The Glycine-Rich Domain Protein GRDP2 Regulates Ovule Development via the Auxin Pathway in Arabidopsis

Abstract

The glycine-rich domain proteins (GRDP) have been functionally implicated in the cell wall structure, biotic, and abiotic stress responses. However, little is known about GRDP genes in female gametophyte development of Arabidopsis. This study shows that GRDP2, a GRDP, plays a crucial role in female gametophyte development. In GRDP2 overexpression lines, grdp2-3, the embryo sacs were arrested at FG1 and no nucleus stages. Furthermore, callose staining shows that cell plate formation during megasporogenesis is disturbed in grdp2-3. In contrast, the pollen development is not affected in grdp2-3. The expression patterns of auxin-specific marker lines in female gametophytes showed that the auxin distribution and transport were significantly changed during megagametogenesis in grdp2-3. In addition, compared with the membrane-localized pattern of PIN1, PIN2, and PIN7 in WT, the signals were detected in the cytoplasm in grdp2-3. Together, our data suggest that GRDP2 plays an essential role in auxin-mediated female gametophyte development.

Keywords: GRDP2; auxin; embryo sac; female gametophyte; ovule development.

FIGURE 1

Phenotype analysis of grdp2 T-DNA mutant. (A) Gene structure of GRDP2 and T-DNA insertion sites of the mutant alleles in grdp2. White boxes show UTRs; gray lines show introns; black boxes show exons. (B) Expression level of the three T-DNA insertion lines as determined by real-time RT-PCR. (C) Seed development in wild-type (WT) plants and the grdp2 mutant alleles. Bar = 1 cm. (D) Seed setting statistics of three T-DNA insertion lines. (E) Alexander’s staining of pollen from WT and three grdp2 mutant alleles. The red color indicates viable pollen. Bar = 50 μm. The asterisks show the significance level (^∗∗∗p < 0.01) as judged by the Student’s t-test.

FIGURE 2

The expression profiles of GRDP2 in Arabidopsis. (A) Real-time PCR to quantify GRDP2 expression levels in different tissue of root, stem, leaf, siliques, and inflorescence. (B) pGRDP2:GUS expression pattern in floral buds. Bars = 1 mm. (C,D) pGRDP2:GUS expression pattern in anthers. Bars = 50 μm. (E,F) pGRDP2:GUS expression pattern in ovules. Bars = 20 μm. (G–J) pGRDP2:GFP expression pattern in different ovule development stages. The developmental stages of the ovules are indicated at the top right corner of each panel. Bars = 20 μm.

FIGURE 3

Female gametogenesis in WT and grdp2-3 plants examined by differential interference contrast microscope (DIC). Bar = 50 μm. (A) Megaspore mother cell (MMC) of WT plants. (B) 98.3% of 121 examined FG1 stage from WT normal ovules. (C–F) Female gametogenesis from FG2 to FG7 was normal in WT plants. (G) Megaspore mother cell (MMC) of grdp2-3 plants. (H) 80.5% of 221 examined FG1 stage ovules from grdp2-3 plants were normal. (I) 19.5% of 221 examined FG1 stage ovules from grdp2-3 plants showed no nucleus. (J) 53.8% of 179 examined FG7 stage ovules from grdp2-3 plants were normal. (K) 12.0% 179 examined FG7 stage ovules from grdp2-3 plants showed single nucleus. (L) 34.2% of 179 examined FG7 stage ovules from grdp2-3 plants showed no nucleus. (M) The phenotype analysis of FG1 stage ovules in WT and grdp2-3 plants. (N) The phenotype analysis of FG7 stage ovules in WT and grdp2-3 plants. CCN, central cell nuclei; EC, egg cell nuclei; FG, female gametophyte; white arrows mean nuclei; white dotted lines mean embryo sac.

FIGURE 4

Phenotype analysis of p35S:GRDP2-OE and pUBQ10:GRDP2-OE lines. (A) Expression levels of GRDP2 in WT, p35S:GRDP2-OE-20^#, p35S:GRDP2-OE-26^#, and p35S:GRDP2-OE-40^# as determined by real-time RT-PCR. (B) Seed development in three p35S:GRDP2-OE line plants. Bar = 1 cm. (C) Seed setting statistic of the WT, p35S:GRDP2-OE-20^#, p35S:GRDP2-OE-26^#, and p35S:GRDP2-OE-40^# lines. (D,E) 10.7% of 316 examined FG7 stage ovules from p35S:GRDP2-OE-26^# line plants showed single nucleus (D), 33.6% of 316 examined FG7 stage ovules from p35S:GRDP2-OE-26^# line plants showed no nucleus (E). (F,G) Alexander’s staining of pollen from WT (F) and p35S:GRDP2-OE-26^# (G) line plants. The red color indicates viable pollen. Bar = 50 μm. (H) Seed development in wild-type (WT) and three pUBQ10:GRDP2-OE line plants. Bar = 1 cm. (I) Expression levels of GRDP2 in WT, pUBQ10:GRDP2-OE-1^#, pUBQ10:GRDP2-OE-6^#, and pUBQ10:GRDP2-OE-10^# as determined by real-time RT-PCR. (J) Seed setting statistics of the WT, pUBQ10:GRDP2-OE-1^#, pUBQ10:GRDP2-OE-6^#, and pUBQ10:GRDP2-OE-10^# lines. (K,L) The FG7 stage ovules from pUBQ10:GRDP2-OE-26^# line plants. (M,N) Alexander’s staining of pollen from WT (M) and pUBQ10:GRDP2-OE-6^# (N) line plants. The red color indicates viable pollen. Bar = 50 μm. Vertical bars represent the mean ± SD as determined by two technical replicates of three biological replicates; asterisks show the significance level judged by Student’s t-test (^∗∗∗p < 0.01). White dotted lines mean embryo sac.

FIGURE 5

Callose deposition during megasporogenesis are defective in grdp2-3 mutant ovules. (A) Quantification of callose staining-positive (+) and callose staining-negative (–) ovules at stage 2IV in more than 100 ovules for each sample. (B) Quantitative profile of callose deposition at various stages of meiosis in more than 100 ovules for each sample. (C) Callose-stained wall deposition in WT ovules at stage 2IV. (D) Callose-stained wall deposition in grdp2-3 ovules at stage 2IV. (E) Abnormal callose staining is observed in grdp2-3 ovules during meiosis.

FIGURE 6

Expression of female gametophyte specific markers. (A,B) pSPL:GUS expression in (A) WT at stage 2-I ovules prior to entering meiosis (165/169) and (B) grdp2-3 stage 2-I ovules (138/142). (C,D) pKNU:KNU-VENUS expression in (C) WT at MMC stage ovules (151/156) and (D) grdp2-3 at MMC stage ovules (129/135). (E) pAKV:H2B-YFP expression in WT at FG6/FG7 stage ovules (163/189), the rest ovules are in the FG5 stage. (F–H) pAKV:H2B-YFP expression in (F) grdp2-3 at FG6/FG7 stage ovules (81/173) ovules are same as the WT; (G) 60/173 ovules have no nucleus; and (H) 32/173 ovule have one nucleus. (I) pMYB98:GFP expression in WT at FG7 stage ovules (133/138). (J,K) pMYB98:GFP expression in grdp2-3 at FG7 stage ovules (J) 69/144 ovules are same as the WT and (K) 75/144 ovules have no GFP signal. (L) pDD45:GFP expression in WT at FG7 stage ovules (103/107). (M,N) pDD45:GFP expression in grdp2-3 at FG7 stage ovules (M) 50/110 ovules are same as the WT and (N) 60/110 ovules have no GFP signal. (O) pDD65:GFP expression in WT at FG7 stage ovules (189/199). (P,Q) pDD65:GFP expression in grdp2-3 at FG7 stage ovules (P) 110/204 ovules are same as the WT and (Q) 96/204 ovules have no GFP signal. Bar = 25 μm. CCN, central cell nuclei; ECN, egg cell nuclei; SCN, synergid cell nuclei; white arrows mean nuclei; white dotted lines mean embryo sac.

FIGURE 7

Auxin distribution in developing ovules from WT and grdp2-3 siliques using DR5:GFP, pPIN1:PIN1-GFP, pPIN7:PIN7-GFP, and YUCCA5-GFP-GUS as an auxin reporter. (A–G) WT DR5:GFP ovules were analyzed at different developmental stages. (A) The cytoplasmatic GFP signal is first detected in the hypodermal L1 cell layer of the ovule primordium from stage 2-II, (E) until stage 3-II. (C) DR5:GFP signal is also detected in the pro-vascular cells of the funiculus from stage 2-V to (G) stage 3-VI. (A’–G’) grdp2-3 DR5:GFP ovules were analyzed at different developmental stages. (B’) The cytoplasmatic GFP signal is first detected in the hypodermal L1 cell layer of the ovule primordium from stage 2-III which is later than WT (A, stage 2-II), and (G’) last until the mature stage 3-VI. White arrows in (E’–G’) showed DR5:GFP signal in the pro-vascular cells of the funiculus is also later than WT. (H–M) WT pPIN1:PIN1-GFP ovules were analyzed at different developmental stages. (H) The cytoplasmatic GFP signal is first detected in the hypodermal L1 cell layer of the ovule primordium and the pro-vascular cells of the funiculus from stage 2-II, until (J) stage 2-V. (white arrows in K–M) GFP signal was only detected in the funiculus from stage 3-II to stage 3-IV. (H’–M’) grdp2-3 pPIN1:PIN1-GFP ovules were analyzed at different developmental stages. Except the hypodermal L1 cell layer of the ovule primordium and the pro-vascular cells of the funiculus, the GFP signal also detected in the nucellus at all developmental stages stage 2-II to stage 3-IV. (N–S) WT pPIN7:PIN7-GFP ovules were analyzed at different developmental stages. (N) The cytoplasmatic GFP signal is only detected in the outer integument of the ovule primordium from stage 2-III, (S) until stage 3-VI. (N’–S’) grdp2-3 pPIN7:PIN7-GFP ovules were analyzed at different developmental stages. (N’) The cytoplasmatic GFP signal can be detected not only in the outer integument of the ovule primordium but also the pro-vascular cells of the funiculus from 2-III, until (S’) stage 3-VI. (T) YUC5:GFP-GUS is expressed only in the chalaza at stage 3-VI. (T’) grdp2-3 YUC5:GFP-GUS ovules were analyzed at stage 3-VI, the GFP expression in the embryo sac which is a big difference with WT. In (A–T) and (A’–T’), cell membranes were stained with FMH 4-64 FX. Scale bars = 25 μm. (U) Relative mRNA levels of auxin-related genes by qRT-PCR. Data are means ± SD (n = 3 biological replicates; ***p < 0.01, Student’s t-test).

FIGURE 8

The expression profiles of GRDP1 in Arabidopsis. (A–E) pGRDP1:GFP expression pattern in different ovule development stages. The developmental stages of the ovules are indicated at the top right corner of each panel. Bars = 20 μm. (F–J) pGRDP1:GUS expression pattern in ovules. Bars = 20 μm. (K,L) pGRDP1:GUS expression pattern in pollen. Bars = 50 μm.

FIGURE 9

Phenotype analysis of pUBQ10:GRDP1-OE lines. (A) Expression levels of GRDP1 in WT, pUBQ10:GRDP1-OE-2^#, pUBQ10:GRDP1-OE-12^#, and pUBQ10:GRDP1-OE-23^# as determined by real-time RT-PCR. (B) Seed development in three pUBQ10:GRDP1-OE line plants. Bar = 0.5 cm. (C) Seed setting statistics of the WT, pUBQ10:GRDP1-OE-2^#, pUBQ10:GRDP1-OE-12^#, and pUBQ10:GRDP1-OE-23^# lines. (D) The FG7 stage ovules from WT plants. (E,F) The FG7 stage ovules from pUBQ10:GRDP1-OE-2^# plants, 24.1% of 196 examined FG7 stage ovules showed single nucleus, 30.6% of 196 examined FG7 stage ovules showed no nucleus. (G) The phenotype analysis of FG7 stage ovules in WT and pUBQ10:GRDP1-OE-2^# plants. Vertical bars represent the mean ± SD as determined by two technical replicates of three biological replicates; asterisks show the significance level judged by Student’s t-test (^∗∗∗p < 0.01). CC, central cell; EC, egg cell. White arrows mean nuclei; white dotted lines mean embryo sac.