The D14 and KAI2 Orthologs of Gymnosperms Sense Strigolactones and KL Mimics, Respectively, and the Signals Are Transduced to Control Downstream Genes.

Strigolactones (SLs), lactone-containing carotenoid derivatives, function as signaling molecules in the rhizosphere, inducing symbiosis with arbuscular mycorrhizal. In addition, as a class of plant hormones, SLs control plant growth and development in flowering plants (angiosperms). Recent studies show that the ancestral function of SLs, which precede terrestrialization of plants, is as rhizosphere signaling molecules. SLs were then recruited as a class of plant hormones through the step-by-step acquisition of signaling components. The D14 gene encoding the SL receptor arose by gene duplication of KARRIKIN INSENSITIVE2 (KAI2), the receptor of karrikins and KAI2 ligand (KL), an unknown ligand, in the common ancestor of seed plants. KL signaling targets SMAX1, a repressor protein. On the other hand, the SL signaling targets SMXL78 subclade repressors, which arose by duplication of SMAX1 in angiosperms. Thus, gymnosperms contain the SL receptor D14 but not SMXL78, the SL signaling-specific repressor proteins. We studied two gymnosperm species, ginkgo (Ginkgo biloba) and Japanese umbrella pine (Sciadopitys verticillata), to clarify whether SLs are perceived and the signals are transduced in gymnosperms. We show that D14 and KAI2 of ginkgo and Japanese umbrella pine specifically perceive an SL analog and KL mimic, respectively. Furthermore, our results suggest that both SL signaling and KL signaling target SMAX1, and the specific localization of the receptor may result in the specificity of the signaling in gymnosperms.

DIENELACTONE HYDROLASE LIKE PROTEIN1 negatively regulates the KAI2-ligand pathway in Marchantia polymorpha.

Karrikins are smoke-derived butenolides that induce seed germination and photomorphogenesis in a wide range of plants.1,2,3 KARRIKIN INSENSITIVE2 (KAI2), a paralog of a strigolactone receptor, perceives karrikins or their metabolized products in Arabidopsis thaliana.4,5,6,7 Furthermore, KAI2 is thought to perceive an unidentified plant hormone, called KAI2 ligand (KL).8,9 KL signal is transduced via the interaction between KAI2, MORE AXILLARY GROWTH2 (MAX2), and SUPPRESSOR of MORE AXILLARY GROWTH2 1 LIKE family proteins (SMXLs), followed by the degradation of SMXLs.4,7,10,11,12,13,14 This signaling pathway is conserved both in A. thaliana and the bryophyte Marchantia polymorpha.14 Although the KL signaling pathway is well characterized, the KL metabolism pathways remain poorly understood. Here, we show that DIENELACTONE HYDROLASE LIKE PROTEIN1 (DLP1) is a negative regulator of the KL pathway in M. polymorpha. The KL signal induces DLP1 expression. DLP1 overexpression lines phenocopied the Mpkai2a and Mpmax2 mutants, while dlp1 mutants phenocopied the Mpsmxl mutants. Mutations in the KL signaling genes largely suppressed these phenotypes, indicating that DLP1 acts upstream of the KL signaling pathway, although DLP1 also has KL pathway-independent functions. DLP1 exhibited enzymatic activity toward a potential substrate, suggesting the possibility that DLP1 works through KL inactivation. Investigation of DLP1 homologs in A. thaliana revealed that they do not play a major role in the KL pathway, suggesting different mechanisms for the KL signal regulation. Our findings provide new insights into the regulation of the KL signal in M. polymorpha and the evolution of the KL pathway in land plants.

Activation of Strigolactone Biosynthesis by the DWARF14-LIKE/KARRIKIN-INSENSITIVE2 Pathway in Mycorrhizal Angiosperms, but Not in Arabidopsis, a Non-mycorrhizal Plant.

Strigolactones (SLs) are a class of plant hormones that regulate many aspects of plant growth and development. SLs also improve symbiosis with arbuscular mycorrhizal fungi (AMF) in the rhizosphere. Recent studies have shown that the DWARF14-LIKE (D14L)/KARRIKIN-INSENSITIVE2 (KAI2) family, paralogs of the SL receptor D14, are required for AMF colonization in several flowering plants, including rice. In this study, we found that (-)-GR5, a 2'S-configured enantiomer of a synthetic SL analog (+)-GR5, significantly activated SL biosynthesis in rice roots via D14L. This result is consistent with a recent report, showing that the D14L pathway positively regulates SL biosynthesis in rice. In fact, the SL levels tended to be lower in the roots of the d14l mutant under both inorganic nutrient-deficient and -sufficient conditions. We also show that the increase in SL levels by (-)-GR5 was observed in other mycorrhizal plant species. In contrast, the KAI2 pathway did not upregulate the SL level and the expression of SL biosynthetic genes in Arabidopsis, a non-mycorrhizal plant. We also examined whether the KAI2 pathway enhances SL biosynthesis in the liverwort Marchantia paleacea, where SL functions as a rhizosphere signaling molecule for AMF. However, the SL level and SL biosynthetic genes were not positively regulated by the KAI2 pathway. These results imply that the activation of SL biosynthesis by the D14L/KAI2 pathway has been evolutionarily acquired after the divergence of bryophytes to efficiently promote symbiosis with AMF, although we cannot exclude the possibility that liverworts have specifically lost this regulatory system.

Control of vegetative reproduction in Marchantia polymorpha by the KAI2-ligand signaling pathway.

In vegetative reproduction of Marchantia polymorpha (M. polymorpha), propagules, called gemmae, are formed in gemma cups. Despite its significance for survival, control of gemma and gemma cup formation by environmental cues is not well understood. We show here that the number of gemmae formed in a gemma cup is a genetic trait. Gemma formation starts from the central region of the floor of the gemma cup, proceeds to the periphery, and terminates when the appropriate number of gemmae is initiated. The MpKARRIKIN INSENSITIVE2 (MpKAI2)-dependent signaling pathway promotes gemma cup formation and gemma initiation. The number of gemmae in a cup is controlled by modulating the ON/OFF switch of the KAI2-dependent signaling. Termination of the signaling results in the accumulation of MpSMXL, a suppressor protein. In the Mpsmxl mutants, gemma initiation continues, leading to the formation of a highly increased number of gemmae in a cup. Consistent with its function, the MpKAI2-dependent signaling pathway is active in gemma cups where gemmae initiate, as well as in the notch region of the mature gemma and midrib of the ventral side of the thallus. In this work, we also show that GEMMA CUP-ASSOCIATED MYB1 works downstream of this signaling pathway to promote gemma cup formation and gemma initiation. We also found that the availability of potassium affects gemma cup formation independently from the KAI2-dependent signaling pathway in M. polymorpha. We propose that the KAI2-dependent signaling pathway functions to optimize vegetative reproduction by adapting to the environment in M. polymorpha.

An ancestral function of strigolactones as symbiotic rhizosphere signals.

In flowering plants, strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), an SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating its origin in the common ancestor of land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

Lipid exchanges drove the evolution of mutualism during plant terrestrialization.

Symbiosis with arbuscular mycorrhizal fungi (AMF) improves plant nutrition in most land plants, and its contribution to the colonization of land by plants has been hypothesized. Here, we identify a conserved transcriptomic response to AMF among land plants, including the activation of lipid metabolism. Using gain of function, we show the transfer of lipids from the liverwort Marchantia paleacea to AMF and its direct regulation by the transcription factor WRINKLED (WRI). Arbuscules, the nutrient-exchange structures, were not formed in loss-of-function wri mutants in M. paleacea, leading to aborted mutualism. Our results show the orthology of the symbiotic transfer of lipids across land plants and demonstrate that mutualism with arbuscular mycorrhizal fungi was present in the most recent ancestor of land plants 450 million years ago.