The mevalonate pathway but not the methylerythritol phosphate pathway is critical for elaioplast and pollen coat development in Arabidopsis

Abstract

Pollen coat components are derived from tapetum cells, which contain elaioplasts derived from plastids and tapetosome derived from the endoplasmic reticulum. In Brassica napus, the main neutral lipids in the elaioplast and tapetosome have been reported to be sterol ester and triacylglycerol, respectively. Isopentenyl pyrophosphate, the structural component of sterol, is produced via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways. Although these two pathways are compartmentalized, partial cross-talk between them has been reported. To investigate the contribution of these two pathways in elaioplast formation, we characterized mutant pollen of these two pathways. We observed the anthers of male sterile hmg1-1 and atipi1 atipi2 mutants ultrastructurally, which were deficient in MVA pathway enzymes. hmg1-1 and atipi1atipi2 showed a shrunken elaioplast inner granule at the bicellular pollen stage. Conversely, in the cla1-1 mutant, which showed a defective MEP pathway, elaioplast development was normal. The pollen of hmg1-1 and atipi1atipi2 was coatless, whereas cla1-1 had a pollen coat. These results indicate that the MVA pathway but not the MEP pathway is critical for elaioplast development though the organelle is derived from plastids.

A mature male gametophyte is covered by exine and a pollen coat. The main component of exine is a sporoporenine polymer that comprises lipids and phenolic derivatives. Exine guards pollen grains chemically and physically (Piffanelli et al. 1998). Conversely, the pollen coat mainly comprises lipids and protein and it has many properties, such as attaching to vector insects to facilitate pollen transmission (Piffanelli et al. 1998), attaching to the dry stigmatic surface to start pollen hydration (Hülskamp et al. 1995; Preuss et al. 1993), and self-incompatibility (Shiba et al. 2001).

The pollen coat is derived from tapetum cells at the innermost cell layer of the anther wall, which contains the elaioplast and tepetosome (Hernández-Pinzón et al. 1999). The elaioplast is an organelle derived from plastids, and the tapetosome is an organelle derived from the endoplasmic reticulum (ER). In Arabidopsis, these organelles develop in tapetum cells when a male gametophyte is in the bicellular pollen stage (Quilichini et al. 2014). In the tricellular pollen stage, tapetum cells disappear and a pollen coat forms at the microspore surface. It is considered that the pollen coat contains sterol ester as the major natural lipid and contains phospholipid as a polar lipid. Various sterol types exist in the pollen of different plant species (Villette et al. 2015). Lipid profiles of tapetum organelles are very different between the elaioplast and tapetosome. In a previous report, the major natural lipids of elaioplasts were identified as sterol ester and wax ester, while that of the tapetosome was triacylglycerol in Brassica napus (Hernández-Pinzón et al. 1999). Arabidopsis knock-down mutants of acetoacethyl CoA synthase (AACT2) (Jin et al. 2012) and HMG-CoA synthase (HMGS) (Ishiguro et al. 2010) also show a pollen coatless phenotype and male sterility. AAC T2 and HMGS are biosynthetic enzymes up-stream of sterol biosynthesis.

Sterol is a kind of isoprenoid. Isoprenoid compounds comprise isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized via the cytosolic MVA pathway and the plastidic methylerythritol phosphate (MEP) pathway (Figure 1). These two pathways are separated by the plastid membrane (Lichtenthaler et al. 1997). Although there is partial metabolic cross-talk between MVA and MEP pathways, the mechanisms remain unclear (Kasahara et al. 2002; Nagata et al. 2002).

Figure 1. Scheme of the isoprenoid biosynthesis pathway. AACT, Acetoacetyl CoA thiolase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, HMG-CoA reductase; IPP, isopentenyl diphosphate; DMAPP, dimethyl allyl diphosphate; IPI, Isopentenyl/dimethylallyl diphosphate isomerase; FPP, farnesyl diphosphate; GA-3P, Glyceraldehyde-3-phosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DX, 1-deoxy-D-xylulose 5-phosphate; MEP, methylerythritol phosphate; and GGPP, geranylgeranyl diphosphate. The white arrow signifies metabolite cross-talk between the cytosolic MVA pathway and plastidic MEP pathway.

It is strange that sterol accumulates in elaioplasts which are derived from plastids (Hernández-Pinzón et al. 1999), although sterol is known as the cytosolic MVA pathway derivative (Newman and Chappell 1999).

In the hmgs/fkp1 mutant, the structure of elaioplasts and the tapetosome is strange, formation of the pollen coat and pollen hydration is inhibited, and the fkp1 mutant exhibits male sterility (Ishiguro et al. 2010). These findings suggest that the sterol of elaioplasts is derived from the MVA pathway.

In a previous study, we isolated the male sterile mutant, which was a defective mutant of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the MVA pathway (Suzuki et al. 2004). In Arabidopsis, HMGR is encoded by two genes, HMG1 and HMG2. In anthers, HMG1 was expressed in the tapetum and microspores, while the expression of HMG2 appeared only in microspores (Suzuki et al. 2009). The hmg1-1 mutant showed an inhibition of pollen germination or pollen tube elongation (Suzuki et al. 2004). Besides the RNAi mutant of AACT2, hmgs/fkp1 mutant, and hmg1 mutant (Ishiguro et al. 2010; Jin et al. 2012; Suzuki et al. 2004), the atipi1atipi2 mutant (Okada et al. 2008) has been isolated as a male sterile mutant, which is defective in the MVA pathway. Isopentenyl pyrophosphate isomerase (IPI) catalyzes the isomerization reaction from IPP to DMAPP. Two genes encoding IPI, IPI1 and IPI2, exist in the Arabidopsis genome. DMAPP is necessary for isoprenoid biosynthesis as an enzyme substrate, such as farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase.

cla1-1 is a null mutant of the first enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase 1 (Estévez et al. 2000). In this mutant, the accumulation of chlorophyll and carotenoids is totally inhibited in the chloroplast and this mutant shows an albino phenotype (Mandel et al. 1996). In this study, we investigated the role of the MEP pathway on elaioplast development using Arabidopsis flowers of cla1-1. We observed the developmental stage of microspores in these mutants and demonstrated the contribution of the MVA pathway but not the MEP pathway on elaioplast development.

We observed and compared the ultrastructure of anthers between wild type (WT) and male sterile mutants of the MVA pathway, hmg1-1 and atipi1atipi2. In the bicellular pollen stage, electron-dense granules in the elaioplast were shrunken in hmg1-1 and atipi1atipi2 (Figure 2A–H). The area of high electron density in this experimental condition was generally considered a lipid-rich area. Thus, these results showed that lipids in the elaioplast were decreased in hmg1-1 and atipi1atipi2. The tapetosome in hmg1-1 and atipi1atipi2 was smaller than that in WT (Figure 2A–H). In the late tricellular pollen stage, a pollen coatless phenotype was observed in hmg1-1 and atipi1atipi2 (Figure 2I–P). These results are consistent with previous results wherein mutants of the MVA pathway, the RNAi mutant of AACT2 (Jin et al. 2012) and hmgs/fkp1 (Ishiguro et al. 2010), showed a pollen coatless phenotype. The elaioplasts in hmgs/fkp1 mutant, hmg1-1 mutant and atipi1atipi2 mutant showed an abnormal shape, although the genes encoding plastidic isoprenoid biosynthetic enzymes are not affected in these mutants (Figure 2A–H). A complete blockage of the MVA pathway resulted in a lethal phenotype (Ishiguro et al. 2010; Jin et al. 2012; Suzuki et al. 2009). Another allele of atipi1atipi2, idi1idi2, has been reported to be lethal (Phillips et al. 2008). Although the molecular mechanism of the difference is unclear, differences in the genetic lines between them may cause this phenotypic variation. atipi1atipi2 was viable probably because 7% WT IPI activity remained in atipi1atipi2 (Okada et al. 2008). However, we demonstrated that 7% WT IPI activity in atipi1atipi2 may be insufficient for normal differentiation of the elaioplast and formation of the pollen coat. These results were also consistent with the results of hmgs/fkp1-1 (Ishiguro et al. 2010).

Figure 2. Abnormal elaioplast and pollen coat of hmg1-1 and atipi1atipi2. The developmental stage of a male gametophyte in WT (WS; A, C, I, K Col-0; E, G, M, O), hmg1-1 (B, D, J, L), and atipi1atipi2 (F, H, N, P) mutants. Tapetal organelle development in the bicellular pollen stage (A–H). Pollen coat in the tricellular pollen stage (I–P). E, elaioplast; T, tapetosome; P, pollen coat. Scale bars=2 µm in A, B, E, F, and m, 1 µm in C, D, G, H, K, L, O, and P, and 5 µm in I, J, and N.

The tapetosome also showed an abnormal phenotype in hmg1-1, atipi1atipi2 (Figure 2A–H), and hmgs/fkp1-1. These results suggest that the cytosolic MVA pathway contributed to the formation of a pollen coat, elaioplast, and tapetosome. A sterol-deficient mutant, such as defective mutants of the MVA pathway, may also show a pollen coatless phenotype because sterol ester, the main component of a pollen coat, was decreased.

The next question is the importance of the products from the MEP pathway in the elaioplast. To investigate the contribution of MEP pathway to elaioplast development, we performed an electron microscopic analysis of the microspore in cla1-1. Most cla1-1 homozygous mutants showed a lethal seedling phenotype when grown on Murashige and Skoog (MS) agar. However, when the seedlings were transferred to 1/2 MS agar supplemented with 1.5% sucrose in a large agripot, only some that grew to the flower stage were obtained (Figure 3A–D). Mature cla1-1 plants showed a sterile phenotype wherein the primary shoot terminated at some buds. At the bicellular pollen stage, the elaioplast and tapetosome of cla1-1 were comparable to that of WT (Figure 2A, C and Figure 3E, F). In the tricellular pollen stage, the pollen coat formed normally (Figure 2I, K and Figure 3G, H), although almost all gametophytes had abnormal vacuoles (Figure 3G) or were crushed in cla1-1 (data not shown). At least the male gametophytes were indistinguishable between WT and cla1-1 at bicellular pollen stage, while normal development may be prevented thereafter in cla1-1 (Supplementary Figure). From these results, we considered that the sterile cla1-1 phenotype may have been caused by an abnormal gametophyte.

Figure 3. Normal elaioplast and pollen coat of cla1-1. A 40-day-old WT (Ws; A) and cla1-1 (B) plant. Close-up view of the plant (C) and flower (D) of cla1-1. Tapetal organelle development of cla1-1 in the bicellular pollen stage (E, F). Pollen coat of cla1-1 in the tricellular pollen stage (G, H). P, pollen coat. The abnormal vacuole is indicated by an asterisk. Scale bars=1 cm in A and B, 2 mm in C, 400 µm in D, 2 µm in E, 1 µm in F, H, and 5 µm in G, I, J.

These results suggest that the MEP pathway, unlike the MVA pathway, was not necessary for the formation of the elaioplast or pollen coat. In the tricellular pollen stage, there were many abnormal gametophytes in the cla1-1 mutant. This result indicated that the cla1-1 mutant had no ability for normal pollen development after the tricellular pollen stage, suggesting that the MEP pathway is important in the formation of the gametophyte but not of the elaioplast.

It has been reported that sterol ester accumulates in the elaioplast in B. napus (Hernández-Pinzón et al. 1999). The abnormal elaioplast, pollen coatless microspores, and male sterility phenotypes were observed not only in hmgs/fkp1-1 (Ishiguro et al. 2010) but also in both hmg1-1 and atipi1atipi2 in this study. Our results strongly support the previous hypothesis that sterol ester in the elaioplast is derived from the cytosolic MVA pathway, even though the elaioplast is derived from plastids. Two possibilities have been proposed: (1) MVA and sterol biosynthesis pathways localize in a special organelle, which is the elaioplast and (2) sterols are produced in cytosol and then transported to the elaioplast. The possibility of (1) may be low as there are more than 11 steps in sterol synthesis from acetyl CoA (Suzuki and Muranaka 2007). Although it is necessary that enzymes with more than 11 synthesis steps are imported to the elaioplast, these 11-step enzymes do not have a plastid-transfer signal. On the contrary, it was reported that ER covered the proplastid in the process of formation of elaioplast from proplastid in Olea europaea (Pacini and Casadoro 1981). It is likely that sterols were transported to the elaioplast from ER. The trafficking of intermediary isoprenoid metabolites between plastids and cytosol has been reported (Hemmerlin et al. 2003; Kasahara et al. 2002; Nagata et al. 2002). Elucidation of the mechanisms how MVA pathway products accumulate in plastids may reveal the developmental mechanisms of the elaioplast in pollen growth stages.

Abbreviations

AACT

acetoacethyl CoA synthase

DMAPP

dimethylallyl pyrophosphate

ER

endoplasmic reticulum

HMGR

3-hydroxy-3-methylglutaryl-CoA reductase

HMGS

HMG-CoA synthase

IPI

isopentenyl pyrophosphate isomerase

IPP

isopentenyl pyrophosphate

MEP

methylerythritol phosphate

MS

Murashige and Skoog

MVA

mevalonate

WT

wild type