Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing second...Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar （Populus deltoides x P. trichocarpa）, which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofluorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubuleassociated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.展开更多
Growth-regulating factors(GRFs)are important regulators of plant development and growth,but their possible roles in xylem development in woody plants remain unclear.Here,we report that Populus alba×Papulus glandu...Growth-regulating factors(GRFs)are important regulators of plant development and growth,but their possible roles in xylem development in woody plants remain unclear.Here,we report that Populus alba×Papulus glandulosa PagGRF12a negatively regulates xylem development in poplar.PagGRF12a is expressed in vascular tissues.Compared to non-transgenic control plants,transgenic poplar plants overexpressing PagGRF12a exhibited reduced xylem width and plants with repressed expression of PagGRF12a exhibited increased xylem width.Xylem NAC domain 1(XND1)encodes a NAC domain transcription factor that regulates xylem development and transcriptional analyses revealed that PagXND1a is highly upregulated in PagGRF12a-overexpressing plants and downregulated in PagGRF12a-suppressed plants,indicating that PagGRF12a may regulate xylem development through PagXND1a.Transient transcriptional assays and chromatin immunoprecipitation-polymerase chain reaction assays confirmed that PagGRF12a directly upregulates PagXND1a.In addition,PagGRF12a interacts with the GRF-Interacting Factor(GIF)PagGIF1b,and this interaction enhances the effects of PagGRF12a on PagXND1a.Our results indicate that PagGRF12a inhibits xylem development by upregulating the expression of PagXND1a.展开更多
Lignin is a major component of plant cell walls and is essential for plant growth and development. Lignin biosynthesis is controlled by a hierarchical regulatory network involving multiple transcription factors. In th...Lignin is a major component of plant cell walls and is essential for plant growth and development. Lignin biosynthesis is controlled by a hierarchical regulatory network involving multiple transcription factors. In this study, we showed that the gene encoding an APETALA 2/ethylene-responsive element binding factor(AP2/ERF) transcription factor, PagERF81,from poplar 84 K(Populus alba × P. glandulosa) is highly expressed in expanding secondary xylem cells. Two independent homozygous Pagerf81 mutant lines created by gene editing, produced significantly more but smaller vessel cells and longer fiber cells with more lignin in cell walls, while PagERF81 overexpression lines had less lignin,compared to non-transgenic controls. Transcriptome and reverse transcription quantitative PCR data revealed that multiple lignin biosynthesis genes including Cinnamoyl CoA reductase 1(PagCCR1),Cinnamyl alcohol dehydrogenase 6(PagCAD6), and 4-Coumarate-CoA ligase-like 9(Pag4CLL9) were upregulated in Pagerf81 mutants, but down-regulated in PagERF81 overexpression lines. In addition, a transient transactivation assay revealed that PagERF81 repressed the transcription of these three genes.Furthermore, yeast one hybrid and electrophoretic mobility shift assays showed that PagERF81 directly bound to a GCC sequence in the PagCCR1 promoter. No known vessel or fiber cell differentiation related genes were differentially expressed, so the smaller vessel cells and longer fiber cells observed in the Pagerf81 lines might be caused by abnormal lignin deposition in the secondary cell walls. This study provides insight into the regulation of lignin biosynthesis, and a molecular tool to engineer wood with high lignin content, which would contribute to the lignin-related chemical industry and carbon sequestration.展开更多
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 endonuc...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.展开更多
Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and perox...Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce （Picea abies （L.） Karst.） laccases utilizing available genomic data. As many as 292 laccase encoding sequences （genes, gene fragments, and pseudogenes） were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five fun-length laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum （pH 3.8-4.2） for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5μM; however, the laccase has a lower catalytic efficiency （Vmax/Km） for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.展开更多
Genetic manipulation of cellulose biosynthesis in trees may provide novel insights into the growth and development of trees. To explore this possibility, the overexpression of an aspen secondary wall-associated cellul...Genetic manipulation of cellulose biosynthesis in trees may provide novel insights into the growth and development of trees. To explore this possibility, the overexpression of an aspen secondary wall-associated cellulose synthase （PtdCesAS） gene was attempted in transgenic aspen （Populus tremuloides L.） and unexpectedly resulted in silencing of the transgene as well as its endogenous counterparts. The main axis of the transgenic aspen plants quickly stopped growing, and weak branches adopted a weeping growth habit. Furthermore, transgenic plants initially developed smaller leaves and a less extensive root system. Secondary xylem （wood） of transgenic aspen plants contained as little as 10% cellulose normalized to dry weight compared to 41% cellulose typically found in normal aspen wood. This massive reduction in cellulose was accompanied by proportional increases in lignin （35%） and non-cellulosic polysaccharides （55%） compared to the 22% lignin and 36% non-cellulosic polysaccharides in control plants. The transgenic stems pro- duced typical collapsed or ＇irregular＇ xylem vessels that had altered secondary wall morphology and contained greatly reduced amounts of crystalline cellulose. These results demonstrate the fundamental role of secondary wall cellulose within the secondary xylem in maintaining the strength and structural integrity required to establish the vertical growth habit in trees.展开更多
基金Supported by the Natural Sciences and Engineering Research Council of Canada, Discovery Grant to LS
文摘Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar （Populus deltoides x P. trichocarpa）, which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofluorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubuleassociated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.
基金This work was supported by the National Natural ScienceFoundation of China(31570676)the National Key Programon Transgenic Research of China(2018ZX08020-002)+3 种基金the Basic Research Fund of Research Institute of Forest,ChineseAcademy of Forestry(RIF-2014-08)the National Key Re-search and Development Program of China(2016YFD0600103)the National Key Program on Transgenic Re-search of China(2018ZX08020-002)the Ten-thousandTalents Program of China for Meng-Zhu Lu.
文摘Growth-regulating factors(GRFs)are important regulators of plant development and growth,but their possible roles in xylem development in woody plants remain unclear.Here,we report that Populus alba×Papulus glandulosa PagGRF12a negatively regulates xylem development in poplar.PagGRF12a is expressed in vascular tissues.Compared to non-transgenic control plants,transgenic poplar plants overexpressing PagGRF12a exhibited reduced xylem width and plants with repressed expression of PagGRF12a exhibited increased xylem width.Xylem NAC domain 1(XND1)encodes a NAC domain transcription factor that regulates xylem development and transcriptional analyses revealed that PagXND1a is highly upregulated in PagGRF12a-overexpressing plants and downregulated in PagGRF12a-suppressed plants,indicating that PagGRF12a may regulate xylem development through PagXND1a.Transient transcriptional assays and chromatin immunoprecipitation-polymerase chain reaction assays confirmed that PagGRF12a directly upregulates PagXND1a.In addition,PagGRF12a interacts with the GRF-Interacting Factor(GIF)PagGIF1b,and this interaction enhances the effects of PagGRF12a on PagXND1a.Our results indicate that PagGRF12a inhibits xylem development by upregulating the expression of PagXND1a.
基金supported by the National Key Scientific Research Project of China (2021YFD2200205)the Ten-Thousand Talents Program of China to Meng-Zhu Lu。
文摘Lignin is a major component of plant cell walls and is essential for plant growth and development. Lignin biosynthesis is controlled by a hierarchical regulatory network involving multiple transcription factors. In this study, we showed that the gene encoding an APETALA 2/ethylene-responsive element binding factor(AP2/ERF) transcription factor, PagERF81,from poplar 84 K(Populus alba × P. glandulosa) is highly expressed in expanding secondary xylem cells. Two independent homozygous Pagerf81 mutant lines created by gene editing, produced significantly more but smaller vessel cells and longer fiber cells with more lignin in cell walls, while PagERF81 overexpression lines had less lignin,compared to non-transgenic controls. Transcriptome and reverse transcription quantitative PCR data revealed that multiple lignin biosynthesis genes including Cinnamoyl CoA reductase 1(PagCCR1),Cinnamyl alcohol dehydrogenase 6(PagCAD6), and 4-Coumarate-CoA ligase-like 9(Pag4CLL9) were upregulated in Pagerf81 mutants, but down-regulated in PagERF81 overexpression lines. In addition, a transient transactivation assay revealed that PagERF81 repressed the transcription of these three genes.Furthermore, yeast one hybrid and electrophoretic mobility shift assays showed that PagERF81 directly bound to a GCC sequence in the PagCCR1 promoter. No known vessel or fiber cell differentiation related genes were differentially expressed, so the smaller vessel cells and longer fiber cells observed in the Pagerf81 lines might be caused by abnormal lignin deposition in the secondary cell walls. This study provides insight into the regulation of lignin biosynthesis, and a molecular tool to engineer wood with high lignin content, which would contribute to the lignin-related chemical industry and carbon sequestration.
基金supported by the National Basic Research Program of China (2012CB114500)the National Natural Science Foundation of China (31070156)
文摘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.
基金supported by University of Helsinki Research Funds (to A.K.)Academy of Finland (grant 251390 to A.K.)Societas pro Fauna et Flora Fennica (to H.A.M.)
文摘Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce （Picea abies （L.） Karst.） laccases utilizing available genomic data. As many as 292 laccase encoding sequences （genes, gene fragments, and pseudogenes） were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five fun-length laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum （pH 3.8-4.2） for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5μM; however, the laccase has a lower catalytic efficiency （Vmax/Km） for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.
文摘Genetic manipulation of cellulose biosynthesis in trees may provide novel insights into the growth and development of trees. To explore this possibility, the overexpression of an aspen secondary wall-associated cellulose synthase （PtdCesAS） gene was attempted in transgenic aspen （Populus tremuloides L.） and unexpectedly resulted in silencing of the transgene as well as its endogenous counterparts. The main axis of the transgenic aspen plants quickly stopped growing, and weak branches adopted a weeping growth habit. Furthermore, transgenic plants initially developed smaller leaves and a less extensive root system. Secondary xylem （wood） of transgenic aspen plants contained as little as 10% cellulose normalized to dry weight compared to 41% cellulose typically found in normal aspen wood. This massive reduction in cellulose was accompanied by proportional increases in lignin （35%） and non-cellulosic polysaccharides （55%） compared to the 22% lignin and 36% non-cellulosic polysaccharides in control plants. The transgenic stems pro- duced typical collapsed or ＇irregular＇ xylem vessels that had altered secondary wall morphology and contained greatly reduced amounts of crystalline cellulose. These results demonstrate the fundamental role of secondary wall cellulose within the secondary xylem in maintaining the strength and structural integrity required to establish the vertical growth habit in trees.
