Phytohormone abscisic acid (ABA) induces the stomatal closure in plants under drought stress, a process requiring actin reorganization. Yet how ABA perception couples with cytoskeletal dynamics in stomatal closure remains unclear. In this study, we report that Arabidopsis cyclase-associated protein 1 (CAP1) functions as a negative regulator of ABA-induced drought responses through modulating actin network organization in Arabidopsis. Further analyses demonstrate that CAP1 interacts with RCAR12 suppresses actin filaments (F-actin) disassembly, whereas ABA disrupts the CAP1-RCAR12 interaction, thus recovering CAP1's depolymerization activity. Further, ABA-activated OST1 (OPEN STOMATA 1) phosphorylates CAP1 in Arabidopsis. OST1-mediated phosphorylation of CAP1 attenuates CAP1 binding to ADF4 and inhibits the ADF4-CAP1 complex. This inhibition leads to F-actin stabilization, which in turn maintains stomata in a closed state. This study demonstrates that CAP1 orchestrates ABA-induced stomatal closure under drought stress in Arabidopsis. Specifically, CAP1 acts by coupling ABA signaling to dynamic reorganization of actin cytoskeleton.

Cell wall matrices undergo continuous remodeling during plant growth and development. The methyl- and de-methyl-esterification of pectic homogalacturonan dynamically influence the physicochemical and mechanical properties of cell walls, and consequently regulate organ morphogenesis. Phloem plays a crucial role in the transport of photoassimilates and other organic molecules from source to sink tissues, and the walls of phloem cells are rich in pectins. However, how pectin metabolism orchestrates phloem architecture and function is unclear. This work demonstrates that the methyl-esterification status and subsequent degradation of pectic homogalacturonan affect flowering time by modulating phloem development and transport capacity, which control the transport of a mobile florigen, FLOWERING LOCUS T protein, in the phloem stream. During this process, auxin signaling is stimulated by pectin de-methyl-esterification to influence vasculogenesis. Together, this data provide new insights into the mechanisms by which pectin chemistry is coordinated with auxin signaling to mediate phloem development, long-distance transport and flowering time.

Hepatocellular carcinoma (HCC) is a common primary malignancy of the liver and has a high mortality. Major facilitator superfamily domain containing 2 (MFSD2A) was previously demonstrated to inhibit tumor progression in several cancers. Here, we elucidated the association between MFSD2A expression and HCC progression and also investigated the underlying mechanism. The online tools were utilized to evaluate MFSD2A expression in HCC samples and predict the prognostic significance of MFSD2A in HCC patients. The biological role of MFSD2A in HCC cellular processes was examined by colony formation, wound healing, transwell, and western blotting. The in vivo role of MFSD2A in HCC was investigated in a xenograft tumor model. The miRNAs and RNA-binding proteins potentially targeting MFSD2A were identified using bioinformatics prediction tools. Luciferase reporter, RNA immunoprecipitation, actinomycin D, and immunofluorescence assays were performed to investigate the molecule mechanisms of MFSD2A. Transforming growth factor (TGF)-β1/Small mothers against decapentaplegic (Smad) signaling was detected using western blot analysis. We found that MFSD2A expression was significantly downregulated in HCC patients and cells and its downregulation predicted a poor prognosis. MFSD2A overexpression repressed HCC cell proliferation, migration, invasion, the epithelial-to-mesenchymal transition in vitro, as well as inhibited HCC tumor growth in vivo. MFSD2A was targeted by miR-3189-3p. High-density lipoprotein binding protein (HDLBP) inhibited MFSD2A expression by binding to and destabilizing MFSD2A mRNA. MFSD2A significantly suppressed activation of TGF-β/Smad signaling in HCC cells. Knockdown of MFSD2A abrogated the inhibitory effect of miR-3189-3p inhibitor on HCC cellular processes, and overexpression of MFSD2A reversed the tumor-promoting effect of HDLBP overexpression. Overall, MFSD2A exerts a tumor-inhibiting effect in HCC via suppression of TGF-β/Smad signaling, suggesting that MFSD2A may be a promising target for HCC therapy.

Plants sense the breakdown products of cell wall components to trigger effective stress responses and to adjust wall synthesis during development. Oligogalacturonides (OGs), derived from pectin degradation, are known to serve as signals for cell wall remodeling and stress responses, while little is known about their perception by plant cells. Here, we characterized a malectin-like domain-containing leucine-rich repeat receptor-like protein kinase MLOP1, which is involved in pectin fragment signal sensing in Arabidopsis thaliana. Mutations of MLOP1 impaired cell wall synthesis and cell elongation in seedling hypocotyls and primary roots. MLOP1 and its homologs were tandem duplicated during evolution. It is expressed in the rapid-growth region of hypocotyl, root elongation zone, leaf and stem vasculature, and silique. MLOP1-GFP is localized to the plasma membrane. The MLOP1 ectodomain is capable to bind with pectic polysaccharide in vitro, and the kinase domain may mediate intracellular signal transduction. Tests on mlop1 mutants indicate its involvement in OG-induced cell defense responses, including phytoalexin synthesis, nitric oxide (NO) accumulation, callose deposition and cytosolic Ca2+ spike. Accordingly, mlop1 mutants showed a high susceptibility to Pseudomonas syringae infection. Furthermore, loss of MLOP1 function alleviated OG-induced inhibition of cellulose synthase complex (CSC) mobility and its density on the plasma membrane. These results indicate that MLOP1 plays a role in pectin fragment signaling and mediates their effects on stress responses and cell wall remodeling.

BACKGROUND:Long noncoding RNAs (LncRNAs) have been demonstrated to have significant roles in the carcinogenesis of hepatocellular carcinoma (HCC). In this work, we sought to determine LncRNA SH3BP5-AS1's function and mechanism in the emergence of HCC. RESULTS:First, we discovered that the advanced tumor stage was strongly correlated with high levels of LncRNA SH3BP5-AS1 expression in HCC. MiR-6838-5p expression was down-regulated and inversely correlated with SH3BP5-AS1 expression. Additionally, overexpression of SH3BP5-AS1 boosted cell invasion, migration, and proliferation. The oncogenic effects of the inhibitor of miR-6838-5p were eliminated when PTPN4 was suppressed, following the identification of PTPN4 as a direct target of miR-6838-5p. In addition, SH3BP5-AS1 promoted cellular glycolysis via miR-6838-5p sponging and PTPN4 activation. Lastly, by directly interacting to the promoter of SH3BP5-AS1, HIF-1α could control the transcription of the gene. CONCLUSIONS:Our research suggests that SH3BP5-AS1 controls miR-6838-5p/PTPN4 in order to act as a new carcinogenic LncRNA during the growth of HCC cells. METHODS:The expression levels of SH3BP5-AS1, miR-6838-5p and PTPN4 were detected by qRT-PCR and Western blot. The effects of LncRNA SH3BP5-AS1/miR-6838-5p/PTPN4 on the proliferation, metastasis and glycolysis of HCC cells were clarified by experimental cellular functionality assays, cell derived xenograft and Glycolysis assay.

Fruit ripening in tomato is a highly coordinated developmental process accompanied with fruit softening, which is closely associated with cell wall degradation and remodeling. Xyloglucan endotransglucosylase/hydrolases (XTHs) are known to play an essential role in cell wall xyloglucan metabolism. Tomato XTH5 exhibits xyloglucan endotransglucosylase (XET) activity in vitro, but the understanding of its biological role in fruit ripening remains unclear. In this study, we revealed that SlXTH5 is highly expressed in mature fruits. Knockout mutant plants of SlXTH5 were generated by CRISPR/Cas9 gene editing strategy in tomato cultivar Micro-Tom. The mutant fruits showed accelerated transition from unripe to ripe process and earlier ethylene accumulation compared to wild type fruits. Although the mutation of SlXTH5 did not affect the size, weight and number of fruits, it indeed increased fruit firmness and extended shelf life, which is probably attributed to the increased cell layer and cell wall thickness of pericarp tissue. Pathogen infection experiment showed the enhanced resistance of mutant fruits to Botrytis cinerea. These results revealed the role of SlXTH5 in fruit ripening process, and provide new insight into how cell wall metabolism and remodeling regulate fruit softening and shelf life.

Xyloglucan, a major hemicellulose, interacts with cellulose and pectin to assemble primary cell walls in plants. Loss of the xyloglucan galactosyltransferase MURUS3 (MUR3) leads to the deficiency of galactosylated xyloglucan and perturbs plant growth. However, it is unclear whether defects in xyloglucan galactosylation influence the synthesis of other wall polysaccharides, cell wall integrity, cytoskeleton behavior and endomembrane homeostasis. We found that mur3-7 etiolated seedlings have reduced cellulose and that CESA genes are down-regulated, the density and mobility of cellulose synthase complexes (CSCs) are reduced, and cellulose microfibrils become discontinuous. Pectin, RGII, and boron contents are reduced in mur3-7 plants, and B-RGII cross-linking is abnormal. Wall porosity and thickness are significantly increased in mur3-7 seedlings. The endomembrane aggregation is also apparent in mur3-7 mutant. Furthermore, mutant seedlings and their actin filaments are more sensitive to LatA treatment. However, all defects in mur3-7 mutants can be substantially restored by exogenous boric acid application. Our study reveals the importance of MUR3-mediated xyloglucan galactosylation for wall structural assembly and cell wall homeostasis, which is required to stabilize the actin cytoskeleton and the endomembrane system.

A synthetic octoploid rapeseed, Y3380, induces maternal doubled haploids when used as a pollen donor to pollinate plant. However, the mechanism underlying doubled haploid formation remains elusive. We speculated that double haploid induction occurs as the inducer line’s chromosomes pass to the maternal egg cell, and the zygote is formed through fertilization. In the process of zygotic mitosis, the paternal chromosome is specifically eliminated. Part of the paternal gene might have infiltrated the maternal genome through homologous exchange during the elimination process. Then, the zygote haploid genome doubles (early haploid doubling, EH phenomenon), and the doubled zygote continues to develop into a complete embryo, finally forming doubled haploid offspring. To test our hypothesis, in the current study, the octoploid Y3380 line was back bred with the 4122-cp4-EPSPS exogenous gene used as a marker into hexaploid Y3380-cp4-EPSPS as paternal material to pollinate three different maternal materials. The fertilization process of crossing between the inducer line and the maternal parent was observed 48 h after pollination, and the fertilization rate reached 97.92% and 98.72%. After 12 d of pollination, the presence of cp4-EPSPS in the embryo was detected by in situ PCR, and at 13–23 d after pollination, the probability of F1 embryos containing cp4-EPSPS gene was up to 97.27%, but then declined gradually to 0% at 23–33 d. At the same time, the expression of cp4-EPSPS was observed by immunofluorescence in the 3rd to 29th day embryo. As the embryos developed, cp4-EPSPS marker genes were constantly lost, accompanied by embryonic death. After 30 d, the presence of cp4-EPSPS was not detected in surviving embryos. Meanwhile, SNP detection of induced offspring confirmed the existence of double haploids, further indicating that the induction process was caused by the loss of specificity of the paternal chromosome. The tetraploid-induced offspring showed infiltration of the induced line gene loci, with heterozygosity and homozygosity. Results indicated that the induced line chromosomes were eliminated during embryonic development, and the maternal haploid chromosomes were synchronously doubled in the embryo. These findings support our hypothesis and lay a theoretical foundation for further localization or cloning of functional genes involved in double haploid induction in rapeseed.

KEYMESSAGE:The putative myristoylome of moss P. patens opens an avenue for studying myristoylation substrates in non-canonical model plants. A myristoylation signal was shown sufficient for membrane targeting and useful for membrane dynamics visualization during cell growth. N-myristoylation (MYR) is one form of lipid modification catalyzed by N-myristoyltransferase that enables protein-membrane association. MYR is highly conserved in all eukaryotes. However, the study of MYR is limited to a few models such as yeasts, humans, and Arabidopsis. Here, using prediction tools, we report the characterization of the putative myristoylome of the moss Physcomitrium patens. We show that basal land plants display a similar signature of MYR to Arabidopsis and may have organism-specific substrates. Phylogenetically, MYR signals have mostly co-evolved with protein function but also exhibit variability in an organism-specific manner. We also demonstrate that the MYR motif of a moss brassinosteroid-signaling kinase is an efficient plasma membrane targeting signal and labels lipid-rich domains in tip-growing cells. Our results provide insights into the myristoylome in a basal land plant and lay the foundation for future studies on MYR and its roles in plant evolution.

Xyloglucan, a major hemicellulose, interacts with cellulose and pectin to assemble primary cell walls in plants. Loss of the xyloglucan galactosyltransferase MURUS3 (MUR3) leads to the deficiency of galactosylated xyloglucan and perturbs plant growth. However, it is unclear whether defects in xyloglucan galactosylation influence the synthesis of other wall polysaccharides, cell wall integrity, cytoskeleton behaviour, and endomembrane homeostasis. Here, we found that in mur3-7 etiolated seedlings cellulose was reduced, CELLULOSE SYNTHASE (CESA) genes were down-regulated, the density and mobility of cellulose synthase complexes (CSCs) were decreased, and cellulose microfibrils become discontinuous. Pectin, rhamnogalacturonan II (RGII), and boron contents were reduced in mur3-7 plants, and B-RGII cross-linking was abnormal. Wall porosity and thickness were significantly increased in mur3-7 seedlings. Endomembrane aggregation was also apparent in the mur3-7 mutant. Furthermore, mutant seedlings and their actin filaments were more sensitive to Latrunculin A (LatA) treatment. However, all defects in mur3-7 mutants were substantially restored by exogenous boric acid application. Our study reveals the importance of MUR3-mediated xyloglucan galactosylation for cell wall structural assembly and homeostasis, which is required for the stabilization of the actin cytoskeleton and the endomembrane system.

When homozygously fertile plants were induced using doubled haploid (DH) induction lines Y3380 and Y3560, the morphology of the induced F1 generation was basically consistent with the female parent, but the fertility was separated, showing characteristics similar to cytoplasmic male sterile (CMS) and maintainer lines. In this study, the morphology, fertility, ploidy, and cytoplasm genotype of the induced progeny were identified, and the results showed that the sterile progeny was polima cytoplasm sterile (pol CMS) and the fertile progeny was nap cytoplasm. The molecular marker and test-cross experimental results showed that the fertile progeny did not carry the restorer gene of pol CMS and the genetic distance between the female parent and the offspring was 0.002. This suggested that those inductions which produced sterile and fertile progeny were coordinated to CMS and maintainer lines. Through the co-linearity analysis of the mitochondrial DNA (mtDNA), it was found that the rearrangement of mtDNA by DH induction was the key factor that caused the transformation of fertility (nap) into sterility (pol). Also, when heterozygous females were induced with DH induction lines, the induction F2 generation also showed the segregation of fertile and sterile lines, and the genetic distance between sterile and fertile lines was approximately 0.075. Therefore, the induction line can induce different types of female parents, and the breeding of the sterile line and the maintainer line can be achieved through the rapid synchronization of sister crosses and self-crosses. The induction of DH inducer in B. napus can provide a new model for the innovation of germplasm resources and open up a new way for its application.

Pectin, cellulose, and hemicelluloses are major components of primary cell walls in plants. In addition to cell adhesion and expansion, pectin plays a central role in seed mucilage. Seed mucilage contains abundant pectic rhamnogalacturonan-I (RG-I) and lower amounts of homogalacturonan (HG), cellulose, and hemicelluloses. Previously, accumulated evidence has addressed the role of pectin RG-I in mucilage production and adherence. However, less is known about the function of pectin HG in seed coat mucilage formation. In this study, we analyzed a novel mutant, designated things fall apart2 (tfa2), which contains a mutation in HG methyltransferase QUASIMODO2 (QUA2). Etiolated tfa2 seedlings display short hypocotyls and adhesion defects similar to qua2 and tumorous shoot development2 (tsd2) alleles, and show seed mucilage defects. The diminished uronic acid content and methylesterification degree of HG in mutant seed mucilage indicate the role of HG in the formation of seed mucilage. Cellulosic rays in mutant mucilage are collapsed. The epidermal cells of seed coat in tfa2 and tsd2 display deformed columellae and reduced radial wall thickness. Under polyethylene glycol treatment, seeds from these three mutant alleles exhibit reduced germination rates. Together, these data emphasize the requirement of pectic HG biosynthesis for the synthesis of seed mucilage, and the functions of different pectin domains together with cellulose in regulating its formation, expansion, and release.

Pectin, cellulose, and hemicellulose constitute the primary cell wall in eudicots and function in multiple developmental processes in plants. Root hairs are outgrowths of specialized epidermal cells that absorb water and nutrients from the soil. Cell wall architecture influences root hair development, but how cell wall remodeling might enable enhanced root hair formation in response to phosphate (P) deficiency remains relatively unclear. Here, we found that POLYGALACTURONASE INVOLVED IN EXPANSION 2 (PGX2) functions in conditional root hair development. Under low P conditions, a PGX2 activation tagged line (PGX2AT) displays bubble-like root hairs and abnormal callose deposition and superoxide accumulation in roots. We found that the polar localization and trafficking of PIN2 are altered in PGX2AT roots in response to P deficiency. We also found that actin filaments were less compact but more stable in PGX2AT root hair cells and that actin filament skewness in PGX2AT root hairs was recovered by treatment with 1-N-naphthylphthalamic acid (NPA), an auxin transport inhibitor. These results demonstrate that activation tagging of PGX2 affects cell wall remodeling, auxin signaling, and actin microfilament orientation, which may cooperatively regulate root hair development in response to P starvation.

Homogalacturonan (HG) is the most abundant pectin subtype in plant cell walls. Although it is a linear homopolymer, its modification states allow for complex molecular encoding. HG metabolism affects its structure, chemical properties, mobility and binding capacity, allowing it to interact dynamically with other polymers during wall assembly and remodelling and to facilitate anisotropic cell growth, cell adhesion and separation, and organ morphogenesis. HGs have also recently been found to function as signalling molecules that transmit information about wall integrity to the cell. Here we highlight recent advances in our understanding of the dual functions of HG as a dynamic structural component of the cell wall and an initiator of intrinsic and environmental signalling. We also predict how HG might interconnect the cell wall, plasma membrane and intracellular components with transcriptional networks to regulate plant growth and development. Homogalacturonans are the most abundant pectin subtype in plant cell walls and also function as signalling molecules. Homogalacturonans thus connect the cell wall and plasma membrane to regulate plant growth and development.

Polyploidization plays a crucial role in the evolution of angiosperm species. Almost all newly formed polyploids encounter genetic or epigenetic instabilities. However, the molecular mechanisms contributing to genomic instability in synthetic polyploids have not been clearly elucidated. Here, we performed a comprehensive transcriptomic and methylomic analysis of natural and synthetic polyploid rapeseeds (Brassica napus). Our results showed that the CHG methylation levels of synthetic rapeseed in different genomic contexts (genes, transposon regions, and repeat regions) were significantly lower than those of natural rapeseed. The total number and length of CHG-DMRs between natural and synthetic polyploids were much greater than those of CG-DMRs and CHH-DMRs, and the genes overlapping with these CHG-DMRs were significantly enriched in DNA damage repair and nucleotide metabolism pathways. These results indicated that CHG methylation may be more sensitive than CG and CHH methylation in regulating the stability of the polyploid genome of B. napus. In addition, many genes involved in DNA damage repair, nucleotide metabolism, and cell cycle control were significantly differentially expressed between natural and synthetic rapeseeds. Our results highlight that the genes related to DNA repair and nucleotide metabolism display differential CHG methylation patterns between natural and synthetic polyploids and reveal the potential connection between the genomic instability of polyploid plants with DNA methylation defects and dysregulation of the DNA repair system. In addition, it was found that the maintenance of CHG methylation in B. napus might be partially regulated by MET1. Our study provides novel insights into the establishment and evolution of polyploid plants and offers a potential idea for improving the genomic stability of newly formed Brassica polyploids.

The primary cell walls of plants provide mechanical strength while maintaining the flexibility needed for cell extension growth. Cell extension involves loosening the bonds between cellulose microfibrils, hemicelluloses and pectins. Pectins have been implicated in this process, but it remains unclear if this depends on the abundance of certain pectins, their modifications, and/or structure. Here, cell wall-related mutants of the model plant Arabidopsis were characterized by biochemical and immunohistochemical methods and Fourier-transform infrared microspectroscopy. Mutants with reduced pectin or hemicellulose content showed no root cell elongation in response to simulated drought stress, in contrast to wild-type plants or mutants with reduced cellulose content. While no association was found between the degrees of pectin methylesterification and cell elongation, cell wall composition analysis suggested an important role of the pectin rhamnogalacturonan II (RGII), which was corroborated in experiments with the RGII-modifying chemical 2β-deoxy-Kdo. The results were complemented by expression analysis of cell wall synthesis genes and microscopic analysis of cell wall porosity. It is concluded that a certain amount of pectin is necessary for stress-induced root cell elongation, and hypotheses regarding the mechanistic basis of this result are formulated.

Plant cell walls contain cellulose embedded in matrix polysaccharides. Understanding carbohydrate structures and interactions is critical to the production of biofuel and biomaterials using these natural resources. Here we present a solid-state NMR study of cellulose and pectin in 13C-labeled cell walls of Arabidopsis wild-type and mutant plants. Using 1D 13C and 2D 13C-13C correlation experiments, we detected a highly branched arabinan structure in qua2 and tsd2 samples, two allelic mutants for a pectin methyltransferase. Both mutants show close physical association between cellulose and the backbones of pectic homogalacturonan and rhamnogalacturonan-I. Relaxation and dipolar order parameters revealed enhanced microsecond dynamics due to polymer disorder in the mutants, but restricted motional amplitudes due to tighter pectin-cellulose associations. These molecular data shed light on polymer structure and packing in these two pectin mutants, helping to elucidate how pectin could influence cell wall architecture at the nanoscale, cell wall mechanics, and plant growth.

为了探究林木植物中果胶甲酯酶PME的功能,本研究中,我们从毛白杨中克隆出拟南芥PME34的同源基因PtoPME34-1,阐述了该基因在毛白杨中的生物学功能.生物信息学分析表明,毛白杨PtoPME34-1基因与毛果杨PtrPME34-1序列相似性为100％,与拟南芥AtPME34序列相似性为93％.组织特异性表达分析显示PtoPME34-1基因在所有组织都有表达,在基部茎、木质部的表达量较高,在老叶表达量最低.蛋白亚细胞定位结果显示,绿色荧光蛋白GFP标记的PtoPME34-1-GFP融合蛋白定位在烟草叶片细胞壁.毛白杨PtoPME34-1基因过表达显著提高植株的抗旱性.毛白杨PtoPME34-1和PtoPME34-2基因的双突变体植株中果胶甲酯化程度升高,且植物抗旱性也显著增加.这些研究结果证实PtoPME34-1在调控植物生长发育和抗旱胁迫中发挥重要作用.

To explore the function of pectin methylesterase (PME) in forest plants, in this study, we cloned the Arabidopsis thaliana PME34 homologous gene PtoPME34-1 from Populus tomentosa, and elucidated the biological function of this gene in Populus tomentosa. Bioinformatics analysis shows that the similarity between the PtoPME34-1 gene of Populus tomentosa and the PtrPME34-1 of Populus tomentosa is 100%, and the similarity of the sequence with AtPME34 of Arabidopsis thaliana was 93%. Tissue specific expression analysis shows that PtoPME34-1 gene is expressed in all tissues, with higher expression levels in the basal stems and xylem, and the lowest expression level in the old leaves. The results of subcellular localization show that GFP- labeled PtoPME34-1-GFP fusion protein is localized in the cell wall of tobacco leaves. The overexpression of PtoPME34-1 gene significantly improved drought resistance of Populus tomentosa. The double mutants of Populus tomentosa PtoPME34-1 and PtoPME34-2 increased the degree of pectin methylesterification level and drought resistance. These results confirm that PtoPME34-1 plays an important role in regulating plant development and drought stress resistance.

Although biotechnologies for astaxanthin production from Haematococcus pluvialis have been developed for decades and many production facilities have been established throughout the world, the production cost is still high. This paper is to evaluate the current production processes and production facilities, to analyze the R&D strategies for process improvement, and to review the recent research advances shedding light on production cost reduction. With these efforts being made, we intent to conclude that the production cost of astaxanthin from Haematococcus might be substantially reduced to the levels comparable to that of chemical astaxanthin through further R&D and the future research might need to focus on strain selection and improvement, cultivation process optimization, innovation of cultivation methodologies, and revolution of extraction technologies.