Junko Kyozuka

Summary: The DWARF14 (D14) gene of rice functions within the signaling pathway of strigolactones, a group of plant hormones that inhibits shoot branching. We isolated a recessive mutant named super apical dormant (sad1‐1) from a suppressor screen of d14‐1. The growth of tillers (vegetative shoot branches) is suppressed in both the d14‐1 sad1‐1 double mutant and the sad1‐1 single mutant. In addition, the sad1‐1 mutant shows pleiotropic defects throughout development. SAD1 encodes an ortholog of RPA34.5, a subunit of RNA polymerase I (Pol I). Consequently, the level of ribosomal RNA (rRNA) is severely reduced in the sad1‐1 mutant. These results indicate that proper ribosome function is a prerequisite for normal development in plants. The Arabidopsis ortholog of SAD1 was previously isolated as a Mediator‐interacting protein. Here we show that SAD1 interacts physically with the Mediator complex through direct binding with OsMED4, a component of the middle module of the Mediator complex in rice. It is known that Mediator interacts with Pol II, which transcribes mRNAs and functions as a central regulator of transcription. This study indicates a novel aspect of Mediator function in Pol I‐controlled rRNA transcription. TFIIF2 and RPC53 are the counterparts of RPA34.5 in Pol II and Pol III, respectively. We demonstrate that the rice orthologs of these proteins also interact with OsMED4. Our results suggest that interaction with MED4 in the Mediator complex is a common feature of the three types of RNA polymerases.

Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant tawawa1-D , the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in TAWAWA1 ( TAW1 ) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences. TAW1 encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs. TAW1 expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the SHORT VEGETATIVE PHASE subfamily of MADS-box genes function downstream of TAW1 . We thus propose that TAW1 is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity.

The architecture of rice is greatly influenced by the growth of tillers, i.e. vegetative shoot branches. OsSPL14, a member of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, and strigolactones (SLs) are known to suppress outgrowth of tiller buds. OsSPL14 also regulates panicle development. We show that OsSPL14 mRNA accumulates in leaf primordia during the vegetative phase and in the primordia of bracts, or modified leaves, in the panicles, but not in the meristems. OsSPL14 is a target of miR156, and accumulation of OsSPL14 transcripts is negatively regulated by miR156. The enhancement of the expression level of OsSPL14 by the introduction of the mOsSPL14 gene, in which the miR156 cleavage site is mutated, resulted in an increase in the plastochron, an acceleration of flowering and a decrease in tiller number in the wild type and in dwarf10-2, an SL-deficient mutant. Our analysis suggests that OsSPL14 and SLs function in parallel pathways to suppress tiller growth. SLs exuded from roots trigger germination of root parasitic plants that can cause severe damage to crop productivity. SL-deficient mutants, however, exhibit an excess branching phenotype which is usually undesirable for productivity. Our results indicate that OsSPL14 can be used to manipulate the branching patterns of SL-deficient mutants. We also confirmed that this strategy is applicable to Arabidopsis. A greater understanding of the OsSPL14 and SL pathways and their interactions may help in the production of root parasite-resistant crops.

In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal.

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.

In rice panicle development, new meristems are generated sequentially in an organized manner and acquire their identity in a time- and position-dependent manner. In the panicle of the panicle phytomer2-1 (pap2-1) mutant, the pattern of meristem initiation is disorganized and newly formed meristems show reduced competency to become spikelet meristems, resulting in the transformation of early arising spikelets into rachis branches. In addition, rudimentary glumes and sterile lemmas, the outermost organs of the spikelet, elongate into a leafy morphology. We propose that PAP2 is a positive regulator of spikelet meristem identity. Map-based cloning revealed that PAP2 encodes OsMADS34, a member of the SEPALLATA (SEP) subfamily of MADS-box proteins. PAP2/OsMADS34 belongs to the LOFSEP subgroup of MADS-box genes that show grass-specific diversification caused by gene duplication events. All five SEP subfamily genes in rice are expressed exclusively during panicle development, while their spatial and temporal expression patterns vary. PAP2 expression starts the earliest among the five SEP genes, and a low but significant level of PAP2 mRNA was detected in the inflorescence meristem, in branch meristems immediately after the transition, and in glume primordia, consistent with its role in the early development of spikelet formation. Our study provides new evidence supporting the hypothesis that the genes of the LOFSEP subgroup control developmental processes that are unique to grass species.

Recent studies using highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones, a group of terpenoid lactones, act as a new hormone class, or its biosynthetic precursors, in inhibiting shoot branching. Here, we provide evidence that DWARF14 (D14) inhibits rice tillering and may act as a new compo-nent of the strigolactone-dependent branching inhibition pathway. The d14 mutant exhibits increased shoot branch-ing with reduced plant height like the previously characterized strigolactone-deficient and -insensitive mutants d10 and d3, respectively. The d10-1 d14-1 double mutant is phenotypically indistinguishable from the d10-1 and d14-1 single mutants, consistent with the idea that D10 and D14 function in the same pathway. However, unlike with d10, the d14 branching phenotype could not be rescued by exogenous strigolactones. In addition, the d14 mutant contained a higher level of 2′-epi-5-deoxystrigol than the wild type. Positional cloning revealed that D14 encodes a protein of the α/β-fold hydrolase superfamily, some members of which play a role in metabolism or signaling of plant hormones. We propose that D14 functions downstream of strigolactone synthesis, as a component of hormone signaling or as an enzyme that participates in the conversion of strigolactones to the bioactive form.