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.

C2H2 zinc finger protein PagIDD15A regulates secondary wall thickening and lignin biosynthesis in poplar.

Wood production is largely determined by the activity of cambial cell proliferation, and the secondary cell wall (SCW) thickening of xylem cells determines the wood property. In this study, we identified an INDETERMINATE DOMAIN (IDD) type C2H2 zinc finger transcription factor PagIDD15A as a regulator of wood formation in Populus alba × Populus glandulosa. Downregulation of PagIDD15A expression by RNA interference (RNAi) inhibited xylem development and xylem cell secondary wall thickening. RNA-seq analysis showed that PagPAL1, PagCCR2 and PagCCoAOMT1 were downregulated in the differentiating xylem of the PagIDD15A-RNAi transgenic plants, showing that PagIDD15A may regulate SCW biosynthesis through inhibiting lignin biosynthesis. The downregulation of PagVND6-B2, PagMYB10 and PagMYC4 and upregulation of PagWRKY12 in the differentiating xylem of RNAi transgenic plants suggest that PagIDD15A may also regulate these transcription factor (TF) genes to affect SCW thickening. RT-qPCR analysis in the phloem-cambium of RNAi transgenic demonstrates that PagIDD15A may regulate the expression of the genes associated with cell proliferation, including, PagSHR (SHORTROOT), PagSCR (SCARECROW), PagCYCD3;1 (CYCLIN D3;1) and PagSMR4 (SIAMESE-RELATED4), to affect the cambial activity. This study provides the knowledge of the IDD-type C2H2 zinc finger protein in regulating wood formation.

PagMYB180 regulates adventitious rooting via a ROS/PCD-dependent pathway in poplar.

The formation of adventitious roots (AR) is an essential step in the vegetative propagation of economically woody species. Reactive oxygen species (ROS) function as signaling molecules in regulating root growth and development. Here, we identified an R2R3-MYB transcription factor PagMYB180 as a regulator of AR formation in hybrid poplar (Populus alba × Populus glandulosa). PagMYB180 was specifically expressed in the vascular tissues of poplar roots, stems and leaves, and its protein was localized in the nucleus and acted as a transcriptional repressor. Both dominant repression and overexpression of PagMYB180 resulted in a significant reduction of AR quantity, a substantial increase of AR length, and an elevation of both the quantity and length of lateral roots (LR) compared to the wild type (WT) plants. Furthermore, PagMYB180 regulates programmed cell death (PCD) in root cortex cells, which is associated with elevated levels of ROS. Transcriptome and reverse transcription-quantitative PCR (RT-qPCR) analyses revealed that a series of differentially expressed genes are related to ROS, PCD and ethylene synthesis. Taken together, these results suggest that PagMYB180 may regulate AR development via a ROS/PCD-dependent pathway in poplar.

Genome-Wide Identification and Expression Profiling of Velvet Complex Transcription Factors in Populus alba × Populus glandulosa.

The discovery of new genes with novel functions is a major driver of adaptive evolutionary innovation in plants. Especially in woody plants, due to genome expansion, new genes evolve to regulate the processes of growth and development. In this study, we characterized the unique VeA transcription factor family in Populus alba × Populus glandulosa, which is associated with secondary metabolism. Twenty VeA genes were characterized systematically on their phylogeny, genomic distribution, gene structure and conserved motif, promoter binding site, and expression profiling. Furthermore, through ChIP-qPCR, Y1H, and effector-reporter assays, it was demonstrated that PagMYB128 directly regulated PagVeA3 to influence the biosynthesis of secondary metabolites. These results provide a basis for further elucidating the function of VeAs gene in poplar and its genetic regulation mechanism.

Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus.

Cell type annotation and lineage construction are two of the most critical tasks conducted in the analyses of single-cell RNA sequencing (scRNA-seq). Four recent scRNA-seq studies of differentiating xylem propose four models on differentiating xylem development in Populus. The differences are mostly caused by the use of different strategies for cell type annotation and subsequent lineage interpretation. Here, we emphasize the necessity of using in situ transcriptomes and anatomical information to construct the most plausible xylem development model.

Transcription factor PagMYB31 positively regulates cambium activity and negatively regulates xylem development in poplar.

Wood formation involves consecutive developmental steps, including cell division of vascular cambium, xylem cell expansion, secondary cell wall (SCW) deposition, and programmed cell death. In this study, we identified PagMYB31 as a coordinator regulating these processes in Populus alba × Populus glandulosa and built a PagMYB31-mediated transcriptional regulatory network. PagMYB31 mutation caused fewer layers of cambial cells, larger fusiform initials, ray initials, vessels, fiber and ray cells, and enhanced xylem cell SCW thickening, showing that PagMYB31 positively regulates cambial cell proliferation and negatively regulates xylem cell expansion and SCW biosynthesis. PagMYB31 repressed xylem cell expansion and SCW thickening through directly inhibiting wall-modifying enzyme genes and the transcription factor genes that activate the whole SCW biosynthetic program, respectively. In cambium, PagMYB31 could promote cambial activity through TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/PHLOEM INTERCALATED WITH XYLEM (PXY) signaling by directly regulating CLAVATA3/ESR-RELATED (CLE) genes, and it could also directly activate WUSCHEL HOMEOBOX RELATED4 (PagWOX4), forming a feedforward regulation. We also observed that PagMYB31 could either promote cell proliferation through the MYB31-MYB72-WOX4 module or inhibit cambial activity through the MYB31-MYB72-VASCULAR CAMBIUM-RELATED MADS2 (VCM2)/PIN-FORMED5 (PIN5) modules, suggesting its role in maintaining the homeostasis of vascular cambium. PagMYB31 could be a potential target to manipulate different developmental stages of wood formation.

Genome-wide identification and expression profiling of B3 transcription factor genes in Populus alba × Populus glandulosa.

B3-domain containing transcription factors (TFs) are well known to play important roles in various developmental processes, including embryogenesis, seed germination, etc. Characterizations and functional studies of the B3 TF superfamily in poplar are still limited, especially on their roles in wood formation. In this study, we conducted comprehensive bioinformatics and expression analysis of B3 TF genes in Populus alba × Populus glandulosa. A total of 160 B3 TF genes were identified in the genome of this hybrid poplar, and their chromosomal locations, syntenic relationships, gene structures, and promoter cis-acting elements were analyzed. Through domain structure and phylogenetic relationship analyses, these proteins were classified into four families LAV, RAV, ARF, and REM. Domain and conservation analyses revealed different gene numbers and different DNA-binding domains among families. Syntenic relationship analysis suggested that approximately 87% of the genes resulted from genome duplication (segmental or tandem), contributing to the expansion of the B3 family in P. alba × P. glandulosa. Phylogeny in seven species revealed the evolutionary relationship of B3 TF genes across different species. B3 domains among the eighteen proteins that were highly expressed in differentiating xylem had a high synteny, suggesting a common ancestor for these seven species. We performed co-expression analysis on the representative genes in two different ages of poplar, followed by pathways analysis. Among those genes co-expressed with four B3 genes, 14 were involved in lignin synthases and secondary cell walls biosynthesis, including PagCOMT2, PagCAD1, PagCCR2, PagCAD1, PagCCoAOMT1, PagSND2, and PagNST1. Our results provide valuable information for the B3 TF family in poplar and show the potential of B3 TF genes in engineering to improve wood properties.

Alternative splicing: transcriptional regulatory network in agroforestry.

Alternative splicing (AS) in plants plays a key role in regulating the expression of numerous transcripts from a single gene in a regulatory pathway. Variable concentrations of growth regulatory hormones and external stimuli trigger alternative splicing to switch among different growth stages and adapt to environmental stresses. In the AS phenomenon, a spliceosome causes differential transcriptional modifications in messenger RNA (mRNAs), resulting in partial or complete retention of one or more introns as compared to fully spliced mRNA. Differentially expressed proteins translated from intron-retaining messenger RNA (mRNAir) perform vital functions in the feedback mechanism. At the post-transcriptional level, AS causes the remodeling of transcription factors (TFs) by the addition or deletion of binding domains to activate and/or repress transcription. In this study, we have summarized the specific role of AS in the regulation of gene expression through repression and activation of the transcriptional regulatory network under external stimuli and switch among developmental stages.

Overexpression of a gibberellin 20-oxidase gene in poplar xylem led to an increase in the size of nanocellulose fibrils and improved paper properties.

Cellulose, the major component of secondary cell walls, is the most abundant renewable long-chain polymer on earth. Nanocellulose has become a prominent nano-reinforcement agent for polymer matrices in various industries. We report the generation of transgenic hybrid poplar overexpressing the Arabidopsis gibberellin 20-oxidase1 gene driven by a xylem-specific promoter to increase gibberellin (GA) biosynthesis in wood. X-ray diffraction (XRD) and sum frequency generation spectroscopic (SFG) analyses showed that cellulose in transgenic trees was less crystalline, but the crystal size was larger. The nanocellulose fibrils prepared from transgenic wood had an increased size compared to those from wild type. When such fibrils were used as a reinforcing agent in sheet paper preparation, the mechanical strength of the paper was significantly enhanced. Engineering the GA pathway can therefore affect nanocellulose properties, providing a new strategy for expanding nanocellulose applications.

Ubiquitinated DA1 negatively regulates vascular cambium activity through modulating the stability of WOX4 in Populus.

Activity of the vascular cambium gives rise to secondary xylem for wood formation in trees. The transcription factor WUSCHEL-related HOMEOBOX4 (WOX4) is a central regulator downstream of the hormone and peptide signaling pathways that maintain cambial activity. However, the genetic regulatory network underlying WOX4-mediated wood formation at the post-transcriptional level remains to be elucidated. In this study, we identified the ubiquitin receptor PagDA1 in hybrid poplar (Populus alba × Populus glandulosa clone 84K) as a negative regulator of wood formation, which restricts cambial activity during secondary growth. Overexpression of PagDA1 in poplar resulted in a relatively reduced xylem due to decreased cambial cell division. By contrast, mutation of PagDA1 by CRISPR/Cas9 resulted in an increased cambial cell activity and promoted xylem formation. Genetic analysis demonstrated that PagDA1 functions antagonistically in a common pathway as PagWOX4 to regulate cambial activity. We propose that PagDA1 physically associates with PagWOX4 and modulates the degradation of PagWOX4 by the 26S proteasome. Moreover, genetic analysis revealed that PagDA1 exerts its negative effect on cambial development by modulating the stability of PagWOX4 in a ubiquitin-dependent manner mediated by the E3 ubiquitin ligase PagDA2. In sum, we have identified a cambial regulatory protein complex, PagDA1-PagWOX4, as a potential target for wood biomass improvement.

The flying spider-monkey tree fern genome provides insights into fern evolution and arborescence.

To date, little is known about the evolution of fern genomes, with only two small genomes published from the heterosporous Salviniales. Here we assembled the genome of Alsophila spinulosa, known as the flying spider-monkey tree fern, onto 69 pseudochromosomes. The remarkable preservation of synteny, despite resulting from an ancient whole-genome duplication over 100 million years ago, is unprecedented in plants and probably speaks to the uniqueness of tree ferns. Our detailed investigations into stem anatomy and lignin biosynthesis shed new light on the evolution of stem formation in tree ferns. We identified a phenolic compound, alsophilin, that is abundant in xylem, and we provided the molecular basis for its biosynthesis. Finally, analysis of demographic history revealed two genetic bottlenecks, resulting in rapid demographic declines of A. spinulosa. The A. spinulosa genome fills a crucial gap in the plant genomic landscape and helps elucidate many unique aspects of tree fern biology.

Determination of Community Structure and Diversity of Seed-Vectored Endophytic Fungi in Alpinia zerumbet.

Endophytic fungi act as seed endosymbiont, thereby playing a very crucial role in the growth and development of seeds. Seed-vectored endophytic fungi establish an everlasting association with seeds and travel from generation to generation. To explore the composition and diversity of endophytic fungi in Alpinia zerumbet seeds, high-throughput Illumina MiSeq sequencing was employed for the following stages: fruit formation period (YSJ1), young fruit period (YSJ2), early mature period (YSJ3), middle mature period (YSJ4), and late mature period (YSJ5). A total of 906,694 sequence reads and 745 operational taxonomic units (OTUs) were obtained and further classified into 8 phyla, 30 classes, 73 orders, 163 families, 302 genera, and 449 species. The highest endophytic fungal diversity was observed at YSJ5. The genera with the highest abundance were Cladosporium, Kodamaea, Hannaella, Mycothermus, Gibberella, Sarocladium, and Neopestalotiopsis. Functional Guild (FUNGuild) analysis revealed that endophytic fungi were undefined saprotroph, plant pathogens, animal pathogen-endophyte-lichen parasite-plant pathogen-wood saprotroph, and soil saprotrophs. Alternaria, Fusarium, Cladosporium, and Sarocladium, which are potential probiotics and can be used as biocontrol agents, were also abundant. This study is part of the Sustainable Development Goals of United Nations Organization (UNO) to "Establish Good Health and Well-Being."

Corrigendum: BEL1-like Homeodomain Protein BLH6a Is a Negative Regulator of CAld5H2 in Sinapyl Alcohol Monolignol Biosynthesis in Poplar.

[This corrects the article DOI: 10.3389/fpls.2021.695223.].

Comprehensive Mechanism of Gene Silencing and Its Role in Plant Growth and Development.

Gene silencing is a negative feedback mechanism that regulates gene expression to define cell fate and also regulates metabolism and gene expression throughout the life of an organism. In plants, gene silencing occurs via transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). TGS obscures transcription via the methylation of 5' untranslated region (5'UTR), whereas PTGS causes the methylation of a coding region to result in transcript degradation. In this review, we summarized the history and molecular mechanisms of gene silencing and underlined its specific role in plant growth and crop production.

Comprehensive Analysis of Bacterial Community Structure and Diversity in Sichuan Dark Tea (Camellia sinensis).

Bacteria and fungi present during pile-fermentation of Sichuan dark tea play a key role in the development of its aesthetic properties, such as color, taste, and fragrance. In our previous study, high-throughput sequencing of dark tea during fermentation revealed Aspergillus was abundant, but scarce knowledge is available about bacterial communities during pile-fermentation. In this study, we rigorously explored bacterial diversity in Sichuan dark tea at each specific stage of piling. Analysis of cluster data revealed 2,948 operational taxonomic units, which were divided into 42 phyla, 98 classes, 247 orders, 461 families, 1,052 genera, and 1,888 species. Certain members of the family Enterobacteriaceae were dominant at early stages of fermentation YC, W1, and W2; Pseudomonas at middle stage W3; and the highest bacterial diversity was observed at the final quality-determining stage W4. Noticeably, probiotics, such as Bacillus, Lactobacillus, Bifidobacterium, and Saccharopolyspora were also significantly higher at the quality-determining stage W4. Our findings might help in precise bacterial inoculation for probiotic food production by increasing the health benefits of Sichuan dark tea. This research also falls under the umbrella of the "Establish Good Health and Well-Being" Sustainable Development Goals of the United Nations Organization.

Vascular Cambium: The Source of Wood Formation.

Wood is the most abundant biomass produced by land plants and is mainly used for timber, pulping, and paper making. Wood (secondary xylem) is derived from vascular cambium, and its formation encompasses a series of developmental processes. Extensive studies in Arabidopsis and trees demonstrate that the initiation of vascular stem cells and the proliferation and differentiation of the cambial derivative cells require a coordination of multiple signals, including hormones and peptides. In this mini review, we described the recent discoveries on the regulation of the three developmental processes by several signals, such as auxin, cytokinins, brassinosteroids, gibberellins, ethylene, TDIF peptide, and their cross talk in Arabidopsis and Populus. There exists a similar but more complex regulatory network orchestrating vascular cambium development in Populus than that in Arabidopsis. We end up with a look at the future research prospects of vascular cambium in perennial woody plants, including interfascicular cambium development and vascular stem cell regulation.

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

High-throughput single-cell RNA sequencing (scRNA-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 scRNA-seq profiled 9,798 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.