MADS78 and MADS79 Are Essential Regulators of Early Seed Development in Rice

Abstract

MADS box transcription factors (TFs) are subdivided into type I and II based on phylogenetic analysis. The type II TFs regulate floral organ identity and flowering time, but type I TFs are relatively less characterized. Here, we report the functional characterization of two type I MADS box TFs in rice (Oryza sativa), MADS78 and MADS79 Transcript abundance of both these genes in developing seed peaked at 48 h after fertilization and was suppressed by 96 h after fertilization, corresponding to syncytial and cellularized stages of endosperm development, respectively. Seeds overexpressing MADS78 and MADS 79 exhibited delayed endosperm cellularization, while CRISPR-Cas9-mediated single knockout mutants showed precocious endosperm cellularization. MADS78 and MADS 79 were indispensable for seed development, as a double knockout mutant failed to make viable seeds. Both MADS78 and 79 interacted with MADS89, another type I MADS box, which enhances nuclear localization. The expression analysis of Fie1, a rice FERTILIZATION-INDEPENDENT SEED-POLYCOMB REPRESSOR COMPLEX2 component, in MADS78 and 79 mutants and vice versa established an antithetical relation, suggesting that Fie1 could be involved in negative regulation of MADS78 and MADS 79 Misregulation of MADS78 and MADS 79 perturbed auxin homeostasis and carbon metabolism, as evident by misregulation of genes involved in auxin transport and signaling as well as starch biosynthesis genes causing structural abnormalities in starch granules at maturity. Collectively, we show that MADS78 and MADS 79 are essential regulators of early seed developmental transition and impact both seed size and quality in rice.

Figure 1.

Expression analysis of MADS78 and MADS79. A and B, RT-qPCR analysis of MADS78 and MADS79 in mature pollen and unfertilized ovary (A) and early seed development (48, 72, and 96 HAF; B) relative to leaf tissue from wild-type plants. For statistical analysis, Student’s t test was used, and different lowercase letters indicate significant differences (P < 0.05). Error bars indicate sd (n = 6; two biological and three technical replicates). C, In situ hybridization of MADS78 and MADS79 in mature anthers, unfertilized ovary, and seeds at 48 and 72 HAF. aw, Anther wall; cv, central vacuole; pg, pollen grain. Bars = 0.2 mm.

Figure 2.

Both MADS78 and MADS79 heterodimerize with MADS87 and MADS89. BiFC assay shows positive interaction of MADS78 (A) and MADS79 (B) with MADS87 and MADS89. For a control, we cotransformed both constructs with an unrelated gene (bZIP76). Bars = 25 µm.

Figure 3.

MADS89 enhances the specificity of nuclear localization for MADS78 and MADS79. In the absence of MADS89, N-terminal GFP fusion constructs of MADS78 and MADS79 localize to nucleus and cytosol. Cotransformation of the respective constructs with MADS89 enhances their localization specifically to the nucleus. Empty vector was used as a control. Bar = 25 µm.

Figure 4.

Overexpression of MADS78 and MADS79 leads to partial spikelet sterility and delayed endosperm cellularization. A, RT-qPCR analysis of MADS78 and MADS79 expression in 11-d-old seedlings and developing seeds (48 and 72 HAF) in the wild-type (WT) and OE mutants for the respective tissue. Wild-type expression was used as a baseline for the corresponding tissue and gene. B, Representative mature panicle images of the OE mutants compared with the wild type. White arrows indicate sterile seeds. Images shown were digitally extracted and scaled for comparison. Bar = 2 cm. C, Quantification of spikelet fertility (%) in the wild type and OE mutants; n = 25 to 40 plants. D, Representative cross sections of developing seeds at 96 HAF from the wild type and OE mutants. cv, Central vacuole; ed, endosperm. Bar = 0.2 µm. For statistical analysis in A and C, Tukey’s test was used: ***P < 0.001 and **P < 0.01. Error bars indicate sd (n = 6; two biological and three technical replicates).

Figure 5.

Single knockout mutants of MADS78 and MADS79 show precocious cellularization. Representative cross sections of developing seeds are shown at 72, 84, and 96 HAF in single knockout mutants of MADS78 and MADS79 compared with the wild type (WT). The red arrowhead points to the central vacuole region (cv). ed, Endosperm. Bar = 0.2 µm.

Figure 6.

Double knockout mutation is lethal. A, Representative mature panicle images of T0 homozygous double knockout mutants compared with the wild type (WT). mads78-79_5 and mads78-79_9 are derived from sgRNA-1, while mads78-79_3 and mads78-79_4 correspond to sgRNA-2 (Supplemental Fig. S11). Bar = 2 cm. B, Representative mature seed images of the T0 homozygous double knockout mutants with husk. Bar = 1 cm. Images shown in A and B were digitally extracted and scaled for comparison.

Figure 7.

FIS-PRC2 potentially regulates MADS78 and MADS79. A, RT-qPCR analysis of MADS78, MADS79, and Fie1 at 48 and 72 HAF in the wild type (WT). Expression levels at 48 HAF were considered as baseline for each gene. B, RT-qPCR analysis of Fie1 in OE and single knockout mutants for MADS78 and MADS79 at 72 and 48 HAF, respectively. C and D, RT-qPCR analysis of MADS78 and MADS79 in OE and single knockout mutants for Fie1 at 48 and 72 HAF, respectively. For B to D, wild type expression was used as a baseline. For statistical analysis, the Holm-Sidak method was used: ***, P < 0.001 and **, P < 0.01. Error bars indicate sd (n = 6; two biological and three technical replicates).

Figure 8.

Misregulation of MADS78 and/or MADS79 disturbs auxin homeostasis. A, Heat map based on normalized read counts for genes involved in auxin homeostasis in the wild type (WT) and single knockout mutants (mads78 and mads79) in developing seeds (72 HAF). The color key represents a range of normalized values. B, MADS78 and MADS79, auxin, and FIS-PRC2 collectively regulate the timely initiation of endosperm cellularization. MADS78 and MADS79 regulate some of the auxin biosynthesis (YUCCAs), signaling (IAAs and GH3s), and transport (ABCB-type transporters) related genes in developing endosperm. In normally developing syncytial stage seeds, auxin is exported from developing endosperm to the seed coat and blocks FIS-PRC2, thus ensuring normal endosperm proliferation and seed coat cell elongation. Seeds deficient in MADS78 and MADS79 have reduced auxin export, thus releasing auxin-mediated FIS-PRC2 suppression. The activated FIS-PRC2 blocks cell elongation and differentiation, which causes cellularization. The accelerated cellularization triggers the activation of auxin biosynthesis genes YUCCA12 and TAR1 that may promote endoreduplication, differentiation, and starch deposition during later stages of seed development. ? indicates additional, uncharacterized factors that might participate in this model.

Figure 9.

Misregulation of MADS78 and MADS79 causes impairment of starch granules. A, Representative images of mature seeds from MADS78 and MADS79 mutants (left column; bar = 1 cm) and their cross sections (right column; bar = 20 µm) using SEM. Images shown were digitally extracted and scaled for comparison. B, RT-qPCR analysis of selected genes involved in carbon metabolism at 72 HAF in OE and knockout mutants relative to the wild type (WT). The genes were selected based on RNA-seq analysis. For statistical analysis, Student’s t test was used: ***, P < 0.001; **, P < 0.01; and *, P < 0.05. Error bars indicate sd (n = 6; two biological and three technical replicates).