Cell wall remodeling in Arabidopsis stamen abscission zones

Abstract

Organ shedding requires cell separation within abscission zones (AZs). Functional genomic AZ studies have been limited by their small size and low incidence. Optimization of laser capture microdissection (LCM) for AZs and other specialized cell types in Arabidopsis¹ allowed recent characterization of the floral stamen AZ transcriptome responding to a developmental shedding cue.² Analyses focused on 551 AZ transcripts (AZ₅₅₁) that were regulated at the highest statistical significance (p ≤ 0.0001) over five stages of stamen development spanning pre-pollination to organ shed.² Here, we seek a fuller understanding of AZ integrity control by relaxing P value restrictions on statistical significance ten-fold to generate an expanded population of 1461 stamen transcripts (AZ₁₄₆₁). Cell wall remodeling functions in AZ₁₄₆₁ are significantly over-represented relative to all transcripts represented on the whole genome GeneChip. Hierarchical clustering of gene expression data corresponding to cell wall-related transcripts suggests a temporal model for AZ remodeling in Arabidopsis stamens destined to abscise.

Multiple Proteins Loosen, Remodel and Separate AZs

Primary cell walls consist of a network of cellulose and hemicellulose embedded in a pectinaceous matrix containing lesser amounts of dissolved solutes and glycoproteins.³ Cellulose microfibrils are coated with hydrogen-bonded xyloglucan that spans adjacent microfibrils.^3,4 Covalent linkages to hemicelluloses and pectins may fortify wall structure.⁵ In Arabidopsis and other dicots, xyloglucan is the major cell wall hemicellulose.

Overrepresentation of cell wall functions in AZ₁₄₆₁ relative to the ATH1 GeneChip was deemed statistically significant using protocols of Cai and Lashbrook.² Figure 1 represents 65 potential cell wall-related genes whose expression is significantly regulated over five floral stages linking pre-pollination to stamen shed. Annotation of gene products follows conventions of refs. ^6–9. Locus identities correspond to gene family members classified under the heading “Assembly, Architecture and Growth” on the Cell Wall Genomics website hosted by Purdue University (http://cellwall.genomics.purdue.edu/families/index.html). One locus ID represents a citrus blight protein homolog that shares amino-terminal structural features of expansins¹⁰ but reportedly lacks cell wall loosening activity.¹¹ Whether this gene product may contribute to cell wall remodeling is therefore contentious. The collective potential for other proteins represented in Figure 1 to modify pectic and hemicellulosic structure of AZcell walls is high.

Figure 1.

Developmental expression profiles of cell wall related genes within AZ₁₄₆₁. Transcripts were subjected to divisive hierarchical clustering. Enzyme families are further described on the Carbohydrate-Active Enzymes website (www.cazy.org) described by Coutinho et al.⁹ Floral stage numbers are defined elsewhere² and in Figure 2. EXP expansin, EXPL expansin-like, GH9 glycosyl hydrolase family 9, GH17 glycosyl hydrolase family 17, PAE pectin acetylesterase, PG polygalacturonase, PL pectate lyase, PME pectin methylesterase, PMEI pectin methylesterase inhibitor protein, RGL rhamnogalacturonan lyase, XTH xyloglucan endotranglucosylase/hydrolase.

In Figure 1, heatmaps score stage-dependent expression of putative cell wall-related transcripts by color. Red represents the lowest transcript level present over five stages. Increasing gene expression is represented by orange and yellow, with white reflecting maximal transcript abundance. Transcripts with similar patterns of gene expression are clustered as per Cai and Lashbrook.² Clusters depicted in dendrogram format on the left face of Figure 1 are further separated by horizontal lines. Eisen et al.,¹² and others have shown that transcripts clustered on the basis of common expression profiles frequently coordinate common functions. We wished to consider whether a temporal model of abscission might be constructed using published information together with the cluster and expression data of Figure 1. We present such a model in Figure 2, based on the premise that the functioning of protein products may be enhanced at those developmental stages where corresponding transcripts exhibit maximum gene expression. Due to likely post-transcriptional control of some AZ genes, this assumption has limitations. However, such an exercise is a reasonable first step towards assessing whether clustered proteins might modify AZ wall structure in a cooperative, ordered manner. Our model facilitates the construction of testable hypotheses for future functional studies that include protein-based methods.

Figure 2.

Model of floral stages at which abscission subfunctions may take place. Floral stage numbers are further defined elsewhere.² Abscission signal receipt is expected to take place before anthesis because abscission regulators have accumulated by S12.² These include the HAESA receptor kinase¹³ and multiple ethylene response factors.² Signaling at or beyond anthesis is evidenced by accumulation at S13 of IDA ligand transcript required for abscission.¹⁴ Evidence that a subset of stamen AZ cells have completely separated by S15c is provided in Cai and Lashbrook.² GH9 is Glycosyl Hydrolase Family 9; GH7 is Glycosyl Hydrolase Family 17.

Genomic Data Suggest Temporal Ordering of AZ Remodeling Processes

Figure 2 incorporates information from Cai and Lashbrook² and other sources^13,14 to predict flower stages at which key abscission events might take place. Below the developmental timeline are placed transcript classes present at maximal levels of abundance as assessed by GeneChip signal intensities. That is, they were previously portrayed by white or near-white zones in Figure 1.

Accumulation of expansins (EXPS) and expansin-like transcripts (EXPLs) before anthesis may initiate cell wall loosening.

Belfield et al.,¹⁵ showed EXP activity to be enhanced in ethylene-treated AZs of Sambucus nigra and identified two cDNAs called SniEXP4 and SniEXP2. Multiple EXP and EXPL genes are expressed in AZ₁₄₆₁ (Fig. 1). Of special interest are AtEXPs that cluster with Sambucus cDNAs in phylogenetic analyses. AtEXPA2 and AtEXPA8 cluster with SniEXP4, while AtEXPA6 is the Arabidopsis sequence closest to SniEXP2.¹⁵ These AtEXP transcripts, as well those for all other AtEXPs and AtEXPLs in Figure 2, exhibit maximal abundance prior to anthesis. They are then significantly downregulated prior to cell separation (Fig. 1). Insofar as developmental abscission signaling is evident at S12,² we postulate that expansin effects on AZ architecture are initiated soon after abscission cue receipt. Expansins likely promote cell wall creep required for earliest loosening of AZs by disrupting cellulose- hemicellulose network stability.^16,17

XTHs and β-1, 3-glucanases may join EXP and EXP-like proteins to mediate early steps of the abscission pathway.

XTHs have dual activities and can strengthen or loosen cell walls in different contexts.⁷ In AZs prior to anthesis, we expect that loosening would predominate. XTH4 and XTH6 are the earliest upregulated AZ XTHs, with XTH6 expression confirming prior observations in abscission layers.¹⁸ Co-production of these XTHs with expansins and expansin-like proteins suggests a potential for synergistic cell wall loosening.

β-1, 3-glucanases within CAZy Glycosyl Hydrolase Family 17 (GH17) mediate diverse processes that include growth, callose deposition and pathogen defense. Well-characterized GH17 proteins related to abscission encode pathogenesis-related (PR) proteins.¹⁹ PR proteins may help prevent pathogen entry into AZ scars resulting from organ shed. At least two AZ transcripts within Figure 1 (At3g04010, At4g16260) probably correspond to PR proteins given their induction by multiple fungal pathogens.²⁰ At3g04010 exhibits highest accumulation between Stages 12–13 whereas At4g16260 reaches highest levels during earliest stages of cell separation. This suggests that AZ defense processes may be initiated during initial loosening and maintained throughout organ shedding.

Cell wall loosening and AZ separation are controlled by distinct complements of pectolytic enzymes.

Pectin degradation is central to cell wall remodeling during abscission given that cellular continuity between AZ cells is maintained by pectin-rich middle lamellae. Pectin remodeling may also establish porosity characteristics necessary for enzyme access to substrates. In AZs, two blocks of pectolytic enzymes are expressed at times we predict to correspond to cell wall loosening and cell separation. These protein groupings are termed Pectolytic I and Pectolytic II. In Figure 2, Pectolytic I-mediated loosening functions predominate during S12–13; Pectolytic II functions occur following S15b when first cell separation is evident.²

β-galactosidases. This class of enzyme has the potential to act on both pectic and hemicellulosic cell wall components.²¹ Here, we assign our sole β-galactosidase, βGAL4, to the Pectolytic I block because structural and enzymic properties suggest it acts on pectin.²² In AZs, βGALs may reduce adhesion between AZ cells or modulate wall porosity.

Polygalacturonases (PGs). Of seven AZ PGs, three are downregulated (At3g16850, At3g07830, At4g23820), three are upregulated (At2g43890, At3g07970, At4g18180), and one shows both up and downregulation (At1g70370). PG transcripts at maximal levels at S12–S13 could play roles in loosening events impacting wall porosity. PGs upregulated thereafter are more likely contributors to actual AZ cell separation. Kim and Patterson²³ previously showed accumulation of At2g43890 transcript to occur during active cell separation. Elevated expression of At1g70370 at both S12 and S15c is consistent with potentially dual roles for its gene product in AZ loosening and separation.

Pectate and rhamnogalacturonan lyases (PLs and RGLs). PL gene expression patterns were similar to those for PG in that some genes were downregulated (At3g07010, At3g55140, At5g48900), and some were upregulated (At3g27400, At4g24780). Two PL transcripts present at highest levels at both S12 and S15c (At4g13210, At4g13710) suggest the potential for dual effects on loosening and cell separation. An RG lyase transcript (At2g22620) of potential significance to AZ separation was also expressed.

Pectin methyl esterases (PMEs) and PME inhibitors (PMEIs). Deesterification of pectic compounds by PMEs generates structurally and functionally distinct pectin classes. In turn, PME activity can be transcriptionally or post-translationally inhibited by PME inhibitors. Multiple PMEs and PMEIs are up and/or downregulated in stamen AZs. Since both PGs and PLs favor deesterified substrates, control of methylesterification level is undoubtedly a key factor in controlling AZ cell wall integrity. In some ripening fruits, PME-mediated de-esterification is necessary for subsequent PG action.²¹ Opposing capacities of PMEs to both loosen and strengthen cell walls also may be contribute to balancing wall disassembly with overall strength during abscission.

Pectin acetylesterases (PAEs). The deacetylation of homogalacturonan polymers by pectin acetylesterases solubilizes pectin and may facilitate PL access to substrate. Two PAEs are expressed at elevated levels early in abscission signal transduction. One of these transcripts, At3g05910, was previously reported to be upregulated in nematode feeding sites very soon after infection.²⁴ This suggests that PAEs potentiate AZ cell wall loosening requirements that are similar to those needed for successful endoparastic infections.

Endo-β-1, 4-glucanases modify hemicellulose-cellulose structure during actual cell separation.

Endo-β-1, 4-glucanases (EGases) cleave internal β-1, 4 linkages in glucan polymers. Substrates include soluble cellulosic polymers including various hemicelluloses. All Arabidopsis EGases are housed in Glycosyl Hydrolase Family 9,²⁵ within one of three structural subclasses.⁸ Expressed in AZs are four EGases of Subclass B; this subclass is expected to be secreted to the extracellular space. Using recently standardized nomenclature,⁸ EGases expressed in AZs include AtGH9B3 (formerly termed EGase9 and AtCel 3), AtGH9B4 (formerly EGase3 and AtCel5), AtGH9B7 (formerly EGase10) and AtGH9B8 (formerly EGase11). Three of these four EGase gene family members are upregulated to maximal levels during actual cell wall separation. An exception was AtGH9B7 transcript, present at highest abundance at both pre-anthesis and S15c. We conclude that EGase contributions to abscission predominate during cell separation, in potential cooperation with Pectolytic II proteins.

AtGH9B3/Cel3 gene expression was previously detected in floral organ AZs²⁶ but expression of its paralog AtGH9B4/Cel5 has been reported to be rootcap-specific.²⁷ High sequence identity between duplicons make cross-hybridization possible,²⁷ and it will be necessary to independently corroborate AtGH9B4 data. However, AtGH9B4 expression in anthers, siliques and mixed stage inflorescences was reported in multiple public gene expression databases we visited. These tissues are known sites for cell separation processes like abscission and dehiscence. We predict that while AtGH9B4 is especially highly expressed in sloughing root caps, it is also expressed in stamen AZs.

XTHs are active in AZs over all stages from pre-pollination to organ shed.

As noted earlier, dual activities of XTHs allow them to perform wall strengthening or loosening functions under different circumstances.⁷ The presence of regulated XTH expression at all times spanning pre-pollination to the inception of stamen detachment suggests that XTHs may be instrumental in balancing AZ loosening needs with cell wall strengthening requirements prior to organ shed. Alternatively, XTHs may trigger loosening processes that are tempered by regulated cell wall biosynthesis. Certainly, the presence of significant wall building events during abscission can be observed within our stamen AZ profiling dataset (M-EXP-1474) available from ArrayExpress (www.ebi.ac.uk/microarray-as/aer/#ae-main).

Abbreviations

ATH1

Arabidopsis genome array (Affymetrix)

AZ

abscission zone

βGAL

beta galactosidase

CAZy

Carbohydrate-Active Enzymes database

EGase

endo-β-1,4-glucanase

EXP

expansin

EXPL

expansin-like

GH9

glycosyl hydrolase family 9

GH17

glycosyl hydrolase family 17

IDA

inflorescence deficient in abscission

LCM

laser capture microdissection

PAE

pectin acetylesterase

PG

polygalacturonase

PL

pectate lyase

PME

pectin methylesterase

PMEI

pectin methylesterase inhibitor protein

RGL

rhamnogalacturonan lyase

S12–S15c

stage 12 to 15c

XTH

xyloglucan endotranglucosylase/hydrolase

Addendum to: Cai S, Lashbrook CC. Stamen abscission zone transcriptome profiling reveals new candidates for abscission control: Enhanced retention of floral organs in transgenic plants overexpressing Arabidopsis Zinc Finger Protein2. Plant Physiol. 2008;146:1305–1321. doi: 10.1104/pp.107.110908.