CRISPR/Cas9-based genome editing of 14 lipid metabolic genes reveals a sporopollenin metabolon ZmPKSB-ZmTKPR1-1/-2 required for pollen exine formation in maize.

Lipid biosynthesis and transport are essential for plant male reproduction. Compared with Arabidopsis and rice, relatively fewer maize lipid metabolic genic male-sterility (GMS) genes have been identified, and the sporopollenin metabolon in maize anther remains unknown. Here, we identified two maize GMS genes, ZmTKPR1-1 and ZmTKPR1-2, by CRISPR/Cas9 mutagenesis of 14 lipid metabolic genes with anther stage-specific expression patterns. Among them, tkpr1-1/-2 double mutants displayed complete male sterility with delayed tapetum degradation and abortive pollen. ZmTKPR1-1 and ZmTKPR1-2 encode tetraketide α-pyrone reductases and have catalytic activities in reducing tetraketide α-pyrone produced by ZmPKSB (polyketide synthase B). Several conserved catalytic sites (S128/130, Y164/166 and K168/170 in ZmTKPR1-1/-2) are essential for their enzymatic activities. Both ZmTKPR1-1 and ZmTKPR1-2 are directly activated by ZmMYB84, and their encoded proteins are localized in both the endoplasmic reticulum and nuclei. Based on protein structure prediction, molecular docking, site-directed mutagenesis and biochemical assays, the sporopollenin biosynthetic metabolon ZmPKSB-ZmTKPR1-1/-2 was identified to control pollen exine formation in maize anther. Although ZmTKPR1-1/-2 and ZmPKSB formed a protein complex, their mutants showed different, even opposite, defective phenotypes of anther cuticle and pollen exine. Our findings discover new maize GMS genes that can contribute to male-sterility line-assisted maize breeding and also provide new insights into the metabolon-regulated sporopollenin biosynthesis in maize anther.

ZmMS1/ZmLBD30-orchestrated transcriptional regulatory networks precisely control pollen exine development.

Because of its significance for plant male fertility and, hence, direct impact on crop yield, pollen exine development has inspired decades of scientific inquiry. However, the molecular mechanism underlying exine formation and thickness remains elusive. In this study, we identified that a previously unrecognized repressor, ZmMS1/ZmLBD30, controls proper pollen exine development in maize. Using an ms1 mutant with aberrantly thickened exine, we cloned a male-sterility gene, ZmMs1, which encodes a tapetum-specific lateral organ boundary domain transcription factor, ZmLBD30. We showed that ZmMs1/ZmLBD30 is initially turned on by a transcriptional activation cascade of ZmbHLH51-ZmMYB84-ZmMS7, and then it serves as a repressor to shut down this cascade via feedback repression to ensure timely tapetal degeneration and proper level of exine. This activation-feedback repression loop regulating male fertility is conserved in maize and sorghum, and similar regulatory mechanism may also exist in other flowering plants such as rice and Arabidopsis. Collectively, these findings reveal a novel regulatory mechanism of pollen exine development by which a long-sought master repressor of upstream activators prevents excessive exine formation.

Triphasic regulation of ZmMs13 encoding an ABCG transporter is sequentially required for callose dissolution, pollen exine and anther cuticle formation in maize.

INTRODUCTION: ATP Binding Cassette G (ABCG) transporters are associated with plant male reproduction, while their regulatory mechanisms underlying anther and pollen development remain largely unknown. OBJECTIVES: Identify and characterize a male-sterility gene ZmMs13 encoding an ABCG transporter in modulating anther and pollen development in maize. METHODS: Phenotypic, cytological observations, and histochemistry staining were performed to characterize the ms13-6060 mutant. Map-based cloning and CRISPR/Cas9 gene editing were used to identify ZmMs13 gene. RNA-seq data and qPCR analyses, phylogenetic and microsynteny analyses, transient dual-luciferase reporter and EMSA assays, subcellular localization, and ATPase activity and lipidomic analyses were carried out to determine the regulatory mechanisms of ZmMs13 gene. RESULTS: Maize ms13-6060 mutant displays complete male sterility with delayed callose degradation, premature tapetal programmed cell death (PCD), and defective pollen exine and anther cuticle formation. ZmMs13 encodes a plasm membrane (PM)- and endoplasmic reticulum (ER)-localized half-size ABCG transporter (ZmABCG2a). The allele of ZmMs13 in ms13-6060 mutant has one amino acid (I311) deletion due to a 3-bp deletion in its fourth exon. The I311 and other conserved amino acid K99 are essential for the ATPase and lipid binding activities of ZmMS13. ZmMs13 is specifically expressed in anthers with three peaks at stages S5, S8b, and S10, which are successively regulated by transcription factors ZmbHLH122, ZmMYB84, and ZmMYB33-1/-2 at these three stages. The triphasic regulation of ZmMs13 is sequentially required for callose dissolution, tapetal PCD and pollen exine development, and anther cuticle formation, corresponding to transcription alterations of callose-, ROS-, PCD-, sporopollenin-, and anther cuticle-related genes in ms13-6060 anthers. CONCLUSION: ms13-6060 mutation with one key amino acid (I311) deletion greatly reduces ZmMS13 ATPase and lipid binding activities and displays multiple effects during maize male reproduction. Our findings provide new insights into molecular mechanisms of ABCG transporters controlling anther and pollen development and male fertility in plants.

The ZmMYB84-ZmPKSB regulatory module controls male fertility through modulating anther cuticle-pollen exine trade-off in maize anthers.

Anther cuticle and pollen exine are two crucial lipid layers that ensure normal pollen development and pollen-stigma interaction for successful fertilization and seed production in plants. Their formation processes share certain common pathways of lipid biosynthesis and transport across four anther wall layers. However, molecular mechanism underlying a trade-off of lipid-metabolic products to promote the proper formation of the two lipid layers remains elusive. Here, we identified and characterized a maize male-sterility mutant pksb, which displayed denser anther cuticle but thinner pollen exine as well as delayed tapetal degeneration compared with its wild type. Based on map-based cloning and CRISPR/Cas9 mutagenesis, we found that the causal gene (ZmPKSB) of pksb mutant encoded an endoplasmic reticulum (ER)-localized polyketide synthase (PKS) with catalytic activities to malonyl-CoA and midchain-fatty acyl-CoA to generate triketide and tetraketide α-pyrone. A conserved catalytic triad (C171, H320 and N353) was essential for its enzymatic activity. ZmPKSB was specifically expressed in maize anthers from stages S8b to S9-10 with its peak at S9 and was directly activated by a transcription factor ZmMYB84. Moreover, loss function of ZmMYB84 resulted in denser anther cuticle but thinner pollen exine similar to the pksb mutant. The ZmMYB84-ZmPKSB regulatory module controlled a trade-off between anther cuticle and pollen exine formation by altering expression of a series of genes related to biosynthesis and transport of sporopollenin, cutin and wax. These findings provide new insights into the fine-tuning regulation of lipid-metabolic balance to precisely promote anther cuticle and pollen exine formation in plants.

The Loss-Function of the Male Sterile Gene ZmMs33/ZmGPAT6 Results in Severely Oxidative Stress and Metabolic Disorder in Maize Anthers.

In plants, oxidative stress and metabolic reprogramming frequently induce male sterility, however our knowledge of the underlying molecular mechanism is far from complete. Here, a maize genic male-sterility (GMS) mutant (ms33-6038) with a loss-of-function of the ZmMs33 gene encoding glycerol-3-phosphate acyltransferase 6 (GPAT6) displayed severe deficiencies in the development of a four-layer anther wall and microspores and excessive reactive oxygen species (ROS) content in anthers. In ms33-6038 anthers, transcriptome analysis identified thousands of differentially expressed genes that were functionally enriched in stress response and primary metabolism pathways. Further investigation revealed that 64 genes involved in ROS production, scavenging, and signaling were specifically changed in expression levels in ms33-6038 anthers compared to the other five investigated GMS lines. The severe oxidative stress triggered premature tapetal autophagy and metabolic reprogramming mediated mainly by the activated SnRK1-bZIP pathway, as well as the TOR and PP2AC pathways, proven by transcriptome analysis. Furthermore, 20 reported maize GMS genes were altered in expression levels in ms33-6038 anthers. The excessive oxidative stress and the metabolic reprogramming resulted in severe phenotypic deficiencies in ms33-6038 anthers. These findings enrich our understanding of the molecular mechanisms by which ROS and metabolic homeostasis impair anther and pollen development in plants.

Genetic Structure and Molecular Mechanisms Underlying the Formation of Tassel, Anther, and Pollen in the Male Inflorescence of Maize (Zea mays L.).

Maize tassel is the male reproductive organ which is located at the plant's apex; both its morphological structure and fertility have a profound impact on maize grain yield. More than 40 functional genes regulating the complex tassel traits have been cloned up to now. However, the detailed molecular mechanisms underlying the whole process, from male inflorescence meristem initiation to tassel morphogenesis, are seldom discussed. Here, we summarize the male inflorescence developmental genes and construct a molecular regulatory network to further reveal the molecular mechanisms underlying tassel-trait formation in maize. Meanwhile, as one of the most frequently studied quantitative traits, hundreds of quantitative trait loci (QTLs) and thousands of quantitative trait nucleotides (QTNs) related to tassel morphology have been identified so far. To reveal the genetic structure of tassel traits, we constructed a consensus physical map for tassel traits by summarizing the genetic studies conducted over the past 20 years, and identified 97 hotspot intervals (HSIs) that can be repeatedly mapped in different labs, which will be helpful for marker-assisted selection (MAS) in improving maize yield as well as for providing theoretical guidance in the subsequent identification of the functional genes modulating tassel morphology. In addition, maize is one of the most successful crops in utilizing heterosis; mining of the genic male sterility (GMS) genes is crucial in developing biotechnology-based male-sterility (BMS) systems for seed production and hybrid breeding. In maize, more than 30 GMS genes have been isolated and characterized, and at least 15 GMS genes have been promptly validated by CRISPR/Cas9 mutagenesis within the past two years. We thus summarize the maize GMS genes and further update the molecular regulatory networks underlying male fertility in maize. Taken together, the identified HSIs, genes and molecular mechanisms underlying tassel morphological structure and male fertility are useful for guiding the subsequent cloning of functional genes and for molecular design breeding in maize. Finally, the strategies concerning efficient and rapid isolation of genes controlling tassel morphological structure and male fertility and their application in maize molecular breeding are also discussed.

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize.

Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.

ZmFAR1 and ZmABCG26 Regulated by microRNA Are Essential for Lipid Metabolism in Maize Anther.

The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1. DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1. These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.

ZmMs25 encoding a plastid-localized fatty acyl reductase is critical for anther and pollen development in maize.

Fatty acyl reductases (FARs) catalyse the reduction of fatty acyl-coenzyme A (CoA) or -acyl carrier protein (ACP) substrates to primary fatty alcohols, which play essential roles in lipid metabolism in plants. However, the mechanism by which FARs are involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticles, abnormal Ubisch body formation, impaired pollen exine formation and complete male sterility. Based on map-based cloning and CRISPR/Cas9 mutagenesis, Zm00001d048337 was identified as ZmMs25, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro. Four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther, and was directly regulated by transcription factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing cutin monomers and internal lipids, and altering the composition of cuticular wax in anthers. Moreover, loss of function of ZmMs25 significantly affected the expression of its four paralogous genes and five cloned lipid metabolic male-sterility genes in maize. These data suggest that ZmMs25 is required for anther development and male fertility, indicating its application potential in maize and other crops.

Molecular regulation of ZmMs7 required for maize male fertility and development of a dominant male-sterility system in multiple species.

Understanding the molecular basis of male sterility and developing practical male-sterility systems are essential for heterosis utilization and commercial hybrid seed production in crops. Here, we report molecular regulation by genic male-sterility gene maize male sterility 7 (ZmMs7) and its application for developing a dominant male-sterility system in multiple species. ZmMs7 is specifically expressed in maize anthers, encodes a plant homeodomain (PHD) finger protein that functions as a transcriptional activator, and plays a key role in tapetal development and pollen exine formation. ZmMs7 can interact with maize nuclear factor Y (NF-Y) subunits to form ZmMs7-NF-YA6-YB2-YC9/12/15 protein complexes that activate target genes by directly binding to CCAAT box in their promoter regions. Premature expression of ZmMs7 in maize by an anther-specific promoter p5126 results in dominant and complete male sterility but normal vegetative growth and female fertility. Early expression of ZmMs7 downstream genes induced by prematurely expressed ZmMs7 leads to abnormal tapetal development and pollen exine formation in p5126-ZmMs7 maize lines. The p5126-ZmMs7 transgenic rice and Arabidopsis plants display similar dominant male sterility. Meanwhile, the mCherry gene coupled with p5126-ZmMs7 facilitates the sorting of dominant sterility seeds based on fluorescent selection. In addition, both the ms7-6007 recessive male-sterility line and p5126-ZmMs7M dominant male-sterility line are highly stable under different genetic germplasms and thus applicable for hybrid maize breeding. Together, our work provides insight into the mechanisms of anther and pollen development and a promising technology for hybrid seed production in crops.

Lipid Metabolism: Critical Roles in Male Fertility and Other Aspects of Reproductive Development in Plants.

Fatty acids and their derivatives are essential building blocks for anther cuticle and pollen wall formation. Disruption of lipid metabolism during anther and pollen development often leads to genic male sterility (GMS). To date, many lipid metabolism-related GMS genes that are involved in the formation of anther cuticle, pollen wall, and subcellular organelle membranes in anther wall layers have been identified and characterized. In this review, we summarize recent progress on characterizing lipid metabolism-related genes and their roles in male fertility and other aspects of reproductive development in plants. On the basis of cloned GMS genes controlling biosynthesis and transport of anther cutin, wax, sporopollenin, and tryphine in Arabidopsis, rice, and maize as well as other plant species, updated lipid metabolic networks underlying anther cuticle development and pollen wall formation were proposed. Through bioinformatics analysis of anther RNA-sequencing datasets from three maize inbred lines (Oh43, W23, and B73), a total of 125 novel lipid metabolism-related genes putatively involved in male fertility in maize were deduced. More, we discuss the pathways regulating lipid metabolism-related GMS genes at the transcriptional and post-transcriptional levels. Finally, we highlight recent findings on lipid metabolism-related genes and their roles in other aspects of plant reproductive development. A comprehensive understanding of lipid metabolism, genes involved, and their roles in plant reproductive development will facilitate the application of lipid metabolism-related genes in gene editing, haploid and callus induction, molecular breeding and hybrid seed production in crops.

Genome-wide analysis of maize GPAT gene family and cytological characterization and breeding application of ZmMs33/ZmGPAT6 gene.

KEY MESSAGE: Genome-wide analysis of maize GPAT gene family, cytological characterization of ZmMs33/ZmGPAT6 gene encoding an ER-localized protein with four conserved motifs, and its molecular breeding application in maize. Glycerol-3-phosphate acyltransferase (GPAT) mediates the initial step of glycerolipid biosynthesis and plays pivotal roles in plant growth and development. Compared with GPAT genes in Arabidopsis, our understanding to maize GPAT gene family is very limited. Recently, ZmMs33 gene has been identified to encode a sn-2 GPAT protein and control maize male fertility in our laboratory (Xie et al. in Theor Appl Genet 131:1363-1378, 2018). However, the functional mechanism of ZmMs33 remains elusive. Here, we reported the genome-wide analysis of maize GPAT gene family and found that 20 maize GPAT genes (ZmGPAT1-20) could be classified into three distinct clades similar to those of ten GPAT genes in Arabidopsis. Expression analyses of these ZmGPAT genes in six tissues and in anther during six developmental stages suggested that some of ZmGPATs may play crucial roles in maize growth and anther development. Among them, ZmGPAT6 corresponds to the ZmMs33 gene. Systemic cytological observations indicated that loss function of ZmMs33/ZmGPAT6 led to defective anther cuticle, arrested degeneration of anther wall layers, abnormal formation of Ubisch bodies and exine and ultimately complete male sterility in maize. The endoplasmic reticulum-localized ZmMs33/ZmGPAT6 possessed four conserved amino acid motifs essential for acyltransferase activity, while ZmMs33/ZmGPAT6 locus and its surrounding genomic region have greatly diversified during evolution of gramineous species. Finally, a multi-control sterility system was developed to produce ms33 male-sterile lines by using a combination strategy of transgene and marker-assisted selection. This work will provide useful information for further deciphering functional mechanism of ZmGPAT genes and facilitate molecular breeding application of ZmMs33/ZmGPAT6 gene in maize.

ZmMs30 Encoding a Novel GDSL Lipase Is Essential for Male Fertility and Valuable for Hybrid Breeding in Maize.

Genic male sterility (GMS) is very useful for hybrid vigor utilization and hybrid seed production. Although a large number of GMS genes have been identified in plants, little is known about the roles of GDSL lipase members in anther and pollen development. Here, we report a maize GMS gene, ZmMs30, which encodes a novel type of GDSL lipase with diverged catalytic residues. Enzyme kinetics and activity assays show that ZmMs30 has lipase activity and prefers to substrates with a short carbon chain. ZmMs30 is specifically expressed in maize anthers during stages 7-9. Loss of ZmMs30 function resulted in defective anther cuticle, irregular foot layer of pollen exine, and complete male sterility. Cytological and lipidomics analyses demonstrate that ZmMs30 is crucial for the aliphatic metabolic pathway required for pollen exine formation and anther cuticle development. Furthermore, we found that male sterility caused by loss of ZmMs30 function was stable in various inbred lines with different genetic background, and that it didn't show any negative effect on maize heterosis and production, suggesting that ZmMs30 is valuable for cross-breeding and hybrid seed production. We then developed a new multi-control sterility system using ZmMs30 and its mutant line, and demonstrated it is feasible for generating desirable GMS lines and valuable for hybrid maize seed production. Taken together, our study sheds new light on the mechanisms of anther and pollen development, and provides a valuable male-sterility system for hybrid breeding maize.

Map-based cloning and characterization of Zea mays male sterility33 (ZmMs33) gene, encoding a glycerol-3-phosphate acyltransferase.

KEY MESSAGE: Map-based cloning of maize ms33 gene showed that ZmMs33 encodes a sn-2 glycerol-3-phosphate acyltransferase, the ortholog of rice OsGPAT3, and it is essential for male fertility in maize. Genetic male sterility has been widely studied for its biological significance and commercial value in hybrid seed production. Although many male-sterile mutants have been identified in maize (Zea mays L.), it is likely that most genes that cause male sterility are unknown. Here, we report a recessive genetic male-sterile mutant, male sterility33 (ms33), which displays small, pale yellow anthers, and complete male sterility. Using a map-based cloning approach, maize GRMZM2G070304 was identified as the ms33 gene (ZmMs33). ZmMs33 encodes a novel sn-2 glycerol-3-phosphate acyltransferase (GPAT) in maize. A functional complementation experiment showed that GRMZM2G070304 can rescue the male-sterile phenotype of the ms33-6029 mutant. GRMZM2G070304 was further confirmed to be the ms33 gene via targeted knockouts induced by the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system. ZmMs33 is preferentially expressed in the immature anther from the quartet to early-vacuolate microspore stages and in root tissues at the fifth leaf growth stage. Phylogenetic analysis indicated that ZmMs33 and OsGPAT3 are evolutionarily conserved for anther and pollen development in monocot species. This study reveals that the monocot-specific GPAT3 protein plays an important role in male fertility in maize, and ZmMs33 and mutants in this gene may have value in maize male-sterile line breeding and hybrid seed production.

Transcriptome analysis of grain-filling caryopses reveals involvement of multiple regulatory pathways in chalky grain formation in rice.

BACKGROUND: Grain endosperm chalkiness of rice is a varietal characteristic that negatively affects not only the appearance and milling properties but also the cooking texture and palatability of cooked rice. However, grain chalkiness is a complex quantitative genetic trait and the molecular mechanisms underlying its formation are poorly understood. RESULTS: A near-isogenic line CSSL50-1 with high chalkiness was compared with its normal parental line Asominori for grain endosperm chalkiness. Physico-biochemical analyses of ripened grains showed that, compared with Asominori, CSSL50-1 contains higher levels of amylose and 8 DP (degree of polymerization) short-chain amylopectin, but lower medium length 12 DP amylopectin. Transcriptome analysis of 15 DAF (day after flowering) caryopses of the isogenic lines identified 623 differential expressed genes (P < 0.01), among which 324 genes are up-regulated and 299 down-regulated. These genes were classified into 18 major categories, with 65.3% of them belong to six major functional groups: signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis. Detailed pathway dissection demonstrated that genes involved in sucrose and starch synthesis are up-regulated, whereas those involved in non-starch polysaccharides are down regulated. Several genes involved in oxidoreductive homeostasis were found to have higher expression levels in CSSL50-1 as well, suggesting potential roles of ROS in grain chalkiness formation. CONCLUSION: Extensive gene expression changes were detected during rice grain chalkiness formation. Over half of these differentially expressed genes are implicated in several important categories of genes, including signal transduction, transcription, carbohydrate metabolism and redox homeostasis, suggesting that chalkiness formation involves multiple metabolic and regulatory pathways.