OsNAC129 Regulates Seed Development and Plant Growth and Participates in the Brassinosteroid Signaling Pathway

Abstract

Grain size and the endosperm starch content determine grain yield and quality in rice. Although these yield components have been intensively studied, their regulatory mechanisms are still largely unknown. In this study, we show that loss-of-function of OsNAC129, a member of the NAC transcription factor gene family that has its highest expression in the immature seed, greatly increased grain length, grain weight, apparent amylose content (AAC), and plant height. Overexpression of OsNAC129 had the opposite effect, significantly decreasing grain width, grain weight, AAC, and plant height. Cytological observation of the outer epidermal cells of the lemma using a scanning electron microscope (SEM) revealed that increased grain length in the osnac129 mutant was due to increased cell length compared with wild-type (WT) plants. The expression of OsPGL1 and OsPGL2, two positive grain-size regulators that control cell elongation, was consistently upregulated in osnac129 mutant plants but downregulated in OsNAC129 overexpression plants. Furthermore, we also found that several starch synthase-encoding genes, including OsGBSSI, were upregulated in the osnac129 mutant and downregulated in the overexpression plants compared with WT plants, implying a negative regulatory role for OsNAC129 both in grain size and starch biosynthesis. Additionally, we found that the expression of OsNAC129 was induced exclusively by abscisic acid (ABA) in seedlings, but OsNAC129-overexpressing plants displayed reduced sensitivity to exogenous brassinolide (BR). Therefore, the results of our study demonstrate that OsNAC129 negatively regulates seed development and plant growth, and further suggest that OsNAC129 participates in the BR signaling pathway.

Keywords: NAC transcription factor; abscisic acid; brassinosteroids; grain size; starch biosynthesis.

Figure 1

OsNAC129 is highly expressed in immature seeds. (A) Expression profiles of OsNAC129 in “Nipponbare.” Total RNA was extracted from roots and shoots at 7 days after germination, from leaves, leaf sheathes, stems, and young panicles before flowering, and from developing seeds from 1 to 25 days after fertilization (DAF). Actin1 was used as the internal control for normalization of gene expression. Data are means ± SD of four biological replicates. (B) OsNAC129 promoter-GUS expression patterns in transgenic plants. GUS expression in root (a) and shoot (b) at 7 days after germination, flag leaf (c), leaf sheath (d), stem (e), and cross section of stem (f) at heading date, younger panicle (g), older panicle (h), flowering spikelet (i), and endosperm at 3 DAF (j), 5 DAF (k), 7 DAF (l), 10 DAF (m), 15 DAF (n), and 25 DAF (o). Scale bars (a–e, g, and h) = 5 mm; scale bar (f) = 1 mm; scale bars (i–o) = 2.5 mm.

Figure 2

The osnac129 mutant has increased grain size and apparent amylose content. (A) Phenotypic observation of osnac129 grain at maturity. Scale bar = 10 mm. (B–E) Show the grain length, grain width, grain thickness, and 1,000-grain weight (TGW) of mature seeds, respectively. Data are means ± SD of three replicates. (F) Brown rice grains and cross sections of grains (upper panel, scale bars = 2.5 mm), and electron micrographs of starch granules (lower panel, scale bars = 10 μm) from the wild-type (WT) and osnac129 mutant. Blue boxes in the upper panels indicate the central area of the mature endosperm, where starch granules were analyzed by scanning electron microscope (SEM), and areas in the bottom panels delineated by dotted lines indicate starch grains. (G,H) Show the apparent amylose content (AAC) and total starch content (TSC) of grains from the WT and osnac129 mutant, respectively. Data are means ± SD of five replicates. ^**p < 0.01, ^***p < 0.001 as determined by Student’s t-test.

Figure 3

Overexpression of OsNAC129 leads to reductions in grain size and apparent amylose content. (A) Phenotypic observation of WT and OE plants mature seeds. Scale bar = 10 mm. (B–D) Show grain length, grain width, and TGW of mature seeds of NIP and two OsNAC129-OE lines, respectively. Data are means ± SD of three replicates. (E,F) Show the AAC and TSC of NIP and the two OsNAC129-OE lines, respectively. Data are means ± SD of five replicates. ^**p < 0.01, ^***p < 0.001 as determined by Student’s t-test.

Figure 4

OsNAC129 simultaneously regulates cell elongation and starch biosynthesis in rice seeds. (A) Observation of the outer epidermal cells of WT and osnac129 mutant lemmas by SEM. Scale bars = 100 μm. (B–D) Show the cell length, cell width, and cell number measurement of the outer epidermal cells of the lemma. Data are means ± SD of three replicates (each replicate consisted of at least 30 cells) in (B,C), and four replicates in (D); ^**p < 0.01, ^***p < 0.001 as determined by Student’s t-test. (E,F) Show the relative expression determined by qRT-PCR of genes reported to be related to grain size by controlling cell elongation in seeds from the WT, osnac129 mutant, and OE plants at 7 DAF. UBQ10 was used as the internal control for normalization of gene expression. Data are means ± SD of four replicates. (G,H) Show qRT-PCR determination of expression of starch synthase-encoding genes in seeds of the WT, osnac129, and OE plants at 7 DAF. UBQ10 was used as the internal control. Data are means ± SD of four replicates; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 as determined by Student’s t-test.

Figure 5

OsNAC129 is an abscisic acid (ABA)-inducible and brassinolide (BR)-related gene. (A) OsNAC129 was exclusively induced by ABA. About 1-week-old NIP seedlings were treated separately with IAA, 6-BA, GA₃, BL, and ABA (50 μM of each phytohormone), and sterile water treatment was the control. Samples were collected, RNA was extracted, and the expression of OsNAC129 was determined by qRT-PCR. Data are means ± SD of four replicates. ND, not detected. (B) About 2-week-old seedlings of WT and the OsNAC129-OE-1 line were treated with three concentrations of BL for 24 h, after which the leaf angles were imaged. Scale bar = 5 cm. (C) Leaf angles of WT and OE-1 seedlings measured by Image J after BL treatment. Data are means ± SD of 10 replicates. ^***p < 0.001 as determined by Student’s t-test.