Content area
Abstract
ABSTRACT
The lack of a mechanistic understanding of the environmental plasticity of secondary cell wall (SCW) biosynthesis restricts large‐scale biomass and bioenergy production on marginal lands. Using
Full text
Introduction
Populus (poplar) is an important renewable resource for lignocellulosic biomass-based bioenergy production. In addition, poplar is a well-established model system for studying woody perennial plant-specific traits, such as wood formation, secondary growth and adaptation to environmental change (Wullschleger et al. 2013). Lignocellulosic biomass is produced through secondary growth and secondary cell wall (SCW) biosynthesis. Secondary growth is supported by the lateral meristem or vascular cambium, whose division and differentiation form the secondary xylem towards the inside of the stem and form the secondary phloem towards the outside of the stem (Du and Groover 2010). After differentiation, SCW is thickened and lignified to form wood.
In plants, SCW biosynthesis is regulated by a hierarchical NAC-MYB gene regulatory network, in which the plant-specific NAC (NAM, ATAF and CUC) transcription factors (TFs) act as master regulators that directly target MYB46/83 TFs, which switch on the biosynthesis of all three major SCW components (lignin, cellulose and xylan) by directly activating the expression of a number of MYB TFs and SCW biosynthetic genes (Xie, Zhang, et al. 2018). For example, Arabidopsis secondary wall-associated NAC domain protein 1/NAC secondary wall thickening promoting factor 3 (SND1/NST3) and its close homologues NST1, NST2, vascular-related NAC domain 6 (VND6) and VND7 were found to form the first layer of the NAC-MYB gene regulatory network and regulate SCW formation in various tissues (Xie, Zhang, et al. 2018). In poplar, a group of NAC TFs named wood-associated NAC domain proteins (WNDs) were found to be functional orthologs of Arabidopsis NAC TFs (Zhong et al. 2010; Zhong and Ye 2010). In particular, PtrWND2B and PtrWND6B have been shown to directly activate the expression of poplar MYB46/83 homologues, many other TFs and synthetic genes involved in SCW biosynthesis (Zhong et al. 2010). Dominant suppression of PtrWND2B or PtrWND6B in poplar reduced cell wall thickness of xylem fibres by over 50% (Zhong et al. 2010).
SCW biosynthesis and its transcriptional regulation in poplar are highly plastic in response to environmental signals (Zinkgraf et al. 2017). The expression of cell wall-associated genes is tightly spatiotemporally co-regulated (Brown et al. 2005). Environmental stresses such as drought and salt have been shown to reduce the total amount of cell wall polysaccharides, vessel size and S/G lignin ratio of poplar stems (Hori et al. 2020). Iron (Fe) is a micronutrient that is essential for plant redox processes and photosynthesis (Briat et al. 2015). Fe deprivation was found to increase SCW deposition and lignification in the root via a REVOLUTA (REV)-controlled gene regulatory network (Taylor-Teeples et al. 2015). However, how Fe deprivation affects SCW formation in aboveground tissues, which are the main source of biomass, is still poorly studied.
The basic helix–loop–helix (bHLH) TF family is one of the largest TF families in plants. The bHLH TFs have been found to play vital roles in plant growth, SCW biosynthesis, iron homeostasis and stress responses (Gao and Dubos 2024). Phylogenetic studies divided the family into 17 major groups (I–XVII) and 31 subfamilies (Gao and Dubos 2024; Pires and Dolan 2010). Among them, members of the bHLH IVb subgroup have been reported to regulate iron homeostasis through various mechanisms. AtbHLH011, upstream regulator of IRT1 (URI/AtbHLH121) and Popeye (PYE/AtbHLH047) are three bHLH IVb TFs in Arabidopsis. AtbHLH011 functions as a negative regulator of FER-like iron deficiency-induced transcription factor (FIT)-dependent Fe uptake (Tanabe et al. 2019). AtbHLH011 has no transcriptional activity but interacts with bHLH IVc TFs to repress their transactivity on bHLH Ib TFs (Tanabe et al. 2019). URI/bHLH121 is expressed throughout the plant body and acts as a direct transcriptional activator of key genes involved in the Fe regulatory network (Gao et al. 2020). URI is phosphorylated upon Fe deficiency, forms heterodimers with bHLH IVc TFs and induces transcription of bHLH Ib TFs, which in turn heterodimerize with FIT and drive transcription of iron-regulated transporter 1 (IRT1) and ferric reduction oxidase 2 (FRO2) to increase Fe uptake (Kim et al. 2019). A shoot iron deficiency signal activates transcription of PYE/AtbHLH047, which encodes a mobile protein to facilitate iron uptake, root-to-shoot translocation and storage via FIT-dependent and/or -independent pathways (Long et al. 2010; Muhammad et al. 2022).
In this study, we demonstrated that Fe deprivation activates SCW biosynthesis via its gene regulatory network in poplar stems. We identified PtrbHLH011, a bHLH IVb TF homologous to AtbHLH011, as a crucial regulator of this response. PtrbHLH011 expression is downregulated during stem wood formation and under iron deprivation conditions in various plant tissues. Overexpression of PtrbHLH011 negatively impacted plant growth, diminished SCW deposition in stems and decreased iron accumulation in leaves. Conversely, knockout of PtrbHLH011 significantly promoted growth, enhanced SCW formation and substantially increased leaf iron content. Integrated analyses combining RNA-seq, transient chromatin-immunoprecipitation sequencing (transient ChIP-seq) and transactivation assay revealed that PtrbHLH011 is a transcriptional repressor that directly targets and represses essential genes involved in SCW biosynthesis, iron homeostasis and flavonoid biosynthesis. Furthermore, we found that PtrbHLH011 recognises these target genes through direct binding to a newly identified cis-regulatory motif (AAAGACA). Collectively, our findings uncovered PtrbHLH011 as a multifunctional regulator coordinating SCW biosynthesis, iron homeostasis and flavonoid biosynthesis, providing mechanistic insights essential for optimising bioenergy crop performance on marginal lands with limited iron availability.
Results
Fe Deprivation Activates
We found that Fe-deprivation stress significantly increases lignin and SCW deposition in poplar stems. The Fe-deprivation treatment was performed by growing poplar seedlings in a hydroponic nutrient solution without iron (0 μM Fe) for 21 days (Methods and Figure S1). In parallel, poplar seedlings grown in a hydroponic nutrient solution with sufficient iron for poplar (10 μM Fe) were used as a control. Consistent with previously reported results (Chen et al. 2019), our Fe-deprivation treatment induced leaf chlorosis and stem growth inhibition (Figure S1A–C). Cross-sections from the 3rd and 10th internodes were then collected for lignin staining with phloroglucinol-HCl. As shown in Figure 1A and Figure S2, ectopic lignin and SCW deposition were observed in the fibre cells of primary xylem and primary phloem of the stems with primary growth (3rd internode). Similarly, in the stems with secondary growth (10th internode), increases in xylem area and SCW thickness of xylem fibres as well as ectopic lignin deposition in phloem fibres were observed under Fe deprivation compared to the control (Figure 1C–E; Figure S2).
[IMAGE OMITTED. SEE PDF]
Consistently, RNA-seq analysis revealed that Fe deprivation activates the expression of various transcriptional activators and synthetic genes for SCW biosynthesis (Figure 1F,G; Figure S1D). The 3rd internode had the most altered transcriptomes by Fe deprivation, with 1463 upregulated and 1367 downregulated genes compared to the control (Figure S3; Data S1, S2). Among the upregulated genes, all the top five enriched gene ontology (GO) terms are relevant to lignin and SCW biosynthesis (Figure 1F). Furthermore, RNA-seq and qRT-PCR results identified a large set of TFs and biosynthesis genes for SCW that were upregulated by Fe deprivation (Figure 1G; Figure S1D). Taken together, our results suggest that Fe deprivation activates SCW biosynthesis through transcriptome reprogramming.
Since transcriptome reprogramming of iron homeostasis is also a key mechanism in response to Fe deprivation (Buckhout et al. 2009), the observed induction of SCW biosynthesis by Fe deprivation suggests that transcriptional co-regulators may exist to control SCW biosynthesis and Fe homeostasis pathways more cost-effectively. bHLH TFs play essential roles in regulating plant Fe homeostasis (Gao and Dubos 2024). By searching AspWood, a high-spatial-resolution RNA-seq database across developing phloem and wood-forming tissues in poplar (Sundell et al. 2017), for bHLH TFs that may also regulate SCW biosynthesis, we identified Potri.005G113400. We named this gene PtrbHLH011 because it encodes a bHLH IVb TF that is a close homologue of AtbHLH011 (Figure 2A; Figure S4A). PtrbHLH011 is expressed in various tissues, including root, leaf and stem (Figure S4B). Across the four replicates in AspWood, PtrbHLH011 exhibits a consistent expression pattern in wood-forming tissues that appears to be opposite to the expression patterns of SCW biosynthesis marker genes PtrWND2B and CELLULOSE SYNTHASE 4 (PtCesA4) (Sundell et al. 2017; Zhong et al. 2010), suggesting that PtrbHLH011 may negatively regulate SCW biosynthesis (Figure 2B). On the other hand, qRT-PCR results showed that PtrbHLH011 expression level was decreased by over 50% under Fe-deprivation treatment in tissues including xylem, phloem, leaf and root (Figure 2C). This expression pattern is similar to its Arabidopsis homologue AtbHLH011, which has been identified as a negative regulator of Fe uptake (Tanabe et al. 2019).
[IMAGE OMITTED. SEE PDF]
To further test whether PtrbHLH011 co-regulates SCW biosynthesis and Fe homeostasis as a negative regulator, we sought to knock out PtrbHLH011/PtabHLH011 in
[IMAGE OMITTED. SEE PDF]
Furthermore, we observed opposite stem and leaf phenotypes in transgenic poplars overexpressing PtrbHLH011 (Figure 4). PtrbHLH011 was overexpressed under the control of CaMV 35S promoter (Figure S5E, Method S1). As shown in Figure 4A, all five independent PtrbHLH011 overexpression (PtrbHLH011 OE) lines showed significantly inhibited growth compared to the empty vector control, with up to 34% reduction in plant height and 37% reduction in stem diameter (Figure 4B). By studying lignin levels in stems of two representative OE lines, we observed an over 65% reduction in SCW thickness in developing xylem (Figure 4C,D; Figure S6B) and an over 45% reduction in lignin level (Figure 4E). In contrast to PtrbHLH011 KO lines, XRF analysis of leaf sections showed the reduction of leaf Fe accumulation in PtrbHLH011 OE lines compared to the empty vector control (Figure 4F).
[IMAGE OMITTED. SEE PDF]
Results of the above phenotypic analyses of PtrbHLH011 KO and OE lines suggest that PtrbHLH011 may be a negative regulator that co-regulates SCW biosynthesis and Fe homeostasis.
The PtrbHLH011 protein is localised in both the cytosol and the nucleus in poplar mesophyll protoplasts (Figure 5A). Furthermore, our protoplast-based transient transactivation assays using the GUS and luciferase reporter system (Xie, Muchero, et al. 2018) revealed that PtrbHLH011 has transcriptional repressor activity but not activator activity (Figure 5B,C). PtrbHLH011 was fused with the Gal4 DNA-binding domain and recruited to the promoter of the GUS reporter gene via the interaction between GD and the Gal4 DNA sequence upstream of GUS. A plasmid overexpressing luciferase (LUC) was co-transfected as an internal standard. To test repressor activity, an additional transactivator plasmid overexpressing the LexA DNA-binding domain (LD) fused with herpes simplex virus VP16 transactivator (VP16) was co-transfected to constitutively activate GUS expression through the interaction between LexA and LD. The normalised GUS activity (GUS/LUC) was used to evaluate the transcriptional activity of PtrbHLH011. As shown in Figure 5B, PtrbHLH011 has a significantly lower GUS/LUC value than the green fluorescent protein (GFP) negative control, indicating transcriptional repressor activity. In contrast, the GUS/LUC value of PtrbHLH011 is similar to that of the GFP negative control and much less than that of the VP16 positive control in the activation assay (Figure 5C), suggesting that PtrbHLH011 does not have transcriptional activator activity.
[IMAGE OMITTED. SEE PDF]
To seek genome-wide targets of PtrbHLH011 repression, we integrated RNA-seq and protoplast-based transient chromatin-immunoprecipitation sequencing (transient ChIP-seq) approaches (Tadesse et al. 2024). By comparing the transcriptomes of PtrbHLH011 OE and empty vector control, we identified 2743 upregulated and 1227 downregulated genes in the leaf tissue, 2172 upregulated and 1854 downregulated genes in the stem tissue (Figure S7A–D; Data S3, S4). In transient ChIP-seq experiments, PtrbHLH011 exhibited the typical binding peak of TFs surrounding the transcription start site (TSS; Figure S7E,F). By filtering downregulated genes in either tissue whose promoters have PtrbHLH011 binding peaks (Figure 5D,E; Data S5), we discovered putative PtrbHLH011 targets that are responsible for SCW biosynthesis, iron homeostasis and flavonoid biosynthesis, including PtrWND2B and PtrMYB92, whose protein products are major activators of SCW biosynthesis (Liu et al. 2021; Zhong et al. 2010); YELLOW STRIPE LIKE 3 (YSL3) and PYE, the Arabidopsis homologues of which are responsible for root-to-shoot iron translocation and long-distance iron signalling (DiDonato Jr. et al. 2004; Kumar et al. 2017; Long et al. 2010; Muhammad et al. 2022); IRON REGULATED TRANSPORTER 3 (PtIRT3) and FERRITIN 1 (FER1) for shoot iron accumulation (Huang and Dai 2015; Lin et al. 2009); and CHALCONE SYNTHASE 1 and 5 (CHS1 and CHS5), which encode the enzyme that catalyses the first step of flavonoid biosynthesis (Zhang et al. 2021).
Noting significant PtrbHLH011 binding peaks in the promoters of these genes (Figure 5E), reporter constructs were generated by inserting the PtrWND2B, PtrMYB92, CHS1, CHS5, YSL3, PYE, PER1 and PtIRT3 promoters between the 35S promoter and the GUS reporter gene, respectively (Figure 5F). The transient transactivation assay confirmed that PtrbHLH011 directly represses the activity of all these promoters (Figure 5F).
The repression of PtrbHLH011 on these eight target genes was further validated in PtrbHLH011 OE and KO plants. qRT-PCR results show that all eight genes except PYE are downregulated in leaf and stem tissues of PtrbHLH011 OE plants (Figure 6A). PYE had a significant reduction in the stem, but not in the leaf (Figure 6A). This could be due to the major function of PYE in iron translocation in the root and stem. In contrast, expressions of tested genes were upregulated in leaf and stem tissues of PtrbHLH011 KO plants (Figure 6B).
[IMAGE OMITTED. SEE PDF]
By investigating cis-regulatory elements enriched in promoters of the eight PtrbHLH011 targets, but not in promoters of genes not directly repressed by PtrbHLH011 in the transactivation assay (Figure S8A, Methods S1), we identified the sequence AAAGACA (Score: 7.6e-003) as a potential cis-regulatory element bound by PtrbHLH011 (Figure 6C). The promoters of the eight PtrbHLH011 targets contain at least one copy of this AAAGACA motif (Figure S8B).
Electrophoretic mobility shift assay (EMSA) confirmed that PtrbHLH011 specifically binds to this DNA motif (Figure 6D). We expressed and purified the full-length PtrbHLH011 protein (Figure 6D) and synthesised 5′ IRDye 800-labelled DNA probes containing three copies of the AAAGACA sequence (Figure 6D; Probe 1). EMSA and the competition assay using 10×, 50× and 100× unlabeled probe of the same sequence showed that the binding was specific, as indicated by the reduction of the bound signal (protein-DNA complex) in the presence of the competitor (Figure 6D). In contrast, the competition using 100× mutated probe that contains three copies of the AGATTTA sequence (Figure 6D; Probe 2) did not reduce the bound signal, further demonstrating the binding specificity.
Discussion
Growing bioenergy crops on marginal lands with low nutrient quality is increasingly utilised as a solution to minimise competition for arable land and food production (Gelfand et al. 2013). However, as the major source of lignocellulosic biomass, SCW biosynthesis is highly plastic in response to environmental stress (Zinkgraf et al. 2017). For the massive production of lignocellulosic biomass on marginal lands that are often exposed to various abiotic stresses, it is crucial to understand how these stresses affect the quantity and quality of SCW. Additionally, the discrepancies between field-grown and greenhouse-grown cell-wall-engineered poplars are increasingly observed (De Meester et al. 2022; Li, Lin, et al. 2023), which are one of the main reasons for hampering the commercialization of wood feedstocks for biofuels worldwide. The lack of knowledge about the adaptation of SCW biosynthesis to environmental stress is a major factor underlying the discrepancies.
Iron is an essential micronutrient for photosynthesis, respiration and other important developmental processes in plants (Briat et al. 2015). However, iron deficiency caused by poor solubility and alkaline soils is widespread and detrimental to crop yield and bioproduct quality. To date, Fe deprivation has only been reported to increase SCW deposition and lignification of roots in the model plant Arabidopsis via the REV-controlled gene regulatory network (Taylor-Teeples et al. 2015). In this study, we demonstrated that iron deprivation upregulates the gene regulatory network of SCW and increases SCW biosynthesis in poplar aboveground stems, the main source of lignocellulosic biomass. We identified a bHLH IVb TF PtrbHLH011 that appears to be involved in this regulation. We found that PtrbHLH011 is a potent inhibitor of SCW biosynthesis by directly targeting PtrWND2B and PtrMYB92, two major activators of SCW biosynthesis that were found to activate the expression of numerous TFs and biosynthetic genes for SCW biosynthesis in poplar (Liu et al. 2021; Zhong et al. 2010, 2011). Under iron deficiency, PtrbHLH011 expression is downregulated to relieve the repression of PtrWND2B and PtrMYB92, which consequently activates SCW biosynthesis. Therefore, we propose that PtrbHLH011 acts as a gatekeeper by limiting SCW biosynthesis under normal growth conditions to prevent growth inhibition caused by excessive lignification (Figure 7). Fe deficiency signal silences PtrbHLH011 expression to enhance lignification of vascular tissue for better water and iron transportation from root to shoot (Figure 7). To further support this functional model, comparative analysis on RNA-seq data shows that 29 out of 33 SCW-related genes upregulated by Fe deprivation, particularly TFs and lignin biosynthetic genes, are downregulated in PtrbHLH011 OE plants (Figure S11A). Complementary qRT-PCR analysis of representative SCW-related genes known to be downstream targets activated by PtrWND2B and/or PtrMYB92 (Liu et al. 2021; Zhong et al. 2010, 2011) validated this pattern, showing downregulation in PtrbHLH011 OE plants and upregulation in PtrbHLH011 KO plants (Figure S11B,C). Among these genes, PtrMYB31/PtaMYB31 (homologue of Arabidopsis MYB69), PtrMYB167/PtaMYB167 (homologue of Arabidopsis MYB52 and MYB54), PtrWND5A/PtaWND5A (homologue of Arabidopsis VND6) and PtrWRKY19/PtaWRKY19 (homologue of Arabidopsis WRKY12) are TFs that regulate SCW biosynthesis (Liu et al. 2022; Ohtani et al. 2011; Zhong and Ye 2009). In addition, 4CL5, C4H1, GAUT12.1 and CesA4 encode enzymes involved in the biosynthesis of lignin, hemicellulose and cellulose (Kumar et al. 2009; Lee et al. 2011; Wang et al. 2018). Meanwhile, it does not exclude the involvement of other TFs since the ectopic lignin deposition in phloem was observed under Fe deprivation treatment but was not clearly observed in PtrbHLH011 KO lines. Screening additional co-regulators could further elucidate the molecular mechanisms and GRNs controlling SCW biosynthesis under Fe deficiency.
[IMAGE OMITTED. SEE PDF]
We showed that PtrbHLH011 is a crucial regulatory node for Fe homeostasis in poplar. Fe is an essential micronutrient for plant growth and development. However, excessive amounts of Fe delivered to cells can be harmful because of the redox properties of Fe (Marschner 2011). We found that PtrbHLH011 directly represses the expression of key genes for Fe deficiency signalling, Fe translocation and shoot Fe accumulation, including PYE, YSL3, PtIRT3 and FER1. PYE is a Fe deprivation-induced bHLH TF and was reported to play an important role in the sense of shoot Fe deprivation signal to increase Fe uptake from soil and root-to-shoot Fe translocation in Arabidopsis (Long et al. 2010; Muhammad et al. 2022). In dicots, YSL proteins are responsible for the mobilisation of nicotianamine (NA)-chelated Fe in the vasculature (DiDonato Jr. et al. 2004; Waters et al. 2006). Arabidopsis homologues of YSL3, AtYSL1 and AtYSL3 have been reported to be required for long-distance Fe deficiency signalling from shoot to root (Kumar et al. 2017). PtIRT3 is an Fe transporter that is constitutively expressed in several plant tissues (Huang and Dai 2015). Its Arabidopsis homologue IRT3 also plays an essential role in maintaining shoot Fe accumulation (Shanmugam et al. 2011). Additionally, overexpression of FER genes has been reported to increase leaf Fe accumulation (Goto et al. 2000; Van Wuytswinkel et al. 1999). To prevent excessive Fe accumulation in leaf cells, PtrbHLH011 limits the expression of YSL3, PYE, PtIRT3 and FER1 under normal growth conditions. This should result in the reduced Fe accumulation in leaf cells of PtrbHLH011 OE plants. This change in leaf Fe accumulation is probably due to the change of root-to-shoot Fe transport since we observed increased Fe accumulation in stems of PtrbHLH011 OE plants (Figure S9). On the other hand, under Fe deprivation conditions, PtrbHLH011 expression is inhibited, which is likely to activate Fe deficiency signalling and mechanisms to improve root-to-shoot Fe transport (Figure 7). To support this, leaf cells of PtrbHLH011 KO plants accumulated more Fe. To further support this hypothesis, how these PtrbHLH011 targets affect Fe deficiency signalling and root-to-shoot Fe transport in poplar remains to be elucidated.
In addition to SCW biosynthesis and Fe homeostasis, our results suggest that PtrbHLH011 co-regulates flavonoid biosynthesis by directly repressing the expression of chalcone synthase genes (e.g., CHS1 and CHS5), which encode the enzyme catalysing the first step of the flavonoid biosynthetic pathway (Zhang et al. 2021). To support this, the soluble phenolic, anthocyanin and salicylic acid contents in the leaves of PtrbHLH011 OE lines showed a significant reduction (Figure S10). Fe deprivation has been reported to disrupt the photosynthetic machinery, resulting in the generation of light-dependent reactive oxygen species (ROS) and leaf photosensitivity (Akmakjian et al. 2021). Consistently, we observed significant GO enrichment of oxidation–reduction and superoxide metabolic processes in genes that were upregulated in Fe deprivation-treated poplar leaves (Figure S3E). Since flavonoids are the main defence metabolites produced through the phenylpropanoid pathway and anthocyanins are a class of flavonoids responsible for leaf colour and stress tolerance (Fini et al. 2011; Li and Ahammed 2023), downregulation of PtrbHLH011 under Fe deprivation appears to protect plants from oxidative stress by reversing the repression of chalcone synthases and increasing flavonoid biosynthesis (Figure 7). Given the role of ferritins in the oxidative stress response (Ravet et al. 2009), regulation of FER1 expression is probably another contributor.
The dramatically increased growth and leaf Fe accumulation in PtrbHLH011 KO plants suggest a viable strategy to enhance biomass production of poplar on iron deficiency marginal lands. As a co-regulator of several essential pathways, direct manipulation of PtrbHLH011 expression or functionality has pleiotropic effects that may result in undesirable phenotypes such as high lignin content-induced biomass recalcitrance. Our discoveries of the regulatory targets and cis-regulatory elements of PtrbHLH011 provide insights for the precise manipulation of PtrbHLH011-target relationships via promoter editing approaches (Zhou et al. 2023) to selectively turn on or off genes for desired phenotypes. This will lay the foundation for new bioengineering strategies to tailor the performance of bioenergy crop performance for human needs.
In summary, our results reveal a regulatory mechanism that controls SCW biosynthesis in response to environmental iron availability. Our mechanistic insights into the functionality of PtrbHLH011 are expected to advance the bioengineering of high-performance bioenergy crops on marginal lands.
Materials and Methods
Plant Materials and Growth Conditions
To generate PtrbHLH011 overexpression lines, full-length Potri.005G113400 CDS (P.trichocarpa v3.1; Phytozome) was synthesised and cloned into the pTwist ENTR Kozak vector (Twist Bioscience). The gene was then subcloned into the Gateway binary destination vector pMDC32 (Curtis and Grossniklaus 2003) using LR Clonase II recombination (Invitrogen) for overexpression. The resulting overexpression cassette is driven by the CaMV 35S promoter. The binary transformation vector was then transformed into Agrobacterium strain GV3101. The
An efficient nCas9-A3A/Y130F-BE3 C-to-T base editing system was used to generate PtrbHLH011 knockout lines (Li, Sretenovic, et al. 2023; Li et al. 2021). The sgRNA was designed using CRISPR-BETS (Wu et al. 2022) to introduce a premature stop codon into the second exon of PtrbHLH011/PtabHLH011 in the
Plants were propagated in a greenhouse maintained at 20°C–24°C and a day length of 16 h.
Hydroponic Treatments
Clonal cuttings of
Histological Analysis
The internodes of the same age or treatment time were collected and cut into 80 μm thick sections using a cryostat (Leica CM1950). Sections were stained with 2% phloroglucinol-HCl or 0.02% toluidine blue O. The stained sections were imaged using the Odyssey M Imaging System (LI-COR) and the LMD7 Laser Microdissection microscope with a 10× objective (Leica). For the fluorescent staining of lignin using Basic Fuchsin, sections were immersed in 0.2% Basic Fuchsin in ClearSee solution (Kurihara et al. 2015) for 1 h. After washing with ClearSee solution three times, sections were imaged using a Zeiss Axioscope 5 microscope with 540–570 nm excitation bandwidth and 600–680 nm emission bandwidth. Cell wall thicknesses of xylem fibre cells in each section were measured using LAS X software (Leica) or Zen software (Zeiss).
Phylogenetic Analysis
For reconstruction of the relatedness between poplar and Arabidopsis homologues, the PtrbHLH011 amino acid sequence was used to search the
X-Ray Fluorescence and Scattering Microscopy
For XRF of leaf sections, freshly collected leaves were dissected into tissue pieces a few mm in size, rinsed with deionised water and fixed in 4% paraformaldehyde (PFA) solution. The tissues were then sectioned to a thickness of 20 μm by using a cryostat (Leica CM1950). Sections were then placed on Silicon Nitride (SiN) membrane windows and air-dried (Lin et al. 2024; Sanchez-Cano et al. 2017). All sections were examined for quality under the light microscope and those with the best integrity were selected for scanning. Room temperature XRF measurements were performed on the 5-ID SRX beamline (Nazaretski et al. 2022) NSLSII. XRF maps were scanned with a step size of 0.5 μm and a dwell time of 0.1 s at 12 keV. Several indigenous elements were detected simultaneously, including Fe, Zn, Ca, K, P, S and so forth. An AXO standard (AXO, Dresden GmbH) was used for XRF quantification (Yang et al. 2019). The spectra were analysed and quantified using the open software PyXRF (Li et al. 2017) to generate elemental distribution maps for each element.
Scattering-based tomographic imaging was used to examine the stem tissues at the LiX beamline at BNL (Yang et al. 2022). Data acquisition and processing were performed as previously described (Yang 2024). The spatially resolved angular scattering intensity profile was divided into a constant background and an azimuthal-angle-dependent component. The ratio between the integrated intensity of these two components was calculated to represent the cellulose crystallinity index (CI). X-ray fluorescence data were acquired simultaneously with scattering data using a single-element silicon drift detector. The emission spectra were decomposed into contributions from known elements using non-negative least squares fitting on the basis of calculated characteristic emission peaks. The self-absorption by the sample was corrected during tomographic reconstruction using the algorithm developed by Ge et al. (2022).
Anthocyanin, Phenolics and Total Lignin Measurements
Anthocyanin content of poplar leaf tissue was quantified as previously described (Nakata and Ohme-Takagi 2014) with modifications. Briefly, 100 mg of leaf tissue from 4-month-old poplar plants was collected and ground using CryoMill (Retsch). The ground tissues were mixed with 1 mL of methanol with 1% HCl and extracted with rotation overnight at 20°C, followed by centrifugation to recover the supernatant. To quantify the anthocyanin content, 200 μL of supernatant of each sample was transferred to a 96-well plate, and the absorbances at 530 nm and 637 nm were measured using the Spark Microplate Reader (Tecan). Each sample was analysed with four technical replicates.
For total soluble phenolics measurement, ultraviolet–visible (UV–Vis) spectrophotometry and high-performance liquid chromatography-mass spectrometry (HPLC-MS) approaches were used. Soluble phenolics were extracted from approximately 100 mg of ground leaf tissue by incubating with 1 mL of 80% methanol overnight at 4°C. For UV–Vis spectra, the phenolic extract was diluted 30 times with methanol. The UV–Vis absorption spectra from 200 to 700 nm (1 nm resolution) of 150 μL of the diluted extractions were measured in a UV-specific 96-well plate (Corning) using the Spark Microplate Reader (Tecan). 150 μL of methanol was measured as the background reference for reading normalisation. For each sample, three biological and three technical replicates were analysed. HPLC-MS was performed as previously described (Dwivedi et al. 2024). The phenolic extracts were dried in a speed vacuum (Labconco) and digested in 2 N HCl for 2 h at 95°C. The acid-digested samples were further extracted with 200 μL of water-saturated ethyl acetate. 100 μL of extracted samples was dried and redissolved in 200 μL of 80% methanol. 1 μL of the sample was injected into a UHPLC–MS system (ThermoFisher Scientific) and resolved with a reverse phase C18 column (Luna, 150 × 2.1 mm2, 1.6 μm, Phenomenex).
For total lignin measurement, cell wall residues (CWRs) were prepared following the method described previously (Dwivedi et al. 2024). The total lignin content was measured using the acetyl bromide method (Foster et al. 2010). Approximately 10 mg of CWRs was incubated with 1 mL of 25% (v/v) acetyl bromide in glacial acetic acid at 50°C for 3 h, with gentle mixing every 15 min. After incubation, the mixture was cooled on ice for 15 min and then diluted with 5 mL of glacial acetic acid. To neutralise the solution, 300 μL of this diluted solution was combined with 400 μL of 2 N NaOH and 300 μL of freshly prepared 0.5 M hydroxylamine hydrochloride. Next, 200 μL of the neutralised solution was transferred to UV-transparent 96-well plates (Corning, Kennebunk), and the absorbance at 280 nm was recorded. An extinction coefficient of 18.21 g−1 cm−1 was used to calculate the total lignin content.
Protoplast Transfection, Subcellular Localization and Transient Transactivation Assays
Poplar mesophyll protoplasts were isolated and transfected as described previously (Xie, Muchero, et al. 2018). Protoplasts were isolated from fully expanded leaves of two-month-old
For subcellular localization analysis, the cDNA of PtrbHLH011 was cloned into a transient expression vector (Xie et al. 2020) for the C-terminal YFP fusion. 5 μg of this plasmid was co-transfected with 5 μg of mCherry-VirD2NLS plasmid (Lee et al. 2008) into 100 μL of protoplast suspension (~100 000 cells). After 16 h of incubation under weak light at 23°C, protoplasts were collected and imaged with a Leica TCS SP5 confocal microscope equipped with 488 and 543 nm laser lines to excite YFP and mCherry, respectively. The emission bandwidth for YFP and mCherry was 500–530 nm and 580–620 nm, respectively. Images were processed using LAS X software (Leica).
Transient transactivation assays were performed as described (Xie et al. 2020). A total of 10 μg of effector, reporter and/or transactivator plasmids were co-transfected into 100 μL of protoplast suspension (~100 000 cells). 100 ng of 35S:Luciferase plasmid was co-transfected for each reaction to normalise GUS activity. After 18 to 20 h incubation in the dark at 23°C, protoplasts were collected and lysed to measure GUS and luciferase activities using the Synergy Neo2 multimode plate reader (BioTek). GUS activity in individual samples was normalised against luciferase activity (GUS/LUC). Three replicates were performed for statistical calculations.
Transient
Transient ChIP-seq was performed as previously described (Tadesse et al. 2024) using protoplasts isolated from
Clean reads were obtained by filtering raw reads to remove adapter sequences and low-quality reads. Because of the use of
Total RNAs were isolated from leaf and stem tissues using the RNeasy Plant Mini Kit (Qiagen) and treated with on-column DNase (Qiagen) to remove genomic DNA contamination. RNA quality was assessed using the 2100 Bioanalyzer (Agilent). For quantitative RT-PCR (qRT-PCR), cDNAs were synthesised using the oligo(dT) primer and RevertAid Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer's instructions. Primers for qRT-PCR are listed in Table S1.
For Fe deprivation RNA-seq, library construction and sequencing were performed by the Joint Genome Institute (JGI) to obtain paired-end reads of 150 bp. For PtrbHLH011 OE RNA-seq, library construction and sequencing were performed by Beijing Genomics Institute (BGI) to obtain paired-end reads of 150 bp. Three biological replicates were initially used for library preparation and sequencing. The library preparation of some stem RNA samples failed probably because of the nature of stem total RNAs. Therefore, only two replicates were sequenced for PtrbHLH011 OE stem. After removing adapters and low-quality sequences, clean reads were aligned to the
To predict the DNA motifs bound by PtrbHLH011, the sequences of the promoters tested in the transactivation assay were analysed using the STREME (Sensitive, Thorough, Rapid, Enriched Motif Elicitation) function of the MEME suite (v5.5.2) (Bailey et al. 2015). Enriched DNA motifs in validated PtrbHLH011 targets (PtrWND2B, PtrMYB92, CHS1, CHS5, YSL3, PYE, FER1 and ZIP4) were identified. Promoter sequences of genes that were not repressed by PtrbHLH011 (ZIP6, TT3, PtrMYB3, SND2-1, SND2-2, MYB5 and MYC2; Figure S7A) were used as control sequences. Promoter sequences are listed in Table S2.
Protein Purification and Near-Infrared Fluorescence Electrophoretic Mobility Shift Assay (
PtrbHLH011 was codon-optimised, cloned into the pET-29b(+) vector for C-terminal 6xHis tag fusion and transformed into
EMSA was performed as previously described (Tadesse et al. 2024) with modifications. DNA oligonucleotides with 5′ IRDye 800 labeling were synthesised by Integrated DNA Technologies (IDT). Equal amounts of forward and reverse DNA oligonucleotides were annealed into a double-stranded DNA probe. 500 ng of purified protein and 2 μL of 5 nM DNA probe was incubated for 20 min at room temperature. In parallel, the same amount of DNA probe was incubated with the protein buffer as the negative control. For competition assays, 10×, 50× or 100× unlabeled DNA probe was added. The reaction mixtures were then resolved in 6% DNA retardation gel (Novex) by electrophoresis at 100 V for 1 h. The gel was then scanned using the Odyssey M Imaging System in the 800 nm channel (LI-COR).
Statistical Analysis
All the data shown in this study were described as mean ± SD. Comparisons between two groups were performed with two-tailed Student's t-tests. Comparisons between multiple groups were performed with one-way ANOVA, followed by Tukey's post hoc test.
Accession Numbers
Sequence data can be found under the following PHYTOZOME accession numbers: (P.trichocarpa v4.1): PtrbHLH011 (Potri.005G113400), PtrWND2B (Potri.002G178700), PtrMYB92 (Potri.001G118800), PtCesA4 (Potri.002G257900), CHS1 (Potri.014G145100), CHS5 (Potri.003G176800), YSL3 (Potri.012G024700), PYE (Potri.015G142700), FER1 (Potri.016G124900), PtIRT3 (Potri.018G053300), ZIP6 (Potri.009G074100), TT3 (Potri.002G033600), PtrMYB3 (Potri.001G267300), SND2-1 (Potri.011G058400), SND2-2 (Potri.007G135300), MYB5 (Potri.001G005100) and MYC2 (Potri.001G142200) and eIF-5A (Potri.018G107300).
Author Contributions
M.X. conceived and designed the research; M.X. wrote the manuscript; D.T., Y.D., N.D., D.K. and K.S. performed experiments; D.T. and G.L. generated transgenic plants; L.Y. and Y.Y. performed and analysed X-ray experiments; C.E.B.-H. performed phylogenetic analysis; M.X. and A.L. analysed the data; G.C., Y.Q., C.-J.L. and K.B. edited the manuscript. All authors reviewed and approved the final version of the manuscript for publication.
Acknowledgements
We thank Katherine Fedotov at Stony Brook University for preparing leaf cross-sections for X-ray fluorescent microscopy experiment. We thank Yanhao Cheng from Y.Q. lab at the University of Maryland for sharing the all-in-one CBE vector pYPQAT13. This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, as part of the Quantitative Plant Science Initiative (QPSI) at Brookhaven National Laboratory (BNL). The Fe deprivation RNA-seq (proposal: 10.46936/10.25585/60001386) was conducted by the U.S. Department of Energy Joint Genome Institute (), a DOE Office of Science User Facility, and is supported by the Office of Science of the U.S. Department of Energy, operated under Contract No. DE-AC02-05CH11231. Flavonoid and lignin analysis was partially supported by the DOE, Office of Science, Office of Basic Energy Sciences, specifically the Physical Biosciences program of the Chemical Sciences, Geosciences and Biosciences Division under contract no. DE-SC0012704 (to C.-J.L.) and by the Joint BioEnergy Institute, one of the Bioenergy Research Centers of the US DOE, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231 (C.E.B.-H.). The XRF experiments used the Submicron Resolution X-ray Spectroscopy (SRX) beamline at 5-ID of the National Synchrotron Light Source II, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. The LiX beamline is part of the Center for BioMolecular Structure (CBMS), which is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1605010). This research used the confocal microscope of the Center for Functional Nanomaterials (CFN), which is a U.S. Department of Energy Office of Science User Facility, at Brookhaven National Laboratory under Contract No. DE-SC0012704. The generation of base edited poplar lines was supported by the DOE BER program (No. DE-SC0023011) to G.C. and Y.Q., and McIntire Stennis Forest Research Program (No. MD-PSLA-24014) to Y.Q. and G.L.
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
The RNA-seq and transient ChIP-seq data that support the findings of this study are openly available on Gene Expression Omnibus (GEO; ), reference no: GSE265823 and GSE265825.
Akmakjian, G. Z., N. Riaz, and M. L. Guerinot. 2021. “Photoprotection During Iron Deficiency Is Mediated by the bHLH Transcription Factors PYE and ILR3.” Proceedings of the National Academy of Sciences, USA 118: e2024918118.
Bailey, T. L., J. Johnson, C. E. Grant, and W. S. Noble. 2015. “The MEME Suite.” Nucleic Acids Research 43: W39–W49.
Briat, J. F., C. Dubos, and F. Gaymard. 2015. “Iron Nutrition, Biomass Production, and Plant Product Quality.” Trends in Plant Science 20: 33–40.
Brown, D. M., L. A. Zeef, J. Ellis, R. Goodacre, and S. R. Turner. 2005. “Identification of Novel Genes in Arabidopsis Involved in Secondary Cell Wall Formation Using Expression Profiling and Reverse Genetics.” Plant Cell 17: 2281–2295.
Buckhout, T. J., T. J. Yang, and W. Schmidt. 2009. “Early Iron‐Deficiency‐Induced Transcriptional Changes in Arabidopsis Roots as Revealed by Microarray Analyses.” BMC Genomics 10: 147.
Chen, H. M., Y. M. Wang, H. L. Yang, Q. Y. Zeng, and Y. J. Liu. 2019. “NRAMP1 Promotes Iron Uptake at the Late Stage of Iron Deficiency in Poplars.” Tree Physiology 39: 1235–1250.
Curtis, M. D., and U. Grossniklaus. 2003. “A Gateway Cloning Vector Set for High‐Throughput Functional Analysis of Genes in Planta.” Plant Physiology 133: 462–469.
De Meester, B., R. Van Acker, M. Wouters, et al. 2022. “Field and Saccharification Performances of Poplars Severely Downregulated in CAD1.” New Phytologist 236: 2075–2090.
DiDonato, R. J., Jr., L. A. Roberts, T. Sanderson, R. B. Eisley, and E. L. Walker. 2004. “Arabidopsis Yellow Stripe‐Like2 (YSL2): A Metal‐Regulated Gene Encoding a Plasma Membrane Transporter of Nicotianamine‐Metal Complexes.” Plant Journal 39: 403–414.
Du, J., and A. Groover. 2010. “Transcriptional Regulation of Secondary Growth and Wood Formation.” Journal of Integrative Plant Biology 52: 17–27.
Dwivedi, N., S. Yamamoto, Y. Zhao, et al. 2024. “Simultaneous Suppression of Lignin, Tricin and Wall‐Bound Phenolic Biosynthesis via the Expression of Monolignol 4‐O‐Methyltransferases in Rice.” Plant Biotechnology Journal 22: 330–346.
Fini, A., C. Brunetti, M. Di Ferdinando, F. Ferrini, and M. Tattini. 2011. “Stress‐Induced Flavonoid Biosynthesis and the Antioxidant Machinery of Plants.” Plant Signaling & Behavior 6: 709–711.
Foster, C. E., T. M. Martin, and M. Pauly. 2010. “Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic Biomass) Part I: Lignin.” Journal of Visualized Experiments 37: e1745.
Gao, F., and C. Dubos. 2024. “The Arabidopsis bHLH Transcription Factor Family.” Trends in Plant Science 29: 668–680.
Gao, F., K. Robe, M. Bettembourg, et al. 2020. “The Transcription Factor bHLH121 Interacts With bHLH105 (ILR3) and Its Closest Homologs to Regulate Iron Homeostasis in Arabidopsis.” Plant Cell 32: 508–524.
Ge, M. Y., X. J. Huang, H. F. Yan, et al. 2022. “Three‐Dimensional Imaging of Grain Boundaries via Quantitative Fluorescence X‐Ray Tomography Analysis.” Communications Materials 3: 37.
Gelfand, I., R. Sahajpal, X. Zhang, R. C. Izaurralde, K. L. Gross, and G. P. Robertson. 2013. “Sustainable Bioenergy Production From Marginal Lands in the US Midwest.” Nature 493: 514–517.
Goto, F., T. Yoshihara, and H. Saiki. 2000. “Iron Accumulation and Enhanced Growth in Transgenic Lettuce Plants Expressing the Iron‐Binding Protein Ferritin.” Theoretical and Applied Genetics 100: 658–664.
Hoang, D. T., O. Chernomor, A. von Haeseler, B. Q. Minh, and L. S. Vinh. 2018. “UFBoot2: Improving the Ultrafast Bootstrap Approximation.” Molecular Biology and Evolution 35: 518–522.
Hori, C., X. Yu, J. C. Mortimer, et al. 2020. “Impact of Abiotic Stress on the Regulation of Cell Wall Biosynthesis in Populus trichocarpa.” Plant Biotechnology 37: 273–283.
Huang, D., and W. Dai. 2015. “Two Iron‐Regulated Transporter (IRT) Genes Showed Differential Expression in Poplar Trees Under Iron or Zinc Deficiency.” Journal of Plant Physiology 186: 59–67.
Kalyaanamoorthy, S., B. Q. Minh, T. K. F. Wong, A. von Haeseler, and L. S. Jermiin. 2017. “ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates.” Nature Methods 14: 587–589.
Katoh, K., and D. M. Standley. 2013. “MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability.” Molecular Biology and Evolution 30: 772–780.
Kim, S. A., I. S. LaCroix, S. A. Gerber, and M. L. Guerinot. 2019. “The Iron Deficiency Response in Arabidopsis Thaliana Requires the Phosphorylated Transcription Factor URI.” Proceedings of the National Academy of Sciences of the United States of America 116: 24933–24942.
Kumar, M., S. Thammannagowda, V. Bulone, et al. 2009. “An Update on the Nomenclature for the Cellulose Synthase Genes in Populus.” Trends in Plant Science 14: 248–254.
Kumar, R. K., H. H. Chu, C. Abundis, et al. 2017. “Iron‐Nicotianamine Transporters Are Required for Proper Long Distance Iron Signaling.” Plant Physiology 175: 1254–1268.
Kurihara, D., Y. Mizuta, Y. Sato, and T. Higashiyama. 2015. “ClearSee: A Rapid Optical Clearing Reagent for Whole‐Plant Fluorescence Imaging.” Development 142: 4168–4179.
Lee, C., Q. Teng, R. Zhong, and Z. H. Ye. 2011. “Molecular Dissection of Xylan Biosynthesis During Wood Formation in Poplar.” Molecular Plant 4: 730–747.
Lee, L. Y., M. J. Fang, L. Y. Kuang, and S. B. Gelvin. 2008. “Vectors for Multi‐Color Bimolecular Fluorescence Complementation to Investigate Protein‐Protein Interactions in Living Plant Cells.” Plant Methods 4: 24.
Li, G., S. Sretenovic, G. Coleman, and Y. Qi. 2023. “Base Editing in Poplar Through an Agrobacterium‐Mediated Transformation Method.” In Plant Genome Engineering: Methods and Protocols, 53–71. Springer.
Li, G., S. Sretenovic, E. Eisenstein, G. Coleman, and Y. Qi. 2021. “Highly Efficient C‐To‐T and A‐To‐G Base Editing in a Populus Hybrid.” Plant Biotechnology Journal 19: 1086–1088.
Li, L., H. Yan, W. Xu, et al. 2017. “PyXRF: Python‐Based X‐Ray Fluorescence Analysis Package.” In X‐Ray Nanoimaging: Instruments and Methods III, 38–45. SPIE.
Li, W., Y.‐C. J. Lin, Y.‐L. Chen, et al. 2023. “Woody Plant Cell Walls: Fundamental and Utilization.” Molecular Plant 17: 112–140.
Li, Z., and G. J. Ahammed. 2023. “Plant Stress Response and Adaptation via Anthocyanins: A Review.” Plant Stress 10: 100230.
Lin, Y. F., H. M. Liang, S. Y. Yang, et al. 2009. “Arabidopsis IRT3 Is a Zinc‐Regulated and Plasma Membrane Localized Zinc/Iron Transporter.” New Phytologist 182: 392–404.
Lin, Z., X. Zhang, P. Nandi, et al. 2024. “Correlative Single‐Cell Hard X‐Ray Computed Tomography and X‐Ray Fluorescence Imaging.” Communications Biology 7: 280.
Liu, B., J. Liu, J. Yu, et al. 2021. “Transcriptional Reprogramming of Xylem Cell Wall Biosynthesis in Tension Wood.” Plant Physiology 186: 250–269.
Liu, H., J. Gao, J. Sun, et al. 2022. “Dimerization of PtrMYB074 and PtrWRKY19 Mediates Transcriptional Activation of PtrbHLH186 for Secondary Xylem Development in Populus trichocarpa.” New Phytologist 234: 918–933.
Liu, L., V. Missirian, M. Zinkgraf, A. Groover, and V. Filkov. 2014. “Evaluation of Experimental Design and Computational Parameter Choices Affecting Analyses of ChIP‐Seq and RNA‐Seq Data in Undomesticated Poplar Trees.” BMC Genomics 15, no. Suppl 5: S3.
Long, T. A., H. Tsukagoshi, W. Busch, B. Lahner, D. E. Salt, and P. N. Benfey. 2010. “The bHLH Transcription Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots.” Plant Cell 22: 2219–2236.
Marschner, H. 2011. Marschner's Mineral Nutrition of Higher Plants. Academic Press.
Miller, M. A., W. Pfeiffer, and T. Schwartz. 2010. “Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees.” In 2010 Gateway Computing Environments Workshop (GCE), 1–8. IEEE.
Muhammad, D., N. M. Clark, S. Haque, C. M. Williams, R. Sozzani, and T. A. Long. 2022. “POPEYE Intercellular Localization Mediates Cell‐Specific Iron Deficiency Responses.” Plant Physiology 190: 2017–2032.
Nakata, M., and M. Ohme‐Takagi. 2014. “Quantification of Anthocyanin Content.” Bio‐Protocol 4: e1098.
Nazaretski, E., D. Coburn, W. Xu, et al. 2022. “A New Kirkpatrick–Baez‐Based Scanning Microscope for the Submicron Resolution X‐Ray Spectroscopy (SRX) Beamline at NSLS‐II.” Journal of Synchrotron Radiation 29: 1284–1291.
Ohtani, M., N. Nishikubo, B. Xu, et al. 2011. “A NAC Domain Protein Family Contributing to the Regulation of Wood Formation in Poplar.” Plant Journal 67: 499–512.
Pires, N., and L. Dolan. 2010. “Origin and Diversification of Basic‐Helix‐Loop‐Helix Proteins in Plants.” Molecular Biology and Evolution 27: 862–874.
Price, M. N., P. S. Dehal, and A. P. Arkin. 2010. “FastTree 2 – Approximately Maximum‐Likelihood Trees for Large Alignments.” PLoS One 5: e9490.
Ravet, K., B. Touraine, J. Boucherez, J. F. Briat, F. Gaymard, and F. Cellier. 2009. “Ferritins Control Interaction Between Iron Homeostasis and Oxidative Stress in Arabidopsis.” Plant Journal 57: 400–412.
Sanchez‐Cano, C., I. Romero‐Canelon, Y. Yang, et al. 2017. “Synchrotron X‐Ray Fluorescence Nanoprobe Reveals Target Sites for Organo‐Osmium Complex in Human Ovarian Cancer Cells.” Chemistry 23: 2512–2516.
Shanmugam, V., J. C. Lo, C. L. Wu, et al. 2011. “Differential Expression and Regulation of Iron‐Regulated Metal Transporters in Arabidopsis Halleri and Arabidopsis thaliana – The Role in Zinc Tolerance.” New Phytologist 190: 125–137.
Sundell, D., N. R. Street, M. Kumar, et al. 2017. “AspWood: High‐Spatial‐Resolution Transcriptome Profiles Reveal Uncharacterized Modularity of Wood Formation in Populus tremula.” Plant Cell 29: 1585–1604.
Tadesse, D., E. F. Yee, T. W. Wolabu, et al. 2024. “Sorghum SbGhd7 Is a Major Regulator of Floral Transition and Directly Represses Genes Crucial for Flowering Activation.” New Phytologist 242: 786–796.
Tanabe, N., M. Noshi, D. Mori, K. Nozawa, M. Tamoi, and S. Shigeoka. 2019. “The Basic Helix‐Loop‐Helix Transcription Factor, bHLH11 Functions in the Iron‐Uptake System in Arabidopsis thaliana.” Journal of Plant Research 132: 93–105.
Taylor‐Teeples, M., L. Lin, M. de Lucas, et al. 2015. “An Arabidopsis Gene Regulatory Network for Secondary Cell Wall Synthesis.” Nature 517: 571–575.
Trifinopoulos, J., L. T. Nguyen, A. von Haeseler, and B. Q. Minh. 2016. “W‐IQ‐TREE: A Fast Online Phylogenetic Tool for Maximum Likelihood Analysis.” Nucleic Acids Research 44: W232–W235.
Tsai, C. J., G. K. Podila, and V. L. Chiang. 1994. “Agrobacterium‐Mediated Transformation of Quaking Aspen (Populus tremuloides) and Regeneration of Transgenic Plants.” Plant Cell Reports 14: 94–97.
Van Wuytswinkel, O., G. Vansuyt, N. Grignon, P. Fourcroy, and J. F. Briat. 1999. “Iron Homeostasis Alteration in Transgenic Tobacco Overexpressing Ferritin.” Plant Journal 17: 93–97.
Wang, J. P., M. L. Matthews, C. M. Williams, et al. 2018. “Improving Wood Properties for Wood Utilization Through Multi‐Omics Integration in Lignin Biosynthesis.” Nature Communications 9: 1579.
Waters, B. M., H. H. Chu, R. J. Didonato, et al. 2006. “Mutations in Arabidopsis Yellow Stripe‐like1 and Yellow Stripe‐like3 Reveal Their Roles in Metal Ion Homeostasis and Loading of Metal Ions in Seeds.” Plant Physiology 141: 1446–1458.
Wu, Y., Y. He, S. Sretenovic, et al. 2022. “CRISPR‐BETS: A Base‐Editing Design Tool for Generating Stop Codons.” Plant Biotechnology Journal 20: 499–510.
Wullschleger, S. D., D. J. Weston, S. P. DiFazio, and G. A. Tuskan. 2013. “Revisiting the Sequencing of the First Tree Genome: Populus trichocarpa.” Tree Physiology 33: 357–364.
Xie, M., W. Muchero, A. C. Bryan, et al. 2018. “A 5‐Enolpyruvylshikimate 3‐Phosphate Synthase Functions as a Transcriptional Repressor in Populus.” Plant Cell 30: 1645–1660.
Xie, M., J. Zhang, T. J. Tschaplinski, G. A. Tuskan, J. G. Chen, and W. Muchero. 2018. “Regulation of Lignin Biosynthesis and Its Role in Growth‐Defense Tradeoffs.” Frontiers in Plant Science 9: 1427.
Xie, M., J. Zhang, T. Yao, et al. 2020. “Arabidopsis C‐Terminal Binding Protein ANGUSTIFOLIA Modulates Transcriptional Co‐Regulation of MYB46 and WRKY33.” New Phytologist 228: 1627–1639.
Yang, L. 2024. “X‐Ray Scattering Based Scanning Tomography for Imaging and Structural Characterization of Cellulose in Plants.” Journal of Synchrotron Radiation 31: 936–947.
Yang, L., J. L. Liu, S. Chodankar, S. Antonelli, and J. DiFabio. 2022. “Scanning Structural Mapping at the Life Science X‐Ray Scattering Beamline.” Journal of Synchrotron Radiation 29: 540–548.
Yang, Y., F. Fus, A. Pacureanu, et al. 2019. “Three‐Dimensional Correlative Imaging of a Malaria‐Infected Cell With a Hard X‐Ray Nanoprobe.” Analytical Chemistry 91: 6549–6554.
Zhang, S., J. Yang, H. Q. Li, V. C. L. Chiang, and Y. J. Fu. 2021. “Cooperative Regulation of Flavonoid and Lignin Biosynthesis in Plants.” Critical Reviews in Plant Sciences 40: 109–126.
Zhong, R., C. Lee, and Z. H. Ye. 2010. “Functional Characterization of Poplar Wood‐Associated NAC Domain Transcription Factors.” Plant Physiology 152: 1044–1055.
Zhong, R., R. L. McCarthy, C. Lee, and Z. H. Ye. 2011. “Dissection of the Transcriptional Program Regulating Secondary Wall Biosynthesis During Wood Formation in Poplar.” Plant Physiology 157: 1452–1468.
Zhong, R., and Z. H. Ye. 2009. “Transcriptional Regulation of Lignin Biosynthesis.” Plant Signaling & Behavior 4: 1028–1034.
Zhong, R., and Z. H. Ye. 2010. “The Poplar PtrWNDs Are Transcriptional Activators of Secondary Cell Wall Biosynthesis.” Plant Signaling & Behavior 5: 469–472.
Zhou, J., G. Liu, Y. Zhao, et al. 2023. “An Efficient CRISPR‐Cas12a Promoter Editing System for Crop Improvement.” Nature Plants 9: 588–604.
Zinkgraf, M., L. Liu, A. Groover, and V. Filkov. 2017. “Identifying Gene Coexpression Networks Underlying the Dynamic Regulation of Wood‐Forming Tissues in Populus Under Diverse Environmental Conditions.” New Phytologist 214: 1464–1478.
© 2025. This work is published under http://creativecommons.org/licenses/by-nc/4.0/ (the "License"). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.