Improved Organic Fertilisers Made from Combinations of Compost, Biochar, and Anaerobic Digestate: Evaluation of Maize Growth and Soil Metrics

[EN] Treated bio-residues can be used as biostimulants in crops within the circular economy approach to reduce the use of traditional fertilisers. In this work, we optimised the combination rates for three types of treated bio-residues (compost, biochar, and anaerobic digestate (AD)) in two microcosm trials, one with a combination of compost and biochar and other with biochar and AD. The crop used was maize, and the variables analysed were plant growth, and soil chemical and biological properties. The combination of bio-residues improved plant growth and soil biological activity to a greater extent than one product alone; that is, compost and biochar performed better than compost alone and biochar, and AD performed better than biochar alone. However, while the concentration in the plant biomass of several essential nutrients for crops increased in the treatments with compost and biochar, and with biochar and AD, compared to the untreated controls, the nitrogen concentration was reduced. This was due to the competition for nitrogen between the plant and the soil microbiome, whose activity was activated. Due to the importance of nitrogen in plant growth, the increase in biomass production could be explained not only by the higher availability of other nutrients but also by the plant-growth-promoting activity exerted by the more active soil microbiome. Further research should focus on validating this hypothesis and unravelling the mechanisms involved. From the environmental site, the presence of biochar in the mixtures of organic residues reduced the soil nitrogen at risk of lixiviation and sequestered carbon, which partially compensated for the increased CO2 emissions because labile forms of carbon were present in the remaining organic residues.S

The Role of INAPERTURATE POLLEN1 as a Pollen Aperture Factor Is Conserved in the Basal Eudicot Eschscholzia californica (Papaveraceae)

This study was supported by the Spanish Ministry of Economy and Competitiveness (project CGL2015-70290-P to VS-S) and by the US National Science Foundation (MCB-1817835 to AD). IM-A was supported by a predoctoral grant (F.P.I. program) from the Spanish Government. BK was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project LTC20050) and DH was supported by Czech Science Foundation (project 21-15856S).Pollen grains show an enormous variety of aperture systems. What genes are involved in the aperture formation pathway and how conserved this pathway is in angiosperms remains largely unknown. INAPERTURATE POLLEN1 (INP1) encodes a protein of unknown function, essential for aperture formation in Arabidopsis, rice and maize. Yet, because INP1 sequences are quite divergent, it is unclear if their function is conserved across angiosperms. Here, we conducted a functional study of the INP1 ortholog from the basal eudicot Eschscholzia californica (EcINP1) using expression analyses, virus-induced gene silencing, pollen germination assay, and transcriptomics. We found that EcINP1 expression peaks at the tetrad stage of pollen development, consistent with its role in aperture formation, which occurs at that stage, and showed, via gene silencing, that the role of INP1 as an important aperture factor extends to basal eudicots. Using germination assays, we demonstrated that, in Eschscholzia, apertures are dispensable for pollen germination. Our comparative transcriptome analysis of wildtype and silenced plants identified over 900 differentially expressed genes, many of them potential candidates for the aperture pathway. Our study substantiates the importance of INP1 homologs for aperture formation across angiosperms and opens up new avenues for functional studies of other aperture candidate genes.Spanish Ministry of Economy and Competitiveness CGL2015-70290-PNational Science Foundation (NSF) MCB-1817835Spanish Government European CommissionMinistry of Education, Youth & Sports - Czech Republic LTC20050Grant Agency of the Czech Republic 21-15856

Combined application of N-fixing PGPB and rice straw mulch compensates N immobilization by straw, improving crop growth

[EN] Background Rice straw, often treated as waste, provides many benefits to crops when used as a mulch. However, straw degradation promotes nitrogen immobilisation due to its high C:N ratio, causing N competition between soil microorganisms and the crop. Currently, sustainable practices to remedy nitrogen immobilisation are hardly being implemented. In microcosm conditions we assessed whether the inoculation with N-fixers could offset the transient nitrogen deficiency caused by straw mulch, thereby harnessing the benefits of straw while mitigating its negative impact on nitrogen depletion and exerting a synergistic effect on crop growth. Results Inoculation with N-fixers increased the nitrogen content in the soil (the increase ranged from 14% up to 90% for NH4 + and from 20% to 60% for NO3 −) and, in most cases, also the nitrogen content in the plant (ranging from 10% to 15% increase), compared to the non-inoculated control. Therefore, inoculation would compensate for the lack of nitrogen caused by nitrogen immobilisation, and this resulted in an increased biomass production by the crop compared with the uninoculated control (the increase ranged from 25% to 85%). In addition, inoculation with N-fixers did not lead to a permanent change in the bacterial community composition, whereas straw addition increased the biodiversity of the soil microbiome. Conclusions The results obtained in microcosm conditions are a first indication that complementing straw mulching with the inoculation of N-fixers could avoid the transient N immobilisation produced during straw degradation. Thus, the benefits of the combination would be a yield increase, while improving the biodiversity of the soil microbiome, stabilising soil temperatures and increasing water soil content.SIThis work was supported by the Spanish Ministry of Science, Innovation and Universities (project RTC-2017-6249-2

Evolution and development of the pollen apertural system in basal eudicots: analysis of the INAPERTURATE POLLEN1 gene and identification of candidate genes determining apertural morphology

Esta tesis doctoral se desarrolla con el objetivo de estudiar la implicación de INP1 en la formación de aperturas en Eschscholzia californica, miembro de Papaveraceae. Por otro lado investigamos genes que posiblemente intervengan en la formación de las aperturas y en el determinismo de su forma. En el Capítulo 1 llevamos a cabo un estudio funcional de EcINP1 en Eschscholzia californica y proponemos genes candidatos a estar implicados en la formación de las aperturas. Analizamos la expresión temporal y espacial de EcINP1, validamos su función generando mutantes mediante silenciamiento génico inducido por virus (VIGS) y comparamos la tasa de germinación y el transcriptoma de plantas salvajes con el de mutantes inp1. EcINP1 presenta su máxima expresión en anteras en estadio de desarrollo del polen de tétradas y su papel como un factor esencial para la 11 formación de las aperturas es conservado. En Eschscholzia californica las aperturas son prescindibles para la germinación del polen. Además, encontramos 971 genes diferencialmente expresados (DEGs) entre plantas salvajes y EcINP1-mutantes, entre los que destacamos los que son potenciales candidatos a intervenir en el proceso de formación de las aperturas, como los homólogos en E. californica de NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1), D6 PROTEIN KINASE LIKE 3 (D6PKL3) y PROTEIN KINASE ASSOCIATED WITH BRX (PAX). Estos genes mostraron patrones de expresión coincidentes con los de EcINP1, que apoyan su implicación en la formación de las aperturas y/o posible interacción de sus productos con la proteína EcINP1. En el Capítulo 2 identificamos genes que podrían estar implicados en el determinismo de la forma de las aperturas. Secuenciamos el transcriptoma de cuatro especies de Papaveraceae, de las que tres no está disponible su genoma de referencia, por lo que generamos un ensamblaje de referencia de novo para cada una de ellas. Filtramos y anotamos los ensamblajes de novo generados. Comparamos el transcriptoma de dos especies con polen colpado con el de otras dos con polen porado y analizamos los DEGs entre ambos grupos. Encontramos 531 DEGs entre los cuales no se encontraba el gen ELMOD_E, el único descrito hasta el momento como un posible factor determinante para la forma de las aperturas (Zhou et al., 2021), por tanto parece que ELMOD_E no regula el cambio de colpo a poro en Papaveraceae. En cambio INP1 si se encontraba diferencialmente expresado. Por otro lado, entre los DEGs encontramos genes implicados en procesos que posiblemente intervienen en la formación de las aperturas, como los relacionados con la síntesis o degradación de la calosa, destacando el factor de transcripción DYSFUNCTIONAL TAPETUM 1 (DYT1) o con la organización de elementos del citoesqueleto.This doctoral thesis is developed with the aim of studying the involvement of INP1 in the formation of apertures in Eschscholzia californica, a member of Papaveraceae. On the other hand, we investigate genes that are possibly involved in the formation of the apertures and in the determinism of their shape. In Chapter 1 we perform a functional study of EcINP1 in Eschscholzia californica and propose candidate genes to be involved in the formation of the apertures. We analysed the temporal and spatial expression of EcINP1, validated its function by generating mutants through virus-induced gene silencing (VIGS) and compared the germination rate and transcriptome of wild-type plants with that of inp1 mutants. EcINP1 is maximally expressed in anthers at the tetrad stage of pollen development and its role as an essential factor for aperture formation is conserved. In Eschscholzia californica the apertures are dispensable for pollen germination. In addition, we found 971 differentially expressed genes (DEGs) between wild-type and 14 EcINP1-mutants, among which we highlight those that are potential candidates for involvement in the aperture formation process, such as the E. californica homologues of NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1), D6 PROTEIN KINASE LIKE 3 (D6PKL3) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX). These genes showed expression patterns coincident with those of EcINP1, supporting their involvement in the formation of the apertures and/or possible interaction of their products with the EcINP1 protein. In Chapter 2 we identified genes that could be involved in aperture shape determinism. We sequenced the transcriptome of four species of Papaveraceae, three of which do not have their reference genome available, so we generated a de novo reference assembly for each of them. We filter and annotate the generated de novo assemblies. We compared the transcriptome of two species with colpate pollen with that of two others with porate pollen and analysed the differentially expressed genes between the two groups. We found 531 DEGs among which ELMOD_E, the only gene described so far as a possible determinant of aperture shape (Zhou et al., 2021), was not found, so ELMOD_E may not be regulating the change from colpo to pore in Papaveraceae. In contrast, INP1 was found to be differentially expressed. On the other hand, among the DEGs we found genes implicated in processes possibly involved in aperture formation, such as genes related to the synthesis or degradation of callose, including the transcription factor DYSFUNCTIONAL TAPETUM 1 (DYT1) or with the organisation of cytoskeletal elements.Tesis Univ. Granada

Identification of candidate genes involved in the determinism of pollen grain aperture morphology by comparative transcriptome analysis in Papaveraceae

Supplementary Tables 3-6, 8-10: Table S3. Trinotate annotation report for Dactylicapnos torulosa. Annotation through blastx and blastp for predict transcript against Swissprot database. Table S4. Trinotate annotation report for Fumaria bracteosa. Annotation through blastx and blastp for predict transcript against Swissprot database. Table S5. Trinotate annotation report for Roemeria refracta. Annotation through blastx and blastp for predict transcript against Swissprot database. Table S6. Eschscholzia californica annotation through blastx against Swissprot database for transcripome transcript aligned to reference. Qseqid, query or source (e.g., gene) sequence id; seqid, subject or target (e.g., reference genome) sequence id; pident, percentage of identical matches; length, alignment length (sequence overlap); mismatch, number of mismatches; gapopen, number of gap openings; qstart, start of alignment in query; qend, end of alignment in query; sstart, start of alignment in subject; send, end of alignment in subject; evalue, expect value; bitscore, bit score. Table S8. Transcripts annotated as transcription factor through PlantTFDB. Table S9. Genes differentially expressed between colpate and porate species. Annotation through BLASTX searching against the SwissProt Database. Table S10. Functional classification of differentially expressed genes (DEGs) between colpate and porate species using Blast2GO software. Sheet 1, Blast2GO output file with annotations and functional classification for each DEG. Sheet2, summary of the number of DEGs for each functional annotation within the three different functional categories, note that each DEG can have several annotation possibilities

Identification of Candidate Genes Involved in the Determinism of Pollen Grain Aperture Morphology by Comparative Transcriptome Analysis in Papaveraceae

In the last decade, certain genes involved in pollen aperture formation have been discovered. However, those involved in pollen aperture shape remain largely unknown. In Arabidopsis, the interaction during the tetrad development stage of one member of the ELMOD protein family, ELMOD_E, with two others, MCR/ELMOD_B and ELMOD_A, can change the morphology of apertures from colpus (elongated) to pore (round). Here, comparative transcriptome analysis is used to identify candidate genes involved in the determination of pollen aperture morphology in Papaveraceae (order Ranunculales). Furthermore, the role of ELMOD genes in the genetic determinism of aperture shape was tested by comparative analysis of their expression levels using RNA-seq data and RT-qPCR. Two pairs of species belonging to two different subfamilies were used. Within each pair, one species has colpate pollen and the other porate (Fumarioideae—Dactylicapnos torulosa, 6-colpate, and Fumaria bracteosa, pantoporate; Papaveroideae—Eschsholzia californica, 5–7 colpate, and Roemeria refracta, 6-porate). The transcriptomes were obtained at the tetrad stage of pollen development. A total of 531 DEGs were found between the colpate and porate pollen species groups. The results from RNA-seq and RT-qPCR indicate that pollen aperture shape is not determined by the relative expression levels of ELMOD family genes in Papaveraceae. However, genes related to callose wall formation or cytoskeleton organisation were found, these processes being involved in pollen aperture formation. In addition, transcriptomes from anthers with pollen during the tetrad stage of three species (D. torulosa, R. refracta, and F. bracteosa) were obtained for the first time. These data will be available for further studies in the field of floral evolution and development