Four Novel Cellulose Synthase (CESA) Genes from Birch (Betula platyphylla Suk.) Involved in Primary and Secondary Cell Wall Biosynthesis

Cellulose synthase (CESA), which is an essential catalyst for the generation of plant cell wall biomass, is mainly encoded by the CesA gene family that contains ten or more members. In this study; four full-length cDNAs encoding CESA were isolated from Betula platyphylla Suk., which is an important timber species, using RT-PCR combined with the RACE method and were named as BplCesA3, −4, −7 and −8. These deduced CESAs contained the same typical domains and regions as their Arabidopsis homologs. The cDNA lengths differed among these four genes, as did the locations of the various protein domains inferred from the deduced amino acid sequences, which shared amino acid sequence identities ranging from only 63.8% to 70.5%. Real-time RT-PCR showed that all four BplCesAs were expressed at different levels in diverse tissues. Results indicated that BplCESA8 might be involved in secondary cell wall biosynthesis and floral development. BplCESA3 appeared in a unique expression pattern and was possibly involved in primary cell wall biosynthesis and seed development; it might also be related to the homogalacturonan synthesis. BplCESA7 and BplCESA4 may be related to the formation of a cellulose synthase complex and participate mainly in secondary cell wall biosynthesis. The extremely low expression abundance of the four BplCESAs in mature pollen suggested very little involvement of them in mature pollen formation in Betula. The distinct expression pattern of the four BplCesAs suggested they might participate in developments of various tissues and that they are possibly controlled by distinct mechanisms in Betula.     

Arabinogalactan Proteins: Focus on the Role in Cellulose Synthesis and Deposition during Plant Cell Wall Biogenesis

Arabinogalactan proteins (AGPs) belong to a family of glycoproteins that are widely present in plants. AGPs are mostly composed of a protein backbone decorated with complex carbohydrate side chains and are usually anchored to the plasma membrane or secreted extracellularly. A trickle of compelling biochemical and genetic evidence has demonstrated that AGPs make exciting candidates for a multitude of vital activities related to plant growth and development. However, because of the diversity of AGPs, functional redundancy of AGP family members, and blunt-force research tools, the precise functions of AGPs and their mechanisms of action remain elusive. In this review, we put together the current knowledge about the characteristics, classification, and identification of AGPs and make a summary of the biological functions of AGPs in multiple phases of plant reproduction and developmental processes. In addition, we especially discuss deeply the potential mechanisms for AGP action in different biological processes via their impacts on cellulose synthesis and deposition based on previous studies. Particularly, five hypothetical models that may explain the AGP involvement in cellulose synthesis and deposition during plant cell wall biogenesis are proposed. AGPs open a new avenue for understanding cellulose synthesis and deposition in plants.     

Genome-Wide Identification,Expansion, and Evolution Analysis of Homeobox Gene Family Reveals TALE Genes Important for Secondary Cell Wall Biosynthesis in Moso Bamboo (Phyllostachys edulis)

The TALE gene family is a subfamily of the homeobox gene family and has been implicated in regulating plant secondary growth. However, reports about the evolutionary history and function of the TALE gene family in bamboo are limited. Here, the homeobox gene families of moso bamboo Olyra latifolia and Bonia amplexicaulis were identified and compared. Many duplication events and obvious expansions were found in the TALE family of woody bamboo. PhTALEs were found to have high syntenies with TALE genes in rice. Through gene co-expression analysis and quantitative real-time PCR analysis, the candidate PhTALEs were thought to be involved in regulating secondary cell wall development of moso bamboo during the fast-growing stage. Among these candidate PhTALEs, orthologs of OsKNAT7, OSH15, and SH5 in moso bamboo may regulate xylan synthesis by regulating the expression of IRX-like genes. These results suggested that PhTALEs may participate in the secondary cell wall deposition in internodes during the fast-growing stage of moso bamboo. The expansion of the TALE gene family may be implicated in the increased lignification of woody bamboo when divergent from herbaceous bamboos.     

Genome-Wide Identification,Characterization and Expression Analysis of the Chalcone Synthase Family in Maize

Members of the chalcone synthase (CHS) family participate in the synthesis of a series of secondary metabolites in plants, fungi and bacteria. The metabolites play important roles in protecting land plants against various environmental stresses during the evolutionary process. Our research was conducted on comprehensive investigation of CHS genes in maize (Zea mays L.), including their phylogenetic relationships, gene structures, chromosomal locations and expression analysis. Fourteen CHS genes (ZmCHS01–14) were identified in the genome of maize, representing one of the largest numbers of CHS family members identified in one organism to date. The gene family was classified into four major classes (classes I–IV) based on their phylogenetic relationships. Most of them contained two exons and one intron. The 14 genes were unevenly located on six chromosomes. Two segmental duplication events were identified, which might contribute to the expansion of the maize CHS gene family to some extent. In addition, quantitative real-time PCR and microarray data analyses suggested that ZmCHS genes exhibited various expression patterns, indicating functional diversification of the ZmCHS genes. Our results will contribute to future studies of the complexity of the CHS gene family in maize and provide valuable information for the systematic analysis of the functions of the CHS gene family.     

Plant Cell Wall Proteomes: The Core of Conserved Protein Families and the Case of Non-Canonical Proteins

Plant cell wall proteins (CWPs) play critical roles during plant development and in response to stresses. Proteomics has revealed their great diversity. With nearly 1000 identified CWPs, the Arabidopsis thaliana cell wall proteome is the best described to date and it covers the main plant organs and cell suspension cultures. Other monocot and dicot plants have been studied as well as bryophytes, such as Physcomitrella patens and Marchantia polymorpha. Although these proteomes were obtained using various flowcharts, they can be searched for the presence of members of a given protein family. Thereby, a core cell wall proteome which does not pretend to be exhaustive, yet could be defined. It comprises: (i) glycoside hydrolases and pectin methyl esterases, (ii) class III peroxidases, (iii) Asp, Ser and Cys proteases, (iv) non-specific lipid transfer proteins, (v) fasciclin arabinogalactan proteins, (vi) purple acid phosphatases and (vii) thaumatins. All the conserved CWP families could represent a set of house-keeping CWPs critical for either the maintenance of the basic cell wall functions, allowing immediate response to environmental stresses or both. Besides, the presence of non-canonical proteins devoid of a predicted signal peptide in cell wall proteomes is discussed in relation to the possible existence of alternative secretion pathways.     

Identification and Fine Mapping of the Candidate Gene Controlling Multi-Inflorescence in Brassica napus

As a desirable agricultural trait, multi-inflorescence (MI) fulfills the requirement of mechanized harvesting and yield increase in rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait remain poorly understood. We previously identified a difference of one pair of dominant genes between the two mapping parental materials. In this study, phenotype and expression analysis indicated that the imbalance of the CLAVATA (CLV)-WUSCHEL (WUS) feedback loop may contribute to the abnormal development of the shoot apical meristem (SAM). BnaMI was fine-mapped to a 55 kb genomic region combining with genotype and phenotype of 5768 BCF₁ individuals using a traditional mapping approach. Through comparative and expression analyses, combined with the annotation in Arabidopsis, five genes in this interval were identified as candidate genes. The present findings may provide assistance in functional analysis of the mechanism associated with multi-inflorescence and yield increase in rapeseed.     

Genome-Wide Identification and Characterization of Polygalacturonase Gene Family in Maize (Zea mays L.)

Polygalacturonase (PG, EC 3.2.1.15) is a crucial enzyme for pectin degradation and is involved in various developmental processes such as fruit ripening, pollen development, cell expansion, and organ abscission. However, information on the PG gene family in the maize (Zea mays L.) genome and the specific members involved in maize anther development are still lacking. In this study, we identified 55 PG family genes from the maize genome and further characterized their evolutionary relationship and expression patterns. Phylogenetic analysis revealed that ZmPGs are grouped into six Clades, and gene structures of the same Clade are highly conserved, suggesting their functional conservation. The ZmPGs are randomly distributed across maize chromosomes, and collinearity analysis showed that many ZmPGs might be derived from tandem duplications and segmental duplications, and these genes are under purifying selection. Furthermore, gene expression analysis provided insights into possible functional divergence among ZmPGs. Based on the RNA-seq data analysis, we found that many ZmPGs are expressed in various tissues while 18 ZmPGs are highly expressed in maize anther, and their detailed expression profiles in different anther developmental stages were further investigated by using RT-qPCR analysis. These results provide valuable information for further functional characterization and application of the ZmPGs in maize.     

The Xyloglucan Endotransglucosylase/Hydrolase Gene XTH22/TCH4 Regulates Plant Growth by Disrupting the Cell Wall Homeostasis in Arabidopsis under Boron Deficiency

TCH4 is a xyloglucan endotransglucosylase/hydrolase (XTH) family member. Extensive studies have shown that XTHs are very important in cell wall homeostasis for plant growth and development. Boron (B), as an essential micronutrient for plants, plays an essential role in the cross-linking of cell wall pectin. However, the effect of B on cell wall organization is unclear. This study aimed to explore the mechanism of plant adaption to B stress by investigating the role of TCH4 in cell wall homeostasis. We conducted both plate and hydroponic cultures of wild-type Col-0 and overexpression and gene knockout lines of XTH22/TCH4 to analyze the phenotype, components, and characteristics of the cell wall using immunofluorescence, atomic force microscopy (AFM), and transmission electron microscopy (TEM). B deficiency induces the expression of TCH4. The overexpression lines of TCH4 presented more sensitivity to B deficiency than the wild-type Col-0, while the knockout lines of TCH4 were more resistant to low B stress. Up-regulation of TCH4 influenced the ratio of chelator-soluble pectin to alkali-soluble pectin and decreased the degree of methylesterification of pectin under B-deficient conditions. Moreover, we found that B deficiency disturbed the arrangement of cellulose, enlarged the gap between cellulose microfibrils, and decreased the mechanical strength of the cell wall, leading to the formation of a thickened and deformed triangular region of the cell wall. These symptoms were more profound in the TCH4 overexpression lines. Consistently, compared with Col-0, the O₂⁻ and MDA contents in the TCH4 overexpression lines increased under B-deficient conditions. This study identified the B-deficiency-induced TCH4 gene, which regulates cell wall homeostasis to influence plant growth under B-deficient conditions.     

The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.     

Differential Polarization Imaging of Plant Cells. Mapping the Anisotropy of Cell Walls and Chloroplasts

Modern light microscopy imaging techniques have substantially advanced our knowledge about the ultrastructure of plant cells and their organelles. Laser-scanning microscopy and digital light microscopy imaging techniques, in general—in addition to their high sensitivity, fast data acquisition, and great versatility of 2D–4D image analyses—also opened the technical possibilities to combine microscopy imaging with spectroscopic measurements. In this review, we focus our attention on differential polarization (DP) imaging techniques and on their applications on plant cell walls and chloroplasts, and show how these techniques provided unique and quantitative information on the anisotropic molecular organization of plant cell constituents: (i) We briefly describe how laser-scanning microscopes (LSMs) and the enhanced-resolution Re-scan Confocal Microscope (RCM of Confocal.nl Ltd. Amsterdam, Netherlands) can be equipped with DP attachments—making them capable of measuring different polarization spectroscopy parameters, parallel with the ‘conventional’ intensity imaging. (ii) We show examples of different faces of the strong anisotropic molecular organization of chloroplast thylakoid membranes. (iii) We illustrate the use of DP imaging of cell walls from a variety of wood samples and demonstrate the use of quantitative analysis. (iv) Finally, we outline the perspectives of further technical developments of micro-spectropolarimetry imaging and its use in plant cell studies.     

Identification of the Karyopherin Superfamily in Maize and Its Functional Cues in Plant Development

Appropriate nucleo-cytoplasmic partitioning of proteins is a vital regulatory mechanism in phytohormone signaling and plant development. However, how this is achieved remains incompletely understood. The Karyopherin (KAP) superfamily is critical for separating the biological processes in the nucleus from those in the cytoplasm. The KAP superfamily is divided into Importin α (IMPα) and Importin β (IMPβ) families and includes the core components in mediating nucleocytoplasmic transport. Recent reports suggest the KAPs play crucial regulatory roles in Arabidopsis development and stress response by regulating the nucleo-cytoplasmic transport of members in hormone signaling. However, the KAP members and their associated molecular mechanisms are still poorly understood in maize. Therefore, we first identified seven IMPα and twenty-seven IMPβ genes in the maize genome and described their evolution traits and the recognition rules for substrates with nuclear localization signals (NLSs) or nuclear export signals (NESs) in plants. Next, we searched for the protein interaction partners of the ZmKAPs and selected the ones with Arabidopsis orthologs functioning in auxin biosynthesis, transport, and signaling to predict their potential function. Finally, we found that several ZmKAPs share similar expression patterns with their interacting proteins, implying their function in root development. Overall, this article focuses on the Karyopherin superfamily in maize and starts with this entry point by systematically comprehending the KAP-mediated nucleo-cytoplasmic transport process in plants, and then predicts the function of the ZmKAPs during maize development, with a perspective on a closely associated regulatory mechanism between the nucleo-cytoplasmic transport and the phytohormone network.     

Revising the Role of Cortical Cytoskeleton during Secretion: Actin and Myosin XI Function in Vesicle Tethering

In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase in transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.     

3,4-Dehydro-L-proline Induces Programmed Cell Death in the Roots of Brachypodium distachyon

As cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs) take part in plant growth and various developmental processes. To fulfil their functions, HRGPs, extensins (EXTs) in particular, undergo the hydroxylation of proline by the prolyl-4-hydroxylases. The activity of these enzymes can be inhibited with 3,4-dehydro-L-proline (3,4-DHP), which enables its application to reveal the functions of the HRGPs. Thus, to study the involvement of HRGPs in the development of root hairs and roots, we treated seedlings of Brachypodium distachyon with 250 µM, 500 µM, and 750 µM of 3,4-DHP. The histological observations showed that the root epidermis cells and the cortex cells beneath them ruptured. The immunostaining experiments using the JIM20 antibody, which recognizes the EXT epitopes, demonstrated the higher abundance of this epitope in the control compared to the treated samples. The transmission electron microscopy analyses revealed morphological and ultrastructural features that are typical for the vacuolar-type of cell death. Using the TUNEL test (terminal deoxynucleotidyl transferase dUTP nick end labelling), we showed an increase in the number of nuclei with damaged DNA in the roots that had been treated with 3,4-DHP compared to the control. Finally, an analysis of two metacaspases’ gene activity revealed an increase in their expression in the treated roots. Altogether, our results show that inhibiting the prolyl-4-hydroxylases with 3,4-DHP results in a vacuolar-type of cell death in roots, thereby highlighting the important role of HRGPs in root hair development and root growth.     

Fine Mapping and Identification of a Candidate Gene of Downy Mildew Resistance,RPF2, in Spinach (Spinacia oleracea L.)

Downy mildew is a major threat to the economic value of spinach. The most effective approach to managing spinach downy mildew is breeding cultivars with resistance genes. The resistance allele RPF2 is effective against races 1–10 and 15 of Peronospora farinosa f. sp. Spinaciae (P. effusa) and is widely used as a resistance gene. However, the gene and the linked marker of RPF2 remain unclear, which limit its utilization. Herein, we located the RPF2 gene in a 0.61 Mb region using a BC₁ population derived from Sp39 (rr) and Sp62 (RR) cultivars via kompetitive allele specific PCR (KASP) markers. Within this region, only one R gene, Spo12821, was identified based on annotation information. The amino acid sequence analysis showed that there were large differences in the length of the LRR domain between the parents. Additionally, a molecular marker, RPF2-IN12821, was developed based on the sequence variation in the Spo12821, and the evaluation in the BC₁ population produced a 100% match with resistance/susceptibility. The finding of the study could be valuable for improving our understanding of the genetic basis of resistance against the downy mildew pathogen and breeding resistance lines in the future.     

Preliminary Expression Analysis of the OSCA Gene Family in Maize and Their Involvement in Temperature Stress

Hyperosmolality-gated calcium-permeable channels (OSCA) are characterized as an osmosensor in plants; they are able to recognize and respond to exogenous and endogenous osmotic changes, and play a vital role in plant growth and adaptability to environmental stress. To explore the potential biological functions of OSCAs in maize, we performed a bioinformatics and expression analysis of the ZmOSCA gene family. Using bioinformatics methods, we identified twelve OSCA genes from the genome database of maize. According to their sequence composition and phylogenetic relationship, the maize OSCA family was classified into four groups (Ⅰ, Ⅱ, Ⅲ, and Ⅳ). Multiple sequence alignment analysis revealed a conserved DUF221 domain in these members. We modeled the calcium binding sites of four OSCA families using the autodocking technique. The expression profiles of ZmOSCA genes were analyzed in different tissues and under diverse abiotic stresses such as drought, salt, high temperature, and chilling using quantitative real-time PCR (qRT-PCR). We found that the expression of twelve ZmOSCA genes is variant in different tissues of maize. Furthermore, abiotic stresses such as drought, salt, high temperature, and chilling differentially induced the expression of twelve ZmOSCA genes. We chose ZmOSCA2.2 and ZmOSCA2.3, which responded most strongly to temperature stress, for prediction of protein interactions. We modeled the calcium binding sites of four OSCA families using autodocking tools, obtaining a number of new results. These results are helpful in understanding the function of the plant OSCA gene family for study of the molecular mechanism of plant osmotic stress and response, as well as exploration of the interaction between osmotic stress, high-temperature stress, and low-temperature stress signal transduction mechanisms. As such, they can provide a theoretical basis for crop breeding.