A new angle for crop improvement? The RING E3 ubiquitin ligase LATA1 impacts tiller angle in rice

Abstract

Rice (Oryza sativa) is the staple crop for more than half the world's population, making yield improvements a critical goal for current breeding efforts. One approach focuses on improving plant architecture, and the tiller angle is a promising trait in this context: a large angle results in a spread-out growth habit that enhances light capture and helps outcompete weeds, while an erect growth habit with a smaller tiller angle allows for dense planting, efficient harvesting and reduces competition between individual plants (Wang et al., 2022). Domestication of wild rice has favoured an erect growth habit, which can be largely attributed to selection for specific alleles of several C2H2 zinc finger transcription factor genes, including PROSTRATE GROWTH 1 (PROG1) (Tan et al., 2008), PROG7 (Hu et al., 2018) and RICE PLANT ARCHITECTURE DOMESTICATION (RPAD) (Wu et al., 2018).

These genes also affect shoot gravitropism (Wang, Gao, Liang, Li, & Wang, 2022), a process more thoroughly studied in Arabidopsis thaliana. The starch-statolith hypothesis states that the direction of gravity is sensed by the sedimentation of starch-filled amyloplasts (Sack, 1997). This sedimentation, possibly sensed by mechanosensitive membranes, activates a signal transduction cascade that results in the asymmetric distribution of the plant hormone auxin across the shoot. High auxin concentrations on the lower side lead to increased cell elongation, causing asymmetric growth and a bending of the shoot (Takahashi et al., 2021). Roles for amyloplastic starch granules and asymmetric auxin distribution have also been established in the control of tiller angle in rice, but only a few genes involved in this process are known.

Lubin Tan's lab at China Agricultural University in Beijing studies the genetic regulation of rice agronomic traits; uncovering the signalling pathways underlying plant architecture thus remains a key area of the group's research. Through an EMS mutagenesis screen, PhD student Jinjian Fan, first author of the highlighted publication, and colleagues identified a mutant with a with a spread-out architecture they named large tiller angle 1 (lata1). Throughout development, this mutant consistently exhibited larger tiller angles than the wild type, with the outermost tiller reaching a 25° angle at the heading stage, compared to an 11° angle in the wild type (Figure 1a). This phenotype was caused by reduced asymmetric growth at the tiller base. As for other tiller angle mutants, the change in angle was associated with altered shoot gravitropism: after rotating seedlings 90°, gravitropic bending was visibly delayed in lata1 seedlings (Figure 1b).

Using bulk segregant analysis, the authors mapped the lata1 phenotype to a single-nucleotide polymorphism (SNP) in a gene located on chromosome 5 that results in a lysine to proline substitution (L139P) in the encoded protein. Complementation studies confirmed that this SNP indeed caused the mutant's spread-out phenotype. In line with these findings, knock-out of the LATA1 gene by CRISPR/Cas9 also resulted in increased tiller angles and impaired gravitropism in both japonica and indica rice cultivars, while LATA1 overexpression did not affect tiller angle, but enhanced the gravitropic response. LATA1 was strongly expressed at the tiller base, and its transcript levels rose upon gravistimulation, further linking the gene to tiller angle and gravitropic phenotypes.

The LATA1 protein is predicted to contain a C3H2C3-type RING zinc finger domain typically found in E3 ubiquitin ligases. LATA1 is homologous to the Arabidopsis E3 ligase SHOOT GRAVITROPISM 9 (SGR9), which is also involved in gravity sensing (Nakamura, Toyota, Tasaka, & Morita, 2011). In vitro ubiquitination assays using recombinantly expressed GST-LATA1 demonstrated that LATA1 has autoubiquitination activity, which was abolished when conserved cysteine residues in the RING domain were mutated. Autoubiquitination activity was also reduced in the GST-lata1 mutant protein, suggesting that the L139P substitution, although located outside the RING domain, impairs E3 ligase activity.

Fan et al. further investigated the link between LATA1 and gravitropism. Using transcript levels of the auxin-responsive gene OsIAA20 as a proxy, they found that the auxin response to gravistimulation was weakened in lata1. Similarly, the auxin content ratio between the lower and upper sides of the shoot increased under gravistimulation in the wild type, but not in lata1 seedlings. Transcript levels of the YUCCA auxin biosynthesis genes were lower in lata1 than in the wild type, and IAA treatment partially restored the mutant's gravitropic response. In addition, lata1 was hypersensitive to treatment with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Taken together, these results suggest that lata1 is impaired in both auxin biosynthesis and lateral auxin transport.

The exact mechanism by which LATA1 affects auxin accumulation and auxin distribution remains unclear. Identifying ubiquitination substrates of LATA1 represents a next logical step in further uncovering its function. Given that lata1 mutants did not differ from wild type in important agronomic traits such as plant height, tiller number, grain number and size, Tan hopes that LATA1 can be leveraged to optimise rice architecture further, using natural variation and precise gene editing to fine-tune tiller angle during development.