Cooperation effects of GA3, IAA and uniconazole-P on tension wood formation and gravitropism in Fraxinus mandshurica seedlings

The cooperation effects of GA3, IAA and uniconazole-P were studied on the gravitropism and wood formation in Fraxinus mandshurica Rupr. var.japonica Maxim. Seedlings using traditional paraffin section technology. Our results are： （1） Gravitropism of stems was strongly inhibited only in B, whereas promoted significantly in D, E, F, H and I treatments; （2） Xylem formation was increased on both sides in H, I and J treatments and on the lower side in E and F which also show the synergistic effect; （3） On the radial direction, cell wall thickness was enhanced on the upper side, whereas decreased on the lower side in C, and was also significantly promoted on the upper in E, G, H, I and J treatments. On the tangential direction, cell wall thickness was increased on the lower side in E, H, I and J treatments respectively; （4） Gelatinous layer of wood fibers was observed on the upper side in all treatments. These results suggest that both GA3 and IAA affected negative gravitropism and wood formation significantly. However, appliedor unapplited-uniconazole-P does not affect G-layer formation, indicating GA does not play the key role on G-layer formation, and ratio of GA3/IAA or IAA may be more important in regulating G-layer formation.

Regulation of applied-hormones on tension wood formation in Fraxinus mandshurica Rupr. var.japonica Maxim. seedlings gravitational response

We investigated the role of GA3, uniconazole-P and IAA on tension wood formation, in particular the vessel features, in Fraxinu smandshurica seedlings. Ninety seedlings were used and treated with applications of GA3 and/or IAA to the apical bud of the stem using a micropipette. Applications of GA3 or GA3 plus IAA with uniconazole-P strongly increased cell number of tension wood in comparison to that of no-uniconazole-P-applied, indicated that GA3 is more efficient than IAA on xylem cell production. Wood quality was also regulated by relative concentration ratio of GA3 to lAA, because of the vessel elements differentiation, density and size were controlled by GA3 and/or IAA on the different levels. These results suggested that the relative concentration ratio of GA3 to IAA and interactions of them are essential in regulating both wood quality and wood quantity, and tension wood formation in this species.

PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar

The biosynthesis of cellulose,lignin,and hemicelluloses in plant secondary cell walls(SCWs)is regulated by a hierarchical transcriptional regulatory network.This network features orthologous transcription factors shared between poplar and Arabidopsis,highlighting a foundational similarity in their genetic regulation.However,knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited.In this study,we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog,AtMYB103.Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development,with fiber cells and vessels displaying thicker walls,and an increase in the levels of cellulose,lignin,and hemicelluloses in the wood.In contrast,plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes.RNA sequencing and reverse transcription–quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression,and chromatin immunoprecipitation along with yeast one-hybrid,and effector–reporter assays showed this regulation was direct.Further analysis revealed that PagSND1(SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1)directly regulates PagMYB128 but not cell wall metabolic genes,highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development,thereby creating a feedforward loop in SCW biosynthesis.

Molecular Dissection of Xylan Biosynthesis during Wood Formation in Poplar

Xylan, being the second most abundant polysaccharide in dicot wood, is considered to be one of the factors contributing to wood biomass recalcitrance for biofuel production. To better utilize wood as biofuel feedstock, it is crucial to functionally characterize all the genes involved in xylan biosynthesis during wood formation. In this report, we investigated roles of poplar families GT43 and GT8 glycosyltransferases in xylan biosynthesis during wood formation. There exist seven GT43 genes in the genome of poplar （Populus trichocarpa）, five of which, namely PtrGT43A, PtrGT43B, PtrGT43C, PtrGT43D, and PtrGT43E, were shown to be highly expressed in the developing wood and their encoded proteins were localized in the Golgi. Comprehensive genetic complementation coupled with chemical analyses demonstrated that overexpression of PtrGT43A/B/E but not PtrGT43C/D was able to rescue the xylan defects conferred by the Arabidopsis irx9 mutant, whereas overexpression of PtrGT43C/D but not PtrGT43A/B/E led to a complementation of the xylan defects in the Arabidopsis irx14 mutant. The essential roles of poplar GT43 members in xylan biosynthesis was further substantiated by RNAi down-regulation of GT43B in the hybrid poplar （Populus alba x tremula） leading to reductions in wall thickness and xylan content in wood, and an elevation in the abundance of the xylan reducing end sequence. Wood digestibility analysis revealed that cellulase digestion released more glucose from the wood of poplar GT43B RNAi lines than the control wood, indicating a decrease in wood biomass recalcitrance. Furthermore, RNAi down-regulation of another poplar wood-associated glycosyltransferase, PoGTSD, was shown to cause decreases in wall thickness and xylan content as well as in the abundance of the xylan reducing end sequence. Together, these findings demonstrate that the poplar GT43 members form two functionally non-redundant groups, namely PtrGT43A/B/E as functional orthologs of Arabidopsis IRX9 and PtrGT43C/D as functional orthologs ofArabidopsis IRX14, all of which are involved in the biosynthesis of xylan backbones, and that the poplar GT8D is essential for the biosynthesis of the xylan reducing end sequence.

Transcriptional Regulation of Secondary Growth and Wood Formation

Secondary growth and wood formation are products of the vascular cambium, a lateral meristem. Although the mechanisms have only recently begun to be uncovered, transcriptional regulation appears increasingly central to the regulation of secondary growth. The importance of transcriptional regulation is illustrated by the correlation of expression of specific classes of genes with related biological processes occurring at specific stages of secondary growth, including cell division, cell expansion, and cell differentiation. At the same time, transcription factors have been characterized that affect specific aspects of secondary growth, including regulation of the cambium and differentiation of cambial daughter cells. In the present review, we summarize evidence pointing to transcription as a major mechanism for regulation of secondary growth, and outline future approaches for comprehensively describing transcriptional networks underlying secondary growth.

Role of GA_3,GA_4 and Uniconazole-P in Controlling Gravitropism and Tension Wood Formation in Fraxinus mandshurica Rupr. var. japonica Maxim. Seedlings

GA3 and GA4 （gibberellins） play an important role in controlling gravitropism and tension wood formation in woody angiosperms. In order to improve our understanding of the role of GA3 and GA4 on xylem cell formation and the G-layer, we studied the effect of GA3 and GA4 and uniconazole-P, which is an inhibitor of GA biosynthesis, on tension wood formation by gravity in Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings. Forty seedlings were divided into two groups; one group was placed upright and the other tilted. Each group was further divided into four sub-groups subjected to the following treatments： 3.43 x 10-9 lunol acetone as control, 5.78 x 10-8 lunol gibberellic acid （GA3）, 6.21 x 10-8 lunol GA4, and 6.86 x 10-8 lunol uniconazole-P. During the experimental period, GAs-treated seedlings exhibited negative gravitropism, whereas application of uniconazole-P inhibited negative gravitropic stem bending. GA3 and GA4 promoted wood fibers that possessed a gelatinous layer on the upper side, whereas uniconazole-P inhibited wood formation but did not inhibit the differentiation of the gelatinous layer in wood fibers on the upper side. These results suggest that： （i） both the formation of gelatinous fibers and the quantity of xylem production are important for the negative gravitropism in horizontally-positioned seedlings; （ii） GA3 and GA4 affect wood production more than differentiation of the gelatinous layer in wood fibers; G-layer development may be regulated by other hormones via the indirect-role of GA3 and GA4 in horizontally-positioned F. mandshurica seedlings rather than the direct effect of GAs; and （iii） the mechanism for upward wood stem bending is different to the newly developed shoot bending in reaction to gravity in this species.

Ethylene Evolution Changes in Tilted Fraxinus mandshurica Rupr. var. japonica Maxim.Seedlings in Relation to Tension Wood Formation

The effects of ethylene on tension wood formation were studied in 3-year-old Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings in two separate experiments. In experiment 1, ethylene evolution of buds and stems was measured using gas chromatography after 0, 2, 4, 7, 14, and 21 d of treatment; in experiment 2, both aminoethoxyvinylglycine （AVG） and AgNO3 were applied to the horizontally-placed stems, and the cell numbers on sites of applications were measured after 40 d. Ethylene evolution from buds was found to be much greater in tilted seedlings than in upright ones. The cell numbers of wood fibers in shoots and 1-year-old stems were reduced in treatments with 12.5×10^-7μmol/L AVG, 12.5×10^-8μmol/L AVG, and 11.8×10^-8μmol/Lmol/L AgNO3; whereas the horizontal and vertical diameters were reduced by treatment of 12.5×10^-7μmol/L AVG. Ethylene evolutions of shoots and 1-year-old stems were inhibited greatly in comparison with the control by applying 12.5×10^-7μmol/L AVG. The formation of a gelatinous layer of wood fibers was affected by neither AVG nor AgNO3 application. These results suggest that ethylene regulates the quantity of wood production, but does not affect G-layer formation in F. mandshurica Rupr. var.japonica Maxim. seedlings.

Wood of trees:Cellular structure,molecular formation,and genetic engineering

Wood is an invaluable asset to human society due to its renewable nature,making it suitable for both sustainable energy production and material manufacturing.Additionally,wood derived from forest trees plays a crucial role in sequestering a significant portion of the carbon dioxide fixed during photosynthesis by terrestrial plants.Nevertheless,with the expansion of the global population and ongoing industrialization,forest coverage has been substantially decreased,resulting in significant challenges for wood production and supply.Wood production practices have changed away from natural forests toward plantation forests.Thus,understanding the underlying genetic mechanisms of wood formation is the foundation for developing high-quality,fast-growing plantation trees.Breeding ideal forest trees for wood production using genetic technologies has attracted the interest of many.Tremendous studies have been carried out in recent years on the molecular,genetic,and cell-biological mechanisms of wood formation,and considerable progress and findings have been achieved.These studies and findings indicate enormous possibilities and prospects for tree improvement.This review will outline and assess the cellular and molecular mechanisms of wood formation,as well as studies on genetically improving forest trees,and address future development prospects.

Comparative transcriptome analyses reveal candidate genes regulating wood quality in Japanese larch(Larix kaempferi)

We studied the molecular mechanism of the quality traits of wood formation in larch.We used the immature latewood cells of two Japanese larch(Larix kaempferi)clones with significant differences in density and in microfibrillar angle(MFA)as materials to analyze their gene expression profiles.A total of 1735 differentially expressed genes were detected in immature latewood cells of the two clones,among which,971 were up-regulated and 764 were down-regulated.Digital gene expression profiling analysis revealed that genes encoding transcription factor members NAC66 and R2R3-MYB4,microtubule-associated protein,actin-related protein,cell wall protein members,arabinogalactan protein,Fasciclin-like arabinogalactan protein and glycine-rich protein,and several cell-wall-synthesis genes affected wood density and MFA by regulating latewood formation at transcriptional level.Our study results represent a basis for selection of quality traits and genetic improvement of larch wood.

Different xylogenesis responses to atmospheric water demand contribute to species coexistence in a mixed pine–oak forest

Seasonal patterns of wood formation(xylogenesis)remain understudied in mixed pine-oak forests despite their contribution to tree coexistence through temporal niche complementarity.Xylogenesis was assessed in three pine species(Pinus cembroides,Pinus leiophylla,Pinus engelmannii)and one oak(Quercus grisea)coexisting in a semi-arid Mexican forest.The main xylogenesis phases(production of cambium cells,radial enlargement,cell-wall thickening and maturation)were related to climate data considering 5-15-day temporal windows.In pines,cambium activity maximized from mid-March to April as temperature and evaporation increased,whereas cell radial enlargement peaked from April to May and was constrained by high evaporation and low precipitation.Cell-wall thickening peaked from June to July and in August-September as maximum temperature and vapour pressure deficit(VPD)increased.Maturation of earlywood and latewood tracheids occurred in May-June and June-July,enhanced by high minimum temperatures and VPD in P.engelmannii and P.leiophylla.In oak,cambial onset started in March,constrained by high minimum temperatures,and vessel radial enlargement and radial increment maximized in April as temperatures and evaporation increased,whereas early wood vessels matured from May to June as VPD increased.Overall,15-day wet conditions enhanced cell radial enlargement in P.leiophylla and P.engelmannii,whereas early-summer high 15-day temperature and VPD drove cell-wall thickening in P.cembroides.Warm night conditions and high evaporation rates during spring and summer enhanced growth.An earlier growth peak in oak and a higher responsiveness to spring-summer water demand in pines contributed to their coexistence.

PagGRF12a interacts with PagGIF1b to regulate secondary xylem development through modulating PagXND1a expression in Populus alba×P.glandulosa

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.

Single-cell RNA sequencing reveals a high-resolution cell atlas of xylem in Populus

High-throughputsingle-cellRNAsequencing(sc RNA-seq) has advantages over traditional RNA-seq to explore spatiotemporal information on gene dynamic expressions in heterogenous tissues. We performed Drop-seq, a method for the dropwise sequestration of single cells for sequencing, on protoplasts from the differentiating xylem of Populus alba × Populus glandulosa. The sc RNA-seq profiled9,798 cells, which were grouped into 12 clusters.Through characterization of differentially expressed genes in each cluster and RNA in situ hybridizations,we identified vessel cells, fiber cells, ray parenchyma cells and xylem precursor cells. Diffusion pseudotime analyses revealed the differentiating trajectory of vessels, fiber cells and ray parenchyma cells and indicated a different differentiation process between vessels and fiber cells, and a similar differentiation process between fiber cells and ray parenchyma cells. We identified marker genes for each cell type(cluster) and key candidate regulators during developmental stages of xylem cell differentiation. Our study generates a high-resolution expression atlas of wood formation at the single cell level and provides valuable information on wood formation.

Transcriptome resources and genome-wide marker development for Japanese larch(Larix kaempferi)

While the differential responses of trees to changes in climatic and environmental conditions have been demonstrated as they age,the underlying mechanisms and age control of tree growth and development are complex and poorly understood particularly at a molecular level.In this paper,we present a transcriptome analysis of Larix kaempferi,a deciduous conifer that is widely-grown in the northern hemisphere and of significant ecological and economic value.Using high-throughput RNA sequencing,we obtained about 26 million reads from the stems of 1-,2-,5-,10-,25-and 50-year-old L.kaempferi trees.Combining these with the published Roche 454 sequencing reads and the expressed sequence tags(both mainly from Larix embryogenic cell cultures),we assembled 26670549 reads into 146786 transcripts,of which we annotated 79182 to support investigations of the molecular basis of tree aging and adaption,somatic embryogenesis and wood formation.Using these sequences we also identified many single-nucleotide polymorphisms,simple sequence repeats,and insertion and deletion markers to assist breeding and genetic diversity studies of Larix.