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The first human prenatal repair of MMC was reported in Tulipan et al. . Cumulative data suggested not only a dramatic improvement in hindbrain herniation but also increased maternal and neonatal risks including preterm labor, uterine dehiscence, and increased risk of fetal and neonatal death among others. Adzick et al. investigated the effects of prenatal repair of MMC via a randomized prospective study. It reported that prenatal surgery for MMC performed before 26 weeks of gestation decreased the risk of death or need for shunting by the age of 12 months and also improved scores on a composite measure of mental and motor function, with adjustment for lesion level, at 30 months of age. Prenatal surgery also improves the degree of hindbrain herniation associated with Chiari II malformation, motor function, and the likelihood of being able to walk independently, as compared with postnatal surgery . Open prenatal repair comes with an increased maternal and neonatal risk including preterm labor, uterine dehiscence, premature rupture of membranes, and increased risk of fetal and neonatal death. The main goal for prenatal repair of MMC is to achieve skin closure to prevent further damage of the placode and arrest the CSF leak.
|
review
| 99.9 |
Despite the 250 mouse mutants with NTDs to date, there has yet to be a significant breakthrough for human NTD gene(s) both causal and/or associated with NTDs that can be used for genetic screening worldwide [4, 7]. The importance of finding candidate gene(s) as a genetic screening tool for potential parents cannot be undervalued as it has been estimated that the total lifetime costs for patients with spina bifida (spinal NTDs) amount to about $1.4 million in the US and more than €500k in Europe, with 37.1% of the total cost attributed to direct medical costs and the remainder in indirect costs, including the needs of the caregiver .
|
review
| 86.7 |
Despite observation of multiplex nonsyndromic NTD cases in multigenerational NTD families as seen in 17 US and 14 Dutch families with more than 1 NTD-affected person, there are other NTD cases that are simplex and sporadic as seen in identical twins with lumbosacral lipomyelomeningocele with no known familiar history of NTDs [201, 202]. This suggests that NTDs have a multifactorial genetic etiology.
|
study
| 99.8 |
To date, the strongest candidate thus far for a potential NTD screening gene is the methylenetetrahydrofolate reductase (MTHFR) C677T (rs1801133) polymorphism in populations of non-Latin origin (meta-analysis study) . In recent meta-analysis study, Zhang et al. support the significant association between C677T and NTDs in case-control studies (22 studies, 2,602 cases, and 4,070 controls) . The second most studied MTHFR variant is A1298C, which did not report any significant increase in risk of NTDs . Another meta-analysis study by Blom et al. (2006) reported increased risk in mothers and associated with NTD infants who are homozygous for C677T variant . In spina bifida case studies, MTHFR C677T variant was clearly reported as associated gene or risk factors in Irish (451 spina bifida patients), mixed USA, mixed UK, and Italian cohort but not in other 180 Dutch patients (Table 3), while A1298C variant was reported with no association to spina bifida cases in Italian, Mexican (Yucatan), and Dutch population (Table 3). MTHFR is the most studied human spina bifida gene, as its role in folate one-carbon metabolism fits into a clear mechanism of NTD. However, the studies have not been well replicated in many other populations across the world, indicating that it is not likely to be either a major contributor or a common factor in NTD globally.
|
study
| 65.75 |
Other genes such as the planar cell polarity (PCP) genes, which have been studied in spina bifida cohorts among Italians, Americans, and the French, are VANGL1 and CESLR1 [44, 82–84]. The noncore PCP gene SCRIB has also been implicated as a spina bifida gene among the American cohort . However, noncore PCP gene association needs to be explored further in larger NTD cohorts. To date, over 100 human spina bifida genes have been used to screen for spina bifida with 48 genes reported as a potential risk factor as listed in Table 3 which was reviewed in Greene et al. ; further candidates since then are NKX2-8, PTCH1, Glypican-5, PARD3, Paraoxonase 1, COMT, AMT, and GLDC genes [16, 17, 206–211]. All of these do not represent a potential global spina bifida gene. Therefore, a strong candidate spina bifida gene(s) for the world population has yet to be discovered.
|
review
| 99.9 |
There exist more than 250 mouse models with neural tube defects, of which 74 are of spina bifida (Table 2) , yet there does not exist a single mouse gene which can be used to screen the orthologous human gene of neural tube defect nor spina bifida to date . That said, it does not mean that the studies on the structural changes afforded by the mouse model cannot be used as a tool to understand human spina bifida. We discuss the various studies on mouse neurulation below and why it is still an invaluable tool for understanding human neurulation.
|
review
| 99.9 |
In vertebrates, the development of the CNS starts with the formation of the neural plate on the dorsal surface of the embryo during late gastrulation [213, 214]. A complex morphogenetic process transforms the neural plate into the hollow neural tube in a process known as “neurulation” . Primary neurulation is responsible for formation of the neural tube throughout the brain and the spinal cord rostral to the mid-sacral level . At more caudal levels, an alternative mechanism (secondary neurulation) operates whereby the neural tube is formed by canalization of a condensed rod of mesenchymal cells in the tail bud .
|
study
| 99.94 |
The process of neurulation in mammals and some other vertebrates is considered discontinuous because it occurs simultaneously at multiple sites along the neuraxis [215–219]. There are three points of de novo neural tube fusion in the mouse, which is the most studied mammalian model (; see Figure 2(a)). Closure 1 occurs adjacent to somite 3 in embryos with 6-7 somites and progresses rostrally and caudally, closure 2 occurs at the midbrain-forebrain boundary at around the 10-somite stage and progresses caudally, and closure 3 occurs at the rostral end of the forebrain, soon after closure 2.
|
study
| 99.94 |
Considering this discontinuous process of neurulation, it can be understood why NTDs are such a complex group of heterogeneous birth defects, with various phenotypic presentations. Failure of closure 1 leads to craniorachischisis (Figure 2(b)); failure of closures 2 and/or 3 causes exencephaly and/or anencephaly, respectively (Figure 2(c)), while failure of neurulation to progress from the site of closure 1 caudally along the spinal axis leads to spina bifida aperta (Figure 2(d)).
|
study
| 94.2 |
During neurulation, the neuroepithelium must undergo various structural changes in order to achieve closure. The advent of molecular biology has allowed scientists to identify the genes that are required for these structural changes to occur. The next section gives a brief overview of the research to date on how gene expression affects structural changes in neural tube development, with an emphasis on gene regulation in the spinal region.
|
review
| 99.9 |
Morphologically, the mouse neural tube undergoes distinct structural changes prior to its closure [7, 215, 221–224]. A summary of the spatiotemporal expression of genes in the mouse neural tube during neurulation is as shown in Table 5. The neuroepithelium narrows and lengthens, a process referred to as convergent extension (Figure 3(a)), in which the polarized cells which form the neuroepithelial plate converge towards the midline, elongate anteroposteriorly, and then intercalate [215, 225].
|
study
| 99.94 |
Convergent extension leads to narrowing and lengthening of the neuroepithelium, a process that has been suggested also to assist neural fold elevation via axial elongation [105, 226–228]. However, the lengthening of the body axis is disrupted by manipulation of gene function required for convergent extension; whilst the neural folds are still able to elevate, convergent extension still fails [227, 229, 230]. Hence, convergent extension and neural fold elevation are separable processes. Elevation of the neural folds at high levels of the spinal neuraxis results from the formation of a median hinge point (MHP) (Figure 3(b)) in a process termed Mode 1 neurulation [215, 231, 232]. The neural folds remain straight along both apical and basal surfaces, resulting in a neural tube with a slit-shaped lumen. Mode 1 neurulation occurs during formation of the spinal neural tube in 6–10-somite stage embryos, as shown in Figures 4(a) and 4(b).
|
study
| 100.0 |
A second set of hinge points are formed dorsolaterally at more caudal levels of the spinal neuraxis, the dorsolateral hinge points (DLHPs), a process that appears to enhance the ability of the apposing tips of the neural folds to come close to each other (Figure 3(c)). Mode 2 occurs during formulation of the spinal neural tube in 12–15-somite stage embryos and generates a diamond-shaped lumen, as depicted in Figures 4(c) and 4(d). In Mode 2, a median hinge point is also present, whereas the remaining portions of the neuroepithelium do not bend. At the 17–27-somite stage, the neural tube closes without a median hinge point, whereas dorsolateral hinge points are retained. This is known as Mode 3 neurulation and generates an almost circular shaped lumen, as shown in Figures 4(e) and 4(f).
|
study
| 100.0 |
Adhesion of the tips of the apposing neural folds is the final step in primary neurulation, enabling the neural tube to complete its closure . The tips of the apposing neural folds and the eventual point of adhesion are reported to contain cell to cell recognition molecules (as demonstrated in red in Figure 3(c)) which may be required for the specific adhesion process to occur [233–243]. This is supported by previous evidence that the cell surface of the neuroepithelium is lined by carbohydrate-rich material that is not observed in the rest of the neuroepithelium . Removal of GPI-anchored proteins from the cell surface during neurulation results in delayed spinal neural tube closure . Interestingly, work performed by Abdul-Aziz et al. and Pyrgaki et al. demonstrated protrusions emanating from the neural fold tips that interdigitate leading to eventual adhesion [244, 245] (Figure 3(d)). Ultimately, the newly formed neural tube undergoes remodelling via apoptosis to enable the neural tube to separate from its surface ectoderm [228, 246] (Figure 3(e)).
|
study
| 100.0 |
Primary neurulation and secondary neurulation are important developmental processes and have been described in many models. In the chick, there does not exist a clear distinction as to when primary neurulation ends and secondary neurulation begins; the lower spinal cord has been described as junctional neurulation, whereby ingression and accretion accompany the process of defining the area which straddles primary and secondary neurulation and is therefore thought to somehow represent human thoracolumbar spina bifida .
|
study
| 95.2 |
In mouse and humans, spina bifida occulta has largely been described as a result of failure of secondary neurulation [3, 215]. However, much has been described of the severity of lipomyelomeningocele [131, 248] in comparison to the somewhat neurologically unperturbed tethered cord phenomenon which is brought on by trapped nerves due to missing vertebral arches . What is evident is that, irrespective of whether or not there is skin covering the neural tube defect lesion, the severity of the condition depends on the level where the site of the lesion is located. Secondary neurulation in the mouse is described as occurring at sacral level 2 . Therefore, to describe lipomyelomeningocele as resulting from failure of secondary neurulation would be artificial.
|
study
| 80.0 |
Planar cell polarity (PCP) is a process in which cells develop with uniform orientation within the plane of an epithelium . The PCP pathway is a noncanonical Wnt pathway [225, 250–252]. Various Wnt molecules are known to play roles in the PCP pathway such as Wnt11 and Wnt5a [250, 253].
|
study
| 96.44 |
PCP signaling has been suggested to be primarily required for cytoskeletal activity, for example, cellular protrusion, cell-cell adhesion, and cell-matrix adhesion . Skin development, body hair orientation, polarization of the sensory epithelium in the inner ear, and the directed movement of mesenchymal cell populations during gastrulation are among the processes requiring proper PCP signaling in vertebrates [227, 254–256]. In vertebrates, function of the PCP pathway appears to be required for convergent extension (CE). Lamellipodia have been the type of cell shown to drive CE. These broad sheet-like protrusions exert traction on adjacent mesodermal cells causing mediolateral intercalation [257–259]. PCP signaling causes the regulation of cytoskeletal organization that redistributes subcellular PCP components asymmetrically causing polarization of these cells . Moreover, components of the signaling cascade converge or are expressed asymmetrically in the lamellipodia [250, 253].
|
review
| 98.3 |
Among the genes implicated in this net movement of cells, known as convergent extension, are 2 asymmetric molecular systems that control PCP behaviour, the “core” genes and the “Fat-Dachsous” PCP system [261, 262]. The “core” genes give rise to multipass transmembrane proteins: Frizzled (Fzd-3, -6, and -7), Van Gogh (Vangl-1 and -2), Flamingo (Celsr-1, -2, and -3), and cytosolic components, Dishevelled (Dvl-1, -2, and -3), Diego (Inversin), and Prickle (Pk-1 and -2) . The Fat-Dachsous (Ft-Ds) pathway includes the large protocadherins Ft and Ds, acting as its ligand, and Four-jointed (Fj) as a Golgi resident transmembrane kinase . Downstream of the PCP system are PPE (Planar Polarity Effector) genes: Inturned (In), Fritz (Frtz), and Fuzzy (Fy) [265, 266]. The Multiple Wing Hairs (mwh) act downstream of both PCP and PPE with Wnt4, Wnt5a, Wnt7a, and Wnt11 as regulators .
|
study
| 99.7 |
Vangl-2 (formerly known as Ltap and Lpp1) has been identified as the causative gene in the loop-tail mouse [105, 268, 269]. Mutations in Celsr-1 cause craniorachischisis in the Crash mouse . The Dvl-1/Dvl-2, Dvl-2/Dvl-3, Dvl-2/Vangl-2, and Fzd-3/Fzd-6 double knockout mice also have severe NTD forms, mainly craniorachischisis and exencephaly [269, 271–273]. The Vangl-1 and Vangl-2 compound heterozygote exhibits craniorachischisis . The noncore PCP genes also exhibit severe NTD in their mouse mutants including Protein Tyrosine Kinase 7 (PTK7), Scribbled PCP protein, the gene responsible for the circle tail mouse phenotype, Scrib, and Dishevelled Binding Antagonist of Beta-Catenin 1 (Dact-1) [252, 270, 274–277]. All of these genes have been implicated in the PCP pathway. Failure of convergent extension results in an open neuraxis (the entire neural tube from midbrain to low spine remains exposed) and a shortened embryo, more commonly described as craniorachischisis.
|
review
| 98.44 |
Dorsoventral patterning in the neural development of vertebrates is controlled by the induction and polarizing properties of the floor plate . Expression of various genes such as sonic hedgehog (Shh), bone morphogenetic protein (BMP) 7, HNF3β, and Vangl-1 emanating from the notochord and floor plate is thought to cause cell specification which influences the morphogenesis of the neural tube [106, 107, 112, 113, 252]. The floor plate and notochord appear to control the pattern of cell types that appear along the dorsoventral axis of the neural tube [226, 278]. Morphogenesis of the spinal neural tube, in particular, the formation of the median hinge point (MHP), is most likely a nonneuroepithelial cell autonomous action as it is dependent on the differentiation of ventral cell types by signals transmitted from axial mesodermal cells of the notochord to overlying neuroepithelial cells [278–284].
|
study
| 99.9 |
Implantation and ablation experiments which manipulated the notochord in both chick and mouse embryos [221, 284–287] verified that the notochord is required for formation of the MHP. It was proposed that the notochord releases a morphogen that may regulate MHP formation. Shh protein is expressed in the notochord at this stage [113, 288] and application of either Shh-expressing cells or purified protein to intermediate neural plate explants leads to induction of the floor plate , suggesting that Shh is the MHP-inducing morphogen. However, MHP formation is not totally abolished in Shh-null mouse embryos, suggesting that other factors from the notochord may also have MHP-inducing properties .
|
study
| 100.0 |
The second site of neural fold bending as described in Section 8.2 and Figure 3(c) is the dorsolateral hinge point (DLHP). Bending of the neuroepithelium at the DLHP is regulated by mutually antagonistic signals external to the neural fold, as reviewed by Greene and Copp [224, 289]. In contrast to midline bending, Shh has been shown to inhibit dorsolateral bending in the mouse consistent with an absence of NTDs in Shh-null embryos. Signal(s) arising from the surface ectoderm (SE) comprise(s) a second antagonistic signal involved in the regulation and formation of the DLHPs . This has been suggested as further evidence that bending of the neural folds involves signaling from the SE. Bone morphogenetic proteins (BMPs) are candidates to mediate this signaling. Three BMPs (BMP2, BMP4, and BMP7) are expressed in the spinal neural tube. BMP2 and BMP7 are expressed in the surface ectoderm adjacent to the open spinal neural tube, while BMP4 is expressed in the surface ectoderm overlying the closed spinal neural tube .
|
study
| 89.75 |
Recent studies suggest that Noggin may also play a role in regulating DLHP formation [292, 293]. Noggin is an inhibitor of BMP signaling and is expressed at the tips of the apposing neural folds [293, 294]. Homozygous mouse embryos null for Noggin exhibit both exencephaly and spina bifida (100%) [292, 295]. However, spina bifida does not arise in homozygous Noggin mutants until embryonic day 11-12 when the neural tube ruptures. The spinal neural tube of homozygous null Noggin embryos during neurulation takes on the appearance of a wavy neural tube before the neural tube reopens , possibly suggesting an unstable initial closure mechanism. Shh works in an antagonistic manner towards Noggin, as does Noggin towards BMP signaling . This suggests that Noggin may facilitate bending of the spinal neural tube by overcoming the inhibitory influence of BMPs.
|
study
| 100.0 |
Stottmann et al. suggest that the spinal defect in Noggin null embryos results from a failure to maintain a closed neural tube due to a defective paraxial mesoderm . Yip et al. also had shown that the mesodermal extracellular matrix plays an important role in maintaining neuroepithelial rigidity of the spinal neural tube during neurulation . Embryos were cultured in the presence of chlorate, which functions to inhibit sulfation of heparan sulphate proteoglycans (HSPGs) in the extracellular matrix of the mesoderm. This treatment not only resulted in an expedited bending of the DLHPs but also elicited an unnatural shape of neural tube due to a convex shaped mesoderm. However, removal of the paraxial mesoderm does not prevent closure of the spinal neural tube .
|
study
| 100.0 |
Interestingly, there are 3 genes which, when mutated, not only affect paraxial mesoderm production in the mouse [109, 292, 298] but also result in an NTD phenotype in the mouse. These are Cyp26, Noggin, and Fgfr1 [94, 109, 293]. The Wnt3a , Lef1/Tcf1 double null and Raldh2 mutants also have defective paraxial mesoderm production, with an abnormal neural tube during neurulation. Whether or not the paraxial mesoderm plays a primary role in successful neurulation in these mutants remains unknown.
|
study
| 99.94 |
Neural tube closure does not depend exclusively on the MHP or DLHPs, since closure can occur in the absence of either, as in Mode 3 and Mode 1 spinal neurulation, respectively. However, cell shape changes of some type, affecting morphogenesis of the spinal neural tube, are clearly required for closure to occur in all species studied, including the mouse . Table 5 demonstrates the lack of specific expression of genes at the DLHPs. However, overlapping gene expression throughout the neuroepithelium and tips of neural folds may facilitate the bending mechanism seen in the DLHPs.
|
study
| 100.0 |
In all animal species studied, a zone of altered cell morphology with numerous rounded cell blebs has been observed along the tips of the spinal neural folds, immediately prior to adhesion. The observed surface alterations may reflect a change in the properties of the cells at the adhesion site which correlate with initial adhesion between the folds [234, 236, 301, 302]. Structural observations of the point of adhesion in human embryos have yet to be reported, possibly due to insufficient or poor preservation of material so that surface structures cannot be observed.
|
study
| 100.0 |
Adhesion is the final process in the sequence of primary neurulation events. Such physical zippering state of the neural tube has been suggested, in previous studies, as evidence that neural tube closure is a continuous process . However, a debate exists as to whether the physical process of neurulation actually equates to continuous zippering or, more accurately, to a button-like process in which neural tube adhesion initially occurs at various slightly separated points along the axis. According to the latter idea, neural tube adhesion is actually a discontinuous process of closure .
|
study
| 99.94 |
PCP regulation may play a role in adhesion and fusion as suggested in both zebrafish and Xenopus studies. Firstly, cell division regulated by PCP signaling leads to rescue of neural tube morphogenesis in the trilobite zebrafish mutant . Secondly, the Xenopus adhesion molecules, NF-protocadherin, and its cytosolic partner TAF1/Set have been suggested to participate in CE after the neural folds are formed. Disruptions in NF-protocadherin and TAF1 can lead to a shortened AP axis that was not evident until stages 22–25, some time after neural tube closure .
|
study
| 100.0 |
Ultrastructures that emanate from the neural folds at the site of closure have been regarded as a secondary process in the frog. This is because wound healing which acts via actin purse-string contraction is thought to be the primary cause of closure in the frog neural tube . Adhesion of the neural tube and epidermis have been suggested to be separate events based upon the observation that the epidermal ectoderm is still able to migrate and cover the open neural tube in both the chick and the frog [302, 305]. However, the issue of whether or not the neural folds could adhere even in the absence of epidermal fusion in both the chick and the frog has yet to be answered.
|
study
| 100.0 |
Adhesion in the neural tube of rodents has been described previously but the mechanism of this highly specialized process is poorly understood [103, 240, 243, 301, 307, 308]. In a recent study, a direct requirement was shown for the binding of a specific ligand (ephrinA5) to a specific type of receptor (EphA7) in order to enable adhesion to occur in the neural tube .
|
study
| 100.0 |
Cell to cell adhesion provides impetus for positional cell migration . This may suggest that PCP driven events in the surface ectoderm may play a role in neural tube closure, as suggested in the chick embryo . Epidermal constriction has also been shown to be crucial for spinal neural tube closure in the frog, while the surface ectoderm was shown to be necessary for spinal neural tube closure in the mouse [287, 311].
|
study
| 100.0 |
Table 4 summarizes the ten mouse mutant models that exhibit a spinal defect alone. Spinal defects encompass mouse mutants with spina bifida (without any other NTD phenotype, e.g., exencephaly and/or craniorachischisis) and abnormal spinal neural tubes with no spina bifida.
|
review
| 99.8 |
All of these mutants have spina bifida, which denotes incomplete closure of the spinal neural tube. A large majority (4 out 6 of these mutants which have only spina bifida) have a second phenotype that is a second neural tube. Vacuolated lens mutant embryos develop spina bifida and, in addition, an ectopic neural tube is observed, ventral to the open neural tube . In Shp2, FGFR1α, and vacuolated lens mutants, an ectopic neural tube is observed during the period of neurulation between E8.5 and E9.5 [94, 96]. In contrast, an ectopic neural tube has only been observed at E12.5 and later stages in Gcm1 mutant embryos .
|
study
| 100.0 |
The prevalence of an ectopic neural tube in 2 out of 6 mutants at E9.5–E10.5 seems to suggest that a second neural tube may be a common occurrence and that this predisposition may be the result of an underlying fault in primary neurulation instead of failure of secondary neurulation.
|
study
| 99.94 |
There are many different examples of mouse mutants in which the caudal neural tube is abnormal but the phenotype differs from spina bifida. In many cases, these are described as spinal neural tube defects [100–103]. Apart from the 3 mutants with only spina bifida (Fgfr1, Shp2, and Gcm1) which have 2 neural tubes with one notochord, 2 other mutants with spinal defect but no spina bifida share the same predicament. These are the EphA2 null mouse and PAK4 null mouse . Another abnormal spinal neural tube phenotype is a wavy spinal neural tube that occurs in the WASP null mouse and the Vinculin null mouse [102, 103]. Vinculin is a large protein that binds multiple cytoskeletal proteins, actin, α-actinin, talin, paxillin, VASP, ponsin, vinexin, and protein kinase C (PKC) which have been suggested to be the adhesion scaffold that connects early adhesion sites to actin-driven protrusive machinery in enabling motility .
|
review
| 97.75 |
Abnormal and ectopic spinal neural tubes may be regarded as variant forms of NTDs as it may be possible that the neural tube reopens after closure due to various reasons. Ectopic neural tube may take on many different variations apart from the expected second or multiple neural tubes. Among them are a neural tube positioned above another neural tube as well as a wavy neural tube phenotype that is observed in many knockout mice with NTDs. The wavy region in these knockout mice has not had its spinal neural tube sectioned; thus it remains unknown whether the neural tube remains adhered. Spina bifida occulta in humans is usually accompanied by various physical abnormalities such as lipoma, rachischisis, hair tufts, ectodermal sinuses, skin pigmentation, or diastematomyelia. These associated defects occur in either syndromic or nonsyndromic NTDs. However, they may be missed and not categorized properly in cases of transgenic mice with possible NTDs. There is only one example of a null mouse in which these abnormalities have been well described which is the Gcm1 mouse mutant that exhibits both open (meningomyelocele) and close (lipoma and diastematomyelia) spina bifida in its litters .
|
study
| 99.75 |
Haploinsufficiency is poorly studied in both man and mouse. Furthermore, the study of the occurrence of spina bifida in genes acting in an additive or subtractive manner is almost unknown. Currently, there are 5 studies in the mouse, which have demonstrated spina bifida and the interaction of the involved genes mechanistically. These include Lrp6 and Wnt5a , Zac1 and Suz12 , Hira and Pax3 , Rybp encompassing Ring1 and YYP1 , and haploinsufficiency of the components in the primary cilium of the hedgehog pathway .
|
study
| 99.94 |
The scenario in humans is somewhat similar in that there are 4 studies to date demonstrating the involvement of haploinsufficiency in the causation of spina bifida. The Pax3 gene and the EphA4 gene act in concert with each other in causing spina bifida due to interstitial deletion at position 2q36 . Furthermore, in the same paper, Goumy et al. suggested that a similar phenomenon occurs in the mouse when taking into account the spina bifida phenotype seen on the Splotch mouse that is affected by both Pax3 and EphA4 , albeit the link between the two in the mouse has yet to be ascertained. The hedgehog pathway has also been implicated in humans, where spina bifida occurs when Patched is perturbed when implicated with Gorlin syndrome . The third and fourth studies implicating human spina bifida involve haploinsufficiency in the region of 13q and 7q .
|
study
| 81.4 |
This review paper aims to probe spina bifida, the surviving form of neural tube defects, closely and to analyze the relationship of what can be learnt from the mouse model of spina bifida and to use that knowledge in order to shine a brighter understanding with regard to the human form.
|
review
| 99.9 |
What is very obvious is that there have been a multitude of genes (74 according to this review) which regulate specifically spina bifida in the mouse. This is a very high number of genes; therefore the take home message would be in our opinion that there are a multitude of genes that can, if perturbed, cause spina bifida. Whether or not these genes cause the condition or are in fact a player in a pool of numerous genes, which can do the job of closing the spinal neural tube, is a tantalising idea. Therefore, we put forth the idea that perhaps these 74 may be working with other genes in their family or other genes which share a common pathway in order to close the neural tube. Furthermore, the idea of gene-gene interaction which promotes heterogeneity among genes is incomplete without also considering the idea of haploinsufficiency of genes, where many mutations in mankind are somehow protected from having a deleterious phenotype by having other genes compensate the job of the gene or genes being perturbed. A very good example of this would be the Vangl-1 and Vangl-2 compound heterozygote mouse mutant which lacks a single allele of both Vangl-1 and Vangl-2; therefore the probability that the 2 genes compensate each other is high and both genes are required in a certain amount of dose, lack of which translates into a neural tube defect phenotype. Therefore, the mouse model which examines the delineation of genes has not completed its true worth until scientists understand the biology of the disease or condition better by also taking into account (i) the amount (the functioning allele) of the said gene and (ii) the interaction with other genes in its family which may be able to compensate its function as well as (iii) the interaction with other genes which share a common pathway. The mouse is a powerful tool to study spina bifida because it is a mammal like humans and its embryology is similar to humans and therefore it is an indispensable tool to mechanistically study the structural changes involved in spinal neural tube closure. The genes involved in spinal neural tube defects may differ in man and mouse; however, parallels may be drawn between the principles of how the genes interact in influencing spinal neural tube closure in both man and mouse.
|
review
| 99.9 |
Starting with the original proposal by Datta and Das1 for a spin-based field-effect transistor, the field of spintronics2 has explored how the spin degrees of freedom can be used for information transfer. More than two decades later, this research has reached the quantum regime3. One motivation for this is the desire for miniaturization which led to the realization of single-electron transistors4, or more generally single-dopant devices5. A second motivation is the potential applications in quantum information and computation. Two guiding proposals in the field involve implementation of quantum gate operations in quantum dots6 and in doped silicon7. Shortly thereafter, molecular magnets have also been proposed8. It was subsequently shown that universal quantum computation can be realized with Heisenberg exchange interaction, known from quantum magnetism, alone910.
|
review
| 99.56 |
Here we put forward a scheme for a quantum spin transistor that may serve as an integral component of quantum information devices. Similarly to the quantum computation proposals, it can be implemented with architectures that realize a Heisenberg spin chain. Various physical realizations of spin chains are being actively explored for short-range quantum state transfer required to integrate and scale-up quantum registers involving many qubits1112131415. In fact, spin chains of the Heisenberg type have been realized in organic and molecular magnets16, quantum dots17, various compounds161819, Josephson junction arrays20, trapped ions2122, in atomic chains on surfaces2324252627 and in thin films or narrow magnetic strips that carry spin waves2829. Combined with conditional dynamics implementing quantum logic gates, spin chains can greatly facilitate large-scale quantum information processing. While many different implementations of the coherent spin transistor may be possible, here we focus on one such proof-of-concept realization with a small ensemble of strongly interacting cold atoms trapped in a tight one-dimensional potential of appropriate shape. Cold atoms have already been used to realize spin chains, and observations of Heisenberg exchange dynamics30, spin impurity dynamics31 and magnon bound states32 have been reported.
|
study
| 99.94 |
Our quantum spin transistor works with an arbitrary spin state at the input port (target spin) which is coherently transferred to the output port, if there are no excited spins in the gate, . However, if the gate contains an excited stationary spin (control spin), , it completely blocks the transfer of the target spin state between the input and output ports. In other words, we have coherent dynamics for the initial state of the system when the gate contains no spin excitation, but complete absence of dynamics for the state when the gate contains a single spin excitation. Our scheme thus realizes a quantum logic operation and it can be used to obtain spatially entangled states of target and control spins, as well as to create Schrödinger cat states for a large number of target spins.
|
other
| 99.9 |
The functionality of the proposed transistor relies on the ability to tune the energy levels of the gate such that one of its states is resonant with the input and output states, resulting in coherent excitation transfer, while the other state of the gate is non-resonant and is used to block the transfer. A system that offers sufficient flexibility to realize this level of control is the Heisenberg XXZ spin- chain in a longitudinal magnetic field. We thus consider a chain of N spin- particles described by the XXZ model Hamiltonian (ħ=1)
|
other
| 98.2 |
where are the Pauli matrices acting on the jth spin, hj determine the energy shifts of the spin-up and spin-down states playing the role of the local magnetic field, Jj are the nearest-neighbour spin–spin interactions, and Δ is the asymmetry parameter: Δ=0 corresponds to the purely spin-exchange XX model, Δ=1 to the homogeneous spin–spin interaction XXX model, while the limit of |Δ|>>1 leads to the Ising model. We assume a spatially symmetric spin chain with Jj=JN−j and hj=hN+1−j with h1,N=0. Note that we do not specify the sign of Jj, which, therefore, can be positive or negative.
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study
| 99.94 |
The input and output ports for the target spin are represented by the first j=1 and last j=N sites of the chain, see Fig. 1a. The inner sites j=2,…, N−1 constitute the gate which may be open (Fig. 1b) or closed (Fig. 1c) for the target spin transfer depending on the absence, , or presence, , of a single control spin excitation. One might think that the shortest possible spin chain to accommodate a gate between the input and output ports would consist of just N=3 spins. As we show in Supplementary Note 1, however, the three-spin chain cannot implement a reusable spin transistor even in the Ising limit >>1 since the control spin excitation at the gate site j=2 is not protected from leakage. We will therefore illustrate the scheme using a chain of N=4 spins, with the gate consisting of spins j=2,3 coupled to each other via the strong exchange constant .
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study
| 100.0 |
Consider a system in the initial state , which we aim to efficiently transfer to the final state using an intermediate resonant state, see Fig. 1b and Supplementary Note 2. The initial and final states have the same energy of , where h ≡ h2,3 (as is the standard convention in quantum optics, we refer to the expectation value of the Hamiltonian in a given state as the energy of this state). In turn, the eigenstates of Hamiltonian (1) in the single excitation space of strongly coupled sites j=2,3 are given by , with the corresponding energies split by 2J2. Then, by a proper choice of the magnetic field, , we can tune the energy of one of the intermediate states into resonance with the initial and final states (for example, for h=h− the resonant state is ). Simultaneously, the other intermediate state does not participate in the transfer since its energy is detuned by 2J2 which is much larger than the exchange coupling of the initial and final states to the intermediate states. The transfer time of the spin excitation between the initial and final states via a single resonant intermediate state is tout=π/|J1| (Fig. 1d).
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| 100.0 |
Next, we place a single spin excitation in one of the eigenstates of the gate. To be specific, for the magnetic field h=h− we place the control spin in state and denote it as . Then the control spin cannot leak out of the gate region and therefore it is stationary. Moreover, if we place a target spin-up at the input port, the resulting state will have the energy which differs significantly from the energies and of the states to which it can couple via a single spin-exchange (assuming Δ≠0, see below and Supplementary Note 2), see Fig. 1c. Therefore, such an initial state will remain stationary and the control spin excitation on the gate will block the transfer of the target spin between the input and output ports, see Fig. 1d. Indeed, the large energy difference will preclude the escape of the gate spin excitation to the output port, while the large energy mismatch will suppress the probability of the second-order exchange of spin excitation between the input and output ports by a factor of <<1 (with Δ=−1, see below and Supplementary Note 2). Exactly the same arguments apply to the initial state with the magnetic field set to h=h+.
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| 100.0 |
In the same spirit, we can construct spin transistors with longer spin chains (see Supplementary Note 3 for N=5), the above case of N=4 being the shortest and simplest one. The general idea illustrated in Fig. 1a–c is as follows: The gate region consists of N−2 strongly coupled spins, for all j∈{2,…, N−2}. Therefore, the single excitation space of the gate has N−2 eigenstates split by . With the magnetic field hj, we tune one of these eigenstates, say , in resonance with the single excitation input and output states. Assuming , all the other eigenstates will remain decoupled during the transfer, and we will have a simple three-level dynamics for a single target spin. Note that if the resonant eigenstate is antisymmetric, the output state will be the same as the input state, independent on the sign of Jj (see also Supplementary Note 2), while if we chose symmetric resonant state , we would obtain the output state which would be identical to the input state upon the application of the Pauli-Z quantum gate ( operation) to the output port. To close the transistor gate, we place a single spin excitation in one of the gate eigenstates from where the control spin cannot leak out since this eigenstate is non-resonant. Simultaneously, the target spin cannot enter the gate region since the double-excitation subspace, to which it is coupled, is shifted in energy due to the spin–spin interaction, resulting in the transfer blockade.
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study
| 100.0 |
A possible system to realize the spin chain Hamiltonian of equation (1) is a cold ensemble of N strongly interacting atoms in a one-dimensional trap of appropriate geometry33343536. Recent experiments have confirmed that spin chains may indeed be realized this way37 with tunable microscopic optical traps38. An outline of the procedure to map this system onto the Heisenberg XXZ spin model is presented in Supplementary Note 4. A pair of suitable internal atomic states can serve as the spin-up and spin-down states, with the microwave or radiofrequency transition frequency further tunable through a global magnetic field inducing spatially-uniform Zeeman shifts and by tightly focused non-resonant laser beams inducing site-selective ac Stark shifts 3139. In the full model, the strong contact interactions between the atoms are described by the dimensionless coefficients >>1 and g↑↑=g↓↓=κg, where the parameter κ>0 is related to the asymmetry parameter of the effective Heisenberg spin- model as . In turn, the exchange constants of the Heisenberg model are proportional to the geometric factors αj which are determined by the single particle solutions of the Schrödinger equation in a one-dimensional confining potential V(x). Hence, the shape of the trapping potential can be used to tune the necessary parameters of the effective spin chain34.
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| 100.0 |
shown in Fig. 2a (see the caption for the parameters a, b and x0). The potential consists of a shallow Gaussian well at the center and a pair of deeper and narrower wells next to the boundaries. The four lowest-energy single particle wavefunctions of potential (2) are also shown in Fig. 2a. The two lower energy states are nearly degenerate and the corresponding wavefunctions have sizable amplitudes at the deep wells near the boundaries, while the two higher energy states have much larger energy separation, with the amplitudes of the corresponding wavefunctions being large in the shallow well in the middle. Accordingly, the effective exchange interactions satisfy J1=J3 and <<1. The dependence of the ratio J2/J1 on the parameters V0 and U in equation (2) are shown in Fig. 2b,c.
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| 100.0 |
The nearly perfect transfer, or complete blockade, of the target spin for the open, or closed, gate as shown in Fig. 1d was obtained for the system parameters as in Fig. 2a. It is however important to quantify the sensitivity of the spin transistor to uncontrolled fluctuations of the parameters. In Fig. 3a we show the fidelities of transfer at time tout=π/|J1| versus the amplitude of random noise affecting the trapping potential or the (effective) magnetic field. We observe that coherent transfer is quite robust with respect to moderate variations in V0, but is rather sensitive to small variations in U and h since they detrimentally affect the gate resonant conditions. Importantly, the gate blockade is virtually unaffected by uncertainties in U, V0, h since it relies on the (large) energy mismatch. In Fig. 3b, we show the dependence of the blockade fidelity on κ which determines the asymmetry parameter Δ of the XXZ model. Clearly, the spin transistor cannot operate when Δ=0, that is, in the absence of the spin–spin interactions, since then the control spin can leak out of the gate, as mentioned above and in Supplementary Note 2.
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study
| 100.0 |
To summarize, we have presented a scheme for a quantum spin transistor realized in a Heisenberg spin chain and proposed and analysed its physical implementation with cold trapped atoms. In our scheme, the presence or absence of a control spin excitation in the gate can block or allow the transfer of an arbitrary target spin state between the input and output ports. If the gate is prepared in a superposition of open and closed states, then the initial state of the system with the target spin-up at the input port will evolve at time tout into the spatially entangled state.
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other
| 99.75 |
Furthermore, if the gate is integrated into a larger system in which the excited spins from the source can be fed (one-by-one or one after the other) into the input port, and the output port is connected to the initially unexcited drain, then the initial gate superposition state will result in a (macroscopically) entangled Schrödinger cat-like state of many spins,
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other
| 99.9 |
We note that with the atomic realization of spin chains, with the spin-up and spin-down states corresponding to the hyperfine (Zeeman) sublevels of the ground electronic state, the preparation of the closed gate state or the coherent superposition of states and (assuming h=h−=0) can be accomplished by applying to a microwave or two-photon (Raman) optical pulse of proper area (π or π/2) and the frequency matching the energy difference δλ=2J2Δ between and . In turn, the initialization of the target spin state at the input port can also be done with resonant microwave or radiofrequency field(s), upon shifting the transition frequency of the atom by a focused laser3139, while the readout at the output port can be done by internal state selective fluorescence using the quantum gas microscope set-up4041. Both the initialization and the readout can be accomplished during time intervals ∼10 μs—short compared with ms transfer time39.
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study
| 92.3 |
Cystic endosalpingiosis is a rare disorder caused by the heterotopical presence of tissue resembling structures of the fallopian tubes . It can be considered part of a wider group of anomalies of embryological origin called müllerianosis consisting in the heterotopic presence of müllerian-derived tissue in pelvic organs, or in distant locations. Although müllerian-derived tissues are sensitive to estrogen and progesterone, reports of cystic endosalpingiosis and other forms of müllerianosis in pregnancy are very scarce. We report a case of florid cystic endosalpingiosis discovered in a pregnant woman during a scheduled cesarean section and review the current knowledge of this disease.
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clinical case
| 99.94 |
A 30-year-old woman with no remarkable past medical history and an uneventful follow-up of a bichorial-biamniotic twin pregnancy attended the hospital day unit at term for fetal growth surveillance and heart rate monitoring. The first twin was in a breech presentation and a cesarean section was scheduled at 39 weeks. During the procedure and after the extraction of both placentas, the uterine fundus and part of the body were seen completely seeded with multitude of cyst-like structures resembling hydatids of Morgagni but with a harder consistency (Figure 1). A sample of the cysts fluid and a couple of entire cysts were sent for anatomopathological study.
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clinical case
| 100.0 |
The results of the cysts biopsy (Figure 2) showed a histology formed by an external serous layer, a well-organized smooth muscle, and an inner layer of tubal cylindrical epithelium with small fibrous stroma papillae, no atypias, and no proliferative activity. Although some decidualized cells were present, no endometrial stroma was found. The immunohistochemistry analysis showed a positive expression for PAX8 (Box-8), CK7, and estrogen and progesterone receptors and a negative expression for CD10, calretinin, and CK20. The proliferative index with Ki67 was below 1%. The cytology showed histiocytes and scarce inflammatory cellularity. The final diagnosis was of florid cystic endosalpingiosis.
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clinical case
| 100.0 |
Three months after the cesarean section, the patient was reevaluated with transvaginal ultrasound (Figure 3). The examination showed that the fundus and part of the uterine body were still covered with multitude of cyst-like structures. The endosalpingiosis lesions did not disappear after pregnancy.
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clinical case
| 100.0 |
Cystic endosalpingiosis is part of müllerianosis, disorders consisting in the heterotopic presence of müllerian-derived tissue [1, 2] in pelvic organs like the uterus , bladder , ovaries , parametrium , uterosacral mesosalpinx , peritoneum , and ureters or in distant locations like the small and large intestine (especially in the appendix) , coledochal duct , axillary nodes , mediastinum , umbilicus , vessels , and spine .
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other
| 99.8 |
Most of the reported cases have been observed in nonpregnant women complaining of pelvic pain [18, 19] and urological , digestive , or neurological symptoms after an ultrasound or MRI examination mimicking diverse kinds of pelvic cystic tumors . Although müllerianosis may contain estrogen and progesterone receptors , reports of cystic endosalpingiosis and other forms of müllerianosis in pregnancy are surprisingly very scarce. They are considered choristomas (masses of normal tissue in an abnormal locations) causing endosalpingiosis, endometriosis, adenomyosis, endocervicosis, leiomyomatosis peritonealis disseminata, and probably vascular leiomyomatosis.
|
review
| 99.8 |
During organogenesis, a number of genes of the WNT family like the WNT4 are activated, producing the necessary signals to conduct the development of the mullerian structures. That is the reason why mutations in the WTN-4 gene cause müllerian duct regression . Recent research has underlined the possibility that, on the other extreme, müllerianosis might be caused by the abnormal reactivation of these genes [26, 27], causing metaplasia of normal tissues like the peritoneum. This would explain why these anomalies appear disseminated in the pelvic and abdominal organs [28, 29] or why Box-8 (PAX8) positive cells appear so frequently in peritoneal washing for diverse gynecological indications . However it is true that another possibility for these findings would be the presence of remnants of müllerian precursor cells included within the developing tissues. Be that as it may, these cells are sensitive to estrogen and progesterone and might proliferate during pregnancy increasing the volume of cyst and thus making them detectable at the end of pregnancy. However, the fact that the lesions did not disappear after pregnancy makes this possibility less likely. In summary, cystic endosalpingiosis is a benign condition that should always be considered, even in pregnancy, when it comes to making the differential diagnosis of a pelvic or systemic multicystic mass.
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study
| 98.25 |
Strigolactones (SLs) are recently characterized phytohormones that play a multitude of roles during plant development and plant–microbial interactions. Initially discovered as germination stimulants of parasitic weeds (Cook et al., 1966), nowadays it is known that SLs regulate plant shoot and root architectures (reviewed in Al‐Babili & Bouwmeester, 2015), leaf senescence (Yamada et al., 2014), responses to biotic and abiotic stresses (Ha et al., 2014; Torres‐Vera et al., 2014), cytoskeletal dynamics, auxin transport (Shinohara et al., 2013; Pandya‐Kumar et al., 2014) and hyphal branching of arbuscular mycorrhizal fungi (AMF) (Akiyama et al., 2005). SLs are carotenoid derivatives synthesized via a pathway starting in plastids with the all‐trans‐β‐carotene/9‐cis‐β‐carotene isomerase D27 (reviewed in Lopez‐Obando et al., 2015). Two dioxygenases, CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7)/MORE AXILLARY GROWTH 3 (MAX3)/DECREASED APICAL DOMINANCE 3 (DAD3) and CCD8/MAX4/DAD1, then synthesize the first bioactive SL precursor, carlactone. As a further step, plant‐specific members of cytochrome P450 mono‐oxygenases, MORE AXILLARY BRANCHES1 (MAX1) and MAX1 homologs produce canonical SLs such as orobanchol and ent‐2′‐epi‐5‐deoxystrigol (Zhang et al., 2014a), respectively most abundant in Petunia hybrida and Oryza sativa (Kretzschmar et al., 2012; Xie et al., 2013), or carlactonoic acid derivatives in Arabidopsis thaliana (Abe et al., 2014; Seto et al., 2014). SL synthesis takes place in several plant tissues: root tips, stem nodes, and along the root and shoot vasculature (Lopez‐Obando et al., 2015). Despite the ubiquitous biosynthesis, grafting experiments and tracking of SLs and of the SL‐mimicking molecule GR24 showed that SLs (or their precursors) move from the root to the shoot (Domagalska & Leyser, 2011; Sasse et al., 2015; Xie et al., 2015). However, wild‐type scions grafted on mutant root stocks do not show SL‐related phenotypes, supporting the hypothesis that shoots can produce enough SLs to regulate their architecture. PLEIOTROPIC DRUG RESISTANCE1 (PDR1) from P. hybrida is the main player for SL shootward transport and SL release to the soil. PDR1 is apically localized in the plasma membrane of cortex cells in root tips and outer‐laterally localized in hypodermal passage cells (HPCs), the entry point of mycorrhizal fungi (Sasse et al., 2015). The high activity of the PDR1 promoter (pPDR1) at the base of lateral axils and the bushy shoots of pdr1 ko mutants suggest that SL transport has an important role in inhibiting lateral bud outgrowth (Kretzschmar et al., 2012).
|
review
| 99.9 |
Biosynthesis and transport of SLs and consequently SL amounts and allocation in planta and in the rhizosphere are regulated by external and internal cues. An important external factor is the availability of inorganic phosphate (Pi) in the soil (reviewed in Brewer et al., 2013). On Pi‐poor substrates, plants react to starvation by inducing SL exudation into the soil as a beacon for AMF; by lateral root formation and root hair elongation for improving the rhizosphere exploration; and by inhibiting shoot lateral branching. It has been shown that PDR1 and DAD1 expression levels are both up‐regulated by low Pi conditions as well as by auxins (Lopez‐Raez et al., 2008; Kretzschmar et al., 2012), thus suggesting that SLs act as integrators of plant nutrient uptake with plant growth regulation.
|
review
| 99.8 |
In modern agriculture, fertilization with phosphorus (P) from mineral sources is required to achieve high crop yields (Tilman, 1999; Roy‐Bolduc & Hijri, 2012). The commonly fertilized P form is soluble Pi, which is readily available to the plant. Arable soils in Europe, parts of Asia and America often contain surplus amounts of P (Cordell et al., 2009). The current, massive input of soluble Pi is not sustainable, as a result of depletion of global P reservoirs and eutrophication of waters by runoff from agricultural lands (Scholz & Wellmer, 2013; Reijnders, 2014). In addition, crops can typically only utilize between 10% and 25% of the fertilized Pi (Cordell et al., 2009), because of its slow diffusion and adherence to soil particles. Plant Pi utilization efficiency can therefore be effectively improved, with simultaneous lowering of environmental risk, by an increase in root surface area realized via lateral roots, root hairs, cluster roots (Neumann & Martinoia, 2002) and mycorrhizal hyphae. SL‐focused strategies, if targeted to tailored crops, might also accelerate Pi uptake by promoting mycorrhiza and root hair growth and can therefore help to increase the availability of Pi for food production while simultaneously increasing the sustainability of crop production.
|
review
| 98.75 |
Here we report that the overexpression of PDR1 causes the reallocation of endogenous SL in both roots and shoots. This change improves plant biomass production on Pi‐poor soils compared with the wild‐type because of enhanced nutrient uptake caused by a larger lateral root system, postponed leaf senescence, higher density and increased length of root hairs, as well as faster mycorrhization. PDR1 overexpression therefore provides a chance to increase plant yield in phosphate‐scarce soils. The possible uses of plants with enhanced SL production and transport are discussed.
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study
| 100.0 |
The cloning of PDR1 OE, a GFP‐PDR1 protein fusion driven by the 35SCaMV promoter, was previously described by Sasse et al. (2015). Three independent PDR1 OE lines have been used for this and previous research and confirmed for GFP‐PDR1 gene and protein expression. P. hybrida W115 (Mitchell) growth was tested in six specific soil mixtures, which differed in Pi content (see details in Table 1) and in the presence or absence of mycorrhizal fungi, in order to study the effects of increased lateral root density and mycorrhiza on plant biomass development and Pi uptake. P. hybrida W115 (wild‐type background of PDR1 OE), PDR1 OE, pdr1 ko (Kretzschmar et al., 2012) and W115 × W138 (wild‐type background of pdr1 ko) plants were grown under long‐day conditions (16 : 8 h, light : dark regime), at 60% relative humidity and 25°C on different soil mixtures. These were: natural soil (from University of Zurich botanical garden, with naturally occurring mycorrhizal fungi); clay granules (Oil Dri US‐Special, Chicago, IL, USA) and mineral soil (subsoil, also known as B‐soil horizon from University of Zurich botanical garden). Also soil mixes were used, as follows: natural soil mix (70% natural soil and 30% mineral soil) and Claymin (50% clay and 50% mineral soil). For clay+ and Claymin+, respective substrates were supplemented with half a teaspoon in 500 ml substrate of a sand inoculum of Rizophagus irregularis, a common AMF frequently used for inoculation studies (Martin et al., 2008). The inoculum was added 2 wk after plant germination with a low Pi inoculation medium prepared as previously described (Reddy et al., 2007), so that a total Pi amount of 0.112 mg was added to the initial 0.46 mg per pot (i.e. a total of 0.001 g l−1). The pot volume used for all experiments was 500 ml. Alternatively, P. hybrida seeds were plated on 0.85% (w/v) Phyto Agar (Duchefa, Haarlem, the Netherlands) medium containing 2.2 g l−1 Murashige and Skoog (half‐strength MS (§ MS)) medium (Duchefa) at 21°C. Low‐Pi MS medium for root experiments contained 0.25 mM instead of 1.25 mM KH2PO4 as in § MS. Clay was chosen against mineral soil and full soil for analyses on the root system architecture because of easier washing away from roots for quantification of mycorrhization while at the same time keeping soil humidity more constant than mineral soil or sand.
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study
| 100.0 |
Mycorrhization is initiated by SL exudation which induces AMF hyphal branching towards the plant root (Akiyama et al., 2005). We therefore assessed whether PDR1 OE plants have higher mycorrhization levels than the wild‐type. As mycorrhization levels are influenced by soil Pi conditions (Breuillin et al., 2010), we quantified mycorrhization in different soils, from the Pi‐richer ‘natural soil mix’ to the Pi‐poorer ‘mineral soil’, going through ‘clay’ and mixed substrates with intermediate Pi amounts (see Fig. 1a, Table 1 and Supporting Information Methods S1; Table S1 for statistical analysis).
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study
| 100.0 |
Biomasses and mycorrhization rates of PDR1 OE and wild‐type Petunia hybrida plants on soils containing different amounts of inorganic phosphate (Pi). (a) Pi abundances in soil, clay and mineral soil. (b) Mycorrhization rates at 4 wk after germination (wag) on natural soil mix in wild‐type (W115, WT), PDR1 OE (OE), the wild‐type background for pdr1 ko (W115 × W138) and pdr1 ko. (c, d) Glomus intraradices (now Rhizophagus irregularis) TUBULIN_1 (GiTUB_1) expression levels in wild‐type and PDR1 OE roots at 6 and 8 wag. (e, f) Glomus intraradices (now Rhizophagus irregularis) TUBULIN_2 (GiTUB_2) expression levels in wild‐type and PDR1 OE roots at 6 and 8 wag. (g) Shoot biomass production on clay+. (h) Shoot biomass production on natural soil mix. Values are means ± SE. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
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study
| 100.0 |
To test if PDR1 expression levels influence mycorrhization, we first quantified it via the grid method on natural soil mix (see the Materials and Methods section). PDR1 OE roots exhibited a mycorrhization rate of 20% compared with 10% for wild‐type plants; pdr1 ko plants scored < 2% (Fig. 1b). As the grid method does not easily allow the quantification of mycorrhization along the whole root, we further scored mycorrhizal rates via qPCR with specific mycorrhizal markers: GiTUB_1 (primers kindly provided by Prof. Didier Reinhardt) and GiTUB_2 (Alkan et al., 2004). On natural soil mix, PDR1 OE plants conserved stronger than the wild‐type GiTUB expression levels until 6 wk after germination (wag) and only on clay+ (100% clay supplied with the AMF Rhizophagus irregularis) up to 8 wag (Fig. 1c–f). In detail, relative to the wild‐type on natural soil mix, on clay+ wild‐type plants showed four‐ to fivefold higher GiTUB_1 expression while PDR1 OE plants showed 60‐ to 150‐fold higher expression at 6 and 8 wag, respectively (Fig. 1c,d). With GiTUB_2 the trend was similar on clay+: wild‐type plants scored 1.3‐ to twofold induction and PDR1 OE plants 22–100 times higher expression levels (Fig. 1e,f), confirming the higher mycorrhization capability of PDR1 OE on low‐Pi soils such as clay+. The different expression levels of GiTUB1_1 and GiTUB_2 are probably a result of the primer specificity. A BLAST analysis with GiTUB_1 hit four different types of the genus Glomus (G. clarum, G. claroideum, G. intraradices, G. diaphanum) while GiTUB_2 are specific for G. intraradices (now Rhizophagus irregularis). With both GiTUB primers, PDR1 OE scored six to 10 times stronger expression on natural soil mix at 6 wag, while at 8 wag the wild‐type scored higher mycorrhization than PDR1 OE, although with low statistical significance (Fig. 1d,f). Via the gridline quantification, we observed partly overlapping but underestimated trends, probably owing to the limited amount of root we could visualize on the total and to mycorrhization spatial heterogeneity (Gamper et al., 2008), both along the root and through the cortex layers of P. hybrida roots. In summary, we observed significantly higher mycorrhization only in PDR1 OE plants compared with the wild‐type at 6 wag on clay+ (Fig. S1a,b). The mycorrhizal structures observed via the grid method at 8 wag comprised mostly arbuscules (Fig. S1c–j) both on natural soil mix (88.6 ± 4.9%) and on clay+ (74.6 ± 8.3%). Of the nonarbuscular structures, no fungal vesicles were present on natural soil mix and 8.06 ± 0.92% (n = 3) of vesicles were present on clay+.
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study
| 100.0 |
Concomitantly with the long‐lasting, high mycorrhization rate of PDR1 OE plants on clay+ and the transient effect on natural soil mix, on clay+ we observed a significantly faster and long‐lasting increase of shoot biomass compared with the wild‐type (Fig. 1g) and a similar and transient increase on natural soil mix at 6 wag (Fig. 1h). As PDR1 OE shoot biomass on natural soil mix is lower than on clay+, the R. irregularis inoculum seems to induce a faster and more efficient mycorrhization than the naturally occurring mycorrhizal mix. To test if low Pi and AMF are both necessary for the growth advantage of PDR1 OE plants, we compared biomass production on full soil (our Pi richest soil) and on clay (without AMF). On full soil and on clay, no significant differences in biomass production could be observed 8 wag (Fig. 2a,b), while on clay+, used as positive control in this experimental setup, both shoot and root biomass production of PDR1 OE was again significantly higher than in the wild‐type (Fig. 2c). Interestingly, by comparing clay and clay+ results, it turned out that PDR1 OE can cope better with mycorrhization compared with the wild‐type, which is exploited to an extent by AMF in low‐Pi conditions (Fig. 2d–g). These results indicate that the faster mycorrhization on clay+ obtained through PDR1 OE is a major trait providing consistent advantage for plant growth, and that PDR1 OE plants can sustain more advantageous tradeoffs with AMF compared with the wild‐type. In order to assess whether the growth advantage on clay+ might be conferred by an enhanced plant Pi status, we determined the expression levels in roots of P. hybrida PHOSPHATE TRANSPORTER 3 (PhPT3) and P. hybrida PHOSPHATE TRANSPORTER 5 (PhPT5), which are known to be strongly up‐regulated by mycorrhization (Breuillin et al., 2010). At 6 wag, the phosphate transporters were up‐regulated in PDR1 OE roots on both natural soil mix and clay+. At 8 wag, PhPT3 and PhPT5 were strongly induced under clay+ conditions and slightly down‐regulated in natural soil mix, according to the results obtained with the mycorrhization quantification via qPCR (Fig. S1k–n).
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study
| 100.0 |
Correlation between genotypes, soils and Pi uptake in Petunia hybrida W115 and PDR1 OE plants. (a–c) FW (root and shoot) on full soil, clay and clay+ 8 wk after germination (wag). (d–g) Representative shoots of W115 and PDR1 OE plants grown on clay (−myc) and clay+ (+myc). Bars, 2 cm. Values are means ± SE. *, P < 0.05; **, P < 0.005.
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study
| 100.0 |
On clay+, neither wild‐type nor PDR1 OE plants showed lateral shoot growth during the first 8 wag (Fig. 2d–g), probably because of the limiting nutrient conditions, and therefore the higher biomass production of PDR1 OE plants is given by main stem and leaves. Interestingly, the main stem and leaves also account for the equal shoot biomass production on full soil (Fig. 2a), where wild‐type plants exhibit a stronger branching compared with PDR1 OE, as previously published (Sasse et al., 2015). We then analyzed in detail the shoot morphology of PDR1 OE on full soil, and we scored larger and rounder foliage surface and thicker stems in PDR1 OE than in the wild‐type (Fig. 3a–d). The leaf length : width ratio in PDR1 OE leaves was smaller than in the wild‐type (Fig. 3e). Epidermal cells in PDR1 OE leaves (middle blade) were larger than in the wild‐type (Fig. 3f,g).
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study
| 100.0 |
Shoot morphology and strigolactone (SL) quantification in wild‐type (W115 (WT)) and PDR1 OE Petunia hybrida plants grown on full soil. (a) Leaf series (from cotyledon to leaf number 15) in 2‐month‐old WT (W115) and PDR1 OE plants. (b) Leaf areas in W115 and PDR1 OE from leaf 5 (l5) to leaf 12 (l12). (c) Representative stem sections (node 9). (d) Significant differences in stem section areas in W115 to PDR1 OE from internode 7 (in7) to internode 9 (in9). (e) Ratio of leaf length : width in WT and PDR1 OE plants. (f) Representative light microscopy pictures of adaxial (ad) and abaxial (ab) epidermal cells in WT and PDR1 OE middle blades. (g) Ratio of PDR1 OE : WT epidermal cell areas. (h) Germination rates of Phelipanche ramosa induced by shoot extracts of WT and PDR1 OE plants. 1 nM GR24 as positive control. (i) PDR1 OE : WT ratio for internode (in) elongation ± mycorrhization. (j) Germination rates of P. ramosa induced by root extracts of WT and PDR1 OE plants. 1 μM GR24 as positive control. Root extracts diluted 10−3 (high) and 10−5 (low). Bars: (a) leaves, 1 cm; (c) stems, 3 mm; (f) epidermal cells, 20 μm. Values are means ± SE. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
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| 100.0 |
To test if altered SL allocations in PDR1 OE shoots are responsible for the observed phenotypes, we assayed SL‐inducible germination of the parasitic weed Phelipanche ramosa using P. hybrida shoot extracts from 1‐month‐old PDR1 OE plantlets, where leaves represent 87% (± 3.5%; n = 5) of the shoot biomass and no lateral buds are formed yet. The germination of Phelipanche ramosa seeds is very sensitive to SLs (up to four orders of magnitude higher than mass spectrometer detection limit; see Guillotin et al., 2017). However, it can be induced not only by SLs but also by isothiocyanates (Auger et al., 2012), present almost exclusively in Brassicaceae (Halkier & Gershenzon, 2006). Still, we tested for the presence of possible inhibitors or activators in our plant extracts. The germination ability of GR24 was assayed ± wild‐type, pdr1 ko and PDR1 OE extracts. In none of the analyzed cases could the negative effect of unknown molecules in the extracts from P. hybrida tissues override the results we scored with pure extracts (Fig. S2a,b). This bioassay operated with PDR1 OE plantlets showed that PDR1 OE leaves contain lower concentrations of SL than the wild‐type (Fig. 3h). By contrast, increased PDR1 OE stem thickness was compatible with SL‐induced cell division in the procambium, as reported in A. thaliana after exogenous GR24 treatments (Agusti et al., 2011). PDR1 OE stems also had longer internodes than those of the wild‐type; with or without mycorrhizal fungi in the soil (Fig. 3i) PDR1 OE internodes were 1.5‐ to two‐fold longer than in the wild‐type. As SL was reported to increase internode elongation in Pisum sativum (de Saint Germain et al., 2013), we propose that this phenotype in P. hybrida is a result of increased SL transport within/towards the procambium.
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The symbiosis between plants and mycorrhizal fungi is a continuous tradeoff as long as both organisms are beneficial to each other (Ryan et al., 2012). Therefore we tested PDR1 OE and wild‐type plants grown on soils with lower Pi concentrations than clay, where high levels of mycorrhization could lead to carbon exploitation from the host plant rather than to beneficial Pi upload from the fungus. On mineral soil (see Table 1), P. hybrida plants could not grow; therefore, we assayed plant biomass production at 8 wag on a substrate mixture called Claymin (50% clay plus 50% mineral soil ± AMF) (see the Materials and Methods section and Table 1). Under these conditions and in the absence of AMF, shoot and root growth of PDR1 OE plants were significantly higher than that of wild‐type plants (Fig. 4a,b,e,f). However, the addition of AMF did not cause significant increases in biomass production (Fig. 4c–f). In Claymin+ growth conditions, the costs of energy supply to the mycorrhiza seem to exceed the benefit obtained from the fungus. In none of the tested substrates could PDR1 OE plants reach the biomass production in full soil: still, on clay+ and Claymin, PDR1 OE plants could produce more shoot and root biomass than the wild‐type up to 8 wag (Fig. 4e,f). Consistently, an increase of Pi uptake is significant only in PDR1 OE plants grown on clay+ or Claymin (Fig. S2c).
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Biomass and gene expression levels in roots and shoots of wild‐type (W115 (WT)) and PDR1 OE Petunia hybrida plants. (a–d) Shoots of W115 and PDR1 OE on Claymin +/– mycorrhizal fungi. (e, f) Shoot and root biomasses in WT and PDR1 OE plants. (g, h) DAD1 and MAX1 expression levels in 6‐wk‐old PDR1 OE roots and shoots. (i) PDR1 expression levels in 6‐wk‐old PDR1 OE plants. (j) MAX1 and DAD1 in stems and leaves of WT and PDR1 OE plants. Bars, 2 cm. Values are means ± SE. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
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The ability of PDR1 OE plants to obtain faster mycorrhization levels and longer/thicker stems suggests that SL biosynthesis might be induced to support the stronger SL transport/exudation driven by PDR1. We analyzed the expression levels of two SL biosynthetic genes, DAD1 and MAX1, in three different PDR1 OE lines (Fig. S2d–f). Compared with the wild‐type, PDR1, DAD1, and MAX1 are significantly up‐regulated (Fig. 4g–i) in shoots and roots of PDR1 OE plants. This result indicates that the overexpression of the transporter induces the SL biosynthesis pathway; SL biosynthesis is feedback‐inhibited by SL; and enhanced SL export from the site of its synthesis releases this feedback inhibition. To test if SL concentrations are changed in PDR1 OE roots, we assayed SL‐inducible germination of the parasitic weed Phelipanche ramosa using P. hybrida root extracts. PDR1 OE root extracts cannot induce P. ramosa germination as strongly as the wild‐type (Fig. 3j), showing that PDR1 OE roots are partially depleted in SL as a result of increased transport to the shoot and/or exudation to the soil. The DAD1 and MAX1 expression results in roots imply that their gene expression can be used as inversely proportional readouts of SL accumulation.
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To further investigate how PDR1 OE shoot phenotypes are related to SL reallocation, we assayed DAD1 and MAX1 expression to obtain an indirect and distinct quantification of SL in wild‐type and PDR1 OE 2‐month‐old (adult) plants. DAD1 and MAX1 were up‐regulated (Fig. 4j) in leaves of PDR1 OE plants, confirming the results obtained in 1‐month‐old PDR1 OE plantlet shoots. By contrast, MAX1 and DAD1 were strongly down‐regulated in PDR1 OE stems (Fig. 4j). This result suggests that SL accumulation in the stem (probably close to the nodes, as inferred from the SL‐related bud phenotype of PDR1 OE plants) might be a result not only of SL reallocation in the stem but also of SL depletion out of the leaves driven by PDR1. To test for the presence of this possible SL transport route from the leaf to the stem, we quantified SL from stem and leaf extracts with the P. ramosa germination assay in wild‐type, PDR1 OE and pdr1 ko leaves.
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Tissue extracts from 2‐month‐old PDR1 OE leaves and stems did not cause any germination of parasitic weeds in three attempts, probably because of low SL concentrations in adult shoots. However, two attempts with one order of magnitude lower dilutions of leaf extracts (see Methods S1) provided germination rates of between 1% and 4% (Fig. 5a). These results confirmed that SL concentrations in PDR1 OE shoots are lower than in the wild‐type, surprisingly also in stems where bud outgrowth is inhibited (Sasse et al., 2015). By contrast, the same experimental setup with extracts from pdr1 ko leaves showed a higher germination rate than in the wild‐type (Fig. S3a), suggesting that PDR1 is necessary for transporting SLs out of the leaf.
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Semiquantitative strigolactone (SL) quantification, 3H‐GR24 transport and leaf senescence in wild‐type (WR) and PDR1 OE Petunia hybrida leaves. (a) Germination rate of Phelipanche ramosa seeds with leaf and stem extracts of WT and PDR1 OE plants. (b, c) Decays min–1 (DPM) of 3H‐GR24 present in leaf unloaded sap relative to 3H‐GR24 leaf content (b) and 3H2O leaf content (c). (d) Senescence‐related leaf phenotypes in 3‐month‐old WT and PDR1 OE plants from the last leaf grown before the transition to flowering time (leaf −1) up to leaf −23. (e–g) Gene expression levels of petunia ORE1like,SAG12like and SAG13like in leaves −5/−6, −9/−10 and −15/−16. Bars, 2 cm. Values are means ± SE. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.
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We then compared SL transport in wild‐type, PDR1 OE and pdr1 ko leaves by quantifying leaf loading and unloading of a radiolabeled SL‐mimicking molecule (3H‐GR24) and we compared leaf senescence, a known SL‐related phenotype (Figs 5b, S3b). Leaves were first incubated for 12 h in 1/2 MS + 3H‐GR24 and then transferred in cold 1/2 MS for an additional 10 h. 3H‐GR24 concentrations were scored after leaf loading and unloading. Wild‐type leaves released 26% (± 1.2%) of the loaded 3H‐GR24, while pdr1 ko only released 18% (± 1.3%); PDR1 OE leaves released 46% (± 7.9%), while their wild‐type counterpart only released 32% (± 0.2%). No differences or opposite trends were scored in control transport experiments with tritiated water (Figs 5c, S3c). These results indicate that PDR1, and not transpiration or phloem flows, regulates SL transport out of the leaves. To test if GR24 and not its metabolites are exported from the leaf via PDR1, we directly quantified GR24 leaf loading and unloading via LC‐MS‐MS analyses (see Methods S1; Fig. S4a,b). The results showed GR24 stability in this time span, as also shown by Akiyama et al. (2010) and that the conditions of this experiment produced no detectable (below our detection limits) GR24 degradation products such as the ABC moiety or the hydrolyzed D‐ring (Fig. S4c,d), thus confirming the positive role of PDR1 in GR24 leaf export. Last but not least, we quantified senescence in PDR1 OE, pdr1 ko and wild‐type leaves by visual examination, gene expression analysis and LC‐MS of nonfluorescent Chl catabolites (NCCs) (Berghold et al., 2004; Christ et al., 2016). Three months after germination, P. hybrida leaves were collected and compared. Wild‐type leaves showed senescence from the 11th top leaf down (leaf −11), while in PDR1 OE, leaf senescence started being visible in marginal spots of the leaf blade in the 15th leaf from the top (leaf −15) (Fig. 5d). Leaves of pdr1 ko plants were significantly smaller than the relative wild‐type background (Fig. S3d,e) and started senescing between leaf −11 and leaf −14, at which point the wild‐type showed no senescing leaves. The gene expression of P. hybrida ORE1‐like, SENESCENCE‐ASSOCIATED GENE 12‐like (SAG12like) and SAG13‐like indicators of leaf senescence (Lohman et al., 1994; Breeze et al., 2011) confirmed that senescence emerges earlier in the wild‐type than in PDR1 OE plants, in leaves not showing wilting phenotypes (Fig. 5e,f). In the older leaves −15/−16 (Fig. 5g) the three genes were strongly up‐regulated in the wild‐type but significantly different only for ORE1like and SAG12like. Leaves −11 and −14 from pdr1 ko plants showed an inverted behavior of the SL‐biosynthetic‐ and senescence‐related genes that we found deregulated in PDR1 OE leaves (Fig. S3f,g). MS analyses conducted on the same tissues confirmed the accumulation of NCC 806 and NCC 892 in wild‐type but not in PDR1 OE leaves (Fig. S5a–d). These results show that PDR1 OE leaves can export to the stem more SL than can the wild‐type, thus releasing MAX1 and DAD1 expression from the SL negative feedback and strongly postponing leaf senescence. Also, with the opposite results from the parallel analyses on pdr1 ko plants, we propose that PDR1 regulates the transport of SL out of the leaves, either to the lateral buds or to the main stem.
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Strigolactones have been described to have an impact on lateral root and root hair formation (Kapulnik et al., 2011; Mayzlish‐Gati et al., 2012), factors that influence plant nutrition. We investigated whether PDR1 OE plants can produce a higher biomass than the wild‐type not only because of faster mycorrhization but also because of altered root structures that are possibly more efficient in nutrient uptake. Indeed, on Claymin in the absence of mycorrhiza, PDR1OE exhibit a higher biomass compared with the wild type (Fig. 4a–f). Using X‐ray computed tomography (see Methods S1 and Table S1) we screened root volumes, surfaces and the amounts of lateral roots on clay+, where we observed a significant increase of PDR1 OE biomass. At 6 wag the tomography did not reveal significant differences between wild‐type and PDR1 OE plants (Fig. S6a–c; Movies S1, S2). By contrast, at 8 wag, significantly more lateral roots could be observed in PDR1 OE plants (Fig. 6a). Interestingly, this difference was also visible in the absence of AMF (Fig. 6b,c; Movies S3, S4), while a significant increase in total root volume and surface was only measured when plants were grown with AMF, suggesting that without the input of AMF, lateral root growth was initiated but did not proceed as quickly. On clay+, a dense disc of lateral roots was present close to the soil surface in PDR1 OE plants, at a depth of 2–4 cm, but not in the wild‐type (Fig. 6d,e). Shallow roots are known to play an important role in nutrient uptake, as in several soils this is the most nutrient‐rich region (Liao et al., 2001; Lynch, 2013). Therefore, we suggest that PDR1 OE, in the presence of AMF, might also have an advantage by better scavenging surface‐close nutrients. These results show that the induction of lateral roots is linked to the mis‐regulation of PDR1 expression and the consequent SL redistribution and not only to a higher rate of mycorrhization. Interestingly, 3‐wk‐old PDR1 OE seedlings grown under sterile conditions also have a higher number of lateral roots than wild‐type seedlings (Fig. 7a). This result confirms that PDR1 overexpression induces lateral roots independently of the growth substrate and of symbioses with soil microbes.
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X‐ray computed tomography on wild‐type (WT) and PDR1 OE Petunia hybrida roots. X‐ray computed tomography on roots of 8‐wk‐old petunia plants. (a) Heat map (root thickness) of WT (left) and PDR1 OE (right) roots grown on clay+. (b) Quantification of lateral roots in WT and PDR1 OE roots ± myc. (c) Ratio of PDR1 OE : WT root volumes and root surfaces +/– myc. (d–g) Digital rendering (via Fiji software) of WT (d, f) and PDR1 OE (e, g) roots: (d, e) top view; (f, g) bottom view. Bars: (a) 4 cm; (d–g) 1 cm. Values are means ± SE. *, P < 0.05.
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Root hair phenotypes in wild‐type (W115 (WT)) and PDR1 OE Petunia hybrida plants. Root hairs of 3‐wk‐old WT and PDR1 OE petunia plantlets grown on half‐strength MS agar plates. (a–c) Quantification of lateral root density, root hair density (area evaluated = 1.6 cm2) and root hair length in WT and PDR1 OE roots. (d, h) WT root segment: (d) differentiated (2 cm from the root tip); (h) above the root tip. (e, i) PDR1 OE root segment: (e) differentiated (2 cm from the root tip); (i) above the root tip. (f, j) WT lateral roots: (f) emerging; (j) elongated. (g, k) PDR1 OE lateral roots: (g) emerging; (k) elongated. In panels (a–k), the asterisk indicates the first root hair from the root tip. Bars, 400 μm. Values in (a–c) are means ± SE. *, P < 0.05.
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Root hairs contribute to nutrient uptake, especially of phosphate, nitrogen and water (Olah et al., 2005). Exogenous applications of GR24 were shown to induce root hair development in Arabidopsis (Kapulnik et al., 2011). Hence we investigated the root hair system in wild‐type and PDR1 OE P. hybrida plants. PDR1 OE seedlings had longer and denser root hairs than the wild‐type, in both main and lateral roots (Fig. 7b–k). Additionally, PDR1 OE root meristems showed root hair formation closer to the main root tip (see asterisks in Fig. 7h–k). To understand whether the root hair phenotype is caused by low nutrient conditions or whether it depends on SL redistribution as a result of PDR1 overexpression, we tested root hair length under high‐ and low‐Pi conditions (see the Materials and Methods section). As expected, wild‐type P. hybrida plants had longer root hairs when grown on low Pi than on high Pi, with a significant length increase of 39.5% (Fig. S7a,b,g). pdr1 ko mutants, on the other hand, had shorter root hairs compared with the wild‐type, independent of the nutrient conditions (Fig. S7c,d,g); however, on low Pi the root hair length still increased by 28.6%. The root hairs of PDR1 OE seedlings were long on both high‐ and low‐Pi media (Fig. S7e–g), thus showing that PDR1 is a major factor determining root hair length in P. hybrida.
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To assess whether PDR1‐dependent SL transport may be involved in root hair elongation, we performed time‐lapse analyses using light‐sheet confocal microscopy (Maizel et al., 2011; Stelzer 2015; von Wangenheim et al., 2016) on emerging lateral roots of seedlings transgenic for pPDR1:nls‐YFP and pPIN‐FORMED 1 (PIN1):nls‐RFP, the latter involved in SL‐regulated auxin transport (Shinohara et al., 2013) and used here as a morphological reference for the vasculature. As P. hybrida roots, owing to their thickness, proved to be unsuitable for this analysis, we chose A. thaliana plants transgenic for the same reporters. The time‐lapse analysis (Movie S5) revealed that pPDR1 is activated in epidermal cells as soon as root hairs elongate, and stops its activity when root hairs are fully elongated (Fig. S3h–k), implying that SL transport plays a key role during root hair elongation but not after root hairs have reached their final size. The sequential appearance of pPDR1:nls‐YFP and pPIN1:nls‐RFP (Fig. S7l,m) suggests a temporal order of hormonal action during root hair elongation.
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Exogenous application of GR24 in Arabidopsis was shown to alter the endocytic recycling of the auxin transporters PIN1 and PIN2 (Shinohara et al., 2013; Pandya‐Kumar et al., 2014). Therefore, we tested whether the increased biosynthesis of endogenous SL in PDR1 OE roots might have an effect on auxin distribution. The expression pattern and intensity of the auxin reporter pDR5:VENUS were investigated using confocal microscopy on 2‐wk‐old seedlings grown on 1/2 MS plates. Full image stacks of P. hybrida root tips showed that the signal intensity of pDR5:VENUS was weaker in PDR1 OE than in wild‐type plants (Fig. 8a, b). The inverted fountain pattern of auxin distribution reported for Arabidopsis (Swarup & Bennett, 2003) is also present in P. hybrida root tips and it is weakened in PDR1 OE roots (Fig. 8c), particularly in the central vasculature (Fig. 8d), where PIN1 is expressed. Cell expansion in the elongation zone (EZ) of PDR1 OE root tips is inhibited (Fig. 8e, f), probably explaining the proximity of the first root hair to the PDR1 OE meristematic zone. These results show that endogenous changes in SL transport and biosynthesis are capable of altering auxin distribution and support the hypothesis that SL is an upstream regulator of auxin transport in the root tip (Ruyter‐Spira et al., 2011).
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pDR5:VENUS patterns and cell morphology in wild‐type (WT) and PDR1 OE Petunia hybrida root tips. (a, b) pDR5:VENUS in 14‐d‐old WT (a) and PDR1 OE root (b). (c) Pattern of pDR5:VENUS in WT (left) and PDR1 OE root tip (right). (d) Digital quantification of pDR5:VENUS fluorescence in WT and PDR1 OE epidermal/cortex cells (epi/cortex) and central root vasculature (vasculature). (e) Representative propidium iodide‐stained root tips. The asterisk is located at the border between the division and the elongation zone (EZ). (f) Quantification of the cell length of the first five cells in the root tip EZ. Bars: 200 μm (a, b); 40 μm (c, e). Values are means ± SE. *, P < 0.05; ***, P < 0.0005.
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The overexpression of PDR1 in P. hybrida plants was previously reported to inhibit shoot lateral branching (Sasse et al., 2015). This result raised the question of whether PDR1 OE plants might increase not only SL transport but also its synthesis. Our results show that SL biosynthesis genes are induced in PDR1 OE roots and shoots, indicating that there is a cross‐regulation between transport and biosynthesis. We hypothesize that the higher amounts of SL transported from the root tip into the soil in PDR1 OE plants release DAD1 and MAX1 from a negative feedback regulation, which might occur in the presence of inhibitory concentrations of SL, thus allowing a higher biosynthesis of SL in shoots and roots. However, this does not necessarily cause higher SL concentrations in all tissues: SL could be even lower, probably because of simultaneously increased transport/export as seen for roots and leaves (see Fig. 9). DAD1 also fits this model in pdr1 ko leaves: it is strongly down‐regulated where SL accumulates. The nonresponsive behavior of MAX1 in pdr1 ko leaves is probably a result of the senescence of pdr1 ko leaves, as MAX1 was reported to be up‐regulated by senescence (Ueda & Kusaba, 2015). PDR1 OE stems seem to diverge from this theory, as stems are low in SL, but also in MAX1 and DAD1. Still, PDR1 OE shoot lateral branching is strongly delayed (Sasse et al., 2015), as if, close to lateral buds, SL concentrations and/or transport are still high enough to inhibit bud outgrowth. Alternatively, the PDR1 OE‐originated redistribution of SL creates plants that are more susceptible to SL in targeted areas such as dormant buds, which might be regulated by the SL ratio between nodes/internodes rather than by the total amount of SL in the stems. It is possible that more sensitive ways of quantifying SLs would allow a finer map of SL distribution in nodes and internodes to be drawn, thus allowing us to understand if local peaks of SL synthesis and distribution are responsible for the regulation of shoot lateral branching. ABC transporters are known to be frequently induced by their substrates (Hwang et al., 2016); however, to our knowledge it has not yet been shown that ABC transporters affect the synthesis of their substrates similarly.
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Proposed model for PDR1 routes of strigolactone (SL) transport in roots and shoots of Petunia hybrida. Model depicting the effects of PDR1 overexpression on SL transport in roots and shoots. PDR1 OE enhances SL exudation from the root to the rhizosphere, thus possibly reducing shoot‐ward SL transport to the stem and dampening SL concentrations in roots and leaves. As a consequence, lateral root inhibition is released, root hair elongation is induced, and mycorrhization and germination of parasitic weed seeds are enhanced. In the shoot, PDR1 drives the reallocation of SL from the leaves to the stem. This SL route is enhanced in PDR1 OE plants, thus promoting larger leaves, longer internodes than the wild‐type, and possibly playing a role in the inhibition of lateral bud outgrowth.
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As we report here in PDR1 OE plants, larger epidermal cells or rounder leaves were previously reported in A. thaliana max2 mutants and in ramosus‐1 (rms) and rms‐2 mutants in P. sativum (Beveridge et al., 1997; Stirnberg et al., 2002). Impaired SL activity, either by knocking out SL receptors or biosynthetic genes, or by enhanced SL exudation into the soil, seems to affect leaf development in a similar manner. Despite the lower SL concentrations we detected in PDR1 OE leaves via the parasitic weed germination assay, PDR1 OE plants are still inhibited in lateral bud outgrowth (Sasse et al., 2015), a phenotype known to be mimicked by applications of GR24 on dormant buds (Gomez‐Roldan et al., 2008). At present, no reliable system is available to quantify SL in certain small tissues, such as lateral axils or nodes, where pPDR1:GUS was shown to be expressed (Kretzschmar et al., 2012), or in internal tissues such as the procambium, where cell division is induced by GR24 (Agusti et al., 2011). So we cannot track whether the SL exported from the leaf via PDR1 accumulates into the stem axils or nodes. We suggest that PDR1 overexpression has different, local effects in shoot tissues, probably because of different SL transport routes and/or different locations of the SL biosynthetic pathway. It was reported that PhCCD7 is strongly expressed in stems (Drummond et al., 2015), while PhMAX1, AtD14 and AtMAX1 (Booker et al., 2005; Drummond et al., 2011; Chevalier et al., 2014) are also present in leaves. The SL source and sink map therefore appears to be tissue‐specific; based on our results, we propose the leaf‐to‐stem route as a new SL transport route that is important in the regulation of SL concentrations in leaves and stems. The function of this route seems to be the regulation of leaf senescence, which is SL‐dependent (Ueda & Kusaba, 2015), but it might also contribute to the SL‐driven inhibition of lateral bud growth.
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PDR1 OE plants grown in full nutrient conditions show several morphological traits common to plants grown in low‐Pi conditions, such as inhibition of lateral shoot growth, induction of lateral roots and root hair development (Zhang et al., 2014a,b). We propose that PDR1 overexpression‐induced synthesis of SL triggers a plant response similar to phosphate starvation, and hence results in plants that are primed to starvation before they experience it. Such behavior could explain why PDR1 OE plants can sequestrate more phosphate from soils, and mycorrhize and produce biomass faster than the wild‐type only in phosphate conditions lower than in full soil, conditions in which wild‐type plants need more time to adapt their architecture to the challenging environment. The observed PDR1 overexpression‐dependent increase in lateral root number particularly affects shallow lateral roots. The topsoil commonly shows higher nutrient (and particularly P) concentrations. Pi resources are limited as well as expensive to explore (Cordell et al., 2009). Therefore plants with higher capacities for Pi uptake are of agricultural interest. Additional field tests will be necessary to obtain a broader view of which conditions and which soils confer a similar advantage to PDR1 OE plants as that seen in glasshouse tests.
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Our results indicate that root hair formation is strongly dependent on exuded SL and/or the presence of active transport of SL by PDR1 through the epidermal layer. Our time‐lapse analyses showed that PDR1 is active from initiation to full elongation of each root hair. As SL was reported to regulate cytoskeletal dynamics (Pandya‐Kumar et al., 2014), we suggest that the extra SL transported by PDR1 allows an extended development of root hairs. We observed a reduced SL concentration within PDR1 OE roots, while pdr1 mutants, which have shorter root hairs compared with the wild‐type, were shown not to differ from the latter in root internal SL concentrations (Kretzschmar et al., 2012). These observations indicate that the PDR1‐triggered SL release into the rhizosphere probably has a stronger impact on root hair formation than does internal SL redistribution.
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The crosstalk between SL and auxins is altered in PDR1 OE plants and thus exerts a role in shaping PDR1 OE plant phenotypes. GR24 treatments cause the removal of the auxin carrier PIN1 from the plasma membrane (Shinohara et al., 2013) and PIN1 protein abundances are down‐regulated in PDR1 OE root tips (Sasse et al., 2015). On the other hand, the auxin carrier PIN2 is positively regulated by SL (Pandya‐Kumar et al., 2014; Sasse et al., 2015). Also, the expression of DAD1 is negatively regulated by SL but positively regulated by auxins (Hayward et al., 2009). Auxin transport/allocation could be influenced in several tissues by PDR1 overexpression, thus also changing DAD1 expression and consequently SL biosynthesis. Analyses of the auxin patterns in PDR1 OE root tips showed that changes in endogenous SL concentrations can alter auxin transport and patterning, thus affecting cell length in the EZ and consequently root hair density close to the root tip. Several pin mutants were indeed reported to inhibit cell elongation in the EZ (Blilou et al., 2005). In root tips SL might influence the abundances of several PIN proteins at the plasma membrane either directly or by acting on auxin flows directed by PIN1 and PIN2. Additional auxin and PIN quantifications in specific plant tissues such as root hairs, lateral roots, lateral buds and leaves might elucidate in detail the mechanisms behind the crosstalk between SL and auxin transporters. Based on the results shown here, we propose that SL regulates cell expansion by changing the efficiency of auxin transport: this change reduces cell expansion (as seen in the EZ of PDR1 OE root tips) or allows cell expansion (as seen in leaf epidermal cells of PDR1 OE plants).
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Despite the costs of producing a larger root system, PDR1 OE plants are still able to produce more shoot biomass when grown on soils that are suboptimal for wild‐type plants. The morphological changes in the root system architecture of PDR1 OE reported here show that many of the phenotypes observed when GR24 is exogenously applied, such as root hair elongation and lateral root induction on low Pi soil, can also be obtained simply by overexpressing the SL transporter, even independently of the soil nutrient conditions. A critical point about exploiting the PDR1 overexpression strategy for field‐grown plants is the possible presence of parasitic weed seeds in some soils and regions (Parker, 2012). Also, a tradeoff with PDR1 OE‐increased biomass production might be a higher sensitivity to drought and salinity stress: SL is reported to induce drought and salinity tolerance in Arabidopsis (Ha et al., 2014). We have preliminary data suggesting that PDR1 OE‐expanded leaf blade might cause higher transpiration in water‐limited conditions compared with the wild‐type. However, mycorrhization is known to alleviate drought stress (Ruiz‐Lozano et al., 2016) and the increased exudation of SLs from PDR1 OE roots induces higher mycorrhization levels than in the wild‐type, which might balance drought and salinity sensitivity. On the other hand, SL‐driven approaches have also been shown to be effective against parasitic weeds, such as suicidal germination, which promotes the germination of parasitic weeds in the absence of host plants (Kgosi et al., 2012; Khosla & Nelson, 2016). Besides, a PDR1 overexpression strategy could provide a solution to improve plant nutrition for crops that are not hosts for parasitic weeds or grown on fields without parasites. PDR1 overexpression may also be combined with two approaches that have already been proposed: overproduction of citrate (Lopez‐Bucio et al., 2000), which was shown to have a positive effect on phosphate nutrition; and/or overexpression of ABCG37/PDR9 (Fourcroy et al., 2014), which was reported to exhibit a positive effect on iron nutrition via coumarin exudation.
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In summary, our studies shed new light on SL transport routes and targets. These could provide a solution for improving plant nutrition and be a strategy for sustainable agriculture on low‐Pi soils, where an increase in the root system volume and/or the symbiosis with mycorrhizal fungi is required to allow the plant to exploit larger soil volumes. Furthermore, screening for accessions with high PDR1 expression could be a new approach to isolate plant varieties with higher mycorrhization efficiency and improved root system architecture.
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L.B., G.L. and E.M. conceived and designed research. G.L., L.B., J.P., R.d.B.F., A.E., C.G., M.S., O.H., J.S., C.M. conducted experiments. L.B., G.L. R.d.B.F. and E.M. analyzed data. L.B., G.L., J.P., R.d.B.F., A.W., E.S. and E.M. wrote the manuscript. All authors read and approved the manuscript.
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