Glucuronosyltransferase Genes Involved in Arabinogalactan Biosynthesis Function in Arabidopsis Growth and Development

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Article Three β-Glucuronosyltransferase Genes Involved in Arabinogalactan Biosynthesis Function in Arabidopsis Growth and Development

Oyeyemi O. Ajayi 1,2, Michael A. Held 2,3 and Allan M. Showalter 1,2,*

1 Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA; [email protected] 2 Molecular and Cellular Biology Program, Ohio University, Athens, OH 45701, USA; [email protected] 3 Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA * Correspondence: [email protected]

Abstract: Arabinogalactan proteins (AGPs) contain arabinogalactan (AG) polysaccharides that are biologically relevant to plant growth processes. Here, the biochemical and physiological roles of three Golgi localized β-glucuronosyltransferase genes (GLCAT14A, GLCAT14B and GLCAT14C) in Arabidopsis thaliana, responsible for the addition of glucuronic acid to AG chains, were further investigated using single, double and triple glcat14 mutant plants. These proteins were localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. Sugar analysis of AGP extracts from Arabidopsis stem, leaf and siliques showed a consistent reduction in glucuronic acid in glcat14 mutants relative to wild type, with concomitant effects resulting in tissue-speciﬁc alterations, especially in arabinose and galactose sugars. Although we observed defects in trichome branching Citation: Ajayi, O.O.; Held, M.A.; in glca14a/b and glca14a/b/c mutants, scanning electron microscope analysis/energy dispersive mi- Showalter, A.M. Three croanalysis (SEM/EDX) showed no difference in the calcium content of trichomes in these mutants β-Glucuronosyltransferase Genes relative to wild type. Immunoblot analyses of the stem and leaf showed a reduction in AGPs as Involved in Arabinogalactan detected with the LM2 antibody in glcat14a/b and glcat14a/b/c mutants relative to wild type. The Biosynthesis Function in Arabidopsis current work exempliﬁes the possibility of conducting structure-function assessment of cell wall Growth and Development. Plants 2021, 10, 1172. https://doi.org/ biosynthetic genes to identify their physiological roles in plant growth and development. 10.3390/plants10061172 Keywords: arabinogalactan; cell wall; calcium; sugar; germination; Arabidopsis; glucuronic acid Academic Editor: Victor Manuel Quesada-Pérez

Received: 24 May 2021 1. Introduction Accepted: 1 June 2021 Plant cell walls are composed of structurally complex heteropolymers that are essential Published: 9 June 2021 to plant development. Arabinogalactan-proteins (AGPs) are a family of complex proteo- glycans found in the plant cell walls of all higher plants [1] and contain a large amount of Publisher’s Note: MDPI stays neutral arabinogalactan polysaccharide (90% w/w) attached to hydroxyproline residues in their with regard to jurisdictional claims in protein cores [2]. AGPs belong to a superfamily of structural macromolecules known as the published maps and institutional afﬁl- hydroxyproline-rich glycoproteins (HRGPs), of which extensins and proline-rich proteins iations. are members with unique carbohydrate moieties central to their biological functions [3]. Out of all the HRGPs, AGPs are the most complex, and their complexity is driven by the heterogeneity of their type II arabinogalactans (AG) and the variety of their protein back- bones [4]. AGPs have N-terminal signal peptides, which are cleaved to allow for secretion, Copyright: © 2021 by the authors. and about half of AGP family members also have C-terminal glycosylphosphatidylinositol Licensee MDPI, Basel, Switzerland. (GPI) anchors to allow for their retention and attachment to the outer leaﬂet of the plasma This article is an open access article membrane [5]. distributed under the terms and AGPs are implicated in an array of plant growth and development processes, in- conditions of the Creative Commons cluding cell expansion, somatic embryogenesis, root and stem growth, salt tolerance, Attribution (CC BY) license (https:// hormone signaling, programmed cell death, male and female gametophyte development, creativecommons.org/licenses/by/ and wounding/defense [4,6]. Structurally, AGs are O—linked to hydroxyproline residues 4.0/).

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in the AGP protein core, and each AG is characterized by the presence of a β-1,3-galactan backbone that is substituted at various O6 positions with β-1,6-galactan side chains [7]. These so-called type II galactan chains are further modified by arabinose (Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl) and glucuronic acid or 4–O–methyl glucuronic acid (collec- tively referred to as GlcA) [8–10]. These various AGP glycosylation reactions are catalyzed by a family of enzymes known as glycosyltransferases (GTs) [9]. While the functional roles of several AGP GTs have been identified and characterized to varying extents using molecular and biochemical approaches, the identity and biological roles of many other AGPs GTs remain to be explored. The AGP glucuronosyltransferases or glucuronic acid transferases (GLCATs) repre- sent one family of AGP GTs, which has been characterized to some extent. As their name implies, this family of enzymes transfer glucuronic acid (GlcA) to type II AGs and are members of the GT14 family of the carbohydrate active enzyme (CAZy). Previous work on ATGLCAT14A-C demonstrated that they transfer GlcA to both β-1,3-galactan and β- 1,6-galactan side chains with distinct substrate preferences [7,11]. Recently, two additional genes/enzymes, GLCAT14D and E, were reported to be involved in the glucuronidation of type II AGs [12]. Furthermore, glcat14a-1 and glcat14a-2, two allelic T-DNA insertion mutants, were reported to display the increased cell elongation phenotype in etiolated seedlings [7], a finding that was disputed in a later study [12]. In addition, multiple developmental defects such as reduced trichome branching and severe reduction in the growth of etiolated seedlings were observed in glcat14a/b/e mutants, including the perturbation of the calcium waves [12]. In another related study, CRISPR-Cas9-generated glcat14a/b and glcat14a/b/c mutants showed delayed seed germination, reductions in root hair length and trichome branching, reduced seed set and developmental defects in seed mucilage and pollen grains [13]. These results reinforce the critical roles that these biosynthetic enzymes play in different aspects of plant growth. Calcium is an essential element needed for the growth and development of plants under both normal (non-stress) and stress conditions. Calcium impacts cell wall rigidity, and low calcium content weakens the cell wall to facilitate cell expansion [14]. Notably, calcium performs multiple roles in plant growth, but its effect is not limited to cell wall and membrane stability, but also serves as a secondary messenger in many aspects of plant biological processes [15]. There are speculations that AG glucuronidation of AGPs is a major source of cytosolic Ca2+, given the negatively charged GlcAs residues’ inherent ability to bind and release calcium in a pH-dependent manner at the periplasmic surface of the plasma membrane [16]. Previous work showed that some of the developmental defects in glcat14a/b/e mutants, specifically the trichome branching defects, were suppressed by increasing the calcium content in the growth medium [12]. This reinforces the interdepen- dence between cell surface apoplastic calcium and GlcA residues in AGPs in regulating important signaling events critical to plant growth and development. Here, we focus on the biochemical and physiological characterization of glcat14 T- DNA insertion mutants of glcat14a, glcat14b, glcat14c, glcat14a/c, glcat14b/c along with CRISPR-Cas9 generated glcat14a/b and glcat14a/b/c mutants. Specifically, we sought to understand the degree to which the alterations in the amounts of GlcA and calcium content accounts for the mutant phenotypes identified in this study using various biochemical and molecular genetic approaches. This will bring us a step closer towards understanding the relative contributions of the GLCAT genes in cell wall integrity maintenance, critical to plant growth processes.

2. Results 2.1. The CAZy GT14 Family This work sought to determine the biochemical and physiological roles of three functionally characterized GLCATs (GLCAT14A, GLCAT14B and GLCAT14C) in Arabidopsis. Previous work provided enzymatic evidence that three GLCATs (GLCAT14A, GLCAT14B, GLCAT14C) out of the 11 putative Arabidopsis GLCAT genes/enzymes in the CAZy Plants 2021, 10, x FOR PEER REVIEW 3 of 19

Plants 2021, 10, 1172 Arabidopsis. Previous work provided enzymatic evidence that three GLCATs 3 of 19 (GLCAT14A, GLCAT14B, GLCAT14C) out of the 11 putative Arabidopsis GLCAT genes/enzymes in the CAZy GT14 family function in glucuronidation of AGPs [11]. More recently, GT14 GLCAT14D family function and GLCAT14E in glucuronidation were reportedof AGPs [11 to]. Morebe involved recently, GLCAT14D in AG and glucuronidationGLCAT14E [12]. were A common reported feature to be in involved verified in and AG putative glucuronidation GLCATs [in12 Arabidopsis]. A common feature is the presencein veriﬁed of a highly and putative conserved GLCATs GLCAT in Arabidopsisdomain [17]. isAdditionally, the presence the of intron a highly-exon conserved genetic architectureGLCAT domain of Arabidopsis [17]. Additionally, CAZy the GT14 intron-exon family members genetic architecture is conserved of andArabidopsis includes CAZythe presence GT14 familyof four members exons and is three conserved introns and (Fig includesure 1B). the Phy presencelogenetic of analysis four exons and showed thatthree GLCAT14A introns (Figure and 1GLCAT14BB). Phylogenetic belong analysis to the showedsame phylogenetic that GLCAT14A clade,and whileGLCAT14B GLCAT14Cbelong, D and to the E sameare phylogenetically phylogenetic clade, distant while fromGLCAT14C GLCAT14A, D and andE areGLCAT14B phylogenetically, consistentdistant with from earlierGLCAT14A reports and[11,12GLCAT14B]. Sequence, consistent analysis with showed earlier reports differences [11,12 ]. in Sequence similaritiesanalysis among showed the GLCAT14 differences genes in,similarities with the lowest among sequence the GLCAT14 similaritygenes, observed with the to lowest se- be 39% (betweenquence similarity AT1G53100 observed and ATGLCAT14D to be 39% (between) and highestAT1G53100 beingand betweenATGLCAT14D AT4G03340) and highest and AT1G03520being between); othersAT4G03340 are nestledand betweenAT1G03520 these );two others extremes are nestled (Table between S2). Interestingly, these two extremes these five(Table enzymes, S2). Interestingly, ATGLCAT14A/B/C/D/E, these ﬁve enzymes, demonstrated ATGLCAT14A/B/C/D/E, to be involved in demonstrated the glucuronidationto be involved of type in II the AGs, glucuronidation have unique of substrate type II AGs, preferences; have unique AT substrateGLCAT14A, preferences; ATGLCAT14BATGLCAT14A, and ATGLCAT14C ATGLCAT14B transfer and ATGLCAT14C GlcA onto both transfer β- 1,3 GlcA and onto β- both1,6 galactanβ- 1,3 and β- 1,6 chains ofgalactan shorter chainslength, of whil shortere AT length,GLCAT14D while ATGLCAT14Dand ATGLCAT14E and ATGLCAT14E have preferences have for preferences longer galactanfor longer side galactan chains [11,12]. side chains [11,12].

Figure 1. Cont.

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Figure 1. ATGLCAT14A (AT5G39990), ATGLCAT14B (AT5G15050), and ATGLCAT14C (AT2G37585) gene/genetic mutant Figure 1. ATGLCAT14A (AT5G39990), ATGLCAT14B (AT5G15050), and ATGLCAT14C (AT2G37585) gene/genetic mutant structure,structure, phylogenetic phylogenetic relationships, and expression. expression. ((AA)) SchematicSchematic drawing drawing of of the theATGLCAT14A ATGLCAT14A(AT5G39990 (AT5G39990), AT-), ATGLCAT14BGLCAT14(BAT5G15050 (AT5G15050) and) andATGLCAT14C ATGLCAT14C(AT2G37585 (AT2G37585) gene) structuregene structure and sites and of T-DNA sites of insertions T-DNA in insertions two independent in two independentlines: atglcat14a-1 lines: andatglcatatglcat14a-214a-1 andare atglcat14a allelic mutants-2 are allelic for ATGLCAT14A mutants for AT; atglcat14b-1GLCAT14Aand; atglcat14batglcat14b-2-1 andare at allelicglcat14b mutants-2 are allelicfor ATGLCAT14B mutants for; ATatglcat14c-1GLCAT14B, and; atatglcat14c-2glcat14c-1, and, generated atglcat14c by-2 CRISPR-Cas9, generated by strategy CRISPR targeting-Cas9 strategy exon 1 targeting by two guide exon RNAs1 by twogRNA1 guide (C1) RNAs and gRNA1 gRNA2 (C1) (C2) and as indicated gRNA2 above(C2) as [ 12indicated] are allelic above mutants [12] are for allelicATGLCAT14C mutants. Exonsfor AT areGLCAT14C represented. Exons by blueare representedboxes; introns by blue are represented boxes; introns by thinare represented gray lines and by thin non-coding gray lines regions and non are-coding represented regions by are orange represented boxes. The by orange T-DNA boxes.insertion The sitesT-DNA for eachinsertion mutant sites are for indicated,each mutant with are primer indicated, binding with sitesprimer indicated binding by sites black indicated arrows. by The black branch arrows. domain The branch domain corresponds to PF02485 from the Pfam database is indicated above. Red arrowheads indicate primer corresponds to PF02485 from the Pfam database is indicated above. Red arrowheads indicate primer binding sites for binding sites for qRT-PCR. (B) Phylogenetic analysis of the GT14 gene family along with their exon-intron structure. (C) qRT-PCR. (B) Phylogenetic analysis of the GT14 gene family along with their exon-intron structure. (C) Relative gene Relative gene expression of GLCAT14A, GLCAT14B, and GLCAT14C in Arabidopsis leaves in various genetic mutant backgroundsexpression of as GLCAT14A, determined GLCAT14B, by qRT-PCR. and Transcript GLCAT14C levels in Arabidopsis were normalized leaves in to various the mean genetic of one mutant reference backgrounds gene, the as Arabidopsisdetermined Actin by qRT-PCR. 2 gene, AtACT2. Transcript Averages levels were of three normalized biological to replicates the mean ± of SE one are reference shown. gene, the Arabidopsis Actin 2 gene, AtACT2. Averages of three biological replicates ± SE are shown. 2.2. glcat14 Mutant Generation and Characterization 2.2. glcat14 Mutant Generation and Characterization Arabidopsis glcat14 T-DNA insertion mutant lines were confirmed for the presence of T-DNAArabidopsis insertionsglcat14 (FigT-DNAure S1A) insertion and were mutant subsequently lines were conﬁrmedused for genetic for the presence crosses to of obtainT-DNA higher insertions order (Figure homozygous S1A) and mutants were subsequently(Figure S1B,C used). Additionally, for genetic results crosses obtained to obtain usinghigher qRT order-PCR homozygous showed the absence mutants of (Figure transcripts S1B,C). in these Additionally, mutants (Fig resultsure obtained1C). We could using notqRT-PCR verify showed the presence the absence of a T of-DN transcriptsA insertion in these in the mutants SALK_051810 (Figure 1 lineC). We belonging could not to verify the presence of a T-DNA insertion in the SALK_051810 line belonging to GLCAT14C, GLCAT14C, but a CRISPR knock-out mutant (glcat14c-2) has a 178 bp deletion [13] in the but a CRISPR knock-out mutant (glcat14c-2) has a 178 bp deletion [13] in the ﬁrst exon first exon (Figure 1A) and biological phenotypes were comparable to those obtained in (Figure1A) and biological phenotypes were comparable to those obtained in the glcat14c-1 the glcat14c-1 mutant (SALK_005705), as subsequently described. mutant (SALK_005705), as subsequently described.

2.3.2.3. Subcellular Subcellular L Localizationocalization of of GLCAT14A, GLCAT14A, GLCAT14B GLCAT14B and and GLCAT14C GLCAT14C InIn the the subcellular subcellular localizationlocalization experimentexperiment involving involving ATGLCAT14A, ATGLCAT14A, ATGLCAT14B ATGLCAT14B and andATGLCAT14C, ATGLCAT14C, we detectedwe detected punctate punctate vesicles vesicles that that co-localized co-localized with with the the Golgi Golgi marker marker sia- sialyltransferaselyltransferase short short cytoplasmic cytoplasmic tail and tail single and single transmembrane transmembrane domain domain fused to fusedenhanced to enhancedGFP (STtmd-GFP) GFP (STtmd (Figure-GFP) S3), indicating (Figure S3 localization), indicating of ATGLCAT14A, localization of ATGLCAT14B ATGLCAT14A, and ATGLCAT14BATGLCAT14C and in the ATGLCAT14C Golgi apparatus. in the Golgi apparatus.

2.4.2.4. glcat14a/b glcat14a/b and and glcat14a/b/c glcat14a/b/c h Hadad Reduced Reduced Immunolabelling Immunolabelling of ofLM2 LM2 bound Bound AGPs AGPs in inStem Stem and Leafand Leaf StructuralStructural modifications modiﬁcations can affectaffect thethe abundanceabundance of of glycan glycan moieties moieties that that make make up up the theplant plant cell cell wall. wall. These These structural structural changes changes can be can delineated be delineated using antibodiesusing antibodies raised againstraised

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Plants 2021, 10, 1172 5 of 19 against glycan epitopes in an immunoblot analysis. The LM2 antibody, which specifically glycanbinds to epitopes β-glucuronic in an immunoblot acid in Type analysis. II AGs in The AGP LM2 protein antibody, cores, which was used speciﬁcally to immunolabel binds to βwild-glucuronic type, glcat14a/b acid in Type and II glcat14a/b/c AGs in AGP leaf protein and cores, stem was protein used extracts to immunolabel separated wild by type, SDS- glcat14a/bPAGE priorand toglcat14a/b/c membraneleaf transfer. and stem As expected, protein extracts LM2 immunolabelling separated by SDS-PAGE generated prior broad to membranesmears, reflective transfer. of As the expected, heavily glycosylated LM2 immunolabelling nature of generatedAGPs, and broad the labelling smears, intensities reﬂective ofof theLM2 heavily-bound glycosylated AGPs were greater nature in of lea AGPs,ves than and in the stem labellings (Figure intensities 2A,B). In of contrast LM2-bound to the AGPswild were type,greater weak in signals leaves thanwith in LM2 stems-bound (Figure AGPs2A,B). were In contrast observed to the in wild glcat14a/b type, weak and signalsglcat14a/b/c with LM2-boundextracts from AGPs leaves were (Fig observedure 2A) in andglcat14a/b stemsand (Figglcat14a/b/cure 2B). Moreover,extracts from the leavesmolecular (Figure size2 A) of and LM2 stems-bound (Figure AGPs2 B). in theMoreover, stem and the the molecular leaf were size similar, of LM2-bound and signal AGPsintensities in the were stem in and the the high leaf molecular were similar, mass and (100 signal–250 intensities kDa) region were of inthe the Western high molecular blot. mass (100–250 kDa) region of the Western blot.

Figure 2. Western blot analysis of protein extracts from stem and leaf of wild type, glcat14a/b and glcat14a/b/c. Proteins Figure 2. Western blot analysis of protein extracts from stem and leaf of wild type, glcat14a/b and glcat14a/b/c. Proteins (25 µ (25µg) g)extracted extracted from from plant plant materials materials were were loaded loaded per per lane lane and and separated separated by by SDS SDS-PAGE-PAGE according to size, PonceauPonceau stained,stained, andand subsequently subsequently transferred transferred to to PVDF PVDF membranes. membranes. LM2 LM2 antibody antibody was was used used for for immunoblotting immunoblotting to target to targetβ-GlcA β-GlcA in leaf in (leafA) and(A) stemand stem (B). Molecular (B). Molecu masslar mass (kDa) (kDa) is indicated is indicated on the on left. the MM,left. MM, Molecular Molecular marker. marker.

2.5.2.5. QuantificationQuantification ofofβ β-D-Gal-Yariv-D-Gal-Yariv Precipitated Precipitated AGPs AGPs in in glcat14 glcat14 Mutants Mutants GivenGiven thethe differentialdifferential expressionexpression ofofGLCATs GLCATsin in different different organs organs and and across across different different developmentaldevelopmental stagesstages [[1717],], wewe quantifiedquantified thethe amountamount ofof YarivYariv precipitable precipitable AGPsAGPs fromfrom glcat14 β differentdifferent organsorgans ofof WTWT and glcat14 mutants. The The ability ability of of β-D-D-Gal-Yariv-Gal-Yariv to to bind bind to to the the β- β-1,3-galactose backbone on AGPs was exploited to isolate and quantify AGP content as 1,3-galactose backbone on AGPs was exploited to isolate and quantify AGP content as described previously [18]. Across the organs examined (rosette leaves, stems and siliques), described previously [18]. Across the organs examined (rosette leaves, stems and siliques), the amounts of Yariv precipitable AGPs in the stems and siliques were greater than those the amounts of Yariv precipitable AGPs in the stems and siliques were greater than those observed in the rosette leaves. Specifically, in contrast to WT, we observed an increase in observed in the rosette leaves. Specifically, in contrast to WT, we observed an increase in Yariv precipitable AGPs in glcat14a, glcat14a/c, glcat14a/b and glcat14a/b/c mutants in the Yariv precipitable AGPs in glcat14a, glcat14a/c, glcat14a/b and glcat14a/b/c mutants in the rosette leaves and stems; however, in the siliques, the glcat14a mutants along with glcat14c, rosette leaves and stems; however, in the siliques, the glcat14a mutants along with glcat14c, glcat14a/c, glcat14a/b and glcat14a/b/c mutants showed an increase in Yariv precipitable glcat14a/c, glcat14a/b and glcat14a/b/c mutants showed an increase in Yariv precipitable AGPs (Figure3). The glcat14b and glcat14b/c mutants, however, did not show an increase in AGP content and were comparable to WT.

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AGPs (Figure 3). The glcat14b and glcat14b/c mutants, however, did not show an increase Plants 2021, 10, 1172in AGP content and were comparable to WT. 6 of 19

Figure 3. Quantification of AGPs in the various glcat mutants in different organs of 40-day-old Arabidopsis plants. Leaves, Figure 3. Quantification of AGPs in the various glcat mutants in different organs of 40-day-old siliques, andArabidopsis stems were obtainedplants. Leaves, from 40-day-old siliques, and Arabidopsis stems were plants. obtainedglcat14a from, glcat14a/c 40-day-,oldglcat14a/b Arabidopsisand glcat14a/b/c showed significant increasesplants. glcat14a in AGP, glcat14a/c content in, glcat14a/b the leaves and and glcat14a/b/c stems, while showedglcat14a significant, glcat14c ,increasesglcat14a/c in, glcat14a/b AGP contentand glcat14a/b/c showed increasesin the leaves in AGP and content stems, in while siliques. glcat14a AGPs, glcat14c were, measuredglcat14a/c, glcat14a/b in micrograms and glcat14a/b/c per gram showed fresh weight. Statistical differences wereincreases determined in AGP by content two-way in siliques. ANOVA, AGPs followed wereby measured the Tukey’s in microgram honestly significants per gram difference fresh weight. test (** p < 0.01); results wereStatistical averages ofdifferences three independent were determined experiments. by two-way ANOVA, followed by the Tukey’s honestly significant difference test (** p < 0.01); results were averages of three independent experiments. 2.6. Monosaccharide Composition Analyses and Calcium Content 2.6. Monosaccharide AGPComposition glycans Analyses were evaluated and Calcium in stems,Content siliques and leaves to provide insights into AGP glycansorgan-specific were evaluated glycan in composition stems, siliques and and to understand leaves to provide to what insights extent the into AGP structural organ-specificmodifications glycan composition would impactand to theunderstand abundance to ofwhat other extent AGP the sugars AGP in structuralglcat14 mutants relative modifications to would WT. When impact the the plant abundance cell wall integrity of other (CWI) AGP system sugars is in compromised, glcat14 mutants adaptive changes relative to WT.in When cellular the and plant cell cell wall wall metabolism integrity (CWI) can alter system wall compositionis compromised, and structureadaptive [19]. Previous changes in cellularwork and on cellglcat14a wall mutantsmetabolism reported can alter a slightwall composition increase in galactosylation,and structure [19 while]. the GlcA Previous workcontent on glcat14a was mutants comparable reported to WT a slight in AG increase extracts in fromgalactosylation 14-day-old, while seedlings the [7]. In acid- GlcA content washydrolyzed comparable AGP to extracts WT in AG investigated extracts from by HPAE-PAD, 14-day-old seedlings we observed [7]. significantIn acid- changes in hydrolyzed AGPsugar extracts amounts investigated relative to by WT. HPAE Specifically,-PAD, we in the observed leaves, andsignificant with the changes exception of glcat14a in sugar amountsand glcat14crelative, to we WT. observed Specifically, an increase in the in leaves, the amounts and with of galactose the exception and minor of changes in glcat14a and glcat14carabinose, we relative observed to the an wild increase type (Table in the S3). amounts While reductions of galactose in andtheamount minor of GlcA were changes in arabinoseobserved relative across to all the genotypes wild type in (Table leaves, S3). we While focused reductions on glcat14a/b in theand amountglcat14a/b/c mutants of GlcA werebecause observed the across leaf trichome all genotypes branching in phenotype leaves, we was focused only found on glcat14a/b in thesemutants and (discussed glcat14a/b/c mutantsbelow) because and was the absent leaf intrichome the glcat14 branchingT-DNA insertionphenotype lines. was In only the leaffound AGPs, in we observed these mutantsa (discussed 47% and 49%below) reduction and was in absent the amounts in the glcat14 of GlcA T- inDNAglcat14a/b insertionand lines.glcat14a/b/c In mutants, respectively, relative to wild type (Figure4A), while for stem AGPs, we observed a 35% and 73% reduction in GlcA content in glcat14a/b and glcat14a/b/c mutants, respectively, relative to wild type; the remaining genotypes had comparable GlcA amounts to WT in

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the leaf AGPs, we observed a 47% and 49% reduction in the amounts of GlcA in glcat14a/b and glcat14a/b/c mutants, respectively, relative to wild type (Figure 4A), while for stem Plants 2021, 10, 1172 AGPs, we observed a 35% and 73% reduction in GlcA content in glcat14a/b and glcat14a/b/c7 of 19 mutants, respectively, relative to wild type; the remaining genotypes had comparable GlcA amounts to WT in stem AGPs (Table S4). With the exception of glcat14a/c and stemglcat14b/c AGPs, which (Table wereS4). With not significantlythe exception different of glcat14a/c fromand WT,glcat14b/c a reduction, which in were galactose not signiﬁcantlycontent in glcat14a/b/c different from in the WT, stem a reduction was unexpected, in galactose given content the significant in glcat14a/b/c increasesin the in stem AGP wascontent unexpected, of glcat14a/b/c given mutants the signiﬁcant in stem increasestissues. In in addition AGP content, minor ofchangesglcat14a/b/c were mutantsobserved inin stemthe amount tissues. of In arabinose addition, in minorglcat14 changes mutants wererelative observed to WT. For in the the amount siliques, of we arabinose observed ina reductionglcat14 mutants in the GlcA relative content to WT. in glcat14 For the mutants, siliques, coupled we observed with a corresponding reduction in the increase GlcA contentin galactose in glcat14 contentmutants, in both coupled single and with high a correspondinger order glcat14 increase mutants in (Table galactose S5A). content It should in bothbe noted single that and some higher residue order amountglcat14 mutantsof GlcA remains (Table S5A). associated It should with be the noted AGPs that in some all of residuethese organs, amount regardless of GlcA remains of the associatedmutant. This with suggest the AGPs the ininvolvement all of these organs,of other regardless GLCATs, ofwhose the mutant. roles remain This to suggest be elucidated. the involvement of other GLCATs, whose roles remain to be elucidated.

Figure 4. Cont.

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Figure 4.4. Biochemical and physiological analyses of wild type and and glcat14glcat14 mutants.mutants. (A) MonosaccharideMonosaccharide composition analysis of leaf AGPs extracted from wild type and glcat14a/b and glcat14a/b/c mutants using HPAE HPAE-PAD.-PAD. (B) Calcium content of leaf AGP ext extractsracts of wild type, glcat14a/b,glcat14a/b, and glcat14a/b/c (expressed(expressed as as % % calcium per per AGPs). AGPs). ( (CC)) HPAE HPAE-PAD-PAD monosaccharide compositioncomposition analysisanalysis ofof stemstem AGPs AGPs extracted extracted from from wild wild type type and andglcat14 glcat14mutants. mutants. (D ()D Calcium) Calcium content content of ofstem stem AGPs AGPs extracted extracted from from wild wild type, type,glcat14a/b glcat14a/b, and,glcat14a/b/c and glcat14a/b/c(expressed (expressed as % calcium as % calcium per AGPs). per ForAGPs). the sugarFor the analysis, sugar analysis, values are relative to total sugar composition (expressed as mol %) of triplicate assays ± SE. For the calcium assay, values are relative to total sugar composition (expressed as mol %) of triplicate assays ± SE. For the calcium assay, values are values are the mean ± standard deviation from three biological replicates. (E) Growth phenotypes of 40-day-old glcat14 ± E glcat14 mutantsthe mean andstandard WT showed deviation growth from reductions three biological in the glcat14 replicates. double ( )and Growth triple phenotypes mutants relative of 40-day-old to WT; Scale barmutants = 12 cm. and (F) WTInflorescence showed growth stem lengths reductions from in 40 the-dayglcat14-old wilddouble-type and and triple glcat14 mutants mutant relative plants. to Asterisks WT; Scale indicate bar = 12 significant cm. (F) Inflorescence differences betweenstem lengths glcat14 from mutants 40-day-old and WT wild-type as defined and byglcat14 one-mutantway ANOVA plants. followed Asterisks by indicate Tukey’s significant multiple differencescomparison between test (Student’sglcat14 tmutants-test, p < and 0.05 WT for assingle defined asterisks, by one-way p < 0.01 ANOVA for double followed asterisks), by Tukey’s n = 15multiple per line comparisonper replicate. test (Student’s t-test, p < 0.05 for single asterisks, p < 0.01 for double asterisks), n = 15 per line per replicate. Calcium is well known as a universal signaling molecule and acts as a ‘second messenger’Calcium in is plants well known [20,21]. as The a universal ability signaling of AGP to molecule bind and and release acts as calcium a ‘second in messen- a pH- dependentger’ in plants manner [20,21 ].in The vitro ability gave of rise AGP to to the bind AGP and Ca release2+ capacitor calcium hypothesis in a pH-dependent [16]. The 2+ intramolecularmanner in vitro calciumgave rise binding to the AGPabilities Ca bycapacitor paired GlcA hypothesis residues [ 16of]. AGPs The intramolecular was exploited tocalcium estimate binding the calcium abilities content by paired of AGPs. GlcA We residues investigated of AGPs whether was exploited there were to estimate significant the alterationscalcium content in calcium of AGPs. bound We by investigated AGPs, possibly whether mediated there by were the reduction significant in alterations the amounts in ofcalcium GlcA boundacross bytissues AGPs, investigated possibly mediated using a bycolorimetric the reduction assay in the[22] amounts. We observed of GlcA that across the calciumtissues investigated content of AGP using extracts a colorimetric in glcat14 assay mutants [22]. We were observed similar that to the the calcium WT in content leaves (Figof AGPure 4B) extracts and insiliquesglcat14 (Tablemutants S5B); were however, similar tothat the was WT not in leavesthe case (Figure for stems.4B) and While siliques the glcat14a calcium(Table S5B); content however, of glcat14a that was was not similar the case to for WT, stems. we Whileobserved the calciuma significant content reduction of in was similar to WT, we observed a significant reduction in calcium content in glcat14b and calcium content in glcat14b and glcat14c single mutants, and also for the higher order glcat14c single mutants, and also for the higher order glcat14 mutants in stem (Figure4D). glcat14 mutants in stem (Figure 4D) 2.7. Both Single and Double glcat14 Mutant Display Pleiotropic Growth Defects 2.7. Both Single and Double glcat14 Mutant Display Pleiotropic Growth Defects Seeds obtained from WT and glcat14 mutant lines were grown on MS plates sup- plementedSeeds withobtained or without from WTABA and to investigate glcat14 mutant the potential lines were roles grownof GLCAT14A, on MS B, plates and supplementedC in seed germination, with or without given that ABAGLCAT14A to investigateand GLCAT14B the potentialare roles both of highly GLCAT14A, expressed B, andin the C micropylar in seed germination endosperm,, given and the th endospermat GLCAT14A cell and wall’s GLCAT14B weakening are is repressed both highly by expressedABA [13]. in The theglcat14 micropylT-DNAar endosperm mutant lines, and germinated the endosperm like WT cell seeds wall’s under weakening standard is repressedgrowth conditions. by ABA [13] However,. The glcat14 when T the-DNA growth mutant media lines were germinated supplemented like WT with seeds 1 µM under ABA, standardwe observed growth that conditions. the glcat14 mutants However, exhibited when the a delay growth in germinationmedia were (Figuresupplemented5). Similarly, with 1seeds µM ABA, grown we under observed light that conditions the glcat14 for9 mutants d showed exhibited no obvious a delay differences in germination in root (Fig growthure 5).relative Similarly, to WT seeds (Figure grown S2A). under Additionally, light conditions dark grown for 9 etiolatedd showed seedlings no obvious in bothdifferencesglcat14 inT-DNA root growth mutant relative lines and to theWTCRISPR (Figure linesS2A). showed Additionally, no obvious dark differencesgrown etiolated in root seedlings growth inrelative both glcat14 to WT (FigureT-DNA S2B).mutant Previous lines and work the reported CRISPR an lines increased showed length no obvious of hypocotyls differences and inroots root in growthglcat14a relativemutants to of WT 5-day (Fig oldure etiolated S2B). Previous seedlings work [7]; however,reported itan is increased noteworthy length that ofour hypocotyls observation and agrees roots within glcat14a earlier mutants work that of 5 showed-day old no etiolated difference seedlings in hypocotyl [7]; however, length itin is etiolated noteworthy seedlings that our except observation for glcat14a/b/e agrees, whichwith earlier were remarkablywork that showed shorter no [12 difference]. We also ininvestigated hypocotyl length the role in of etiolated GLCAT14A, seedlings B, and except C in for plant glcat14a/b/e height, and, which our resultswere remarkably show that

Plants 2021, 10, x FOR PEER REVIEW 9 of 19 Plants 2021, 10, 1172 9 of 19

shorter [12]. We also investigated the role of GLCAT14A, B, and C in plant height, and ourglcat14a/c results showand glcat14b/c that glcat14a/cand and CRISPR glcat14b/c lines and (glcat14a/b CRISPR and linesglcat14a/b/c (glcat14a/b )and were signiﬁcantly glcat14a/b/cshorter)than were thesignificantly wild type shorter plants than (Figure the wild4E,F). type plants (Figure 4E,F).

FigureFigure 5. Germination 5. Germination percentages percentages of the glcat14 of mutants the glcat14 grownmutants in the presence grown of in1 µM the ABA. presence of 1 µM ABA. GerminationGermination percentages percentages of glcat14 of mglcat14utants mutantsshowed delayed showed germination delayed compared germination to wild compared type to wild type in in normal ½ MS supplemented with 1 μM ABA, which were grown for 12 days following 3 days 1 of stratificationnormal /2 atMS 4 ° supplementedC (See methods). Twenty with 1-fiveµM seeds ABA, of whicheach genotype were grown were used for for 12 days following 3 days of germination,stratification with atthr 4ee◦ replicates.C (See methods). Twenty-five seeds of each genotype were used for germination, with three replicates. Previous work observed that CRISPR-Cas9 triple knockouts of GLCAT14A-C [13] showed Previousdefects in trichome work observed branching. thatA recent CRISPR-Cas9 study similarly triple identified knockouts ATGLCAT14D of GLCAT14A-C [13] andshowed ATGLCAT14E defects as in being trichome involved branching. in ensuring A recent normal study trichome similarly development; identified this ATGLCAT14D study also found that trichome defects in glcat14 mutants were suppressed in a Ca2+ concentrationand ATGLCAT14E-dependent manner as being [12]. involved Using SEM, in we ensuring examined normal the trichome trichome defects development; of this 2+ glcat14a/bstudy and also glcat14a/b/c found that and trichome conducted defectsan elemental in glcat14 compositionmutants analysis were directed suppressed at in a Ca estimatingconcentration-dependent the amount of calcium manner using the [ 12energy]. Using dispersive SEM, X we-ray examined(EDX) microanalysis the trichome defects of techniqueglcat14a/b [23].and Energyglcat14a/b/c dispersiveand X-ray conducted (EDX) microanalysis an elemental is an compositionanalytical technique analysis directed at commonlyestimating used the to give amount semiquantitative of calcium measurements using the energy of surface dispersive elements X-ray within (EDX) the microanalysis surfacetechnique layer of [23 1].–2 Energy microns dispersive in thickness X-ray [24]. While (EDX) the microanalysis significant majority is an of analytical the technique trichomes in glcat14a/b and glcat14a/b/c are one-branched instead of the two-branched commonly used to give semiquantitative measurements of surface elements within the sur- trichomes characteristics of WT (Figure 6A; Table 1), EDX spectra of investigated genotypesface layer for of elemental 1–2 microns calcium in showed thickness no [ significant24]. While difference the significants in the normalizedmajority of the trichomes massin glcat14a/bof calcium relativeand glcat14a/b/c to WT (Figareure one-branched6D). It is noteworthy instead that of none the of two-branched the single and trichomes characteristics of WT (Figure6A; Table1), EDX spectra of investigated genotypes for elemental calcium showed no significant differences in the normalized mass of calcium relative to WT (Figure6D ). It is noteworthy that none of the single and double glcat14 T-DNA insertion mutants had defects in trichome branching, as the trichome defects were only observed in glcat14a/b and glcat14a/b/c mutants. Plants 2021, 10, x FOR PEER REVIEW 10 of 19

Plants 2021, 10, 1172 double glcat14 T-DNA insertion mutants had defects in trichome branching, 10as of the19 trichome defects were only observed in glcat14a/b and glcat14a/b/c mutants.

FigureFigure 6. 6.SEM SEM and and SEM/EDX SEM/EDX elemental elemental calcium calcium analyses analyses of trichomesof trichomes of wildof wild type, type,glcat14a/b glcat14a/b,, and glcat14a/b/cand glcat14a/b/cmutants. mutants. (A) SEM(A) imagesSEM images of glcat14a/b of glcat14a/band glcat14a/b/c and glcat14a/b/cmutants showingmutants defects showing in trichome defects branching in trichome relative branching to WT. relative (B) Representative to WT. (B) imageRepresentative of the sampling image pointsof the sampling for SEM/EDX points elemental for SEM/EDX analysis elemental of trichomes. analysis ( Cof) trichomes. Representative (C) Representative spectral plots forspectral the plots for the elemental composition of trichomes. (D) Normalized mass of calcium in WT and glcat14a/b and glcat14a/b/c elemental composition of trichomes. (D) Normalized mass of calcium in WT and glcat14a/b and glcat14a/b/c mutants showed mutants showed no significant difference between WT and the glcat14 mutants. no significant difference between WT and the glcat14 mutants. Table 1. Percentage of trichome branches in wild type, glcat14a/b and glcat14a/b/c. Table 1. Percentage of trichome branches in wild type, glcat14a/b and glcat14a/b/c. Number of Trichome Branches Genotype 1 Number of Trichome2 Branches 3 Total GenotypeWild Type 3.19 1 92.6 2 4.26 3 Total94 Wildglcat14a/b Type 3.1972.06 92.626.47 4.261.47 9468 glcat14a/b/cglcat14a/b 72.0690.2 26.477.84 1.471.96 6851 glcat14a/b/c 90.2 7.84 1.96 51 2.8. glcat14a/c Mutants Had Reduced Pollen Germination and Misshaped Pollen 2.8. glcat14a/c Mutants Had Reduced Pollen Germination and Misshaped Pollen Scanning electron micrographs and in vitro pollen germination experiments were conductedScanning to examine electron micrographsthe pollen morphology and in vitro andpollen pollen germination germination experiments of various glcat14 were conductedT-DNA mutants. to examine Previous the pollen work showed morphology that glcat14a/b and pollen and germination glcat14a/b/c ofmutants various displayedglcat14 T-DNAsignificant mutants. increases Previous in defective work showed pollen that withglcat14a/b concomitantand glcat14a/b/c effects on mutantspollen germination displayed significant[13]. In this increases study, inwe defective observed pollen that within addition concomitant to glcat14a/b effects on and pollen glcat14a/b/c germination mutants, [13]. Inpollen this study, from wethe observedglcat14a/c thatmutant in addition also demonstrated to glcat14a/b aand significantlyglcat14a/b/c lowermutants, germination pollen fromrate thecomparedglcat14a/c to WTmutant (Fig alsoure 7 demonstratedB), which could a significantly be reflective lower of the germination significant increase rate com- in pareddefective to WT pollen (Figure in glcat14a/c7B), which mutants could bein both reflective the in of vitro the significantpollen germination increase (Fig in defectiveure 7A,C) glcat14a/c pollenand SEM in (Figuremutants 7E). Although in both thethein pollen vitro pollen tube lengths germination of all (Figure glcat147A,C) mutants and SEM were (Figure7E) . Although the pollen tube lengths of all glcat14 mutants were comparable to comparable to WT (Figure 7D), an observation that is consistent with earlier studies [13], WT (Figure7D), an observation that is consistent with earlier studies [ 13], some pollen some pollen tubes of glcat14a/c mutants displayed tip swelling relative to WT (Figure 7A) tubes of glcat14a/c mutants displayed tip swelling relative to WT (Figure7A) coupled with coupled with slight alterations in the reticulate pattern of Arabidopsis pollen (Figure 7E). slight alterations in the reticulate pattern of Arabidopsis pollen (Figure7E). The role of The role of AGPs in pollen germination was demonstrated in previous work on agp6 and AGPs in pollen germination was demonstrated in previous work on agp6 and agp11 and agp11 and agp6/11 double mutants, which displayed reduced pollen germination [25], agp6/11 double mutants, which displayed reduced pollen germination [25], while another report in Torenia fournieri demonstrated that a disaccharide sugar, β-methyl-glucuronosyl galactose (4-Me-GlcA-β-1,6-Gal), present on AGPs, makes pollen tubes competent for ovule

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Plants 2021, 10, 1172 while another report in Torenia fournieri demonstrated that a disaccharide sugar,11 of 19β- methyl-glucuronosyl galactose (4-Me-GlcA-β-1,6-Gal), present on AGPs, makes pollen tubes competent for ovule targeting/guidance [26]. Considering that large amounts of targeting/guidance [26]. Considering that large amounts of pollen grains are produced pollen grains are produced during Arabidopsis sexual reproduction, there appears to be during Arabidopsis sexual reproduction, there appears to be a signiﬁcant proportion that area signif competenticant proportion to ensure fertilization, that are competent given that to theensure silique fertilization, lengths and given seed setthat in theglcat14a/c silique mutantslengths and were seed comparable set in glcat14a/c to WT (datamutants not were shown). comparable to WT (data not shown).

Figure 7. Pollen phenotypes of selected glcat14 mutants. (A) In vitro pollen germination showed defective pollen (arrow- Figureheads) 7. Pollen and pollenphenotypes tube tipof swellingselected glcat14 (arrow; mutants. inset in A) (A in) In the vitroglcat14a/c pollenmutant germination compared showed to WT. defective (B) Pollen pollen germination (arrowheads) (%) and pollenin tubeglcat14 tipmutants swelling and (arrow wild; inset type showedin A) in decreasedthe glcat14a/c pollen mutant germination compared in glcat14a/cto WT. (Bmutants) Pollen relativegermination to WT. (%) (C in) Proportion glcat14 mutants and wildof defective type showed pollen decreased was signiﬁcantly pollen germination higher in glcat14a/c in glcat14a/cmutants mutants relative relative to WT. to ( DWT.) Pollen (C) Proportion tube lengths of weredefective similar pollen in was significantlyglcat14 mutants higher in and glcat14a/c wild type. mutants All measurements relative to WT. were (D) takenPollen 3 tube h after lengths incubation were similar of pollen in grainsglcat14 on mutants pollen and germination wild type. All media. Approximately 150 pollen grains were measured for each genotype with three replicates. (* p < 0.05; ** p < 0.01); (E) SEM images of glcat14a/c mutants showed misshaped pollen and slight alterations in the reticulate structure relative to WT.

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3. Discussion A large number of Arabidopsis genes are predicted to contain glycosyltransferases that are involved in cell wall biosynthesis [27]. Despite these predictions, very few candidate genes have been functionally characterized. Genetic strategies deployed to elucidate glycosyltransferase functions have been hampered by functional redundancy, as many genes have been identified to play similar roles in the same and/or different organs/tissues. As a result, multiple genes have to be disrupted before biochemical or physiological roles of these cell wall genes can be deduced. Previous work identified eleven GLCAT genes in Arabidopsis that belong to the CAZy GT14 family [17,28]; these genes may be involved in the glucuronidation of Type II AGs, given their conserved genetic architecture and the conserved GLCAT domain present in genes [17]. While the functional roles of some GLCAT genes have been identified (i.e., GLCAT14A-E; Figure1B), the functional roles of the remaining putative GLCATs remain to be verified and studied further. Work done previously using CRISPR-Cas9 knockouts of GLCAT14A-C showed severe pleiotropic growth defects, especially in the higher order mutants [13], while subsequent work by another group extended this work and identified developmental defects in additional Arabidopsis glcat14 mutants that were partially suppressed by supplementing the growth medium with calcium [12]. In this report, T-DNA insertion mutants and CRISPR lines of GLCAT14A-C were utilized to corroborate previous findings and to obtain additional insights into the biochemical and physiological functions of GLCAT14A-C in Arabidopsis. Specifically, we sought to understand the degree to which the alterations in the amount of GlcA and calcium content accounts for the mutant phenotypes using biochemical and molecular genetic approaches. Plant cell walls are important dynamic structures that need to be monitored properly to ensure their functional integrity [29]. Alterations in the polymer structure often trigger compensatory changes in the cell wall during normal growth or in changing environments. Strong support for the continuous cellular surveillance mechanism aimed at optimizing plant cell function exists [30]. The cell wall integrity maintenance mechanism constantly monitors and perceives changes in biological processes and coordinates activities to pro- mote plant growth through the restructuring of glycan moieties [31,32]. In order to examine whether there are alterations in AGP abundance in glcat14 mutants relative to WT, we quantified the amount of AGPs in rosette leaves, stems, and siliques using the β-Gal Yariv reagent that selectively binds to AGPs [18]. We observed that the amount of glycosylated AGPs was altered in the various organs of the glcat14 mutants, with increased amounts of Yariv-precipitable AGP detected in siliques and stems compared to rosette leaves (Figure3) . Moreover, an increase in AGP content was detected in glcat14a, glcat14a/c, glcat14a/b and glcat14a/b/c mutants in the rosette leaves and stems, while in the siliques, glcat14a, glcat14c, glcat14a/c, glcat14a/b and glcat14a/b/c mutants showed an increase in AGP content. Al- though previous work reported an increase in Yariv-precipitable AGPs in CRISPR-Cas9 generated glcat14ab and glcat14abc [13], our findings indicate that the genetic knock out of GLCAT14A plays a role in the increased Yariv precipitable AGP content observed for glcat14a/c, glcat14a/b and glcat14a/b/c mutants. In addition, the increase in Yariv-precipitable AGP observed among glcat14 mutants relative to the wild type might be a compensatory response that is necessary for maintaining cell wall integrity and establishing normal plant physiological functions. Considering the observed alterations in AGP content across the investigated organs in the glcat14 mutants, we conducted monosaccharide composition and Western blot analyses to gain additional insights into changes in the glycosylation processes in these mutants. We observed organ-specific glycosylation responses, reflected by organ-specific alterations in the galactose content in glcat14 mutants investigated. Previous work on the sugar analysis of AGP extracts of the aerial parts of glcat14 mutants showed an increase in the galactose content in glcat14ab and glcat14abc [13], but our findings indicate that this pattern is not the same for all organs. While we observed an increase in galactose contents in the leaves (Figure4A) and siliques (Table S3) for glcat14a/b and glcat14a/b/c, we observed Plants 2021, 10, 1172 13 of 19

a significant reduction in galactose content in the stem for glcat14a/b/c mutants relative to WT (Figure4C ). Overall, the observed increase in galactose content in the glcat14 T- DNA mutant and CRISPR mutant lines (Tables S3–S5A) centers around the idea that GlcA residues serve as a “cap” that can terminate the elongation of galactan chains and hence the absence of such GlcA residues could lead to an increase in the length of galactan chains [7]; however, this idea was challenged elsewhere [12]. Our data indicate that organ-specific cell wall changes do occur in response to structural changes, to ensure that plant cell walls can perform their biological functions in different situations [32]. Immunolabelling of β-GlcAs of Type II AGs showed weak immunolabelling with LM2, an antibody that targets β-GlcAs of Type II AGs, in glcat14a/b and glcat14a/b/c stem and leaf extracts compared to the wild type which had increased LM2 binding. This finding corroborates the observed reduction in GlcA in the monosaccharide composition analysis, supporting the argument that other GLCATs may be involved in the AG glucuronidation processes. The LM2 signal was detected in the high molecular mass region (Figure2A,B), which might be a due to AGPs’ interaction with other cell wall polymers, as demonstrated in APAP1 mutants [33]. We observed weak binding of LM2 antibody in stem compared to the leaf, and this differences in LM2 binding may be due to the micro-heterogeneity of the AGP glycan in stem and leaf. Arabidopsis GLCAT14A-C are highly expressed during seed germination [17]. More- over, GLCAT14A and GLCAT14B are primarily expressed in the micropylar endosperm during seed development [34]. We observed significant delays in seed germination in the presence of 1 µm ABA in glcat14 mutants relative to WT (Figure5), a finding that is consistent with an earlier report [13]. ABA is reported to influence the cytosolic calcium oscillations and impact many physiological processes, such as seed germination and environmental stress responses [35,36], and such oscillations in cytoplasmic calcium concentrations could be mediated by calcium that is bound by GlcA residues in AGPs [16]. In any case, further research is needed to verify this idea. Following the localization of ATGLCAT14A in the Golgi apparatus [7], we investigated the subcellular localizations of ATGLCAT14B and ATGLCAT14C. We observed that ATGLCAT14B-EYFP and ATGLCAT14C-EYFP signals, in addition to that of ATGLCAT14A- EYFP, were detected in punctate vesicles that co-localized with STtmd–GFP (Figure S3A–C), indicating localization of ATGLCAT14A, ATGLCAT14B and ATGLCAT14C in the Golgi apparatus. These observations are consistent with the idea that that ATGLCAT14A, AT- GLCAT14B and ATGLCAT14C function in the glucuronidation of AGPs at unique positions in the type II glycan structure [12], and therefore are expected to be localized to the Golgi. The importance of GLCATs in leaf development is reflected by the significant reduction in GlcA content in glcat14a/b and glcat14a/b/c mutants relative to WT, as revealed by monosaccharide composition and Western blot analyses (Figures2A and4A). Using SEM, we observed defects in trichome branching in glcat14a/b and glcat14a/b/c mutants (Figure6A ), and this finding is consistent with earlier work [12,13]. While most of the glcat14a/b and glcat14a/b/c trichomes were single-branched trichomes (Table1), a much more severe trichome branching defect was reported for glcat14a/b/e mutants, which could be partially suppressed by supplementing the growth media with calcium [12]. Energy dispersive X-ray (EDX) analysis is an analytical technique commonly used to provide semi-quantitative measurements of surface elements within a surface layer of 1–2 microns in thickness [24]. We utilized this technique to estimate whether there were differences in the amount of calcium in trichomes of glcat14a/b and glcat14a/b/c mutants. We found that the normalized calcium masses of trichomes of glcat14a/b and glcat14a/b/c mutants were comparable to wild type based on the SEM-EDX technique (Figure6D). Similarly, the calcium contents of leaf AGP extracts obtained from glcat14a/b and glcat14a/b/c mutants were similar to WT (Figure4B). Despite this finding, we cannot exclude the possibility that calcium plays a role in establishing normal trichome branching and may be involved during the earlier stages of trichome development, as demonstrated earlier [12]. It is worth mentioning that most GlcA residues in leaf AGPs are methylated [10], and it remains to be Plants 2021, 10, 1172 14 of 19

determined whether certain GlcA residues added by particular GLCATs are preferentially methylated or involved in calcium binding and how such modifications impact trichome development in Arabidopsis. This investigation into understanding the roles of GLCAT14A, B, and C in plant growth and development identified significant reductions in plant height in glcat14a/c, glcat14b/c T-DNA insertion mutant lines relative to WT (Figure4E,F). Previous work showed that glcat14bc, glcat14ab and glcat14abc were shorter than WT [13], while severe growth defects were also reported for glcat14a/b/d and glcat14a/b/e [12]. We found that glcat14a/c mutants were also significantly shorter than the WT but were not as severe as those observed in glcat14a/b and glcat14a/b/c mutants (Figure4E,F). The importance of calcium was recently demonstrated by the observed hypersensitivity of the inflorescence stem lengths of some glcat14 mutants to low calcium concentrations [12]. In addition, the significant reductions in calcium were only observed in the stem, while the calcium contents of glcat14 mutants in leaves and siliques were comparable to WT. Considering the complex framework of plant cell wall glycans, the maintenance of cell wall structure and expansion may not be limited to the interaction of Ca2+ with AGP and/or pectins, as other possible interactions of calcium may exist with cell wall-modifying enzymes [20,21].

4. Materials and Methods 4.1. Plant Materials Arabidopsis T–DNA insertion lines atglcat14a–1 (SALK_064313) and atglcat14a–2 (SALK_043905), atglcat14b-1 (SALK_080923) and atglcat14b-2 (SALK_117005), atglcat14c-1 (SALK_005705) and atglcat14c-2 (SALK_051810) were selected using the SIGnaL database (http://signal.salk.edu/; accessed on 26 May 2017) and were obtained from the ABRC (Arabidopsis Biological Research Centre) (http://abrc.osu.edu/; accessed on 26 May 2017) [37]. We could not verify the presence of a T-DNA insertion in SALK_051810, but a CRISPR knock-out mutant (glcat14c-2) in the first exon generated a 178 bp deletion [13] and was included in this study as an allelic mutant that exhibited similar phenotypes to atglcat14c-1 (SALK_005705). Arabidopsis ecotype Col–0 was used as the wild-type for comparison. Homozygous lines were identified by PCR using primer sets identified using the T-DNA primer design tool provided by the Salk Institute (Figure1A; Table S1). Homozygous glcat14a–1 and glcat14c-1 were crossed, and the F1 generation was selfed before screening the F2 generation to obtain glcat14a/c. Similarly, glcat14b–1 and glcat14c-1 were crossed, and the F1 generation was selfed before screening the F2 generation to obtain glcat14b/c (Figure S1). CRISPR-Cas9 generated glcat14a/b and glcat14a/b/c mutants (henceforth, referred simply as glcat14a/b and glcat14a/b/c) were previously generated in our lab [13] and included in this study to gain broader insight into the biological roles of the GLCATs. For the purpose of this work, glcat14a/b and glcat14a/b/c, generated by the CRISPR-Cas9 approach, are referred to as glcat14 CRISPR lines, while the other genotypes involving T-DNA insertion mutants are referred to as glcat14 T-DNA insertion lines.

4.2. Quantitative RT-PCR To verify whether gene transcripts exist in Arabidopsis glcat14 T-DNA insertion lines, total RNA was extracted from Arabidopsis leaves using Trizol (Life Technologies, Grand Island, NY, USA). RNA (1 µg) was used for ﬁrst-strand cDNA synthesis along with an oligo (dT20) primer and SuperScript III reverse transcriptase (Thermo Scientiﬁc). The qPCR was performed using PerfeCTa SYBR Green SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) following methods described elsewhere [13]. Expression levels were calculated relative to the Arabidopsis ACTIN2 gene.

4.3. Bioinformatics Phylogenetic analysis of CAZy GT14 family members was conducted using PhyML (http://www.phylogeny.fr/one_task.cgi?task_type=phyml, accessed on 30 May 2021), Plants 2021, 10, 1172 15 of 19

while the Gene Structure Display Server was used to determine the genetic architecture of Arabidopsis GT14 gene family (http://gsds.gao-lab.org/, accessed on 30 May 2021).

4.4. AGPs Quantiﬁcation Using β-D-Gal-Yariv Reagent AGPs were extracted from rosette leaves, stems, and siliques of 40-day-old WT, glcat14 T-DNA insertion and CRISPR lines, using the β-D-Gal-Yariv precipitation method described elsewhere [38]. The dissolved AGPs were quantiﬁed by measuring absorbance at OD420. Different concentrations of gum arabic (G9752, Sigma-Aldrich, St. Louis, MO, USA) dissolved in 1% CaCl2 were used to make the standard curve. Measurements for each genotype were done in triplicate.

4.5. Monosaccharide Composition Analysis by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD) Following the extraction of AGPs as described above, ﬁfty microliters of 10 mg/mL AGP were hydrolyzed using 2 N TFA at 121 ◦C for 90 min. TFA was removed by evapo- ration with N2 gas. Samples were dissolved in 500 µL milli-Q water containing 0.2 mM cellobiose as an internal standard. A standard sugar mixture (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid) was used for making the standard curve. Monosaccharide compositions were calculated as molar percentages (mol %). All samples and standards were subjected to high pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) using a Dionex PA-20 column (Thermo Fisher Scientiﬁc, Sunnyvale, CA, USA), essentially as described previously [39].

4.6. Calcium Binding Assay Following the extraction of AGPs, calcium binding assays were conducted as previously described [13]. Brieﬂy, 10 µL of 10 mg/mL AGP extracted from glcat14 T-DNA insertion and glcat14 CRISPR lines were used for the calcium binding assay. A commercial calcium colorimetric assay kit (MAK022, Sigma-Aldrich, St. Louis, MO, USA) was used for calcium measurement following the manufacturer’s protocol. In this assay, calcium ions from the AGP extracts form a complex with the o-cresolphthalein, which causes a color change from transparent to pink. The amount of calcium was determined using a UV spectrometer at OD575 and a standard curve made with different concentrations of CaCl2.

4.7. Seed Germination One-month post-harvested seeds of WT and allelic glcat14 mutant lines (glcat14a-1, glcat14a-2, glcat14b-1, glcat14b-2, glcat14c-1, glcat14c-2, glcat14a/c, glcat14b/c, glcat14a/b, and glcat14a/b/c) were used for germination experiments. Following seed sterilization, seeds ◦ were stratified at 4 C for 3 d in the dark and were sown on 1/2 MS and 1% sucrose agar plates supplemented with or without 1 µM ABA. Germination percentages were counted from 1 to 12 d after sowing and approximately 25 seeds were sown for each genotype with three replicates. In another experiment to evaluate the growth of etiolated seedlings of respective genotypes, stratified seeds sown on MS plates were exposed to 4 h of fluorescent white light at 20 ◦C to synchronize germination before wrapping the plates in aluminum foil for growth in the darkness for 5 d at 20 ◦C. Root and hypocotyl length measurements at 5 d were taken and values were compared to wild type.

4.8. Trichome Morphology The ﬁrst two leaves of 14-day-old WT, glcat14a/b and glcat14a/b/c mutants were im- mersed in methanol and leaf tissues were critical point dried following methods described earlier [40] before examining using scanning electron micrography (SEM). Trichomes were examined using a JEOL JSM-6390 scanning electron microscope (Hitachi High-Technologies, Tokyo, Japan) and leaf tissues were mounted on aluminum stubs using double adhesive tapestubs and sputter coated with a palladium alloy using a Cressington 208C high- Plants 2021, 10, 1172 16 of 19

resolution sputter coater (Ted Pella Inc., Redding, CA, USA). Scanning electron microscope energy dispersive X-ray (SEM/EDX) analysis was used as a semi quantitative approach to estimate the calcium content in the trichomes of WT, glcat14a/b and glcat14a/b/c mutants following methods described previously [23].

4.9. In Vitro Pollen Germination Flowers from 30-day-old WT and glcat14a, glcat14b, glcat14c, glcat14a/c, glcat14b/c plants were used for an in vitro pollen germination assay. Pollen germination medium contained 10% sucrose, 0.01% boric acid, 1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM KCl, 0.03% casein enzymatic hydrolysate, 0.01% myo-inositol, 0.1 mM spermidine, 10 mM GABA, 500 µM methyl jasmonate, and 1% low-melting agarose. Pollen from each genotype was incubated on pollen germination medium. Pollen shape, pollen germination rate, and pollen tube length were measured 3 h after incubation with a Nikon phot-lab2 microscope at 10× magniﬁcation. More than 150 pollen and pollen tubes were measured in an individual experiment with three replicates.

4.10. Western Blotting Analysis The total protein extraction from leaves and stems of wild type, glcat14a/b and glcat14a/b/c mutants was performed following methods described previously [41]. Total protein supernatants from the samples extracted were quantified by the Bradford method [42]. Twenty-five micrograms (25 µg) of crude protein extract of wild type, glcat14a/b and glcat14a/b/c mutants were separated using 10% mini-PROTEAN® TGX™ (456-1033; Bio- Rad, Hercules, CA, USA, https://www.bio-rad.com, accessed on 30 May 2021), gel elec- trophoresis and transferred onto a 0.45-µm Immun-Blot low florescence PVDF membrane (1620260; Bio-Rad) using the wet transfer system at a constant current of 100V for 45 min. The blots were blocked for 1 h (3% BSA, in 1× Tris-buffered saline with Tween (150 mM NaCl, 20 mM Tris base, 0.1% Tween 20; TBST)), probed with 1:10 LM2 monoclonal rat IgM primary antibody (Carbosource, https://www.ccrc.uga.edu, accessed on 30 May 2021) in blocking buffer for 1 h, washed three times with 1× TBST for 15 min, probed with goat anti-rat IgG H + L secondary antibody (PI31629; FisherScientific), washed thrice with 1× TBST for 10 min, once with 1× TBS for 10 min, and treated with chemiluminescent substrate (Clarity TM ECL substrate, 1705060S; Biorad). A ChemiDoc imaging system (Bio-Rad) was used for image acquisition. PVDF membranes were incubated for 10 min with Ponceau S solution (0.1% (w/v) Ponceau S; 5% (v/v) acetic acid) to stain for total proteins.

4.11. Cloning Procedures and Subcellular Localizations of GLCAT14A, GLCAT14B and GLCAT14C Full-length sequenced GLCAT14A, GLCAT14B and GLCAT14C CDS, flanked by the sites KpnI were subcloned into pSAT6 vector to generate pSAT6-GLCAT14A-EYFP and pSAT6-GLCAT14C-EYFP constructs containing PI-PSPI restriction sites. Following the digestion of the new constructs (pSAT6-GLCAT14A-EYFP, pSAT6-GLCAT14B-EYFP and pSAT6-GLCAT14C-EYFP) with PI-PSPI, the fragment was introduced into the pPZP-RCS- nptII binary vector [43]. The binary vector containing the constructs was transformed into Agrobacterium strain GV3101 before infiltration using 5-week old tobacco leaves grown at 22–24 ◦C[44]. Golgi co-localization was confirmed by using the Golgi marker sialyltrans- ferase short cytoplasmic tail and single transmembrane domain fused to enhanced GFP (STtmd-GFP). Transformed plants were incubated under normal growth conditions and imaged 3 days post-infiltration using an upright Zeiss LSM 510 META laser scanning con- focal microscope (Jena, Germany), with a 40× oil immersion lens and an argon laser. Both GFP and E-YFP were imaged using 458 and 488 nm excitation wavelengths, respectively.

5. Conclusions This study corroborates and extends our understanding of the molecular dynamics of GlcA and calcium in AGPs and how they inﬂuence plant physiological processes. Although we observed a signiﬁcant reduction in glucuronidation that differs across organs Plants 2021, 10, 1172 17 of 19

in glcat14 mutants, it remains to be determined which of the GlcA residues added by the various GLCATs are modified by calcium or methyl groups or are cross-linked with other structurally complex heteropolymers such as pectins [33] to perform their cellular function. An understanding of which GlcA residues are modified and/or unmodified and how such modifications (or lack of modifications or crosslinking with other cell wall polymers) impact plant physiological functions remains to be established. Future research aiming to address such questions could potentially increase our understanding of AGP-Ca2+ function and its possible multifunctional role in plant growth processes.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10061172/s1, Figure S1: PCR screening of glcat14 T-DNA insertion mutants. Figure S2: Growth measurements of glcat14 mutants and wild type. Figure S3: Subcellular localization of AtGLCAT14A, AtGLCAT14B and AtGLCAT14C. Table S1: List of primers used for mutant characterization using PCR. Table S2: Amino acid sequence similarity matrix among GT14 family. Table S3: Monosaccharide composition analysis of Yariv-precipitated AGPs extracted from rosette leaves of 40-day-old glcat14 mutants and WT (Col-0). Table S4: Monosaccharide composition analysis of AGPs extracted from stems of 40-day-old glcat14 mutants and WT (Col-0). Table S5: Monosaccharide composition analysis of AGPs extracted from siliques of 40-day-old glcat14 mutants and WT (Col-0). Author Contributions: Conceptualization, O.O.A. and A.M.S.; methodology, O.O.A., M.A.H. and A.M.S.; validation, O.O.A., M.A.H. and A.M.S.; formal analysis, O.O.A.; investigation, O.O.A.; resources, O.O.A. and A.M.S.; data curation, O.O.A.; writing—original draft preparation, O.O.A.; writing—review and editing, O.O.A. and A.M.S. and M.A.H.; visualization, O.O.A.; supervision, O.O.A. and A.M.S.; project administration, O.O.A. and A.M.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: Not applicable. Acknowledgments: We thank Alexander Ford, Rowan Andrews, Helena Hitler, Savannah Bisson Weiheng Yu and Ashton Smith for helping with the mutant screening and phenotypic analyses. We also thank Yuan Zhang for proofreading and providing quality feedback on the manuscript. This work was supported by an Ohio University (OU) Student Enhancement Award; an OU College of Arts and Science Graduate Student Research Fund award; and an OU Nanoscale and Quantum Phenomena Institute (NQPI) fellowship to O.O.A. M.A.H. would like to acknowledge funding from the NIFA-USDA-AFRI Exploratory Research Program, Award Number 2019-67030-29670. This work was supported by a facility constructed with funds from Research Facilities Improvement Program Grant Number C06 RR-014575-01 from the National Center for Research Resources, National Institutes of Health. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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