Auxin Transporter OsPIN1b, a Novel Regulator of Leaf Inclination in Rice (Oryza sativa L.).

Leaf inclination is one of the most important components of the ideal architecture, which effects yield gain. Leaf inclination was shown that is mainly regulated by brassinosteroid (BR) and auxin signaling. Here, we reveal a novel regulator of leaf inclination, auxin transporter OsPIN1b. Two CRISPR-Cas9 homozygous mutants, ospin1b-1 and ospin1b-2, with smaller leaf inclination compared to the wild-type, Nipponbare (WT/NIP), while overexpression lines, OE-OsPIN1b-1 and OE-OsPIN1b-2 have opposite phenotype. Further cell biological observation showed that in the adaxial region, OE-OsPIN1b-1 has significant bulge compared to WT/NIP and ospin1b-1, indicating that the increase in the adaxial cell division results in the enlarging of the leaf inclination in OE-OsPIN1b-1. The OsPIN1b was localized on the plasma membrane, and the free IAA contents in the lamina joint of ospin1b mutants were significantly increased while they were decreased in OE-OsPIN1b lines, suggesting that OsPIN1b might action an auxin transporter such as AtPIN1 to alter IAA content and leaf inclination. Furthermore, the OsPIN1b expression was induced by exogenous epibrassinolide (24-eBL) and IAA, and ospin1b mutants are insensitive to BR or IAA treatment, indicating that the effecting leaf inclination is regulated by OsPIN1b. This study contributes a new gene resource for molecular design breeding of rice architecture.

OsARF4 regulates leaf inclination via auxin and brassinosteroid pathways in rice.

Leaf inclination is a vital agronomic trait and is important for plant architecture that affects photosynthetic efficiency and grain yield. To understand the molecular mechanisms underlying regulation of leaf inclination, we constructed an auxin response factor (arf) rice mutant-osarf4-showing increased leaf inclination using CRISPR/Cas9 gene editing technology. OsARF4 encodes a nuclear protein that is expressed in the lamina joint (LJ) at different developmental stages in rice. Histological analysis indicated that an increase in cell differentiation on the adaxial side resulted in increased leaf inclination in the osarf4 mutants; however, OsARF4-overexpressing lines showed a decrease in leaf inclination, resulting in erect leaves. Additionally, a decrease in the content and distribution of indole-3-acetic acid (IAA) in osarf4 mutant led to a greater leaf inclination, whereas the OsARF4-overexpressing lines showed the opposite phenotype with increased IAA content. RNA-sequencing analysis revealed that the expression of genes related to brassinosteroid (BR) biosynthesis and response was different in the mutants and overexpressing lines, suggesting that OsARF4 participates in the BR signaling pathway. Moreover, BR sensitivity assay revealed that OsARF4-overexpressing lines were more sensitive to exogenous BR treatment than the mutants. In conclusion, OsARF4, a transcription factor in auxin signaling, participates in leaf inclination regulation and links auxin and BR signaling pathways. Our results provide a novel insight into l leaf inclination regulation, and have significant implications for improving rice architecture and grain yield.

OsRLR4 binds to the OsAUX1 promoter to negatively regulate primary root development in rice.

Root architecture is one of the most important agronomic traits that determines rice crop yield. The primary root (PR) absorbs mineral nutrients and provides mechanical support; however, the molecular mechanisms of PR elongation remain unclear in rice. Here, the two loss-of-function T-DNA insertion mutants of root length regulator 4 (OsRLR4), osrlr4-1 and osrlr4-2 with longer PR, and three OsRLR4 overexpression lines, OE-OsRLR4-1/-2/-3 with shorter PR compared to the wild type/Hwayoung (WT/HY), were identified. OsRLR4 is one of five members of the PRAF subfamily of the regulator chromosome condensation 1 (RCC1) family. Phylogenetic analysis of OsRLR4 from wild and cultivated rice indicated that it is under selective sweeps, suggesting its potential role in domestication. OsRLR4 controls PR development by regulating auxin accumulation in the PR tip and thus the root apical meristem activity. A series of biochemical and genetic analyses demonstrated that OsRLR4 functions directly upstream of the auxin transporter OsAUX1. Moreover, OsRLR4 interacts with the TRITHORAX-like protein OsTrx1 to promote H3K4me3 deposition at the OsAUX1 promoter, thus altering its transcription level. This work provides insight into the cooperation of auxin and epigenetic modifications in regulating root architecture and provides a genetic resource for plant architecture breeding.

A novel miR167a-OsARF6-OsAUX3 module regulates grain length and weight in rice.

Grain size is one of the most important factors that control rice yield, as it is associated with grain weight (GW). To date, dozens of rice genes that regulate grain size have been isolated; however, the regulatory mechanism underlying GW control is not fully understood. Here, the quantitative trait locus qGL5 for grain length (GL) and GW was identified in recombinant inbred lines of 9311 and Nipponbare (NPB) and fine mapped to a candidate gene, OsAUX3. Sequence variations between 9311 and NPB in the OsAUX3 promoter and loss of function of OsAUX3 led to higher GL and GW. RNA sequencing, gene expression quantification, dual-luciferase reporter assays, chromatin immunoprecipitation-quantitative PCR, and yeast one-hybrid assays demonstrated that OsARF6 is an upstream transcription factor regulating the expression of OsAUX3. OsARF6 binds directly to the auxin response elements of the OsAUX3 promoter, covering a single-nucleotide polymorphism site between 9311 and NPB/Dongjin/Hwayoung, and thereby controls GL by altering longitudinal expansion and auxin distribution/content in glume cells. Furthermore, we showed that miR167a positively regulate GL and GW by directing OsARF6 mRNA silencing. Taken together, our study reveals that a novel miR167a-OsARF6-OsAUX3 module regulates GL and GW in rice, providing a potential target for the improvement of rice yield.

The auxin influx carrier, OsAUX3, regulates rice root development and responses to aluminium stress.

In rice, there are five members of the auxin carrier AUXIN1/LIKE AUX1 family; however, the biological functions of the other four members besides OsAUX1 remain unknown. Here, by using CRISPR/Cas9, we constructed two independent OsAUX3 knock-down lines, osaux3-1 and osaux3-2, in wild-type rice, Hwayoung (WT/HY) and Dongjin (WT/DJ). osaux3-1 and osaux3-2 have shorter primary roots (PRs), decreased lateral root (LR) density, and longer root hairs (RHs) compared with their WT. OsAUX3 expression in PRs, LRs, and RHs further supports that OsAUX3 plays a critical role in the regulation of root development. OsAUX3 locates at the plasma membrane and functions as an auxin influx carrier affecting acropetal auxin transport. OsAUX3 is up-regulated in the root apex under aluminium (Al) stress, and osaux3-2 is insensitive to Al treatments. Furthermore, 1-naphthylacetic acid accented the sensitivity of WT/DJ and osaux3-2 to respond to Al stress. Auxin concentrations, Al contents, and Al-induced reactive oxygen species-mediated damage in osaux3-2 under Al stress are lower than in WT, indicating that OsAUX3 is involved in Al-induced inhibition of root growth. This study uncovers a novel pathway alleviating Al-induced oxidative damage by inhibition of acropetal auxin transport and provides a new option for engineering Al-tolerant rice species.