Huaitong Wu

Quercus texana seeds are recalcitrant and thus highly sensitive to desiccation, which makes storage difficult. For practical seed handling, it is important to define their safe water content and to understand how water is distributed during dehydration. The present study utilized magnetic resonance imaging (MRI) and nuclear magnetic resonance (NMR) technologies to investigate the migration and phases of water, respectively, revealing the underlying reasons for the recalcitrance of Q. texana seeds. The water content of fresh Q. texana seeds was found to be 39.6% and the germination percentage was 93.3%. As the water content decreased, the germination percentage decreased continuously, reaching 0% at a water content of 13.0%. At 20.0% water content, the germination percentage was 71.7%. MRI showed that water was primarily stored in the embryo axis and cotyledon center in fresh Q. texana seeds. Water loss occurs in the following order during seed dehydration: embryo axis, cotyledon center, cotyledon periphery, and cotyledon end. However, water in the radicle region persisted until seed water content decreased to 15.0%, at which point no signal was detected. The NMR T₂ relaxation spectrum indicated the presence of bound water (T₂₁ = 0.01–5.44 ms) and free water (T₂₂ = 7.19–1401.93 ms) in the seeds. During the dehydration process, most of the water was lost as free water, and the T₂₂ shifted to longer times. Concurrently, the bound water shifted to shorter T₂₁ times. Overall, for practical purposes, seed water should be maintained at or above 20.0%. MRI further showed that water loss from the radicle plays a decisive role in the decline of seed germination, and that protecting the region of radicle and the cupule scar can effectively retard water loss. Furthermore, the bound-water content is positively correlated with seed germination.

Invertase (INV) irreversibly converts sucrose to glucose and fructose during processes such as differentiation and organ development in plants, especially during the development of trichomes. Systematic identification and analysis of INVs in Salicaceae remain limited. Here, INV genes in Populus deltoides and Salix suchowensis were investigated, and their chromosomal localization, collinearity, gene structures, cis-regulatory elements, and phylogenetic relationships were comprehensively analyzed. Twenty and seventeen INVs were found, respectively, in P. deltoides and S. suchowensis, most of which were derived from a common ancestor and exhibited similar chromosomal distribution and high collinearity. Orthologs between the two species showed conservation of gene structures and promoter regulatory elements. Multi-species phylogenetic analysis identified an evolutionary clade associated with seed fiber development in P. deltoides and S. suchowensis. Further evaluation of INV expression in female catkins at various stages of seed fiber formation verified the predominance of PdeVINV1, PdeVINV2, PdeVINV3, and PdeVINV4 in P. deltoides, as well as SsuVINV1 and SsuVINV2 in S. suchowensis, during critical phases of catkin fiber differentiation. These genes are likely to have significant regulatory roles in the initiation and development of catkin fiber cells. These findings provide a reference for future functional studies of INVs.

Stamens are vital reproductive organs in angiosperms, essential for plant growth, reproduction, and development. The genetic regulation and molecular mechanisms underlying stamen development are, however, complex and varied among different plant species. MSL-lncRNAs, a gene specific to the Y chromosome of Populus deltoides, is predominantly expressed in male flower buds. Heterologous expression of MSL-lncRNAs in Arabidopsis thaliana resulted in an increase in both stamen and anther count, without affecting pistil development or seed set. To reveal the molecular regulatory network influenced by MSL-lncRNAs on stamen development, we conducted transcriptome sequencing of flowers from both wild-type and MSL-lncRNAs-overexpressing Arabidopsis. A total of 678 differentially expressed genes were identified between wild-type and transgenic Arabidopsis. Among these, 20 were classified as transcription factors, suggesting a role for these regulatory proteins in stamen development. GO enrichment analysis revealed that the differentially expressed genes were significantly associated with processes such as pollen formation, polysaccharide catabolic processes, and secondary metabolism. KEGG pathway analysis indicated that MSL-lncRNAs might promote stamen development by upregulating genes involved in the phenylpropanoid biosynthesis pathway. The top three upregulated genes, all featuring the DUF295 domain, were found to harbor an F-box motif at their N-termini, which is implicated in stamen development. Additionally, in transgenic Arabidopsis flowers, genes implicated in tapetum formation and anther development were also observed to be upregulated, implying a potential role for MSL-lncRNAs in modulating pollen development through the positive regulation of these genes. The findings from this study establish a theoretical framework for elucidating the genetic control exerted by MSL-lncRNAs over stamen and pollen development.

Expansins are a group of cell wall enzyme proteins that help to loosen cell walls by breaking hydrogen bonds between cellulose microfibrils and hemicellulose. Expansins are essential plant proteins that are involved in several key processes, including seed germination, the growth of pollen tubes and root hairs, fruit ripening and abscission processes. Currently, there is a lack of knowledge concerning the role of expansins in woody plants. In this study, we analyzed expansin genes using Populus genome as the study target. Thirty-six members of the expansin gene family were identified in Populus that were divided into four subfamilies (EXPA, EXPB, EXLA and EXLB). We analyzed the molecular structure, chromosome localization, evolutionary relationships and tissue specificity of these genes and investigated expression changes in responses to phytohormone and abiotic stresses of the expansin genes of Populus tremula L. (PtEXs). Molecular structure analysis revealed that each PtEX protein had several conserved motifs and all of the PtEXs genes had multiple exons. Chromosome structure analysis showed that the expansin gene family is distributed on 14 chromosomes. The PtEXs gene family expansion patterns showed segmental duplication. Transcriptome data of Populus revealed that 36 PtEXs genes were differently expressed in different tissues. Cis-element analysis showed that the PtEXs were closely associated with plant development and responses to phytohormone and abiotic stress. Quantitative real-time PCR showed that abscisic acid (ABA) and low-temperature treatment affected the expression of some PtEXs genes, suggesting that these genes are involved in responses to phytohormone and abiotic stress. This study provides a further understanding of the expansin gene family in Populus and forms a basis for future functional research studies.

Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs) containing both AGP-like glycated domains and fasciclin (FAS) domains, which are involved in plant growth and development and synthesis of the cell wall. However, these proteins have not been identified or analyzed in willow, Salix, the sister genus of Populus. In this study, we performed a whole genome study of the FLA gene family of Salix suchowensis and compared it with the FLA gene family of Populus deltoides. The results showed the presence of 40 and 46 FLA genes in P. deltoides and S. suchowensis, distributed on 17 and 16 chromosomes, respectively. Four pairs of tandem repeat genes were found in willow, while poplar had no tandem repeat genes. Twelve and thirteen pairs of duplicated gene fragments were identified in poplar and willow, respectively. The multispecies phylogenetic tree showed that the FLA gene family could be divided into four groups (I–IV), with Group 1 showing significant expansion in woody plants. A gene expression analysis showed that PdeFLA19/27 in Group I of poplar was highly expressed, specifically during the secondary growth period of the stem and the rapid elongation of seed hairs. In the Group I genes of S. suchowensis, SsuFLA25/26/28 was also highly expressed during the secondary growth period, whereas increased expression of SsuFLA35 was associated with seed hair tissue. These results provide important clues about the differences in the FLA gene family during the evolution of herbs and woody plants, and suggest that the FLA gene family may play an essential role in regulating the secondary growth of woody plants. It also provides a reference for further studies on the regulation of secondary growth and seed hair development by FLA genes in poplar and willow.

Differentiation of plant epidermal cells is a keen research topic in plant biology. Our study on Populus deltoides revealed that epidermal cells of the female flower placenta protruded to form catkin fibers immediately after pollination. We discovered that PdeMIXTA04 was explicitly expressed in placenta of female poplar flowers. Heterologous expression of PdeMIXTA04 in Arabidopsis demonstrated that it significantly promoted the leaf epidermal cells to differentiate into trichomes. Compared with the wild type, significant increases in trichome density and trichome branches were observed on leaves of all 35S:PdeMIXTA04 transgenic lines. Furthermore, transformation of PdeMIXTA04 in the trichomeless Arabidopsis mutant (gl1) restored trichome development to that of the wild type. GUS expression in poplar, driven by the promoter of PdeMIXTA04 , also confirmed trichome-specific expression. We then screened a yeast library with PdeMIXTA04 and captured two interacting genes, PdeMYC and PdeWD40 . Interactions between these two proteins were verified by yeast two-hybrid (Y2H), biomolecular fluorescence complementation (BiFC), dual-luciferase (dual-LUC), and pull-down assays, indicating that PdeMIXTA04 functions through the MYB-BHLH-WD40 (MBW) ternary complex. Our work presents evidence of PdeMIXTA04 as a candidate gene for editing to resolve catkins associated pollution and provides distinctive understanding of the molecular mechanism triggering differentiation of plant epidermal cells.

Salix is a dioecious plant. Research on the molecular regulation mechanism of male and female inflorescence differentiation and development is necessary to analyze sex differentiation in the willow and the underlying mechanisms of unisexual flower development. However, at present, there are no reference genes suitable for stable expression in the process of willow inflorescence development. In this study, Salix suchowensis was used as the research material, nine candidate reference genes (α-TUB1, α-TUB2, ACT, H2A, DnaJ, CDC2, GAPDH, TIP41, β-TUB) were selected, and qRT-PCR technology was used to detect the expression of each candidate reference gene in female and male flowers at different developmental stages and using five algorithms (geNorm, Normfinder, Delta Ct, BestKeeper, and RefFinder) to comprehensively evaluate the stability of candidate reference genes. The results showed that ACT and DnaJ were stably expressed in all samples and could be used as reference genes. In addition, the reliability of the screening results was further verified via an expression pattern analysis of the CFS gene that encodes flower specific transcription factor in different samples. The stable reference genes selected in this study provide the basis for future research on the expression analysis of functional genes related to the development of male and female flowers of S. suchowensis.

The MYB transcription factor (TF) family is one of the largest plant transcription factor gene family playing vital roles in plant growth and development, including defense, cell differentiation, secondary metabolism, and responses to biotic and abiotic stresses. As a model tree species of woody plants, in recent years, the identification and functional prediction of certain MYB family members in the poplar genome have been reported. However, to date, the characterization of the gene family in the genome of the poplar’s sister species willow has not been done, nor are the differences and similarities between the poplar and willow genomes understood. In this study, we conducted the first genome-wide investigation of the R2R3 MYB subfamily in the willow, identifying 216 R2R3 MYB gene members, and combined with the poplar R2R3 MYB genes, performed the first comparative analysis of R2R3 MYB genes between the poplar and willow. We identified 81 and 86 pairs of R2R3 MYB paralogs in the poplar and willow, respectively. There were 17 pairs of tandem repeat genes in the willow, indicating active duplication of willow R2R3 MYB genes. A further 166 pairs of poplar and willow orthologs were identified by collinear and synonymous analysis. The findings support the duplication of R2R3 MYB genes in the ancestral species, with most of the R2R3 MYB genes being retained during the evolutionary process. The phylogenetic trees of the R2R3 MYB genes of 10 different species were drawn. The functions of the poplar and willow R2R3 MYB genes were predicted using reported functional groupings and clustering by OrthoFinder. Identified 5 subgroups in general expanded in woody species, three subgroups were predicted to be related to lignin synthesis, and we further speculate that the other two subgroups also play a role in wood formation. We analyzed the expression patterns of the GAMYB gene of subgroup 18 (S18) related to pollen development in the male flower buds of poplar and willow at different developmental stages by qRT-PCR. The results showed that the GAMYB gene was specifically expressed in the male flower bud from pollen formation to maturity, and that the expression first increased and then decreased. Both the specificity of tissue expression specificity and conservation indicated that GAMYB played an important role in pollen development in both poplar and willow and was an ideal candidate gene for the analysis of male flower development-related functions of the two species.

Almost all plants in the genus Populus are dioecious (i.e. trees are either male or female), but it is unknown whether dioecy evolved in a common ancestor or independently in different subgenera. Here, we sequence the small peritelomeric X- and Y-linked regions of P. deltoides chromosome XIX. Two genes are present only in the Y-linked region. One is a duplication of a non-Y-linked, female-specifically expressed response regulator, which produces siRNAs that block this gene’s expression, repressing femaleness. The other is an LTR/Gypsy transposable element family member, which generates long non-coding RNAs. Overexpression of this gene in A. thaliana promotes androecium development. We also find both genes in the sex-determining region of P. simonii, a different poplar subgenus, which suggests that they are both stable components of poplar sex-determining systems. By contrast, only the duplicated response regulator gene is present in the sex-linked regions of P. davidiana and P. tremula. Therefore, findings in our study suggest dioecy may have evolved independently in different poplar subgenera.

Identification of the particular genes in an R genes supercluster underlying resistance to the rust fungus Melampsora larici-populina in poplar genome remains challenging. Based on the de novo assembly of the Populus deltoides genome, all of the detected major genetic loci conferring resistance to M. larici-populina were confined to a 3.5-Mb region on chromosome 19. The transcriptomes of the resistant and susceptible genotypes were sequenced for a timespan from 0 to 168 hours postinoculation. By mapping the differentially expressed genes to the target genomic region, we identified two constitutive expression R genes and one inducible expression R gene that might confer resistance to M. larici-populina. Nucleotide variations were predicted based on the reconstructed haplotypes for each allele of the candidate genes. We also confirmed that salicylic acid was the phytohormone mediating signal transduction pathways, and PR-1 was identified as a key gene inhibiting rust reproduction. Finally, quantitative reverse transcription PCR assay revealed consistent expressions with the RNA-sequencing data for the detected key genes. This study presents an efficient approach for the identification of particular genes underlying phenotype of interest by the combination of genetic mapping, transcriptome profiling, and candidate gene sequences dissection. The identified key genes would be useful for host resistance diagnosis and for molecular breeding of elite poplar cultivars exhibiting resistance to M. larici-populina infection. The detected R genes are also valuable for testing whether the combination of individual R genes can induce durable quantitative resistance.