Application of ethanol alleviates heat damage to leaf growth and yield in tomato.

Chemical priming has emerged as a promising area in agricultural research. Our previous studies have demonstrated that pretreatment with a low concentration of ethanol enhances abiotic stress tolerance in Arabidopsis and cassava. Here, we show that ethanol treatment induces heat stress tolerance in tomato (Solanum lycopersicon L.) plants. Seedlings of the tomato cultivar 'Micro-Tom' were pretreated with ethanol solution and then subjected to heat stress. The survival rates of the ethanol-pretreated plants were significantly higher than those of the water-treated control plants. Similarly, the fruit numbers of the ethanol-pretreated plants were greater than those of the water-treated ones. Transcriptome analysis identified sets of genes that were differentially expressed in shoots and roots of seedlings and in mature green fruits of ethanol-pretreated plants compared with those in water-treated plants. Gene ontology analysis using these genes showed that stress-related gene ontology terms were found in the set of ethanol-induced genes. Metabolome analysis revealed that the contents of a wide range of metabolites differed between water- and ethanol-treated samples. They included sugars such as trehalose, sucrose, glucose, and fructose. From our results, we speculate that ethanol-induced heat stress tolerance in tomato is mainly the result of increased expression of stress-related genes encoding late embryogenesis abundant (LEA) proteins, reactive oxygen species (ROS) elimination enzymes, and activated gluconeogenesis. Our results will be useful for establishing ethanol-based chemical priming technology to reduce heat stress damage in crops, especially in Solanaceae.

Genome editing and molecular analyses of an Arabidopsis transcription factor, LATE FLOWERING.

Correct flower organ formation at the right timing is one of the most important strategies for plants to achieve reproductive success. Ectopic overexpression of LATE FLOWERING (LATE) is known to induce late flowering, partly through suppressing expression of the florigen-encoding gene FLOWERING LOCUS T (FT) in Arabidopsis. LATE is one of the C2H2 zinc finger transcription factors, and it has a canonical transcriptional repression domain called the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif at the end of its C terminus. Therefore, LATE is considered a transcriptional repressor, but its molecular function remains unclear. Our genome-edited late mutants exhibited no distinct phenotype, even in flowering, indicating the presence of redundancy from other factors. To reveal the molecular function of LATE and factors working with it, we investigated its transcriptional activity and interactions with other proteins. Transactivation activity assay showed that LATE possesses transcriptional repression ability, which appears to be attributable to both the EAR motif and other sequences. Yeast two-hybrid assay showed the EAR motif-mediated interaction of LATE with TOPLESS, a transcriptional corepressor. Moreover, LATE could also interact with CRABS CLAW (CRC), one of the most important regulators of floral meristem determinacy, through sequences in LATE other than the EAR motif. Our findings demonstrated the possibility that LATE can form a transcriptional repression complex with CRC for floral meristem determinacy.

Enhancement of Secondary Cell Wall Formation in Poplar Xylem Using a Self-Reinforced System of Secondary Cell Wall-Related Transcription Factors.

The secondary cell wall (SCW) in the xylem is one of the largest sink organs of carbon in woody plants, and is considered a promising sustainable bioresource for biofuels and biomaterials. To enhance SCW formation in poplar (Populus sp.) xylem, we developed a self-reinforced system of SCW-related transcription factors from Arabidopsis thaliana, involving VASCULAR-RELATED NAC-DOMAIN7 (VND7), SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN 1/NAC SECONDARY WALL THICKENING-PROMOTING FACTOR3 (SND1/NST3), and MYB46. In this system, these transcription factors were fused with the transactivation domain VP16 and expressed under the control of the Populus trichocarpa CesA18 (PtCesA18) gene promoter, creating the chimeric genes PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16. The PtCesA18 promoter is active in tissues generating SCWs, and can be regulated by AtVND7, AtSND1, and AtMYB46; thus, the expression levels of PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16 are expected to be boosted in SCW-generating tissues. In the transgenic hybrid aspens (Populus tremula × tremuloides T89) expressing PtCesA18pro::AtSND1:VP16 or PtCesA18pro::AtMYB46:VP16 grown in sterile half-strength Murashige and Skoog growth medium, SCW thickening was significantly enhanced in the secondary xylem cells, while the PtCesA18pro::AtVND7:VP16 plants showed stunted xylem formation, possibly because of the enhanced programmed cell death (PCD) in the xylem regions. After acclimation, the transgenic plants were transferred from the sterile growth medium to pots of soil in the greenhouse, where only the PtCesA18pro::AtMYB46:VP16 aspens survived. A nuclear magnetic resonance footprinting cell wall analysis and enzymatic saccharification analysis demonstrated that PtCesA18pro::AtMYB46:VP16 influences cell wall properties such as the ratio of syringyl (S) and guaiacyl (G) units of lignin, the abundance of the lignin ß-aryl ether and resinol bonds, and hemicellulose acetylation levels. Together, these data indicate that we have created a self-reinforced system using SCW-related transcription factors to enhance SCW accumulation.

Transcriptome analysis of Pará rubber tree (H. brasiliensis) seedlings under ethylene stimulation.

BACKGROUND: Natural rubber (cis-1,4-polyioprene, NR) is an indispensable industrial raw material obtained from the Pará rubber tree (H. brasiliensis). Natural rubber cannot be replaced by synthetic rubber compounds because of the superior resilience, elasticity, abrasion resistance, efficient heat dispersion, and impact resistance of NR. In NR production, latex is harvested by periodical tapping of the trunk bark. Ethylene enhances and prolongs latex flow and latex regeneration. Ethephon, which is an ethylene-releasing compound, applied to the trunk before tapping usually results in a 1.5- to 2-fold increase in latex yield. However, intense mechanical damage to bark tissues by excessive tapping and/or over-stimulation with ethephon induces severe oxidative stress in laticifer cells, which often causes tapping panel dryness (TPD) syndrome. To enhance NR production without causing TPD, an improved understanding of the molecular mechanism of the ethylene response in the Pará rubber tree is required. Therefore, we investigated gene expression in response to ethephon treatment using Pará rubber tree seedlings as a model system. RESULTS: After ethephon treatment, 3270 genes showed significant differences in expression compared with the mock treatment. Genes associated with carotenoids, flavonoids, and abscisic acid biosynthesis were significantly upregulated by ethephon treatment, which might contribute to an increase in latex flow. Genes associated with secondary cell wall formation were downregulated, which might be because of the reduced sugar supply. Given that sucrose is an important molecule for NR production, a trade-off may arise between NR production and cell wall formation for plant growth and for wound healing at the tapping panel. CONCLUSIONS: Dynamic changes in gene expression occur specifically in response to ethephon treatment. Certain genes identified may potentially contribute to latex production or TPD suppression. These data provide valuable information to understand the mechanism of ethylene stimulation, and will contribute to improved management practices and/or molecular breeding to attain higher yields of latex from Pará rubber trees.

Involvement of VNS NAC-domain transcription factors in tracheid formation in Pinus taeda.

Vascular plants have two types of water-conducting cells, xylem vessel cells (in angiosperms) and tracheid cells (in ferns and gymnosperms). These cells are commonly characterized by secondary cell wall (SCW) formation and programmed cell death (PCD), which increase the efficiency of water conduction. The differentiation of xylem vessel cells is regulated by a set of NAC (NAM, ATAF1/2 and CUC2) transcription factors, called the VASCULAR-RELATED NAC-DOMAIN (VND) family, in Arabidopsis thaliana Linne. The VNDs regulate the transcriptional induction of genes required for SCW formation and PCD. However, information on the transcriptional regulation of tracheid cell differentiation is still limited. Here, we performed functional analysis of loblolly pine (Pinus taeda Linne) VND homologs (PtaVNS, for VND, NST/SND, SMB-related protein). We identified five PtaVNS genes in the loblolly pine genome, and four of these PtaVNS genes were highly expressed in tissues with tracheid cells, such as shoot apices and developing xylem. Transient overexpression of PtaVNS genes induced xylem vessel cell-like patterning of SCW deposition in tobacco (Nicotiana benthamiana Domin) leaves, and up-regulated the promoter activities of loblolly pine genes homologous to SCW-related MYB transcription factor genes and cellulose synthase genes, as well as to cysteine protease genes for PCD. Collectively, our data indicated that PtaVNS proteins possess transcriptional activity to induce the molecular programs required for tracheid formation, i.e., SCW formation and PCD. Moreover, these findings suggest that the VNS-MYB-based transcriptional network regulating water-conducting cell differentiation in angiosperm and moss plants is conserved in gymnosperms.

Proteomic analysis of xylem vessel cell differentiation in VND7-inducible tobacco BY-2 cells by two-dimensional gel electrophoresis.

The xylem vessel is an essential structure for water conduction in vascular plants. Xylem vessel cells deposit thick secondary cell walls and undergo programmed cell death, to function as water-conducting elements. Since the discovery of the plant-specific NAC domain-type VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors, which function as master switches of xylem vessel cell differentiation in Arabidopsis, much has been learned about the transcriptional regulatory network of xylem vessel cell differentiation. However, little is known about proteome dynamics during xylem vessel cell differentiation. Here, we performed two-dimensional electrophoresis-based proteomic analysis of xylem vessel cell differentiation using a transgenic tobacco BY-2 cell line carrying the VND7-inducible system (BY-2/35S::VND7-VP16-GR), in which synchronous trans-differentiation into xylem vessel cells can be induced by the application of a glucocorticoid. Of the 47 spots revealed by gel electrophoresis, we successfully identified 40 proteins. Seventeen proteins, including several well-characterized proteins such as a cysteine protease and serine carboxypeptidase (involved in programmed cell death), were upregulated after 24 h of induction. However, previous transcriptomic analysis showed that only eight of these proteins are upregulated at the transcriptional level during xylem vessel cell differentiation in BY-2/35S::VND7-VP16-GR cells. These findings suggest that post-transcriptional regulation strongly affects proteomic dynamics during xylem vessel cell differentiation.

Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells.

The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation.

NSR1/MYR2 is a negative regulator of ASN1 expression and its possible involvement in regulation of nitrogen reutilization in Arabidopsis.

Nitrogen (N) is a major macronutrient that is essential for plant growth. It is important for us to understand the key genes that are involved in the regulation of N utilization. In this study, we focused on a GARP-type transcription factor known as NSR1/MYR2, which has been reported to be induced under N-deficient conditions. Our results demonstrated that NSR1/MYR2 has a transcriptional repression activity and is specifically expressed in vascular tissues, especially in phloem throughout the plant under daily light-dark cycle regulation. The overexpression of NSR1/MYR2 delays nutrient starvation- and dark-triggered senescence in the mature leaves of excised whole aerial parts of Arabidopsis plants. Furthermore, the expression of asparagine synthetase 1 (ASN1), which plays an important role in N remobilization and reallocation, i.e. N reutilization, in Arabidopsis, is negatively regulated by NSR1/MYR2, since the expressions of NSR1/MYR2 and ASN1 were reciprocally regulated during the light-dark cycle and ASN1 expression was down-regulated in overexpressors of NSR1/MYR2 and up-regulated in T-DNA insertion mutants of NSR1/MYR2. Therefore, the present results suggest that NSR1/MYR2 plays a role in N reutilization as a negative regulator through controlling ASN1 expression.

Primary Metabolism during Biosynthesis of Secondary Wall Polymers of Protoxylem Vessel Elements.

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell's metabolic activity for the biosynthesis of secondary wall polymers.

NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants.

Plant cells biosynthesize primary cell walls (PCW) in all cells and produce secondary cell walls (SCWs) in specific cell types that conduct water and/or provide mechanical support, such as xylem vessels and fibers. The characteristic mechanical stiffness, chemical recalcitrance, and hydrophobic nature of SCWs result from the organization of SCW-specific biopolymers, i.e., highly ordered cellulose, hemicellulose, and lignin. Synthesis of these SCW-specific biopolymers requires SCW-specific enzymes that are regulated by SCW-specific transcription factors. In this review, we summarize our current knowledge of the transcriptional regulation of SCW formation in plant cells. Advances in research on SCW biosynthesis during the past decade have expanded our understanding of the transcriptional regulation of SCW formation, particularly the functions of the NAC and MYB transcription factors. Focusing on the NAC-MYB-based transcriptional network, we discuss the regulatory systems that evolved in land plants to modify the cell wall to serve as a key component of structures that conduct water and provide mechanical support.

Importance of Reelin C-terminal region in the development and maintenance of the postnatal cerebral cortex and its regulation by specific proteolysis.

During brain development, Reelin exerts a variety of effects in a context-dependent manner, whereas its underlying molecular mechanisms remain poorly understood. We previously showed that the C-terminal region (CTR) of Reelin is required for efficient induction of phosphorylation of Dab1, an essential adaptor protein for canonical Reelin signaling. However, the physiological significance of the Reelin CTR in vivo remains unexplored. To dissect out Reelin functions, we made a knock-in (KI) mouse in which the Reelin CTR is deleted. The amount of Dab1, an indication of canonical Reelin signaling strength, is increased in the KI mouse, indicating that the CTR is necessary for efficient induction of Dab1 phosphorylation in vivo. Formation of layer structures during embryonic development is normal in the KI mouse. Intriguingly, the marginal zone (MZ) of the cerebral cortex becomes narrower at postnatal stages because upper-layer neurons invade the MZ and their apical dendrites are misoriented and poorly branched. Furthermore, Reelin undergoes proteolytic cleavage by proprotein convertases at a site located 6 residues from the C terminus, and it was suggested that this cleavage abrogates the Reelin binding to the neuronal cell membrane. Results from ectopic expression of mutant Reelin proteins in utero suggest that the dendrite development and maintenance of the MZ require Reelin protein with an intact CTR. These results provide a novel model regarding Reelin functions involving its CTR, which is not required for neuronal migration during embryonic stages but is required for the development and maintenance of the MZ in the postnatal cerebral cortex.

Multiple classes of transcription factors regulate the expression of VASCULAR-RELATED NAC-DOMAIN7, a master switch of xylem vessel differentiation.

The secondary cell walls of xylem cells, including vessel elements, provide mechanical strength and contribute to the conduction of water and minerals. VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a NAC-domain transcription factor that regulates the expression of genes required for xylem vessel element formation. Transient expression assays using 68 transcription factors that are expressed during xylem vessel differentiation showed that 14 transcription factors, including VND1-VND7, are putative positive regulators of VND7 expression. Electrophoretic mobility shift assays revealed that all seven VND proteins bound to the VND7 promoter region at its SMBE/TERE motif, indicating that VND7 is a direct target of all of the VND transcription factors. Overexpression of VND1-VND5, GATA12 and ANAC075, newly identified transcription factors that function upstream of VND7, resulted in ectopic xylem vessel element formation. These data suggest that VND7 transcription is a regulatory target of multiple classes of transcription factors.

Contribution of NAC transcription factors to plant adaptation to land.

The development of cells specialized for water conduction or support is a striking innovation of plants that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of these cells in vascular plants. However, the path by which plants with these cells have evolved from their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting-like cells. Our results show conservation of transcriptional regulation and cellular function between moss and Arabidopsis thaliana water-conducting cells. The conserved genetic basis suggests roles for NAC proteins in the adaptation of plants to land.

DWARF50 (D50), a rice (Oryza sativa L.) gene encoding inositol polyphosphate 5-phosphatase, is required for proper development of intercalary meristem.

Rice internodes are vital for supporting high-yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell-producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5-phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization.

VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system.

We previously showed that the VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 genes, which encode NAM/ATAF/CUC domain protein transcription factors, act as key regulators of xylem vessel differentiation. Here, we report a glucocorticoid-mediated posttranslational induction system of VND6 and VND7. In this system, VND6 or VND7 is expressed as a fused protein with the activation domain of the herpes virus VP16 protein and hormone-binding domain of the animal glucocorticoid receptor, and the protein's activity is induced by treatment with dexamethasone (DEX), a glucocorticoid derivative. Upon DEX treatment, transgenic Arabidopsis (Arabidopsis thaliana) plants carrying the chimeric gene exhibited transdifferentiation of various types of cells into xylem vessel elements, and the plants died. Many genes involved in xylem vessel differentiation, such as secondary wall biosynthesis and programmed cell death, were up-regulated in these plants after DEX treatment. Chemical analysis showed that xylan, a major hemicellulose component of the dicot secondary cell wall, was increased in the transgenic plants after DEX treatment. This induction system worked in poplar (Populus tremula x tremuloides) trees and in suspension cultures of cells from Arabidopsis and tobacco (Nicotiana tabacum); more than 90% of the tobacco BY-2 cells expressing VND7-VP16-GR transdifferentiated into xylem vessel elements after DEX treatment. These data demonstrate that the induction systems controlling VND6 and VND7 activities can be used as powerful tools for understanding xylem cell differentiation.

Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components.

The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.

C-terminal region-dependent change of antibody-binding to the Eighth Reelin repeat reflects the signaling activity of Reelin.

Reelin is a secreted glycoprotein that plays pivotal roles in the development and function of the brain, but how it activates downstream intracellular signaling is not fully understood. We have recently reported that the highly conserved C-terminal region (CTR) of Reelin is required for its full signaling activity, although the underlying mechanism remains unknown. During biochemical study of Reelin, we serendipitously found that one commercially available anti-Reelin antibody G20 can bind to CTR-lacking mutant Reelin proteins, but not wild-type Reelin, on Western blotting. The G20 epitope resides in the last 19 residues of Reelin-repeat 8 (RR8), and neither posttranslational modification nor proteolysis can explain this effect. Furthermore, when an unrelated sequence, such as FLAG-tag, is inserted between RR8 and CTR, the reactivity of the corresponding antibody greatly decreases. These results suggest that RR8 and CTR form a tight structure that makes the surrounding sequence inaccessible to an antibody. Taking advantage of this phenomenon, we show the existence of CTR-lacking Reelin isoform in vivo for the first time and estimate its contribution to the total amount of secreted Reelin. Importantly, the extent to which Reelin mutants react with G20 is inversely correlated with their signaling activity, indicating that the CTR-induced structural change of RR8 is a prerequisite for downstream signaling activation, presumably via binding to a certain neuronal membrane molecule(s).

Mechanism and significance of specific proteolytic cleavage of Reelin.

Reelin is a secreted glycoprotein essential for normal brain development and function. In the extracellular milieu, Reelin is subject to specific cleavage at two (N-t and C-t) sites. The N-t cleavage of Reelin is implicated in psychiatric and Alzheimer's diseases, but the molecular mechanism and physiological significance of this cleavage are not completely understood. Particularly, whether the N-t cleavage affects the signaling activity of Reelin remains controversial. Here, we show that the protease in charge of the N-t cleavage of Reelin requires the activity of certain proprotein convertase family for maturation and has strong affinity for heparin. By taking advantage of these observations, we for the first time succeeded in obtaining "Uncleaved" and "Completely Cleaved" Reelin proteins. The N-t cleavage splits Reelin into two distinct fragments and virtually abolishes its signaling activity. These findings provide an important biochemical basis for the function of Reelin proteolysis in brain development and function.

Structure of a receptor-binding fragment of reelin and mutational analysis reveal a recognition mechanism similar to endocytic receptors.

Reelin, a large secreted protein implicated in the cortical development of the mammalian brain, is composed of eight tandem concatenations of "reelin repeats" and binds to neuronal receptors belonging to the low-density lipoprotein receptor gene family. We found that both receptor-binding and subsequent Dab1 phosphorylation occur solely in the segment spanning the fifth and sixth reelin repeats (R5-6). Monomeric fragment exhibited a suboptimal level of signaling activity and artificial oligomerization resulted in a 10-fold increase in activity, indicating the critical importance of higher-order multimerization in physiological reelin. A 2.0-A crystal structure from the R5-6 fragment revealed not only a unique domain arrangement wherein two repeats were aligned side by side with the same orientation, but also the unexpected presence of bound Zn ions. Structure-guided alanine mutagenesis of R5-6 revealed that two Lys residues (Lys-2360 and Lys-2467) constitute a central binding site for the low-density lipoprotein receptor class A module in the receptor, indicating a strong similarity to the ligand recognition mode shared among the endocytic lipoprotein receptors.

The extremely conserved C-terminal region of Reelin is not necessary for secretion but is required for efficient activation of downstream signaling.

Reelin is a very large secreted glycoprotein essential for correct development of the mammalian brain. It is also implicated in higher functions and diseases of human brain. However, whether or not secretion of Reelin is regulated and how Reelin transmits signals remain largely unknown. Reelin protein is composed of an N-terminal F-spondin-like domain, Reelin repeats, and a short and highly basic C-terminal region (CTR). The primary sequence of CTR is almost completely conserved among vertebrates except fishes, indicating its importance. A prevailing idea regarding the function of CTR is that it is required for the secretion of Reelin, although this remains unproven. Here we aimed to clarify the function of Reelin CTR. Neither deleting most of CTR nor replacing CTR with unrelated amino acids affected secretion efficiency, indicating that CTR is not absolutely required for the secretion of Reelin. We also found that Reelin mutants without CTR were less potent in activating the downstream signaling in cortical neurons. Although these mutants were able to bind to the Reelin receptor ectodomain as efficiently as wild-type Reelin, quite interestingly, their ability to bind to the isolated cell membrane bearing Reelin receptors or receptor-expressing cells (including cortical neurons) was much weaker than that of wild-type Reelin. Therefore, it is concluded that the CTR of Reelin is not essential for its secretion but is required for efficient activation of downstream signaling events, presumably via binding to an unidentified "co-receptor" molecule(s) on the cell membrane.