scholarly journals Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

2020 ◽  
Author(s):  
Guillermo Garcia-Gimenez ◽  
Abdellah Barakate ◽  
Pauline Smith ◽  
Jennifer Stephens ◽  
Shi F. Khor ◽  
...  
Keyword(s):  
Cell Wall ◽  
Reverse Genetics ◽  
Grain Morphology ◽  
Single Amino Acid ◽  
Cell Wall Polysaccharide ◽  
Thousand Grain Weight ◽  
Knock Out ◽  
Targeted Mutation ◽  
Length Width ◽  
Complex Relationships

SummaryBarley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-β-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-β-glucan content. (1,3;1,4)-β-Glucan was absent in the grain of cslf6 knock-out lines whereas cslf9 knock-out lines had similar (1,3;1,4)-β-glucan content to WT. However, cslf9 mutants showed changes in the abundance of other cell wall-related monosaccharides compared to WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knock-outs and to a lesser extent TGW in cslf9 knock-outs. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-β-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-β-glucan in mature grain.

scholarly journals Targeted mutation of barley (1,3;1,4)‐β‐glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

2020 ◽  
Vol 104 (4) ◽  
pp. 1009-1022
Author(s):  
Guillermo Garcia‐Gimenez ◽  
Abdellah Barakate ◽  
Pauline Smith ◽  
Jennifer Stephens ◽  
Shi F. Khor ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Polysaccharide Content ◽  
Targeted Mutation ◽  
Complex Relationships

scholarly journals The synthesis of xyloglucan, an abundant plant cell wall polysaccharide, requires CSLC function

2020 ◽  
Vol 117 (33) ◽  
pp. 20316-20324 ◽  
Author(s):  
Sang-Jin Kim ◽  
Balakumaran Chandrasekar ◽  
Anne C. Rea ◽  
Linda Danhof ◽  
Starla Zemelis-Durfee ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Reverse Genetics ◽  
Plant Cell Wall ◽  
Phylogenetic Analyses ◽  
Higher Order ◽  
Wall Structure ◽  
Cell Wall Polysaccharide ◽  
Apparent Lack

Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role ofArabidopsiscellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the fiveArabidopsis CSLCgenes had normal levels of XyG. However, higher-ordercslcmutants had significantly reduced XyG levels, and a mutant with disruptions in all fiveCSLCgenes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, thecslcquintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the fiveCSLCgenes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that theCSLCgenes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of theCSLCgenes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function.

scholarly journals Microtubule-associated IQD9 guides cellulose synthase velocity to shape seed mucilage

2021 ◽  
Author(s):  
Bo Yang ◽  
Gina Stamm ◽  
Katharina Bürstenbinder ◽  
Cătălin Voiniciuc
Keyword(s):  
Cell Wall ◽  
Reverse Genetics ◽  
Cortical Microtubule ◽  
Genetic Regulation ◽  
Cellulose Synthase ◽  
Cell Wall Polysaccharide ◽  
Cell Wall Polysaccharides ◽  
Polysaccharide Biosynthesis ◽  
Highly Expressed Genes ◽  
Seed Mucilage

Arabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes is guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis (SCE). Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles for cell wall polysaccharide biosynthesis and cortical microtubule (MT) organization. Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. Double mutant analyses revealed that their closest paralogs (IQD10 and KLCR2, respectively) are not required for mucilage biosynthesis. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. Similar to the previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein, IQD9 is required to maintain the velocity of cellulose synthases. Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in guiding the distribution of cell wall polysaccharides. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.

scholarly journals Discovery of putative Golgi S-Adenosyl methionine transporters reveals the importance of plant cell wall polysaccharide methylation

2021 ◽  
Author(s):  
Henry Temple ◽  
Pyae Phyo ◽  
Weibing Yang ◽  
Jan J Lyczakowski ◽  
Alberto Echevarria-Poza ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Cell Walls ◽  
Plant Cell Wall ◽  
Intact Cell ◽  
Major Facilitator Superfamily ◽  
Wall Structure ◽  
Cell Wall Polysaccharide ◽  
Knock Out ◽  
Adenosyl Methionine

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, to date, no Golgi SAM transporter has been identified in plants. In this work, we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knock-out of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesised polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, the NMR revealed the existence of pectin egg-box structures in intact cell walls, and showed that their formation is enhanced by reduced methyl-esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters that import SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

scholarly journals Crystal structure to 2.45 Å resolution of a monoclonal Fab specific for theBrucellaA cell wall polysaccharide antigen

1993 ◽  
Vol 2 (7) ◽  
pp. 1106-1113 ◽  
Author(s):  
D. R. Rose ◽  
M. Przybylska ◽  
R. J. To ◽  
C. S. Kayden ◽  
E. Vorberg ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Polysaccharide Antigen

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals PUL-Mediated Plant Cell Wall Polysaccharide Utilization in the Gut Bacteroidetes

2021 ◽  
Vol 22 (6) ◽  
pp. 3077
Author(s):  
Zhenzhen Hao ◽  
Xiaolu Wang ◽  
Haomeng Yang ◽  
Tao Tu ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Microbial Community ◽  
Gut Microbiota ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cell Wall Polysaccharide ◽  
Prominent Feature ◽  
Cell Wall Polysaccharides ◽  
Gut Microbial Community

Plant cell wall polysaccharides (PCWP) are abundantly present in the food of humans and feed of livestock. Mammalians by themselves cannot degrade PCWP but rather depend on microbes resident in the gut intestine for deconstruction. The dominant Bacteroidetes in the gut microbial community are such bacteria with PCWP-degrading ability. The polysaccharide utilization systems (PUL) responsible for PCWP degradation and utilization are a prominent feature of Bacteroidetes. In recent years, there have been tremendous efforts in elucidating how PULs assist Bacteroidetes to assimilate carbon and acquire energy from PCWP. Here, we will review the PUL-mediated plant cell wall polysaccharides utilization in the gut Bacteroidetes focusing on cellulose, xylan, mannan, and pectin utilization and discuss how the mechanisms can be exploited to modulate the gut microbiota.

scholarly journals UDP-glucose dehydrogenases of maize: a role in cell wall pentose biosynthesis

2005 ◽  
Vol 391 (2) ◽  
pp. 409-415 ◽  
Author(s):  
Anna Kärkönen ◽  
Alain Murigneux ◽  
Jean-Pierre Martinant ◽  
Elodie Pepey ◽  
Christophe Tatout ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sequence Similarity ◽  
Glucose Dehydrogenase ◽  
Soya Bean ◽  
Amino Acid Sequences ◽  
Cell Wall Polysaccharide ◽  
Polysaccharide Biosynthesis ◽  
Polysaccharide Synthesis ◽  
Ethanol Dehydrogenase ◽  
Adh Activity

UDPGDH (UDP-D-glucose dehydrogenase) oxidizes UDP-Glc (UDP-D-glucose) to UDP-GlcA (UDP-D-glucuronate), the precursor of UDP-D-xylose and UDP-L-arabinose, major cell wall polysaccharide precursors. Maize (Zea mays L.) has at least two putative UDPGDH genes (A and B), according to sequence similarity to a soya bean UDPGDH gene. The predicted maize amino acid sequences have 95% similarity to that of soya bean. Maize mutants with a Mu-element insertion in UDPGDH-A or UDPGDH-B were isolated (udpgdh-A1 and udpgdh-B1 respectively) and studied for changes in wall polysaccharide biosynthesis. The udpgdh-A1 and udpgdh-B1 homozygotes showed no visible phenotype but exhibited 90 and 60–70% less UDPGDH activity respectively than wild-types in a radiochemical assay with 30 μM UDP-glucose. Ethanol dehydrogenase (ADH) activity varied independently of UDPGDH activity, supporting the hypothesis that ADH and UDPGDH activities are due to different enzymes in maize. When extracts from wild-types and udpgdh-A1 homozygotes were assayed with increasing concentrations of UDP-Glc, at least two isoforms of UDPGDH were detected, having Km values of approx. 380 and 950 μM for UDP-Glc. Leaf and stem non-cellulosic polysaccharides had lower Ara/Gal and Xyl/Gal ratios in udpgdh-A1 homozygotes than in wild-types, whereas udpgdh-B1 homozygotes exhibited more variability among individual plants, suggesting that UDPGDH-A activity has a more important role than UDPGDH-B in UDP-GlcA synthesis. The fact that mutation of a UDPGDH gene interferes with polysaccharide synthesis suggests a greater importance for the sugar nucleotide oxidation pathway than for the myo-inositol pathway in UDP-GlcA biosynthesis during post-germinative growth of maize.

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