BiP3 supports the early stages of female gametogenesis in the absence of BiP1 and BiP2 in Arabidopsis thaliana

Abstract

Immunoglobulin binding protein (BiP) is an essential heat shock protein 70 (Hsp70) in the endoplasmic reticulum (ER) that functions in various processes including protein translocation, protein folding and quality control. Arabidopsis thaliana harbors ubiquitously expressed genes BIP1 and BIP2, as well as BIP3, which is induced only by ER stress. Recently, we reported that these BIP genes are expressed in male gametophytes and cooperate with each other to support male gametogenesis and pollen competitiveness. Here, we report that the BIP genes cooperate to support female gametogenesis. As reported previously, the bip1 bip2 double mutation causes defects in the fusion of polar nuclei during female gametogenesis. By contrast, the bip triple mutant female gametophytes exhibited defects during the early stages of female gametophyte development, which suggests that BIP3 supports the early stages of female gametophyte development, but not polar nuclear fusion, in the absence of BiP1 and BiP2.

Abbreviations

BiP

immunoglobulin binding protein

CLSM

confocal laser-scanning microscopy

ER

endoplasmic reticulum

GUS

β-glucuronidase

Hsp70

heat shock protein 70

BiP is a molecular chaperone Hsp70 in the ER that plays key roles in protein translocation across the ER membrane, protein folding and quality control in the ER.¹ Unlike yeasts and mammals, which have single BIP genes, flowering plants carry multiple BIP genes; A. thaliana has 3 BIP genes.² While BIP1 and BIP2 are ubiquitously expressed in plant tissues and encode proteins that are 99% identical to each other, BIP3 encodes a less conserved BiP paralog (80% identical to BiP1 and BiP2), which is expressed only under ER-stress conditions in most tissues.

We recently reported that the functions of BiP3 are comparable to those of BiP1 and BiP2. The three BIP genes in A. thaliana cooperate with each other to ensure ER homeostasis in cells with active secretions, such as developing pollen grains and actively growing pollen tubes.³ We previously reported that BiP also functions in female gametophyte development. The bip1 bip2 double mutant female gametophytes are defective in the fusion of polar nuclei in the central cell during female gametogenesis.⁴ Since expression of BIP3 under the control of the BIP1 promoter suppresses the polar nuclear fusion defect as well as male competitiveness, cooperation of multiple BIP genes may well occur during female gametogenesis.

To examine the roles of multiple BIP genes in female gametogenesis, we performed confocal laser-scanning microscopy (CLSM) analyses of mature female gametophytes with various combinations of the bip1, bip2 and bip3 mutant alleles. A. thaliana produces Polygonum-type female gametophytes consisting of one egg cell, one central cell, 2 synergid cells and 3 antipodal cells within ovules.⁵ A single megaspore produced by meiosis undergoes 3 rounds of nuclear divisions without cytokinesis, producing an 8-nucleate female gametophyte. Cellularization takes place after the third nuclear division, producing a 7-celled female gametophyte. In the central cell, 2 polar nuclei fuse with each other to produce a diploid secondary nucleus in the central cell. Since the bip1 bip2 double mutant female gametophyte causes defects in polar nuclear fusion, the polar nuclei remained unfused (Fig. 1B) in ∼50% of ovules from bip1–2/+ bip2–1/bip2–1 plants (Fig. 1G), which is similar to that reported for bip1–4/+ bip2–1/bip2–1 plants.⁴

Figure 1.

The bip1 bip2 bip3 triple mutation causes defects during early female gametophyte development. (A–F) CLSM analyses of ovules from mature pistils of bip1/+ bip2/bip2 bip3/+ plants. Six types of ovules were observed. (A) Ovule containing a normal female gametophyte with a large secondary nucleus generated by the fusion of 2 polar nuclei. (B) Ovule containing a female gametophyte with unfused polar nuclei in the central cell. Ovule containing a 4-nucleate female gametophyte (C), a 2-nucleate female gametophyte (D), a one-nucleate female gametophyte (E) and a degenerated female gametophyte (F). Arrowheads indicate nuclei of female gametophyte arrested during the early stages of development. Abbreviations: scn, secondary nucleus; upn, unfused polar nucleus; ecn, egg cell nucleus; syn, synergid cell nucleus. Scale bars represent 20 μm. (G) Percentages of types of ovules shown in (A–F) observed in plants with the indicated genotypes.

By contrast, additional developmental defects were observed in ovules from bip1/+ bip2/bip2 bip3/+ plants. Although ∼50% of ovules were normal (Fig. 1A), ∼25% of ovules had unfused polar nuclei (Fig. 1B). The remaining ovules appeared to show defects in the earlier developmental stages. We observed 4 nucleate (Fig. 1C), 2 nucleate (Fig. 1D), one nucleate (Fig. 1E) and even degenerated (Fig. 1F) female gametophytes. We tested different alleles of the bip1 mutant and obtained similar results (Fig. 1G). The bip1/+ bip2/bip2 bip3/+ plants are expected to produce female gametophytes with bip1 bip2 bip3, bip1 bip2, bip2 bip3 and bip2 mutations with a 1:1:1:1 segregation ratio. Since bip2 bip3 double mutant female gametophytes develop normally,⁴ the observed ratio of female gametophytes showing defects in the earlier stages correlates well with the expected percentage of female gametophytes carrying the bip1 bip2 bip3 triple mutation. These results indicate that depletion of all 3 BIP genes causes defects in the early stages of female gametogenesis.

We next examined the expression of the BIP genes in mature ovules using transgenic lines harboring the β -glucuronidase (GUS) gene driven by the BIP promoters (1 kb upstream of the coding region of each BIP gene).³ Mature ovules of the transgenic lines were subjected to GUS staining. GUS signals were observed throughout the ovules of pBIP1::GUS plants, with strong signals around the micropylar region (Fig. 2A). Shorter GUS staining treatments (∼30 min) revealed predominant GUS expression throughout the female gametophytes (Fig. 2B; compare to the drawing of a mature ovule shown in Fig. 2F). Similar results were obtained when we analyzed ovules from pBIP2::GUS plants (Fig. 2C and D). These results indicate that BiP1 and BiP2 are expressed in mature female gametophytes. By contrast, GUS signals were restricted to the antipodal cell region in ovules from pBIP3::GUS plants, even under longer (3 h) staining conditions (Fig. 2E). These results indicate that BiP3 expression is very weak in most parts of the female gametophyte, including the central cell.

Figure 2.

Expression patterns of Arabidopsis BIP genes in ovules. (A to E) GUS-stained ovules of pBIP1::GUS (A and B), pBIP2::GUS (C and D) and pBIP3::GUS (E) plants are shown. Ovules were observed after GUS staining for 3 hours (A, C, and E) or 30 min (B and D). Scale bars represent 20 μm. (F) Schematic drawing of a mature ovule of A. thaliana. Abbreviations: mp, micropylar region; cp, chalazal region; cc, central cell; ec, egg cell; syn, synergid cell; ac, antipodal cell.

We also analyzed the expression of the BIP1 and BIP3 genes in developing female gametophytes. Developing ovules from pBIP1::GUS and pBIP3::GUS plants were subjected to GUS staining and were examined by differential interference contrast microscopy and CLSM. As observed in mature ovules, GUS expression was observed in 4-nucleate and 8-nucleate/7-celled female gametophytes of the pBIP1::GUS plants with shorter (∼30 min) GUS staining conditions (Fig. 3C and 3D). In the case of one-nucleate female gametophytes of the pBIP1::GUS plants, GUS signals were observed only under longer GUS staining treatments (2 h), suggesting that BIP1 expression is relatively low during the early stages of female gametogenesis (Fig. 3A and B). By contrast, GUS signal was not observed in one-nucleate and 4-nucleate female gametophytes of the pBIP3::GUS plants under even longer (6 h and 3 h) staining conditions (Fig. 3E and F). GUS signals were observed in the antipodal cell region in an 8-nucleate/7-celled female gametophyte (Fig. 3G). These results show little or no expression of BIP3 during the early stages of female gametogenesis.

Figure 3.

Expression patterns of the BIP1 and BiP3 genes in the early stages of female gametogenesis. GUS-stained ovules from developing pistils of the pBIP1::GUS (A to D) and the pBIP3::GUS (E to G) plants are shown. Ovules were observed by CLSM and differential interference contrast microscopy after GUS staining for 30 min (B to D), 2 hours (A), 3 hours (F and G) and 6 hours (E). Ovules with a one-nucleate female gametophyte (A, B, and E), with a 4-nucleate female gametophyte (C and F) and with an 8-nucleate/7-celled female gametophyte (D and G) are shown. Left and right pictures are CLSM and differential interference contrast microscopy images of a GUS-stained ovule, respectively. Arrowheads indicate nuclei in developing female gametophytes. Abbreviations: upn, unfused polar nucleus; ecn, egg cell nucleus; syn, synergid cell nucleus. Scale bars represent 20 μm.

Our results show that BIP3 can support the early stages of female gametogenesis in the absence of BIP1 and BIP2 in A. thaliana, which resembles what was demonstrated for male gametogenesis.⁴ Although we did not detect BIP3 expression during early stages of female gametogenesis, upregulation of BIP3 expression in cells lacking BiP1 and BiP2 could support development of the bip1 bip2 female gametophytes during the early developmental stages. Nevertheless, the bip1 bip2 female gametophytes were defective in polar nuclear fusion. Since BiP3 expression from the BIP1 promoter suppressed the polar nuclear fusion defect of the bip1 bip2 double mutant female gametophytes,⁴ the observed polar nuclear fusion defect was not due to differences in chaperone activities between BiP3 and BiP1/2. We observed that BIP1 expression is higher in the late stages of female gametogenesis, which could reflect higher cellular demand of BiP proteins in these stages. Most likely, expression level of BiP3 driven by its own promoter in the central cell is too low enough to support polar nuclear fusion in the absence of BiP1 and BiP2. Mechanisms underlying this differential BIP gene expression are currently unknown. Differential tissue-specific BIP gene expression was also reported in soybean.⁷ Further analysis should demonstrate the significance and mechanisms of differential expression of multiple BIP genes for plant growth and development.

Methods

Plant materials and growth conditions

The Arabidopsis thaliana quartet1–2 mutant (CS8846) served as the wild type in this study. Seeds of the bip mutants CS853146 (bip1–2), CS801763 (bip1–3), CS856879 (bip1–4), CS842467 (bip2–1) and SALK_024133 (bip3–1) were obtained from the Arabidopsis Biological Resource Center at Ohio State University. Primer sets designed using the SIGnAL iSect tool (http://signal.salk.edu) were utilized to verify T-DNA insertions in the bip mutants. The pBIP1::GUS, pBIP2::GUS and pBIP3::GUS transgenic plants used for the promoter-GUS assay were described previously.⁴ Plants were grown in soil at 22°C under continuous light.

Microscopy

Preparation and analysis of ovules for CLSM were described by Christensen et al.⁶ GUS staining of mature ovules was performed as previously described.⁴ To analyze GUS-stained ovules by CLSM, GUS-stained ovules were fixed with 4% glutalaldehyde, 12.5 mM sodium cacodylate, pH 6.9 for more than 3 d at 4°C. Samples were dehydrated, cleared and analyzed by differential interference contrast microscopy and CLSM.⁶

Funding

This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grants-in-Aid for Scientific Research on Priority Areas (no. 1685101 to S.N.); Grants-in-Aid for Scientific Research on Innovative Areas (nos. 23120512 and 25120711 to S.N.); Grants-in-Aid for Scientific Research (no. 23570051 to S.N.)]. D.M. was supported by grant number 6526 from the Japan Society for the Promotion of Science Fellowships.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank D. Preuss for materials and members of the Endo Laboratory for useful discussions.