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

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

Systematic Production of Inactivating and NonInactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli

In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (DspoT) mutants obtained by transducing a DspoT allele from DrelADspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 DspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of DspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 DspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the DspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the DspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetaseFil: Montero, Manuel. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Rahimpour, Mehdi. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Viale, Alejandro Miguel. Gobierno de Navarra. Instituto de Agrobiotecnología; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Almagro, Goizeder. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Eydallin, Gustavo. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Sevilla, Angel. Universidad de Murcia; EspañaFil: Canovas, Manuel. Universidad de Murcia; EspañaFil: Bernal, Cristina. Universidad de Murcia; EspañaFil: Lozano, Ana Belen. Universidad de Murcia; EspañaFil: Muñoz, Francisco Jose. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Bora Fernandez, Edurne. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Bahaji, Abdellatif. Gobierno de Navarra. Instituto de Agrobiotecnología; EspañaFil: Mori, Hirotada. Nara Institute of Science and Technology. Graduate School of Biological Sciences; JapónFil: Codoñer, Francisco M.. Lifesequencing SL. Valencia; EspañaFil: Potueza Romeo, Javier. Gobierno de Navarra. Instituto de Agrobiotecnología; Españ

Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways

Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.A.F.C was funded by a contract from Ministerio Economía y Competitividad-MINECO, reference 20132RC947, Spain; I.R.A. is funded by a PhD studentship from Universidad Pública de Navarra, Spain; J.M. was funded by PhD studentship BES-2013-062644 from MINECO; S.M. is funded by a postdoctoral contract from CIBERES; L.C. was funded by a contract from MINECO, reference CS_NAV_IDAB_005, Spain; T.L.B. is the recipient of a PhD fellowship funded by the Department for Employment and Learning (Northern Ireland, UK). This work has been funded by grants from MINECO SAF2012-31166 and SAF2015-66520-R, from Health Department, Regional Govern from Navarra, Spain, reference 03/2016, and from SEPAR 31/2015 to J.G.; and by grant from MINECO DPI2015-64221 to COdS. CIBER is an initiative from Instituto de Salud Carlos III (ISCIII), Madrid, Spain

Reply to Smith et al.: No evidence to challenge the current paradigm on starch and cellulose biosynthesis involving sucrose synthase activity

In our opinion, no pressing biological evidence has been presented by Barratt et al. to challenge the current paradigm on cellulose and starch metabolism involving SUS activity. In this context, we must emphasize that Angeles-Núñez and Tiessen have shown that SUS2 and SUS3 are required for channeling carbon toward ADP-glucose and starch in Arabidopsis seeds.This work was supported by Grant BIO2010-18239 from the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain)

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

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