Tiller Outgrowth Research Articles

Heading Date 3a Stimulates Tiller Bud Outgrowth in Oryza sativa L. through Strigolactone Signaling Pathway.

Heading date 3a (Hd3a, a FLOWERING LOCUS T (FT) ortholog from rice) is well known for its important role in rice (Oryza sativa L.), controlling floral transition under short-day (SD) conditions. Although the effect of Hd3a on promoting branching has been found, the underlying mechanism remains largely unknown. In this report, we overexpressed an Hd3a and BirAG (encoding a biotin ligase) fusion gene in rice, and found that early flowering and tiller bud outgrowth was promoted in BHd3aOE transgenic plants. On the contrary, knockout of Hd3a delayed flowering and tiller bud outgrowth. By using the BioID method, we identified multiple Hd3a proximal proteins. Among them, D14, D53, TPR1, TPR2, and TPRs are central components of the strigolactone signaling pathway, which has an inhibitory effect on rice tillering. The interaction between Hd3a, on the one hand, and D14 and D53 was further confirmed by the bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H), and co-immunoprecipitation (Co-IP) methods. We also found that Hd3a prevented the degradation of D53 induced by rac-GR24 (a strigolactone analog) in rice protoplasts. RT-qPCR assay showed that the expression levels of genes involved in strigolactone biosynthesis and signal transduction were altered significantly between WT and Hd3a overexpression (Hd3aOE) or mutant (hd3a) plants. OsFC1, a downstream target of the strigolactone signaling transduction pathway in controlling rice tillering, was downregulated significantly in Hd3aOE plants, whereas it was upregulated in hd3a lines. Collectively, these results indicate that Hd3a promotes tiller bud outgrowth in rice by attenuating the negative effect of strigolactone signaling on tillering and highlight a novel molecular network regulating rice tiller outgrowth by Hd3a.

Belowground growth strategies of native and invasive rhizomatous perennial grasses in response to precipitation variability, clipping, and competition

Invasive clonal species may exhibit different growth strategies than their native clonal competitors. In this study, we examined the spatial distribution of tiller outgrowth and the bud bank by comparing the investment in phalanx versus guerilla growth of a native and invasive perennial grass in North America. We also examined the effect of altered precipitation frequency, clipping, and competition on their clonal growth strategies. Investment in phalanx and guerilla growth was assessed by examining live propagule and tiller production from the plant crown versus its rhizomes. Although invasive Bromus inermis and native Pascopyrum smithii exhibited similar clonal growth strategies as young seedlings, their clonal growth strategies significantly differed by the end of their first growing season. Pascopyrum smithii invested in dual phalanx and guerilla tiller outgrowth and bud placement, and B. inermis primarily invested in phalanx tiller outgrowth and bud placement. Competition rather than intra-annual precipitation variability and clipping altered the clonal growth strategy of these species. Intra- and inter- specific competition did not alter tiller outgrowth for either species. However, inter-specific competition caused both species to alter their bud placement. Bromus inermis shifted more buds from phalanx to guerilla positions while P. smithii shifted in the opposite direction. This may enable invasive B. inermis to expand while confining native P. smithii to more localized areas in the future. Clonal growth strategies appear to be species specific and responsive to inter-specific competition. Investigating the belowground bud aspect of clonal growth can reveal the mechanism driving the future aboveground clonal growth strategy of native and invasive rhizomatous grasses and help inform the patterns of invasion within a plant community.

Modelling the dynamics and phenotypic consequences of tiller outgrowth and cessation in sorghum

Abstract Tillering affects canopy leaf area, and hence crop growth via capture of light, water and nutrients. Depending on the season, variation in tillering can result in increased or decreased yield. Reduced tillering has been associated with water-saving and enhanced yield in water-limited conditions. The objective of this study was to develop a generic model of the dynamics of tillering in sorghum incorporating key genetic and environmental controls. The dynamic of tillering was defined in four key phases—pre-tillering, tiller emergence, cessation of tiller emergence and cessation of tiller growth. Tillering commenced at full expansion of leaf four and thereafter was synchronized with leaf appearance. The potential total number of tillers (TTN) was dependent on a genetic propensity to tiller and an index of assimilate availability dependent on the shoot source–sink balance. Cessation of tiller emergence could occur before TTN depending on extent of competition from neighbours. Subsequent cessation of growth of emerged tillers was related to the extent of internal competition for assimilate among plant organs, resulting in prediction of final fertile tiller number (FTN). The model predicted tillering dynamics well in an experiment with a range in plant density. Plausibility simulations of FTN conducted for diverse field conditions in the Australian sorghum belt reflected expectations. The model is able to predict FTN as an emergent property. Its utility to explore GxMxE crop adaptation landscapes, guide molecular discovery, provide a generic template for other cereals and link to advanced methods for enhancing genetic gain in crops were discussed.

Strigolactones and Cytokinin Interaction in Buds in the Control of Rice Tillering.

Shoot branching is among the most crucial morphological traits in rice (Oryza sativa L.) and is physiologically modulated by auxins, cytokinins (CKs), and strigolactones (SLs) cumulatively in rice. A number of studies focused on the interplay of these three hormones in regulating rice tiller extension. The present study primarily aimed at determining the impact of different treatments, which were used to regulate rice tiller and axillary bud development on node 2 at the tillering stage and full heading stage, respectively. Transcription levels of several genes were quantified through qRT-PCR analysis, and an endogenous auxin and four types of CKs were determined through LC-MS/MS. Both nutrient deficiency and exogenous SL supply were found to inhibit rice tiller outgrowth by reducing the CK content in the tiller buds. Furthermore, supplying the inhibitor of both exogenous SLs and endogenous SL synthesis could also affect the expression level of OsCKX genes but not the OsIPT genes. Comparison of OsCKX gene expression pattern under exogenous SL and CK supply suggested that the induction of OsCKX expression was most likely via a CK-induced independent pathway. These results combined with the expression of CK type-A RR genes in bud support a role for SLs in regulating bud outgrowth through the regulation of local CK levels. SL functioned antagonistically with CK in regulating the outgrowth of buds on node 2, by promoting the OsCKX gene expression in buds.

Higher nitrogen content and auxin export from rice tiller enhance low-ammonium-dependent tiller outgrowth

In the early growth stage, nutrient uptake by rice roots is weak. However, rice tillering at this stage would require high N input. Thus, it is vital to clarify the mechanism involved in tillering capacity with low N inputs. In this report, two widely-planted japonica cultivars (cvs Yangyujing 2 and Nanjing 45) were selected mainly because, unlike cv. Nanjing 45, cv. Yangyujing 2 shows low-N-induced tiller outgrowth. Responses of tillers in two rice cultivars to mixture of N forms versus sole NH4+ supply were similar, suggesting that NH4+ plays a pivotal role in N-modulated rice tillering. Under low NH4+ supply, higher expression of OsAMT1.2, OsAMT1.3, OsGS1;2, and OsGS2 was recorded in the roots of cv. Yangyujing 2 in comparison with cv. Nanjing 45, ultimately resulting in higher N content and dry weight in cv. Yangyujing 2. Stronger 3H-IAA export from tiller stems was observed in cv. Yangyujing 2, mainly due to higher expression level of auxin efflux transporters. Moreover, tillers in auxin efflux transporter mutant ospin9 did not respond to NH4+ supply relative to wild-type plants. These findings can be used in the molecular breeding of rice varieties to simultaneously improve rice population productivity and reduce N fertilizer input.

Tiller Outgrowth in Rice (Oryza sativa L.) is Controlled by OsGT1, Which Acts Downstream of FC1 in a PhyB-Independent Manner

Tillering is one of the most important determinants of biomass and yield in rice (Oryza sativa L.). The capacity of plants to develop tillers from primordial meristems or buds is determined not only by the genotype but also by environmental cues. Here, we characterized the function of rice grassy tiller1 (OsGT1) and its interaction with other genetic and biological factors involved in tiller bud outgrowth in rice by generating OsGT1 RNA interference (RNAi) and overexpression (OX) lines. The tiller number was increased in OsGT1-RNAi mutants but strongly suppressed in OsGT1-OX lines. Expression analysis of OsGT1 in rice phyB mutants and in genotypes carrying various genetic combinations of GT1 RNAi and phyB demonstrated that OsGT1 is not involved in phyB-mediated suppression of tiller development in rice. Expression analysis of fine culm1 (fc1), a rice tb1 homolog, and molecular assays demonstrated that FC1 enhances the expression of OsGT1 by directly binding to its promoter. Comparison of the transcriptomic profiles of fc1 and OsGT1-RNAi mutants revealed differentially expressed genes (DEGs) common to both genotypes. Finally, analysis of tillering phenotypes of OX and RNAi seedlings treated with various phytohormones implied a possible role of OsGT1 in strigolactone-mediated tiller outgrowth. Overall, this study enhances our understanding of the diverse mechanisms of tiller development in grasses.

OsSHI1 Regulates Plant Architecture Through Modulating the Transcriptional Activity of IPA1 in Rice.

Tillering and panicle branching are important determinants of plant architecture and yield potential in rice (Oryza sativa). IDEAL PLANT ARCHITECTURE1 (IPA1) encodesSQUAMOSA PROMOTER BINDING PROTEIN-LIKE14, which acts as a key transcription factor regulating tiller outgrowth and panicle branching by directly activating the expression of O. sativa TEOSINTE BRANCHED1 (OsTB1) and O. sativa DENSE AND ERECT PANICLE1 (OsDEP1), thereby influencing grain yield in rice. Here, we report the identification of a rice mutant named shi1 that is characterized by dramatically reduced tiller number, enhanced culm strength, and increased panicle branch number. Map-based cloning revealed that O. sativa SHORT INTERNODES1 (OsSHI1) encodes a plant-specific transcription factor of the SHI family with a characteristic family-specific IGGH domain and a conserved zinc-finger DNA binding domain. Consistent with the mutant phenotype, OsSHI1 is predominantly expressed in axillary buds and young panicle, and its encoded protein is exclusively targeted to the nucleus. We show that OsSHI1 physically interacts with IPA1 both in vitro and in vivo. Moreover, OsSHI1 could bind directly to the promoter regions of both OsTB1 and OsDEP1 through a previously unrecognized cis-element (T/GCTCTAC motif). OsSHI1 repressed the transcriptional activation activity of IPA1 by affecting its DNA binding activity toward the promoters of both OsTB1 and OsDEP1, resulting in increased tiller number and diminished panicle size. Taken together, our results demonstrate that OsSHI1 regulates plant architecture through modulating the transcriptional activity of IPA1 and provide insight into the establishment of plant architecture in rice.

OsASN1 Plays a Critical Role in Asparagine-Dependent Rice Development.

Asparagine is one of the important amino acids for long-distance transport of nitrogen (N) in plants. However, little is known about the effect of asparagine on plant development, especially in crops. Here, a new T-DNA insertion mutant, asparagine synthetase 1 (asn1), was isolated and showed a different plant height, root length, and tiller number compared with wild type (WT). In asn1, the amount of asparagine decreased sharply while the total nitrogen (N) absorption was not influenced. In later stages, asn1 showed reduced tiller number, which resulted in suppressed tiller bud outgrowth. The relative expression of many genes involved in the asparagine metabolic pathways declined in accordance with the decreased amino acid concentration. The CRISPR/Cas9 mutant lines of OsASN1 showed similar phenotype with asn1. These results suggest that OsASN1 is involved in the regulation of rice development and is specific for tiller outgrowth.

Reduction in sucrose contents by downregulation of fructose-1,6-bisphosphatase 2 causes tiller outgrowth cessation in rice mutants lacking glutamine synthetase1;2

BackgroundOur previous transcriptomic analysis revealed that downregulation of nitrogen and carbon metabolism in the basal portions of the shoots inhibited cytosolic glutamine synthetase1;2 (GS1;2), which severely reduced rice tiller number. In the present study, we used rice mutants lacking GS1;2 (gs1;2 mutants) to determine the contribution of carbon metabolism to tiller growth.ResultsMetabolomic analysis indicated the effects of carbon metabolism disorder such as reductions in the levels of sugar metabolites (e.g., sucrose and glucose 6-phosphate) in the shoot basal portions of the gs1;2 mutant seedlings. Decrease in sucrose caused by the lack of GS1;2 was successfully restored to the wild-type levels by introducing OsGS1;2 cDNA into the mutants. In the basal portions of the shoots, the lack of GS1;2 caused low expression of cytosolic fructose 1,6-bisphosphatase2 (OscFBP2), which is a key cytosolic sucrose synthesis enzyme; it is especially important in the phloem companion cells of the nodal vascular anastomoses. NH4+ supply upregulated OscFBP2 expression in the shoot basal portions of the wild type but not in those of the gs1;2 mutants. Rice mutants lacking cFBPase2 presented with ~ 30% reduction in total cFBPase activity in the basal portions of their shoots. These mutants displayed reductions in sucrose levels of the basal portions of their shoots but not in their leaf blades. They also had relatively lower tiller numbers at the early growth stage.ConclusionsMetabolomic analysis revealed that the lack of GS1;2 reduced sucrose metabolism in the basal portions of the shoots. Our results indicated that sucrose reduction was caused by the downregulation of OscFBP2 expression in the basal portions of the gs1;2 mutant shoots. The reduction in sucrose content caused by the lack of cFBPase2 resulted in lower tiller number at the early growth stage. Therefore, adequate sucrose supply via cFBPase2 may be necessary for tiller growth in the basal portions of rice shoots.

Chlorophyll a fluorescence analysis can detect phosphorus deficiency under field conditions and is an effective tool to prevent grain yield reductions in spring barley (Hordeum vulgare L.)

Phosphorus (P) is an essential macronutrient with major impacts on global crop productivity. Recent work showed that chlorophyll a fluorescence analysis can be used as a sensitive indicator of latent P deficiency across different plant species. Here, we demonstrate that chlorophyll a fluorescence OJIP transients are a powerful tool for early detection of P deficiency directly in the field. Barley was grown in a P responsive field. One treatment received 30 kg P ha−1 at sowing, four treatments were fertilized with P at 26, 35, 46 or 56 days after sowing (DAS), respectively, and the final treatment did not receive any P throughout the experiment. Chlorophyll a fluorescence measurements, multi-elemental leaf analysis, and growth stage evaluation were performed 26, 35, 46, 56, and 69 DAS. Phosphorus deficiency during early vegetative growth irreversibly affected plant development including tiller outgrowth and grain yields. However, in the present study, yield reduction could be avoided if short-term P deficiency was corrected by application of P fertilizer no later than 35 days after sowing, when plants had not yet entered the tillering stage. The chlorophyll a fluorescence OJIP transients were able to detect latent P deficiency in this critical phase, thereby providing an opportunity for avoiding a potential yield reduction of up to 27 hkg ha−1. It was further noted, that chlorophyll a fluorescence analysis and P leaf tissue analysis should be performed during early vegetative growth, as probable remobilization of P within the plant during tillering and shoot differentiation masks the effects of P deficiency at the single leaf level. It is concluded that chlorophyll a fluorescence analysis provides a unique opportunity for a timely detection and correction of P deficiency under field conditions to prevent yield reductions.

Developmental dynamics of stem starch accumulation in Sorghum bicolor.

Sweet sorghums were identified that accumulate up to ~9% of their total stem dry weight as starch. Starch accumulated preferentially in stem pith parenchyma in close proximity to vascular bundles. Stem starch accumulated slowly between floral initiation and anthesis and more rapidly between anthesis and 43 days post‐anthesis before declining in parallel with tiller outgrowth. Genes involved in stem starch metabolism were identified through phylogenetic approaches and RNA‐seq analysis of Della stem gene expression during the starch accumulation phase of development. Genes differentially expressed in stems were identified that are involved in starch biosynthesis (i.e., AGPase SS/LS, starch synthases, starch‐branching enzymes), degradation (i.e., glucan‐water dikinase, β‐amylase, disproportionating enzyme, alpha‐glucan phosphorylase) and amyloplast sugar transport (glucose‐6‐P translocator). Transcripts encoding AGPase SS and LS subunits with plastid localization were differentially induced during stem starch accumulation indicating that ADP‐glucose for starch biosynthesis is primarily generated in stem plastids. Cytosolic heteroglucan metabolism may play a role in stem sucrose/starch accumulation because genes encoding cytosolic forms of the disproportionating enzyme and alpha‐glucan phosphorylase were induced in parallel with stem sucrose/starch accumulation. Information on the stem starch pathway obtained in this study will be useful for engineering sorghum stems with elevated starch thereby improving forage quality and the efficiency of biomass conversion to biofuels and bio‐products.

Phytohormone inhibitor treatments phenocopy brassinosteroid-gibberellin dwarf mutant interactions in maize.

SummaryPhytohormone biosynthesis produces metabolites with profound effects on plant growth and development. Modulation of hormone levels during developmental events, in response to the environment, by genetic polymorphism, or by chemical application, can reveal the plant processes most responsive to a phytohormone. Applications of chemical inhibitors and subsequent measurements of specific phytohormones can determine whether, and which, phytohormone is affected by a molecule. In many cases, the sensitivity of biochemical testing has determined multiple pathways affected by a single inhibitor. Genetic studies are not subject to this problem, and a wealth of data about the morphological impacts of hormone biosynthetic inhibition have accumulated through the study of enzyme mutants. In this work, we sought to assess the specificity of three triazole inhibitors of cytochrome P450s by determining their abilities to recapitulate the phenotypes of single and double mutants affected in the production of brassinosteroid (BR) and gibberellin (GA) biosynthesis. The GA biosynthetic inhibitors uniconazole (UCZ) and paclobutrazol (PAC) were applied to the BR biosynthetic mutant nana plant2 (na2), and all double‐mutant phenotypes were recovered in the UCZ treatment. PAC was unable to suppress the retention of pistils in the tassels of na2 mutant plants. The BR biosynthetic inhibitor propiconazole (PCZ) suppressed tiller outgrowth in the GA biosynthetic mutant dwarf5 (d5). All treatments were additive with genetic mutants for effects on plant height. Due to additional measurements performed here but not in previous studies of the double mutants, we detected new interactions between GA and BR biosynthesis affecting the days to tassel emergence and tassel branching. These experiments, a refinement of our previous model, and a discussion of the extension of this type of work are presented.

OsIAA6, a member of the rice Aux/IAA gene family, is involved in drought tolerance and tiller outgrowth

Auxin signaling is a fundamental part of many plant growth processes and stress responses and operates through Aux/IAA protein degradation and the transmission of the signal via auxin response factors (ARFs). A total of 31 Aux/IAA genes have been identified in rice (Oryza sativa), some of which are induced by drought stress. However, the mechanistic link between Aux/IAA expression and drought responses is not well understood. In this study we found that the rice Aux/IAA gene OsIAA6 is highly induced by drought stress and that its overexpression in transgenic rice improved drought tolerance, likely via the regulation of auxin biosynthesis genes. We observed that OsIAA6 was specifically expressed in the axillary meristem of the basal stem, which is the tissue that gives rise to tillers. A knock-down mutant of OsIAA6 showed abnormal tiller outgrowth, apparently due to the regulation of the auxin transporter OsPIN1 and the rice tillering inhibitor OsTB1. Our results confirm that the OsIAA6 gene is involved in drought stress responses and the control of tiller outgrowth.

The alteration in the architecture of a T-DNA insertion rice mutant osmtd1 is caused by up-regulation of MicroRNA156f.

Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter‐Binding Protein‐Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T‐DNA insertion mutant Osmtd1 (Oryza sativa multi‐tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T‐DNA insertion in Osmtd1. Further analysis revealed that the T‐DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild‐type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.

Alteration of osa-miR156e expression affects rice plant architecture and strigolactones (SLs) pathway.

Overexpressing osa--miR156e in rice produced a bushy mutant and osa--miR156e regulation of tillering may do this through the strigolactones (SLs) pathway. Appropriate downregulation of osa--miR156 expression contributed to the improvement of plant architecture. Tillering is one of the main determinants for rice architecture and yield. In this study, a bushy mutant of rice was identified with increased tiller number, reduced plant height, prolonged heading date, low seed setting, and small panicle size due to a T-DNA insertion which essentially elevated the expression of osa-miR156e. Transgenic plants with constitutive expression of osa-miR156e also had the bushy phenotype, which showed osa-miR156 may control apical dominance and tiller outgrowth via regulating the strigolactones signaling pathway. Furthermore, the extent of impaired morphology was correlated with the expression level of osa-miR156e. In an attempt to genetically improve rice architecture, ectopic expression of osa-miR156e under the GAL4-UAS system or OsTB1 promoter was conducted. According to agronomic trait analysis, pTB1:osa-miR156e transgenic plants significantly improved the grain yield per plant compared to plants overexpressing osa-miR156e, even though the yield was still inferior to the wild type, making it a very interesting albeit negative result. Our results suggested that osa-miR156 could serve as a potential tool for modifying rice plant architecture through genetic manipulation of the osa-miR156 expression level.

The making of a bushy grass with a branched flowering stem

In Arabidopsis thaliana, a eudicot species, the transcription factor LFY is expressed throughout the floral meristem and promotes their formation. The expression pattern of the rice LFY homolog-RFL shows distinct differences from that of its Arabidopsis counterpart. In the March issue of PNAS (2008) we have shown the temporally-regulated high-level expression of RFL in the apical meristem is necessary for its transition to an inflorescence meristem and thus to initiate flowering. RFL controls the time taken for flowering, by activating integrators of flowering signals such as OsSOC1 and RFT1. Further, the dynamic pattern of RFL expression in the branching inflorescence meristem (panicle) and in vegetative axillary meristems (tiller buds) is required for panicle branching and tiller outgrowth. Thus RFL functions determine the architecture of the rice plant. Here we propose a plausible model for a regulatory feedback loop between RFL and OsSOC1/RFT1 in controlling the vegetative to flowering phase transition. We discuss the possibility that non-cell autonomous RFL functions may also regulate signaling the net outcome of which determines the rice plant body plan.Addendum to: Rao NN, Prasad K, Kumar PR, Vijayraghavan U. Distinct regulatory role for RFL, the rice LFY homolog, in determining flowering time and plant architecture. Proc Natl Acad Sci USA 2008; 105:3646-51.