HPLC-MS/MS Analyses Show That the Near-Starchless aps1 and pgm Leaves Accumulate Wild Type Levels of ADPglucose: Further Evidence for the Occurrence of Important ADPglucose Biosynthetic Pathway(s) Alternative to the pPGI-pPGM-AGP Pathway

In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.This research was partially supported by the grants [BIO2010-18239] from the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) and [IIM010491.RI1] from the Government of Navarra, and by Iden Biotechnology. This research was also supported by Scientific Research on Innovative Areas [22114507] and Grants-in-Aid for Scientific Research (B) [22380186] from the Ministry of Education, Culture, Sports, Science and Technology, Japan.Peer Reviewe

Volatile compounds emitted by diverse phytopathogenic microorganisms promote plant growth and flowering through cytokinin action

Sánchez-López, Ángela María et al.It is known that volatile emissions from some beneficial rhizosphere microorganisms promote plant growth. Here we show that volatile compounds (VCs) emitted by phylogenetically diverse rhizosphere and non-rhizhosphere bacteria and fungi (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote growth and flowering of various plant species, including crops. In Arabidopsis plants exposed to VCs emitted by the phytopathogen Alternaria alternata, changes included enhancement of photosynthesis and accumulation of high levels of cytokinins (CKs) and sugars. Evidence obtained using transgenic Arabidopsis plants with altered CK status show that CKs play essential roles in this phenomenon, because growth and flowering responses to the VCs were reduced in mutants with CK-deficiency (35S:AtCKX1) or low receptor sensitivity (ahk2/3). Further, we demonstrate that the plant responses to fungal VCs are light-dependent. Transcriptomic analyses of Arabidopsis leaves exposed to A. alternata VCs revealed changes in the expression of light- and CK-responsive genes involved in photosynthesis, growth and flowering. Notably, many genes differentially expressed in plants treated with fungal VCs were also differentially expressed in plants exposed to VCs emitted by the plant growth promoting rhizobacterium Bacillus subtilis GB03, suggesting that plants react to microbial VCs through highly conserved regulatory mechanisms.This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) (grant numbers BIO2010-18239 and BIO2013- 49125-C2-1-P), the Government of Navarra (grant number IIM010491.RI1), the I-Link0939 project from the Ministerio de Economía y Competitividad, the Ministry of Education, Youth and Sports of the Czech Republic (Grant L01204 from the National Program of Sustainability) and Palacky University institutional support. AM S-L and P G-G gratefully acknowledge predoctoral fellowships from the Spanish Ministry of Science and Innovation. M B and G A acknowledge post-doctoral fellowships awarded by the Public University of Navarra.Peer reviewe

Spontaneous suppressor mutations can compensate the total absence of SpoT in Escherichia coli

Póster presentado en el XXIII Congreso Nacional de Microbiología (SEM), celebrado en Salamanca del 11 al 14 de julio de 2011.Peer Reviewe

Cell-free microbial culture filtrates as candidate biostimulants to enhance plant growth and yield and activate soil- and plant-associated beneficial microbiota

In this work we compiled information on current and emerging microbial-based fertilization practices, especially the use of cell-free microbial culture filtrates (CFs), to promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identified limitations to bring microbial CFs to the market as biostimulants. In nature, plants act as metaorganisms, hosting microorganisms that communicate with the plants by exchanging semiochemicals through the phytosphere. Such symbiotic interactions are of high importance not only for plant yield and quality, but also for functioning of the soil microbiota. One environmentally sustainable practice to increasing crop productivity and/or protecting plants from (a)biotic stresses while reducing the excessive and inappropriate application of agrochemicals is based on the use of inoculants of beneficial microorganisms. However, this technology has a number of limitations, including inconsistencies in the field, specific growth requirements and host compatibility. Beneficial microorganisms release diffusible substances that promote plant growth and enhance yield and stress tolerance. Recently, evidence has been provided that this capacity also extends to phytopathogens. Consistently, soil application of microbial cell-free culture filtrates (CFs) has been found to promote growth and enhance the yield of horticultural crops. Recent studies have shown that the response of plants to soil application of microbial CFs is associated with strong proliferation of the resident beneficial soil microbiota. Therefore, the use of microbial CFs to enhance both crop yield and stress tolerance, and to activate beneficial soil microbiota could be a safe, efficient and environmentally friendly approach to minimize shortfalls related to the technology of microbial inoculation. In this review, we compile information on microbial CFs and the main constituents (especially volatile compounds) that promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identify challenges and limitations for their use as biostimulants to bring them to the market and we propose remedial actions and give suggestions for future work.This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grant PID2019-104685GB-100)

Subcellular localization of enzymes in carbohydrate metabolism in the effort to improve production parameters

Trabajo presentado en el Olomouc Biotech 2011 Plant Biotechnology (Green for Good), celebrado en Olomouc (República Checa) del 19 al 22 de junio de 2011.Peer Reviewe

Microbial volatiles induced accumulation of exceptionally high levels of starch in leaves involves a photocontrolled process wherein starch synthases class III and IV and NTRC-mediated changes in redox status of plastidial enzymes play important roles

XIX Reunión de la Sociedad Española de Fisiología Vegetal y XII Congreso Hispano-Luso de Fisiología Vegetal, celebrado en Castelló de la Plana del 21 al 24 de junio de 2011.Peer Reviewe

Microbial volatile emissions promote accumulation of exceptionally high levels of starch in leaves in mono- and di-cotyledonous plants

Trabajo presentado en la X Reunión de Biología Molecular de plantas, celebrada en Valencia del 8 al 10 de julio de 2010.Peer Reviewe

Soil application of volatile compounds emitted by Alternaria alternata enhance yield of pepper plants cultured under greenhouse conditions

Póster presentado en el 2nd World Congress on the use of Biostimulants in Agriculture, celebrado en Florencia del 16 al 19 de noviembre de 2015.Peer Reviewe

Characterization of multiple SPS knockout mutants reveals redundant functions of the four Arabidopsis sucrose phosphate synthase isoforms in plant viability, and strongly indicates that enhanced respiration and accelerated starch turnover can alleviate the blockage of sucrose biosynthesis

We characterized multiple knock-out mutants of the four Arabidopsis sucrose phosphate synthase (SPSA1, SPSA2, SPSB and SPSC) isoforms. Despite their reduced SPS activity, spsa1/spsa2, spsa1/spsb, spsa2/spsb, spsa2/spsc, spsb/spsc, spsa1/spsa2/spsb and spsa2/spsb/spsc mutants displayed wild type (WT) vegetative and reproductive morphology, and showed WT photosynthetic capacity and respiration. In contrast, growth of rosettes, flowers and siliques of the spsa1/spsc and spsa1/spsa2/spsc mutants was reduced compared with WT plants. Furthermore, these plants displayed a high dark respiration phenotype. spsa1/spsb/spsc and spsa1/spsa2/spsb/spsc seeds poorly germinated and produced aberrant and sterile plants. Leaves of all viable sps mutants, except spsa1/spsc and spsa1/spsa2/spsc, accumulated WT levels of nonstructural carbohydrates. spsa1/spsc leaves possessed high levels of metabolic intermediates and activities of enzymes of the glycolytic and tricarboxylic acid cycle pathways, and accumulated high levels of metabolic intermediates of the nocturnal starch-to-sucrose conversion process, even under continuous light conditions. Results presented in this work show that SPS is essential for plant viability, reveal redundant functions of the four SPS isoforms in processes that are important for plant growth and nonstructural carbohydrate metabolism, and strongly indicate that accelerated starch turnover and enhanced respiration can alleviate the blockage of sucrose biosynthesis in spsa1/spsc leaves.This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) [grant numbers BIO2010-18239, BIO2013-49125-C2-1-P, BIO2008-02292 and BIO2011-28847-C02-02]. A.M.S-L. acknowledges a predoctoral fellowship from the Spanish Ministry of Science and Innovation. M.B. acknowledges a post-doctoral fellowship from the Public University of Navarra.Peer Reviewe