Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis

[EN] Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plant Arabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, including ROF2, which encodes a peptidyl-prolyl cis-trans isomerase of the FK506-binding protein class. Loss of function of ROF2, and especially double mutation of ROF2 and the closely related gene ROF1, results in acid sensitivity. Over-expression of ROF2 confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H+-ATPase) is indirect: over-expression of ROF2 activates K+ uptake, causing depolarization of the plasma membrane, which activates the electrogenic H+ pump. The depolarization of ROF2 over-expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. As ROF2 induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.This work was supported by grants BFU2008-00604 from the Ministerio de Ciencia e Innovacion (Madrid, Spain) and PROMETEO/2010/ 038 of the 'Conselleria de Educacion' (Valencia, Spain). We thank Dr Eugenio Grau (Sequencing Service, Instituto de Biologia Molecular y Celular de Plantas, Valencia, Spain) for sequencing of the various genes, and Dr Vicente Fornes (Instituto de Tecnologia Quimica, Valencia, Spain) for assistance with atomic absorption spectrophotometry. None of the authors has a conflict of interest to declare.Bissoli, G.; Niñoles Rodenes, R.; Fresquet Corrales, S.; Palombieri, S.; Bueso Ródenas, E.; Rubio, L.; Garcia-Sanchez, MJ.... (2012). Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis. Plant Journal. 70(4):704-716. https://doi.org/10.1111/j.1365-313X.2012.04921.xS70471670

A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions

[EN] The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H+ efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit H+ efflux (H+ -ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H+ -ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H+ efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to K+ efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H+ -ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H+ -ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H+ -ATPase while the guard-cell kinase SnRK2.6/OST1 did not.This work was funded by grants BFU2011-22526 (to RS) and BIO2011-23446 (to PLR) of the Spanish 'Ministerio de Economia y Competitividad', Madrid, Spain, and grant PROMETEO/2010/038 (to RS) of the 'Generalitat Valenciana', Valencia, Spain. MGG was funded by a JAE-DOC contract of the Spanish 'Consejo Superior de Investigaciones Cientificas', Madrid, Spain. We thank Dr Toshinori Kinoshita (Nagoya University, Nagoya, Japan) for the rabbit antibody against the last 9 aa of AHA2 H+-ATPase with the penultimate Thr947 phosphorylated. We also thank the Proteomics Facility of the 'Centro Nacional de Biotecnologia', Madrid, Spain, for the attempts to identify the phosphorylation site of the H+-ATPase.Planes Ferrer, MD.; Niñoles Rodenes, R.; Rubio, L.; Bissoli, G.; Bueso Ródenas, E.; Garcia-Sanchez, MJ.; Alejandro Martínez, S.... (2015). A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions. Journal of Experimental Botany. 66(3):813-825. https://doi.org/10.1093/jxb/eru442S81382566

La tolerancia a litio del mutante cat2 de arabidopsis revela una estrecha relación entre estrés oxidativo y etileno

Con el fin de investigar los efectos en la homeostasis de iones de un aumento en la concentración celular de peróxido de hidrógeno, se aisló un mutante de inserción de T-DNA en el gen CATALASA 2 de Arabidopsis. El mutante cat2-1 presenta una reducción del 80% de la catalasa de hoja en comparación con genotipos silvestres y acumula más peróxido de hidrógeno en condiciones sin estrés. Además de presentar un tamaño reducido, un color verde más pálido y gran reducción en el número de raíces secundarias, el mutante cat2-1 presenta una marcada sensibilidad a peróxido de hidrógeno, cloruro sódico, norespermidina, alta intensidad lumínica y estrés por frío. Por otra parte, el mutante cat2-1 presenta una tolerancia parcial cuando es crecido en medios con cloruro de litio en comparación con el genotipo silvestre. Este novedoso fenotipo no puede ser explicado por cambios en el transporte de este catión. Realmente, la toma de litio y de otros cationes tóxicos como sodio y norespermidina es mayor en el mutante cat2-1, mientras que los niveles de potasio de la planta son inferiores. La tolerancia a litio de este mutante parece ser resultado de una insensibilidad a la inhibitoria respuesta de etileno producida por el catión y a su reducida capacidad de producción de etileno. De acuerdo con esto, la inducción por etileno de genes de respuesta tales como PR4 y EBP/ERF72 es menor en el mutante cat2-1. Mutantes insensibles a etileno como etr1-1 y ein3-3 son tolerantes a litio y la inhibición de la biosíntesis de etileno con 2-aminoisobutirato protege contra la toxicidad del litio. Análisis de la expresión génica con micromatrices indican que la expresión de genes relacionados al transporte de cationes y a la síntesis y percepción de etileno no están alterados en el mutante cat2-1, lo que nos hace suponer que el peróxido de hidrógeno modula estos procesos a nivel de proteína. Todos estos resultados descubren una estrecha relación entre estrés oxidativo, la homeostasis de cationes y elBueso Ródenas, E. (2008). La tolerancia a litio del mutante cat2 de arabidopsis revela una estrecha relación entre estrés oxidativo y etileno [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/2341Palanci

A forward genetic approach in Arabidopsis thaliana identifies a RING-type ubiquitin ligase as a novel determinant of seed longevity

[EN] Seed longevity is important to preserve crops and wild plants and it is limited by progressive cellular damage (aging) during storage. The induction of cellular stress defenses and the formation of the seed coat are crucial protecting events during seed development, a process mediated in Arabidopsis thaliana by the transcription factors LEC1, LEC2, FUS3 and the abscisic acid-activated ABI3. In order to identify novel determinants of seed longevity we have screened an activation-tagging mutant collection of Arabidopsis and isolated a dominant mutant with increased seed longevity under both natural and accelerated aging conditions. Molecular characterization indicates that the mutant phenotype is caused by over-expression of the At2g26130 gene encoding a RING-type zinc finger putative ubiquitin ligase. Loss of function of this gene in a T-DNA insertion mutant resulted in decreased seed longevity. We named this important gene for seed longevity RSL1 (from Ring finger of Seed Longevity1) and we could demonstrate ubiquitin ligase activity with the recombinant protein. Morphological alterations in shoot tissues of the RSL1 over-expressing plants and analysis of gibberellins levels suggest that RSL1 may increase gibberellins responses by some unknown mechanism. These results validate the forward genetic approach to seed longevity and anticipate the identification of many novel determinants of this important trait. (C) 2013 Elsevier Ireland Ltd. All rights reserved.This work was funded by grant EUI2009-03985 of the Spanish "Ministerio de Economia y Competividad" (MINECO, Madrid). J.M.-B. was supported by a contract (Juan de la Cierva) of the Spanish MINECO. M.G.-G. was supported by a contract (JAE-DOC) of the Spanish Consejo Superior de Investigaciones Cientificas (CSIC, Madrid). We thank Dr Judy Callis for kindly lending us the 6xHis-AtUBC8 plas mid.Bueso Ródenas, E.; Ibanez, C.; Sayas, E.; Muñoz Bertomeu, J.; González Guzmán, M.; Rodríguez Egea, PL.; Serrano Salom, R. (2014). A forward genetic approach in Arabidopsis thaliana identifies a RING-type ubiquitin ligase as a novel determinant of seed longevity. Plant Science. 215:110-116. https://doi.org/10.1016/j.plantsci.2013.11.004S11011621

A dominant-negative form of Arabidopsis AP-3 #-adaptin improves intracellular pH homeostasis

[EN] Intracellular pH (pHi) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pHi regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid-tolerant 1-1D (wat1-1D) are due to the expression of a truncated form of AP-3 -adaptin (encoded by the PAT2 gene) that behaves as a as dominant-negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pHi and a more depolarized plasma membrane electrical potential than wild-type cells. Additional phenotypes of wat1-1D roots include increased rates of acetate efflux, K+ uptake and H+ efflux, the latter reflecting the in vivo activity of the plasma membrane H+-ATPase. The in vitro activity of the enzyme was not increased but, as the H+-ATPase is electrogenic, the increased ion permeability would allow a higher rate of H+ efflux. The AP-3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1-1D mutant can be explained if loss of function of the AP-3 -adaptin causes activation of channels or transporters for organic anions (acetate) and for K+ at the plasma membrane, perhaps through miss-localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.This work was supported by grants BFU2008-00604 from the 'Ministerio de Ciencia e Innovacion' (Madrid, Spain) and from PROME-TEO/2010/038 of the 'Conselleria de Educacion' (Valencia, Spain). We thank Frans J. Maathuis (York, United Kingdom) for the pA7-TPK1-GFP plasmid, Enrico Martinoia (Zurich, Switzerland) for the pART7-ALMT9-GFP plasmid and for the tdt mutant, Karin Schumacher (Heidelberg, Germany) for the vha-a2 vha-a3 mutant, Jose M. Pardo (Sevilla, Spain) for the nhx2-1 mutant, NASC for the akt1-1 mutant, Roberto A. Gaxiola (Tempe, Arizona) for the AVP1 over-expression line and Wei-Hua Wu (Beijing, China) for the AKT1 over-expression line. R. Ninoles was supported by a JAE-preDOC contract ('Consejo Superior de Investigaciones Cientificas', Madrid, Spain).Niñoles Rodenes, R.; Rubio, L.; García Sánchez, MJ.; Fernandez, JA.; Bueso Ródenas, E.; Alejandro Martínez, S.; Serrano Salom, R. (2013). A dominant-negative form of Arabidopsis AP-3 #-adaptin improves intracellular pH homeostasis. Plant Journal. 74(4):557-568. https://doi.org/10.1111/tpj.12138S55756874

ARABIDOPSIS THALIANA HOMEOBOX25 uncovers a role for gibberellins in seed longevity

[EN] Seed longevity is crucial for agriculture and plant genetic diversity, but it is limited by cellular damage during storage. Seeds are protected against aging by cellular defenses and by structures such as the seed coat. We have screened an activation-tagging mutant collection of Arabidopsis (Arabidopsis thaliana) and selected four dominant mutants with improved seed longevity (isl1-1D to isl4-1D) under both natural and accelerated aging conditions. In the isl1-1D mutant, characterized in this work, overexpression of the transcription factor ARABIDOPSIS THALIANA HOMEOBOX25 (ATHB25; At5g65410) increases the expression of GIBBERELLIC ACID3-OXIDASE2, encoding a gibberellin (GA) biosynthetic enzyme, and the levels of GA(1) and GA(4) are higher (3.2- and 1.4-fold, respectively) in the mutant than in the wild type. The morphological and seed longevity phenotypes of the athb25-1D mutant were recapitulated in transgenic plants with moderate (4- to 6-fold) overexpression of ATHB25. Simultaneous knockdown of ATHB25, ATHB22, and ATHB31 expression decreases seed longevity, as does loss of ATHB25 and ATHB22 function in a double mutant line. Seeds from wild-type plants treated with GA and from a quintuple DELLA mutant (with constitutive GA signaling) are more tolerant to aging, providing additional evidence for a role of GA in seed longevity. A correlation was observed in several genotypes between seed longevity and mucilage formation at the seed surface, suggesting that GA may act by reinforcing the seed coat. This mechanism was supported by the observation of a maternal effect in reciprocal crosses between the wild type and the athb25-1D mutant.This work was supported by the Spanish Ministerio de Economia y Competividad (grant no. EUI2009-03985 and Juan de la Cierva contract to J.M-B.) as well as by the Consejo Nacional de Ciencia y Tecnologia and Direccion General de Asuntos del Personal Academico, Universidad Nacional Autonoma de Mexico (sabbatical fellowship to F.C.).Bueso Ródenas, E.; Muñoz Bertomeu, J.; Campos, F.; Brunaud, V.; Martinez, L.; Sayas Montañana, EM.; Ballester Fuentes, P.... (2014). ARABIDOPSIS THALIANA HOMEOBOX25 uncovers a role for gibberellins in seed longevity. Plant Physiology. 164(2):999-1010. https://doi.org/10.1104/pp.113.232223S9991010164

A fungal transcription factor gene is expressed in plants from its own promoter and improves drought tolerance

[EN] A fungal gene encoding a transcription factor is expressed from its own promoter in Arabidopsis phloem and improves drought tolerance by reducing transpiration and increasing osmotic potential. Horizontal gene transfer from unrelated organisms has occurred in the course of plant evolution, suggesting that some foreign genes may be useful to plants. The CtHSR1 gene, previously isolated from the halophytic yeast Candida tropicalis, encodes a heat-shock transcription factor-related protein. CtHSR1, with expression driven by its own promoter or by the Arabidopsis UBQ10 promoter, was introduced into the model plant Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation and the resulting transgenic plants were more tolerant to drought than controls. Fusions of the CtHSR1 promoter with beta-glucuronidase reporter gene indicated that this fungal promoter drives expression to phloem tissues. A chimera of CtHSR1 and green fluorescence protein is localized at the cell nucleus. The physiological mechanism of drought tolerance in transgenic plants is based on reduced transpiration (which correlates with decreased opening of stomata and increased levels of jasmonic acid) and increased osmotic potential (which correlates with increased proline accumulation). Transcriptomic analysis indicates that the CtHSR1 transgenic plants overexpressed a hundred of genes, including many relevant to stress defense such as LOX4 (involved in jasmonic acid synthesis) and P5CS1 (involved in proline biosynthesis). The promoters of the induced genes were enriched in upstream activating sequences for water stress induction. These results demonstrate that genes from unrelated organisms can have functional expression in plants from its own promoter and expand the possibilities of useful transgenes for plant biotechnology.We acknowledge support by Grants BFU2011-22526 of the Spanish MICINN (Madrid, Spain) and PROMETEO II 2014-041 of Generalitat Valenciana (Valencia, Spain). J. M.-B. was supported by a Juan de la Cierva contract of the Spanish MICINN. A. A. was supported by a short-term EMBO fellowship to visit the laboratory of R. Serrano. We thank Dr. Jose Maria Belles (IBMCP, Valencia, Spain) for assistance in the determination of sugars, Dr. Isabel Lopez-Diaz and Dr. Esther Carrera for the hormone analysis carried out at the Plant Hormone Quantification Service of IBMCP and Prof. Jorg Kudla (Westfalische Wilhelms-Universitat, Munster, Germany) for the pGPTVII.Hyg.PUBQ10::MCS plasmid.Martínez Macías, F.; Arif, A.; González Nebauer, S.; Bueso Ródenas, E.; Ali, R.; Montesinos De Lago, C.; Brunaud, V.... (2015). A fungal transcription factor gene is expressed in plants from its own promoter and improves drought tolerance. Planta. 242(1):39-52. https://doi.org/10.1007/s00425-015-2285-5S39522421Acharya BR, Assmann SM (2009) Hormone interactions in stomatal function. Plant Mol Biol 69:451–462Alba MM, Tompa P, Veitia RA (2007) Amino acid repeats and the structure and evolution of proteins. Genome Dyn 3:119–130Alejandro S, Rodríguez PL, Bellés JM, Yenush L, García-Sanchez MJ, Fernández JA, Serrano R (2007) An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast-xylem interface. EMBO J 26:3203–3215Ali R, Pascual-Ahuir A, Marquez JA, Malik KA, Serrano R (2001) Identification of Candida tropicalis HSR1, a gene of the heat-shock factor-related family, which confers salt tolerance in Saccharomyces cerevisiae. Yeast 18:605–610Bates L, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. CR Acad Sci Paris/Life Sci 316:1194–1199Benfey PN, Chua NH (1990) The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250:959–966Bernard V, Lecharny A, Brunaud V (2010) Improved detection of motifs with preferential location in promoters. Genome 53:739–752Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721Bissoli G, Niñoles R, Fresquet S, Palombieri S, Bueso E, Rubio L, García- Sánchez MJ, Fernández JA, Mulet JM, Serrano R (2012) Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis. Plant J 70:704–716Bock R (2009) The give-and-take of DNA: horizontal gene transfer in plants. Trends Plant Sci 15:11–22Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254Bueso E, Alejandro S, Carbonell P, Perez-Amador MA, Fayos J, Bellés JM, Rodriguez PL, Serrano R (2007) The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene. Plant J 52:1052–1065Bueso E, Muñoz-Bertomeu J, Campos F, Brunaud V, Martínez L, Sayas E, Ballester P, Yenush L, Serrano R (2014) ARABIDOPSIS THALIANA HOMEOBOX25 uncovers a role for gibberellins in seed longevity. Plant Physiol 164:999–1010Cheng MC, Liao PM, Kuo WW, Lin TP (2013) The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals. Plant Physiol 162:1566–1582Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17Grefen C, Donald N, Hashimoto K, Kudla J, Schumacher K, Blatt MR (2010) A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J 64:355–365Han G, Wang M, Yuan F, Sui N, Song J, Wang B (2014) The CCCH zinc finger protein gene AtZFP1 improves salt resistance in Arabidopsis thaliana. Plant Mol Biol 86:237–253Hentrich M, Böttcher C, Düchting P, Cheng Y, Zhao Y, Berkowitz O, Masle J, Medina J, Pollmann S (2013) The jasmonic acid signaling pathway is linked to auxin homeostasis through the modulation of YUCCA8 and YUCCA9 gene expression. Plant J 74:626–637Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoot RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180Kavi Kishor PB, Sreenivasulu N (2014) Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant, Cell Environ 37:300–311Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396Landsman D, Wolffe AP (1995) Common sequence and structural features in the heat- shock factor and Ets families of DNA binding domains. Trends Biochem Sci 20:225–226Montillet J-L, Hirt H (2013) New checkpoints in stomatal defense. Trends Plant Sci 18:295–297Naleway JJ (1992) Histochemical, spectrophotometric, and fluorometric GUS substrates. In: Gallagher SR (ed) GUS protocols. Using the GUS gene as a reporter of gene expression. Academic Press, San Diego, pp 61–76Norris SR, Meyer SE, Callis J (1993) The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol Biol 21:895–906Ozalvo R, Cabrera J, Escobar C, Christensen SA, Borrego EJ, Kolomiets MV, Castresana C, Iberkleid I, Brown Horowitz S (2014) Two closely related members of Arabidopsis 13-lipoxygenases (13-LOXs), LOX3 and LOX4, reveal distinct functions in response to plant-parasitic nematode infection. Mol Plant Pathol 15:319–332Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45Ramanjulu S, Bartels D (2002) Drought- and desiccation-induced modulation of gene expression in plants. Plant, Cell Environ 25:141–151Reed CJ, Lewis H, Trejo E, Winston V, Evilia C (2013) Protein adaptations in archaeal extremophiles. Archaea 2013:373275. doi: 10.1155/2013/373275Saha P, Chakraborti D, Sarkar A, Dutta I, Basu D, Das A (2007) Characterization of vascular-specific RSs1 and rolC promoters for their utilization in engineering plants to develop resistance against hemipteran insect pests. Planta 226:429–442Schönknecht G, Weber AP, Lercher MJ (2014) Horizontal gene acquisitions by eukaryotes as drivers of adaptive evolution. BioEssays 36:9–20Sorger PK (1991) Heat shock factor and the heat shock response. Cell 65:363–366Sun X, Li Y, Cai H, Bai X, Ji W, Ding X, Zhu Y (2012) The Arabidopsis AtbZIP1 transcription factor is a positive regulator of plant tolerance to salt, osmotic and drought stresses. J Plant Res 125:429–438Székely G, Abrahám E, Cséplo A, Rigó G, Zsigmond L, Csiszár J, Ayaydin F, Strizhov N, Jásik J, Schmelzer E, Koncz C, Szabados L (2008) Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. Plant J 53:11–28Talianova M, Janousek B (2011) What can we learn from tobacco and other solanaceae about horizontal gene transfer? Am J Bot 98:1231–1242Tyedmers J, Mogk A, Bukau B (2010) Cellular strategies for controlling protein aggregation. Mol Cell Biol 11:777–778Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33:949–956Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40:428–438Yamaguchi-Shinozaki K, Shinozaki K (2005) Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci 10:88–94Yang T, Poovaiah BW (2002) A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants. J Biol Chem 277:45049–45058Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551Yilmaz A, Mejia-Guerra MK, Kurz K, Liang X, Welch L, Grotewold E (2011) AGRIS: the Arabidopsis gene regulatory information server, an update. Nucleic Acids Res 39:D1118–D1122Yin Y, Beachy R (1997) Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice. Plant J 12:1179–118

Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis

This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of gamma-aminobutyrate, which in turn affect plant development.This work was supported by the Spanish Government and the European Union (Fondo Europeo de Desarrollo Regional grant no. BFU2012-31519 to J.M.-B., Formacion del Personal Investigador fellowship to S.R.-T., and Agencia Espanola de Cooperacion Internacional fellowship to A.D.A.), by the Valencian Regional Government (PROMETEO grant no. II/2014/052), and by the University of Valencia (Atraccio de Talent fellowship to M.F.-T.).Anoman, AD.; Muñoz Bertomeu, J.; Rosa-Tellez, S.; Flores-Tornero, M.; Serrano Salom, R.; Bueso Ródenas, E.; Fernie, AR.... (2015). Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis. Plant Physiology. 169(3):1619-1637. https://doi.org/10.1104/pp.15.00696S16191637169