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.     

PAY1 improves plant architecture and enhances grain yield in rice

Plant architecture, a complex of the important agronomic traits that determine grain yield, is a primary target of artificial selection of rice domestication and improvement. Some important genes affecting plant architecture and grain yield have been isolated and characterized in recent decades; however, their underlying mechanism remains to be elucidated. Here, we report genetic identification and functional analysis of the PLANT ARCHITECTURE AND YIELD 1 (PAY1) gene in rice, which affects plant architecture and grain yield in rice. Transgenic plants over‐expressing PAY1 had twice the number of grains per panicle and consequently produced nearly 38% more grain yield per plant than control plants. Mechanistically, PAY1 could improve plant architecture via affecting polar auxin transport activity and altering endogenous indole‐3‐acetic acid distribution. Furthermore, introgression of PAY1 into elite rice cultivars, using marker‐assisted background selection, dramatically increased grain yield compared with the recipient parents. Overall, these results demonstrated that PAY1 could be a new beneficial genetic resource for shaping ideal plant architecture and breeding high‐yielding rice varieties.     

The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance

The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN‐FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t–GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1‐N‐naphthylphthalamic acid (NPA) treatment. The tissue‐specific expression patterns of OsPIN3t were also investigated using a β‐glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild‐type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants.     

CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF11, controls panicle architecture and seed size in rice

Panicle architecture and seed size are important agronomic traits that directly determine grain yield in rice (Oryza sativa L.). Although a number of key genes controlling panicle architecture and seed size have been cloned and characterized in recent years, their genetic and molecular mechanisms remain unclear. In this study, we identified a mutant that produced panicles with fascicled primary branching and reduced seeds in size. We isolated the underlying CLUSTERED PRIMARY BRANCH 1 (CPB1) gene, a new allele of DWARF11 (D11) encoding a cytochrome P450 protein involved in brassinosteroid (BR) biosynthesis pathway. Genetic transformation experiments confirmed that a His360Leu amino acid substitution residing in the highly conserved region of CPB1/D11 was responsible for the panicle architecture and seed size changes in the cpb1 mutants. Overexpression of CPB1/D11 under the background of cpb1 mutant not only rescued normal panicle architecture and plant height, but also had a larger leaf angle and seed size than the controls. Furthermore, the CPB1/D11 transgenic plants driven by panicle‐specific promoters can enlarge seed size and enhance grain yield without affecting other favourable agronomic traits. These results demonstrated that the specific mutation in CPB1/D11 influenced development of panicle architecture and seed size, and manipulation of CPB1/D11 expression using the panicle‐specific promoter could be used to increase seed size, leading to grain yield improvement in rice.     

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild‐type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss‐of‐function mutations, or in a dominant (gain‐of‐function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.     

Identification and characterization of OsEBS,a gene involved in enhanced plant biomass and spikelet number in rice

Common wild rice (Oryza rufipogon Griff.) is an important genetic reservoir for rice improvement. We investigated a quantitative trait locus (QTL), qGP5‐1, which is related to plant height, leaf size and panicle architecture, using a set of introgression lines of O. rufipogon in the background of the Indica cultivar Guichao2 (Oryza sativa L.). We cloned and characterized qGP5‐1 and confirmed that the newly identified gene OsEBS (enhancing biomass and spikelet number) increased plant height, leaf size and spikelet number per panicle, leading to an increase in total grain yield per plant. Our results showed that the increased size of vegetative organs in OsEBS‐expressed plants was enormously caused by increasing cell number. Sequence alignment showed that OsEBS protein contains a region with high similarity to the N‐terminal conserved ATPase domain of Hsp70, but it lacks the C‐terminal regions of the peptide‐binding domain and the C‐terminal lid. More results indicated that OsEBS gene did not have typical characteristics of Hsp70 in this study. Furthermore, Arabidopsis (Arabidopsis thaliana) transformed with OsEBS showed a similar phenotype to OsEBS‐transgenic rice, indicating a conserved function of OsEBS among plant species. Together, we report the cloning and characterization of OsEBS, a new QTL that controls rice biomass and spikelet number, through map‐based cloning, and it may have utility in improving grain yield in rice.     

OsPIN2, which encodes a member of the auxin efflux carrier proteins,is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice

Auxin flow is important for different root developmental processes such as root formation, emergence, elongation and gravitropism. However, the detailed information about the mechanisms regulating the auxin flow is less well understood in rice. We characterized the auxin transport‐related mutants, Ospin‐formed2‐1 (Ospin2‐1) and Ospin2‐2, which exhibited curly root phenotypes and altered lateral root formation patterns in rice. The OsPIN2 gene encodes a member of the auxin efflux carrier proteins that possibly regulates the basipetal auxin flow from the root tip toward the root elongation zone. According to DR5‐driven GUS expression, there is an asymmetric auxin distribution in the mutants that corresponded with the asymmetric cell elongation pattern in the mutant root tip. Auxin transport inhibitor, N‐1‐naphthylphthalamic acid and Ospin2‐1 Osiaa13 double mutant rescued the curly root phenotype indicating that this phenotype results from a defect in proper auxin distribution. The typical curly root phenotype was not observed when Ospin2‐1 was grown in distilled water as an alternative to tap water, although higher auxin levels were found at the root tip region of the mutant than that of the wild‐type. Therefore, the lateral root formation zone in the mutant was shifted basipetally compared with the wild‐type. These results reflect that an altered auxin flow in the root tip region is responsible for root elongation growth and lateral root formation patterns in rice.     

Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11‐controlled root development

Potassium (K) deficiency in plants confines root growth and decreases root‐to‐shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL‐related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low‐K‐enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11‐regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K‐deficiency‐enhanced expression of several RR genes encoding type‐A cytokinin‐responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K‐deficient soil increased total K uptake by 72% and grain yield by 24%–32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.     

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.     

Improving panicle exsertion of rice cytoplasmic male sterile line by combination of artificial microRNA and artificial target mimic

The adoption of hybrid rice caused the second leap in rice yield after the ‘green revolution’ and contributes substantially to food security of China and the world. However, almost all cytoplasmic male sterile lines (A lines) as females of hybrid rice have a natural deficiency of ‘panicle enclosure’, which blocks pollination between the A line and the fertility restorer line as the male (R line) of hybrid rice and decreases seed yield. In hybrid rice seed production, exogenous ‘920’ (the active ingredient is gibberellin A₃) must be applied to eliminate or alleviate panicle enclosure of the A line; however, this not only increases production cost and pollutes the environment, it also decreases seed quality. In this study, we designed a transgenic approach to improve plant height and panicle exsertion of the A line to facilitate hybrid rice production and maintain the semi‐dwarf plant type of the hybrid. This approach comprising two components—artificial microRNA (amiRNA) and artificial target mimicry—can manipulate the differential expression of the endogenous Eui1 gene that is associated with rice internode elongation in the A line and the hybrid. amiRNA is a recently developed gene silencing method with high specificity, while target mimicry is a natural mechanism inhibiting the miRNA function that was also recently characterized. This approach provides a paradigm to tune the expression of endogenous genes to achieve the desired phenotype by combining amiRNA and artificial target mimicry technologies.     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.     

Altered expression of the PTR/NRT1 homologue OsPTR9 affects nitrogen utilization efficiency,growth and grain yield in rice

The plant PTR/NRT1 (peptide transporter/nitrate transporter 1) gene family comprises di/tripeptide and low‐affinity nitrate transporters; some members also recognize other substrates such as carboxylates, phytohormones (auxin and abscisic acid), or defence compounds (glucosinolates). Little is known about the members of this gene family in rice (Oryza sativa L.). Here, we report the influence of altered OsPTR9 expression on nitrogen utilization efficiency, growth, and grain yield. OsPTR9 expression is regulated by exogenous nitrogen and by the day‐night cycle. Elevated expression of OsPTR9 in transgenic rice plants resulted in enhanced ammonium uptake, promotion of lateral root formation and increased grain yield. On the other hand, down‐regulation of OsPTR9 in a T‐DNA insertion line (osptr9) and in OsPTR9‐RNAi rice plants had the opposite effect. These results suggest that OsPTR9 might hold potential for improving nitrogen utilization efficiency and grain yield in rice breeding.     

PpeTAC1 promotes the horizontal growth of branches in peach trees and is a member of a functionally conserved gene family found in diverse plants species

Trees are capable of tremendous architectural plasticity, allowing them to maximize their light exposure under highly competitive environments. One key component of tree architecture is the branch angle, yet little is known about the molecular basis for the spatial patterning of branches in trees. Here, we report the identification of a candidate gene for the br mutation in Prunus persica (peach) associated with vertically oriented growth of branches, referred to as ‘pillar’ or ‘broomy’. Ppa010082, annotated as hypothetical protein in the peach genome sequence, was identified as a candidate gene for br using a next generation sequence‐based mapping approach. Sequence similarity searches identified rice TAC1 (tiller angle control 1) as a putative ortholog, and we thus named it PpeTAC1. In monocots, TAC1 is known to lead to less compact growth by increasing the tiller angle. In Arabidopsis, an attac1 mutant showed more vertical branch growth angles, suggesting that the gene functions universally to promote the horizontal growth of branches. TAC1 genes belong to a gene family (here named IGT for a shared conserved motif) found in all plant genomes, consisting of two clades: one containing TAC1‐like genes; the other containing LAZY1, which contains an EAR motif, and promotes vertical shoot growth in Oryza sativa (rice) and Arabidopsis through influencing polar auxin transport. The data suggest that IGT genes are ancient, and play conserved roles in determining shoot growth angles in plants. Understanding how IGT genes modulate branch angles will provide insights into how different architectural growth habits evolved in terrestrial 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.     

A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice

Auxin transport affects a variety of important growth and developmental processes in plants, including the regulation of shoot and root branching. The asymmetrical localization of auxin influx and efflux carriers within the plasma membrane establishes the auxin gradient and facilitates its transport. REH1, a rice EIR1 (Arabidopsis ethylene insensitive root 1)-like gene, is a putative auxin efflux carrier. Phylogenetic analysis of 32 members of the PIN family, taken from across different species, showed that in terms of evolutionary relationship, OsPIN1 is closer to the PIN1 family than to the PIN2 family. It is, therefore, renamed as OsPIN1 in this study. OsPIN1 was expressed in the vascular tissues and root primordial in a manner similar to AtPIN1. Adventitious root emergence and development were significantly inhibited in the OsPIN1 RNA interference (RNAi) transgenic plants, which was similar to the phenotype of NPA (N-1-naphthylphalamic acid, an auxin-transport inhibitor)-treated wild-type plants. alpha-naphthylacetic acid (alpha-NAA) treatment was able to rescue the mutated phenotypes occurring in the RNAi plants. Overexpression or suppression of the OsPIN1 expression through a transgenic approach resulted in changes of tiller numbers and shoot/root ratio. Taken together, these data suggest that OsPIN1 plays an important role in auxin-dependent adventitious root emergence and tillering.