Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants

Abstract

PAE8 and PAE9 have pectin acetylesterase activity and together remove one-third of the cell wall acetate associated with pectin formation in Arabidopsis leaves. In pae8 and pae9 mutants, substantial amounts of acetate accumulate in cell walls. In addition, the inflorescence stem height is decreased. Pectic polysaccharides constitute a significant part of the primary cell walls in dicotyledonous angiosperms. This diverse group of polysaccharides has been implicated in several physiological processes including cell-to-cell adhesion and pathogenesis. Several pectic polysaccharides contain acetyl-moieties directly affecting their physical properties such as gelling capacity, an important trait for the food industry. In order to gain further insight into the biological role of pectin acetylation, a reverse genetics approach was used to investigate the function of genes that are members of the Pectin AcetylEsterase gene family (PAE) in Arabidopsis. Mutations in two members of the PAE family (PAE8 and PAE9) lead to cell walls with an approximately 20 % increase in acetate content. High-molecular-weight fractions enriched in pectic rhamnogalacturonan I (RGI) extracted from the mutants had increased acetate content. In addition, the pae8 mutant displayed increased acetate content also in low-molecular-weight pectic fractions. The pae8/pae9-2 double mutant exhibited an additive effect by increasing wall acetate content by up to 37 %, suggesting that the two genes are not redundant and act on acetyl-substituents of different pectic domains. The pae8 and pae8/pae9-2 mutants exhibit reduced inflorescence growth underscoring the role of pectic acetylation in plant development. When heterologously expressed and purified, both gene products were shown to release acetate from the corresponding mutant pectic fractions in vitro. PAEs play a significant role in modulating the acetylation state of pectic polymers in the wall, highlighting the importance of apoplastic metabolism for the plant cell and plant growth.

Fig. 1

Phylogenetic tree of gene family related to a pectin acetylesterase. Tree constructed using the maximum likelihood method with the Seaview4 software package, which used muscle and PhyML for alignment and tree, respectively. VLAE acetylesterase expressed in the developing corm of Amorphophallus konjac. Bar indicates relative distance between protein sequences. Black stars highlight the genes focused on in the present study

Fig. 2

RT-PCR of pectin acetylesterase T-DNA insertion lines. RT-PCR for PAE3 transcript (pae3-1, SALK_066524C; pae3-2, SALK_137505C; 3-week-old leaves); PAE5 (pae5-1, SALK_140555; pae5-2, SALK_052303C; 3-week-old leaves); PAE6 (pae6-1, SALK_020618; pae6-2, SALK_134907; 19-day-old leaves); PAE8 (pae8, SALK_132026; 2-week-old leaves); PAE9 (pae9-1, SALK_046973C; pae9-2, GABI-803G08; 3-week-old leaves); PAE7 (pae7-1, SALK_093502C; pae7-2, GABI_272B08, 19-day-old leaves); PAE10 (pae10-1, SALK_043807; pae10-2, SAIL_802_C05; 3-week-old leaves); PAE11 (pae11-1, SALK_049340.48.65.x; pae11-2, GABI_505H02; 19-day-old leaves); PAE12 (pae12-1, GABI_018A02; pae12-2, GABI_646F06; 3-week-old leaves). PTB—housekeeping gene. WT = Col-0 plants of same age as insertion lines being tested. Primers used for each reaction are listed in Suppl. Table S2

Fig. 3

Gene models for PAE8 and PAE9 and Q-RT-PCR of complementation and overexpression lines. a Gene model for PAE8 and PAE9; boxes indicate exons (gray, translated regions); black line in between exons indicate introns. Vertical black triangles indicate position of T-DNA insertions in relation to translation start site (pae8 SALK_132026, +2,046 bp; pae9-1, SALK_046973C, +2,712 bp; pae9-2, GABI-803G08, +1,949 bp). Small black triangles indicate primer positions used for RT-PCR. b Relative quantity of the PAE8/PAE9 transcript determined by Q-RT-PCR in 35-/30-day-old leaves of T3 complementation lines. Error bars indicate minimum and maximum variation. PTB gene expression was used as an internal control for normalization. WT = Col-0 35/30-day-old leaves. n = 6

Fig. 4

Acetate content of wall (a), pectin extract, and remaining residue (b) of PAE mutant lines. Error bars indicate standard deviation; letters indicate statistical significant differences based on one-way ANOVA (P < 0.05); n ≥ 4

Fig. 5

Inflorescence stem heights and stem acetate content. a General appearance of 35-day-old Arabidopsis plants; white bar 5 cm. b Inflorescence height in cm; n ≥ 26. c Acetate content in the second internode of 35-day-old stems; n ≥ 6. Error bars indicate standard deviation; letters indicate statistical significant differences based on one-way ANOVA (P < 0.05)

Fig. 6

Size exclusion chromatography of pectic extracts. Pectic extracts of PAE mutant lines (right titles) were applied to size exclusion chromatography. Fractions were collected according to depicted scheme (roman numerals); dashed bars indicate fraction borders. Black arrows indicate elution time of dextran standards. ox9 overexpression of the PAE9 coding sequence. comp-1 complementation with the native promoter and genomic sequence of PAE8. n ≥ 5

Fig. 7

PAE8 releases acetate in vitro from pae8 pectin fractions. a Percentage acetate released from pectin fractions (I–V) and Amorphophallus konjac acetylated glucomannan by heterologously expressed PAE8. Alkali would release 100 % of acetate in those fractions. Pectic fractions were generated from three independent pae8 biological replicates. The same protein content was used for PAE8 and EV activity assays. Asterisk indicates significant differences based on t test (P < 0.015; n = 3); EV protein extracts purified from tobacco plants transformed with an empty vector; PAE8 protein extract purified from tobacco plants transformed with PAE8:6XHIS. b Western blot showing the presence of PAE8 protein (~55 kDa) derived from tobacco plants transformed with the PAE8:6XHIS construct. +control = multi-tag positive control (Life Technologies). EV native protein extracts purified from tobacco plants transformed with an empty vector. M Magic Marker^TM XP (Life Technologies)

Fig. 8

PAE9 releases acetate in vitro from pae9 pectin fractions. a Percentage acetate released from pectin fraction (I–V) and Amorphophallus konjac acetylated glucomannan by heterologously expressed PAE9. Alkali would release 100 % of acetate in those fractions. Pectic fractions were generated from three independent pae9-1 biological replicates. The same protein content was used for PAE9 and EV activity assays. Asterisk indicates significant differences based on t test (P < 0.015; n = 3); EV protein extracts purified from tobacco plants transformed with an empty vector; PAE9 protein extract purified from tobacco plants transformed with PAE9:6XHIS. b Western blot showing the presence of PAE9 protein (~55 kDa) derived from tobacco plants transformed with the PAE9:6XHIS construct. EV native protein extracts purified from tobacco plants transformed with an empty vector. Marker Magic Marker^TM XP (Life Technologies)