Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha.

Gibberellins (GAs) are key phytohormones that regulate growth, development, and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthesis intermediates such as ent-kaurenoic acid (KA) are also produced by bryophytes. Here, we show that in the liverwort Marchantia polymorpha, KA and GA12 are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently exhibited altered thallus growth allometry, delayed initiation of gametogenesis, and abnormal morphology of gamete-bearing structures (gametangiophores). By chemical treatments and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, we confirmed that these phenotypes were caused by the deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism in M. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but were co-opted independently to regulate responses to light quality change in different plant lineages during the past 450 million years of evolution.

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.

Fertilization controls tiller numbers via transcriptional regulation of a MAX1-like gene in rice cultivation.

Fertilization controls various aspects of cereal growth such as tiller number, leaf size, and panicle size. However, despite such benefits, global chemical fertilizer use must be reduced to achieve sustainable agriculture. Here, based on field transcriptome data from leaf samples collected during rice cultivation, we identify fertilizer responsive genes and focus on Os1900, a gene orthologous to Arabidopsis thaliana MAX1, which is involved in strigolactone biosynthesis. Elaborate genetic and biochemical analyses using CRISPR/Cas9 mutants reveal that Os1900 together with another MAX1-like gene, Os5100, play a critical role in controlling the conversion of carlactone into carlactonoic acid during strigolactone biosynthesis and tillering in rice. Detailed analyses of a series of Os1900 promoter deletion mutations suggest that fertilization controls tiller number in rice through transcriptional regulation of Os1900, and that a few promoter mutations alone can increase tiller numbers and grain yields even under minor-fertilizer conditions, whereas a single defective os1900 mutation does not increase tillers under normal fertilizer condition. Such Os1900 promoter mutations have potential uses in breeding programs for sustainable rice production.

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.

A carlactonoic acid methyltransferase that contributes to the inhibition of shoot branching in Arabidopsis.

SignificanceStrigolactones (SLs) are a group of apocarotenoid hormones, which regulates shoot branching and other diverse developmental processes in plants. The major bioactive form(s) of SLs as endogenous hormones has not yet been clarified. Here, we identify an Arabidopsis methyltransferase, CLAMT, responsible for the conversion of an inactive precursor to a biologically active SL that can interact with the SL receptor in vitro. Reverse genetic analysis showed that this enzyme plays an essential role in inhibiting shoot branching. This mutant also contributed to specifying the SL-related metabolites that could move from root to shoot in grafting experiments. Our work has identified a key enzyme necessary for the production of the bioactive form(s) of SLs.

Strigolactone biosynthesis, transport and perception.

Strigolactones (SLs) are plant hormones that regulate diverse developmental processes and environmental responses. They are also known to be root-derived chemical signals that regulate symbiotic and parasitic interactions with arbuscular mycorrhizal fungi and root parasitic plants, respectively. Since the discovery of the hormonal function of SLs in 2008, there has been much progress in the SL research field. In particular, a number of breakthroughs have been achieved in our understanding of SL biosynthesis, transport and perception. The discovery of the hormonal function of SL was quite valuable not only as the identification of a new class of plant hormones, but also as the discovery of the long-sought-after SL biosynthetic and response mutants. These mutants in several plant species provided us the genetic resources to address fundamental questions regarding SL biosynthesis and perception. Such mutants were further characterized later, and biochemical analyses of these genetically identified factors have uncovered the outline of SL biosynthesis and perception so far. Moreover, new genes involved in SL transport have been discovered through reverse genetic analyses. In this review, we summarize recent advances in SL research with a focus on biosynthesis, transport and perception.

Mechanism of delayed seed germination caused by high temperature during grain filling in rice (Oryza sativa L.).

High temperature during grain filling considerably reduces yield and quality in rice (Oryza sativa L.); however, how high temperature affects seed germination of the next generation is not yet well understood. Here, we report that seeds from plants exposed to high temperature during the grain filling stage germinated significantly later than seeds from unstressed plants. This delay remained even after dormancy release treatments, suggesting that it was not due to primary seed dormancy determined during grain filling. In imbibed embryos of heat-stressed seeds, expression of abscisic acid (ABA) biosynthesis genes (OsNCEDs) was higher than in those of control seeds, whereas that of ABA catabolism genes (OsABA8'OHs) was lower. In the aleurone layer, despite no change in GA signaling as evidenced by no effect of heat stress on OsGAMYB gene expression, the transcripts of α-amylase genes OsAmy1C, OsAmy3B, and OsAmy3E were significantly down-regulated in heat-stressed seeds in comparison with controls. Changes in promoter methylation levels were consistent with transcriptional changes of ABA catabolism-related and α-amylase genes. These data suggest that high temperature during grain filling results in DNA methylation of ABA catabolism-related and α-amylase gene promoters, delaying germination of heat-stressed seeds.

Chemical screening of novel strigolactone agonists that specifically interact with DWARF14 protein.

Strigolactones (SLs) are a class of plant hormones that regulate shoot branching as well as being known as root-derived signals for parasitic and symbiotic interactions. The physical interaction between SLs and the DWARF14 (D14) receptor family can be examined by differential scanning fluorimetry (DSF) that monitors the changes in protein melting temperature (Tm). The Tm of D14 is lowered by bioactive SLs in DSF analysis. In this report, we screened the compounds that lower the Tm of Arabidopsis D14 (AtD14) as potential candidates for SL agonists using DSF analysis. Subsequent physiological analyzes revealed that 113D10 acts as a novel SL agonist in a D14-dependent manner. Intriguingly, 113D10 has a chemical structure different from natural SLs in that it does not possess an enol ether bond that connects to a methylbutenolide moiety. Moreover, 113D10 does not stimulate seed germination of root parasitic plants. Accordingly, 113D10 can be a useful tool for SL studies and agricultural applications.

Strigolactone perception and deactivation by a hydrolase receptor DWARF14.

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/ß-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.

Structural Basis of Karrikin and Non-natural Strigolactone Perception in Physcomitrella patens.

In plants, strigolactones are perceived by the dual receptor-hydrolase DWARF14 (D14). D14 belongs to the superfamily of α/ß hydrolases and is structurally similar to the karrikin receptor KARRIKIN INSENSITIVE 2 (KAI2). The moss Physcomitrella patens is an ideal model system for studying this receptor family, because it includes 11 highly related family members with unknown ligand specificity. We present the crystal structures of three Physcomitrella D14/KAI2-like proteins and describe a loop-based mechanism that leads to a permanent widening of the hydrophobic substrate gorge. We have identified protein clades that specifically perceive the karrikin KAR1 and the non-natural strigolactone isomer (-)-5-deoxystrigol in a highly stereoselective manner.

Agrobacterium tumefaciens Enhances Biosynthesis of Two Distinct Auxins in the Formation of Crown Galls.

The plant pathogen Agrobacterium tumefaciens infects plants and introduces the transferred-DNA (T-DNA) region of the Ti-plasmid into nuclear DNA of host plants to induce the formation of tumors (crown galls). The T-DNA region carries iaaM and iaaH genes for synthesis of the plant hormone auxin, indole-3-acetic acid (IAA). It has been demonstrated that the iaaM gene encodes a tryptophan 2-monooxygenase which catalyzes the conversion of tryptophan to indole-3-acetamide (IAM), and the iaaH gene encodes an amidase for subsequent conversion of IAM to IAA. In this article, we demonstrate that A. tumefaciens enhances the production of both IAA and phenylacetic acid (PAA), another auxin which does not show polar transport characteristics, in the formation of crown galls. Using liquid chromatography-tandem mass spectroscopy, we found that the endogenous levels of phenylacetamide (PAM) and PAA metabolites, as well as IAM and IAA metabolites, are remarkably increased in crown galls formed on the stem of tomato plants, implying that two distinct auxins are simultaneously synthesized via the IaaM-IaaH pathway. Moreover, we found that the induction of the iaaM gene dramatically elevated the levels of PAM, PAA and its metabolites, along with IAM, IAA and its metabolites, in Arabidopsis and barley. From these results, we conclude that A. tumefaciens enhances biosynthesis of two distinct auxins in the formation of crown galls.

An allelic series at the KARRIKIN INSENSITIVE 2 locus of Arabidopsis thaliana decouples ligand hydrolysis and receptor degradation from downstream signalling.

Karrikins are butenolide compounds present in post-fire environments that can stimulate seed germination in many species, including Arabidopsis thaliana. Plants also produce endogenous butenolide compounds that serve as hormones, namely strigolactones (SLs). The receptor for karrikins (KARRIKIN INSENSITIVE 2; KAI2) and the receptor for SLs (DWARF14; D14) are homologous proteins that share many similarities. The mode of action of D14 as a dual enzyme receptor protein is well established, but the nature of KAI2-dependent signalling and its function as a receptor are not fully understood. To expand our knowledge of how KAI2 operates, we screened ethyl methanesulphonate (EMS)-mutagenized populations of A. thaliana for mutants with kai2-like phenotypes and isolated 13 new kai2 alleles. Among these alleles, kai2-10 encoded a D184N protein variant that was stable in planta. Differential scanning fluorimetry assays indicated that the KAI2 D184N protein could interact normally with bioactive ligands. We developed a KAI2-active version of the fluorescent strigolactone analogue Yoshimulactone Green to show that KAI2 D184N exhibits normal rates of ligand hydrolysis. KAI2 D184N degraded in response to treatment with exogenous ligands, suggesting that receptor degradation is a consequence of ligand binding and hydrolysis, but is insufficient for signalling activity. Remarkably, KAI2 D184N degradation was hypersensitive to karrikins, but showed a normal response to strigolactone analogues, implying that these butenolides may interact differently with KAI2. These results demonstrate that the enzymatic and signalling functions of KAI2 can be decoupled, and provide important insights into the mechanistic events that underpin butenolide signalling in plants.

Ethylene-gibberellin signaling underlies adaptation of rice to periodic flooding.

Most plants do poorly when flooded. Certain rice varieties, known as deepwater rice, survive periodic flooding and consequent oxygen deficiency by activating internode growth of stems to keep above the water. Here, we identify the gibberellin biosynthesis gene, SD1 (SEMIDWARF1), whose loss-of-function allele catapulted the rice Green Revolution, as being responsible for submergence-induced internode elongation. When submerged, plants carrying the deepwater rice-specific SD1 haplotype amplify a signaling relay in which the SD1 gene is transcriptionally activated by an ethylene-responsive transcription factor, OsEIL1a. The SD1 protein directs increased synthesis of gibberellins, largely GA4, which promote internode elongation. Evolutionary analysis shows that the deepwater rice-specific haplotype was derived from standing variation in wild rice and selected for deepwater rice cultivation in Bangladesh.

Growth- and stress-related defects associated with wall hypoacetylation are strigolactone-dependent.

Mutants affected in the Arabidopsis TBL29/ESK1 xylan O-acetyltransferase display a strong reduction in total wall O-acetylation accompanied by a dwarfed plant stature, collapsed xylem morphology, and enhanced freezing tolerance. A newly identified tbl29/esk1 suppressor mutation reduces the expression of the MAX4 gene, affecting the biosynthesis of methyl carlactonoate (MeCLA), an active strigolactone (SL). Genetic and biochemical evidence suggests that blocking the biosynthesis of this SL is sufficient to recover all developmental and stress-related defects associated with the TBL29/ESK1 loss of function without affecting its direct effect-reduced wall O-acetylation. Altered levels of the MAX4 SL biosynthetic gene, reduced branch number, and higher levels of MeCLA, were also found in tbl29/esk1 plants consistent with a constitutive activation of the SL pathway. These results suggest that the reduction in O-acetyl substituents in xylan is not directly responsible for the observed tbl29/esk1 phenotypes. Alternatively, plants may perceive defects in the structure of wall polymers and/or wall architecture activating the SL hormonal pathway as a compensatory mechanism.

Yucasin DF, a potent and persistent inhibitor of auxin biosynthesis in plants.

The plant hormone auxin plays a crucial role in plant growth and development. Indole-3-acetic acid (IAA), a natural auxin, is mainly biosynthesized by two sequential enzyme reactions catalyzed by TAA1 and YUCCA (YUC). TAA1 is involved in the conversion of tryptophan to IPA, and YUC catalyzes the conversion of IPA to IAA. We previously demonstrated that yucasin inhibits AtYUC1 enzyme activity and suppress high-auxin phenotype of YUC overexpression plants, although yucasin displayed weak effects on the auxin-related phenotype of wild-type plants. To develop more potent YUC inhibitors, various derivatives of yucasin were synthesized, and their structure-activity relationships were investigated. Yucasin difluorinated analog (YDF) (5-[2,6-difluorophenyl]-2,4-dihydro-[1,2,4]-triazole-3-thione) was identified to be a more potent YUC inhibitor than the original yucasin. YDF caused an auxin-deficient phenotype in Arabidopsis wild-type plants that was restored with auxin application. YDF was found to be highly stable regarding metabolic conversion in vivo, accounting for the potent activity of the inhibition of IAA biosynthesis in planta. Photoaffinity labeling experiments demonstrated that yucasin-type inhibitors bind to the active site of AtYUC1. YDF is a promising auxin biosynthesis inhibitor and is a useful chemical tool for plant biology and agrochemical studies.

Involvement of STH7 in light-adapted development in Arabidopsis thaliana promoted by both strigolactone and karrikin.

Strigolactones (SLs) and karrikins (KARs) regulate photomorphogenesis. GR24, a synthetic SL and KAR1, a KAR, inhibit the hypocotyl elongation of Arabidopsis thaliana in a weak light. GR24 and KAR1 up-regulate the expression of STH7, encoding a transcription factor belonging to the double B-box zinc finger subfamily. In this study, we used STH7-overexpressing (STH7ox) lines and functionally defective STH7 (STH7-SRDX) mutants to investigate roles of SLs and KARs in photomorphogenesis of Arabidopsis. Hypocotyl elongation of STH7-SRDX mutants was less sensitive to both GR24 and KAR1 treatment than that of wild-type Arabidopsis under weak light conditions. Furthermore, the chlorophyll and anthocyanin content was increased in STH7ox lines when de-etiolated with light and GR24-treated plants had enhanced anthocyanin production. GR24 and KAR1 treatment significantly increased the expression level of photosynthesis-related genes LHCB1 and rbcS. The results strongly suggest that SL and KAR induce photomorphogenesis of Arabidopsis in an STH7-dependent manner.

Distinct Characteristics of Indole-3-Acetic Acid and Phenylacetic Acid, Two Common Auxins in Plants.

The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins.

Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation.

The colonization of land by plants was a key event in the evolution of life. Here we report the draft genome sequence of the filamentous terrestrial alga Klebsormidium flaccidum (Division Charophyta, Order Klebsormidiales) to elucidate the early transition step from aquatic algae to land plants. Comparison of the genome sequence with that of other algae and land plants demonstrate that K. flaccidum acquired many genes specific to land plants. We demonstrate that K. flaccidum indeed produces several plant hormones and homologues of some of the signalling intermediates required for hormone actions in higher plants. The K. flaccidum genome also encodes a primitive system to protect against the harmful effects of high-intensity light. The presence of these plant-related systems in K. flaccidum suggests that, during evolution, this alga acquired the fundamental machinery required for adaptation to terrestrial environments.

Chemical screening of an inhibitor for gibberellin receptors based on a yeast two-hybrid system.

We applied a yeast two-hybrid (Y2H) system to the high-throughput monitoring of two proteins' interaction, a receptor for phytohormone gibberellin (GA) and its direct signal transducer DELLA. With this system, we screened inhibitors to the interaction. As a result, we discovered a chemical, 3-(2-thienylsulfonyl)pyrazine-2-carbonitrile (TSPC), and we confirmed that TSPC is an inhibitor for GA perception by in vitro and in planta evaluations.