LncRNA SH3BP5-AS1 promotes hepatocellular carcinoma progression by sponging miR-6838-5p and activation of PTPN4.

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.

Paternal chromosome elimination of inducer triggers induction of double haploids in Brassica napus.

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.

Galactosylation of xyloglucan is essential for the stabilization of the actin cytoskeleton and endomembrane system through the proper assembly of cell walls.

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.

The putative myristoylome of Physcomitrium patens reveals conserved features of myristoylation in basal land plants.

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.

Rapid and Synchronous Breeding of Cytoplasmic Male Sterile and Maintainer Line Through Mitochondrial DNA Rearrangement Using Doubled Haploid Inducer in Brassica napus.

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.

The Root Hair Development of Pectin Polygalacturonase PGX2 Activation Tagging Line in Response to Phosphate Deficiency.

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.

Pectin methyltransferase QUASIMODO2 functions in the formation of seed coat mucilage in Arabidopsis.

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.

Dynamics of pectic homogalacturonan in cellular morphogenesis and adhesion, wall integrity sensing and plant development.

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.

A pectin methyltransferase modulates polysaccharide dynamics and interactions in Arabidopsis primary cell walls: Evidence from solid-state NMR.

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.

DNA repair- and nucleotide metabolism-related genes exhibit differential CHG methylation patterns in natural and synthetic polyploids (Brassica napus L.).

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.

Reduced pectin content of cell walls prevents stress-induced root cell elongation in Arabidopsis.

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 2ß-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.

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Biotechnological production of astaxanthin from the microalga Haematococcus pluvialis.

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.

Effects of hepatic arterial infusion chemotherapy in the treatment of hepatocellular carcinoma: A meta-analysis.

BACKGROUND: The potential benefits and safety of hepatic arterial infusion chemotherapy (HAIC) for the treatment of patients with hepatocellular carcinoma (HCC) remains inconsistent. Therefore, we conducted this meta-analysis of evaluate the efficacy and safety of HAIC in the treatment of HCC. METHODS: A comprehensive literature search was performed using PubMed, Embase, Web of Science, and the Cochrane library to identify eligible studies that compared HAIC with other therapies for patients with HCC. The main outcomes of our interest, including overall survival (OS), disease free survival (DFS), objective response rate (ORR), disease control rate (DCR), and adverse events, were calculated using the meta-analysis. The pooled estimates were expressed with hazard ratio (HR) with 95%confidence intervals (95%CIs) or risk ratio (RR) with 95%CIs. RESULTS: A total of 13 studies met the inclusion criteria and were included in this meta-analysis. Pooled estimates showed that, HAIC was associated with significantly improved OS (HR = 0.61, 95%CI: 0.48, 0.77; P < .001) and DFS (HR = 0.66, 95%CI: 0.52, 0.84; P = .001) as compared with other therapies. The ORR (RR = 2.28, 95%CI: 1.77, 2.94; P < .001) and DCR (RR = 1.47, 95%CI: 1.23, 1.77; P < .001) were also significantly higher in HAIC group than in control group. Most of the common adverse events were comparably occurred in the 2 groups, except for nausea/vomiting, hypoalbuminemia, pain, anemia and hepatic toxicity. Subgroup analysis suggested that, the improved OS and DFS associated with HAIC were only observed in patients with colorectal liver metastases (CRLM), or advanced HCC, but not in those with unresectable HCC or pancreatic liver metastases. CONCLUSION: Based on the present data, HAIC showed benefit effect in HCC patients, with pronged OS and DFS, as well as increased ORR and DCR. These benefit effects were more obvious in CRLM or advanced HCC patients. However, considering the potential limitations, more large-scale, randomized trials are needed to verify our findings.

The Coix Genome Provides Insights into Panicoideae Evolution and Papery Hull Domestication.

Coix is a grass crop domesticated as early as the Neolithic era. It is still widely cultivated for both highly nutritional food and medicinal use. However, the genetic study and breeding of this crop are hindered by the lack of a sequenced genome. Here, we report de novo sequencing and assembly of the 1619-Mb genome of Coix, and annotation of 75.39% repeats and 39 629 protein-coding genes. Comparative genomics analysis showed that Coix is more closely related to sorghum than maize, but intriguingly only Coix and maize had a recent genome duplication event, which was not detected in sorghum. We further constructed a genetic map and mapped several important traits, especially the strength of hull. Selection of papery hull (thin: easy dehulling) from the stony hull (thick: difficult dehulling) in wild progenitors was a key step in Coix domestication. The papery hull makes seed easier to process and germinate. Anatomic and global transcriptome analysis revealed that the papery hull is a result of inhibition of cell division and wall biogenesis. We also successfully demonstrated that seed hull pressure resistance is controlled by two major quantitative trait loci (QTLs), which are associated with hull thickness and color, respectively. The two QTLs were further fine mapped within intervals of 250 kb and 146 kb, respectively. These resources provide a platform for evolutionary studies and will facilitate molecular breeding of this important crop.

Overexpression of a pectin methylesterase gene PtoPME35 from Populus tomentosa influences stomatal function and drought tolerance in Arabidopsis thaliana.

Poplar is a superior forestation species with high adaptability. The woody tissue of poplar is mainly derived from cell wall. Cell wall formation determines cell shape and woody growth. Pectin is rich in primary cell wall, but it is also involved in the regulation of wood formation. In our study, we cloned a gene from poplar (Populus tomentos), designed as PtoPME35, which encodes a putative pectin methylesterase. PtoPME35 has higher sequence similarity with Arabidopsis AtPME35. Gene expression analysis shows that PtoPME35 has a constitutive expression pattern in multiple tissues, with the highest expression in stem. Subcellular localization result indicates that PtoPME35 is localized to the cell wall. To elucidate the biological function of PtoPME35 in vivo, we generated overexpression plants in poplar and Arabidopsis. The degree of pectin methylesterification is decreased in PtoPME35-overexpressing transgenic poplar, although no obvious phenotypes were displayed. In PtoPME35-overexpressing Arabidopsis plants, stomatal opening is inhibited and water loss rate is decreased under the drought condition. Moreover, the expression levels of drought-stress responsive genes were higher with mannitol treatment in PtoPME35-overexpressing Arabidopsis plants than in wild type controls. Accordingly, these results suggest that PtoPME35 may regulate osmotic stress responses by modulating stomatal functions.

POLYGALACTURONASE INVOLVED IN EXPANSION3 Functions in Seedling Development, Rosette Growth, and Stomatal Dynamics in Arabidopsis thaliana.

Plant cell separation and expansion require pectin degradation by endogenous pectinases such as polygalacturonases, few of which have been functionally characterized. Stomata are a unique system to study both processes because stomatal maturation involves limited separation between sister guard cells and stomatal responses require reversible guard cell elongation and contraction. However, the molecular mechanisms for how stomatal pores form and how guard cell walls facilitate dynamic stomatal responses remain poorly understood. We characterized POLYGALACTURONASE INVOLVED IN EXPANSION3 (PGX3), which is expressed in expanding tissues and guard cells. PGX3-GFP localizes to the cell wall and is enriched at sites of stomatal pore initiation in cotyledons. In seedlings, ablating or overexpressing PGX3 affects both cotyledon shape and the spacing and pore dimensions of developing stomata. In adult plants, PGX3 affects rosette size. Although stomata in true leaves display normal density and morphology when PGX3 expression is altered, loss of PGX3 prevents smooth stomatal closure, and overexpression of PGX3 accelerates stomatal opening. These phenotypes correspond with changes in pectin molecular mass and abundance that can affect wall mechanics. Together, these results demonstrate that PGX3-mediated pectin degradation affects stomatal development in cotyledons, promotes rosette expansion, and modulates guard cell mechanics in adult plants.

Effects of Pectin Molecular Weight Changes on the Structure, Dynamics, and Polysaccharide Interactions of Primary Cell Walls of Arabidopsis thaliana: Insights from Solid-State NMR.

Significant cellulose-pectin interactions in plant cell walls have been reported recently based on 2D 13C solid-state NMR spectra of intact cell walls, but how these interactions affect cell growth has not been probed. Here, we characterize two Arabidopsis thaliana lines with altered expression of the POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1) gene, which encodes a polygalacturonase that cleaves homogalacturonan (HG). PGX1AT plants overexpress PGX1, have HG with lower molecular weight, and grow larger, whereas pgx1-2 knockout plants have HG with higher molecular weight and grow smaller. Quantitative 13C solid-state NMR spectra show that PGX1AT cell walls have lower galacturonic acid and xylose contents and higher HG methyl esterification than controls, whereas high molecular weight pgx1-2 walls have similar galacturonic acid content and methyl esterification as controls. 1H-transferred 13C INEPT spectra indicate that the interfibrillar HG backbones are more aggregated whereas the RG-I side chains are more dispersed in PGX1AT cell walls than in pgx1-2 walls. In contrast, the pectins that are close to cellulose become more mobile and have weaker cross peaks with cellulose in PGX1AT walls than in pgx1-2 walls. Together, these results show that polygalacturonase-mediated plant growth is accompanied by increased esterification and decreased cross-linking of HG, increased aggregation of interfibrillar HG, and weaker HG-cellulose interactions. These structural and dynamical differences give molecular insights into how pectins influence wall dynamics during cell growth.

Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation, leaf expansion, stem lignification, mechanical stiffening, and lodging.

Pectin is the most abundant component of primary cell walls in eudicot plants. The modification and degradation of pectin affects multiple processes during plant development, including cell expansion, organ initiation, and cell separation. However, the extent to which pectin degradation by polygalacturonases affects stem development and secondary wall formation remains unclear. Using an activation tag screen, we identified a transgenic Arabidopsis thaliana line with longer etiolated hypocotyls, which overexpresses a gene encoding a polygalacturonase. We designated this gene as POLYGALACTURONASE INVOLVED IN EXPANSION2 (PGX2), and the corresponding activation tagged line as PGX2AT . PGX2 is widely expressed in young seedlings and in roots, stems, leaves, flowers, and siliques of adult plants. PGX2-GFP localizes to the cell wall, and PGX2AT plants show higher total polygalacturonase activity and smaller pectin molecular masses than wild-type controls, supporting a function for this protein in apoplastic pectin degradation. A heterologously expressed, truncated version of PGX2 also displays polygalacturonase activity in vitro. Like previously identified PGX1AT plants, PGX2AT plants have longer hypocotyls and larger rosette leaves, but they also uniquely display early flowering, earlier stem lignification, and lodging stems with enhanced mechanical stiffness that is possibly due to decreased stem thickness. Together, these results indicate that PGX2 both functions in cell expansion and influences secondary wall formation, providing a possible link between these two developmental processes.