Junko Kyozuka

Inhibition of shoot branching by new terpenoid plant hormones

Shoot branching is a major determinant of plant architecture and is highly regulated by endogenous and environmental cues. Two classes of hormones, auxin and cytokinin, have long been known to have an important involvement in controlling shoot branching. Previous studies using a series of mutants with enhanced shoot branching suggested the existence of a third class of hormone(s) that is derived from carotenoids, but its chemical identity has been unknown. Here we show that levels of strigolactones, a group of terpenoid lactones, are significantly reduced in some of the branching mutants. Furthermore, application of strigolactones inhibits shoot branching in these mutants. Strigolactones were previously found in root exudates acting as communication chemicals with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Thus, we propose that strigolactones act as a new hormone class—or their biosynthetic precursors—in regulating above-ground plant architecture, and also have a function in underground communication with other neighbouring organisms.

Direct control of shoot meristem activity by a cytokinin-activating enzyme

The growth of plants depends on continuous function of the meristems. Shoot meristems are responsible for all the post-embryonic aerial organs, such as leaves, stems and flowers. It has been assumed that the phytohormone cytokinin has a positive role in shoot meristem function. A severe reduction in the size of meristems in a mutant that is defective in all of its cytokinin receptors has provided compelling evidence that cytokinin is required for meristem activity. Here, we report a novel regulation of meristem activity, which is executed by the meristem-specific activation of cytokinins. The LONELY GUY (LOG) gene of rice is required to maintain meristem activity and its loss of function causes premature termination of the shoot meristem. LOG encodes a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. Revising the long-held idea of multistep reactions, LOG directly converts inactive cytokinin nucleotides to the free-base forms, which are biologically active, by its cytokinin-specific phosphoribohydrolase activity. LOG messenger RNA is specifically localized in shoot meristem tips, indicating the activation of cytokinins in a specific developmental domain. We propose the fine-tuning of concentrations and the spatial distribution of bioactive cytokinins by a cytokinin-activating enzyme as a mechanism that regulates meristem activity.

High frequency plant regeneration from rice protoplasts by novel nurse culture methods

SummaryNovel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.

LAX and SPA: Major regulators of shoot branching in rice

The aerial architecture of plants is determined primarily by the pattern of shoot branching. Although shoot apical meristem initiation during embryogenesis has been extensively studied by molecular genetic approaches using Arabidopsis, little is known about the genetic mechanisms controlling axillary meristem initiation, mainly because of the insufficient number of mutants that specifically alter it. We identified the LAX PANICLE (LAX) and SMALL PANICLE (SPA) genes as the main regulators of axillary meristem formation in rice. LAX encodes a basic helix–loop–helix transcription factor and is expressed in the boundary between the shoot apical meristem and the region of new meristem formation. This pattern of LAX expression was repeatedly observed in every axillary meristem, consistent with our observation that LAX is involved in the formation of all types of axillary meristems throughout the ontogeny of a rice plant. Ectopic LAX expression in rice caused pleiotropic effects, including dwarfing, an altered pattern of stem elongation, darker color, bending of the lamina joint, absence of the midribs of leaves, and severe sterility.

The role of barren stalk1 in the architecture of maize

The architecture of higher plants is established through the activity of lateral meristems—small groups of stem cells formed during vegetative and reproductive development. Lateral meristems generate branches and inflorescence structures, which define the overall form of a plant, and are largely responsible for the evolution of different plant architectures. Here, we report the isolation of the barren stalk1 gene, which encodes a non-canonical basic helix–loop–helix protein required for the initiation of all aerial lateral meristems in maize. barren stalk1 represents one of the earliest genes involved in the patterning of maize inflorescences, and, together with the teosinte branched1 gene, it regulates vegetative lateral meristem development. The architecture of maize has been a major target of selection for early agriculturalists and modern farmers, because it influences harvesting, breeding strategies and mechanization. By sampling nucleotide diversity in the barren stalk1 region, we show that two haplotypes entered the maize gene pool from its wild progenitor, teosinte, and that only one was incorporated throughout modern inbreds, suggesting that barren stalk1 was selected for agronomic purposes.

FRIZZY PANICLE is required to prevent the formation of axillary meristems and to establish floral meristem identity in rice spikelets

Inflorescences of grass species have a distinct morphology in which florets are grouped in compact branches called spikelets. Although many studies have shown that the molecular and genetic mechanisms that control floret organ formation are conserved between monocots and dicots, little is known about the genetic pathway leading to spikelet formation. In the frizzy panicle (fzp) mutant of rice, the formation of florets is replaced by sequential rounds of branching. Detailed analyses revealed that several rudimentary glumes are formed in each ectopic branch, indicating that meristems acquire spikelet identity. However, instead of proceeding to floret formation, axillary meristems are formed in the axils of rudimentary glumes and they either arrest or develop into branches of higher order. The fzp mutant phenotype suggests that FZP is required to prevent the formation of axillary meristems within the spikelet meristem and permit the subsequent establishment of floral meristem identity. The FZP gene was isolated by transposon tagging. FZP encodes an ERF transcription factor and is the rice ortholog of the maize BD1 gene. Consistent with observations from phenotypic analyses, FZP expression was found to be restricted to the time of rudimentary glumes differentiation in a half-ring domain at the base of which the rudimentary glume primordium emerged.

New genes in the strigolactone-related shoot branching pathway

Shoot branching is controlled by the formation and subsequent outgrowth of axillary buds in the axils of leaves. Axillary buds are indeterminate structures that can be arrested and await endogenous or environmental cues for outgrowth. A major breakthrough in this area of plant development has been the discovery that a specific group of terpenoid lactones, named strigolactones, can directly or indirectly, inhibit axillary bud outgrowth. Since that discovery, new branching mutants have been identified with reduced strigolactone levels or which are defective in strigolactone regulation or response. DWARF27 and DWARF14 probably act on strigolactone biosynthesis and strigolactone metabolism or signal transduction, respectively. Auxin signaling mutants have also been useful in demonstrating that strigolactone levels are mediated by a classical auxin signal transduction pathway. The discovery and characterization of these mutants is an important first step toward understanding the mechanisms of strigolactone biosynthesis and signaling and their importance in regulating shoot branching.

FINE CULM1 (FC1) Works Downstream of Strigolactones to Inhibit the Outgrowth of Axillary Buds in Rice

Recent studies of highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones (SLs) act as hormones that inhibit shoot branching. The identification of genes that work downstream of SLs is required for a better understanding of how SLs control the growth of axillary buds. We found that the increased tillering phenotype of fine culm1 (fc1) mutants of rice is not rescued by the application of 1 μM GR24, a synthetic SL analog. Treatment with a high concentration of GR24 (10 μM) causes suppression of tiller growth in wild-type plants, but is not effective on fc1 mutants, implying that proper FC1 functioning is required for SLs to inhibit bud growth. Overexpression of FC1 partially rescued d3-2 defects in the tiller growth and plant height. An in situ hybridization analysis showed that FC1 mRNA accumulates in axillary buds, the shoot apical meristem, young leaves, vascular tissues and the tips of crown roots. FC1 mRNA expression was not significantly affected by GR24, suggesting that transcriptional induction may not be the mechanism by which SLs affect FC1 functioning. On the other hand, the expression level of FC1 is negatively regulated by cytokinin treatment. We propose that FC1 acts as an integrator of multiple signaling pathways and is essential to the fine-tuning of shoot branching in rice.

Structures of D14 and D14L in the strigolactone and karrikin signaling pathways

Strigolactones (SLs) are plant hormones that inhibit shoot branching. DWARF14 (D14) inhibits rice tillering and is an SL receptor candidate in the branching inhibition pathway, whereas the close homologue DWARF14‐LIKE (D14L) participates in the signaling pathway of karrikins (KARs), which are derived from burnt vegetation as smoke stimulants of seed germination. We provide the first evidence for direct binding of the bioactive SL analogue GR24 to D14. Isothermal titration calorimetry measurements show a D14–GR24 binding affinity in the sub‐micromolar range. Similarly, bioactive KAR1 directly binds D14L in the micromolar range. The crystal structure of rice D14 shows a compact α‐/β‐fold hydrolase domain forming a deep ligand‐binding pocket capable of accommodating GR24. Insertion of four α‐helices between β6 strand and αD helix forms the helical cap of the pocket, although the pocket is open to the solvent. The pocket contains the conserved catalytic triad Ser‐His‐Asp aligned with the oxyanion hole, suggesting hydrolase activity. Although these structural characteristics are conserved in D14L, the D14L pocket is smaller than that of D14. The KAR‐insensitive mutation kai2‐1 is located at the prominent long β6‐αD1 loop, which is characteristic in D14 and D14L, but not in related α‐/β‐fold hydrolases.

Low-affinity cation transporter (OsLCT1) regulates cadmium transport into rice grains

Accumulation of cadmium (Cd) in rice (Oryza sativa L.) grains poses a potential health problem, especially in Asia. Most Cd in rice grains accumulates through phloem transport, but the molecular mechanism of this transport has not been revealed. In this study, we identified a rice Cd transporter, OsLCT1, involved in Cd transport to the grains. OsLCT1-GFP was localized at the plasma membrane in plant cells, and OsLCT1 showed Cd efflux activity in yeast. In rice plants, strong OsLCT1 expression was observed in leaf blades and nodes during the reproductive stage. In the uppermost node, OsLCT1 transcripts were detected around large vascular bundles and in diffuse vascular bundles. RNAi-mediated knockdown of OsLCT1 did not affect xylem-mediated Cd transport but reduced phloem-mediated Cd transport. The knockdown plants of OsLCT1 accumulated approximately half as much Cd in the grains as did the control plants. The content of other metals in rice grains and plant growth were not negatively affected by OsLCT1 suppression. These results suggest that OsLCT1 functions at the nodes in Cd transport into grains and that in a standard japonica cultivar, the regulation of OsLCT1 enables the generation of “low-Cd rice” without negative effects on agronomical traits. These findings identify a transporter gene for phloem Cd transport in plants.