Unveiling the complexity of strigolactones: exploring structural diversity, biosynthesis pathways, and signaling mechanisms.

Strigolactone is the collective name for compounds containing a butenolide as a part of their structure, first discovered as compounds that induce seed germination of root parasitic plants. They were later found to be rhizosphere signaling molecules that induce hyphal branching of arbuscular mycorrhizal fungi, and, finally, they emerged as a class of plant hormones. Strigolactones are found in root exudates, where they display a great variability in their chemical structure. Their structure varies among plant species, and multiple strigolactones can exist in one species. Over 30 strigolactones have been identified, yet the chemical structure of the strigolactone that functions as an endogenous hormone and is found in the above-ground parts of plants remains unknown. We discuss our current knowledge of the synthetic pathways of diverse strigolactones and their regulation, as well as recent progress in identifying strigolactones as plant hormones. Strigolactone is perceived by the DWARF14 (D14), receptor, an α/ß hydrolase which originated by gene duplication of KARRIKIN INSENSITIVE 2 (KAI2). D14 and KAI2 signaling pathways are partially overlapping paralogous pathways. Progress in understanding the signaling mechanisms mediated by two α/ß hydrolase receptors as well as remaining challenges in the field of strigolactone research are reviewed.

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.

Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface.

Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

Environmental control of rice flowering time.

Correct measurement of environmental parameters is fundamental for plant fitness and survival, as well as for timing developmental transitions, including the switch from vegetative to reproductive growth. Important parameters that affect flowering time include day length (photoperiod) and temperature. Their response pathways have been best described in Arabidopsis, which currently offers a detailed conceptual framework and serves as a comparison for other species. Rice, the focus of this review, also possesses a photoperiodic flowering pathway, but 150 million years of divergent evolution in very different environments have diversified its molecular architecture. The ambient temperature perception pathway is strongly intertwined with the photoperiod pathway and essentially converges on the same genes to modify flowering time. When observing network topologies, it is evident that the rice flowering network is centered on EARLY HEADING DATE 1, a rice-specific transcriptional regulator. Here, we summarize the most important features of the rice photoperiodic flowering network, with an emphasis on its uniqueness, and discuss its connections with hormonal, temperature perception, and stress pathways.

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.

Two florigens and a florigen-like protein form a triple regulatory module at the shoot apical meristem to promote reproductive transitions in rice.

Many plant species monitor and respond to changes in day length (photoperiod) for aligning reproduction with a favourable season. Day length is measured in leaves and, when appropriate, leads to the production of floral stimuli called florigens that are transmitted to the shoot apical meristem to initiate inflorescence development1. Rice possesses two florigens encoded by HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1)2. Here we show that the arrival of Hd3a and RFT1 at the shoot apical meristem activates FLOWERING LOCUS T-LIKE 1 (FT-L1), encoding a florigen-like protein that shows features partially differentiating it from typical florigens. FT-L1 potentiates the effects of Hd3a and RFT1 during the conversion of the vegetative meristem into an inflorescence meristem and organizes panicle branching by imposing increasing determinacy to distal meristems. A module comprising Hd3a, RFT1 and FT-L1 thus enables the initiation and balanced progression of panicle development towards determinacy.

Precise and heritable gene targeting in rice using a sequential transformation strategy.

Gene targeting (GT) is a powerful tool for modifying endogenous genomic sequences of interest, such as sequence replacement and gene knockin. Although the efficiency of GT is extremely low in higher plants, engineered sequence-specific nucleases (SSNs)-mediated double-strand breaks (DSBs) can improve GT frequency. We recently reported a CRISPR-Cas9-mediated approach for heritable GT in Arabidopsis, called the "sequential transformation" strategy. For efficient establishment of GT via the sequential transformation method, strong Cas9 activity and robust DSBs are required in the plant cells being infected with Agrobacterium carrying sgRNA and donor DNA. Accordingly, we generated two independent parental lines with maize Ubiquitin 1 promoter-driven Cas9 and established sequential transformation-mediated GT in the Japonica rice cultivar Oryza sativa Nipponbare. We achieved precise GFP knockin into the endogenous OsFTL1 and OsROS1a loci. We believe that our GT technology could be widely utilized in rice research and breeding applications.

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.

ABERRANT PANICLE ORGANIZATION2 controls multiple steps in panicle formation through common direct-target genes.

At the transition from vegetative to reproductive growth in rice (Oryza sativa), a developmental program change occurs, resulting in panicle (rice inflorescence) formation. The initial event of the transition is the change of the shoot apical meristem to an inflorescence meristem (IM), accompanied by a rapid increase in the meristem size. Suppression of leaf growth also occurs, resulting in the formation of bracts. The IM generates branch meristems (BMs), indeterminate meristems that reiteratively generate next-order meristems. All meristems eventually acquire a determinate spikelet meristem identity and terminate after producing a floret. ABERRANT PANICLE ORGANIZATION2 (APO2) is the rice ortholog of Arabidopsis (Arabidopsis thaliana) LEAFY (LFY), a plant-specific transcription factor (TF). APO2 is a positive regulator of panicle branch formation. Here, we show that APO2 is also required to increase the meristem size of the IM and suppress bract outgrowth. We identified genes directly and indirectly regulated by APO2 and identified APO2-binding sites. These analyses showed that APO2 directly controls known regulators of panicle development, including SQUAMOSA PROMOTER BINDING PROTEIN LIKE14 and NECK LEAF1. Furthermore, we revealed that a set of genes act as downstream regulators of APO2 in controlling meristem cell proliferation during reproductive transition, bract suppression, and panicle branch formation. Our findings indicate that APO2 acts as a master regulator of rice panicle development by regulating multiple steps in the reproductive transition through directly controlling a set of genes.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

Origins and evolution of the dual functions of strigolactones as rhizosphere signaling molecules and plant hormones.

Strigolactones (SLs) play roles as a class of plant hormones and rhizosphere signaling chemicals that induce hyphal branching of arbuscular mycorrhizal fungi and seed germination of parasitic plants. Therefore, SLs have dual functions. Recent progress in genome sequencing and genetic studies of bryophytes and algae has begun to shed light on the origin and evolution of these two functions of SLs.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

The bryophytes Physcomitrium patens and Marchantia polymorpha as model systems for studying evolutionary cell and developmental biology in plants.

Bryophytes are nonvascular spore-forming plants. Unlike in flowering plants, the gametophyte (haploid) generation of bryophytes dominates the sporophyte (diploid) generation. A comparison of bryophytes with flowering plants allows us to answer some fundamental questions raised in evolutionary cell and developmental biology. The moss Physcomitrium patens was the first bryophyte with a sequenced genome. Many cell and developmental studies have been conducted in this species using gene targeting by homologous recombination. The liverwort Marchantia polymorpha has recently emerged as an excellent model system with low genomic redundancy in most of its regulatory pathways. With the development of molecular genetic tools such as efficient genome editing, both P. patens and M. polymorpha have provided many valuable insights. Here, we review these advances with a special focus on polarity formation at the cell and tissue levels. We examine current knowledge regarding the cellular mechanisms of polarized cell elongation and cell division, including symmetric and asymmetric cell division. We also examine the role of polar auxin transport in mosses and liverworts. Finally, we discuss the future of evolutionary cell and developmental biological studies in plants.

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.

Major components of the KARRIKIN INSENSITIVE2-dependent signaling pathway are conserved in the liverwort Marchantia polymorpha.

KARRIKIN INSENSITIVE2 (KAI2) was first identified as a receptor of karrikins, smoke-derived germination stimulants. KAI2 is also considered a receptor of an unidentified endogenous molecule called the KAI2 ligand. Upon KAI2 activation, signals are transmitted through the degradation of D53/SMXL proteins via MAX2-dependent ubiquitination. Although components in the KAI2-dependent signaling pathway, namely MpKAI2A and MpKAI2B, MpMAX2, and MpSMXL, exist in the genome of the liverwort Marchantia polymorpha, their functions remain unknown. Here, we show that early thallus growth is retarded and gemma dormancy in the dark is suppressed in Mpkai2a and Mpmax2 loss-of-function mutants. These defects are counteracted in Mpkai2a Mpsmxl and Mpmax2 Mpsmxl double mutants indicating that MpKAI2A, MpMAX2, and MpSMXL act in the same genetic pathway. Introduction of MpSMXLd53, in which a domain required for degradation is mutated, into wild-type plants mimicks Mpkai2a and Mpmax2 plants. In addition, the detection of citrine fluorescence in Nicotiana benthamiana cells transiently expressing a SMXL-Citrine fusion protein requires treatment with MG132, a proteasome inhibitor. These findings imply that MpSMXL is subjected to degradation, and that the degradation of MpSMXL is crucial for MpKAI2A-dependent signaling in M. polymorpha. Therefore, we claim that the basic mechanisms in the KAI2-dependent signaling pathway are conserved in M. polymorpha.

Plant stem cell research is uncovering the secrets of longevity and persistent growth.

Plant stem cells have several extraordinary features: they are generated de novo during development and regeneration, maintain their pluripotency, and produce another stem cell niche in an orderly manner. This enables plants to survive for an extended period and to continuously make new organs, representing a clear difference in their developmental program from animals. To uncover regulatory principles governing plant stem cell characteristics, our research project 'Principles of pluripotent stem cells underlying plant vitality' was launched in 2017, supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese government. Through a collaboration involving 28 research groups, we aim to identify key factors that trigger epigenetic reprogramming and global changes in gene networks, and thereby contribute to stem cell generation. Pluripotent stem cells in the shoot apical meristem are controlled by cytokinin and auxin, which also play a crucial role in terminating stem cell activity in the floral meristem; therefore, we are focusing on biosynthesis, metabolism, transport, perception, and signaling of these hormones. Besides, we are uncovering the mechanisms of asymmetric cell division and of stem cell death and replenishment under DNA stress, which will illuminate plant-specific features in preserving stemness. Our technology support groups expand single-cell omics to describe stem cell behavior in a spatiotemporal context, and provide correlative light and electron microscopic technology to enable live imaging of cell and subcellular dynamics at high spatiotemporal resolution. In this perspective, we discuss future directions of our ongoing projects and related research fields.