Regulation of expression of starch synthesis genes by ethylene and ABA in relation to the development of rice inferior and superior spikelets

Abstract

Later-flowering spikelets in a rice panicle, referred to as the inferior spikelets, are usually poorly filled and often limit the yield potential of some rice cultivars. The physiological and molecular mechanism for such poor grain filling remains unclear. In this study the differentially expressed genes in starch synthesis and hormone signalling between inferior and superior spikelets were comprehensively analysed and their relationships with grain filling was investigated. DNA microarray and real-time PCR analysis revealed that a group of starch metabolism-related genes showed enhanced expression profiles and had higher transcript levels in superior spikelets than in inferior ones at the early and middle grain-filling stages. Expression of the abscisic acid (ABA) synthesis genes, 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED5, and the ethylene synthesis genes, 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO1) and ACO3, declined with development of the caryopses. Meanwhile, if compared with inferior spikelets, expression of these genes in superior spikelets decreased faster and had lower transcript profiles, especially for ethylene. ABA concentration and ethylene evolution rate showed similar trends to their gene expression. Exogenous supply of ABA reduced the sucrose synthase (SUS) mRNA level and its enzyme activity in detached rice grains, while exogenously supplied ethephon (an ethylene-releasing reagent) suppressed the expression of most starch synthesis genes; that is, SUS, ADP-glucose pyrophosphorylase (AGPase), and soluble starch synthase (SSS), and down-regulated their enzyme activities. In summary, it is concluded that the relatively high concentrations of ethylene and ABA in inferior spikelets suppress the expression of starch synthesis genes and their enzyme activities and consequently lead to a low grain-filling rate.

Fig. 1.

Grain weight (A), grain filling rate (B), starch accumulation (C), and soluble carbohydrate content (D) in rice spikelets. The indica cultivar YD-6 was field grown. Superior spikelets are those which flowered on the first 2 d within a panicle and inferior spikelets are those which flowered on the last 2 d within a panicle. Vertical bars represent the SEM (n=3).

Fig. 2.

Hierarchical clustering of starch metabolism-related genes of rice during grain filling. Horizontal rows represent individual genes and vertical rows represent individual samples. I3, inferior spikelets at 3 DPA; S3, superior spikelets at 3 DPA; I9, inferior spikelets at 9 DPA; S9, superior spikelets at 9 DPA. Red and blue indicate the transcript level above and below the median, respectively, for that gene across all samples. The clustering is performed on the log₂-transformed expression values.

Fig. 3.

Expression of genes related to starch metabolism in developing caryopses determined by RT-PCR analysis. RNA was extracted from superior and inferior spikelets at 1, 3, 6, 9, 15, and 21 DPA and then reverse transcribed for RT-PCR analysis. The PCR primers are shown in Supplementary Table S5 at JXB online.

Fig. 4.

A pathway description of the differentially expressed starch metabolism-related genes and their ratios between superior and inferior spikelets at 9 DPA. The ratio means the fold difference in the respective gene expression of superior to inferior spikelets, which was calculated according to microarray data, as described in Supplementary Table S3 at JXB online.

Fig. 5.

Changes in the expression profiles of ABA synthesis genes NCED1 and NCED5 and the ethylene synthesis genes ACO1 and ACO3 in superior and inferior spikelets during grain filling of rice. Transcript levels were quantified by qRT-PCR as described in the Materials and methods. Values are means with the SD (n=3).

Fig. 6.

Levels of ABA content and ethylene evolution rate in the superior and inferior spikelets during grain filling of rice. ABA content and the ethylene evolution rate were assayed as described in the Materials and methods. Values are means with the SD (n=3).

Fig. 7.

Effects of applied ABA, fluridone, ethephon, and cobaltous nitrate on the expression profiles of starch metabolism-related genes in the grains of detached rice ears. Rice spikelets were detached at 9 DPA and then cultured in solutions with 20 μM ABA, 20 μM fluridone, 5 mM ethephon, or 50 μM cobaltous nitrate. RNAs were extracted from the grains on the top four branches. Values are means with the SD (n=3).

Fig. 8.

Effects of applied ABA, fluridone, ethephon, and cobaltous nitrate on the activities of SUS, AGPase, SSS, and GBSS in the grains of detached rice ears. Rice spikelets were detached at 9 DPA and then cultured in solutions with 20 μM ABA, 20 μM fluridone, 5 mM ethephon, or 50 μM cobaltous nitrate. The grains on the top four branches were sampled for the present study. Values are means with the SD (n=3).