Distinctive expression patterns and roles of the miRNA393/TIR1 homolog module in regulating flag leaf inclination and primary and crown root growth in rice (Oryza sativa)

? MicroRNA (miRNA)-mediated regulation of auxin signaling components plays a critical role in plant development. miRNA expression and functional diversity contribute to the complexity of regulatory networks of miRNA/target modules. ? This study functionally characterizes two members of the rice (Oryza sativa) miR393 family and their target genes, OsTIR1 and OsAFB2 (AUXIN SIGNALING F-BOX), the two closest homologs of Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 (TIR1). ? We found that the miR393 family members possess distinctive expression patterns, with miR393a expressed mainly in the crown and lateral root primordia, as well as the coleoptile tip, and miR393b expressed in the shoot apical meristem. Transgenic plants overexpressing miR393a/b displayed a severe phenotype with hallmarks of altered auxin signaling, mainly including enlarged flag leaf inclination and altered primary and crown root growth. Furthermore, OsAFB2- and OsTIR1-suppressed lines exhibited increased inclination of flag leaves at the booting stage, resembling miR393-overexpressing plants. Moreover, yeast two-hybrid and bimolecular fluorescence complementation assays showed that OsTIR1 and OsAFB2 interact with OsIAA1. ? Expression diversification of miRNA393 implies the potential role of miRNA regulation during species evolution. The conserved mechanisms of the miR393/target module indicate the fundamental importance of the miR393-mediated regulation of auxin signal transduction in rice.     

Regulation of leaf morphology by microRNA394 and its target LEAF CURLING RESPONSIVENESS

The present study identified Arabidopsis miR394 and its target, an F-box (SKP1-Cullin/CDC53-F-box) gene At1g27340 (here referred to as LEAF CURLING RESPONSIVENESS, LCR), for regulation of leaf curling-related morphology. The loss-of-function lcr mutants exhibit pleiotropic defects with semi-dwarfism, altered leaf shape and a shorter stem. Overexpression of an miR394-resistant version of LCR under the 35S promoter (35S:m5LCR) and target mimicry MIM394 resulted in a curled-down leaf defect. Conversely, transgenic plants overexpressing 35S:MIR394a/b display a curled-up leaf phenotype. Detailed analyses show that there is a certain level of LCR that is optimal for leaf morphology, but lower or higher levels lead to abnormal leaf development, indicating that expression of miR394 in the leaf lamina is necessary for proper leaf morphology. Because the phytohormone auxin plays a crucial role in leaf morphogenesis and patterning, the DR5-GUS reporter gene was used to monitor the auxin response. We show that DR5 expression patterns in lcr and 35S::m5LCR plants were significantly different from those in the wild type. Also, overexpression of LCR in 35S::m5LCR plants drastically decreased the expression of the auxin-responsive genes IAA3, AXR3 and IAMT1, whereas increased expression of the genes was found in 35S::MIR394a plants. These results indicate that miR394 and its target LCR are involved in the regulation of leaf development.     

AUXIN RESPONSE FACTORS 6 and 17 control the flag leaf angle in rice by regulating secondary cell wall biosynthesis of lamina joints

Flag leaf angle impacts the photosynthetic capacity of densely grown plants and is thus an important agronomic breeding trait for crop architecture and yield. The hormone auxin plays a key role in regulating this trait, yet the underlying molecular and cellular mechanisms remain unclear. Here, we report that two rice (Oryza sativa) auxin response factors (ARFs), OsARF6 and OsARF17, which are highly expressed in lamina joint tissues, control flag leaf angle in response to auxin. Loss-of-function double osarf6 osarf17 mutants displayed reduced secondary cell wall levels of lamina joint sclerenchymatous cells (Scs), resulting in an exaggerated flag leaf angle and decreased grain yield under dense planting conditions. Mechanical measurements indicated that the mutant lamina joint tissues were too weak to support the weight of the flag leaf blade, resembling the phenotype of the rice increased leaf angle1 (ila1) mutant. We demonstrate that OsARF6 and OsARF17 directly bind to the ILA1 promoter independently and synergistically to activate its expression. In addition, auxin-induced ILA1 expression was dependent on OsARF6 and OsARF17. Collectively, our study reveals a mechanism that integrates auxin signaling with the secondary cell wall composition to determine flag leaf angle, providing breeding targets in rice, and potentially other cereals, for this key trait.

Two auxin response genes influence the secondary cell wall biosynthesis and the strength of lamina joints, thereby contributing to the adjustment of flag leaf angles.     

Harnessing Genetic Variation in Leaf Angle to Increase Productivity of Sorghum bicolor

The efficiency with which a plant intercepts solar radiation is determined primarily by its architecture. Understanding the genetic regulation of plant architecture and how changes in architecture affect performance can be used to improve plant productivity. Leaf inclination angle, the angle at which a leaf emerges with respect to the stem, is a feature of plant architecture that influences how a plant canopy intercepts solar radiation. Here we identify extensive genetic variation for leaf inclination angle in the crop plant Sorghum bicolor, a C4 grass species used for the production of grain, forage, and bioenergy. Multiple genetic loci that regulate leaf inclination angle were identified in recombinant inbred line populations of grain and bioenergy sorghum. Alleles of sorghum dwarf-3, a gene encoding a P-glycoprotein involved in polar auxin transport, are shown to change leaf inclination angle by up to 34° (0.59 rad). The impact of heritable variation in leaf inclination angle on light interception in sorghum canopies was assessed using functional-structural plant models and field experiments. Smaller leaf inclination angles caused solar radiation to penetrate deeper into the canopy, and the resulting redistribution of light is predicted to increase the biomass yield potential of bioenergy sorghum by at least 3%. These results show that sorghum leaf angle is a heritable trait regulated by multiple loci and that genetic variation in leaf angle can be used to modify plant architecture to improve sorghum crop performance.     

Auxin‐mediated lamina growth in tomato leaves is restricted by two parallel mechanisms

In the development of tomato compound leaves, local auxin maxima points, separated by the expression of the Aux/IAA protein SlIAA9/ENTIRE (E), direct the formation of discrete leaflets along the leaf margin. The local auxin maxima promote leaflet initiation, while E acts between leaflets to inhibit auxin response and lamina growth, enabling leaflet separation. Here, we show that a group of auxin response factors (ARFs), which are targeted by miR160, antagonizes auxin response and lamina growth in conjunction with E. In wild‐type leaf primordia, the miR160‐targeted ARFs SlARF10A and SlARF17 are expressed in leaflets, and SlmiR160 is expressed in provascular tissues. Leaf overexpression of the miR160‐targeted ARFs SlARF10A, SlARF10B or SlARF17, led to reduced lamina and increased leaf complexity, and suppressed auxin response in young leaves. In agreement, leaf overexpression of miR160 resulted in simplified leaves due to ectopic lamina growth between leaflets, reminiscent of e leaves. Genetic interactions suggest that E and miR160‐targeted ARFs act partially redundantly but are both required for local inhibition of lamina growth between initiating leaflets. These results show that different types of auxin signal antagonists act cooperatively to ensure leaflet separation in tomato leaf margins.     

Genetic modification of plant architecture and variety improvement in rice

The structure of the aerial part of a plant, referred to as plant architecture, is subject to strict genetic control, and grain production in cereal crops is governed by an array of agronomic traits. Rice is one of the most important cereal crops and is also a model plant for molecular biological research. Recently, significant progress has been made in isolating and collecting rice mutants that exhibit altered plant architecture. In this article we summarize the recent progress in understanding the basic patterning mechanisms involved in the regulation of tillering (branching) pattern, stem structure and leaf arrangement in rice plants. We discuss the relationship between the genetic modification of plant architecture and the improvement of pivotal agronomic traits in rice.     

The auxin response factor,OsARF19, controls rice leaf angles through positively regulating OsGH3‐5 and OsBRI1

Auxin and brassinosteroid (BR) are important phytohormones for controlling lamina inclination implicated in plant architecture and grain yield. But the molecular mechanism of auxin and BR crosstalk for regulating lamina inclination remains unknown. Auxin response factors (ARFs) control various aspects of plant growth and development. We here report that OsARF19‐overexpression rice lines show an enlarged lamina inclination due to increase of its adaxial cell division. OsARF19 is expressed in various organs including lamina joint and strongly induced by auxin and BR. Chromatin immunoprecipitation (ChIP) and yeast one‐hybrid assays demonstrate that OsARF19 binds to the promoter of OsGH3‐5 and brassinosteroid insensitive 1 (OsBRI1) directing their expression. OsGH3‐5‐overexpression lines show a similar phenotype as OsARF19‐O1. Free auxin contents in the lamina joint of OsGH3‐5‐O1 or OsARF19‐O1 are reduced. OsGH3‐5 is localized at the endoplasmic retieulum (ER) matching reduction of the free auxin contents in OsGH3‐5‐O1. osarf19‐TDNA and osgh3‐5‐Tos17 mutants without erected leaves show a function redundancy with other members of their gene family. OsARF19‐overexpression lines are sensitive to exogenous BR treatment and alter the expressions of genes related to BR signalling. These findings provide novel insights into auxin and BR signalling, and might have significant implications for improving plant architecture of monocot crops.     

Phytohormones signaling and crosstalk regulating leaf angle in rice

Leaf angle is an important agronomic trait in rice (Oryza sativa L.). It affects both the efficiency of sunlight capture and nitrogen reservoirs. The erect leaf phenotype is suited for high-density planting and thus increasing crop yields. Many genes regulate leaf angle by affecting leaf structure, such as the lamina joint, mechanical tissues, and the midrib. Signaling of brassinosteroids (BR), auxin (IAA), and gibberellins (GA) plays important roles in the regulation of lamina joint bending in rice. In addition, the biosynthesis and signaling of BR are known to have dominant effects on leaf angle development. In this review, we summarize the factors and genes associated with the development of leaf angle in rice, outline the regulatory mechanisms based on the signaling of BR, IAA, and GA, and discuss the contribution of crosstalk between BR and IAA or GA in the formation of leaf angle. Promising lines of research in the transgenic engineering of rice leaf angle to increase grain yield are proposed.     

OsPIN5b modulates rice (Oryza sativa) plant architecture and yield by changing auxin homeostasis,transport and distribution

Plant architecture attributes such as tillering, plant height and panicle size are important agronomic traits that determine rice (Oryza sativa) productivity. Here, we report that altered auxin content, transport and distribution affect these traits, and hence rice yield. Overexpression of the auxin efflux carrier‐like gene OsPIN5b causes pleiotropic effects, mainly reducing plant height, leaf and tiller number, shoot and root biomass, seed‐setting rate, panicle length and yield parameters. Conversely, reduced expression of OsPIN5b results in higher tiller number, more vigorous root system, longer panicles and increased yield. We show that OsPIN5b is an endoplasmic reticulum (ER) ‐localized protein that participates in auxin homeostasis, transport and distribution in vivo. This work describes an example of an auxin‐related gene where modulating its expression can simultaneously improve plant architecture and yield potential in rice, and reveals an important effect of hormonal signaling on these traits.     

Suppression of auxin signalling promotes rice susceptibility to Rice black streaked dwarf virus infection

Auxin plays a fundamental role in plant growth and development, and also influences plant defence against various pathogens. Previous studies have examined the different roles of the auxin pathway during infection by biotrophic bacteria and necrotrophic fungi. We now show that the auxin signalling pathway was markedly down-regulated following infection of rice by Rice black streaked dwarf virus (RBSDV), a dsRNA virus. Repression of the auxin receptor TIR1 by a mutant overexpressing miR393 increased rice susceptibility to RBSDV. Mutants overexpressing the auxin signalling repressors OsIAA20 and OsIAA31 were also more susceptible to RBSDV. The induction of jasmonic acid (JA) pathway genes in response to RBSDV was supressed in auxin signalling mutants, suggesting that activation of the JA pathway may be part of the auxin signalling-mediated rice defence against RBSDV. More importantly, our results also revealed that OsRboh-mediated reactive oxygen species levels played important roles in this defence. The results offer novel insights into the regulatory mechanisms of auxin signalling in the rice–RBSDV interaction.     

Brassinosteroid-induced rice lamina joint inclination and its relation to indole-3-acetic acid and ethylene

Brassinosteroid (BR)-induced rice (Oriza sativa L.) lamina joint (RLJ) inclination and its relationship to indole-3-acetic acid (IAA) and ethylene were investigated using BR isolated from beeswax. The effect of BR on RLJ inclination was time- and concentration-dependent. Etiolated lamina were more sensitive to BR than green lamina. The BR-induced inclination was accompanied by increased lamina fresh weight, total water content, free-water content, proton extrusion and ethylene production, and decreased bound-water content. Lamina dry weight was not changed. The inclination was due to greater expansion of the adaxial cells relative to the dorsal cells in the lamina joint. This response was caused by BR and/or BR-induced signal(s) that were transported from the leaf sheath to the leaf blade. Both BR-induced RLJ inclination and ethylene production were inhibited by cobalt chloride (CoCl₂), an inhibitor of ACC oxidase. BR-induced inclination was much higher than that of IAA, and was inhibited by high concentration of 2,3,5-triiodobenzoic acid (TIBA), an inhibitor of IAA transport. A synergistic effect was observed between BR and IAA. These results suggest that the effects of BR on RLJ inclination and pulvinus cell expansion may be resulted from BR-increased water potential and proton extrusion in the lamina. The BR-induced RLJ inclination may involve the action of ethylene but may be independent of IAA.Abbreviations BR brassinolide or brassinosteroid(s) - IAA indole-3-acetic acid - TIBA 2,3,5-triiodobenzoic acid - RLJ rice lamina joint