Integrative Dissection of Lignin Composition in Tartary Buckwheat Seed Hulls for Enhanced Dehulling Efficiency.

The rigid hull encasing Tartary buckwheat seeds necessitates a laborious dehulling process before flour milling, resulting in considerable nutrient loss. Investigation of lignin composition is pivotal in understanding the structural properties of tartary buckwheat seeds hulls, as lignin is key determinant of rigidity in plant cell walls, thus directly impacting the dehulling process. Here, the lignin composition of seed hulls from 274 Tartary buckwheat accessions is analyzed, unveiling a unique lignin chemotype primarily consisting of G lignin, a common feature in gymnosperms. Furthermore, the hardness of the seed hull showed a strong negative correlation with the S lignin content. Genome-wide detection of selective sweeps uncovered that genes governing the biosynthesis of S lignin, specifically two caffeic acid O-methyltransferases (COMTs) and one ferulate 5-hydroxylases, are selected during domestication. This likely contributed to the increased S lignin content and decreased hardness of seed hulls from more domesticated varieties. Genome-wide association studies identified robust associations between FtCOMT1 and the accumulation of S lignin in seed hull. Transgenic Arabidopsis comt1 plants expressing FtCOMT1 successfully reinstated S lignin content, confirming its conserved function across plant species. These findings provide valuable metabolic and genetic insights for the potential redesign of Tartary buckwheat seed hulls.

Comparative genomic survey and functional analysis of DKKL1 during spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis).

A feature of the Chinese soft-shelled turtle (Pelodiscus sinensis) is seasonal spermatogenesis; however, the underlying molecular mechanism is not well clarified. Here, we firstly cloned and characterized P. sinensis DKKL1, and then performed comparative genomic studies, expression analysis, and functional validation. P. sinensis DKKL1 had 2 putative N-glycosylation sites and 16 phosphorylation sites. DKKL1 also had classic transmembrane structures that were extracellularly localized. DKKL1's genetic distance was close to turtles, followed by amphibians and mammals, but its genetic distance was far from fishes. DKKL1 genes from different species shared distinct genomic characteristics. Meanwhile, they were also relatively conserved among themselves, at least from the perspective of classes. Notably, the transcription factors associated with spermatogenesis were also identified, containing CTCF, EWSR1, and FOXL2. DKKL1 exhibited sexually dimorphic expression only in adult gonads, which was significantly higher than that in other somatic tissues (P < 0.001), and was barely expressed in embryonic gonads. DKKL1 transcripts showed a strong signal in sperm, while faint signals were detected in other male germ cells. DKKL1 in adult testes progressively increased per month (P < 0.05), displaying a seasonal expression trait. DKKL1 was significantly downregulated in testes cells after the sex hormones (17ß-estradiol and 17α-methyltestosterone) and Wnt/ß-catenin inhibitor treatment (P < 0.05). Likewise, the Wnt/ß-catenin inhibitor treatment dramatically repressed CTCF, EWSR1, and FOXL2 expression. Conversely, they were markedly upregulated after the 17ß-estradiol and 17α-methyltestosterone treatment, suggesting that the three transcription factors might bind to different promoter regions, thereby negatively regulating DKKL1 transcription in response to the changes in the estrogen and androgen pathways, and positively controlling DKKL1 transcription in answer to the alterations in the Wnt/ß-catenin pathway. Knockdown of DKKL1 significantly reduced the relative expression of HMGB2 and SPATS1 (P < 0.01), suggesting that it may be involved in seasonal spermatogenesis of P. sinensis through a positive regulatory interaction with these two genes. Overall, our findings provide novel insights into the genome evolution and potential functions of seasonal spermatogenesis of P. sinensis DKKL1.

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.

Vacuolar H+ -ATPase subunit VAB3 regulates cell growth and ion homeostasis in Arabidopsis.

Vacuolar H+ -ATPase (V-ATPase) has diverse functions related to plant development and growth. It creates the turgor pressure that drives cell growth by generating the energy needed for the active transport of solutes across the tonoplast. V-ATPase is a large protein complex made up of multiheteromeric subunits, some of which have unknown functions. In this study, a forward genetics-based strategy was employed to identify the vab3 mutant, which displayed resistance to isoxaben, a cellulose synthase inhibitor that could induce excessive transverse cell expansion. Map-based cloning and genetic complementary assays demonstrated that V-ATPase B subunit 3 (VAB3) is associated with the observed insensitivity of the mutant to isoxaben. Analysis of the vab3 mutant revealed defective ionic homeostasis and hypersensitivity to salt stress. Treatment with a V-ATPase inhibitor exacerbated ionic tolerance and cell elongation defects in the vab3 mutant. Notably, exogenous low-dose Ca2+ or Na+ could partially restore isoxaben resistance of the vab3 mutant, suggesting a relationship between VAB3-regulated cell growth and ion homeostasis. Taken together, the results of this study suggest that the V-ATPase subunit VAB3 is required for cell growth and ion homeostasis in Arabidopsis.

MiR-296-3p inhibits cell proliferation by the SOX4-Wnt/ßcatenin pathway in triple-negative breast cancer.

MicroRNAs (miRNAs) have been demonstrated to play critical roles in the tumorigenesis of triple-negative breast cancer (TNBC). In this work, we addressed the specific role of miR-296-3p in TNBC. The levels of miR-296-3p and SOX4 were determined using RT-qPCR. The function of miR-296-3p overexpression on TNBC cell proliferation, migration, invasion, cancer stem cell (CSC)-like properties, and Wnt pathway activation was investigated by MTT, EdU, wound healing, Transwell, sphere formation assays and western blot. Mechanistic investigations, including luciferase reporter, RNA pull-down, and RIP assays, were con-ducted to explore the regulatory mechanisms of miR-296-3p. We found that miR-296-3p was downregulated in TNBC tissues and cells. Overexpression of miR-296-3p suppressed TNBC cell proliferation, migration, invasion, and CSC-like properties. Furthermore, miR-296-3p could bind to SOX4 and negatively modulate SOX4 expression. In addition, miR-296-3p was verified to inhibit Wnt/ß-catenin pathway by downregulating SOX4. Moreover, overexpression of SOX4 or activation of Wnt pathway rescued the miR-296-3p upregulation-mediated suppressive effect on cellular processes in TNBC. In conclusion, miR-296-3p inhibits Wnt/ß-catenin pathway by targeting SOX4 and exerts anti-tumor effects in TNBC.

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.

A small molecule inhibits cell elongation by modulating cell wall polysaccharide composition in Arabidopsis.

The plant primary cell wall is comprised of pectin, cellulose and hemicelluloses, whose dynamic interactions play essential roles in plant cell elongation. Through a chemical genetics screening, we identified a small molecule, named cell wall modulator (CWM), which disrupted cell growth and deformed cell shape in etiolated Arabidopsis hypocotyl. A pectin defective mutant qua2, identified from screening an Arabidopsis EMS mutant library, showed a reduced sensitivity to CWM treatment. On the other hand, pectinase treatment suppressed the CWM induced phenotype. Furthermore, cellulose content was decreased in response to CWM treatment, while the cellulose synthesis mutants ixr1 and ixr2 were hypersensitive to CWM. Together, the study identified a small molecule CWM that induced a modification of the cell wall in elongating cells, likely through interfering with pectin modification. This molecule may be used as a tool to study cell wall remodeling during plant growth.

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.

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.