The cellular and molecular processes of lenticel development during tree stem growth.

The lenticel is a channel-like structure that facilitates oxygen, carbon dioxide, and water vapor exchange on secondary growth tissue, such as a tree stem. Although the structure of lenticel has been described, there is limited understanding regarding the impact of this secondary structure on secondary growth as well as the cellular and metabolic processes underlying its formation. The study reveals the essential role of the lenticel in the process of tree secondary growth and the cellular and metabolic processes that take place during its formation. Under the stomata, lenticel development occurs when cells divide and differentiate into a structure of disconnected cells with air spaces between them. During lenticel formation, specific metabolic pathways and wax biosynthesis are activated. The SERK (somatic embryogenesis receptor kinase) gene controls lenticel density, and serk1serk3serk5 triple mutants enhance lenticel initiation. The findings shed light on the cellular and metabolic processes involved in lenticel formation, laying the groundwork for further mechanistic elucidation of their development, function, and genetic regulation in trees.

Tyrosylprotein Sulfotransferase Positively Regulates Symbiotic Nodulation and Root Growth.

Posttranslational tyrosine sulfation of peptides and proteins is catalysed by tyrosylprotein sulfotransferases (TPSTs). In Arabidopsis, tyrosine sulfation is essential for the activities of peptide hormones, such as phytosulfokine (PSK) and root meristem growth factor (RGF). Here, we identified a TPST-encoding gene, MtTPST, from model legume Medicago truncatula. MtTPST expression was detected in all organs, with the highest level in root nodules. A promoter:GUS assay revealed that MtTPST was highly expressed in the root apical meristem, nodule primordium and nodule apical meristem. The loss-of-function mutant mttpst exhibited a stunted phenotype with short roots and reduced nodule number and size. Application of both of the sulfated peptides PSK and RGF3 partially restored the defective root length of mttpst. The reduction in symbiotic nodulation in mttpst was partially recovered by treatment with sulfated PSK peptide. MtTPST-PSK module functions downstream of the Nod factor signalling to promote nodule initiation via regulating accumulation and/or signalling of cytokinin and auxin. Additionally, the small-nodule phenotype of mttpst, which resulted from decreased apical meristematic activity, was partially complemented by sulfated RGF3 treatment. Together, these results demonstrate that MtTPST, through its substrates PSK, RGF3 and other sulfated peptide(s), positively regulates nodule development and root growth.

Advancements of CRISPR-Mediated Base Editing in Crops and Potential Applications in Populus.

Base editing represents a cutting-edge genome editing technique that utilizes the CRISPR system to guide base deaminases with high precision to specific genomic sites, facilitating the targeted alteration of individual nucleotides. Unlike traditional gene editing approaches, base editing does not require DNA double-strand breaks or donor templates. It functions independently of the cellular DNA repair machinery, offering significant advantages in terms of both efficiency and accuracy. In this review, we summarize the core design principles of various DNA base editors, their distinctive editing characteristics, and tactics to refine their efficacy. We also summarize their applications in crop genetic improvement and explore their potential contributions to forest genetic engineering.

Acetyl Bromide Method for Total Lignin Content Determination in Plant Biomass.

Lignin is a tough biopolymer that gives plants strength and protection. It is also a major obstacle for converting plant biomass into biofuels because it prevents enzymes from accessing the sugar-rich fibers. To optimize biofuel production, we need to measure the lignin content in plant tissues accurately and efficiently. In this protocol, we describe a simple and reliable method to measure the total lignin content in plant tissues. The method uses acetyl bromide, a chemical that dissolves lignin into soluble derivatives and makes it possible to detect them by their absorbance at 280 nm. The method consists of two steps: first, we obtained destarched cell wall material from the plant samples, and second, we treat the cell wall material with acetyl bromide and measure the absorbance of the lignin solution. This method can capture all types of lignin and works well with different plant tissues.

OsTCP19 coordinates inhibition of lignin biosynthesis and promotion of cellulose biosynthesis to modify lodging resistance in rice.

Lignin and cellulose are two essential elements of plant secondary cell walls that shape the mechanical characteristics of the culm to prevent lodging. However, how the regulation of the lignin and cellulose composition is combined to achieve optimal mechanical characteristics is unclear. Here, we show that increasing OsTCP19 expression in rice coordinately repressed lignin biosynthesis and promoted cellulose biosynthesis, resulting in enhanced lodging resistance. In contrast, repression of OsTCP19 coordinately promoted lignin biosynthesis and inhibited cellulose biosynthesis, leading to greater susceptibility to lodging. We found that OsTCP19 binds to the promoters of both MYB108 and MYB103L to increase their expression, with the former being responsible for repressing lignin biosynthesis and the latter for promoting cellulose biosynthesis. Moreover, up-regulation of OsTCP19 in fibers improved grain yield and lodging resistance. Thus, our results identify the OsTCP19-OsMYB108/OsMYB103L module as a key regulator of lignin and cellulose production in rice, and open up the possibility for precisely manipulating lignin-cellulose composition to improve culm mechanical properties for lodging resistance.

The MPK6-LTF1L1 module regulates lignin biosynthesis in rice through a distinct mechanism from Populus LTF1.

Lignin is a complex polymer that provides structural support and defense to plants. It is synthesized in the secondary cell walls of specialized cells. Through regulates its stability, LTF1 acts as a switch to control lignin biosynthesis in Populus, a dicot plant. However, how lignin biosynthesis is regulated in rice, a monocot plant, remains unclear. By employing genetic, cellular, and chemical approaches, we discovered that LTF1L1, a rice homolog of LTF1, regulates lignin biosynthesis through a distinct mechanism from Populus LTF1. Knockout of LTF1L1 increased lignin synthesis in the sclerenchyma cells of rice stems, while overexpression of LTF1L1 decreased it. LTF1L1 is phosphorylated by OsMPK6 at Ser169, which did not affect its stability but impaired its ability to repress the expression of lignin biosynthesis genes. This was supported by the non-phosphorylated mutant of LTF1L1 (LTF1L1S169A), which displayed a stronger repressive effect on lignin biosynthesis in both rice and Populus. Our findings reveal that LTF1L1 acts as a negative regulator of lignin biosynthesis via a distinct mechanism from that of LTF1 in Populus and highlight the evolutionary diversity in the regulation of lignin biosynthesis in plants.

Chromosome-scale genomes of commercial timber trees (Ochroma pyramidale, Mesua ferrea, and Tectona grandis).

Wood is the most important natural and endlessly renewable source of energy. Despite the ecological and economic importance of wood, many aspects of its formation have not yet been investigated. We performed chromosome-scale genome assemblies of three timber trees (Ochroma pyramidale, Mesua ferrea, and Tectona grandis) which exhibit different wood properties such as wood density, hardness, growth rate, and fiber cell wall thickness. The combination of 10X, stLFR, Hi-Fi sequencing and HiC data led us to assemble high-quality genomes evident by scaffold N50 length of 55.97 Mb (O. pyramidale), 22.37 Mb (M. ferrea) and 14.55 Mb (T. grandis) with >97% BUSCO completeness of the assemblies. A total of 35774, 24027, and 44813 protein-coding genes were identified in M. ferrea, T. grandis and O. pyramidale, respectively. The data generated in this study is anticipated to serve as a valuable genetic resource and will promote comparative genomic analyses, and it is of practical importance in gaining a further understanding of the wood properties in non-model woody species.

Screening genome-editing knockouts reveals the receptor-like kinase ASX role in regulations of secondary xylem development in Populus.

In trees, secondary xylem development is essential for the growth of perennial stem increments. Many signals regulate the process of development, but our knowledge of the molecular components involved in signal transduction is still limited. In this study, we identified Attenuation of Secondary Xylem (ASX) knockouts by screening genome-editing knockouts of xylem-expressed receptor-like kinases (RLKs) in Populus. The ASX role in secondary xylem development in Populus was discovered using biochemical, cellular, and genomic analyses. The ASX knockout plants had abnormal secondary stem growth but had little effect on shoot apical primary growth. ASX and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)2/4 were co-precipitated in developing xylem. Through their interaction, ASX is phosphorylated by SERK. Transcriptome analysis of developing xylem revealed that ASX deficiency inhibited the transcriptional activity of genes involved in xylem differentiation and secondary cell wall formation. By forming a complex, ASX and SERK may function as a signaling module for signal transduction required in the regulation of secondary xylem development in trees. This study shows that ASX, which encodes a RLKs, is required for secondary xylem development and sheds light on regulatory signals found in tree stem secondary growth.

An MKP-MAPK protein phosphorylation cascade controls vascular immunity in plants.

Global crop production is greatly reduced by vascular diseases. These diseases include bacterial blight of rice and crucifer black rot caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas campestris pv. campestris (Xcc). The molecular mechanisms that activate vascular defense against such pathogens remains underexplored. Here, we show that an Arabidopsis MAPK phosphatase 1 (MKP1) mutant has increased host susceptibility to the adapted pathogen Xcc and is compromised in nonhost resistance to the rice pathogen Xoo. MKP1 regulates MAPK-mediated phosphorylation of the transcription factor MYB4 that negatively regulates vascular lignification through inhibiting lignin biosynthesis. Induction of lignin biosynthesis is, therefore, an important part of vascular-specific immunity. The role of MKP-MAPK-MYB signaling in lignin biosynthesis and vascular resistance to Xoo is conserved in rice, indicating that these factors form a tissue-specific defense regulatory network. Our study likely reveals a major vascular immune mechanism that underlies tissue-specific disease resistance against bacterial pathogens in plants.

A Comparative Analysis of Transcription Networks Active in Juvenile and Mature Wood in Populus.

Juvenile wood (JW) and mature wood (MW) have distinct physical and chemical characters, resulting from wood formation at different development phases over tree lifespan. However, the regulatory mechanisms that distinguish or modulate the characteristics of JW and MW in relation to each other have not been mapped. In this study, by employing the Populus trees with an identical genetic background, we carried out RNA sequencing (RNA-seq) and whole genome bisulfite sequencing (WGBS) in JW and MW forming tissue and analyzed the transcriptional programs in association with the wood formation in different phrases. JW and MW of Populus displayed different wood properties, including higher content of cellulose and hemicelluloses, less lignin, and longer and larger fiber cells and vessel elements in MW as compared with JW. Significant differences in transcriptional programs and patterns of DNA methylation were detected between JW and MW. The differences were concentrated in gene networks involved in regulating hormonal signaling pathways responsible for auxin distribution and brassinosteroids biosynthesis as well as genes active in regulating cell expansion and secondary cell wall biosynthesis. An observed correlation between gene expression profiling and DNA methylation indicated that DNA methylation affected expression of the genes related to auxin distribution and brassinosteroids signal transduction, cell expansion in JW, and MW formation. The results suggest that auxin distribution, brassinosteroids biosynthesis, and signaling be the critical molecular modules in formation of JW and MW. DNA methylation plays a role in formatting the molecular modules which contribute to the transcriptional programs of wood formation in different development phases. The study sheds light into better understanding of the molecular networks underlying regulation of wood properties which would be informative for genetic manipulation for improvement of wood formation.

Cell-Specific Suppression of 4-Coumarate-CoA Ligase Gene Reveals Differential Effect of Lignin on Cell Physiological Function in Populus.

Lignin is a main component of the secondary cell wall in vessels and fibers of xylem tissue. However, the significance of lignin in cell physiology during plant growth is unclear. In this study, we generated lignin-modified Populus via cell-specific downregulation of the 4-coumarate-CoA ligase gene (4CL). The transgenic plants with selective lignin modification in vessel elements or fiber cells allowed us to investigate how lignin affects the physiology of vessel or fiber cells in relation to plant growth. Results showed that vessel-specific suppression of lignin biosynthesis resulted in deformed vessels and normal fibers, while fiber-specific suppression of lignin biosynthesis led to less-lignified fibers and normal vessels. Further analyses revealed that the efficiency of long distance water transport was severely affected in transgenics with vessel-specific lignin modification, while minimal effect was detected in transgenics with fiber-specific lignin modification. Vessel-specific lignin reduction led to high susceptibility to drought stress and poor growth in field, likely due to vessel defects in long distance transport of water. The distinct physiological significance of lignin in different cell types provides insights into the selective modification of lignin for improvement of lignocellulosic biomass utilization.

Phenylpropanoid Derivatives Are Essential Components of Sporopollenin in Vascular Plants.

The outer wall of pollen and spores, namely the exine, is composed of sporopollenin, which is highly resistant to chemical reagents and enzymes. In this study, we demonstrated that phenylpropanoid pathway derivatives are essential components of sporopollenin in seed plants. Spectral analyses showed that the autofluorescence of Lilium and Arabidopsis sporopollenin is similar to that of lignin. Thioacidolysis and NMR analyses of pollen from Lilium and Cryptomeria further revealed that the sporopollenin of seed plants contains phenylpropanoid derivatives, including p-hydroxybenzoate (p-BA), p-coumarate (p-CA), ferulate (FA), and lignin guaiacyl (G) units. The phenylpropanoid pathway is expressed in the tapetum in Arabidopsis, consistent with the fact that the sporopollenin precursor originates from the tapetum. Further germination and comet assays showed that this pathway plays an important role in protection of pollen against UV radiation. In the pteridophyte plant species Ophioglossum vulgatum and Lycopodium clavata, phenylpropanoid derivatives including p-BA and p-CA were also detected, but G units were not. Taken together, our results indicate that phenylpropanoid derivatives are essential for sporopollenin synthesis in vascular plants. In addition, sporopollenin autofluorescence spectra of bryophytes, such as Physcomitrella and Haplocladium, exhibit distinct characteristics compared with those of vascular plants, indicating the diversity of sporopollenin among land plants.

Fibre-specific regulation of lignin biosynthesis improves biomass quality in Populus.

Lignin is a major component of cell wall biomass and decisively affects biomass utilisation. Engineering of lignin biosynthesis is extensively studied, while lignin modification often causes growth defects. We developed a strategy for cell-type-specific modification of lignin to achieve improvements in cell wall property without growth penalty. We targeted a lignin-related transcription factor, LTF1, for modification of lignin biosynthesis. LTF1 can be engineered to a nonphosphorylation form which is introduced into Populus under the control of either a vessel-specific or fibre-specific promoter. The transgenics with lignin suppression in vessels showed severe dwarfism and thin-walled vessels, while the transgenics with lignin suppression in fibres displayed vigorous growth with normal vessels under phytotron, glasshouse and field conditions. In-depth lignin structural analyses revealed that such cell-type-specific downregulation of lignin biosynthesis led to the alteration of overall lignin composition in xylem tissues reflecting the population of distinctive lignin polymers produced in vessel and fibre cells. This study demonstrates that fibre-specific suppression of lignin biosynthesis resulted in the improvement of wood biomass quality and saccharification efficiency and presents an effective strategy to precisely regulate lignin biosynthesis with desired growth performance.

A xylem-produced peptide PtrCLE20 inhibits vascular cambium activity in Populus.

In trees, lateral growth of the stem occurs through cell divisions in the vascular cambium. Vascular cambium activity is regulated by endogenous developmental programmes and environmental cues. However, the underlying mechanisms that regulate cambium activity are largely unknown. Genomic, biochemical and genetic approaches were used here to elucidate the role of PtrCLE20, a CLAVATA3 (CLV3)/embryo surrounding region (ESR)-related peptide gene, in the regulation of lateral growth in Populus. Fifty-two peptides encoded by CLE genes were identified in the genome of Populus trichocarpa. Among them PtrCLE20 transcripts were detected in developing xylem while the PtrCLE20 peptide was mainly localized in vascular cambium cells. PtrCLE20 acted in repressing vascular cambium activity indicated by that upregulation of PtrCLE20 resulted in fewer layers of vascular cambium cells with repressed expression of the genes related to cell dividing activity. PtrCLE20 peptide also showed a repression effect on the root growth of Populus and Arabidopsis, likely through inhibiting meristematic cell dividing activity. Together, the results suggest that PtrCLE20 peptide, produced from developing xylem cells, plays a role in regulating lateral growth by repression of cambium activity in trees.

Phosphorylation of LTF1, an MYB Transcription Factor in Populus, Acts as a Sensory Switch Regulating Lignin Biosynthesis in Wood Cells.

Lignin is specifically deposited in plant secondary cell walls, and initiation of lignin biosynthesis is regulated by a variety of developmental and environmental signals. However, the mechanisms governing the regulation of lignin biosynthesis remain to be elucidated. In this study, we identified a lignin biosynthesis-associated transcription factor (LTF) from Populus, LTF1, which binds the promoter of a key lignin biosynthetic gene encoding 4-coumarate-CoA ligase (4CL). We showed that LTF1 in its unphosphorylated state functions as a regulator restraining lignin biosynthesis. When LTF1 becomes phosphorylated by PdMPK6 in response to external stimuli such as wounding, it undergoes degradation through a proteasome pathway, resulting in activation of lignification. Expression of a phosphorylation-null mutant version of LTF1 led to stable protein accumulation and persistent attenuation of lignification in wood cells. Taken together, our study reveals a mechanism whereby LTF1 phosphorylation acts as a sensory switch to regulate lignin biosynthesis in response to environmental stimuli. The discovery of novel modulators and mechanisms modifying lignin biosynthesis has important implications for improving the utilization of cell-wall biomass.

The Receptor-Like Kinase AtVRLK1 Regulates Secondary Cell Wall Thickening.

During the growth and development of land plants, some specialized cells, such as tracheary elements, undergo secondary cell wall thickening. Secondary cell walls contain additional lignin, compared with primary cell walls, thus providing mechanical strength and potentially improving defenses against pathogens. However, the molecular mechanisms that initiate wall thickening are unknown. In this study, we identified an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like kinase, encoded by AtVRLK1 (Vascular-Related Receptor-Like Kinase1), that is expressed specifically in cells undergoing secondary cell wall thickening. Suppression of AtVRLK1 expression resulted in a range of phenotypes that included retarded early elongation of the inflorescence stem, shorter fibers, slower root growth, and shorter flower filaments. In contrast, up-regulation of AtVRLK1 led to longer fiber cells, reduced secondary cell wall thickening in fiber and vessel cells, and defects in anther dehiscence. Molecular and cellular analyses showed that down-regulation of AtVRLK1 promoted secondary cell wall thickening and up-regulation of AtVRLK1 enhanced cell elongation and inhibited secondary cell wall thickening. We propose that AtVRLK1 functions as a signaling component in coordinating cell elongation and cell wall thickening during growth and development.

The Receptor-Like Cytoplasmic Kinase STRK1 Phosphorylates and Activates CatC, Thereby Regulating H2O2 Homeostasis and Improving Salt Tolerance in Rice.

Salt stress can significantly affect plant growth and agricultural productivity. Receptor-like kinases (RLKs) are believed to play essential roles in plant growth, development, and responses to abiotic stresses. Here, we identify a receptor-like cytoplasmic kinase, salt tolerance receptor-like cytoplasmic kinase 1 (STRK1), from rice (Oryza sativa) that positively regulates salt and oxidative stress tolerance. Our results show that STRK1 anchors and interacts with CatC at the plasma membrane via palmitoylation. CatC is phosphorylated mainly at Tyr-210 and is activated by STRK1. The phosphorylation mimic form CatCY210D exhibits higher catalase activity both in vitro and in planta, and salt stress enhances STRK1-mediated tyrosine phosphorylation on CatC. Compared with wild-type plants, STRK1-overexpressing plants exhibited higher catalase activity and lower accumulation of H2O2 as well as higher tolerance to salt and oxidative stress. Our findings demonstrate that STRK1 improves salt and oxidative tolerance by phosphorylating and activating CatC and thereby regulating H2O2 homeostasis. Moreover, overexpression of STRK1 in rice not only improved growth at the seedling stage but also markedly limited the grain yield loss under salt stress conditions. Together, these results offer an opportunity to improve rice grain yield under salt stress.

Shortened Basal Internodes Encodes a Gibberellin 2-Oxidase and Contributes to Lodging Resistance in Rice.

Breeding semi-dwarf varieties to improve lodging resistance has been proven to be enormously successful in increasing grain yield since the advent of the "green revolution." However, the breeding of the majority of semi-dwarf rice varieties in Asia has been dependent mainly on genetic introduction of the mutant alleles of SD1, which encodes a gibberellin (GA) 20-oxidase, OsGA20ox2, for catalyzing GA biosynthesis. Here, we report a new rice lodging-resistance gene, Shortened Basal Internodes (SBI), which encodes a gibberellin 2-oxidase and specifically controls the elongation of culm basal internodes through deactivating GA activity. SBI is predominantly expressed in culm basal internodes. Genetic analyses indicate that SBI is a semi-dominant gene affecting rice height and lodging resistance. SBI allelic variants display different activities and are associated with the height of rice varieties. Breeding with higher activity of the SBI allele generates new rice varieties with improved lodging resistance and increased yield. The discovery of the SBI provides a desirable gene resource for producing semi-dwarf rice phenotypes and offers an effective strategy for breeding rice varieties with enhanced lodging resistance and high yield.

OsREM4.1 Interacts with OsSERK1 to Coordinate the Interlinking between Abscisic Acid and Brassinosteroid Signaling in Rice.

Crosstalk among phytohormones is crucial for balancing plant growth and adjustment to various environments. Abscisic acid (ABA) and brassinosteroids (BRs) exhibit antagonistic interactions during many plant development processes, but little is known about the molecular mechanism mediating those interactions. Here, we identified a rice (Oryza sativa) remorin gene, OsREM4.1, whose expression is upregulated by ABA through the transcriptional activator OsbZIP23. OsREM4.1, in return, negatively regulates BR signaling output. We discovered that OsREM4.1 interacts with OsSERK1 to inhibit its interaction with rice BR receptor OsBRI1. Moreover, OsBRI1 could phosphorylate OsREM4.1 to reduce the binding affinity of OsREM4.1 to OsSERK1. These results demonstrate that OsREM4.1 is transcriptionally regulated by ABA and functions as an OsBRI1 substrate and OsSERK1-interacting protein to inhibit the formation and subsequent activation of the OsBRI1-OsSERK1 receptor complex. Our findings provide insight into the mechanism by which the antagonistic interactions between ABA and BRs are coordinated in rice.

Suppression of PtrDUF579-3 Expression Causes Structural Changes of the Glucuronoxylan in Populus.

DUF579 (domain unknown function 579) genes have been reported to play diverse roles in cell wall biosynthesis, such as in glucuronoxylan (GX) synthesis. As GX is a major type of hemicelluloses in hard wood species, how DUF579 genes function in wood formation remains to be demonstrated in planta. This study reports a Populus DUF579 gene, PtrDUF579-3, which is characterized for its function in wood cell wall formation. PtrDUF579-3 is localized in Golgi apparatus and catalyzes methylation of the glucuronic acid (GlcA) in GX biosynthesis. Suppression of PtrDUF579-3 expression in Populus caused a reduction in both the GlcA side chain number and GlcA side chain methylation on the GX backbone. The modified GX polymer through PtrDUF579-3 suppression was more susceptible to acid treatment and the PtrDUF579-3 suppressed plants displayed enhanced cellulose digestibility. These results suggest that PtrDUF579-3 is involved in GX biosynthesis and GX structure can be modified through PtrDUF579-3 suppression in Populus.