Unravelling the proteomic profile of rice meiocytes during early meiosis

Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier.This research was supported by grants ERC-StG-243118 from the FP7 and The European Regional Development Fund (FEDER) from the European Union.Peer Reviewe

Novel Bread Wheat Lines Enriched in Carotenoids Carrying Hordeum chilense Chromosome Arms in the ph1b Background

The use of crop wild relative species to improve major crops performance is well established. Hordeum chilense has a high potential as a genetic donor to increase the carotenoid content of wheat. Crosses between the 7Hch H. chilense substitution lines in wheat and the wheat pairing homoeologous1b (ph1b) mutant allowed the development of wheat-H. chilense translocation lines for both 7Hchα and 7Hchβ chromosome arms in the wheat background. These translocation lines were characterized by in situ hybridization and using molecular markers. In addition, reverse phase chromatography (HPLC) analysis was carried out to evaluate the carotenoid content and both 7Hchα∙7AL and 7AS∙7Hchβ disomic translocation lines. The carotenoid content in 7Hchα∙7AL and 7AS∙7Hchβ disomic translocation lines was higher than the wheat-7Hch addition line and double amount of carotenoids than the wheat itself. A proteomic analysis confirmed that the presence of chromosome 7Hch introgressions in wheat scarcely altered the proteomic profile of the wheat flour. The Psy1 (Phytoene Synthase1) gene, which is the first committed step in the carotenoid biosynthetic pathway, was also cytogenetically mapped on the 7Hchα chromosome arm. These new wheat-H. chilense translocation lines can be used as a powerful tool in wheat breeding programs to enrich the diet in bioactive compounds.This work was supported by the ERC Starting Grant 243118 (http://www.ias.csic.es/ercstg-superwheatcrops/).Peer reviewe

Identification of a GCC transcription factor responding to fruit colour change events in citrus through the transcriptomic analyses of two mutants

14 páginas, 6 figuras, 3 tablas.[Background]: External ripening in Citrus fruits is morphologically characterized by a colour shift from green to orange due to the degradation of chlorophylls and the accumulation of carotenoid pigments. Although numerous genes coding for enzymes involved in such biochemical pathways have been identified, the molecular control of this process has been scarcely studied. In this work we used the Citrus clementina mutants 39B3 and 39E7, showing delayed colour break, to isolate genes potentially related to the regulation of peel ripening and its physiological or biochemical effects. [Results]: Pigment analyses revealed different profiles of carotenoid and chlorophyll modification in 39B3 and 39E7 mutants. Flavedo from 39B3 fruits showed an overall delay in carotenoid accumulation and chlorophyll degradation, while the flavedo of 39E7 was devoid of the apocarotenoid β-citraurin among other carotenoid alterations. A Citrus microarray containing about 20,000 cDNA fragments was used to identify genes that were differentially expressed during colour change in the flavedo of 39B3 and 39E7 mutants respect to the parental variety. The results highlighted 73 and 90 genes that were respectively up- and down-regulated in both mutants. CcGCC1 gene, coding for a GCC type transcriptional factor, was found to be down-regulated. CcGCC1 expression was strongly induced at the onset of colour change in the flavedo of parental clementine fruit. Moreover, treatment of fruits with gibberellins, a retardant of external ripening, delayed both colour break and CcGCC1 overexpression. [Conclusions]: In this work, the citrus fruit ripening mutants 39B3 and 39E7 have been characterized at the phenotypic, biochemical and transcriptomic level. A defective synthesis of the apocarotenoid β-citraurin has been proposed to cause the yellowish colour of fully ripe 39E7 flavedo. The analyses of the mutant transcriptomes revealed that colour change during peel ripening was strongly associated with a major mobilization of mineral elements and with other previously known metabolic and photosynthetic changes. The expression of CcGCC1 was associated with peel ripening since CcGCC1 down-regulation correlated with a delay in colour break induced by genetic, developmental and hormonal causes.Work was supported by grants AGL2007-65437-C04-01/AGR (Centro de Genómica) and AGL2009-11558 (L. Zacarías and M. J. Rodrigo) from the Ministerio de Educación y Ciencia of Spain.Peer reviewe

Involvement of ethylene in color changes and carotenoid biosynthesis in loquat fruit (Eriobotrya japonica Lindl. cv. Algerie)

[EN] In loquat (Eriobotrya japonica Lindl cv. Algerie) fruit, despite the non-climacteric ripening behaviour, evidence suggest that ethylene may participate in the regulation of several ripening- and postharvest-related processes. Color changes and carotenoid profile were analyzed in fruit at three developmental stages (breaker, yellow and colored fruits). At early stages, the fruit peel contained phytoene, phytofluene and other typical chloroplastic carotenoids that decreased during ripening, to accumulate ß-carotene, violaxanthin and ß-cryptoxanthin in mature fruits. In the pulp, carotenoid concentration increased during ripening to become predominant phytoene, followed by ß-carotene and ß-cryptoxanthin. Expression of the carotenoid biosynthetic genes (PSY, PDS, ZDS, CYCB and BCH) was downregulated in the peel during maturation, but increased in the pulp with the exception of BCH. The involvement of ethylene in the regulation of pigmentation was further evaluated by treating fruits at the three ripening stages with ethylene or its action inhibitor 1-MCP. At breaker fruit, ethylene accelerated and 1-MCP delayed fruit coloration, but the effect was progressively lost as fruit matured. Ethylene and 1-MCP produced different changes in carotenoids content and gene expression in peel and pulp. Application of ethylene enhanced ß-carotene content in both tissues whereas ß-cryptoxanthin was only stimulated in the pulp. 1-MCP suppressed these changes in carotenoid composition in the pulp but had little effect in the peel. A differential transcriptional level the pulp was more responsive to downregulated gene expression than the peel. Collectively, results indicate that: 1) ethylene is involved in the regulation of pigmentation and carotenoid biosynthesis in loquat fruits, 2) a differential regulation of carotenoid biosynthesis and response to ethylene appear to operate in the peel and the pulp, and 3) ß-carotene hydroxylase (BCH) is a key step in the regulation of carotenoid content and composition in both tissues of loquat fruit.Dr. E. Alos was recipient a post-doctoral contract JAE-Doc-CSIC (Fondo Social Europeo). The financial support of the research grants FP7-PEOPLE-2011-CIG-2011-303652 (Marie Curie Actions, European Union), AGL-2015-70218 (Ministerio Economia y Competitividad, Spain), GV/2012/036 (GeneralitatValenciana, Spain) and PROMETEO-II 2014/27 (Generalitat Valenciana) are gratefully acknowledged. MJR and LZ are members of Eurocaroten (COST_Action CA15136) and CaRed (Spanish Carotenoid Network, BIO2015-71703-REDT and BIO2017-90877-REDT).Alós, E.; Martinez Fuentes, A.; Reig Valor, C.; Mesejo Conejos, C.; Zacarias, L.; Agustí Fonfría, M.; Rodrigo-Esteve, MJ. (2019). Involvement of ethylene in color changes and carotenoid biosynthesis in loquat fruit (Eriobotrya japonica Lindl. cv. Algerie). Postharvest Biology and Technology. 149:129-138. https://doi.org/10.1016/j.postharvbio.2018.11.022S12913814

Analysis of 13000 unique Citrus clusters associated with fruit quality, production and salinity tolerance

BACKGROUND: Improvement of Citrus, the most economically important fruit crop in the world, is extremely slow and inherently costly because of the long-term nature of tree breeding and an unusual combination of reproductive characteristics. Aside from disease resistance, major commercial traits in Citrus are improved fruit quality, higher yield and tolerance to environmental stresses, especially salinity. RESULTS: A normalized full length and 9 standard cDNA libraries were generated, representing particular treatments and tissues from selected varieties (Citrus clementina and C. sinensis) and rootstocks (C. reshni, and C. sinenis × Poncirus trifoliata) differing in fruit quality, resistance to abscission, and tolerance to salinity. The goal of this work was to provide a large expressed sequence tag (EST) collection enriched with transcripts related to these well appreciated agronomical traits. Towards this end, more than 54000 ESTs derived from these libraries were analyzed and annotated. Assembly of 52626 useful sequences generated 15664 putative transcription units distributed in 7120 contigs, and 8544 singletons. BLAST annotation produced significant hits for more than 80% of the hypothetical transcription units and suggested that 647 of these might be Citrus specific unigenes. The unigene set, composed of ~13000 putative different transcripts, including more than 5000 novel Citrus genes, was assigned with putative functions based on similarity, GO annotations and protein domains CONCLUSION: Comparative genomics with Arabidopsis revealed the presence of putative conserved orthologs and single copy genes in Citrus and also the occurrence of both gene duplication events and increased number of genes for specific pathways. In addition, phylogenetic analysis performed on the ammonium transporter family and glycosyl transferase family 20 suggested the existence of Citrus paralogs. Analysis of the Citrus gene space showed that the most important metabolic pathways known to affect fruit quality were represented in the unigene set. Overall, the similarity analyses indicated that the sequences of the genes belonging to these varieties and rootstocks were essentially identical, suggesting that the differential behaviour of these species cannot be attributed to major sequence divergences. This Citrus EST assembly contributes both crucial information to discover genes of agronomical interest and tools for genetic and genomic analyses, such as the development of new markers and microarrays

Effect of 7Hch Hordeum chilense Chromosome Introgressions on the Wheat Endosperm Proteomic Profile

Hordeum chilense is an excellent genetic resource for wheat breeding due to its potential to improve breadmaking quality and nutritional value and provide resistance to some biotic and abiotic stresses. Hexaploid wheat lines carrying chromosome 7Hch introgressions, namely, chromosome additions of the whole chromosome 7Hch or the 7Hchα or the 7Hchβ chromosome arms, and chromosome substitutions of the homeologous chromosomes 7A, 7B, or 7D by chromosome 7Hch were compared by 2D-PAGE analysis to study the effect of these alien introgressions on the wheat endosperm proteome. The addition of the 7Hchα chromosome arm did not alter the profile of most glutenins and gliadins, but showed higher quantities of puroindolines and lower xylanase inhibitors, which might improve also resistance to plant pathogens. On the other hand, (7A)7Hch or (7D)7Hch substitution lines showed enhanced avenin-like b proteins and triticin levels but reduced puroindolines, which could be desirable to improve dough properties and nutritional value and increase kernel hardness in wheat.This research was supported by Grant ERC-StG-243118 from the FP7 and The European Regional Development Fund (FEDER) from the European Union.Peer reviewe

Effect of 7Hch Hordeum chilense Chromosome Introgressions on the Wheat Endosperm Proteomic Profile

Hordeum chilense is an excellent genetic resource for wheat breeding due to its potential to improve breadmaking quality and nutritional value and provide resistance to some biotic and abiotic stresses. Hexaploid wheat lines carrying chromosome 7Hch introgressions, namely, chromosome additions of the whole chromosome 7Hch or the 7Hchα or the 7Hchβ chromosome arms, and chromosome substitutions of the homeologous chromosomes 7A, 7B, or 7D by chromosome 7Hch were compared by 2D-PAGE analysis to study the effect of these alien introgressions on the wheat endosperm proteome. The addition of the 7Hchα chromosome arm did not alter the profile of most glutenins and gliadins, but showed higher quantities of puroindolines and lower xylanase inhibitors, which might improve also resistance to plant pathogens. On the other hand, (7A)7Hch or (7D)7Hch substitution lines showed enhanced avenin-like b proteins and triticin levels but reduced puroindolines, which could be desirable to improve dough properties and nutritional value and increase kernel hardness in wheat.This research was supported by Grant ERC-StG-243118 from the FP7 and The European Regional Development Fund (FEDER) from the European Union.Peer reviewe

Light avoidance reduces ascorbic acid accumulation in the peel of Citrus fruit

Citrus fruits are highly consumed worldwide and represent one of the most important sources of ascorbic acid (AsA). However, information about the molecular mechanisms regulating AsA accumulation in Citrus fruit and the effects of environmental factors is scarce. In this study we have investigated the effect of fruit shading on AsA content and the expression of AsA biosynthetic, degrading and recycling genes in fruits of different Citrus species. Immature-green fruits were covered at the end of the cell enlargement phase and AsA concentration in the flavedo declined and remained at low levels as compared with light-exposed fruits. Fruit shading marginally altered the expression of genes from the l-galactose pathway and this effect was variable in the four Citrus species. However, specific isoforms (GalUR8 or GalUR12) from the l-galacturonic acid pathway were significantly repressed paralleling the reduction in AsA concentration. No significant effect of shading was detected in transcription of genes of the myo-inositol and l-gulose pathways as well as recycling and degradation. Collectively, results indicate that light avoidance inhibited accumulation of AsA in the flavedo of Citrus fruits and suggest that the l-galacturonic acid pathway has a relevant contribution to AsA content in this tissue.This work was supported by research grants FP7-PEOPLE-2011-CIG-303652 (Marie Curie Actions, European Union), Proyectos de I + D para Grupos de Investigación Emergentes GV/2012/036 (Generalitat Valenciana, Spain) and AGL 2012-34573 (CICyT, Ministerio de Educación y Ciencia, Spain). Financial support by PROMETEOII/2014/027 (Generalitat Valenciana) is also acknowledged. We acknowledge the Citrus Germplasm Bank for experimental material and the technical assistance of Amparo Beneyto. JL was recipient of a JAE-Predoc (CSIC-FSE) pre-doctoral contract and EA was recipient of a JAE-doc (CSIC-FSE) contract.Peer reviewe

Pseudomonas fluorescens PICF7 displays an endophytic lifestyle in cultivated cereals and enhances yield in barley

Pseudomonas fluorescens PICF7, an indigenous inhabitant of olive roots, displays an endophytic lifestyle in this woody crop and exerts biocontrol against the fungal phytopathogen Verticillium dahliae. Here we report microscopy evidence that the strain PICF7 is also able to colonize and persist on or in wheat and barley root tissues. Root colonization of both cereal species followed a similar pattern to that previously reported in olive, including inner colonization of the root hairs. This demonstrates that strain PICF7 can colonize root systems of distant botanical species. Barley plants germinated from PICF7-treated seeds showed enhanced vegetative growth. Moreover, significant increases in the number of grains (up to 19.5%) and grain weight (up to 20.5%) per plant were scored in this species. In contrast, growth and yield were not significantly affected in wheat plants by the presence of PICF7. Proteomics analysis of the root systems revealed that different proteins were exclusively found depending on the presence or absence of PICF7 and only one protein with hydrogen ion transmembrane transporter activity was exclusively found in both PICF7-inoculated barley and wheat plants but not in the controls.This work was supported by grants from and (Convocatoria Proyectos de Excelencia from Junta de Andalucía, Spain), both co-funded by from the European Union (UE).Peer reviewe

Ascorbic acid content and transcriptional profiling of genes involved in its metabolism during development of petals, leaves, and fruits of orange (Citrus sinensis cv. valencia late)

Citrus fruit is one of the most important contributors to the ascorbic acid (AsA) intake in humans. Here, we report a comparative analysis of AsA content and transcriptional changes of genes related to its metabolism during development of petals, leaves and fruits of Valencia Late oranges (Citrus sinensis). Petals of close flowers and at anthesis contained the highest concentration of AsA. In fruits, AsA content in the flavedo reached a maximum at color break, whereas the pulp accumulated lower levels and experienced minor fluctuations during development. AsA levels in leaves were similar to those in the flavedo at breaker stage. The transcriptional profiling of AsA biosynthetic, degradation, and recycling genes revealed a complex and specific interplay of the different pathways for each tissue. The D-galacturonic acid pathway appeared to be relevant in petals, whereas in leaves the L-galactose pathway (GGP and GME) also contributed to AsA accumulation. In the flavedo, AsA content was positively correlated with the expression of GGP of the L-galactose pathway and negatively with DHAR1 gene of the recycling pathway. In the pulp, AsA appeared to be mainly controlled by the coordination among the D-galacturonic acid pathway and the MIOX and GalDH genes. Analysis of the promoters of AsA metabolism genes revealed a number of cis-acting elements related to developmental signals, but their functionalities remain to be investigated.This work was supported by research grants FP7-PEOPLE-2011-CIG-2011-303652 (Marie Curie Actions, European Union), Proyectos de I+D para Grupos de Investigación Emergentes GV/2012/036 (Generalitat Valenciana, Spain). The financial support of the projects AGL2015-70218 (Ministerio Economía y Competitividad, Spain) and PROMETEOII/2014/027 (Generalitat Valenciana) are also acknowledged. Enriqueta Alós was recipient of a JAE-Doc (CSIC) post-doctoral contract that was co-funded by Fondo Social Europeo de Desarrollo Regional (FEDER).Peer reviewe