scholarly journals CRISPR-Cas9 multiplex genome editing of the hydroxyproline-O-galactosyltransferase gene family alters arabinogalactan-protein glycosylation and function in Arabidopsis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yuan Zhang ◽  
Michael A. Held ◽  
Dasmeet Kaur ◽  
Allan M. Showalter
Keyword(s):  
Gene Family ◽  
Female Gametophyte ◽  
Gene Editing ◽  
Seed Set ◽  
Higher Order ◽  
Protein Glycosylation ◽  
Arabinogalactan Protein ◽  
Composition Analysis ◽  
Biological Functions ◽  
Knockout Mutants

Abstract Background Arabinogalactan-proteins (AGPs) are a class of hydroxyproline-rich proteins (HRGPs) that are heavily glycosylated (> 90%) with type II arabinogalactans (AGs). AGPs are implicated in various plant growth and development processes including cell expansion, somatic embryogenesis, root and stem growth, salt tolerance, hormone signaling, male and female gametophyte development, and defense. To date, eight Hyp-O-galactosyltransferases (GALT2–6, HPGT1–3) have been identified; these enzymes are responsible for adding the first sugar, galactose, onto AGPs. Due to gene redundancy among the GALTs, single or double galt genetic knockout mutants are often not sufficient to fully reveal their biological functions. Results Here, we report the successful application of CRISPR-Cas9 gene editing/multiplexing technology to generate higher-order knockout mutants of five members of the GALT gene family (GALT2–6). AGPs analysis of higher-order galt mutants (galt2 galt5, galt3 galt4 galt6, and galt2 galt3 galt4 galt5 gal6) demonstrated significantly less glycosylated AGPs in rosette leaves, stems, and siliques compared to the corresponding wild-type organs. Monosaccharide composition analysis of AGPs isolated from rosette leaves revealed significant decreases in arabinose and galactose in all the higher-order galt mutants. Phenotypic analyses revealed that mutation of two or more GALT genes was able to overcome the growth inhibitory effect of β-D-Gal-Yariv reagent, which specifically binds to β-1,3-galactan backbones on AGPs. In addition, the galt2 galt3 galt4 galt5 gal6 mutant exhibited reduced overall growth, impaired root growth, abnormal pollen, shorter siliques, and reduced seed set. Reciprocal crossing experiments demonstrated that galt2 galt3 galt4 galt5 gal6 mutants had defects in the female gametophyte which were responsible for reduced seed set. Conclusions Our CRISPR/Cas9 gene editing/multiplexing approach provides a simpler and faster way to generate higher-order mutants for functional characterization compared to conventional genetic crossing of T-DNA mutant lines. Higher-order galt mutants produced and characterized in this study provide insight into the relationship between sugar decorations and the various biological functions attributed to AGPs in plants.

scholarly journals Functional characterization of hydroxyproline-O-galactosyltransferases for Arabidopsis arabinogalactan-protein synthesis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dasmeet Kaur ◽  
Michael A. Held ◽  
Mountain R. Smith ◽  
Allan M. Showalter
Keyword(s):  
Cell Wall ◽  
Seed Set ◽  
Pollen Viability ◽  
Expression Patterns ◽  
Functional Characterization ◽  
Primary Root ◽  
Arabinogalactan Protein ◽  
Root Tip ◽  
Composition Analysis ◽  
Genetic Redundancy

Abstract Background Arabinogalactan-proteins (AGPs) are structurally complex hydroxyproline-rich cell wall glycoproteins ubiquitous in the plant kingdom. AGPs biosynthesis involves a series of post-translational modifications including the addition of type II arabinogalactans to non-contiguous Hyp residues. To date, eight Hyp-galactosyltransferases (Hyp-GALTs; GALT2-GALT9) belonging to CAZy GT31, are known to catalyze the addition of the first galactose residues to AGP protein backbones and enable subsequent AGP glycosylation. The extent of genetic redundancy, however, remains to be elucidated for the Hyp-GALT gene family. Results To examine their gene redundancy and functions, we generated various multiple gene knock-outs, including a triple mutant (galt5 galt8 galt9), two quadruple mutants (galt2 galt5 galt7 galt8, galt2 galt5 galt7 galt9), and one quintuple mutant (galt2 galt5 galt7 galt8 galt9), and comprehensively examined their biochemical and physiological phenotypes. The key findings include: AGP precipitations with β-Yariv reagent showed that GALT2, GALT5, GALT7, GALT8 and GALT9 act redundantly with respect to AGP glycosylation in cauline and rosette leaves, while the activity of GALT7, GALT8 and GALT9 dominate in the stem, silique and flowers. Monosaccharide composition analysis showed that galactose was decreased in the silique and root AGPs of the Hyp-GALT mutants. TEM analysis of 25789 quintuple mutant stems indicated cell wall defects coincident with the observed developmental and growth impairment in these Hyp-GALT mutants. Correlated with expression patterns, galt2, galt5, galt7, galt8, and galt9 display equal additive effects on insensitivity to β-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, galt7, galt8, and galt9 contributed more to primary root growth and root tip swelling under salt stress, whereas galt2 and galt5 played more important roles in seed morphology, germination defects and seed set. Pollen defects likely contributed to the reduced seed set in these mutants. Conclusion Additive and pleiotropic effects of GALT2, GALT5, GALT7, GALT8 and GALT9 on vegetative and reproductive growth phenotypes were teased apart via generation of different combinations of Hyp-GALT knock-out mutants. Taken together, the generation of higher order Hyp-GALT mutants demonstrate the functional importance of AG polysaccharides decorating the AGPs with respect to various aspects of plant growth and development.

Protein glycosylation in extracellular vesicles: Structural characterization and biological functions

2021 ◽  
Vol 135 ◽  
pp. 226-246
Author(s):  
Janaina Macedo-da-Silva ◽  
Verônica F. Santiago ◽  
Livia Rosa-Fernandes ◽  
Claudio R.F. Marinho ◽  
Giuseppe Palmisano
Keyword(s):  
Extracellular Vesicles ◽  
Structural Characterization ◽  
Protein Glycosylation ◽  
Biological Functions

scholarly journals Genome-wide identification and expression analysis of the VQ gene family in soybean (Glycine max)

2019 ◽  
Author(s):  
Yongbin Wang ◽  
Zhenfeng Jiang ◽  
Zhenxiang Li ◽  
Yuanling Zhao ◽  
Weiwei Tan ◽  
...  
Keyword(s):  
Glycine Max ◽  
Plant Growth ◽  
Gene Family ◽  
Stress Responses ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Biological Functions ◽  
Conserved Domain ◽  
Genome Wide ◽  
Development Processes

Background. VQ proteins, the plant-specific transcription factors, are involved in the regulation of plant growth, development, and stress responses; however, few articles systematic reported VQ genes in the soybean. Methods. In total, we identified 75 GmVQ genes, which were classified into 7 groups (Ⅰ-Ⅶ). Conserved domain analysis indicated that VQ gene family members all contained the VQ domains. The VQ genes from the same evolutionary branches of soybean shared similar motifs and structures. Promoter analysis revealed cis-elements related to stress responses, phytohormone responses and controlling physical and reproductive growth. Based on the RNA-seq and qRT-PCR analysis, GmVQ genes were expressed in nine tissues suggested their putative function in many aspects of plant growth and development, and response to stresses in Glycine max. Results. The present study provided basic information for further analysis of the biological functions of GmVQ proteins in various development processes.

scholarly journals Identification and Expression Profiling of Protein Phosphatases (PP2C) Gene Family in Gossypium hirsutum L.

2019 ◽  
Vol 20 (6) ◽  
pp. 1395 ◽  
Author(s):  
Hamna Shazadee ◽  
Nadeem Khan ◽  
Jingjing Wang ◽  
Chencan Wang ◽  
Jianguo Zeng ◽  
...  
Keyword(s):  
Gossypium Hirsutum ◽  
Gene Family ◽  
Expression Profiling ◽  
Critical Role ◽  
Fiber Development ◽  
Chromosomal Mapping ◽  
Composition Analysis ◽  
Specific Gene ◽  
Specific Response ◽  
Pp2c Gene

The protein phosphatase (PP2C) gene family, known to participate in cellular processes, is one of the momentous and conserved plant-specific gene families that regulate signal transduction in eukaryotic organisms. Recently, PP2Cs were identified in Arabidopsis and various other crop species, but analysis of PP2C in cotton is yet to be reported. In the current research, we found 87 (Gossypium arboreum), 147 (Gossypium barbadense), 181 (Gossypium hirsutum), and 99 (Gossypium raimondii) PP2C-encoding genes in total from the cotton genome. Herein, we provide a comprehensive analysis of the PP2C gene family in cotton, such as gene structure organization, gene duplications, expression profiling, chromosomal mapping, protein motif organization, and phylogenetic relationships of each species. Phylogenetic analysis further categorized PP2C genes into 12 subgroups based on conserved domain composition analysis. Moreover, we observed a strong signature of purifying selection among duplicated pairs (i.e., segmental and dispersed) of Gossypium hirsutum. We also observed the tissue-specific response of GhPP2C genes in organ and fiber development by comparing the RNA-sequence (RNA-seq) data reported on different organs. The qRT-PCR validation of 30 GhPP2C genes suggested their critical role in cotton by exposure to heat, cold, drought, and salt stress treatments. Hence, our findings provide an overview of the PP2C gene family in cotton based on various bioinformatic tools that demonstrated their critical role in organ and fiber development, and abiotic stress tolerance, thereby contributing to the genetic improvement of cotton for the resistant cultivar.

The Classical Arabinogalactan Protein Gene Family of Arabidopsis

2000 ◽  
Vol 12 (9) ◽  
pp. 1751 ◽  
Author(s):  
Carolyn J. Schultz ◽  
Kim L. Johnson ◽  
Graeme Currie ◽  
Antony Bacic
Keyword(s):  
Gene Family ◽  
Protein Gene ◽  
Arabinogalactan Protein

scholarly journals Attenuation of Salmonella enterica Serovar Typhimurium by Altering Biological Functions of Murein Lipoprotein and Lipopolysaccharide

2005 ◽  
Vol 73 (12) ◽  
pp. 8433-8436 ◽  
Author(s):  
A. A. Fadl ◽  
J. Sha ◽  
G. R. Klimpel ◽  
J. P. Olano ◽  
C. L. Galindo ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Biological Functions ◽  
Double Knockout ◽  
In Vivo Models ◽  
Background Strain ◽  
Knockout Mutants ◽  
Serovar Typhimurium

ABSTRACT We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.

scholarly journals The impact of O-glycan chemistry on the stability of intrinsically disordered proteins

2018 ◽  
Vol 9 (15) ◽  
pp. 3710-3715 ◽  
Author(s):  
Erica T. Prates ◽  
Xiaoyang Guan ◽  
Yaohao Li ◽  
Xinfeng Wang ◽  
Patrick K. Chaffey ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Protein Glycosylation ◽  
Disordered Proteins ◽  
Biological Functions ◽  
Post Translational Modification ◽  
Intrinsically Disordered ◽  
P Protein ◽  
The Stability ◽  
The Impact

Protein glycosylation is a diverse post-translational modification that serves myriad biological functions.

scholarly journals CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Chao Sui ◽  
Dandan Jiang ◽  
Xiangju Wu ◽  
Xiaoyan Cong ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Editing ◽  
Rna Degradation ◽  
Type I ◽  
Rnase L ◽  
Agarose Gel Electrophoresis ◽  
Biological Functions ◽  
Wild Type ◽  
Induction Of Apoptosis

Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine.

Sexual reproduction in western red cedar (Thuja plicata)

1980 ◽  
Vol 58 (12) ◽  
pp. 1376-1393 ◽  
Author(s):  
John N. Owens ◽  
M. Molder
Keyword(s):  
Sexual Reproduction ◽  
Seed Production ◽  
Low Temperatures ◽  
Female Gametophyte ◽  
Seed Set ◽  
Thuja Plicata ◽  
Insect Damage ◽  
Red Cedar ◽  
Pollination Drop ◽  
Micropylar Canal

Pollen cones and seed cones ended dormancy in mid-February, microsporogenesis occurred in late February, and pollination occurred for about 1 week in early March. Pollen was shed at the two-celled stage. Pollination drops were exuded from only a few ovules at one time. Pollen contacting the pollination drop was rapidly taken in. The pollination drop was withdrawn into the micropyle which was later sealed by enlargement of cells lining the micropylar canal. Megasporogenesis occurred in late February but female gametophytes did not mature and fertilization did not occur until late May. An archegonial complex formed containing seven to nine archegonia, of which several usually were fertilized. Proembryo development varied depending upon the size and shape of the archegonia. Usually, a 12-celled, three-tiered proembryo formed by mid-June. Cleavage polyembryony was not observed. Embryos were mature by mid-August and most seed was shed in September and October.The potential seed set was only 16 seeds per cone and filled seed averaged only 2.6 per cone. Most potential seed was lost because of early ovule abortion from unknown causes, insect damage, or low temperatures at or shortly after pollination. Some potential seeds were lost because the ovules were not pollinated or the embryos aborted. These seeds were soft but nearly normal appearing and contained spongy female gametophyte tissue. Methods of maximizing seed production are suggested.

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