Cell wall remodeling in Arabidopsis stamen abscission zones

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.

This work was supported by the U.S. Environmental Protection Agency (Cooperative Agreement no. CR-832811-01). Genomic data is available as Accession E-MEXP-1474 at ArrayExpress: www.ebi.ac.uk/microarray-as/aer/#ae-main.