Dynamic Changes of Pectin Epitopes in Cell Walls during the Development of the Procambium-Cambium Continuum in Poplar

Abstract

The change of pectin epitopes during procambium-cambium continuum development was investigated by immunolocalization in poplar. The monoclonal antibody JIM5 labels homogalacturonan (HGA) with a low degree of esterification, and the monoclonal antibody JIM7 labels HGA with a high degree of methyl-esterification. Arabinan, rather than galactan, and HGA with low degree of esterification were located in the cell walls of procambial, while HGA with a low degree of esterification was located in the tangential walls, and galactan was located in both the tangential and radial walls of procambial, yet nearly no arabinan was located in the tangential walls of the cambial cells. The changes in pectin distribution took place when periclinal divisions appeared within a procambial trace. The distribution difference of pectin epitopes was also present in procambium-cambium derivatives. The arabinan existed in all cell walls of primary xylem, but was absent from the tangential walls of secondary xylem cells. The galactan existed only in mature primary phloem. Furthermore, 19 pectin methylesterases (PMEs) genes were identified by RNA sequencing, six genes presented highly differentially and were supposed to be involved in the cell wall esterification process. The results provide direct evidence of the dynamic changes of pectin epitopes during the development of the procambium-cambium continuum in poplar.

Keywords: PME; Populus tomentosa; cambium; pectin; procambium.

Figure 1

Cross sections of apex and stem at different developmental stages during the procambium–cambium continuum in P. tomentosa. (a) at the 1 mm level below apex, procambial cells are in the procambium stage, and their divisions were irregular; (b) 2 mm below apex, protophloem differentiated on the cortical side of the strand, procambial cells were still in the procambium stage; (c) 3 mm below apex, the initiating layer appeared, and the strand was protophloem dominated; (d) 8 mm below apex, protoxylem occurred at site internal to the initiating layer; (e) 25 mm below apex, the initiating layer had been united into metacambium; (f) 200 mm below apex, typical cambium was present. pp Protophloem; px protoxylem; pc procambium; il initiating layer; mx metaxylem; mp metaphloem; mc metacambium; ca cambium; sp secondary phloem; sx secondary xylem. The arrows in the figure indicate the tissues examined. Bar: 10 μm in (a,b); 20 μm in (c–f).

Figure 2

Immunofluorescent labeling without the primary mABs in partial sections at different stages of the procambium–cambium continuum. (A) 1 mm level below apex; (B) 2 mm below apex; (C) 20 mm below apex; (D) 200 mm below apex. pp protophloem; px protoxylem; pc procambium; mx metaxylem; mp metaphloem; mc metacambium; ca cambium; sp secondary phloem; sx secondary xylem. Bar: 10 μm in (A–C); 20 μm in (D).

Figure 3

Immunofluorescent labeling with JIM5 antibodies in partial sections at different stages of the procambium–cambium continuum. (A) 1 mm level below apex; (B) 2 mm below apex; (C) 20 mm below apex; (D) 200 mm below apex. The arrows in the figure indicate the signal detected. pp protophloem; px protoxylem; pc procambium; mx metaxylem; mp metaphloem; mc metacambium; ca cambium; sp secondary phloem; sx secondary xylem. Bar: 10 μm in (A–C); 20 μm in (D).

Figure 4

Immunofluorescent labeling with JIM7 antibodies in partial sections at different stages of the procambium–cambium continuum. (A) 1 mm level below apex; (B) 2 mm below apex; (C) 20 mm below apex; (D) 200 mm below apex. The arrows in the figure indicate the signal detected. pp protophloem; px protoxylem; pc procambium; mx metaxylem; mp metaphloem; mc metacambium; ca cambium; sp secondary phloem; sx secondary xylem. Bar: 10 μm in (A–C); 20 μm in (D).

Figure 5

Immunofluorescent labeling with LM5 antibodies in partial section at different stages of the procambium–cambium continuum. (A) At the 1 mm level below apex, the cell walls of procambium were not labeled with LM5; (B) 2 mm below apex, it was faintly labeled; (C) 20 mm below apex; (D) 200 mm below apex, cambium and new-formed secondary xylem were intensively labeled with LM5, while secondary phloem was not. The arrows in the figure indicate the signal detected. pp protophloem; px protoxylem; pc procambium; mx metaxylem; mp metaphloem; mc metacambium; ca cambium; sp secondary phloem; sx secondary xylem. Bar: 10 μm in (A–C); 20 μm in (D).

Figure 6

Immunofluorescent labeling with LM6 antibodies in partial sections at different stages of the procambium–cambium continuum. (A) At the 1 mm level below apex, the cell walls of procambium were labeled with LM6; (B) 2 mm below apex, protophloem and protoxylem were labeled with LM6, while the nascent tangential walls produced by periclinal divisions in the initiating layer were not labeled; (C) 20 mm below apex, the tangential walls of metacambium and metaxylem were unlabeled by LM6, while the labelling intensity increased in metaphloem; (D) 200 mm below apex, only the radial wall of cambium was faintly labeled. The labelling intensity increased in the secondary phloem side, but decreased in the secondary xylem side. The arrows in the figure indicate the signal detected. pp protophloem; px protoxylem; pc procambium; mx metaxylem; mp metaphloem; mc metacambium; ca cambium; sp secondary phloem; sx secondary xylem. Bar 10 μm in (A–C); 20 μm in (D).

Figure 7

A N-J phylogenetic tree was constructed according to the PME genes sequence similarity of Arabidopsis and P. trichocarpa. The phylogenetic tree was built using the biology software MEGA version 6, adopting genetic distance building method (neighbor-joining, NJ), and processing the confidence analysis based on bootstrap by re-sampling 1000 times [40]. The scale (0.5) in the figure presents the bootstrapt value.

Figure 8

The expression pattern of highly differentially expressed PMEs genes (A) Potri.014G117100.1; (B) Potri.004G128900.1; (C) Potri.018G068400.1; (D) Potri.008G104800.1; (E) Potri.003G076900.1; (F) Potri.007G015700.1) in poplar procambium–cambium continuum development. pc procambium; il initiating layer; mc metacambium; ca cambium; sp secondary phloem; sx secondary xylem.