Genome-wide profiling of circular RNAs,alternative splicing,and R-loops in stem-differentiating xylem of Populus trichocarpa

Circular RNAs(circRNAs)are a recently dis-covered type of non‐coding RNA derived from pre‐mRNAs.R‐loops consist of a DNA:RNA hybrid andthe associated single‐stranded DNA.InArabi-dopsis thaliana,circRNA:DNA R‐loops regulatealternative splicing(AS)ofSEPALLATA3(SEP3).However,the occurrence and functions ofcircRNAs and R‐loops inPopulus trichocarpaarelargely unexplored.Here,we performed circRNA‐enriched sequencing in the stem‐differentiatingxylem(SDX)ofP.trichocarpaand identified 2,742distinct circRNAs,including circ‐CESA4,circ‐IRX7,and circ‐GUX1,which are generated from genesinvolved in cellulose,and hemicellulose biosyn-thesis,respectively.To investigate the roles ofcircRNAs in modulating alternative splicing(AS),we detected 7,836 AS events using PacBio Iso‐Seq and identified 634 circRNAs that overlappedwith 699 AS events.Furthermore,using DNA:RNAhybrid immunoprecipitation followed by se-quencing(DRIP‐seq),we identified 8,932 R‐looppeaks that overlapped with 181 circRNAs and 672AS events.Notably,several SDX‐related circRNAsoverlapped with R‐loop peaks,pointing to theirpossible roles in modulating AS in SDX.Indeed,overexpressing circ‐IRX7increased the levels ofR‐loop structures and decreased the frequency ofintron retention in linearIRX7transcripts.Thisstudy provides a valuable R‐loop atlas resourceand uncovers the interplay between circRNAs andAS in SDX ofP.trichocarpa.

Dynamic Changes in Distribution of Lignin and Hemicelluloses in Cell Walls During Differentiation of Secondary Xylem in Eucommia ulmoides

The dynamic changes in the distribution of lignin and hemicelluloses (xylans and xyloglucans) in cell walls during the differentiation of secondary xylem in Eucommia ulmoides Oliv. were studied by means of ultraviolet light microscopy and transmission electron microscopy combined with immunogold labelling. In the cambial zone and cell expansion zone, xyloglucans were localized both in the tangential and radial walls, but no xylans or lignin were found in these regions. With the formation of secondary wall S-1 layer, lignin occurred in the cell corners and middle lamella, while xylans appeared in S-1 layer, and xyloglucans were localized in the primary walls and middle lamella. In pace with the formation of secondary wall S-2 and S-3 layer, lignification extended to S-1, S-2 and S-3 layer in sequence, showing a patchy style of lignin deposition. Concurrently, xylans distributed in the whole secondary walls and xyloglucans, on the other hand, still localized in the primary walls and middle lamella. The results indicated that along with the formation and lignification of the secondary wall, great changes had taken place in the cell walls. Different parts of cell walls, such as cell corners, middle lamella, primary walls and various layers of secondary walls, had different kinds of hemicelluloses, which formed various cell wall architecture combined with lignin and other cell wall components.

Programmed Cell Death During Secondary Xylem Differentiation in Eucommia ulmoides

Programmed cell death (PCD) during secondary xylem differentiation in Eucommia ulmoides Oliv. was examined using electron microscopy and by investigation of DNA fragmentation and degradation of caspase-like proteases (CLPs). DNA ladders were detected in developing secondary xylem by gel electrophoresis. DNA fragmentation was further confirmed by using the TdT-mediated dUTP nick-end labeling (TUNEL) method. Western blotting analysis showed that CLPs (caspase-8- and caspase-3-like proteases) and PARP (poly (ADP-ribose) polymerase) were degraded during secondary xylem differentiation. The results thus indicated that secondary xylem differentiation in E ulmoides was a typical process of PCD and the degradation of CLPs might be a constitutive PCD event during secondary xylem differentiation.

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.