A High-Throughput Screening System for Populus Wood-Associated Transcription Factors and Its Application to Lignin Regulation

Wood formation of trees is a complex and costly developmental process, whose regulatory network is involved in the protein-protein and protein-DNA interactions. To detect such interactions in wood development, we developed a high-throughput screening system with 517 Gal4-AD-wood-associated transcription factors (TFs) library from Populus alba × P. glandulosa cv “84K.” This system can be used for screening the upstream regulators and interacting proteins of targets by mating-based yeast-one hybrid (Y1H) and yeast-two-hybrid (Y2H) method, respectively. Multiple regulatory modules of lignin biosynthesis were identified based on this Populus system. Five TFs interacted with the 500-bp promoter fragment of PHENYLALANINE AMMONIA-LYASE 2 (PAL2), the first rate-limiting enzyme gene in the lignin biosynthesis pathway, and 10 TFs interacted with PaMYB4/LTF1, a key regulator of lignin biosynthesis. Some of these interactions were further validated by EMSA and BiFC assays. The TF-PaPAL2 promoter interaction and TF-PaMYB4 interaction revealed a complex mechanism governing the regulation of lignin synthesis in wood cells. Our high-throughput Y1H/Y2H screening system may be an efficient tool for studying regulatory network of wood formation in tree species.

Integrated transcriptome and proteome analysis reveals brassinosteroid-mediated regulation of cambium initiation and patterning in woody stem.

Wood formation involves sequential developmental events requiring the coordination of multiple hormones. Brassinosteroids (BRs) play a key role in wood development, but little is known about the cellular and molecular processes that underlie wood formation in tree species. Here, we generated transgenic poplar lines with edited PdBRI1 genes, which are orthologs of Arabidopsis vascular-enriched BR receptors, and showed how inhibition of BR signaling influences wood development at the mRNA and/or proteome level. Six Populus PdBRI1 genes formed three gene pairs, each of which was highly expressed in basal stems. Simultaneous mutation of PdBRI1–1, −2, −3 and − 6, which are orthologs of the Arabidopsis vascular-enriched BR receptors BRI1, BRL1 and BRL3, resulted in severe growth defects. In particular, the stems of these mutant lines displayed a discontinuous cambial ring and patterning defects in derived secondary vascular tissues. Abnormal cambial formation within the cortical parenchyma was also observed in the stems of pdbri1–1;2;3;6. Transgenic poplar plants expressing edited versions of PdBRI1–1 or PdBRI1–1;2;6 exhibited phenotypic alterations in stem development at 4.5 months of growth, indicating that there is functional redundancy among these PdBRI1 genes. Integrated analysis of the transcriptome and proteome of pdbri1–1;2;3;6 stems revealed differential expression of a number of genes/proteins associated with wood development and hormones. Concordant (16%) and discordant (84%) regulation of mRNA and protein expression, including wood-associated mRNA/protein expression, was found in pdbri1–1;2;3;6 stems. This study found a dual role of BRs in procambial cell division and xylem differentiation and provides insights into the multiple layers of gene regulation that contribute to wood formation in Populus.

Cell-wall fluorescence highlights the phases of xylogenesis

The monitoring of xylogenesis makes it possible to follow tree growth responses to stress factors in real-time, by observing the course of wood cell division and differentiation. Proper microscopy techniques are of key importance to exactly identify the xylem cells during the different phases of differentiation. We aimed to apply epifluorescence microscopy to follow the lignification process during the different phases of xylogenesis in Mediterranean softwood and hardwood. Microcores from trees of Pinus halepensis Mill. and Arbutus unedo L. were collected at a site in southern Italy, during the period June-December. Fluorescence imaging of sections stained with a water solution of safranin and Astra blue clearly highlighted the contrast between lignified and un-lignified tissue. The proposed methodology is useful to quickly and unambiguously detect the different stages of cell differentiation, as well as the progress in the lignification process. Moreover, it proved to be easily applied to demanding wood materials, such as Mediterranean woods and can be helpful to better track stress responses and the development of anomalies during wood formation, such as intra-annual density fluctuations.

Full-length transcriptomic identification of R2R3-MYB family genes related to secondary cell wall development in Cunninghamia lanceolata (Chinese fir)

Background R2R3-MYB is a class of transcription factor crucial in regulating secondary cell wall development during wood formation. The regulation of wood formation in gymnosperm has been understudied due to its large genome size. Using Single-Molecule Real-Time sequencing, we obtained full-length transcriptomic libraries from the developmental stem of Cunninghamia lanceolata, a perennial conifer known as Chinese fir. The R2R3-MYB of C. lanceolata (hereafter named as ClMYB) associated with secondary wall development were identified based on phylogenetic analysis, expression studies and functional study on transgenic line. Results The evolutionary relationship of 52 ClMYBs with those from Arabidopsis thaliana, Eucalyptus grandis, Populus trichocarpa, Oryza sativa, two gymnosperm species, Pinus taeda, and Picea glauca were established by neighbour-joining phylogenetic analysis. A large number of ClMYBs resided in the woody-expanded subgroups that predominated with the members from woody dicots. In contrast, the woody-preferential subgroup strictly carrying the members of woody dicots contained only one candidate. The results suggest that the woody-expanded subgroup emerges before the gymnosperm/angiosperm split, while most of the woody-preferential subgroups are likely lineage-specific to woody dicots. Nine candidates shared the same subgroups with the A. thaliana orthologs, with known function in regulating secondary wall development. Gene expression analysis inferred that ClMYB1/2/3/4/5/26/27/49/51 might participate in secondary wall development, among which ClMYB1/2/5/26/27/49 were significantly upregulated in the highly lignified compression wood region, reinforcing their regulatory role associated with secondary wall development. ClMYB1 was experimentally proven a transcriptional activator that localised in the nucleus. The overexpression of ClMYB1 in Nicotiana benthamiana resulted in an increased lignin deposition in the stems. The members of subgroup S4, ClMYB3/4/5 shared the ERF-associated amphiphilic repression motif with AtMYB4, which is known to repress the metabolism of phenylpropanoid derived compounds. They also carried a core motif specific to gymnosperm lineage, suggesting divergence of the regulatory process compared to the angiosperms. Conclusions This work will enrich the collection of full-length gymnosperm-specific R2R3-MYBs related to stem development and contribute to understanding their evolutionary relationship with angiosperm species.

PmMYB4, a Transcriptional Activator from Pinus massoniana, Regulates Secondary Cell Wall Formation and Lignin Biosynthesis

Wood formation originates in the biosynthesis of lignin and further leads to secondary cell wall (SCW) biosynthesis in woody plants. Masson pine (Pinus massoniana Lamb) is an economically important industrial timber tree, and its wood yield affects the stable development of the paper industry. However, the regulatory mechanisms of SCW formation in Masson pine are still unclear. In this study, we characterized PmMYB4, which is a Pinus massoniana MYB gene involved in SCW biosynthesis. The open reading frame (ORF) of PmMYB4 was 1473 bp, which encoded a 490 aa protein and contained two distinctive R2 and R3 MYB domains. It was shown to be a transcription factor, with the highest expression in semi-lignified stems. We overexpressed PmMYB4 in tobacco. The results indicated that PmMYB4 overexpression increased lignin deposition, SCW thickness, and the expression of genes involved in SCW formation. Further analysis indicated that PmMYB4 bound to AC-box motifs and might directly activate the promoters of genes (PmPAL and PmCCoAOMT) involved in SCW biosynthesis. In addition, PmMYB4-OE(over expression) transgenic lines had higher lignin and cellulose contents and gene expression than control plants, indicating that PmMYB4 regulates SCW mainly by targeting lignin biosynthetic genes. In summary, this study illustrated the MYB-induced SCW mechanism in Masson pine and will facilitate enhanced lignin and cellulose synthesis in genetically engineered trees.

A Talk between Flavonoids and Hormones to Reorient the Growth of Gymnosperms

Plants reorient the growth of affected organs in response to the loss of gravity vector. In trees, this phenomenon has received special attention due to its importance for the forestry industry of conifer species. Sustainable management is a key factor in improving wood quality. It is of paramount importance to understand the molecular and genetic mechanisms underlying wood formation, together with the hormonal and environmental factors that affect wood formation and quality. Hormones are related to the modulation of vertical growth rectification. Many studies have resulted in a model that proposes differential growth in the stem due to unequal auxin and jasmonate allocation. Furthermore, many studies have suggested that in auxin distribution, flavonoids act as molecular controllers. It is well known that flavonoids affect auxin flux, and this is a new area of study to understand the intracellular concentrations and how these compounds can control the gravitropic response. In this review, we focused on different molecular aspects related to the hormonal role in flavonoid homeostasis and what has been done in conifer trees to identify molecular players that could take part during the gravitropic response and reduce low-quality wood formation.

Origin of Intra-annual Density Fluctuations in a Semi-arid Area of Northwestern China

Intra-annual density fluctuation (IADF) is a structural modification of the tree ring in response to fluctuations in the weather. The expected changes in monsoon flow would lead to heterogeneous moisture conditions during the growing season and increase the occurrence of IADF in trees of the arid ecosystems of continental Asia. To reveal the timings and physiological mechanisms behind IADF formation, we monitored cambial activity and wood formation in Chinese pine (Pinus tabuliformis) during 2017–2019 at three sites in semi-arid China. We compared the dynamics of xylem formation under a drought event, testing the hypothesis that drought affects the process of cell enlargement and thus induces the production of IADF. Wood microcores collected weekly from April to October were used for anatomical analyses to estimate the timings of cambial activity, and the phases of enlargement, wall thickening, and lignification of the xylem. The first cells started enlargement from late April to early May. The last latewood cells completed differentiation in mid-September. Trees produced IADF in 2018. During that year, a drought in June limited cell production in the cambium, only 36% of the xylem cells being formed in IADF trees, compared to 68% in normal tree rings. IADF cells enlarged under drought in early July and started wall thickening during the rainfall events of late July. The drought restricted cell enlargement and affected wall thickening, resulting in narrow cells with wide walls. Cambium and cell enlargement recovered from the abundant rainfall, producing a new layer with large earlywood tracheids. IADF is a specific adaptation of trees to cope with water deficit events occurring during xylem formation. Our findings confirmed the hypothesis that the June-July drought induces latewood-like IADFs by limiting the process of cell enlargement in the xylem. Our finding suggests a higher occurrence of IADF in trees of arid and semi-arid climates of continental Asia if the changes to monsoon flows result in more frequent drought events during the earlywood formation in June.

Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.