Sylvia Morais de Sousa

A role for root morphology and related candidate genes in P acquisition efficiency in maize

Phosphorus (P) is an essential nutrient for plants and is acquired from the rhizosphere solution as inorganic phosphate. P is one of the least available mineral nutrients, particularly in highly weathered, tropical soils, and can substantially limit plant growth. The aim of this work was to study a possible effect of root morphology and the expression pattern of related candidate genes on P efficiency in maize. Our field phenotyping results under low and high P conditions enabled us to identify two contrasting genotypes for P acquisition efficiency that were used for the root traits studies. Root morphology was assessed in a paper pouch system to investigate root traits that could be involved in P acquisition efficiency. The genes, Rtcs, Bk2 and Rth3, which are known to be involved in root morphology, showed higher expression in the P efficient line relative to the P inefficient line. Overall, root traits showed high heritability and a low coefficient of variation. Principal component analysis revealed that out of the 24 root traits analysed, only four root traits were needed to adequately represent the diversity among genotypes. The information generated by this study will be useful for establishing early selection strategies for P efficiency in maize, which are needed to support subsequent molecular and physiological studies.

Structural and kinetic characterization of a maize aldose reductase

The aldo-keto reductases (AKRs) are classified as oxidoreductases and are found in organisms from prokaryotes to eukaryotes. The AKR superfamily consists of more than 120 proteins that are distributed throughout 14 families. Very few plant AKRs have been characterized and their biological functions remain largely unknown. Previous work suggests that AKRs may participate in stress tolerance by detoxifying reactive aldehyde species. In maize endosperm, the presence of an aldose reductase (AR; EC 1.1.1.21) enzyme has also been hypothesized based on the extensive metabolism of sorbitol. This manuscript identifies and characterizes an AKR from maize (Zea mays L.) with features of an AR. The cDNA clone, classified as AKR4C7, was expressed as a recombinant His-tag fusion protein in Escherichia coli. The product was purified by immobilized metal affinity chromatography followed by anion exchange chromatography. Circular dichroism spectrometry and SAXS analysis indicated that the AKR4C7 protein was stable, remained folded throughout the purification process, and formed monomers of a globular shape, with a molecular envelope similar to human AR. Maize AKR4C7 could utilize dl-glyceraldehyde and some pentoses as substrates. Although the maize AKR4C7 was able to convert sorbitol to glucose, the low affinity for this substrate indicated that AKR4C7 was probably a minimal contributor to sorbitol metabolism in maize seeds. Polyclonal antisera raised against AKR4C7 recognized at least three AR-like polypeptides in maize kernels, consistent with the presence of a small gene family. Diverse functions may have evolved for maize AKRs in association with specific physiological requirements of kernel development.

Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase.

Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 A and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 A was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.

A comparative structural analysis reveals distinctive features of co-factor binding and substrate specificity in plant aldo-keto reductases.

Plant aldo-keto reductases of the AKR4C subfamily play key roles during stress and are attractive targets for developing stress-tolerant crops. However, these AKR4Cs show little to no activity with previously-envisioned sugar substrates. We hypothesized a structural basis for the distinctive cofactor binding and substrate specificity of these plant enzymes. To test this, we solved the crystal structure of a novel AKR4C subfamily member, the AKR4C7 from maize, in the apo form and in complex with NADP(+). The binary complex revealed an intermediate state of cofactor binding that preceded closure of Loop B, and also indicated that conformational changes upon substrate binding are required to induce a catalytically-favorable conformation of the active-site pocket. Comparative structural analyses of homologues (AKR1B1, AKR4C8 and AKR4C9) showed that evolutionary redesign of plant AKR4Cs weakened interactions that stabilize the closed conformation of Loop B. This in turn decreased cofactor affinity and altered configuration of the substrate-binding site. We propose that these structural modifications contribute to impairment of sugar reductase activity in favor of other substrates in the plant AKR4C subgroup, and that catalysis involves a three-step process relevant to other AKRs.

Endophytic Bacillus strains enhance pearl millet growth and nutrient uptake under low-P

Bacterial endophytes are considered to have a beneficial effect on host plants, improving their growth by different mechanisms. The objective of this study was to investigate the capacity of four endophytic Bacillus strains to solubilize iron phosphate (Fe-P), produce siderophores and indole-acetic acid (IAA) in vitro, and to evaluate their plant growth promotion ability in greenhouse conditions by inoculation into pearl millet cultivated in a P-deficient soils without P fertilization, with Araxá rock phosphate or soluble triple superphosphate. All strains solubilized Fe-P and three of them produced carboxylate-type siderophores and high levels of IAA in the presence of tryptophan. Positive effect of inoculation of some of these strains on shoot and root dry weight and the N P K content of plants cultivated in soil with no P fertilization might result from the synergistic combination of multiple plant growth promoting (PGP) traits. Specifically, while B1923 enhanced shoot and root dry weight and root N P content of plants cultivated with no P added, B2084 and B2088 strains showed positive performance on biomass production and accumulation of N P K in the shoot, indicating that they have higher potential to be microbial biofertilizer candidates for commercial applications in the absence of fertilization.

The role of root morphology and architecture in phosphorus acquisition: physiological, genetic, and molecular basis

Low-phosphorus (P) availability caused by P fixation on soil clay minerals is a serious constraint for agricultural production and food security, particularly in the humid tropics and subtropics. Here we look at the underlying basis of root phenotypes that can potentially enhance a crop’s ability to acquire P under low availability in the soil. In the context of P acquisition and root system architecture (RSA) and morphology we discuss the following: (1) the molecular determinants, (2) the role of microRNAs (miRNAs), (3) possible quantitative trait loci (QTL) influencing root traits and grain yield in the field and (4) we also discuss recent advances in high throughput root system imaging and root architecture quantification methods. We aim at providing a repertoire of possible targets and strategies that can be explored as part of novel molecular breeding strategies to speed the generation of cultivars that are adapted to low P availability in the soil.

AB0581 Two years existence of reuma.pt/vasculitis – the portuguese registry of vasculitis

Background The vasculitides are a group of relatively rare diseases with different manifestations and outcomes. New therapeutic options have led to the need for long-term registries. The Rheumatic Diseases Portuguese Register, Reuma.pt, is an electronic clinical record, created in 2008, which currently includes specific protocols for 11 diseases and >16000 patients registered from 79 national and international rheumatology centres. Since October 2014, a dedicated protocol to vasculitis has been created as part of the European Vasculitis Society initiative of having compatible European registries. Objectives To describe the structure of Reuma.pt/Vasculitis and characterize the patients registered over the last two years. Methods We developed a dedicated web-based software to enable prospective collection and central storage of anonymised data from patients with vasculitis. All Portuguese rheumatology centres were invited to participate. Data regarding demographics, diagnosis, classification criteria, imaging and laboratory tests, outcome measures of prognosis, damage, disease activity and quality of life, and treatment were collected. We performed a cross-sectional descriptive analysis of all patients registered up to January 2017. Results A total of 492 patients, with 1114 visits, from 11 centres were registered in Reuma.pt/Vasculitis. The mean age was 53±20 years at last visit; 68% were females. The diagnoses followed the 2012 Chapel Hill Consensus nomenclature (Table 1). The most common diagnoses were Behçets disease (BD) (39%) and giant cell arteritis (GCA) (20%). Patients with BD met the International Study Group 1990 criteria, the International Criteria for BD 2006 and 2013 in 84%, 95% and 95% of cases, respectively. Patients with GCA met the 1990 American College of Rheumatology criteria in 95% of cases. Data on vascular ultrasound was available in 74% of patients; 73% compatible with the diagnosis. Assessment of the Birmingham Vasculitis Activity Score (BVAS) and Vasculitis Damage Index (VDI) was available for all vasculitides and the Five Factor Score calculation of survival rate for ANCA associated vasculitis (AAV) and polyarteritis nodosa (PAN). The mean BVAS at first visit was 18±7 for AAV and 15±9 for PAN; the mean VDI at last visit was 3±2 for AAV and 2±2 for PAN. Health related quality of life assessments (SF-36, EQD5, FACIT and HADS) were also collected. Treatment registry with the disease assessment variables shown in graphics was available for all patients; only 6% were under biologic treatment. Conclusions Reuma.pt/Vasculitis is a registry adapted for routine care, allowing an efficient data repository at a national level with the potential to link with other international databases. It facilitates research, trials recruitment, service planning and benchmarking. Disclosure of Interest None declared

SAT0237 Nailfold Capillaroscopy Findings in Scleroderma Patients – Prognostic Implications

Background Nailfold capillaroscopy (NCP) is a useful tool for the diagnosis and follow-up of systemic rheumatic diseases and represents one of the best methods to evaluate microvascular abnormalities. Objectives To characterize NCP findings of patients with Systemic Sclerosis (SSc), and understand how NCP associates with the presence of digital ulcers, gastrointestinal involvement, pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD). Methods NCP findings of adult patients with SSc followed-up in our center were reviewed. Demographic and clinical features were collected. Nonparametric tests were used to determine potential associations between capillaroscopy findings/patterns and the presence of digital ulcers, gastrointestinal involvement, PAH and ILD. Results In total, 52 out of 103 patients with SSc had at least one NCP result available. Forty five were female (86.5%) and seven male (13.5%), the age was 56.6 ±13.2 years and disease duration 10.9 ±10.5 years. Thirty one (59.6%) had limited cutaneous SSc, seven (13.5%) diffuse cutaneous SSc, ten (19,2%) VEDOSS, three (5.8%) overlap syndromes and one (1.9%) SSc sine scleroderma. (table 1). The most frequent NCP findings were hemorrhages (54.7%) megacapillaries (54.7%), and capillary dilatations (52.8%). We found that digital ulcers were significantly associated with the existence of avascular areas (p=0.02), and with neoangiogenesis (p=0.03) in univariate analysis, but were not associated with any capillaroscopy pattern. Looking to interstitial lung disease, there is a trend for association with the presence of avascular areas (p=0.056). Only one patient had PAH confirmed by right heart catheterization. In this case NCP showed giant capillaries, neoangiogenesis and avascular areas. There was also a trend for association between avascular areas and higher values of NT pro BNP (p=0.078). Gastrointestinal involvement was not significantly associated with NCP findings (table 2).Table 1. Distribution of patients according to NCP and disease subtypes Diffuse cutaneous Limited cutaneous Overlap syndrome VEDOSS Sine scleroderma (n=7) (n=31) (n=3) (n=10) (n=1) Early pattern n (%) 0 (0) 9 (29) 1 (33) 6 (60) 0 (0) Active pattern n (%) 1 (14.2) 5 (16.1) 1 (33) 2 (20) 0 (0) Late pattern n (%) 3 (42.9) 7 (22.6) 0 (0) 0 (0) 0 (0) Nonspecific n (%) 3 (42.9) 8 (25.8) 1 (33) 1 (10) 1 (100) Normal NCP n (%) 0 (0) 2 (6.5) 0 (0) 1 (11) 0 (0) Conclusions In our study the presence of avascular areas and neoangiogenesis in NCP was significantly associated with the existence of digital ulcers. Also, ILD and higher values of NT-proBNP were more common in patients who present avascular areas. These results suggest that abnormalities detected in the NCP might help predict organ involvement, although long term follow up and greater numbers of patients are needed in order to confirm the prognostic value of NCP findings. References Mannarino E, Pasqualini L, Fedeli F, Scricciolo V, Innocente S. Nailfold capillaroscopy in the screening and diagnosis of Raynauds phenomenon. Angiology 1994; 45: 37–42 Disclosure of Interest None declared

FRI0428 The Weaker Sex: Characterization of Gender Disparities in A Nationwide Lupus Registry (REUMA.PT LES): Table 1.

Background Systemic lupus erythematosus (SLE) is characterized by female predominance with male to female ratio around 1:10. Differences regarding clinical manifestations, disease activity, damage and mortality between men and women with SLE have been reported. Overall it is recognized that gender may affect SLE phenotype, but results concerning disease severity and prognosis are still a matter of debate. Objectives Characterization of Portuguese SLE male patients, focusing demographic, clinical, and laboratorial features. Methods All SLE patients from the Portuguese Lupus Register, Reuma.pt/LES were included. Demographic, clinical and therapeutic data were analyzed upon records from the last visit. Student t-tests, chi-square tests and Fishers exact tests were used to compare male and female patients. Analyses were further adjusted to age and disease duration. Results Of the 1510 SLE patients registered in Reuma.pt/LES, 122 (8%) are men. Male patients had later onset (39.4±20.6y vs 35.6±14.1y; p=0.005) and shorter disease duration (10.7±7.6y vs 14.1±9.0 y; p=0.0001). Mean current age, racial distribution and education level was similar in the two groups. Serositis, renal involvement and hemolytic anemia were more prevalent in men while, photosensitivity, alopecia, oral ulcers and arthritis were more commonly found in women (Table 1). Thyroid disease was more frequent in women (11.4 vs 2.3%). Cardiovascular risk factors had a similar distribution between these groups. Accumulated damage assessed by the SLICC damage index (SDI) and disease activity, assessed by SLEDAI-2K at last visit were similar in the two groups, with adjustment to age and disease duration. Antimalarial drugs and steroids were used more frequently in women. Table 1. Characteristics of SLE in male and female patients Men (n=122) Women (n=1389) P Photosensitivity 36 (32.4) 620 (49.9) <0.001* Alopecia 7 (6.7) 310 (26.8) <0.001* Oral ulcers 20 (17.7) 395 (31.9) 0.008* Arthritis 65 (57.0) 906 (72.5) <0.001* Serositis 36 (32.1) 236 (18.9) 0.001* Renal Involvement 49 (44.1) 344 (28.1) <0.001* Neurologic disorder 6 (5.4) 59 (4.8) 0.448 Hemolytic anemia 18 (16.4) 122 (9.8) 0.031* Anti-dsDNA positivity 96 (84.96) 929 (74.7) 0.020 Anti-SSA positivity 15 (27.8) 688 (39.1) 0.064 SLEDAI-2K 2.3±3.0 2.6±3.1 0.650 SLICC 0.82±1.3 0.71±1.22 0.126 * Statistically significant differences, adjusted to age and disease duration. Conclusions Male patients with SLE are older at disease onset and present a distinct phenotype with less cutaneous, mucous membranes and articular manifestations. However, disease outcome evaluated by the SDI is comparable in men and women, which is in line with observations from other European cohorts. The acknowledgement of the effect of gender on disease manifestations may help physicians in the timely introduction of an appropriate care. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4615

beta-thalassemia intermedia in a Brazilian patient with - 101(C > T) and codon 39 (C > T) mutations

CONTEXT We verified molecular alterations in a 72-year-old Brazilian male patient with a clinical course of homozygous beta-thalassemia intermedia, who had undergone splenectomy and was surviving without regular blood transfusions. The blood cell count revealed microcytic and hypochromic anemia (hemoglobin = 6.5 g/dl, mean cell volume = 74 fl, mean cell hemoglobin = 24 pg) and hemoglobin electrophoresis showed fetal hemoglobin = 1.3%, hemoglobin A2 = 6.78% and hemoglobin A = 79.4%. OBJECTIVE To identify mutations in a patient with the symptoms of beta-thalassemia intermedia. DESIGN Molecular inquiry into the mutations possibly responsible for the clinical picture described. SETTING The structural molecular biology and genetic engineering center of the Universidade Estadual de Campinas, Campinas, Brazil. PROCEDURES DNA extraction was performed on the patients blood samples. The polymerase chain reaction (PCR) was done using five specific primers that amplified exons and the promoter region of the beta globin gene. The samples were sequenced and then analyzed via computer programs. RESULTS Two mutations that cause the disease were found: -101 (C > T) and codon 39 (C > T). CONCLUSIONS This case represents the first description of -101 (C > T) mutation in a Brazilian population and it is associated with a benign clinical course.