Immunolocalization of pectic polysaccharides during abscission in pea seeds (Pisum sativum L.) and in abscission less def pea mutant seeds

Abstract

Background: In pea seeds (Pisum sativum L.), the presence of the Def locus determines abscission event between its funicle and the seed coat. Cell wall remodeling is a necessary condition for abscission of pea seed. The changes in cell wall components in wild type (WT) pea seed with Def loci showing seed abscission and in abscission less def mutant peas were studied to identify the factors determining abscission and non-abscission event.

Methods: Changes in pectic polysaccharides components were investigated in WT and def mutant pea seeds using immunolabeling techniques. Pectic monoclonal antibodies (1 → 4)-β-D-galactan (LM5), (1 → 5)-α-L-arabinan(LM6), partially de-methyl esterified homogalacturonan (HG) (JIM5) and methyl esterified HG (JIM7) were used for this study.

Results: Prior to abscission zone (AZ) development, galactan and arabinan reduced in the predestined AZ of the pea seed and disappeared during the abscission process. The AZ cells had partially de-methyl esterified HG while other areas had highly methyl esterified HG. A strong JIM5 labeling in the def mutant may be related to cell wall rigidity in the mature def mutants. In addition, the appearance of pectic epitopes in two F3 populations resulting from cross between WT and def mutant parents was studied. As a result, we identified that homozygous dominant lines (Def/Def) showing abscission and homozygous recessive lines (def/def) showing non-abscission had similar immunolabeling pattern to their parents. However, the heterogeneous lines (Def/def) showed various immunolabeling pattern and the segregation pattern of the Def locus.

Conclusions: Through the study of the complexity and variability of pectins in plant cell walls as well as understanding the segregation patterns of the Def locus using immunolabeling techniques, we conclude that cell wall remodeling occurs in the abscission process and de-methyl esterification may play a role in the non-abscission event in def mutant. Overall, this study contributes new insights into understanding the structural and architectural organization of the cell walls during abscission.

Keywords: Abscission; Def mutant; Immunolocalization; Pea (Pisum sativum L.); Pectic polysaccharide.

Fig. 1

Light micrographs of pea seed sections stained with toluidine blue to show structural differences at intervening area between funicle and seed coat in wild type and def mutant peas. a JI116 tall WT young pea at 10.1. b JI116 tall WT mature pea at 2.1. c JI2822 dwarf WT young pea at 4.1. d JI2822 dwarf WT mature pea at 1.1. e JI1184 tall def mutant young pea at 8.1. f JI1184 tall def mutant mature pea at 2.1. g JI3020 dwart def mutant young pea at 3.1. h JI3020 dwarf def mutant mature pea at 1.2. AZ abscission zone; FN funicle; SE seed; ALZ abscission less zone. Arrows indicate the AZ. Scale bars 310 µm

Fig. 2

Light micrographs of sections stained with toluidine blue to show structural differences (a, f, k and p) and micrographs of indirect immunofluorescence detection of pectic epitopes in AZ of tall wild type (JI116) and in ALZ of tall def mutant (JI1184) pea seeds. a–e Tall wild type young seeds (JI116 10.1). f–j Tall def mutant young seed (JI1184 8.1). k–o Tall wild type mature seed (JI116 2.1). p–t Tall def mutant mature seed (JI1184 2.2). b, g, l and q Immunolabeling with LM5 galactan. c, h, m and r Immunolabeling with LM6 arabinan. d, i, n and s Immunolabeling with JIM5. e, j, o and t Immunolabeling with JIM7. PL Palisade layer, CPL Counter palisade layer, AZ abscission zone; FN funicle; SE seed; ALZ abscission less zone. Arrows indicated the AZ. Scale bars 100 μm

Fig. 3

Light micrographs of sections stained with toluidine blue to show structural differences (a, f, k and p) and micrographs of indirect immunofluorescence detection of pectic epitopes in the AZ of dwarf wild type (JI2822) and ALZ of dwarf def mutant (JI3020) pea seeds. a–e Dwarf wild type young seeds (JI2822). f–j Dwarf def mutant young seed (JI3020). k–o Dwarf wild type mature seed (JI2822). p–t Dwarf def mutant mature seed (JI3020). b, g, l and q Immunolabeling with LM5 galactan. c, h, m and r Immunolabeling with LM6 arabinan. d, i, n and s Immunolabeling with JIM5. e, j, o and t Immunolabeling with JIM7. PL palisade layer; CPL counter palisade layer; AZ abscission zone; FN funicle; SE seed; ALZ abscission less zone. Arrows indicated the AZ. Scale bars 100 μm

Fig. 4

Micrographs of indirect immunofluorescence detection of pectic epitopes JIM5 and JIM7 in wild type (JI116 and JI2822) and def mutant (JI1184 and JI3020) pea seeds. Prior to labeling, longitudinal sections were treated with 0.05 M Na₂CO₃ for de-esterification of homogalacturonan. a–h Immunolabeling with JIM5. i–p Immunolabeling with JIM7. a and i Tall wild type young pea seeds (JI116 at 10.1). b and j Tall wild type mature pea seeds (JI116 at 2.1). c and k Dwarf wild type young pea seeds (JI2822 at 4.1). d and i Dwarf wild type mature pea seeds (JI2822 at 1.1). e and m Tall def mutant young pea seeds (JI1184 at 8.1). f and n Tall def mutant mature pea seeds (JI1184 at 2.2). g and o Dwarf def mutant young pea seeds (JI3020 3.1).h and p Dwarf def mutant mature pea seeds (JI3020 1.2). PL palisade layer; CPL counter palisade layer; AZ abscission zone; FN funicle, SE seed; ALZ abscission less zone. Arrows indicated the AZ. Scale bars 100 μm

Fig. 5

Micrographs of indirect immunofluorescence detection of pectic epitopes LM5 galactan and LM6 arabinan in F₃ population from cross of dwarf wild type JI 2822 and tall def mutant JI 1184 at pod number 2 stage (a–f) and F₃ population from cross of the dwarf wild type JI 2822 and dwarf def mutant JI 3020 (g–p). a–c and g–k Immunolabelling with LM5 galactan. d–f and l–p Immunolabelling with LM6 arabinan. a and d Homozygous dominant (Def/Def) F₃ line 11 showing the presence of double palisade layers as in the wild type phenotype. b and e Homozygous recessive (def/def) F₃ line 18 showing the presence of the ALZ as in def mutant phenotypes. c and f Heterozygous (Def/def) F₃ line 14 showing the presence of PL and CPL. g and l Homozygous dominant (Def/Def) F₃ line 1 showing the presence both PL and CPL as in the WT phenotypes. h and m Homozygous recessive (def/def) F₃ line 33 showing the ALZ as in parents def mutant phenotypes. i and p Heterozygous (Def/def) F₃ line 77. i and n Young seed at pod stage 3.1. j and o Intermediated seed at pod stage 2.1. k and p Mature seed at pod stage 1.1. PL palisade layer; CPL counter palisade layer; AZ abscission zone; FN funicle; SE seed. Scale bars 50 μm