BUSCADOR RECOLECTA

[EN] Zea mays Brittle1-1 (ZmBT1-1) is an essential component of the starch biosynthetic machinery in maize endosperms, enabling ADPglucose transport from cytosol to amyloplast in exchange for AMP or ADP. Although ZmBT1-1 has been long considered to be an amyloplast-specific marker, evidence has been provided that ZmBT1-1 is dually localized to plastids and mitochondria (Bahaji et al., 2011b). The mitochondrial localization of ZmBT1-1 suggested that this protein may have as-yet unidentified function(s). To understand the mitochondrial ZmBT1-1 function(s), we produced and characterized transgenic Zmbt1-1 plants expressing ZmBT1-1 delivered specifically to mitochondria. Metabolic and differential proteomic analyses showed down-regulation of sucrose synthase (SuSy)-mediated channeling of sucrose into starch metabolism, and up-regulation of the conversion of sucrose breakdown products generated by cell wall invertase (CWI) into ethanol and alanine, in Zmbt1-1 endosperms compared to wild-type. Electron microscopic analyses of Zmbt1-1 endosperm cells showed gross alterations in the mitochondrial ultrastructure. Notably, the protein expression pattern, metabolic profile, and aberrant mitochondrial ultrastructure of Zmbt1-1 endosperms were rescued by delivering ZmBT1-1 specifically to mitochondria. Results presented here provide evidence that the reduced starch content in Zmbt1-1 endosperms is at least partly due to (i) mitochondrial dysfunction, (ii) enhanced CWI-mediated channeling of sucrose into ethanol and alanine metabolism, and (iii) reduced SuSy-mediated channeling of sucrose into starch metabolism due to the lack of mitochondrial ZmBT1-1. Our results also strongly indicate that (a) mitochondrial ZmBT1-1 is an important determinant of the metabolic fate of sucrose entering the endosperm cells, and (b) plastidic ZmBT1-1 is not the sole ADPglucose transporter in maize endosperm amyloplasts. The possible involvement of mitochondrial ZmBT1-1 in exchange between intramitochondrial AMP and cytosolic ADP is discussed., This research was partially supported by the grants BIO2010-18239, BI2013-49125-C2-2-P and BIO2016-78747-P from the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) and by the ERDF project Plants as a tool for sustainable global development (No. CZ.02.1.01/0.0/0.0/16_019/0000827).

ADP-glucose is the precursor of glycogen biosynthesis in bacteria, and a compound abundant in the starchy plant organs ingested by many mammals. Here we show that the enteric species Escherichia coli is capable of scavenging exogenous ADP-glucose for use as a glycosyl donor in glycogen biosynthesis and feed the adenine nucleotide pool. To unravel the molecular mechanisms involved in this process, we screened the E. coli single-gene deletion mutants of the Keio collection for glycogen content in ADP-glucose-containing culture medium. In comparison to wild-type (WT) cells, individual ∆nupC and ∆nupG mutants lacking the cAMP/CRP responsive inner-membrane nucleoside transporters NupC and NupG displayed reduced glycogen contents and slow ADP-glucose incorporation. In concordance, ∆cya and ∆crp mutants accumulated low levels of glycogen and slowly incorporated ADP-glucose. Two-thirds of the glycogen-excess mutants identified during screening lacked functions that underlie envelope biogenesis and integrity, including the RpoE specific RseA anti-sigma factor. These mutants exhibited higher ADP-glucose uptake than WT cells. The incorporation of either ∆crp, ∆nupG or ∆nupC null alleles sharply reduced the ADP-glucose incorporation and glycogen content initially witnessed in ∆rseA cells. Overall, the data showed that E. coli incorporates extracellular ADP-glucose through a cAMP/CRP-regulated process involving the NupC and NupG nucleoside transporters that is facilitated under envelope stress conditions., This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo
de Desarrollo Regional (Spain) (grant numbers BIO2013-49125-C2-1-P and BIO2016-78747-P). A.M.V. is a
Career Researcher of the Consejo Nacional de Investigaciones Cientificas y Técnicas de Argentina (CONICET)
and Professor of Micribiology at the National University of Rosario (U.N.R., Argentina). A.M.V. expresses his
gratitude to the Ministerio de Educación y Cultura, the Consejo Superior de Investigaciones Científicas, and the
Public University of Navarra for financial support.

The plastid-localized phosphoglucose isomerase isoform PGI1 is an important determinant of growth in Arabidopsis thaliana, likely due to its involvement in the biosynthesis of plastidial isoprenoid-derived hormones. Here, we investigated whether PGI1 also influences seed yields. PGI1 is strongly expressed in maturing seed embryos and vascular tissues. PGI1-null pgi1-2 plants had ∼60% lower seed yields than wild-type plants, with reduced numbers of inflorescences and thus fewer siliques and seeds per plant. These traits were associated with low bioactive gibberellin (GA) contents. Accordingly, wild-type phe-notypes were restored by exogenous GA application. pgi1-2 seeds were lighter and accumulated ∼50% less fatty acids (FAs) and ∼35% less protein than wild-type seeds. Seeds of cytokinin-deficient plants overexpressing CYTOKININ OXIDASE/DE-HYDROGENASE1 (35S:AtCKX1) and GA-deficient ga20ox1 ga20ox2 mutants did not accumulate low levels of FAs, and exogenous application of the cytokinin 6-benzylaminopurine and GAs did not rescue the reduced weight and FA content of pgi1-2 seeds. Seeds from reciprocal crosses between pgi1-2 and wild-type plants accumulated wild-type levels of FAs and proteins. Therefore, PGI1 is an important determinant of Arabidopsis seed yield due to its involvement in two processes: GA-mediated reproductive development and the metabolic conversion of plastidial glucose-6-phosphate to storage reserves in the embryo., This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) (grant numbers BIO2013-49125-C2-1-P and BIO2016-78747-P), the Ministry of Education, Youth and Sports of the Czech Republic (grant LO1204 from the National Program of Sustainability I), the Government of Navarra (ref. P1004 PROMEBIO), the Università degli Studi di Milano (UNIMI-RTD-A, Linea2-DBS 2017-2018) and the H2020-MSCA-RISE project (ExpoSeed GA-691109).

A 'box-in-box' cocultivation system was used to investigate plant responses to microbial volatile compounds (VCs) and to evaluate the contributions of organic and inorganic VCs (VOCs and VICs, respectively) to these responses. Arabidopsis plants were exposed to VCs emitted by adjacent Alternaria alternata and Penicillium aurantiogriseum cultures, with and without charcoal filtration. No VOCs were detected in the headspace of growth chambers containing fungal cultures with charcoal filters. However, these growth chambers exhibited elevated CO2 and bioactive CO and NO headspace concentrations. Independently of charcoal filtration, VCs from both fungal phytopathogens promoted growth and distinct developmental changes. Plants cultured at CO2 levels observed in growth boxes containing fungal cultures were identical to those cultured at ambient CO2. Plants exposed to charcoal-filtered fungal VCs, nonfiltered VCs, or superelevated CO2 levels exhibited transcriptional changes resembling those induced by increased irradiance. Thus, in the 'box-in-box'' system, (a) fungal VICs other than CO2 and/or VOCs not detected by our analytical systems strongly influence the plants' responses to fungal VCs, (b) different microorganisms release VCs with distinct action potentials, (c) transcriptional changes in VC-exposed plants are mainly due to enhanced photosynthesis signaling, and (d) regulation of some plant responses to fungal VCs is primarily posttranscriptional., Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional, Grant/Award Numbers: BIO2013-49125-C2-1-P, BIO2016-78747-P and BIO2013-49125-C2-1-; Government of Navarra, Grant/Award Numbers: P1044 AGROESTI and P1004 PROMEBIO

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1–NPP6. Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)–Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2–GFP and NPP6–GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER–Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs., This research was supported by the Japan Society for the Promotion of Sciences [KAKENHI Grants-in-Aid for Scientific Research (A) (15H02486) to T.M.]; the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) [grants BIO2010-18239 and BIO2013-49125-C2-1-P]; the Government of Navarra [grant IIQ14067.RI1].

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs., 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-C2-1-P] and by the Government of Navarra [grant number IIM010491.RI1], the Ministry of Education, Youth and Sports of the Czech Republich [Grant L01204 from the National Program of Sustainability] and the European Social Fund and the state budget of the Czech Republic [project POST-UP, reg. No. CZ.1.07/2.3.00/30.0004]. AMS-L acknowledges a predoctoral fellowship from the Spanish Ministry of Science and Innovation. MB acknowledges a post-doctoral fellowship from the Public University of Navarra.

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.

El crecimiento y el desarrollo de las plantas están afectados por microorganismos presentes en la filosfera, la rizosfera y/o la endosfera. En la fase de precolonización, antes de que se establezca un contacto físico con la planta, bacterias y hongos beneficiosos sintetizan una gran cantidad de sustancias que fomentan el crecimiento de la planta. Tales sustancias fomentan además la formación de raíces laterales y el crecimiento de pelos radiculares, mejorando así la capacidad exploratoria de las raíces para obtener agua y minerales del suelo y aumentando la superficie de la raíz y, por tanto, su predisposición para ser colonizada e infectada. Estos microorganismos también emiten un gran número de compuestos volátiles (VCs), con masa molecular inferior a 300 Da y alta presión de vapor, que promueven el crecimiento de la planta y la fotosíntesis y modulan la arquitectura de la raíz. Recientemente, el grupo de investigación en el que he realizado mis investigaciones demostró que esta capacidad no está restringida a microorganismos beneficiosos, sino que también se extiende a patógenos. Esta tesis doctoral se ha llevado a cabo con la doble intención de identificar la naturaleza de los VCs microbianos con propiedades bioestimulantes y profundizar en el conocimiento de los mecanismos implicados en la respuesta de las raíces a los VCs emitidos por microorganismos patógenos., Plants’ growth and development are influenced by microorganisms occurring either aboveground in the phyllosphere, underground in the rhizosphere and/or in the endosphere inside the vascular transport system and apoplastic space. In the precolonization phase, before direct contact with plants occurs, beneficial bacteria and fungi synthesize a multitude of substances that promote growth and cause massive lateral root formation and enhanced root hair growth, thus improving the root´s exploratory capacity for water and minerals and predisposing plants for microbial colonization and infection. These microorganisms also emit a large number of volatile compounds (VCs) with molecular masses of less than 300 Da and high vapour pressure that promote growth and photosynthesis, and modulate root system architecture in both host and non-host plants. We have recently demonstrated that this capacity is not restricted to beneficial microbes, but also extends to phytopathogens. This thesis has been focused on investigating the nature of VCs involved in plant´s response, and deepen in the knowledge of the mechanisms responsible for that response in roots., Beca predoctoral FPI (referencia BES-2014-068741) del Ministerio de Ciencia, Innovación y Universidades. Proyectos BIO2013-49125-C2-1-P y BIO2016-78747-P de la Comisión Interministerial de Ciencia y Tecnología., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Incluye material complementario, Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do
not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in
leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic
phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of
its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-
Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to
microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in
the presence or absence of VCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen
promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2
leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote
global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the
observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic
microorganisms by triggering pPGI-independent mechanisms., This work was supported by the Comisión Interministerial de
Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional,
Spain (grant nos. BIO2010–18239 and BIO2013–49125–C2–1–P), by
the Government of Navarra (grant no. IIM010491.RI1), by the
I-Link0939 project from the Ministerio de Economía y Competitividad,
by the Ministry of Education, Youth, and Sports of the Czech
Republic (grant no. LO1204 from the National Program of Sustainability),
by Palacky University institutional support, by predoctoral
fellowships from the Spanish Ministry of Science and Innovation (to
A.M.S.-L. and P.G.-G.), and by postdoctoral fellowships from the
Public University of Navarra (to M.B. and G.A.).

Although there is a great wealth of data supporting the occurrence of simultaneous synthesis
and breakdown of storage carbohydrate in many organisms, previous 13CO2 pulse-chase
based studies indicated that starch degradation does not operate in illuminated Arabidopsis
leaves. Here we show that leaves of gwd, sex4, bam4, bam1/bam3 and amy3/isa3/lda starch
breakdown mutants accumulate higher levels of starch than wild type (WT) leaves when cultured
under continuous light (CL) conditions. We also show that leaves of CL grown dpe1
plants impaired in the plastidic disproportionating enzyme accumulate higher levels of maltotriose
than WT leaves, the overall data providing evidence for the occurrence of extensive
starch degradation in illuminated leaves. Moreover, we show that leaves of CL grown mex1/
pglct plants impaired in the chloroplastic maltose and glucose transporters display a severe
dwarf phenotype and accumulate high levels of maltose, strongly indicating that the MEX1
and pGlcT transporters are involved in the export of starch breakdown products to the cytosol
to support growth during illumination. To investigate whether starch breakdown products can
be recycled back to starch during illumination through a mechanism involving ADP-glucose
pyrophosphorylase (AGP) we conducted kinetic analyses of the stable isotope carbon composition
(δ13C) in starch of leaves of 13CO2 pulsed-chased WT and AGP lacking aps1 plants.
Notably, the rate of increase of δ13C in starch of aps1 leaves during the pulse was exceedingly
higher than that of WT leaves. Furthermore, δ13C decline in starch of aps1 leaves during
the chase was much faster than that of WT leaves, which provides strong evidence for the
occurrence of AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis
leaves., Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) Grant numbers: BIO2010-18239 and BIO2013-49125-C2-1-P.

Incluye material complementario, 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 LO1204 from the National Program of Sustainability) and Palacky University institutional support. A.M. 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.