Phenotypic differentiation between wild and domesticated varieties of <Emphasis Type="Italic">Crescentia cujete</Emphasis>L. and culturally relevant uses of their fruits as bowls in the Yucatan Peninsula,Mexico

Background

Selection criteria are important for analyzing domestication of perennial plant species, which experience a selection pressure throughout several human generations. We analyze the preferred morphological characteristics of Crescentia cujete fruits, which are used as bowls by the Maya of Yucatan, according to the uses they are given and the phenotypic consequences of artificial selection between one wild and three domesticated varieties.

Methods

We performed 40 semi-structured interviews in seven communities. We calculated Sutrop’s salience index (S) of five classes of ceremonial and daily life uses, and of each item from the two most salient classes. We sampled 238 bowls at homes of people interviewed and compared their shape, volume and thickness with 139 fruits collected in homegardens and 179 from the wild. Morphology of varieties was assessed in fruit (n?=?114 trees) and vegetative characters (n?=?136 trees). Differences between varieties were evaluated through linear discriminant analysis (LDA).

Results

Use of bowls as containers for the Day of the Dead offerings was the most salient class (S?=?0.489) with chocolate as its most salient beverage (S?=?0.491), followed by consumption of daily beverages (S?=?0.423), especially maize-based pozol (S?=?0.412). The sacred saka’ and balche' are offered in different sized bowls during agricultural and domestic rituals. Roundness was the most relevant character for these uses, as bowls from households showed a strong selection towards round shapes compared with wild and homegarden fruits. Larger fruits from domesticated varieties were also preferred over small wild fruits, although in the household different sizes of the domesticated varieties are useful. LDA separated wild from domesticated trees (p?<?0.001) according to both fruit and vegetative variables, but domesticated varieties were not different among themselves.

Conclusions

The association between C. cujete bowls and traditional beverages in ritual and daily life situations has driven for centuries the selection of preferred fruit morphology in this tree. Selection of fruit roundness and volume has allowed for the differentiation between the wild variety and the three domesticated ones, counteracting gene flow among them. By choosing the best fruits from domesticated varieties propagated in homegardens, the Maya people model the domestication process of this important tree in their culture.

     

Identification and Characterisation CRN Effectors in Phytophthora capsici Shows Modularity and Functional Diversity

Phytophthora species secrete a large array of effectors during infection of their host plants. The Crinkler (CRN) gene family encodes a ubiquitous but understudied class of effectors with possible but as of yet unknown roles in infection. To appreciate CRN effector function in Phytophthora, we devised a simple Crn gene identification and annotation pipeline to improve effector prediction rates. We predicted 84 full-length CRN coding genes and assessed CRN effector domain diversity in sequenced Oomycete genomes. These analyses revealed evidence of CRN domain innovation in Phytophthora and expansion in the Peronosporales. We performed gene expression analyses to validate and define two classes of CRN effectors, each possibly contributing to infection at different stages. CRN localisation studies revealed that P. capsici CRN effector domains target the nucleus and accumulate in specific sub-nuclear compartments. Phenotypic analyses showed that few CRN domains induce necrosis when expressed in planta and that one cell death inducing effector, enhances P. capsici virulence on Nicotiana benthamiana. These results suggest that the CRN protein family form an important class of intracellular effectors that target the host nucleus during infection. These results combined with domain expansion in hemi-biotrophic and necrotrophic pathogens, suggests specific contributions to pathogen lifestyles. This work will bolster CRN identification efforts in other sequenced oomycete species and set the stage for future functional studies towards understanding CRN effector functions.     

Maternal Conditions and Perinatal Characteristics Associated with Autism Spectrum Disorder and Intellectual Disability

Background

As well as being highly comorbid conditions, autism spectrum disorders (ASD) and intellectual disability (ID) share a number of clinically-relevant phenomena. This raises questions about similarities and overlap in diagnosis and aetiological pathways that may exist for both conditions.

Aims

To examine maternal conditions and perinatal factors for children diagnosed with an ASD, with or without ID, and children with ID of unknown cause, compared with unaffected children.

Methods

The study population comprised all live singleton births in Western Australia (WA) between January 1984 and December 1999 (N = 383,153). Univariate and multivariate multinomial logistic regression models were applied using a blocked modelling approach to assess the effect of maternal conditions, sociodemographic factors, labour and delivery characteristics and neonatal outcomes.

Results

In univariate analyses mild-moderate ID was associated with pregnancy hypertension, asthma, urinary tract infection, some types of ante-partum haemorrhage, any type of preterm birth, elective C-sections, breech presentation, poor fetal growth and need for resuscitation at birth, with all factors showing an increased risk. Severe ID was positively associated with poor fetal growth and need for resuscitation, as well as any labour or delivery complication. In the multivariate analysis no maternal conditions or perinatal factors were associated with an increased risk of ASD without ID. However, pregnancy hypertension and small head circumference were associated with a reduced risk (OR = 0.64, 95% CI: 0.43, 0.94; OR = 0.58, 95% CI: 0.34, 0.96, respectively). For ASD with ID, threatened abortion before 20 weeks gestation and poor fetal growth were associated with an increased risk.

Conclusion

Findings show that indicators of a poor intrauterine environment are associated with an elevated risk of ID, while for ASD, and particularly ASD without ID, the associations are much weaker. As such, these findings highlight the importance of accounting for the absence or presence of ID when examining ASD, if we are to improve our understanding of the causal pathways associated with these conditions.     

Impacts on Coralligenous Outcrop Biodiversity of a Dramatic Coastal Storm

Extreme events are rare, stochastic perturbations that can cause abrupt and dramatic ecological change within a short period of time relative to the lifespan of organisms. Studies over time provide exceptional opportunities to detect the effects of extreme climatic events and to measure their impacts by quantifying rates of change at population and community levels. In this study, we show how an extreme storm event affected the dynamics of benthic coralligenous outcrops in the NW Mediterranean Sea using data acquired before (2006–2008) and after the impact (2009–2010) at four different sites. Storms of comparable severity have been documented to occur occasionally within periods of 50 years in the Mediterranean Sea. We assessed the effects derived from the storm comparing changes in benthic community composition at sites exposed to and sheltered from this extreme event. The sites analyzed showed different damage from severe to negligible. The most exposed and impacted site experienced a major shift immediately after the storm, represented by changes in the species richness and beta diversity of benthic species. This site also showed higher compositional variability immediately after the storm and over the following year. The loss of cover of benthic species resulted between 22% and 58%. The damage across these species (e.g. calcareous algae, sponges, anthozoans, bryozoans, tunicates) was uneven, and those with fragile forms were the most impacted, showing cover losses up to 50 to 100%. Interestingly, small patches survived after the storm and began to grow slightly during the following year. In contrast, sheltered sites showed no significant changes in all the studied parameters, indicating no variations due to the storm. This study provides new insights into the responses to large and rare extreme events of Mediterranean communities with low dynamics and long-lived species, which are among the most threatened by the effects of global change.     

Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF''s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF''s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.Glial cell line-derived neurotrophic factor (GDNF) is the founding member of the four ligands in the GDNF family, which belong to the transforming growth factor-β superfamily.¹ GDNF was characterized as a potent survival factor for many neurons in culture such as dopaminergic, motor, sympathetic, parasympathetic, sensory and enteric neurons.^{1, 2} In addition, in dopaminergic neuron cultures GDNF stimulates neuronal differentiation, neurite outgrowth, synapse formation and dopamine release.^{1, 2}As degeneration of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNpc) represents a major hallmark of Parkinson disease (PD), the most common neurodegenerative movement disorder, GDNF has raised considerable interest as a therapeutic molecule for the treatment of PD.^{3, 4, 5} PD affects >2% of individuals over the age of 60 years, but no curative treatment is available to date, mainly due to a lack of understanding disease etiology.^{6, 7, 8} Preclinical studies in the established 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) rodent and primate models of PD demonstrated a substantial neuroprotection and regeneration effect by striatal provided GDNF or its close relative neurturin.^{3, 4, 9} However, clinical phase II trials on PD patients using GDNF or neurturin did so far not convincingly recapitulate their beneficial effects on the dopaminergic system in humans most likely due to technical problems and the selection of advanced PD patients.^{10, 11, 12, 13}GDNF signaling is highly complex as this neurotrophic factor can bind to a variety of receptors, thus being able to induce pleiotropic effects. GDNF efficiently binds to the GPI-linked GDNF family receptor α1 (GFRα1).^{1, 2} It has been shown that the GDNF/GFRα1 complex can activate not only the canonical GDNF receptor Ret, a receptor tyrosine kinase which signals through the sarcoma protein (Src)/rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, NF-κB (nuclear factor ''kappa-light-chain-enhancer'' of activated B cells), JNK (c-Jun N-terminal kinases) and PLCγ (phospholipase γ) pathway, but also with other signaling inducing receptors.^{1, 2, 4, 5, 13} So far, at least four alternative GDNF receptors have been described which are all expressed in midbrain dopaminergic neurons, NCAM,^{14, 15} the integrins αV and βI,^{14, 16} syndecan 3¹⁷ and N-cadherin.¹⁸ Interestingly, Ret is not essential during pre- and postnatal development of the mouse dopaminergic system,^{19, 20, 21, 22, 23} but specifically required for the maintenance of SNpc dopaminergic neurons and their striatal innervation in aged mice.^{23, 24, 25} In contrast, GDNF seems most likely under physiological conditions to be dispensable during development and maintenance of midbrain dopaminergic neurons in mice, although conflicting results exist.^{26, 27, 28} Thus, Ret might be activated by a GDNF-independent mechanism to stimulate SNpc dopaminergic neuron survival. In addition, the in vivo function of the alternative GDNF receptors in the dopaminergic system under physiological and pathophysiological conditions, like PD, and their dependence on GDNF has not yet been addressed in detail. This raised the important question which GDNF receptor might be required to mediate GDNF''s reported neuroprotective and regenerative effect in the dopaminergic system in PD animal models and potentially in PD patients.^{5, 29}Previously, we showed in dopaminergic neuron-specific Ret knockout mice that Ret receptor loss does not result in a higher vulnerability of midbrain dopaminergic neurons against MPTP but to less resprouting of left over dopaminergic neuron axons in the striatum after MPTP intoxication.³⁰ In adult mice endogenous GDNF levels are rather low.^{26, 31} Therefore, we could not rule out in that study the possibility, that higher levels of GDNF—as also used in the clinical GDNF trials in PD patients—might have neuroprotective and regenerating effects even in the absence of the Ret receptor. Here we addressed now this question by viral overexpression of GDNF in MPTP-treated mice lacking expression of Ret again specifically in dopaminergic neurons.^{23, 30} We found that in the absence of Ret in dopaminergic neurons even a substantial overexpression of GDNF in the striatum does not have a neuroprotective and regenerative effect. Thus, despite the expression of alternative GDNF receptors on midbrain dopaminergic neurons, the presence of the canonical GDNF receptor Ret seems to be mandatory for mediating GDNF''s beneficial survival and axonal resprouting effect in these neurons.     

Single and Serial Fetal Biometry to Detect Preterm and Term Small- and Large-for-Gestational-Age Neonates: A Longitudinal Cohort Study

ObjectivesTo assess the value of single and serial fetal biometry for the prediction of small- (SGA) and large-for-gestational-age (LGA) neonates delivered preterm or at term.MethodsA cohort study of 3,971 women with singleton pregnancies was conducted from the first trimester until delivery with 3,440 pregnancies (17,334 scans) meeting the following inclusion criteria: 1) delivery of a live neonate after 33 gestational weeks and 2) two or more ultrasound examinations with fetal biometry parameters obtained at ≤36 weeks. Primary outcomes were SGA (<5^th centile) and LGA (>95^th centile) at birth based on INTERGROWTH-21^st gender-specific standards. Fetus-specific estimated fetal weight (EFW) trajectories were calculated by linear mixed-effects models using data up to a fixed gestational age (GA) cutoff (28, 32, or 36 weeks) for fetuses having two or more measurements before the GA cutoff and not already delivered. A screen test positive for single biometry was based on Z-scores of EFW at the last scan before each GA cut-off so that the false positive rate (FPR) was 10%. Similarly, a screen test positive for the longitudinal analysis was based on the projected (extrapolated) EFW at 40 weeks from all available measurements before each cutoff for each fetus.ResultsFetal abdominal and head circumference measurements, as well as birth weights in the Detroit population, matched well to the INTERGROWTH-21^st standards, yet this was not the case for biparietal diameter (BPD) and femur length (FL) (up to 9% and 10% discrepancy for mean and confidence intervals, respectively), mainly due to differences in the measurement technique. Single biometry based on EFW at the last scan at ≤32 weeks (GA IQR: 27.4–30.9 weeks) had a sensitivity of 50% and 53% (FPR = 10%) to detect preterm and term SGA and LGA neonates, respectively (AUC of 82% both). For the detection of LGA using data up to 32- and 36-week cutoffs, single biometry analysis had higher sensitivity than longitudinal analysis (52% vs 46% and 62% vs 52%, respectively; both p<0.05). Restricting the analysis to subjects with the last observation taken within two weeks from the cutoff, the sensitivity for detection of LGA, but not SGA, increased to 65% and 72% for single biometry at the 32- and 36-week cutoffs, respectively. SGA screening performance was higher for preterm (<37 weeks) than for term cases (73% vs 46% sensitivity; p<0.05) for single biometry at ≤32 weeks.ConclusionsWhen growth abnormalities are defined based on birth weight, growth velocity (captured in the longitudinal analysis) does not provide additional information when compared to the last measurement for predicting SGA and LGA neonates, with both approaches detecting one-half of the neonates (FPR = 10%) from data collected at ≤32 weeks. Unlike for SGA, LGA detection can be improved if ultrasound scans are scheduled as close as possible to the gestational-age cutoff when a decision regarding the clinical management of the patient needs to be made. Screening performance for SGA is higher for neonates that will be delivered preterm.     

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B₁, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B₁ and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network.Sphingolipids play critical roles in plant growth and development as essential components of endomembranes, including the plasma membrane, where they constitute more than 40% of the total lipid (Sperling et al., 2005; Cacas et al., 2016). Sphingolipids also are highly enriched in detergent-insoluble membrane fractions of the plasma membrane that form microdomains for proteins with important cell surface activities, including cell wall biosynthesis and hormone transport (Cacas et al., 2012, 2016; Perraki et al., 2012; Bayer et al., 2014). In addition, sphingolipids, particularly those with very-long-chain fatty acids (VLCFAs), are integrally associated with Golgi-mediated protein trafficking that underlies processes related to the growth of plant cells (Bach et al., 2008, 2011; Markham et al., 2011; Melser et al., 2011). Furthermore, sphingolipids function through their bioactive long-chain base (LCB) and ceramide metabolites to initiate programmed cell death (PCD), important for mediating plant pathogen resistance through the hypersensitive response (Greenberg et al., 2000; Liang et al., 2003; Shi et al., 2007; Bi et al., 2014; Simanshu et al., 2014).Sphingolipid biosynthesis is highly regulated in all eukaryotes. In plants, the maintenance of sphingolipid homeostasis is vital to ensure sufficient sphingolipids for growth (Chen et al., 2006; Kimberlin et al., 2013) while restricting the accumulation of PCD-inducing ceramides and LCBs until required for processes such as the pathogen-triggered hypersensitive response. Serine palmitoyltransferase (SPT), which catalyzes the first step in LCB synthesis, is generally believed to be the primary control point for sphingolipid homeostasis (Hanada, 2003). SPT synthesizes LCBs, unique components of sphingolipids, by catalyzing a pyridoxal phosphate-dependent condensation of Ser and palmitoyl (16:0)-CoA in plants (Markham et al., 2013). Similar to other eukaryotes, the Arabidopsis (Arabidopsis thaliana) SPT is a heterodimer consisting of LCB1 and LCB2 subunits (Chen et al., 2006; Dietrich et al., 2008; Teng et al., 2008). Research to date has shown that SPT is regulated primarily by posttranslational mechanisms involving physical interactions with noncatalytic, membrane-associated proteins that confer positive and negative regulation of SPT activity (Han et al., 2009, 2010; Breslow et al., 2010). These proteins include a 56-amino acid small subunit of SPT (ssSPT) in Arabidopsis, which was recently shown to stimulate SPT activity and to be essential for generating sufficient amounts of sphingolipids for pollen and sporophytic cell viability (Kimberlin et al., 2013).Evidence from yeast and mammalian research points to a more critical role for proteins termed ORMs (for orosomucoid-like proteins) in sphingolipid homeostatic regulation (Breslow et al., 2010; Han et al., 2010). The Saccharomyces cerevisiae Orm1p and Orm2p negatively regulate SPT through reversible phosphorylation of these polypeptides in response to intracellular sphingolipid levels (Breslow et al., 2010; Han et al., 2010; Roelants et al., 2011; Gururaj et al., 2013; Muir et al., 2014). Phosphorylation/dephosphorylation of ORMs in S. cerevisiae presumably affects the higher order assembly of SPT to mediate flux through this enzyme for LCB synthesis (Breslow, 2013). In this sphingolipid homeostatic regulatory mechanism, the S. cerevisiae Orm1p and Orm2p are phosphorylated at their N termini by Ypk1, a TORC2-dependent protein kinase (Han et al., 2010; Roelants et al., 2011). The absence of this phosphorylation domain in mammalian and plant ORM homologs brings into question the nature of SPT reversible regulation by ORMs in other eukaryotic systems (Hjelmqvist et al., 2002).Sphingolipid synthesis also is mediated by the N-acylation of LCBs by ceramide synthases to form ceramides, the hydrophobic backbone of the major plant glycosphingolipids, glucosylceramide (GlcCer) and glycosyl inositolphosphoceramide (GIPC). Two functionally distinct classes of ceramide synthases occur in Arabidopsis, designated class I and class II (Chen et al., 2008). Class I ceramide synthase activity resulting from the Longevity Assurance Gene One Homolog2 (LOH2)-encoded ceramide synthase acylates, almost exclusively, LCBs containing two hydroxyl groups (dihydroxy LCBs) with 16:0-CoA to form C16 ceramides, which are used primarily for GlcCer synthesis (Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016). Class II ceramide synthase activities resulting from the LOH1- and LOH3-encoded ceramide synthases are most active in the acylation of LCBs containing three hydroxyl groups (trihydroxy LCBs) with VLCFA-CoAs, including primarily C₂₄ and C₂₆ acyl-CoAs (Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016). Class II (LOH1 and LOH3) ceramide synthase activity is essential for producing VLCFA-containing glycosphingolipids to support the growth of plant cells, whereas class I (LOH2) ceramide synthase activity is nonessential under normal growth conditions (Markham et al., 2011; Luttgeharm et al., 2015b). It was speculated recently that LOH2 ceramide synthase functions, in part, as a safety valve to acylate excess LCBs for glycosylation, resulting in a less cytotoxic form (Luttgeharm et al., 2015b; Msanne et al., 2015). Recent studies have shown that the Lag1/Lac1 components of the S. cerevisiae ceramide synthase are phosphorylated by Ypk1, and this phosphorylation stimulates ceramide synthase activity in response to heat and reduced intracellular sphingolipid levels (Muir et al., 2014). This finding points to possible coordinated regulation of ORM-mediated SPT and ceramide synthase activities to regulate sphingolipid homeostasis, which is likely more complicated in plants and mammals due to the occurrence of functionally distinct ceramide synthases in these systems (Stiban et al., 2010; Markham et al., 2011; Ternes et al., 2011; Luttgeharm et al., 2016).RNA interference (RNAi) suppression of ORM genes in rice (Oryza sativa) has been shown to affect pollen viability (Chueasiri et al., 2014), but no mechanistic characterization of ORM proteins in plants has yet to be reported. Here, we describe two Arabidopsis ORMs, AtORM1 and AtORM2, that suppress SPT activity through direct interaction with the LCB1/LCB2 heterodimer. We also show that strong up-regulation of AtORM expression impairs growth. In addition, up- or down-regulation of ORMs is shown to differentially affect the sensitivity of Arabidopsis to the PCD-inducing mycotoxin fumonisin B₁ (FB₁), a ceramide synthase inhibitor, and to differentially affect the activities of class I and II ceramide synthases as a possible additional mechanism for regulating sphingolipid homeostasis.