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.

The role of Arabidopsis 5PTase13 in root gravitropism through modulation of vesicle trafficking

Inositol polyphosphate 5-phosphatases （5PTases） are enzymes of phosphatidylinositol metabolism that affect various aspects of plant growth and development. Arabidopsis 5PTasel3 regulates auxin homeostasis and hormonerelated cotyledon vein development, and here we demonstrate that its knockout mutant 5pt13 has elevated sensitivity to gravistimulation in root gravitropic responses. The altered responses of 5pt13 mutants to 1-N-naphthylphthalamic acid （an auxin transport inhibitor） indicate that 5PTase13 might be involved in the regulation of auxin transport. Indeed, the auxin efflux carrier PIN2 is expressed more broadly under 5PTasel3 deficiency, and observations of the internalization of the membrane-selective dye FM4-64 reveal altered vesicle trafficking in 5pt13 mutants. Compared with wild-type, 5pt13 mutant seedlings are less sensitive to the inhibition by brefeldin A of vesicle cycling, seedling growth, and the intracellular cycling of the PIN1 and PIN2 proteins. Further, auxin redistribution upon gravitropic stimulation is stimulated under 5PTasel3 deficiency. These results suggest that 5PTasel3 may modulate auxin transport by regulating vesicle trafficking and thereby play a role in root gravitropism.

Identification of a Gravitropism-Deficient Mutant in Rice

A gravitropism-deficient mutant M96 was isolated from a mutant bank, generated by ethyl methane sulfonate(EMS) mutagenesis of indica rice accession ZJ100. The mutant was characterized as prostrate growth at the beginning of germination, and the prostrate growth phenotype ran through the whole life duration. Tiller angle and tiller number of M96 increased significantly in comparison with the wild type. Tissue section observation analysis indicated that asymmetric stem growth around the second node occurred in M96. Genetic analysis and gene mapping showed that M96 was controlled by a single recessive nuclear gene, tentatively termed as gravitropism-deficient M96(gd M96), which was mapped to a region of 506 kb flanked by markers RM5960 and In Del8 on the long arm of chromosome 11. Sequencing analysis of the open reading frames in this region revealed a nucleotide substitution from G to T in the third exon of LOC_Os11g29840. Additionally, real-time fluorescence quantitative PCR analysis showed that the expression level of LOC_Os11g29840 in the stems was much higher than in the roots and leaves in M96. Furthermore, the expression level was more than four times in M96 stem than in the wild type stem. Our results suggested that the mutant gene was likely a new allele to the reported gene LAZY1. Isolation of this new allele would facilitate the further characterization of LAZY1.

Characterization of wavy root 1,an agravitropism allele,reveals the functions of OsPIN2 in fine regulation of auxin transport and distribution and in ABA biosynthesis and response in rice(Oryza sativa L.)

Root system architecture is influenced by gravity.How the root senses gravity and directs its orientation,so-called gravitropism,is not only a fundamental question in plant biology but also theoretically important for genetic improvement of crop root architecture.However,the mechanism has not been elucidated in most crops.We characterized a rice agravitropism allele,wavy root 1(war1),a loss-of-function allele in OsPIN2,which encodes an auxin efflux transporter.With loss of OsPIN2 function,war1 leads to altered root system architecture including wavy root,larger root distribution angle,and shallower root system due to the loss of gravitropic perception in root tips.In the war1 mutant,polar auxin transport was disrupted in the root tip,leading to abnormal auxin levels and disturbed auxin transport and distribution in columella cells.Amyloplast sedimentation,an important process in gravitropic sensing,was also decreased in root tip columella cells.The results indicated that OsPIN2 controls gravitropism by finely regulating auxin transport,distribution and levels,and amyloplast sedimentation in root tips.We identified a novel role of OsPIN2 in regulating ABA biosynthesis and response pathways.Loss of OsPIN2 function in the war1 resulted in increased sensitivity to ABA in seed germination,increased ABA level,changes in ABA-associated genes in roots,and decreased drought tolerance in the seedlings.These results suggest that the auxin transporter OsPIN2 not only modulates auxin transport to control root gravitropism,but also functions in ABA signaling to affect seed germination and root development,probably by mediating crosstalk between auxin and ABA pathways.

Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles

The phytochrome gene family, which is in Arabidopsis thaliana, consists of phytochromes A-E（phyA to phyE）, regulates plant responses to ambient light environments. PhyA and phyB have been characterized in detail, but studies on phyC to phyE have reported discrepant functions. In this study, we show that phyD regulates the Arabidopsis gravitropic response by inhibiting negative gravitropism of hypocotyls under red light condition. PhyD had only a limited effect on the gravitropic response of roots in red light condition. PhyD also enhanced phyB-regulated gravitropic responses in hypocotyls. Moreover, the regulation of hypocotyl gravitropic responses by phyD was dependent upon the red light fluence rate.

Light-stabilized GIL1 suppresses PIN3 activity to inhibit hypocotyl gravitropism

Light and gravity coordinately regulate the directional growth of plants.Arabidopsis Gravitropic in the Light 1(GIL1)inhibits the negative gravitropism of hypocotyls in red and far-red light,but the underlying molecular mechanisms remain elusive.Our study found that GIL1 is a plasma membrane-localized protein.In endodermal cells of the upper part of hypocotyls,GIL1 controls the negative gravitropism of hypocotyls.GIL1 directly interacts with PIN3 and inhibits the auxin transport activity of PIN3.Mutation of PIN3 suppresses the abnormal gravitropic response of gil1 mutant.The GIL1 protein is unstable in darkness but it is stabilized by red and far-red light.Together,our data suggest that light-stabilized GIL1 inhibits the negative gravitropism of hypocotyls by suppressing the activity of the auxin transporter PIN3,thereby enhancing the emergence of young seedlings from the soil.

LAZY4 acts additively with the starch-statolithdependent gravity-sensing pathway to regulate shoot gravitropism and tiller angle in rice

Rice tiller angle is a key agronomic trait that has significant effects on the establishment of a high-yield rice population.However,the molecular mechanism underlying the control of rice tiller angle remains to be clarified.Here,we characterized the novel tiller-angle gene LAZY4(LA4)in rice through map-based cloning.LA4 encodes a C3H2C3-type RING zinc-finger E3 ligase localized in the nucleus,and an in vitro ubiquitination assay revealed that the conserved RING finger domain is essential for its E3 ligase activity.We found that expression of LA4 can be induced by gravistimulation and that loss of LA4 function leads to defective shoot gravitropism caused by impaired asymmetric auxin redistribution upon gravistimulation.Genetic analysis demonstrated that LA4 acts in a distinct pathway from the starch biosynthesis regulators LA2 and LA3,which function in the starch-statolith-dependent pathway.Further genetic analysis showed that LA4 regulates shoot gravitropism and tiller angle by acting upstream of LA1 to mediate lateral auxin transport upon gravistimulation.Our studies reveal that LA4 regulates shoot gravitropism and tiller angle upstream of LA1 through a novel pathway independent of the LA2-LA3-mediated gravity-sensing mechanism,providing new insights into the rice tiller-angle regulatory network.

The IDD Transcription Factors:Their Functions in Plant Development and Environmental Response

INDETERMINATE-DOMAIN proteins(IDDs)are a plant-specific transcription factor family characterized by a conserved ID domain with four zinc finger motifs.Previous studies have demonstrated that IDDs coordinate a diversity of physiological processes and functions in plant growth and development,including floral transition,plant architecture,seed and root development,and hormone signaling.In this review,we especially summarized the latest knowledge on the functions and working models of IDD members in Arabidopsis,rice,and maize,particularly focusing on their role in the regulatory network of biotic and abiotic environmental responses,such as gravity,temperature,water,and pathogens.Understanding these mechanisms underlying the function of IDD proteins in these processes is important for improving crop yields by manipulating their activity.Overall,the review offers valuable insights into the functions and mechanisms of IDD proteins in plants,providing a foundation for further research and potential applications in agriculture.

OsBRXL4 Regulates Shoot Gravitropism and Rice Tiller Angle through Affecting LAZY1 Nuclear Localization

Rice tiller angle is a key agronomic trait that contributes to ideal plant architecture and grain production.LAZY1 (LA1) was previously shown to control tiller angle via affecting shoot gravitropism,but the underlying molecular mechanism remains largely unknown.In this study,we identified an LA1-interacting protein named Brevis Radix Like 4 (OsBRXL4).We showed that the interaction between OsBRXL4 and LA1 occurs at the plasma membrane and that their interaction determines nuclear localization of LA1.We found that nuclear localization of LA1 is essential for its function,which is different from AtLA1,its Arabidopsis ortho.log.Overexpression of OsBRXL4 leads to a prostrate growth phenotype,whereas OsBRXLs RNAi plants,in which the expression levels of OsBRXLI,OsBRXL4,and OsBRXL5 were decreased,display a compact phenotype.Further genetic analysis also supported that OsBRXL4 controls rice tiller angle by affecting nuclear localization of LA1.Consistently,we demonstrated that OsBRXL4 regulates the shoot gravitropism through affecting polar auxin transport as did LA1.Taken together,our study not only identifies OsBRXL4 as a regulatory component of rice tiller angle but also provides new insights into genetic regulation of rice plant architecture.

Control of Negative Gravitropism and Tension Wood Formation by Gibberellic Acid and Indole Acetic Acid in Fraxinus mandshurica Rupr. var. japonica Maxim Seedlings

In the present study, we Investigated the role of glbberelllc acid （GA3） and Indole acetic acid （IAA） In the gravity response of stems and tension wood formation using two-year-old stems of Fraxinus mandshurica Rupr. var. Japonica Maxim seedlings. Forty-five seedlings were used and divided Into nine groups that Included five seedlings In each group. Seedlings were treated with applications of GA3 alone at concentrations of 2.89×10^-8 and 2.89×10^-7 μmol/L, IAA alone at concentrations of 5.71×10^-8 and 5.71×10^-7 μmol/L, or their combination to the apical bud of the stem using a mlcroplpette. Seedlings were positioned horizontally after the first treatment. The same treatments were repeated six times per week. At the end of the experiment, all seedlings were harvested. Then, stem segments were cut under a light microscope. Application of exogenous GA3 at the higher concentration stimulated the upward bending of stems, whereas exogenous IAA had no effect. A synergistic effect of GA3 and IAA on upward stem bending was observed following application of the two combinations of GA3 and IAA. Moreover, application of exogenous GA3 at the higher dose stimulated wood formation on both the upper and lower sides of the stems, whereas the mixture of GA3 and IAA had a synerglstic effect on wood formation In horizontal stems. Application of exogenous IAA alone at the lower concentration （5.71×10^-8 μmol/L） or application of a mixture of the higher concentrations of GA3 （2.89×10^-7 μmol/L） and IAA （5.71×10^-7 μmol/L） Inhibited the development of gelatinous fibers （the G-layer） of tension wood on the upper side of the horizontal stems. The differentiation of gelatinous fibers of tension wood was not Inhibited by GA3 when It was applied alone, whereas the development of the gelatinous fibers of tension wood was strongly affected by the application of IAA. The findings of the present study suggest that the development of the G-layer Is not related to the dose of GA3, but needs a relatively lower concentration of IAA.

Role of GA_3,GA_4 and Uniconazole-P in Controlling Gravitropism and Tension Wood Formation in Fraxinus mandshurica Rupr. var. japonica Maxim. Seedlings

GA3 and GA4 （gibberellins） play an important role in controlling gravitropism and tension wood formation in woody angiosperms. In order to improve our understanding of the role of GA3 and GA4 on xylem cell formation and the G-layer, we studied the effect of GA3 and GA4 and uniconazole-P, which is an inhibitor of GA biosynthesis, on tension wood formation by gravity in Fraxinus mandshurica Rupr. var. japonica Maxim. seedlings. Forty seedlings were divided into two groups; one group was placed upright and the other tilted. Each group was further divided into four sub-groups subjected to the following treatments： 3.43 x 10-9 lunol acetone as control, 5.78 x 10-8 lunol gibberellic acid （GA3）, 6.21 x 10-8 lunol GA4, and 6.86 x 10-8 lunol uniconazole-P. During the experimental period, GAs-treated seedlings exhibited negative gravitropism, whereas application of uniconazole-P inhibited negative gravitropic stem bending. GA3 and GA4 promoted wood fibers that possessed a gelatinous layer on the upper side, whereas uniconazole-P inhibited wood formation but did not inhibit the differentiation of the gelatinous layer in wood fibers on the upper side. These results suggest that： （i） both the formation of gelatinous fibers and the quantity of xylem production are important for the negative gravitropism in horizontally-positioned seedlings; （ii） GA3 and GA4 affect wood production more than differentiation of the gelatinous layer in wood fibers; G-layer development may be regulated by other hormones via the indirect-role of GA3 and GA4 in horizontally-positioned F. mandshurica seedlings rather than the direct effect of GAs; and （iii） the mechanism for upward wood stem bending is different to the newly developed shoot bending in reaction to gravity in this species.

A feedback regulation between ARF7-mediated auxin signaling and auxin homeostasis involving MES17 affects plant gravitropism

Gravitropism is an essential adaptive response of land plants.Asymmetric auxin gradients across plant organs,interpreted by multiple auxin signaling components including AUXIN RESPONSE FACTOR7(ARF7),trigger differential growth and bending response.However,how this fundamental process is strictly maintained in nature remains unclear.Here,we report that gravity stimulates the transcription of METHYL ESTERASE17(MES17)along the lower side of the hypocotyl via ARF7-dependent auxin signaling.The asymmetric distribution of MES17,a methyltransferase that converts auxin from its inactive form methyl indole-3-acetic acid ester(MeI AA)to its biologically active form free-IAA,enhanced the gradient of active auxin across the hypocotyl,which in turn reversely amplified the asymmetric auxin responses and differential growth that shape gravitropic bending.Taken together,our findings reveal the novel role of MES17-mediated auxin homeostasis in gravitropic responses and identify an ARF7-triggered feedback mechanism that reinforces the asymmetric distribution of active auxin and strictly controls gravitropism in plants.

A new genetic factor for root gravitropism in rice (Oryza sativa L.)

Root gravitropism is one of the important factors to determine root architecture. To understand the mechanism underlying root gravitropism, we isolated a rice （Xiushui63） mutant defective in root gravitropism, designated as glsl. Vertical sections of root caps revealed that glsl mutant displayed normal distribution of amyloplast in the columella cells compared with the wild type. The glsl mutant was less sensitive to 2,4-dichlorophenoxyacetic acid （2,4-D） and ct-naphthaleneacetic acid （NAA） than the wild type. Genetic analysis indicated that the phenotype ofglsl mutant was caused by a single recessive mutation, which is mapped in a 255-kb region between RM16253 and CAPS1 on the short arm of chromosome 4.

Transcriptional regulation of MdPIN7 by MdARF19 during gravityinduced formation of adventitious root GSA in self-rooted apple stock

Self-rooted apple stock is widely used for apple production.However,the shallowness of the adventitious roots in self-rooted apple stock leads to poor performance in the barren orchards of China.This is because of the considerable difference in the development of a gravitropic set-point angle(GSA)between self-rooted apple stock and seedling rootstock.Therefore,it is crucial to study the molecular mechanism of adventitious root GSA in self-rooted apple stock for breeding self-rooted and deep-rooted apple rootstock cultivars.An apple auxin response factor MdARF19 functioned to establish the adventitious root GSA of self-rooted apple stock in response to gravity and auxin signals.MdARF19 bound directly to the MdPIN7 promoter,activating its transcriptional expression and thus regulating the formation of the adventitious root GSA in 12-2 self-rooted apple stock.However,MdARF19 influenced the expression of auxin efflux carriers(MdPIN3 and MdPIN10)and the establishment of adventitious root GSA of self-rooted apple stock in response to gravity signals by direct activation of MdFLP.Our findings provide new information on the transcriptional regulation of MdPIN7 by auxin response factor MdARF19 in the regulation of the adventitious root GSA of self-rooted apple stock in response to gravity and auxin signals.

PROG1 acts upstream of LAZY1 to regulate rice tiller angle as a repressor

Rice tiller angle,as a component of plant architecture,affects rice grain yield via plant density.However,the molecular mechanism underlying rice tiller angle remains elusive.We report that the key domestication gene PROSTRATE GROWTH 1(PROG1)controls rice tiller angle by regulating shoot gravitropism and LAZY1(LA1)-mediated asymmetric distribution of auxin.Acting as a transcriptional repressor,PROG1 negatively regulates the expression of LA1 in light-grown rice seedlings.Overexpression of LA1 partially rescued the larger tiller angle of the PROG1 complementation transgenic plant(prog1-D).Double-mutant analysis showed that PROG1 acts upstream of LA1 to regulate shoot gravitropism and tiller angle.Mutation of Suppressors of lazy1(SOL1),encoding DWARF3(D3)acting in the strigolactone signal pathway,suppressed the large tiller angle of prog1-D by rescuing the transcription of LA1.The discovery of a light-sensitive PROG1-LA1 transcription regulatory module controlling rice shoot gravitropism and tiller angle sheds light on the genetic control of rice tiller angle.

Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

Auxin distribution during embryogenesis and seed germination were studied with transgenic Arabidopsis plants expressing GUS gene driven by a synthetic DR5 promoter,an auxin responsive promoter. The results showed that GUS activity is higher in ends of hypophysis and cotyledon primordia of heart-, torpedo- and cotyledon-stage embryos, leaf tip area, lateral root primordia, root apex and cotyledon of young seedlings. And GUS accumulated in root apex of the seedlings grown on auxin transport inhibitor containing media. All these suggested that above-mentioned part of the organs and tissues have a higher level of auxin, and auxin polar transport inhibitor could cause the accumulation of auxin in root apex. And auxin transport inhibitor also resulted in aberration of Arabidopsis leaf pattern formation, root gravitropism and elongation.

Brassinosteroid-regulated plant growth and development and gene expression in soybean

Brassinosteroids(BRs) are endogenous phytohormones that play important roles in regulating plant growth and development.In this study, we evaluated the effects of brassinolide(BL, one of the active BRs) on soybean and identified roles of the hormone in regulating multiple aspects of plant growth and development.BL application promoted hypocotyl and epicotyl elongation in the light but blocked epicotyl elongation in the dark.High levels of castasterone and BL accumulated in light-grown plants.BL disrupted shoot negative gravitropism, whereas gibberellin did not.BL delayed leaf senescence.Transcriptome analysis showed that BL induced cell wall-modifying genes and auxin-associated genes but suppressed a class of WRKY genes involved in senescence and stress responses,showing the complex roles of BRs in multiple biological processes.

Preliminary study on a gravity－insensitive rice mutant

A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

The chloroplast-localized protein LTA1 regulates tiller angle and yield of rice

Plant architecture strongly influences rice grain yield.We report the cloning and characterization of the LTA1 gene,which simultaneously controls tiller angle and yield of rice.LTA1 encodes a chloroplastlocalized protein with a conserved YbaB DNA-binding domain,and is highly expressed in photosynthetic tissues including leaves and leaf sheaths.Disrupting the function of LTA1 leads to large tiller angle and yield reduction of rice.LTA1 affects the gravity response by mediating the distribution of endogenous auxin,thereby regulating the tiller angle.An lta1 mutant showed abnormal chloroplast development and decreased chlorophyll content and photosynthetic rate,in turn leading to reduction of rice yield.Our findings shed light on the genetic basis of tiller angle and provide a potential gene resource for the improvement of plant architecture and rice yield.

A Botanist’s Cognitive View on Plant Growth: Cross-Talk between Developmental and Sensitivity Networks

An alteration in plant phenotypes assisted by their responses to the environmental stimuli (=tropism) has been fundamental to understand the “plant sensitivity ” that plays a crucial role in plants’ adaptive success. Plants succeed through the deployment of moderators controlling polar auxin-transport determining organ bending. Stimulus-specific effectors can be synthesized by the outer peripheral cells at the bending sites where they target highly conserved cellular processes and potentially persuade the plant sensitivity at large. Remarkably, the peripheral cells require different time-intervals to achieve the threshold expression-levels of stimulus-specific molecular responders. After stimulus perception, tropic curvatures (especially at growing root-apices) are duly coordinated via integrated chemical and electrical signalling which is the key to cellular communications. Thus, the acquired phenotypic alterations are the perplexed outcome of plant’s developmental pace, complemented by the sensitivity. A novel aspect of this study is to advance our understanding of plant developmental-programming and the extent of plant-sensitivity, determining the plant growth and their future applications.