Complete blockage of the mevalonate pathway results in male gametophyte lethality

Abstract

Plants have two isoprenoid biosynthetic pathways: the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, possible metabolic cross-talk between these pathways has prompted intense research. Although many studies have shown the existence of such cross-talk using feeding experiments, it remains to be determined if native cross-talk, rather than exogenously applied metabolites, can compensate for complete blockage of the MVA pathway. Previously, Arabidopsis mutants for HMG1 and HMG2 encoding HMG-CoA reductase (HMGR) were isolated. Although it was shown that HMGR1 is a functional HMGR, the enzyme activity of HMGR2 has not been confirmed. It is demonstrated here that HMG2 encodes a functional reductase with similar activity to HMGR1, using enzyme assays and complementation experiments. To estimate the contribution of native cross-talk, an attempt was made to block the MVA pathway by making double mutants lacking both HMG1 and HMG2, but no double homozygotes were detected in the progeny of self-pollinated HMG1/hmg1 hmg2/hmg2 plants. hmg1 hmg2 male gametophytes appeared to be lethal based on crossing experiments, and microscopy indicated that approximately 50% of the microspores from the HMG1/hmg1 hmg2/hmg2 plant appeared shrunken and exhibited poorly defined endoplasmic reticulum membranes. In situ hybridization showed that HMG1 transcripts were expressed in both the tapetum and microspores, while HMG2 mRNA appeared only in microspores. It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametophyte development.

Fig. 1.

Outline of the two isoprenoid biosynthetic pathways in an Arabidopsis cell: the MVA and MEP pathways. Blue arrows indicate metabolic flow between the cytosol and plastid. Two genes, HMG1 and HMG2, encoding HMGR are shown in red letters. MVA, mevalonate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GAP, glyceraldehyde 3-phosphate.

Fig. 2.

HMGR enzyme activity and expression of hmgr mutants and complementation lines of hmg1-1. (A) Relative HMGR activity in microsome fractions prepared from 2-week-old seedlings of WS, hmg1-1, Col, hmg2-1, lines 1.1 and 1.4, and lines 2.13 and 2.23. The activity in WS plants, 151±6.9 pmol min⁻¹ mg⁻¹ protein, was given a value of 1. Values are the average of three experiments. (B) Relative expression of HMG1 (gray box) and HMG2 (white box) in 2-week-old seedlings of WS, hmg1-1, lines 1.1 and 1.4 (both have HMG1 promoter::HMG1S in an hmg1-1 background), and lines 2.13 and 2.23 (both have HMG1 promoter::HMG2 in an hmg1-1 background). The expression in WS plants was given a value of 1.

Fig. 3.

Phenotypic analyses of hmg1-1 complementation lines. (A) Left to right, upper figure: mature WS, hmg1-1, 1.1, 1.4, 2.13, and 2.23 plants. Scale bar indicates 5 cm. Left to right, lower figure: close-up photos of inflorescences of WS, hmg1-1, 1.1, 1.4, 2.13, and 2.23 plants. Scale bar indicates 1 cm. (B) Total sterols (μg 100mg⁻¹ dry weight) in WS, hmg1-1, 1.1, 1.4, 2.13, and 2.23 plants. (C) The effect of HMG1S and HMG2 expressed in hmg1-1 plants on XTR9 and extensin-like-protein expression. Total RNA was extracted from 2-week-old seedlings of WS (1), hmg1-1 (2), 1.1 (3), 1.4 (4), 2.13 (5), and 2.23 (6) plants. EF-1 transcripts were amplified as a control.

Fig. 4.

Outline of the crossing experiment. The results of self-pollination of HMG1/hmg1-1 HMG2/HMG2 plants (A) and crossing of HMG1/hmg1-2 hmg2-1/hmg2-1 as the female parent pollinated with pollen from HMG1/hmg1-1 hmg2-1/hmg2-1 plants (B) are shown. The resultant segregation of the F₁ generation is shown.

Fig. 5.

Photographs of the anthers and pollen grains. (A) Anther of HMG1/HMG1 hmg2-1/hmg2-1 plant. (B) Anther of a HMG1/hmg1-1 hmg2-1/hmg2-1 plant. (C) Tetrad in HMG1/HMG1 HMG2/HMG2 qrt1-1/qrt1-1 plant. (D) Tetrad in HMG1/hmg1-1 hmg2-1/hmg2-1 qrt1-1/qrt1-1 plant. (E) DAPI-stained tetrad shown in (D). Arrows indicate abnormal, shrunken pollen grains. Scale bars indicate 10 μm.

Fig. 6.

Ultrastructural analysis of the male gametophyte. Normal male gametophytes (A, E, H) and abnormal male gametophytes (B, C, D, F, G, I) in HMG1/hmg1-1 hmg2-1/hmg2-1 plants. Abbreviations: ex, exine; pc, pollen coat; v, vacuole; m, mitochondria; p, plastid. Arrows indicate abnormal membrane structures of apparent endoplasmic reticulum origin. Scale bars indicate 10 μm (A, B, C, D), 1 μm (E, F, G), or 200 nm (H, I).

Fig. 7.

Comparison of HMG1 and HMG2 expression in anthers using in situ hybridization. Sections of an anther hybridized with antisense HMG1 (A) or HMG2 (B) probes. Sections of an anther hybridized with sense HMG1 (C) or HMG2 (D) probes as a control. Arrows and arrowheads indicate the tapetum and microspores, respectively. Scale bars indicates 10 μm.