Functional delineation of rice MADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis

Abstract

Rice MADS29 has recently been reported to cause programmed cell death of maternal tissues, the nucellus, and the nucellar projection during early stages of seed development. However, analyses involving OsMADS29 protein expression domains and characterization of OsMADS29 gain-of-function and knockdown phenotypes revealed novel aspects of its function in maintaining hormone homeostasis, which may have a role in the development of embryo and plastid differentiation and starch filling in endosperm cells. The MADS29 transcripts accumulated to high levels soon after fertilization; however, protein accumulation was found to be delayed by at least 4 days. Immunolocalization studies revealed that the protein accumulated initially in the dorsal-vascular trace and the outer layers of endosperm, and subsequently in the embryo and aleurone and subaleurone layers of the endosperm. Ectopic expression of MADS29 resulted in a severely dwarfed phenotype, exhibiting elevated levels of cytokinin, thereby suggesting that cytokinin biosynthesis pathway could be one of the major targets of OsMADS29. Overexpression of OsMADS29 in heterologous BY2 cells was found to mimic the effects of exogenous application of cytokinins that causes differentiation of proplastids to starch-containing amyloplasts and activation of genes involved in the starch biosynthesis pathway. Suppression of MADS29 expression by RNAi severely affected seed set. The surviving seeds were smaller in size, with developmental abnormalities in the embryo and reduced size of endosperm cells, which also contained loosely packed starch granules. Microarray analysis of overexpression and knockdown lines exhibited altered expression of genes involved in plastid biogenesis, starch biosynthesis, cytokinin signalling and biosynthesis.

Keywords: Cytokinin; MADS box; Oryza sativa; embryo; endosperm; seed development.; starch.

Fig. 1.

Temporal and spatial expression of MADS29 across developmental stages of rice. (A) Relative transcript levels of MADS29 using real-time PCR; bars indicate standard error (n = 3). (B) Microarray-based expression profile of MADS29; bar indicates log₂ expression values. (C) Western blot analysis of MADS29 using MADS29 antibody across rice developmental stages. ML, mature leaf; MR, mature root; P1–P6, panicle stages; S1–S5, seed stages; SDL, seedling. (D) Immunolocalization of MADS29 expression in the developing embryo at 4, 6, 8, 14, and 27 days after pollination (DAP); bars: 100 µm (4, 6, and 8 DAP), 400 µm (14 and 27 DAP). (E) Immunolocalization of MADS29 expression in the developing endosperm at 1, 4, 6, 8, 14, and 27 DAP. The nucellar region and the aleurone region at 8 DAP are shown separately: bars: 100 µm (4, 6, and 8 DAP), 500 µm (14 and 27 DAP). Al, aleurone; Col, coleoptile; Dv, dorsal vascular bundle; Em, embryo; En, endosperm; Epi, epiblast; Esr, embryo-surrounding region; Ne, nucellar epidermis; Np, nucellar projection; Nu, nucellus; Rad, radicle; Sal, subaleurone; Scu, scutellum; Sh, shoot.

Fig. 2.

Characterization of the MADS29 overexpression (MADS29^OX) phenotype in transgenic rice. (A) Relative transcript abundance of MADS29 in MADS29^OX lines; bars indicate standard error (n = 3). (B) Morphology of MADS29^OX lines; inset shows an emerging panicle; bar: 5cm. (C) Floral organs of a single MADS29^OX floret; bar: 1mm. (D) Comparison of intercalary meristematic zone in wild-type and MADS29^OX leaf; bar: 100 µm. (E) Hormone (auxin/cytokinin) profile in leaves of wild-type and MADS29^OX plants; tZ, trans-zeatin; IAA, indole acetic acid.

Fig. 3.

OsMADS29 expression in BY2 cells. (A) BY2:MADS29^OX stable transgenic cells showing development of multiple organelles under DIC mode; bar: 50 µm. (B) I₂KI staining of BY2 cells to detect amyloplasts: AM, amyloplast; NU, nucleus; bars, 50 µm. (C) qPCR analysis to determine MADS29 and AgpS transcript levels in wild-type BY2 and transgenic BY2:MADS29^OX lines; bars indicate standard error (n = 3) (this figure is available in colour at JXB online).

Fig. 4.

RNAi-mediated OsMADS29 knockdown phenotype. (A) Comparison of wild-type and MADS29^KD vegetative development; bar: 7cm. (B) OsMADS29 transcript accumulation in wild-type and MADS29^KD lines as determined by qPCR; bars indicate standard error (n = 3). (C) Comparison of dry seed weight between wild-type and MADS29^KD lines; bars indicate standard error (n = 45). (D) Left panel: representative mature seeds from wild-type and three MADS29^KD lines, bar: 1mm; right panel: comparison of grain length between wild-type and MADS29^KD lines where MADS29 silencing results in decreased length of mature grain; bars indicate standard error (n = 20). (E) Scanning electron micrographs of half-split endosperms from wild-type and MADS29^KD plants showing organization of cells and sizes and structures of starch granules. Starch granules in representative cells have been pseudocoloured purple using Adobe Photoshop; bar: 10 µm. (F) Toluidine-blue-stained longitudinal sections of embryos from wild-type and MADS29^KD lines showing reduction in size and developmental abnormalities in the MADS29^KD lines; bar, 200 µm.

Fig. 5.

Microarray-based comparison of MADS29^OX and WT leaf transcriptomes. (A) Total number of 2-fold upregulated genes (red) and downregulated genes (green) in MADS29^OX leaf samples in comparison to the WT (P ≤ 0.05, n = 3). (B) Functional categorization of up- and downregulated genes into biological process GO classes in MADS29^OX leaves; values on the pie chart and in parentheses are percentage and number of genes represented in each category; categories with <1% share of the differentially expressed genes are cumulatively represented in the ‘Others’ category. (C) Proportion of differentially expressed genes included in two major ‘cellular component’ GO categories, ‘membrane’ and ‘plastids’; values in parentheses show the number of genes; red and green arrows depict up and downregulation, respectively. (D) Real-time quantitative PCR validation of selected genes from the highlighted pathways; transcript profiles of low- and high-abundance transcripts are shown in upper and lower panels, respectively; bars indicate standard error (n = 3) (this figure is available in colour at JXB online).

Fig. 6.

Comparison of MADS29^KD and wild-type S3-stage seed transcriptomes. (A) Total number of 2-fold upregulated genes (red) and downregulated genes (green) in MADS29^KD seed samples in comparison to the WT (P ≤ 0.05, n = 3). (B) Differentially expressed genes showing inverse correlation between the MADS29^OX lines (large arrows) and the MADS29^KD lines (inset arrows). (C) Functional categorization of up- and downregulated genes into biological process GO classes in MADS29^KD seeds; values on the pie chart and in parentheses are percentage and number of genes represented in each category; categories with <1% share of the differentially expressed genes are not shown individually but are included in the ‘Others’ category. (D) Proportion of differentially expressed genes included in two major ‘cellular component’ GO categories, ‘membrane’ and ‘plastids’; values in parentheses show the number of genes; red and green arrows depict up- and downregulation, respectively. (E) Real-time quantitative PCR validation of microarray data for selected genes from the highlighted pathways; bars indicate standard error (n = 3) (this figure is available in colour at JXB online).

Fig. 7.

A hypothetical model representing MADS29 function in seed development. An earlier report by Yin and Xue (2012) described MADS29’s role in programmed cell death of maternal tissues, the nucellus, and the nucellar projection during early stages of seed development (blue rectangle). The current data suggest that MADS29 expression affects cytokinin levels in homologous (rice) as well as heterologous (tobacco BY-2 cell line) conditions during post-5-DAP seed development (peach rectangle). Transcriptome analyses of MADS29^OX and MADS29^KD plants followed by quantitative PCR-based validation of candidate genes point towards MADS29’s involvement in plastid biogenesis and starch metabolism, either directly or indirectly via the cytokinin pathway. OsMADS29-mediated alterations in auxin and cytokinin homeostasis may also affect cell division and differentiation in the embryo (this figure is available in colour at JXB online).