode × P. bolleana Lauche).ResultsPdRanBP was predominantly expressed in leaf buds and tissues undergoing secondary wall expansion, including immature xylem and immature phloem in the stem. Overexpression of PdRanBP in poplar increased the number of sylleptic branches and the proportion of cells in the G2 phase of the cell cycle, retarded plant growth, consistently decreased the size of the secondary xylem and secondary phloem zones, and reduced the expression levels of cell wall biosynthesis genes. The downregulation of PdRanBP facilitated secondary wall expansion and increased stem height, the sizes of the xylem and phloem zones, and the expression levels of cell wall biosynthesis genes.ConclusionsThese results suggest that PdRanBP influences the apical and radial growth of poplar trees and that PdRanBP may regulate cell division during cell cycle progression. Taken together, our results demonstrated that PdRanBP is a nuclear, vascular tissue development-associated protein in P. deltoides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0403-4) contains supplementary material, which is available to authorized users.

Transcriptome analysis of immature xylem in the Chinese fir at different developmental phases.

Background.Chinese fir [Cunninghamia lanceolata (Lamb.) Hook.] is one of the most important native tree species for timber production in southern China. An understanding of overall fast growing stage, stem growth stage and senescence stage cambium transcriptome variation is lacking. We used transcriptome sequencing to identify the repertoire of genes expressed during development of xylem tissue in Chinese fir, aiming to delineate the molecular mechanisms of wood formation. Results. We carried out transcriptome sequencing at three different cultivation ages (7Y, 15Y and 21Y) generating 68.71 million reads (13.88 Gbp). A total of 140,486 unigenes with a mean size of 568.64 base pairs (bp) were obtained via de novo assembly. Of these, 27,427 unigenes (19.52%) were further annotated by comparison to public protein databases. A total of 5,331 (3.79%) unigenes were mapped into 118 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Differentially expressed genes (DEG) analysis identified 3, 16 and 5,899 DEGs from the comparison of 7Y vs. 15Y, 7Y vs. 21Y and 15Y vs. 21Y, respectively, in the immature xylem tissues, including 2,638 significantly up-regulated and 3,280 significantly down-regulated genes. Besides, five NAC transcription factors, 190 MYB transcription factors, and 34 WRKY transcription factors were identified respectively from Chinese fir transcriptome.Conclusion. Our results revealed the active transcriptional pathways and identified the DEGs at different cultivation phases of Chinese fir wood formation. This transcriptome dataset will aid in understanding and carrying out future studies on the molecular basis of Chinese fir wood formation and contribute to future artificial production and applications.

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 four Eucalyptus genes potentially involved in cell wall biosynthesis and evolutionarily related to SHINE transcription factors

Recently, a new Arabidopsis thaliana master regulator of plant cell wall biosynthesis was characterized. It was named SHINE transcription factor (SHINE TF). This work searched for homologous genes in Eucalyptus grandis genome draft. RNAseq data, phylogeny analysis and qRT-PCR experiments were performed to complement SHINE gene analysis. By similarity searches using A. thaliana SHINE genes, four sequences were identified in Eucalyptus. Two of them contain all conserved motifs and characteristic features of this family, being assumed as true SHINE TFs and named EgrSHN1 and EgrSHN2. The other two sequences contain an incomplete 'mm' motif and were not considered true SHINE TFs, being further referred as Egr33m and Egr40m. Expression analysis revealed that EgrSHN1 is more expressed in flowers than in leaves and immature xylem, and both EgrSHN1 and EgrSHN2 are absent from adult xylem RNAseq libraries. This expression profile is similar to A. thaliana orthologues. On the other hand, Egr33m and Egr40m expression was detected in adult xylems. The phylogenetic studies indicate that both EgrSHNs were originated by gene duplication events which, together with gene loss, are hypothesized as com- mon events in SHINE evolution. In conclusion, it is pos- sible that the overexpression of SHINE genes in Eucalyptus xylem can generate information about wood formation processes, allowing an effective increase in forest planta- tion productivity.

Ultrastructural effects of infection caused by Tobacco rattle virus transmitted by Trichodorus primitivus in potato and tobacco tissues

Tobacco rattle virus (TRV), genus Tobravirus, is able to infect a broad range of plant species, possesses worldwide distribution and naturally infects a very large number of cultivated as well as ornamental plants. Members of the plant ectoparasitic nematode genera Trichodorus and Paratrichodorus transmit TRV in a semi-persistent way in a non-replicative process, where virus particles are transferred to the host plant through vector feeding on root epidermal and root hair cells. Our investigations concentrated on ultrastructural and anatomical effects of TRV strain PSG infection in situ following introduction into potato and tobacco tissues by Trichodorus primitivus. Our anatomical observations indicated necrotic changes of rhizodermis and cell wall deformations in primary cortex parenchyma and external phloem layers as a consequence of the interactions between Trichodorus–TRV–host plants. Ultrastructural analyses revealed TRV particles in rhizodermis, cortex and vascular roots of potato and tobacco tissues. These results indicate that TRV strain PSG was transferred into roots and also transported from cell to cell in all root tissues. Complete TRV PSG particles of two lengths were documented in companion cells, phloem fibre and parenchyma, as well as for the first time in immature and mature xylem tracheary elements and xylem parenchyma. Our findings suggested that TRV was systemically transported from the place of direct transfer by the vector to above-ground plant organs (especially leaves). The presence of TRV PSG particles in mesophyll and vascular leaf tissues confirmed our thesis. Moreover, we concluded that TRV systemic movement occurred not only in the phloem, but especially in the xylem, because virus particles were more frequently observed in xylem parenchyma and xylem tracheary elements.

Genetic dissection of transcript, metabolite, growth and wood property traits in an F2 pseudo-backcross pedigree of Eucalyptus grandis x E. urophylla

Background E. grandis is used extensively for the production of pulp and paper due to its rapid growth, good form and ease of vegetative propagation. E. urophylla exhibits tolerance to fungal diseases that limit the growth of E. grandis in tropical and subtropical regions. Interspecific hybrids of these two species are, therefore, commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions (e.g., South Africa, Congo and Brazil). These hybrids often exhibit superior growth and wood quality compared to the pure species, but the underlying genetic basis of the observed hybrid superiority remains unclear. Phenotypic variation observed in interspecific mapping populations has been used to identify QTLs in several genetic linkage studies in Eucalyptus. However, QTL intervals have generally been wide (20 to 30 cM) and may include several hundred genes. To bridge the gap between fine mapping and QTL validation studies, the expression levels of genes and metabolites in individuals from the segregating population can be treated as quantitative traits and used for eQTL and mQTL mapping, respectively. Co-localization of wood property, expression and metabolite QTLs will facilitate the identification of positional candidate genes and other components of regulatory networks underlying phenotypic variation. Methods To identify genetic factors controlling growth and wood property traits in Eucalyptus, an F2 pseudo-backcross mapping family derived from a cross between an F1 hybrid (GUSAP1, E. grandisxE. urophylla, Sappi Forest Research) and an E. urophylla parent (USAP1), consisting of 555 individuals, was used for genetic linkage map construction using microsatellite and DArT markers. Phenotypic trait assessment included physical measurements of tree diameter and wood density performed on 319 three-year-old individuals. Klason (acid-soluble & -insoluble) lignin and cell wall sugar content were determined for a selection of 100 backcross progeny and used for near-infrared analysis (NIRA) calibration. NIRA predictions for glucose, xylose, arabinose, cellulose and total lignin content, as well as pulp yield were made for all 315 individuals. Total lignin and S:G ratios were also separately measured for the 315 individuals. Immature xylem tissues, collected from 192 backcross progeny, were used for metabolite profiling (ORNL, Oak Ridge, TN) and Illumina mRNA-Seq (PE50, BGI Americas) quantification of transcript levels.

Cloning and expression analysis of an E-class MADS-box gene from Populus deltoides

An E-class MADS-box gene, PdMADS2, was isolated from Populus deltoides Bartr. ex Marsh male floral buds by RT-PCR. As shown by relative–quantitative real-time polymerase chain reaction analysis, the expression of PdMADS2 was high in apical and floral buds, intermediate in immature xylem and roots and low in mature leaves of adult male P. deltoides. In developing male floral buds, PdMADS2 expression was high and remained constant from July to September, increased significantly on February 6 and quickly fell to a very low level with the maturation of the flowers. In male inflorescences, PdMADS2 expression was abundant in the perianth cups and inflorescence peduncles; it was not detected in mature pollen. In female inflorescences, PdMADS2 was highly expressed in both ovaries and inflorescence peduncles. Our results suggest that PdMADS2 plays an important role in the development of inflorescence meristems and flower organs in poplar.

A MADS-box gene of Populus deltoides expressed during flower development and in vegetative organs

A MADS-box gene (PdPI) was isolated from a cDNA library constructed from male flower buds of Populus deltoides Bartr. ex Marsh. According to an analysis of genomic DNA structure and putative protein structure, and a phylogenetic study, PdPI is an ortholog of the Arabidopsis PI gene. Relative-quantitative real-time polymerase chain reaction analysis showed that PdPI has a broader expression pattern than PI in Arabidopsis. PdPI was strongly expressed in floral buds and roots and weakly expressed in immature xylem, leaves and apical buds of the male P. deltoides tree. In male inflorescences, PdPI expression was abundant in the perianth and anther, and weak in the peduncle and mature pollen. The large differences in PdPI expression at various phases of male floral bud development were closely related to the development of flower organs (perianth and stamen) and pollen. PdPI was also expressed in female inflorescences. Our results suggest that PdPI has multiple functions in the development of P. deltoides.

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.