LAZY1 controls rice shoot gravitropism through regulating polar auxin transport

Tiller angle of rice （Oryza sativa L.） is an important agronomic trait that contributes to grain production, and has long attracted attentions of breeders for achieving ideal plant architecture to improve grain yield. Although enormous efforts have been made over the past decades to study mutants with extremely spreading or compact tillers, the molecular mechanism underlying the control of tiller angle of cereal crops remains unknown. Here we report the cloning of the LAZY1 （LA1） gene that regulates shoot gravitropism by which the rice tiller angle is controlled. We show that LA1, a novel grass-specific gene, is temporally and spatially expressed, and plays a negative role in polar auxin transport （PAT）. Loss-of-function of LA1 enhances PAT greatly and thus alters the endogenous IAA distribution in shoots, leading to the reduced gravitropism, and therefore the tiller-spreading phenotype of rice plants.     

TAC4 controls tiller angle by regulating the endogenous auxin content and distribution in rice

Tiller angle, an important component of plant architecture, greatly influences the grain yield of rice (Oryza sativa L.). Here, we identified Tiller Angle Control 4 (TAC4) as a novel regulator of rice tiller angle. TAC4 encodes a plant‐specific, highly conserved nuclear protein. The loss of TAC4 function leads to a significant increase in the tiller angle. TAC4 can regulate rice shoot gravitropism by increasing the indole acetic acid content and affecting the auxin distribution. A sequence analysis revealed that TAC4 has undergone a bottleneck and become fixed in indica cultivars during domestication and improvement. Our findings facilitate an increased understanding of the regulatory mechanisms of tiller angle and also provide a potential gene resource for the improvement of rice plant architecture.     

植物重力反应的分子调控机制

重力是调节植物生长发育和形态建成的重要环境因子。植物感受到重力刺激后可以通过重力反应来协调自身各个器官的生长方向与重力方向之间的最适角度。植物重力反应过程分为重力信号的感受、重力信号的转导、生长素不对称分布的形成和重力反应器官的弯曲生长4个阶段。近年来,随着大量重力反应缺陷突变体的鉴定及其控制基因的功能解析,重力信号的感受和生长素不对称分布的分子机制等方面的研究取得了重要进展。作为植物适应环境变化的重要手段之一,重力反应还可以通过调节水稻(Oryza sativa L.)的分蘖角度实现对水稻株型和产量的调控。因此,研究植物的重力反应,不仅有助于解析植物生长发育的调控机制,对于作物株型的改良也具有重要的指导意义。然而,重力反应的分子机制及其调控网络仍不清楚。本文综述了近年来植物重力反应的调控机理及其调控水稻分蘖角度的作用机制,并对该领域未来的研究方向和热点进行了展望。     

OsmiR167a‐targeted auxin response factors modulate tiller angle via fine‐tuning auxin distribution in rice

Rice tiller angle determines plant growth density and further contributes grain production. Although a few genes have been characterized to regulate tiller angle in rice, the molecular mechanism underlying the control of tiller angle via microRNA is poorly understood. Here, we report that rice tiller angle is controlled by OsmiR167a‐targeted auxin response factors OsARF12, OsARF17 and OsARF25. In the overexpression of OsMIR167a plants, the expression of OsARF12, OsARF17 and OsARF25 was severely repressed and displayed larger tiller angle as well as the osarf12/osarf17 and osarf12/ osarf25 plants. In addition, those plants showed compromised abnormal auxin distribution and less sensitive to gravity. We also demonstrate that OsARF12, OsARF17 and OsARF25 function redundantly and might be involved in HSFA2D and LAZY1‐dependent asymmetric auxin distribution pathway to control rice tiller angle. Our results reveal that OsmiR167a represses its targets, OsARF12, OsARF17 and OsARF25, to control rice tiller angle by fine‐tuning auxin asymmetric distribution in shoots.     

Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways

We identified the gene responsible for three allelic lazy1 mutations of Japonica rice (Oryza sativa L.) by map-based cloning, complementation and RNA interference. Sequence analysis and database searches indicated that the wild-type gene (LAZY1) encodes a novel and unique protein (LAZY1) and that rice has no homologous gene. Two lazy1 mutants were LAZY1 null. Confirming and advancing the previously reported results on lazy1 mutants, we found the following. (i) Gravitropism is impaired, but only partially, in lazy1 coleoptiles. (ii) Circumnutation, observed in dark-grown coleoptiles, is totally absent from lazy1 coleoptiles. (iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissue-specific manner in gravity-sensitive shoot tissues (i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is little expressed in roots. (v) The gravitropic response of lazy1 coleoptiles is kinetically separable from that absent from lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of auxin, found in wild-type coleoptiles, does not occur in lazy1 coleoptiles. Based on the genetic and physiological evidence obtained, it is concluded that LAZY1 is specifically involved in shoot gravitropism and that LAZY1-dependent and -independent signaling pathways occur in coleoptiles. It is further concluded that, in coleoptiles, only the LAZY1-dependent gravity signaling involves asymmetric distribution of auxin between the two lateral halves and is required for circumnutation.     

Gravity sensing and signaling

Gravitropism has attracted much attention from plant biologists. Recent studies have provided molecular evidence supporting two long-surviving hypotheses about the mechanism of gravitropism: the starch-statolith hypothesis and the Cholodney-Went hypothesis. Amyloplast movement along the gravity vector within gravity-sensing cells in the root and shoot is the most likely trigger of subsequent intracellular signaling. Several possible events leading from this signaling to differential auxin distribution within the sensing cells have been suggested recently.     

Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana

Shoots of higher plants exhibit negative gravitropism. However, little is known about the mechanism or site of gravity perception in shoots. We have identified two loci that are essential for normal shoot gravitropism in Arabidopsis thaliana . Genetic analysis demonstrated that the shoot gravitropism mutants sgr1 and sgr7 are allelic to the radial pattern mutants, scr and shr , respectively. Characterization of the aerial phenotype of these mutants revealed that the primary defect is the absence of a normal endodermis in hypocotyls and inflorescence stems. This indicates that the endodermis is essential for shoot gravitropism and strongly suggests that this cell layer functions as the gravity-sensing cell layer in dicotyledonous plant shoots. These results also demonstrate that, in addition to their previously characterized role in root radial patterning, SCR and SHR regulate the radial organization of the shoot axial organs in Arabidopsis .      

The Endosperm-Specific Expression of <i>YUCCA</i> Genes Enhances Rice Grain Filling

Grain filling is a crucial process that affects yield in rice (Oryza sativa L.). Auxin biosynthesis and signaling are closely related to rice yield; therefore, it is important to understand the effects of auxin biosynthesis on rice grain filling to improve crop yield. In this study, we used physiological and molecular strategies to identify the roles of auxin in rice grain filling. Exogenous application of auxin (IAA) or auxin analogues (2, 4-D) to young spikelets and flag leaves improved the seed-setting rate and yield per spike. Furthermore, real-time quantitative PCR assays confirmed that nine members of the OsYUCCA family of auxin biosynthetic genes were upregulated during grain filling, implication that auxin biosynthesis plays a major role in grain development. The specific expression of either Arabidopsis AtYUCCA1 or OsYUCCA2 in the endosperm or leaves resulted in increased expression of OsIAA genes and auxin content of seeds, as well as increased grain filling and seed-setting rate. This result establishes that the auxin content in grains and leaves is important for grain development. Our findings further highlight the potential applications for improving rice yield by elevating targeted gene expression in specific tissues.     

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.     

AtLAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence

The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T‐DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch‐angle phenotype and the gene consequently named AtLAZY1. Time‐resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch‐angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch‐angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery.     

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.     

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.     

Over-expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1

Crop architecture parameters such as tiller number, angle and plant height are important agronomic traits that have been considered for breeding programmes. Auxin distribution within the plant has long been recognized to alter architecture. The rice (Oryza sativa L.) genome contains 12 putative PIN genes encoding auxin efflux transporters, including four PIN1 and one PIN2 genes. Here, we report that over-expression of OsPIN2 through a transgenic approach in rice (Japonica cv. Nipponbare) led to a shorter plant height, more tillers and a larger tiller angle when compared with wild type (WT). The expression patterns of the auxin reporter DR5::GUS and quantification of auxin distribution showed that OsPIN2 over-expression increased auxin transport from the shoot to the root-shoot junction, resulting in a non-tissue-specific accumulation of more free auxin at the root-shoot junction relative to WT. Over-expression of OsPIN2 enhanced auxin transport from shoots to roots, but did not alter the polar auxin pattern in the roots. Transgenic plants were less sensitive to N-1-naphthylphthalamic acid, an auxin transport inhibitor, than WT in their root growth. OsPIN2-over-expressing plants had suppressed the expression of a gravitropism-related gene OsLazy1 in the shoots, but unaltered expression of OsPIN1b and OsTAC1, which were reported as tiller angle controllers in rice. The data suggest that OsPIN2 has a distinct auxin-dependent regulation pathway together with OsPIN1b and OsTAC1 controlling rice shoot architecture. Altering OsPIN2 expression by genetic transformation can be directly used for modifying rice architecture.     

Tiller formation in rice is altered by overexpression of OsIAGLU gene encoding an IAA-conjugating enzyme or exogenous treatment of free IAA

Optimization of plant architecture is important for cultivation and yield of cereal crops in the field. Tillering is an essential factor used to determine the overall architecture of cereal crops. It has long been recognized that the development of branching patterns is controlled by the level and distribution of auxin within a plant. To better understand the relationship between auxin levels and tillering in rice, we examined rice plants with increased or decreased levels of free IAA. To decrease IAA levels, we selected the rice IAA-glucose synthase gene (OsIAGLU) from the rice genome database based on high sequence homology with IAA-glucose synthase from maize (ZmIAGLU), which is known to generate IAAglucose conjugate from free IAA. The OsIAGLU gene driven by the Cauliflower Mosaic Virus 35S promoter was transformed into a rice cultivar to generate transgenic rice plants constitutively over-expressing this gene. The number of tillers and panicles significantly increased in the transgenic lines compared to the wild-type plants, while plant height and panicle length decreased. These results indicate that decreased levels of free IAA likely enhance tiller formation in rice. To increase levels of free IAA, we treated rice plants with three different concentrations of exogenous IAA (1 μM, 10 μM and 100 μM) twice a week by spraying. Exogenous IAA treatment at concentrations of 10 μM and 100 μM significantly reduced tiller number in three different rice cultivars. These results indicate that exogenously applied IAA inhibits shoot branching in rice. Overall, auxin tightly controls tiller formation in rice in a negative way.     

FLOURY ENDOSPERM16 encoding a NAD‐dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice

Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map‐based cloning and complementation tests demonstrated that FLO16 encodes a NAD‐dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis‐related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.     

Growth Responses and Allocation of Assimilates of Rice Seedlings by Paclobutrazol and Gibberellin Treatment

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)methyl-4,4-dimethyl-2-(1h-1,2,4-trizol-1-yl)penten-3-ol] effectively decreased vegetative growth of rice (Oryza sativa L.) seedlings and increased the chlorophyll content. The number of veins in a leaf, the calculated number of stomata per leaf, and the length of guard cells were not altered by the paclobutrazol treatment, suggesting an effect on cell elongation. The allocation pattern of carbohydrates was changed by either gibberellin (GA) or paclobutrazol treatment. GA₃ induced more shoot growth and less accumulation of starch than the control and paclobutrazol-treated seedlings. Photosynthetic ability was not affected by either paclobutrazol or GA₃ treatment. Paclobutrazol-treated plants allocated a smaller amount of photosynthates for vegetative shoot growth and stored more as starch in the crowns than the control and GA₃-treated plants. The same starch degrading activity in the crown tissue of paclobutrazol-treated seedlings as in control plants suggests that the accumulated starch is utilized in a normal activity for growth including leaf emergence, tiller formation, and root production, resulting in improved seedling quality. Received May 30, 1996; accepted December 10, 1996