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

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.

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Functional Characterization of Hydroxyproline-O-Galactosyltransferases For Arabidopsis Arabinogalactan-Proteins synthesis

10.21203/rs.3.rs-785512/v1 ◽

2021 ◽

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 Proteins ◽

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 (galt5galt8galt9), two quadruple mutants (galt2galt5galt7galt8, galt2galt5galt7galt9), and one quintuple mutant (galt2galt5galt7galt8galt9), 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. The AGP profile of 25789 quintuple mutant stems indicated changes in AGP profiles compared to WT. Additionally, 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.

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The Arabidopsis Root Tip (Phospho)Proteomes at Growth-Promoting versus Growth-Repressing Conditions Reveal Novel Root Growth Regulators

Cells ◽

10.3390/cells10071665 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1665

Author(s):

Natalia Nikonorova ◽

Evan Murphy ◽

Cassio Flavio Fonseca de Lima ◽

Shanshuo Zhu ◽

Brigitte van de Cotte ◽

...

Keyword(s):

Cell Wall ◽

Root Growth ◽

Growth Regulators ◽

Growth Regulation ◽

Phosphorylation Site ◽

Primary Root ◽

Root Tip ◽

Arabidopsis Root ◽

Growth Promoting ◽

The Impact

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies have revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different concentrations of the synthetic auxin NAA that establish growth-promoting and -repressing conditions on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data, we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results, together with previously published studies, suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip.

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Genome-Wide Identification and Expression Profile Analysis of the Phospholipase C Gene Family in Wheat (Triticum aestivum L.)

Plants ◽

10.3390/plants9070885 ◽

2020 ◽

Vol 9 (7) ◽

pp. 885

Author(s):

Xianguo Wang ◽

Yang Liu ◽

Zheng Li ◽

Xiang Gao ◽

Jian Dong ◽

...

Keyword(s):

Gene Family ◽

Phospholipase C ◽

Profile Analysis ◽

Expression Patterns ◽

Functional Characterization ◽

Primary Root ◽

In Silico Analysis ◽

Triticum Aestivum L ◽

Stress Conditions ◽

Phosphatase Domain

Phospholipid-hydrolyzing enzymes include members of the phospholipase C (PLC) family that play important roles in regulating plant growth and responding to stress. In the present study, a systematic in silico analysis of the wheat PLC gene family revealed a total of 26 wheat PLC genes (TaPLCs). Phylogenetic and sequence alignment analyses divided the wheat PLC genes into 2 subfamilies, TaPI-PLC (containing the typical X, Y, and C2 domains) and TaNPC (containing a phosphatase domain). TaPLC expression patterns differed among tissues, organs, and under abiotic stress conditions. The transcript levels of 8 TaPLC genes were validated through qPCR analyses. Most of the TaPLC genes were sensitive to salt stress and were up-regulated rapidly, and some were sensitive to low temperatures and drought. Overexpression of TaPI-PLC1-2B significantly improved resistance to salt and drought stress in Arabidopsis, and the primary root of P1-OE was significantly longer than that of the wild type under stress conditions. Our results not only provide comprehensive information for understanding the PLC gene family in wheat, but can also provide a solid foundation for functional characterization of the wheat PLC gene family.

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

BMC Plant Biology ◽

10.1186/s12870-020-02791-9 ◽

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.

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Genome-wide identification and characterization of SnRK family genes in Brassica napus

10.21203/rs.3.rs-17109/v1 ◽

2020 ◽

Author(s):

Weizhuo Zhu ◽

Dezhi Wu ◽

Lixi Jiang ◽

Lingzhen Ye

Keyword(s):

Brassica Napus ◽

Abiotic Stresses ◽

Expression Patterns ◽

Functional Characterization ◽

Composition Analysis ◽

Distribution Analysis ◽

Element Analysis ◽

Genome Wide ◽

Identification And Characterization

Abstract Background: Sucrose non-fermenting 1 related protein kinases (SnRK) play crucial roles in responding to biotic and abiotic stresses through activating protein phosphorylation pathways. However, little information of SnRK genes was available in Brassica napus, one of important oil crops. Recently, the released sequences of the reference genome of B.napus provide a good chance to perform genome-wide identification and characterization of BnSnRK gene family in the rapeseed.Results: Totally 114 SnRK genes distributed on 19 chromosomes were identified in the genome of B.napus and classified into three subfamilies on the basis of phylogenetic analysis and the domain types. According to gene structure and motif composition analysis, the BnSnRK sequences showed obvious divergence among three subfamilies. Gene duplication and synteny between the genomes of the rapeseed and Arabidopsis were also analyzed to provide insights into the evolutionary characteristics of BnSnRK family genes. Cis-element analysis revealed that BnSnRKs may response to diverse environmental stresses. Moreover, the expression patterns of BnSnRKs in various tissues and under diverse abiotic stresses were distinct difference. Besides, Single Nucleotide Polymorphisms (SNP) distribution analysis suggests the function disparity of BnSnRK family genes in different genotypes of the rapeseed.Conclusion: We examined genomic structures, evolution features, expression patterns and SNP distribution of 114 BnSnRKs. The results provide valuable information for functional characterization of BnSnRK genes in future studies.

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Identification and Functional Characterization of Apple MdCKX5.2 in Root Development and Abiotic Stress Tolerance

Horticulturae ◽

10.3390/horticulturae8010062 ◽

2022 ◽

Vol 8 (1) ◽

pp. 62

Author(s):

Yang Liu ◽

Xun Wang ◽

Xiaofei Wang ◽

Wensheng Gao ◽

Chunxiang You

Keyword(s):

Expression Analysis ◽

Root Development ◽

Expression Profiles ◽

Abiotic Stress Tolerance ◽

Expression Patterns ◽

Functional Characterization ◽

Primary Root ◽

Cytokinin Oxidase ◽

Lateral Root Development ◽

Exogenous Cytokinin

Cytokinin oxidase/dehydrogenases (CKXs) are the key enzymes in cytokinin degradation and have been widely studied in model plants. Little is known about apple’s (Malus×domestica) CKX genes. Here, using genome-wide analysis, we identified 10 MdCKX genes in apple. The phylogenetics, chromosome locations, and genome structures were then tested. Expression analysis showed that MdCKX genes had different expression profiles in apple, pointing to the different roles. Meanwhile, relative expression analysis showed that these genes have different expression patterns in response to several exogenous cytokinin factors, including trans-zeatin (ZT), thidiazuron (TDZ), and N6-furfuryladenine (KT). Finally, we introduced the MdCKX5.2 gene into Arabidopsis to evaluate its functions, and the results suggested the transgenic Arabidopsis displayed phenotypes related to promoting primary root and lateral root development, response to exogenous ZT, and conferring to drought and salt tolerant. Taken together, our results provide insights on the possible application of the MdCKX5.2 gene for molecular breeding in apples.

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Genome-Wide Analysis of Glycoside Hydrolase Family 35 Genes and Their Potential Roles in Cell Wall Development in Medicago truncatula

Plants ◽

10.3390/plants10081639 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1639

Author(s):

Junfeng Yang ◽

Qian Li ◽

Wenxuan Du ◽

Yu Yao ◽

Guoan Shen ◽

...

Keyword(s):

Cell Wall ◽

Medicago Truncatula ◽

Glycoside Hydrolase ◽

Expression Patterns ◽

Functional Characterization ◽

Glycoside Hydrolase Family ◽

Forage Crops ◽

Specific Expression ◽

Hydrolase Family ◽

Expression Module

Plant β-galactosidases (BGAL) function in various cell wall biogeneses and modifications, and they belong to the glycoside hydrolase family. However, the roles of BGAL family members in Medicago truncatula cell wall remodeling remain unclear. In this study, a total of 25 MtBGAL members of the glycoside hydrolase gene family 35 were identified, and they were clustered into nine sub-families. Many cis-acting elements possibly related to MeJA and abscisic acid responses were identified in the promoter region of the MtBGAL genes. Transcript analyses showed that these MtBGAL genes exhibited distinct expression patterns in various tissues and developing stem internodes. Furthermore, a stem-specific expression module associated with cell wall metabolic pathways was identified by weighted correlation network analysis (WGCNA). In particular, MtBGAL1 and MtBGAL23 within the stem-specific expression module were highly expressed in mature stems. In addition, several genes involved in lignin, cellulose, hemicellulose and pectin pathways were co-expressed with MtBGAL1 and MtBGAL23. It was also found that MtBGAL1 and MtBGAL23 were localized to the cell wall at the subcellular level, indicating their roles in the modification of cell wall metabolites in Medicago. As a whole, these results will be useful for further functional characterization and utilization of BGAL genes in cell wall modifications aiming to improve the quality of legume forage crops.

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Auxin-triggered changes in the Arabidopsis root tip (phospho)proteome reveal novel root growth regulators

10.1101/2021.03.04.433936 ◽

2021 ◽

Author(s):

Natalia Nikonorova ◽

Evan Murphy ◽

Cassio Flavio Fonseca de Lima ◽

Shanshuo Zhu ◽

Brigitte van de Cotte ◽

...

Keyword(s):

Cell Wall ◽

Root Growth ◽

Growth Regulators ◽

Growth Regulation ◽

Phosphorylation Site ◽

Primary Root ◽

Root Tip ◽

Arabidopsis Root ◽

Transcriptional Reprogramming ◽

The Impact

ABSTRACTAuxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different auxin concentrations on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results together with previously published studies suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31phosphorylation site for growth regulation in theArabidopsisroot tip.ONE SENTENCE SUMMARYAn auxin-triggered Arabidopsis root tip (phospho)proteome reveals novel root growth regulators

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Genome-wide identification and characterization of SnRK family genes in Brassica napus

10.21203/rs.3.rs-17109/v2 ◽

2020 ◽

Author(s):

Weizhuo Zhu ◽

Dezhi Wu ◽

Lixi Jiang ◽

Lingzhen Ye

Keyword(s):

Brassica Napus ◽

Abiotic Stresses ◽

Expression Patterns ◽

Functional Characterization ◽

Composition Analysis ◽

Distribution Analysis ◽

Element Analysis ◽

Genome Wide ◽

Identification And Characterization

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FASCICLIN-LIKE 18 Is a New Player Regulating Root Elongation in Arabidopsis thaliana

Frontiers in Plant Science ◽

10.3389/fpls.2021.645286 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hewot Allelign Ashagre ◽

David Zaltzman ◽

Anat Idan-Molakandov ◽

Hila Romano ◽

Oren Tzfadia ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Cell Wall ◽

Root Elongation ◽

Genetic Interaction ◽

Lateral Roots ◽

Primary Root ◽

Arabinogalactan Protein ◽

Wild Type ◽

Synthesis Inhibitor ◽

High Sucrose

The plasticity of root development represents a key trait that enables plants to adapt to diverse environmental cues. The pattern of cell wall deposition, alongside other parameters, affects the extent, and direction of root growth. In this study, we report that FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 18 (FLA18) plays a role during root elongation in Arabidopsis thaliana. Using root-specific co-expression analysis, we identified FLA18 to be co-expressed with a sub-set of genes required for root elongation. FLA18 encodes for a putative extra-cellular arabinogalactan protein from the FLA-gene family. Two independent T-DNA insertion lines, named fla18-1 and fla18-2, display short and swollen lateral roots (LRs) when grown on sensitizing condition of high-sucrose containing medium. Unlike fla4/salt overly sensitive 5 (sos5), previously shown to display short and swollen primary root (PR) and LRs under these conditions, the PR of the fla18 mutants is slightly longer compared to the wild-type. Overexpression of the FLA18 CDS complemented the fla18 root phenotype. Genetic interaction between either of the fla18 alleles and sos5 reveals a more severe perturbation of anisotropic growth in both PR and LRs, as compared to the single mutants and the wild-type under restrictive conditions of high sucrose or high-salt containing medium. Additionally, under salt-stress conditions, fla18sos5 had a small, chlorotic shoot phenotype, that was not observed in any of the single mutants or the wild type. As previously shown for sos5, the fla18-1 and fla18-1sos5 root-elongation phenotype is suppressed by abscisic acid (ABA) and display hypersensitivity to the ABA synthesis inhibitor, Fluridon. Last, similar to other cell wall mutants, fla18 root elongation is hypersensitive to the cellulose synthase inhibitor, Isoxaben. Altogether, the presented data assign a new role for FLA18 in the regulation of root elongation. Future studies of the unique vs. redundant roles of FLA proteins during root elongation is anticipated to shed a new light on the regulation of root architecture during plant adaptation to different growth conditions.

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