Fine-Tuning of Eui1: Breaking the Bottleneck in Hybrid Rice Seed Production

Shaopei Gao and Chengcai Chu

Gibberellins (GAs) are essential phytohormones regulating many aspects of plant growth and development. Manipulation of endogenous GA status by genetic alteration or exogenous application of GA or GA biosynthesis inhibitors are common strategies to optimize plant growth and crop yields. Rice (Oryza sativa) is a major source of calories and mineral nutrients, feeding more than half of the human population. With the challenges of feeding an increasing population and decreasing availability of arable land, improving productivity and yields of rice crop has always been an ultimate goal for breeders. In the 1960s, semi-dwarf rice varieties were bred and spread rapidly, leading to record yield increase throughout Asia, which is so-called “green revolution”. Genetic basis for rice “green revolution” stems from a mutated GA20ox-2 gene (at the sd1 locus) encoding GA biosynthesis enzyme (Sasaki et al., 2002). In the 1970s, the successful application of hybrid rice revolutionized rice production by 20% increase. Hybrid rice is thus considered as one of the biggest contributors for yield gains in last 40 years.

GAs are also indispensible for hybrid rice seed production. Male sterility, in either three-line system or two-line system, is most important in the hybrid rice seed production. However, male sterile lines often have a common defect in the elongation of the uppermost internode, leading eventually to incomplete panicle exsertion, which blocks pollination (Figure 1). To this end, large amount of exogenous GAs have to be used, which not only increases the cost, but also increases the probability of seed germination on the panicle (pre-harvest sprouting), affecting the quality of hybrid seeds.

Figure 1. Hybrid rice seed production. (A) Four genetic elements of hybrid rice seed production: maintainer line, restorer line, cytoplasmic male sterility (CMS) line, and eui1 mutant. CMS line is maintained by crossing with the maintainer line. The hybrid seeds from the cross between CMS line and restorer line have hybrid vigor. Almost all CMS lines have a natural deficiency of panicle enclosure, which blocks pollination between CMS line and restorer line and decreases seed yield. The eui1 mutant for enhanced panicle exsertion is therefore regarded as the fourth genetic element of hybrid seed production. Bar, 10 cm. (B) Commercial hybrid rice seed production in field. CMS lines are regularly interspaced by the restorer lines for better pollination.

In 1981, a recessive rice mutant eui1 (elongated uppermost internode1) was characterized by the extremely elongated uppermost internode and excess panicle exsertion at the heading stage (Rutger and Carnahan, 1981). Because of its prospective application to amend panicle enclosure, eui1 locus has been incorporated into many male sterile lines, and this recessive trait, along with male sterile line, maintainer line and restorer line are referred to as four genetic elements for hybrid rice seed production (Chen et al., 2013; Rutger and Carnahan, 1981; Yang et al., 2005).  In 2006, Eui1 gene was isolated and characterized, and it encodes a cytochrome P450 monooxygenase that inactivates 13-H GAs by epoxidation (Luo et al., 2006; Zhu et al., 2006). In rice, there are two main bioactive GAs, GA₁ and GA₄. GA₁ is a predominantly bioactive GA form in vegetative tissues, while GA₄ is predominantly present in reproductive tissues, especially in anthers. Eui1 gene is highly expressed in anthers and spikelets, but it is relatively low in the uppermost internode (Magome et al., 2013). The deactivation of bioactive GA₄ in anthers is catalyzed by Eui1, which epoxidizes GAs and regulates the influx of GA₄ into the stems from panicles (Gao et al., 2016). When Eui1 loses its function, increased amounts of GA₄ flow into the stems, leading to the dramatic elongation of uppermost internode. However, panicle exsertion in eui1 mutants varies significantly among different genetic backgrounds and excess elongation of uppermost internode makes rice much more susceptible to lodging (Luo et al., 2006). Therefore, fully understanding the molecular mechanism through which Eui1 activity is regulated will provide a more flexible way to fine-tune panicle exsertion, which may greatly help breeders in hybrid rice seed production.

To maintain homeostasis of endogenous bioactive GAs, rice itself has also evolved the capacity to fine-tune the expression of Eui1. Previously studies suggest that Eui1 expression is positively regulated by transcription factors OsAP2-39 and HOX12, which directly bind to the promoter region of Eui1 (Gao et al., 2016; Yaish et al., 2010). In addition, OsDCL3a could produce transposable element-associated 24-nt siRNAs to suppress the Eui1 expression (Wei et al., 2014).

A recent study in this issue of Molecular Plant by Prof. Letian Chen’s group and their collaborators has brought us much closer to elucidating the regulatory network of Eui1 expression (Xie et al., 2018). In this study, Xie et al. discovered a highly informative rice mutant, dwarf Eui1 (dEui1). At the heading stage, dEui1 plant exhibits a severe enclosed-panicle phenotype due to the shorter uppermost internode. The phenotypic defects in dEui1 are caused by elevated expression of Eui1 resulting from T-DNA replacement of the 135-bp fragment in the Eui1 intron. Complementary experiment indicated that loss of the 8-bp RY-containing cis-element (5’-CATGCATG-3’, herein named SE1) in the intron of Eui1 greatly increased Eui1 expression. RY motif (core sequence 5’-CATGCA-3’) could serve as a binding site of B3-domain proteins (Reidt et al., 2000). Interestingly, SE1 recruits the repressor complex, which consists of two B3 repressors (OsVAL2 and OsGD1), a co-repressor (OsSAP18), and a histone deacetylase (OsHDA710). This complex maintains a low level of histone acetylation in Eui1 chromatin, especially in the intron, and a low level of Eui1 expression, thus leading to normal growth of plants. In the mutants that lack the intronic SE1, the repressor complex fails to bind the chromatin of Eui1 and thus does not erase the acetylation from histones in the intronic region. As a result, the histone acetylation level remains high, resulting in elevated expression of Eui1 and eventually a dwarf phenotype.

By far, few silencing elements have been identified in the gene introns of monocotyledonous plants, the mechanisms by which intronic silencing element function remain largely elusive. This work provides very solid evidences for the biological function of intronic silencing element by recruiting repressor complexes for histone modification and chromatin remodeling at Eui1 locus, revealing another layer of regulation in Eui1. Epialleles with certain number of SE1 elements in the intron by precise genome editing may reduce basal level of Eui1 expression to certain levels and enhance panicle exsertion at will in male sterile lines. Therefore, this finding not only provides novel molecular insights into the function of non-coding DNAs, but also greatly facilitates the optimization of panicle exsertion through fine-tuning of Eui1 expression, which ultimately breaks the bottleneck in hybrid rice seed production.

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Gao S and Chu C (2018) Fine-tuning of Eui1: Breaking the bottleneck in hybrid rice seed production. Molecular Plant  Doi: 10.1016/j.molp.2018.04.001.