Leaf-derived ABA regulates rice seed development via a transporter-mediated and temperature-sensitive mechanism

Abstract

Long-distance transport of the phytohormone abscisic acid (ABA) has been studied for ~50 years, yet its mechanistic basis and biological significance remain very poorly understood. Here, we show that leaf-derived ABA controls rice seed development in a temperature-dependent manner and is regulated by defective grain-filling 1 (DG1), a multidrug and toxic compound extrusion transporter that effluxes ABA at nodes and rachilla. Specifically, ABA is biosynthesized in both WT and dg1 leaves, but only WT caryopses accumulate leaf-derived ABA. Our demonstration that leaf-derived ABA activates starch synthesis genes explains the incompletely filled and floury seed phenotypes in dg1 Both the DG1-mediated long-distance ABA transport efficiency and grain-filling phenotypes are temperature sensitive. Moreover, we extended these mechanistic insights to other cereals by observing similar grain-filling defects in a maize DG1 ortholog mutant. Our study demonstrates that rice uses a leaf-to-caryopsis ABA transport-based mechanism to ensure normal seed development in response to variable temperatures.

Fig. 1. DG1 encoding a MATE transporter regulates seed development.

(A) Comparison of WT and the dg1 mutant seeds showing various degrees of grain-filling defects. Scale bars, 1 cm. (B) Transverse sections of representative fully filled mature seeds. Scale bar, 1 mm. (C and D) The starch granules observed via scanning electron microscopy in magnified regions of the WT and dg1 black squares in (B), respectively. Scale bars, 10 μm. (E) One thousand–grain weight comparison of filled seeds [including incompletely filled seeds (IFS)] between WT and dg1. Data are presented as means ± SD (n = 5; **P < 0.01, Student’s t test). (F) Diagram of the DG1 gene and the mutation causing the dg1 phenotype. The positions of ko-1 and ko-2 represent the two knockout sites targeted using a CRISPR-Cas9 approach. (G and H) Phenotypic comparison of mature seeds (G) and transverse sections of fully filled seeds (H) from WT Nipponbare (Nip) and the two DG1 knockout lines. Scale bars, 1 cm (G) and 1 mm (H). (I) One thousand–grain weight comparison of filled seeds (including IFS) from two knockout lines. Data are means ± SD (n = 5; **P < 0.01, Student’s t test). FFS, fully filled seeds; Photo credit: Binhua Hu.

Fig. 2. DG1 functions as an ABA efflux transporter.

(A) ABA efflux activity analysis using Xenopus oocytes. The complementary RNA (cRNA) for DG1 or for the known ABA transporter AtDTX50 (positive control), or water as a negative control, were injected into oocyte cells, which were then injected with ABA. The ABA concentration in the incubation buffer was measured before injection and after 10, 20, and 30 min of incubation. Data are means ±SD (n = 3). (B) ABA efflux transport activity analysis using ³H-ABA and protoplasts of WT (Nip), a knockout line (ko-1), and an overexpression line: DG1-OE (35S::DG1^cDNA-eGFP). The radioactivity in the protoplast incubation buffer was measured before loading and after 10, 20, and 30 min of incubation (presented as the proportional increases in the ³H-ABA signal for the three incubation intervals). Data are means ± SD (n = 3; **P < 0.01, Student’s t test). (C to E) Plasma membrane localization of a DG1-EGFP fusion protein (C) in the root tip of the overexpression line transgenic plant. (F to H) The root tip of WT Nipponbare was used as the negative control. FM4-64 dye (red) stains plasma membranes. Scale bar, 20 μm. Photo credit: Guohua Zhang.

Fig. 3. Long-distance leaf-to-caryopsis ABA transport is regulated by DG1.

(A to C) Determination of ABA content in caryopsis (A) at 3 and 5 DAP and stem (B) and flag leaf (C) at 0, 3, and 5 DAP. Data are means ± SD (n = 5; **P < 0.01, Student’s t test). FW, fresh weight. (D and E) Determination of ABA precursor content between WT and dg1 and the content of 9′-cis-neoxanthin (D) and 9-cis-violaxanthin (E) in caryopsis at 3 and 5 DAP and stem and flag leaf at 0, 3, and 5 DAP. Data are means ± SD (n = 5). (F and G) ³H-ABA feeding of WT, dg1, and complementation line leaves. Radioactivity in caryopses, stems, and leaves were measured before feeding, as well as at 12 and 24 hours after feeding (F). The increased radioactivity in each tissue were calculated for the 0- to 12-hour and 12- to 24-hour periods; these data were used to determine the distribution of increased radioactivity in each of the tissues and to analyze the ABA transport efficiency from leaf to stem and from stem to caryopsis for WT and dg1 plants. Data are means ± SD (n = 3; **P < 0.01, Student’s t test for WT and dg1). Schematic diagrams for ³H-ABA feeding of leaves (G).

Fig. 4. Expression pattern analysis of DG1 using immunofluorescence.

(A to O) Expression pattern of DG1 in node, leaf, and rachilla. Node I (A to G), flag leaf (H to K), or rachilla (L to O) at 3 DAP of representative proDG1:GUS lines were used for immunofluorescence. (E to G) Respectively represent magnified regions of white squares in (B to D). The arrow in (E) indicates the parenchyma cells positioned between the DVBs and EVBs of the node; the arrows in (I) and (M) indicate the phloem of leaf vascular bundles and the central vascular bundle of rachilla, respectively. The red dash lines in (A), (H), and (L) indicate the transverse section position for immunofluorescence in node I, flag leaf, and rachilla, respectively. Scale bars, 1 cm (A, H, and L), 200 μm (B to G), and 100 μm (I to K and M to O). Photo credit: Guohua Zhang and Peng Qin.

Fig. 5. DG1-mediated long-distance ABA transport and seed development are sensitive to temperature.

(A) Two growth temperature courses for WT, dg1, and complementation line plants initially grown in the field before being moved into a temperature-controlled greenhouse before flowering. (B) Phenotypic comparison in the extent of grain-filling at average ~24°C/day versus ~30°C/day, dg1 plants exhibited temperature-dependent incompletely filled seeds. Photo credit: Guohua Zhang. (C) DG1 expression in caryopses of WT plants grew at ~24°C/day temperature condition in (A) was significantly increased at 35°C for 2 hours of treatment. Data are means ± SD (n = 4; **P < 0.01, Student’s t test). Scale bars, 10 mm. (D and E) ³H-ABA feeding of WT, dg1, and complementation line stems at 24° and 30°C. Radioactivity in caryopses and stems was assessed before feeding, as well as at 1, 2, and 4 hours after feeding of panicles at the two temperature conditions (D). The increased radioactivity in stems and caryopses at 0- to 1-hour, 1- to 2-hour, and 2- to 4-hour intervals were then calculated to facilitate quantification of long-distance ABA transport efficiency from stem-to-caryopsis at different temperatures. Data are means ± SD (n = 3; different letters indicate significant differences; P < 0.05, Duncan multiple range test for each interval). Schematic diagram illustrating the experiments for ³H-ABA feeding from stems (E).

Fig. 6. ABA induces expression of starch synthesis–related genes, and disruption of the DG1 ortholog mutant in maize causes grain-filling phenotypes.

(A to J) Exogenous application of ABA to WT and dg1 stems induces expression of starch synthesis–related transcription factors (A to E) and enzymes (F to J) in WT caryopses but not in dg1 caryopses. 0 h, before treatment; W–2 h, after 2 hours of water treatment; ABA–2 h, after 2 hours of ABA treatment. Data are means ± SD (n = 5; **P < 0.01, Student’s t test). (K) Gene model of maize DG1 ortholog (ZmDG1) showing the site of Mu insertion (Zmdg1). (L to N) Comparison of grain-filling phenotypes between WT and Zmdg1 mutant that were pollinated on the same day, highlighting the obviously slower grain-filling rate and incompletely filled kernels [red arrows in (M)] in the mutant. (M) and (N) respectively represent the magnified regions of WT and the Zmdg1 mutant in (L). Scale bars, 3 cm (L) and 1 cm (M and N). Photo credit: Guohua Zhang.

Fig. 7. A model illustrating the function of leaf-derived ABA in regulating seed development in rice.

A model of long-distance ABA transport: When DG1 facilities leaf-synthesized ABA loading into leaf phloem and then ABA arrives at the EVB in nodes along with transpiration flow, ABA is unloaded from the EVB into parenchyma cell (PC) by as-yet-unidentified transporters; this PC ABA is then exported into the DVB and stem by DG1 and other minor ABA transporters. The ABA arrives at the basal region (rachilla) of the caryopsis along with transpiration flow, after which DG1 unloads ABA from the rachilla into the caryopsis. The efficiency of leaf-to-caryopsis long-distance ABA transport increases with rising temperature. After arriving in caryopses, leaf-derived ABA directly actives the expression of genes essential for starch synthesis, thus regulating seed development.