Plasmodesmata callose binding protein 2 contributes to the regulation of cambium/phloem formation and auxin response during the tissue reunion process in incised Arabidopsis stem.

Plants are exposed to a variety of biotic and abiotic stresses, including wounding at the stem. The healing process (tissue reunion) begins immediately after stem wounding. The plant hormone auxin plays an important role during tissue reunion. In decapitated stems, auxin transport from the shoot apex is reduced and tissue reunion does not occur but is restored by application of indole-3-acetic acid (IAA). In this study, we found that plasmodesmata callose binding protein 2 (PDCB2) affects the expansion of the cambium/phloem region via changes in auxin response during the process of tissue reunion. PDCB2 was expressed in the cortex and endodermis on the incised side of stems 1-3 days after incision. PDCB2-knockout plants showed reduced callose deposition at plasmodesmata and DR5::GUS activity in the endodermis/cortex in the upper region of the incision accompanied by an increase in size of the cambium/phloem region during tissue reunion. In addition, PIN(PIN-FORMED)3, which is involved in lateral auxin transport, was induced by auxin in the cambium/phloem and endodermis/cortex in the upper part of the incision in wild type, but its expression of PIN3 was decreased in pdcb2 mutant. Our results suggest that PDCB2 contributes to the regulation of cambium/phloem development via auxin response.

Characterization of zinc uptake and translocation visualized with positron-emitting 65Zn tracer and analysis of transport-related gene expression in two Lotus japonicus accessions.

BACKGROUND AND AIMS: Zinc (Zn) is an essential element for humans and plants. However, Zn deficiency is widespread and 25 % of the world's population is at risk of Zn deficiency. To overcome the deficiency of Zn intake, crops with high Zn content are required. However, most crop-producing areas have Zn-deficient soils, therefore crops with excellent Zn uptake/transport characteristics (i.e. high Zn efficiency) are needed. Our objective was to identify the crucial factors responsible for high Zn efficiency in the legume Lotus japonicus. METHODS: We evaluated Zn efficiency by static and real-time visualization of radioactive Zn (65Zn) uptake/transport in two L. japonicus accessions, MG-20 and B-129, that differ in Zn efficiency. The combination of visualization methods verified the dynamics of Zn accumulation and transport within the plant. We compared gene expression under a normal Zn concentration (control) and Zn deficiency to evaluate genetic factors that may determine the differential Zn efficiency of the accessions. KEY RESULTS: The accession B-129 accumulated almost twice the amount of Zn as MG-20. In the static 65Zn images, 65Zn accumulated in meristematic tissues, such as root tips and the shoot apex, in both accessions. The positron-emitting tracer imaging system (PETIS), which follows the transport process in real time, revealed that 65Zn transport to the shoot was more rapid in B-129 than in MG-20. Many genes associated with Zn uptake and transport were more highly expressed in B-129 than in MG-20 under the control condition. These gene expression patterns under Zn deficiency differed from those under the control Zn condition. CONCLUSIONS: PETIS confirmed that the real-time transport of 65Zn to the shoot was faster in B-129 than in MG-20. The high Zn efficiency of B-129 may be due to the elevated expression of a suite of Zn uptake- and transport-related genes.

WIND transcription factors orchestrate wound-induced callus formation, vascular reconnection and defense response in Arabidopsis.

Wounding triggers de novo organogenesis, vascular reconnection and defense response but how wound stress evoke such a diverse array of physiological responses remains unknown. We previously identified AP2/ERF transcription factors, WOUND INDUCED DEDIFFERENTIATION1 (WIND1) and its homologs, WIND2, WIND3 and WIND4, as key regulators of wound-induced cellular reprogramming in Arabidopsis. To understand how WIND transcription factors promote downstream events, we performed time-course transcriptome analyses after WIND1 induction. We observed a significant overlap between WIND1-induced genes and genes implicated in cellular reprogramming, vascular formation and pathogen response. We demonstrated that WIND transcription factors induce several reprogramming genes to promote callus formation at wound sites. We, in addition, showed that WIND transcription factors promote tracheary element formation, vascular reconnection and resistance to Pseudomonas syringae pv. tomato DC3000. These results indicate that WIND transcription factors function as key regulators of wound-induced responses by promoting dynamic transcriptional alterations. This study provides deeper mechanistic insights into how plants control multiple physiological responses after wounding.

RAP2.6L and jasmonic acid-responsive genes are expressed upon Arabidopsis hypocotyl grafting but are not needed for cell proliferation related to healing.

KEY MESSAGE: Jasmonic acid and RAP2.6L are induced upon wounding but are not involved in cell proliferation during healing in Arabidopsis hypocotyls. Plants produce jasmonic acid in response to wounding, but its role in healing, if any, has not been determined. Previously, the jasmonic acid-induced transcription factor, RAP2.6L, related to APETALA 2.6-like, was identified as a spatially expressed factor involved in tissue reunion in partially incised flowering stems of Arabidopsis. In the present study, we investigated the function of JA and RAP2.6L on wound healing using an Arabidopsis hypocotyl-grafting system, in which separated tissues are reattached by vascular tissue cell proliferation. The jasmonic acid-responsive genes AOS and JAZ10 were transiently expressed immediately after grafting. We confirmed that the endogenous content of jasmonic acid-Ile, which is the bioactive form of jasmonic acid, increased in hypocotyls 1 h after grafting. Morphological analysis of the grafted tissue revealed that vascular tissue cell proliferation occurred in a similar manner in wild-type Arabidopsis, the jasmonic acid-deficient mutant aos, the jasmonic acid-insensitive mutant coi1, and in Arabidopsis that had been exogenously treated with jasmonic acid. RAP2.6L expression was also induced during graft healing. Because RAP2.6L expression occurred during graft healing in aos and coi1, its expression must be regulated via a jasmonic acid-independent pathway. The rap2.6L mutant and dominant repressor transformants for RAP2.6L showed normal cell proliferation during graft healing. Taken together, our results suggest that JA and RAP2.6L, induced by grafting, are not necessary for cell proliferation process in healing.

Differential Cellular Control by Cotyledon-Derived Phytohormones Involved in Graft Reunion of Arabidopsis Hypocotyls.

When wounding or grafting interrupts the original connection of plant tissue, cell proliferation is induced and the divided tissue is reunited. Previous studies suggested that gibberellin derived from the cotyledon is required for tissue reunion in cucumber and tomato incised hypocotyls, and tissue reunion of Arabidopsis incised flowering stems is controlled by auxin. Differences in the hormone requirements of the tissue reunion process between Arabidopsis and cucumber might be due to differences in organs or species. In this study, we performed morphological and gene expression analyses of graft union in Arabidopsis hypocotyl. We found that removal of the cotyledon and treatment of the cotyledon with the auxin transport inhibitor triiodobenzoic acid (TIBA) suppressed cell proliferation of vascular tissue during graft union formation. These treatments also suppressed expression of IAA5, ANAC071, ANAC096 and CYCB1;1. ANAC071 is involved in the tissue reunion process. The anac071 anac096 double mutant suppressed cell proliferation more so than either of the single mutants. On the other hand, paclobutrazol treatment or deficiency of gibberellin biosynthesis genes suppressed expansion of cortex cells, and exogenous gibberellin treatment or rga/gai mutations that lack the negative regulator of gibberellin reversed this inhibition. The up-regulation of the key gibberellin biosynthesis gene GA20ox1 during graft union formation was prevented by cotyledon removal or TIBA treatment. These data suggest that auxin regulates cell proliferation of vascular tissue and expansion of cortex cells by promoting gibberellin biosynthesis during graft attachment. We hypothesize that the cotyledon-derived phytohormones are essential for graft reunion of the hypocotyl, processed in a cell type-specific manner, in Arabidopsis.

CLE6 expression recovers gibberellin deficiency to promote shoot growth in Arabidopsis.

Small peptides act as local signals during plant development, but few studies have examined their interaction with phytohormone signaling. Here, we show that application of gibberellin (GA) to Arabidopsis shoots induces substantial accumulation of transcripts encoded by CLE6, a member of the CLAVATA/ESR-RELATED (CLE) gene family, in the root stele, followed by promotion of organ growth by CLE6 in GA-deficient plants. The long-distance effect of GA4 was demonstrated by the observation that its application to the shoot apex of the GA-deficient mutant ga3ox1/ga3ox2 rescued the short-root phenotype. Microarray analysis was used to identify root-expressed genes that respond to systemic application of GA, and CLE6 was selected for further analysis. CLE6 was highly expressed in roots at the young seedling stage, and CLE6 promoter activity was strong in hypocotyls and roots, especially in root stele cells at branch points. Application of CLE6 peptide had no obvious effect on the growth and development of GA-deficient mutant plants. Nonetheless, the fact that ectopic over-expression of CLE6 in the GA-deficient mutant promoted root growth and branching, petiole elongation, bolting rate and stem length showed that CLE6 expression partially compensates for the GA deficiency. Reciprocal grafting of GA-deficient mutant plants to 35S::CLE6 transformants complemented the shoot phenotype associated with GA deficiency, demonstrating the systemic effect of CLE6 from root to shoot. These data suggest that root-expressed CLE6 is systemically involved in shoot growth under GA action in Arabidopsis.

XTH20 and XTH19 regulated by ANAC071 under auxin flow are involved in cell proliferation in incised Arabidopsis inflorescence stems.

One week after partial incision of Arabidopsis inflorescence stems, the repair process in damaged tissue includes pith cell proliferation. Auxin is a key factor driving this process, and ANAC071, a transcription factor gene, is upregulated in the distal region of the incised stem. Here we show that XTH20 and the closely related XTH19, members of xyloglucan endotransglucosylase/hydrolases family catalyzing molecular grafting and/or hydrolysis of cell wall xyloglucans, were also upregulated in the distal part of the incised stem, similar to ANAC071. XTH19 was expressed in the proximal incision region after 3 days or after auxin application to the decapitated stem. Horizontal positioning of the plant with the incised side up resulted in decreased ProDR 5 :GUS, ANAC071, XTH20, and XTH19 expression and reduced pith cell proliferation. In incised stems of Pro35S :ANAC071-SRDX plants, expression of XTH20 and XTH19 was substantially and moderately decreased, respectively. XTH20 and XTH19 expression and pith cell proliferation were suppressed in anac071 plants and were increased in Pro35S :ANAC071 plants. Pith cell proliferation was also inhibited in the xth20xth19 double mutant. Furthermore, ANAC071 bound to the XTH20 and XTH19 promoters to induce their expression. This study revealed XTH20 and XTH19 induction by auxin via ANAC071 in the distal part of an incised stem and their involvement in cell proliferation in the tissue reunion process.

Gibberellin-induced expression of Fe uptake-related genes in Arabidopsis.

In dicots, iron (Fe) is acquired from the soil by IRT1 (IRON-REGULATED TRANSPORTER 1) and FRO2 (FERRIC REDUCTION OXIDASE 2) that are localized at the root epidermis. IRT1 and FRO2 expression is induced by local and systemic signals under Fe-deficient conditions in Arabidopsis thaliana. In this study, the expression of IRT1, FRO2, bHLH038 and bHLH39 (the latter two of which control IRT1 and FRO2 expression) was promoted by GA4 treatment of gibberellin (GA) deficient ga3ox1 ga3ox2 mutants. In contrast, the expression of FIT, which encodes a transcription factor necessary for IRT1 and FRO2 induction under Fe deficiency, was not induced by the application of GA4. The induction of those genes triggered by shoot-applied GA4 was observed, even in the fit-2 mutant which had reduced endogenous GA levels caused by treatment with paclobutrazol (PBZ), a GA biosynthesis inhibitor. These results suggested that FIT was not a key regulator in the GA responses under Fe-sufficient conditions. On the other hand, among Fe uptake-related genes, the expression of IRT1, bHLH038 and bHLH39 was lower in ga3ox1 ga3ox2 compared with the wild type (WT) under Fe-sufficient conditions, but the expression of all Fe uptake-related genes decreased under Fe-deficient conditions. Additionally, the PBZ treatment decreased IRT1 expression in the WT under Fe-deficient conditions, but not in the fit-2 mutant. These data suggest the contribution of GA to the induction of Fe uptake-related genes under Fe-sufficient and Fe-deficient conditions, possibly in FIT-independent and FIT-dependent manners, respectively.

Retrograde thrombectomy of acute common carotid artery occlusion with mobile thrombus: illustrative cases.

BACKGROUND: Acute embolic occlusion of the common carotid artery (CCA) alone is rare. However, once it occurs, recanalization is challenging due to the large volume of the clot, larger diameter of the CCA, and risk of procedure-related distal embolism into the intracranial arteries. OBSERVATIONS: The authors report two cases of acute embolic occlusion of CCA alone, caused by a cardiac embolus trapped at the proximal end of a preexisting atherosclerotic plaque at the cervical carotid bifurcation. In both cases, the CCA was successfully recanalized using retrograde thrombectomy in a hybrid operating room. In case 1, a 78-year-old male with acute right CCA occlusion underwent retrograde thrombectomy, where the cervical carotid bifurcation was exposed and incised, and the entire embolus was retrieved with forceps. Despite successful revascularization, massive bleeding from the CCA just after the retrieval remained a concern. In case 2, a 79-year-old female with acute right CCA occlusion underwent retrograde thrombectomy in the same manner. Because manual retrieval failed, a Fogarty balloon catheter inserted from the arteriotomy successfully retrieved the entire thrombus with minimal blood loss. LESSONS: Retrograde thrombectomy through the arteriotomy of the cervical carotid bifurcation safely and effectively recanalizes acute embolic occlusion of the CCA alone.

Structure-ATPase Activity Relationship of Rhodamine Derivatives as Potent Inhibitors of P-Glycoprotein CmABCB1.

Understanding the transport and inhibition mechanisms of substrates by P-glycoprotein (P-gp) is one of the important approaches in addressing multidrug resistance (MDR). In this study, we evaluated a variety of rhodamine derivatives as potential P-gp inhibitors targeting CmABCB1, a P-gp homologue, with a focus on their ATPase activity. Notably, a Q-rhodamine derivative with an o,o'-dimethoxybenzyl ester moiety (RhQ-DMB) demonstrated superior affinity and inhibitory activity, which was further confirmed by a drug susceptibility assay in yeast strains expressing CmABCB1. Results from a tryptophan fluorescence quenching experiment using a CmABCB1 mutant suggested that RhQ-DMB effectively enters and binds to the inner chamber of CmABCB1. These findings underscore the promising potential of RhQ-DMB as a tool for future studies aimed at elucidating the substrate-bound state of CmABCB1.

Vertebral artery wall inflammation suspected as the cause of cryptogenic ischemic stroke developing during the recovery period of COVID-19.

Coronavirus disease-2019 (COVID-19) can cause acute ischemic stroke (AIS) due to large vessel occlusion (LVO). Some cases of COVID-19-related LVO are known to be resistant to mechanical thrombectomy and have different characteristics from non-COVID-19-related LVO. Inflammation of the occluded arterial wall is suspected as one of the causes of such differences, but the exact mechanism is not fully understood. A 52-year-old man suffered from AIS due to left vertebral artery (VA) occlusion during the recovery period after mild COVID-19. Successful recanalization of the left VA was achieved with antithrombotic therapy, but a late and reversible edematous lesion appeared in part of the brainstem adjacent to the left VA, with abnormal enhancement in both the left VA wall and medulla oblongata on postcontrast magnetic resonance imaging. We suggest that the left VA wall inflammation, induced by COVID-19, caused the ischemic stroke and extended to the brainstem, and an incidental thrombosed unruptured aneurysm of the left VA accelerated these changes. This case provides the first evidence of LVO after COVID-19 in which the pathological conditions in the brainstem adjacent to the affected artery could be observed with neuroimaging and inflammation of the arterial wall was indirectly confirmed. Physicians should be aware that unconventional ischemic stroke may develop in some patients during the recovery period after COVID-19.

Cell-wall damage activates DOF transcription factors to promote wound healing and tissue regeneration in Arabidopsis thaliana.

Wound healing is a fundamental property of plants and animals that requires recognition of cellular damage to initiate regeneration. In plants, wounding activates a defense response via the production of jasmonic acid and a regeneration response via the hormone auxin and several ethylene response factor (ERF) and NAC domain-containing protein (ANAC) transcription factors. To better understand how plants recognize damage and initiate healing, we searched for factors upregulated during the horticulturally relevant process of plant grafting and found four related DNA binding with one finger (DOF) transcription factors, HIGH CAMBIAL ACTIVITY2 (HCA2), TARGET OF MONOPTEROS6 (TMO6), DOF2.1, and DOF6, whose expression rapidly activated at the Arabidopsis graft junction. Grafting or wounding a quadruple hca2, tmo6, dof2.1, dof6 mutant inhibited vascular and cell-wall-related gene expression. Furthermore, the quadruple dof mutant reduced callus formation, tissue attachment, vascular regeneration, and pectin methylesterification in response to wounding. We also found that activation of DOF gene expression after wounding required auxin, but hormone treatment alone was insufficient for their induction. However, modifying cell walls by enzymatic digestion of cellulose or pectin greatly enhanced TMO6 and HCA2 expression, whereas genetic modifications to the pectin or cellulose matrix using the PECTIN METHYLESTERASE INHIBITOR5 overexpression line or korrigan1 mutant altered TMO6 and HCA2 expression. Changes to the cellulose or pectin matrix were also sufficient to activate the wound-associated ERF115 and ANAC096 transcription factors, suggesting that cell-wall damage represents a common mechanism for wound perception and the promotion of tissue regeneration.

Crystal structure of CmABCB1 multi-drug exporter in lipidic mesophase revealed by LCP-SFX.

CmABCB1 is a Cyanidioschyzon merolae homolog of human ABCB1, a well known ATP-binding cassette (ABC) transporter responsible for multi-drug resistance in various cancers. Three-dimensional structures of ABCB1 homologs have revealed the snapshots of inward- and outward-facing states of the transporters in action. However, sufficient information to establish the sequential movements of the open-close cycles of the alternating-access model is still lacking. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has proven its worth in determining novel structures and recording sequential conformational changes of proteins at room temperature, especially for medically important membrane proteins, but it has never been applied to ABC transporters. In this study, 7.7 mono-acyl-glycerol with cholesterol as the host lipid was used and obtained well diffracting microcrystals of the 130 kDa CmABCB1 dimer. Successful SFX experiments were performed by adjusting the viscosity of the crystal suspension of the sponge phase with hy-droxy-propyl methyl-cellulose and using the high-viscosity sample injector for data collection at the SACLA beamline. An outward-facing structure of CmABCB1 at a maximum resolution of 2.22 Šis reported, determined by SFX experiments with crystals formed in the lipidic cubic phase (LCP-SFX), which has never been applied to ABC transporters. In the type I crystal, CmABCB1 dimers interact with adjacent molecules via not only the nucleotide-binding domains but also the transmembrane domains (TMDs); such an interaction was not observed in the previous type II crystal. Although most parts of the structure are similar to those in the previous type II structure, the substrate-exit region of the TMD adopts a different configuration in the type I structure. This difference between the two types of structures reflects the flexibility of the substrate-exit region of CmABCB1, which might be essential for the smooth release of various substrates from the transporter.

The crystal structure of the CmABCB1 G132V mutant, which favors the outward-facing state, reveals the mechanism of the pivotal joint between TM1 and TM3.

CmABCB1 is a homologue of human P-glycoprotein, which extrudes various substrates by iterative cycles of conformational changes between the inward- and outward-facing states. Comparison of the inward- and outward-facing structures of CmABCB1 suggested that pivotal joints in the transmembrane domain regulate the tilt of transmembrane helices. Transmembrane helix 1 (TM1) forms a tight helix-helix contact with TM3 at the TM1-3 joint. Mutation of Gly132 to valine at the TM1-3 joint, G132V, caused a 10-fold increase in ATPase activity, but the mechanism underlying this change remains unclear. Here, we report a crystal structure of the outward-facing state of the CmABCB1 G132V mutant at a 2.15 Å resolution. We observed structural displacements between the outward-facing states of G132V and the previous one at the region around the TM1-3 joint, and a significant expansion at the extracellular gate. We hypothesize that steric hindrance caused by the Val substitution shifted the conformational equilibrium toward the outward-facing state, favoring the dimeric state of the nucleotide-binding domains and thereby increasing the ATPase activity of the G132V mutant.

Wound-inducible ANAC071 and ANAC096 transcription factors promote cambial cell formation in incised Arabidopsis flowering stems.

ANAC071 and its homolog ANAC096 are plant-specific transcription factors required for the initiation of cell division during wound healing in incised Arabidopsis flowering stems and Arabidopsis hypocotyl grafts; however, the mechanism remains mostly unknown. In this study, we showed that wound-induced cambium formation involved cell proliferation and the promoter activity of TDR/PXY (cambium-related gene) in the incised stem. Prior to the wound-induced cambium formation, both ANAC071 and ANAC096 were expressed at these sites. anac-multiple mutants significantly decreased wound-induced cambium formation in the incised stems and suppressed the conversion from mesophyll cells to cambial cells in an ectopic vascular cell induction culture system (VISUAL). Our results suggest that ANAC071 and ANAC096 are redundantly involved in the process of "cambialization", the conversion from differentiated cells to cambial cells, and these cambium-like cells proliferate and provide cells in wound tissue during the tissue-reunion process.

Acute Deterioration in a Patient with Bilateral Chronic Subdural Hematomas Associated with Intracranial Hypotension Treated with an Epidural Blood Patch.

BACKGROUND: Intracranial hypotension due to cerebrospinal fluid (CSF) leak is often associated with secondary chronic subdural hematoma (CSDH). Although epidural blood patch (EBP) treatment for the CSF leak site has been reported to result in spontaneous regression of the CSDH in most cases, it is still debatable whether blocking CSF leak first in the patients with intracranial hematoma is always safe. CASE DESCRIPTION: A 72-year-old woman presented with orthostatic headache after a head injury and was diagnosed with intracranial hypotension. Computed tomography myelography and radioisotope cisternography failed to reveal the CSF leak point. The overflow leak test, a novel diagnostic method for intracranial hypotension, revealed a leakage at the cervical spine. Bilateral CSDHs were also observed by a computed tomography scan of the head. We performed EBP at the cervical spine and anticipated subsequent regression of the CSDH by normalizing intrathecal pressure. However, the patient became delirious the morning after EBP, and an emergency burr hole trepanation was performed. The patient's consciousness fully recovered, and her orthostatic headache improved as well. CONCLUSIONS: This case presentation demonstrated that in the case of intracranial hypotension with secondary CSDH, performing EBP and waiting for subsequent spontaneous regression of CSDH are not necessarily safe. Immediate burr hole trepanation should be prepared for the subsequent rapid symptomatic change of the CSDH after EBP.

Inward- and outward-facing X-ray crystal structures of homodimeric P-glycoprotein CmABCB1.

P-glycoprotein extrudes a large variety of xenobiotics from the cell, thereby protecting tissues from their toxic effects. The machinery underlying unidirectional multidrug pumping remains unknown, largely due to the lack of high-resolution structural information regarding the alternate conformational states of the molecule. Here we report a pair of structures of homodimeric P-glycoprotein: an outward-facing conformational state with bound nucleotide and an inward-facing apo state, at resolutions of 1.9 Å and 3.0 Å, respectively. Features that can be clearly visualized at this high resolution include ATP binding with octahedral coordination of Mg2+; an inner chamber that significantly changes in volume with the aid of tight connections among transmembrane helices (TM) 1, 3, and 6; a glutamate-arginine interaction that stabilizes the outward-facing conformation; and extensive interactions between TM1 and TM3, a property that distinguishes multidrug transporters from floppases. These structural elements are proposed to participate in the mechanism of the transporter.

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.