Pectin methylesterase gene AtPMEPCRA contributes to physiological adaptation to simulated and spaceflight microgravity in Arabidopsis

Abstract

Pectin is biosynthesized in a highly methylated form and is partially de-methylated by pectin methylesterase (PME) activity. Plant PMEs play a critical role in cell wall remodeling in many physiological processes. Here, we studied Arabidopsis seedlings, which had been exposed to simulated or actual microgravity. Simulated microgravity inhibited total PME activity in Arabidopsis seedlings. We identified that AtPMEPCRA expression played a major role in the microgravity-induced inhibition of PME activity. atpmepcra mutants did not exhibit the enlarged leaf area of Arabidopsis seedlings observed under spaceflight microgravity. The downregulation of AtPMEPCRA expression in response to microgravity was due, in part, to changes in methylation patterns. The sexual offspring of the plants grown during spaceflight retained the methylation changes at AtPMEPCRA locus for one generation and thus contribute to the physiological adaptation to microgravity among F₁ offspring seed generation. We conclude that AtPMEPCRA contributes to the spaceflight-induced transgenerational responses in Arabidopsis.

Keywords: Biological sciences; Plant biology; Plant physiology; Space sciences.

Graphical abstract

Figure 1

Seedling pectin methylesterase (PME) activity and AtPMEPCRA expression were inhibited under simulated microgravity treatment (A) Wild-type Col-0 plants were exposed to simulated microgravity in a 3D clinostat. Root growth after 8-day simulated microgravity treatment was nondirectional, but root length was not significantly different from that of gravity-treated control plants. (B) Seedlings were harvested after a few days of simulated microgravity treatment. The total protein was extracted in the control and simulated microgravity seedlings at each time point, and the activity of PME was determined. Bars indicate SE. ∗ indicates significant differences (p＜0.05), using Student’s t test. (C) qRT-PCR analysis of gene expression pattern of some PME subfamily genes in Arabidopsis after exposure to simulated microgravity for 8 days. Bars indicates SE from three independent experiments, and three technical repetitions were performed for each replicate. ∗ indicates significant differences (p＜0.05), using Student’s t test.

Figure 2

Effect of simulated microgravity on PME activity in mutants blocked with respect to different plant hormone pathways (A) Plants of wild type and mutants defective in jasmonic acid (JA) perception (coi1), ethylene (ET) signaling (ein2), salicylic acid (SA) signaling (pad4), and (B) abscisic acid (ABA) biosynthesis (nced3, nced2nced3) and ABA signaling (abi4) were exposed to simulated microgravity. Seedlings were harvested from 0 days to 8 days after treatment began and total PME activity was assayed. (C) Inhibition of AtPMEPCRA expression level by simulated microgravity was significantly attenuated in the ABA biosynthesis nced2nced3 double mutants. Bar indicates SE from three independent biological replicates. ∗ indicates significant differences (p＜0.05), using Student’s t test.

Figure 3

Identification of atpmepcra transfer DNA insertion mutants and molecular analysis of the AtPMEPCRA gene (A) Organization of the AtPMEPCRA gene. The pink boxes show the positions and sizes of the exons. The green boxes show the positions and sizes of the introns. Each triangle indicates a site of transfer DNA insertion. (B) Identification of relative AtPMEPCRA gene expression level in Col-0 and mutants. Bar indicates SE. ∗ indicates significant differences (p＜0.05), using Student’s t test. (C) proGUS analysis of AtPMEPCRA expression patterns in various organs: leaf vein, cotyledon, peduncle, root tip, root, and silique.

Figure 4

Sensitivity of AtPMEPCRA pectin methylesterase (PME) activity to pH, temperature, and pectin methylesterase inhibitor (AtPMEI1) (A) The response to pH of purified recombinant AtPMEPCRA and Botrytis cinerea PME (f-PME) was compared. Bar indicates SE. ∗ indicates significant differences (p＜0.05), using Student’s t test. (B) Thermal stability of purified recombinant AtPMEPCRA. The activity at 30°C was set at 100%, and the activities incubated at different temperatures were expressed relative to that at 30°C. Bar indicates SE. ∗ indicates significant differences (p＜0.05), using Student’s t test. (C) Inhibition of purified recombinant AtPMEPCRA by AtPMEI1. Values are expressed as means ± SE from three independent biological replicates. ∗ indicates significant differences (p＜0.05), using Student’s t test.

Figure 5

Comparison of mean leaf area of seedlings of Arabidopsis Col-0 wild type and atpmepcra transfer DNA insertion mutants grown under microgravity or gravity (A) Seedlings were grown on Earth (exposed to gravity) or in space on the SJ-10 satellite (exposed to microgravity) for 11 days. Bar: 1 cm. (B) The data shown are the mean values ±SD obtained for at least eight leaves from the plants presented in (A). The leaf area was measured using ImageJ. Bar indicates means ± SE from three independent biological replicates. ∗ indicates significant differences between the same genotype exposed to gravity of microgravity (p＜0.05), using Student’s t test.

Figure 6

Analysis of spaceflight-induced heritable DNA methylation of AtPMEPCRA and analysis of the AtPMEPCRA expression levels in the offspring (A) Visualization of bisulfite sequencing PCR (BSP) analysis of AtPMEPCRA putative spaceflight-induced heritable differential methylated regions (DMRs). Visualization of methylation data was performed, using the Integrated Genome Viewer (IGV) (http://software.roadinstitute.org/software/igv). The inherited methylation level of CpG islands was verified using BSP analysis. (B and C) (B) Expression levels of AtPMEPCRA in the F₁ and F₂ (C) offspring of plants grown in space or on Earth. Parent plants were grown in space or on Earth for 11 days, and seed was collected from plants on Earth. Error bars are SE. ∗ indicates statistically significant difference using Student’s two-tailed t test (p < 0.05).

Figure 7

Comparison of the silique length, root length, flowering time, and gravitropic orientation analysis in F₁ offspring of plants of Col-0 and atpmepcra transfer DNA insertion mutants grown in space or on Earth (A and B) The silique length and (C and D) root length of the F₁ offspring of plants grown for 11 days in space or on Earth. Lengths were measured using ImageJ software. The data shown in b. and d. are the mean ± SE values obtained for about 21 plants per sample. The red bar indicates significantly different. (E–G) Analysis of the response to 90° change in gravitropic orientation analysis of the 8-day-old F₁ offspring seedlings of the plants grown in space or on Earth. (H) Flowering time analysis of the F₁ offspring of plants grown for 11 days in space or on Earth. (I) RT-qPCR analysis of FT expression levels. The data shown are the mean ± SE (n > 10). ∗ indicates a statistically significant difference between the samples, using Student’s t test (p < 0.05). Bar indicates 1 cm.

Figure 8

Analysis of gene expression levels and ABA response in the F₁ offspring of plants of Col-0 and atpmepcra transfer DNA insertion mutants grown for 11 days in space or on Earth (A) Expression level of AtPMEPCRA in F₁ generation of plants grown for 11 days in space or on Earth. B, c. Visualization of ABA response in the F₁ generation. (B) and F₂ generation. (C) of plants grown for 11 days in space or on Earth in the presence of various concentrations of ABA (n > 40). Bars indicate 1 cm. (D) Time course of seed germination of wild type and atpmepcra transfer DNA insertion mutants in the absence (CK) or the presence of various concentrations of ABA (n > 30 per sample). (E) Expression levels of ABA-associated genes in F₁ generation of plants grown for 11 days in space or on Earth. Error bars indicate SE. ∗ indicates statistically significant difference using Student’s t test (p < 0.05).