The translation elongation factor eEF-1Bβ1 is involved in cell wall biosynthesis and plant development in Arabidopsis thaliana

Abstract

The eukaryotic translation elongation factor eEF-1Bβ1 (EF1Bβ) is a guanine nucleotide exchange factor that plays an important role in translation elongation. In this study, we show that the EF1Bβ protein is localized in the plasma membrane and cytoplasm, and that the transcripts should be expressed in most tissue types in seedlings. Sectioning of the inflorescence stem revealed that EF1Bβ predominantly localizes to the xylem vessels and in the interfascicular cambium. EF1Bβ gene silencing in efβ caused a dwarf phenotype with 38% and 20% reduction in total lignin and crystalline cellulose, respectively. This loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type plants, but no changes were detected in a gain-of-function mutant transformed with the EF1Bβ gene. Histochemical analysis showed a reduced vascular apparatus, including smaller xylem vessels in the inflorescence stem of the loss-of-function mutant. Over-expression of EF1Bβ in an eli1 mutant background restored a WT phenotype and abolished ectopic lignin deposition as well as cell expansion defects in the mutant. Taken together, these data strongly suggest a role for EF1Bβ in plant development and cell wall formation in Arabidopsis.

Figure 1. Confocal images showing localization of EF1Bβ-YFP (A and E) in Arabidopsis root tip cells plasmolyzed with 0.75 M sorbitol.

SynaptoRed (SR) was used as a plasma membrane marker (B and F). Panels C and G are the merged images of the YFP and SR channels and D and H show the DIC images. Lower panels (E-H) show a close-up of root tip cells. Bars  =  10 µm. Arrowheads (A and E) highlight the YFP localization in the cell periphery.

Figure 2. Expression pattern of EF1Bβ gene in Arabidopsis seedlings, inflorescence stem and reproductive organs.

Transgenic Arabidopsis plants expressing EF1Bβpromoter-UidA reporter fusion were examined histochemically for GUS activity. Seedling (A), hypocotyl (B), root tip (C), root hairs (D), trichomes (E), cross section of inflorescence stem (F), flower (G), silique epidermis (H), and fully developed seed (I). Cross section of the stem (F) shows the expression of EF1Bβ in xylem vessels (x) and interfascicular fibers (if).

Figure 3. Analysis of EF1Bβ expression in different tissues of Arabidopsis, including 5-day-old seedling, hypocotyl and roots from 14-day-old plants, rosette leaves from 4-week-old plants, and inflorescence stem, root and flower from 6-week-old plants.

Data represent mean transcript abundance ± SD relative to EF1α and elF4A1 from three independent experiments each replicated three times. ** indicates significant difference relative to stem transcript level at P≤0.01.

Figure 4. Phenotypes of Arabidopsis wild type, efβ and EFβOX-complemented plants.

Complemented mutant plant was transformed with a 35S::EF1Bβ cDNA.

Figure 5. EF1Bβ expression in inflorescence stem of WT and efβ (A), and of WT and EFβOX (B).

Data represent mean transcript abundance ± SD relative to EF1α and elF4A1 from three independent experiments each replicated three times. ** indicates significant difference at P ≤ 0.01.

Figure 6. Histochemical analysis of lignin in basal stem cross sections.

WT (A,D,G), efβ (B,E,H), and EFβOX (C,F,I) plants stained with either phloroglucinol (A–C), Mäule (D-F), or Toluidine Blue staining (G–I). In (H), arrow indicates unusual cell shape in efβ stem.

Figure 7. Total lignin and crystalline cellulose in inflorescence stems of EFβOX and efβ plants.

Lignin is shown relative to that of the WT (A). Cellulose is expressed as µg cellulose mg⁻¹ dry CWM (B). Data presented are means ± SD for three independent experiments, each replicated three times. * and ** indicate significant differences at P≤0.05 and 0.01, respectively.

Figure 8. Expression of lignin (A) and cellulose-related (B) genes in WT, EFβOX and efβ stems.

Data presented as mean transcript abundance ± SD relative to EF1α and elF4A1of three independent experiments each replicated three times. * indicates significant difference relative to transcript levels in the efβ mutant at P≤0.05.

Figure 9

Phenotypes of Arabidopsis wild type, eli1 and EFβOX-complemented plants (A). Expression of EF1Bβ in WT and eli1-EFβOX inflorescence stems (B). Data presented as mean transcript abundance ± SD relative to EF1α and elF4A1 of three independent experiments and each replicated three times. ** indicates significant differences relative to WT transcript levels at P ≤ 0.01. Phloroglucinol staining of roots (C) and hypocotyls (D) from 5-day-old dark grown seedlings showing ectopic lignification in eli1 but not in eli1-EFBOX or in WT background. Cross sections of the base of the inflorescence stem of 6-week-old plants; WT (E,H), eli1 (F,I), and eli1-EFβOX (G,J) stained with phloroglucinol (E-G) and Toluidine Blue (H-J).