Instituto de Bioquímica Vegetal y Fotosíntesis

AUTORES: Daniel J Klionsky1745,1749*, Kotb Abdelmohsen840, Akihisa Abe1237, Md Joynal Abedin1762, Hagai Abeliovich425,
\nAbraham Acevedo Arozena789, Hiroaki Adachi1800, Christopher M Adams1669, Peter D Adams57, Khosrow Adeli1981,
\nPeter J Adhihetty1625, Sharon G Adler700, Galila Agam67, Rajesh Agarwal1587, Manish K Aghi1537, Maria Agnello1826,
\nPatrizia Agostinis664, Patricia V Aguilar1960, Julio Aguirre-Ghiso784,786, Edoardo M Airoldi89,422, Slimane Ait-Si-Ali1376,
\nTakahiko Akematsu2010, Emmanuel T Akporiaye1097, Mohamed Al-Rubeai1394, Guillermo M Albaiceta1294,
\nChris Albanese363, Diego Albani561, Matthew L Albert517, Jesus Aldudo128, Hana Alg€ul1164, Mehrdad Alirezaei1198,
\nIraide Alloza642,888, Alexandru Almasan206, Maylin Almonte-Beceril524, Emad S Alnemri1212, Covadonga Alonso544,
\nNihal Altan-Bonnet848, Dario C Altieri1205, Silvia Alvarez1497, Lydia Alvarez-Erviti1395, Sandro Alves107,
\nGiuseppina Amadoro860, Atsuo Amano930, Consuelo Amantini1554, Santiago Ambrosio1458, Ivano Amelio756,
\nAmal O Amer918, Mohamed Amessou2089, Angelika Amon726, Zhenyi An1538, Frank A Anania291, Stig U Andersen6,
\nUsha P Andley2079, Catherine K Andreadi1690, Nathalie Andrieu-Abadie502, Alberto Anel2027, David K Ann58,
\nShailendra Anoopkumar-Dukie388, Manuela Antonioli832,858, Hiroshi Aoki1791, Nadezda Apostolova2007,
\nSaveria Aquila1500, Katia Aquilano1876, Koichi Araki292, Eli Arama2098, Agustin Aranda456, Jun Araya591,
\nAlexandre Arcaro1472, Esperanza Arias26, Hirokazu Arimoto1225, Aileen R Ariosa1749, Jane L Armstrong1930,
\nThierry Arnould1773, Ivica Arsov2120, Katsuhiko Asanuma675, Valerie Askanas1924, Eric Asselin1867, Ryuichiro Atarashi794,
\nSally S Atherton369, Julie D Atkin713, Laura D Attardi1131, Patrick Auberger1787, Georg Auburger379, Laure Aurelian1727,
\nRiccardo Autelli1992, Laura Avagliano1029,1755, Maria Laura Avantaggiati364, Limor Avrahami1166, Suresh Awale1986,
\nNeelam Azad404, Tiziana Bachetti568, Jonathan M Backer28, Dong-Hun Bae1933, Jae-sung Bae677, Ok-Nam Bae409,
\nSoo Han Bae2117, Eric H Baehrecke1729, Seung-Hoon Baek17, Stephen Baghdiguian1368,
\nAgnieszka Bagniewska-Zadworna2, Hua Bai90, Jie Bai667, Xue-Yuan Bai1133, Yannick Bailly884,
\nKithiganahalli Narayanaswamy Balaji473, Walter Balduini2002, Andrea Ballabio316, Rena Balzan1711, Rajkumar Banerjee239,
\nG abor B anhegyi1052, Haijun Bao2109, Benoit Barbeau1363, Maria D Barrachina2007, Esther Barreiro467, Bonnie Bartel997,
\nAlberto Bartolom e222, Diane C Bassham550, Maria Teresa Bassi1046, Robert C Bast Jr1273, Alakananda Basu1798,
\nMaria Teresa Batista1578, Henri Batoko1336, Maurizio Battino970, Kyle Bauckman2085, Bradley L Baumgarner1909,
\nK Ulrich Bayer1594, Rupert Beale1553, Jean-Fran¸cois Beaulieu1360, George R. Beck Jr48,294, Christoph Becker336,
\nJ David Beckham1595, Pierre-Andr e B edard749, Patrick J Bednarski301, Thomas J Begley1135, Christian Behl1419,
\nChristian Behrends757, Georg MN Behrens406, Kevin E Behrns1627, Eloy Bejarano26, Amine Belaid490,
\nFrancesca Belleudi1041, Giovanni B enard497, Guy Berchem706, Daniele Bergamaschi983, Matteo Bergami1401,
\nBen Berkhout1441, Laura Berliocchi714, Am elie Bernard1749, Monique Bernard1354, Francesca Bernassola1880,
\nAnne Bertolotti791, Amanda S Bess272, S ebastien Besteiro1351, Saverio Bettuzzi1828, Savita Bhalla913,
\nShalmoli Bhattacharyya973, Sujit K Bhutia838, Caroline Biagosch1159, Michele Wolfe Bianchi520,1378,1381,
\nMartine Biard-Piechaczyk210, Viktor Billes298, Claudia Bincoletto1314, Baris Bingol350, Sara W Bird1128, Marc Bitoun1112,
\nIvana Bjedov1258, Craig Blackstone843, Lionel Blanc1183, Guillermo A Blanco1496, Heidi Kiil Blomhoff1812,
\nEmilio Boada-Romero1297, Stefan B€ockler1464, Marianne Boes1423, Kathleen Boesze-Battaglia1835, Lawrence H Boise286,287,
\nAlessandra Bolino2063, Andrea Boman693, Paolo Bonaldo1823, Matteo Bordi897, J€urgen Bosch608, Luis M Botana1308,
\nJoelle Botti1375, German Bou1405, Marina Bouch e1038, Marion Bouchecareilh1331, Marie-Jos ee Boucher1901,
\nMichael E Boulton481, Sebastien G Bouret1926, Patricia Boya133, Micha€el Boyer-Guittaut1345, Peter V Bozhkov1141,
\nNathan Brady374, Vania MM Braga469, Claudio Brancolini1997, Gerhard H Braus353, Jos e M Bravo-San Pedro299,393,508,1374,
\nLisa A Brennan322, Emery H Bresnick2022, Patrick Brest490, Dave Bridges1939, Marie-Agn es Bringer124, Marisa Brini1822,
\nGlauber C Brito1311, Bertha Brodin631, Paul S Brookes1872, Eric J Brown352, Karen Brown1690, Hal E Broxmeyer480,
\nAlain Bruhat486,1339, Patricia Chakur Brum1893, John H Brumell446, Nicola Brunetti-Pierri315,1171,
\nRobert J Bryson-Richardson781, Shilpa Buch1777, Alastair M Buchan1819, Hikmet Budak1022, Dmitry V Bulavin118,505,1789,
\nScott J Bultman1792, Geert Bultynck665, Vladimir Bumbasirevic1470, Yan Burelle1356, Robert E Burke216,217,
\nMargit Burmeister1750, Peter B€utikofer1473, Laura Caberlotto1987, Ken Cadwell896, Monika Cahova112, Dongsheng Cai24,
\nJingjing Cai2099, Qian Cai1018, Sara Calatayud2007, Nadine Camougrand1343, Michelangelo Campanella1700,
\nGrant R Campbell1525, Matthew Campbell1249, Silvia Campello556,1876, Robin Candau1769, Isabella Caniggia1983,
\nLavinia Cantoni560, Lizhi Cao116, Allan B Caplan1656, Michele Caraglia1051, Claudio Cardinali1043, Sandra Morais Cardoso1579, Jennifer S Carew208, Laura A Carleton874, Cathleen R Carlin101, Silvia Carloni2002,
\nSven R Carlsson1267, Didac Carmona-Gutierrez1643, Leticia AM Carneiro312, Oliana Carnevali971, Serena Carra1318,
\nAlice Carrier120, Bernadette Carroll900, Caty Casas1324, Josefina Casas1116, Giuliana Cassinelli324, Perrine Castets1462,
\nSusana Castro-Obregon214, Gabriella Cavallini1841, Isabella Ceccherini568, Francesco Cecconi253,555,1884,
\nArthur I Cederbaum459, Valent ın Ce~na199,1281, Simone Cenci1323,2064, Claudia Cerella444, Davide Cervia1996,
\nSilvia Cetrullo1478, Hassan Chaachouay2028, Han-Jung Chae187, Andrei S Chagin634, Chee-Yin Chai626,628,
\nGopal Chakrabarti1502, Georgios Chamilos1601, Edmond YW Chan1142, Matthew TV Chan181, Dhyan Chandra1003,
\nPallavi Chandra548, Chih-Peng Chang818, Raymond Chuen-Chung Chang1653, Ta Yuan Chang345, John C Chatham1434,
\nSaurabh Chatterjee1910, Santosh Chauhan527, Yongsheng Che62, Michael E Cheetham1263, Rajkumar Cheluvappa1783,
\nChun-Jung Chen1153, Gang Chen598,1676, Guang-Chao Chen9, Guoqiang Chen1078, Hongzhuan Chen1077, Jeff W Chen1514,
\nJian-Kang Chen370,371, Min Chen249, Mingzhou Chen2104, Peiwen Chen1823, Qi Chen1674, Quan Chen172,
\nShang-Der Chen138, Si Chen325, Steve S-L Chen10, Wei Chen2125, Wei-Jung Chen829, Wen Qiang Chen979, Wenli Chen1113,
\nXiangmei Chen1133, Yau-Hung Chen1157, Ye-Guang Chen1250, Yin Chen1447, Yingyu Chen953,955, Yongshun Chen2135,
\nYu-Jen Chen712, Yue-Qin Chen1145, Yujie Chen1208, Zhen Chen339, Zhong Chen2123, Alan Cheng1702,
\nChristopher HK Cheng184, Hua Cheng1728, Heesun Cheong814, Sara Cherry1836, Jason Chesney1703,
\nChun Hei Antonio Cheung817, Eric Chevet1359, Hsiang Cheng Chi140, Sung-Gil Chi656, Fulvio Chiacchiera308,
\nHui-Ling Chiang958, Roberto Chiarelli1826, Mario Chiariello235,567,577, Marcello Chieppa835, Lih-Shen Chin290,
\nMario Chiong1285, Gigi NC Chiu878, Dong-Hyung Cho676, Ssang-Goo Cho650, William C Cho982, Yong-Yeon Cho105,
\nYoung-Seok Cho1064, Augustine MK Choi2095, Eui-Ju Choi656, Eun-Kyoung Choi387,400,685, Jayoung Choi1563,
\nMary E Choi2093, Seung-Il Choi2116, Tsui-Fen Chou412, Salem Chouaib395, Divaker Choubey1574, Vinay Choubey1936,
\nKuan-Chih Chow822, Kamal Chowdhury730, Charleen T Chu1856, Tsung-Hsien Chuang827, Taehoon Chun657,
\nHyewon Chung652, Taijoon Chung978, Yuen-Li Chung1194, Yong-Joon Chwae18, Valentina Cianfanelli254,
\nRoberto Ciarcia1775, Iwona A Ciechomska886, Maria Rosa Ciriolo1876, Mara Cirone1042, Sofie Claerhout1694,
\nMichael J Clague1698, Joan Cl aria1457, Peter GH Clarke1687, Robert Clarke361, Emilio Clementi1045,1398, C edric Cleyrat1781,
\nMiriam Cnop1366, Eliana M Coccia574, Tiziana Cocco1459, Patrice Codogno1375, J€orn Coers271, Ezra EW Cohen1533,
\nDavid Colecchia235,567,577, Luisa Coletto25, N uria S Coll123, Emma Colucci-Guyon516, Sergio Comincini1829,
\nMaria Condello578, Katherine L Cook2073, Graham H Coombs1929, Cynthia D Cooper2076, J Mark Cooper1395,
\nIsabelle Coppens601, Maria Tiziana Corasaniti1387, Marco Corazzari485,1884, Ramon Corbalan1566,
\nElisabeth Corcelle-Termeau251, Mario D Cordero1899, Cristina Corral-Ramos1289, Olga Corti507,1109, Andrea Cossarizza1767,
\nPaola Costelli1993, Safia Costes1518, Susan L Cotman721, Ana Coto-Montes946, Sandra Cottet566,1688, Eduardo Couve1301,
\nLori R Covey1015, L Ashley Cowart762, Jeffery S Cox1536, Fraser P Coxon1427, Carolyn B Coyne1846, Mark S Cragg1919,
\nRolf J Craven1679, Tiziana Crepaldi1995, Jose L Crespo1300, Alfredo Criollo1285, Valeria Crippa558, Maria Teresa Cruz1576,
\nAna Maria Cuervo26, Jose M Cuezva1277, Taixing Cui1907, Pedro R Cutillas987, Mark J Czaja27, Maria F Czyzyk-Krzeska1572,
\nRuben K Dagda2068, Uta Dahmen1404, Chunsun Dai800, Wenjie Dai1187, Yun Dai2059, Kevin N Dalby1940,
\nLuisa Dalla Valle1822, Guillaume Dalmasso1340, Marcello D’Amelio557, Markus Damme188, Arlette Darfeuille-Michaud1340,
\nCatherine Dargemont950, Victor M Darley-Usmar1433, Srinivasan Dasarathy205, Biplab Dasgupta202, Srikanta Dash1254,
\nCrispin R Dass242, Hazel Marie Davey8, Lester M Davids1560, David D avila227, Roger J Davis1731, Ted M Dawson604,
\nValina L Dawson606, Paula Daza1898, Jackie de Belleroche470, Paul de Figueiredo1180,1182,
\nRegina Celia Bressan Queiroz de Figueiredo135, Jos e de la Fuente1023, Luisa De Martino1775,
\nAntonella De Matteis1171, Guido RY De Meyer1443, Angelo De Milito631, Mauro De Santi2002,

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Nitrogen is a quantitatively important bioelement which is incorporated into the biosphere through assimilatory processes carried out by microorganisms and plants. Numerous nitrogencontaining compounds can be used by different organisms as sources of nitrogen. These include, for instance, inorganic ions like nitrate or ammonium and simple organic compounds like urea, amino acids, and some nitrogen-containing bases. Additionally, many bacteria are capable of fixing N 2. Nitrogen control is a phenomenon that occurs widely among microorganisms and consists of repression of the pathways of assimilation of some nitrogen sources when some other, more easily assimilated source of nitrogen is available to the cells. Ammonium is the preferred nitrogen source for most bacteria, but glutamine is also a very good source of nitrogen for many microorganisms. Two thoroughly investigated nitrogen control systems are the NtrB-NtrC two-component regulatory system found in enterics and some other proteobacteria (80) and the GATA family global nitrogen control transcription factors of yeast and some fungi (75). Novel nitrogen control systems have, however, been identified in bacteria other than the proteobacteria, like Bacillus subtilis (26), Corynebacterium glutamicum (52), and the cyanobacteria. The cyanobacterial system is the subject of this review. The cyanobacteria are prokaryotes that belong to the Bacteria domain and are characterized by the ability to perform oxygenic photosynthesis. Cyanobacteria have a wide ecological distribution, and they occupy a range of habitats, which includes vast oceanic areas, temperate soils, and freshwater lakes, and even extreme habitats like arid deserts, frigid lakes, or hot springs. Photoautotrophy, fixing CO 2 through the Calvin cycle, is the dominant mode of growth of these organisms (109). A salient feature of the intermediary metabolism of cyanobacteria is their lack of 2-oxoglutarate dehydrogenase (109). As a consequence, they use 2-oxoglutarate mainly as a substrate for the incorporation of nitrogen, a metabolic arrangement that may have regulatory consequences. Notwithstanding their rather homogeneous metabolism, cyanobacteria exhibit remarkable morphological diversity, being found as either unicellular or filamentous forms and exhibiting a number of cell differentiation processes, some of which take place in response to defined environmental cues, as is the case for the differentiation of N 2-fixing heterocysts (109). Nitrogen control in cyanobacteria is mediated by NtcA, a transcriptional regulator which belongs to the CAP (the catabolite gene activator or cyclic AMP [cAMP] receptor protein) family and is therefore different from the well-characterized Ntr system. Interestingly, however, the signal transduction P II protein, which plays a key role in Ntr regulation, is found in cyanobacteria but with characteristics which differentiate it from proteobacterial P II. In the following paragraphs, we shall first briefly summarize our current knowledge of the cyanobacterial nitrogen assimilation pathways and of what is known about their regulation at the protein level. This description will introduce most of the known cyanobacterial nitrogen assimilation genes. We shall then describe the ntcA gene and the NtcA protein themselves to finally discuss NtcA function through a survey of the NtcA-regulated genes which participate in simple nitrogen assimilation pathways or in heterocyst differentiation and function.

The transcriptional regulator CONSTANS (CO) promotes flowering of Arabidopsis under long summer days (LDs) but not under short winter days (SDs). Post-translational regulation of CO is crucial for this response by stabilizing the protein at the end of a LD, whereas promoting its degradation throughout the night under LD and SD. We show that mutations in CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a component of a ubiquitin ligase, cause extreme early flowering under SDs, and that this is largely dependent on CO activity. Furthermore, transcription of the CO target gene FT is increased in cop1 mutants and decreased in plants overexpressing COP1 in phloem companion cells. COP1 and CO interact in vivo and in vitro through the C-terminal region of CO. COP1 promotes CO degradation mainly in the dark, so that in cop1 mutants CO protein but not CO mRNA abundance is dramatically increased during the night. However, in the morning CO degradation occurs independently of COP1 by a phytochrome B-dependent mechanism. Thus, COP1 contributes to day length perception by reducing the abundance of CO during the night and thereby delaying flowering under SDs.

GATA transcription factors are a group of DNA binding proteins broadly distributed in eukaryotes. The GATA factors DNA binding domain is a class IV zinc finger motif in the form CX(2)CX(17-20)CX(2)C followed by a basic region. In plants, GATA DNA motifs have been implicated in light-dependent and nitrate-dependent control of transcription. Herein, we show that the Arabidopsis and the rice (Oryza sativa) genomes present 29 and 28 loci, respectively, that encode for putative GATA factors. A phylogenetic analysis of the 57 GATA factors encoding genes, as well as the study of their intron-exon structure, indicates the existence of seven subfamilies of GATA genes. Some of these subfamilies are represented in both species but others are exclusive for one of them. In addition to the GATA zinc finger motif, polypeptides of the different subfamilies are characterized by the presence of additional domains such as an acidic domain, a CCT (CONSTANS, CO-like, and TOC1) domain, or a transposase-like domain also found in FAR1 and FHY3. Subfamily VI comprises genes that encode putative bi-zinc finger polypeptides, also found in metazoan and fungi, and a tri-zinc finger protein which has not been previously reported in eukaryotes. The phylogeny of the GATA zinc finger motif, excluding flanking regions, evidenced the existence of four classes of GATA zinc fingers, three of them containing 18 residues in the zinc finger loop and one containing a 20-residue loop. Our results support multiple models of evolution of the GATA gene family in plants including gene duplication and exon shuffling.

The target of rapamycin (TOR) is a conserved Ser/Thr kinase that controls cell growth by activating an array of anabolic processes including protein synthesis, transcription and ribosome biogenesis, and by inhibiting catabolic processes such as mRNA degradation and autophagy. Control of autophagy by TOR occurs primarily at the induction step, and involves activation of the ATG1 kinase, a conserved component of the autophagic machinery. A substantial number of genes participating in autophagy have been originally identified in yeast. Most of these genes have mammalian homologues and many have apparent homologues in plants, indicating that autophagy is conserved among eukaryotes. The recent identification of TOR as a key element in cell growth control in plants and algae opens the way for future studies to investigate whether this signaling pathway may also control autophagy in photosynthetic organisms.

Modern agriculture relies on mineral fertilization. Unlike other major macronutrients, potassium (K+) is not incorporated into organic matter but remains as soluble ion in the cell sap contributing up to 10% of the dry organic matter. Consequently, K+ constitutes a chief osmoticum to drive cellular expansion and organ movements, such as stomata aperture. Moreover, K+ transport is critical for the control of cytoplasmic and luminal pH in endosomes, regulation of membrane potential, and enzyme activity. Not surprisingly, plants have evolved a large ensemble of K+ transporters with defined functions in nutrient uptake by roots, storage in vacuoles, and ion translocation between tissues and organs. This review describes critical transport proteins governing K+ nutrition, their regulation and coordinated activity, and summarizes our current understanding of signaling pathways activated by K+ starvation.

Cysteine (Cys) occupies a central position in plant metabolism due to its biochemical functions. Arabidopsis (Arabidopsis thaliana) cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes that catalyze the biosynthesis of Cys. Because they are localized in the cytosol, plastids, and mitochondria, this results in multiple subcellular Cys pools. Much progress has been made on the most abundant OASTL enzymes; however, information on the less abundant OASTL-like proteins has been scarce. To unequivocally establish the enzymatic reaction catalyzed by the minor cytosolic OASTL isoform CS-LIKE (for Cys synthase-like; At5g28030), we expressed this enzyme in bacteria and characterized the purified recombinant protein. Our results demonstrate that CS-LIKE catalyzes the desulfuration of L-Cys to sulfide plus ammonia and pyruvate. Thus, CS-LIKE is a novel L-Cys desulfhydrase (EC 4.4.1.1), and we propose to designate it DES1. The impact and functionality of DES1 in Cys metabolism was revealed by the phenotype of the T-DNA insertion mutants des1-1 and des1-2. Mutation of the DES1 gene leads to premature leaf senescence, as demonstrated by the increased expression of senescence-associated genes and transcription factors. Also, the absence of DES1 significantly reduces the total Cys desulfuration activity in leaves, and there is a concomitant increase in the total Cys content. As a consequence, the expression levels of sulfur-responsive genes are deregulated, and the mutant plants show enhanced antioxidant defenses and tolerance to conditions that promote oxidative stress. Our results suggest that DES1 from Arabidopsis is an L-Cys desulfhydrase involved in maintaining Cys homeostasis, mainly at late developmental stages or under environmental perturbations.

Plants contain three thioredoxin systems. Chloroplast thioredoxins are reduced by ferredoxin-thioredoxin reductase, whereas the cytosolic and mitochondrial thioredoxins are reduced by NADPH thioredoxin reductase (NTR). There is high similarity among NTRs from plants, lower eukaryotes, and bacteria, which are different from mammal NTR. Here we describe the OsNTRC gene from rice encoding a novel NTR with a thioredoxin-like domain at the C terminus, hence, a putative NTR/thioredoxin system in a single polypeptide. Orthologous genes were found in other plants and cyanobacteria, but not in bacteria, yeast, or mammals. Full-length OsNTRC and constructs with truncated NTR and thioredoxin domains were expressed in Escherichia coli as His-tagged polypeptides, and a polyclonal antibody specifically cross-reacting with the OsNTRC enzyme was raised. An in vitro activity assay showed that OsNTRC is a bifunctional enzyme with both NTR and thioredoxin activity but is not an NTR/thioredoxin system. Although the OsNTRC gene was expressed in roots and shoots of rice seedlings, the protein was exclusively found in shoots and mature leaves. Moreover, fractionation experiments showed that OsNTRC is localized to the chloroplast. An Arabidopsis NTRC knock-out mutant showed growth inhibition and hypersensitivity to methyl viologen, drought, and salt stress. These results suggest that the NTRC gene is involved in plant protection against oxidative stress. Plants contain three thioredoxin systems. Chloroplast thioredoxins are reduced by ferredoxin-thioredoxin reductase, whereas the cytosolic and mitochondrial thioredoxins are reduced by NADPH thioredoxin reductase (NTR). There is high similarity among NTRs from plants, lower eukaryotes, and bacteria, which are different from mammal NTR. Here we describe the OsNTRC gene from rice encoding a novel NTR with a thioredoxin-like domain at the C terminus, hence, a putative NTR/thioredoxin system in a single polypeptide. Orthologous genes were found in other plants and cyanobacteria, but not in bacteria, yeast, or mammals. Full-length OsNTRC and constructs with truncated NTR and thioredoxin domains were expressed in Escherichia coli as His-tagged polypeptides, and a polyclonal antibody specifically cross-reacting with the OsNTRC enzyme was raised. An in vitro activity assay showed that OsNTRC is a bifunctional enzyme with both NTR and thioredoxin activity but is not an NTR/thioredoxin system. Although the OsNTRC gene was expressed in roots and shoots of rice seedlings, the protein was exclusively found in shoots and mature leaves. Moreover, fractionation experiments showed that OsNTRC is localized to the chloroplast. An Arabidopsis NTRC knock-out mutant showed growth inhibition and hypersensitivity to methyl viologen, drought, and salt stress. These results suggest that the NTRC gene is involved in plant protection against oxidative stress. Thioredoxins are small 12–13-kDa proteins with a redox-active disulfide bridge and are widely distributed in all types of organisms (1Schürmann P. Jacquot J.-P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 371-400Crossref PubMed Scopus (326) Google Scholar). Because of their disulfide/dithiol interchange activity, thioredoxins interact with target proteins and are involved in the regulation of a large number of cellular processes (2Balmer Y. Buchanan B.B. Trends Plant Sci. 2002; 7: 191-193Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Plants contain more types of thioredoxin systems than any other organism. The cytosolic system in plants is composed of type h thioredoxins reduced by NADPH in a reaction catalyzed by NADPH thioredoxin reductase (NTR) 1The abbreviations used are: NTR, NADPH thioredoxin reductase; OsNTR, Oryza sativa NTR; AtNTR, Arabidopsis thaliana NTR; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); Trx, thioredoxin. (1Schürmann P. Jacquot J.-P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 371-400Crossref PubMed Scopus (326) Google Scholar). In addition, plants contain other thioredoxin systems localized in organelles. In chloroplasts, the thioredoxin system includes type f, m, and x thioredoxins (3Collin V. Issakidis-Bourguet E. Marchand C. Hirasawa M. Lancelin J.-M. Knaff D.B. Miginiac-Maslow M. J. Biol. Chem. 2003; 278: 23747-23752Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), which are reduced by ferredoxin in a reaction catalyzed by ferredoxin thioredoxin reductase (1Schürmann P. Jacquot J.-P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 371-400Crossref PubMed Scopus (326) Google Scholar). This system is involved in light/dark regulation of chloroplast enzymes (1Schürmann P. Jacquot J.-P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 371-400Crossref PubMed Scopus (326) Google Scholar, 4Ruelland E. Miginiac-Maslow M. 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Spyrou G. J. Biol. Chem. 1999; 274: 6366-6373Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 11Spyrou G. Enmark E. Miranda-Vizuete A. Gustafsson J-A. J. Biol. Chem. 1997; 272: 2936-2941Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 12Miranda-Vizuete A. Damdimopoulus A.E. Pedrajas J.R. Gustafsson J.A. Spirou G. Eur. J. Biochem. 1999; 261: 405-412Crossref PubMed Scopus (149) Google Scholar). The plant mitochondrial system is formed by a novel type of thioredoxin (type o), which is reduced by an NADPH-dependent thioredoxin reductase similar to the cytosolic enzyme (9Laloi C. Rayapuram N. Chartier Y. Grienenberger J.-N. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (223) Google Scholar). Although NTR is universally distributed from bacteria to mammals, two forms of this enzyme have evolved (13Williams C.H. Arscott L.D. Muller S. Lennon B.W. Ludwig M.L. Wang P.-F. Veine D.M. Becker K. Schirmer R.H. Eur. J. Biochem. 2000; 267: 6110-6117Crossref PubMed Scopus (284) Google Scholar): (i) a low molecular mass NTR formed by a homodimer of 35-kDa subunits that is found in bacteria and yeast and is very similar to the plant enzyme (14Jacquot J.-P. Rivera-Madrid R. Marinho P. Kollavora M. LeMarechal P. Miginiac-Maslow M. Meyer Y. J. Mol. Biol. 1994; 235: 1357-1363Crossref PubMed Scopus (129) Google Scholar, 15Dai S. Saarinen M. Ramaswamy S. Meyer Y. Jacquot J.-P. Eklund H. J. Mol. Biol. 1996; 264: 1044-1057Crossref PubMed Scopus (91) Google Scholar, 16Serrato A.J. Pérez-Ruiz J.M. Cejudo F.J. Biochem. J. 2002; 217: 392-399Google Scholar), and (ii) the mammalian NTR, which is also homodimer formed by subunits of 55 kDa with an unusual selenocysteine as the penultimate residue at the C terminus (17Gladishev V.N. Jeang K.T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6146-6151Crossref PubMed Scopus (410) Google Scholar). Taking advantage of the recently sequenced rice genome, we have identified the OsNTRC gene encoding a novel NTR with an extension at the C terminus containing a putative thioredoxin active site. In this paper we report on the cloning of this gene, its expression in Escherichia coli, and the biochemical characterization of the purified recombinant protein as well as truncated polypeptides containing the NTR and Trx domains of the enzyme, showing that it is a bifunctional enzyme with both NTR and thioredoxin activity but not an NTR/thioredoxin system when assayed with insulin as the substrate. Surprisingly, this novel NTR is localized in chloroplasts and, to our knowledge, is the first description of a chloroplast-localized NTR. The identification and characterization of an NTRC knock-out mutant of Arabidopsis suggest that this novel NTR is involved in protection against oxidative stress. Plant Material—Rice (Oryza sativa L. ssp. japonica cv. Nipponbare) grains were sterilized and germinated at 25 °C on filter paper soaked with water. Chloroplasts were isolated as described in (18Price C.A. Hadjeb N. Newman L. Reardon E.M. Gelvin S.B. Schilperoort R.A. Plant Molecular Biology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 1-15Crossref Google Scholar) from rice leaves of 36-day-old plants. Arabidopsis wild-type and the T-DNA insertion mutant SALK_012208 (19Alonso J.M. Stepanova A.N. Leisse T.J. Kim C.J. Chen H. Shinn P. Stevenson D.K. Zimmerman J. Barajas P. Cheuk R. Gadrinab C. Heller C. Jeske A. Koesema E. Meyers C.C. Parker H. Prednis L. Ansari Y. Choy N. Deen H. Geralt M. Hazari N. Hom E. Karnes M. Mulholland C. Ndubaku R. Schmidt I. Guzman P. Aguilar-Henonin L. Schmid M. Weigel D. Carter D.E. Marchand T. Risseeuw E. Brogden D. Zeko A. Crosby W.L. Berry C.C. Ecker J.R. Science. 2003; 301: 653-657Crossref PubMed Scopus (4127) Google Scholar) were grown as reported previously (20Sánchez R. Cejudo F.J. Plant Physiol. 2003; 132: 949-957Crossref PubMed Scopus (112) Google Scholar). The T-DNA homozygous line was selected after PCR analysis with oligonucleotides in the T-DNA (5′-TGGTTCACGTAGTGGGCCATCG-3′) and the AtNTRC gene (5′-TCACCAACATGTGGCCC-3′ and 5′-TCGGGTGTGAAGATGAAGAA-3′). Isolation of OsNTRC cDNA—Total RNA (1 μg) from 5-day-old rice seedling shoots was reverse transcribed in the presence of oligo(dT) and 200 units of reverse transcriptase (Invitrogen). An aliquot (1 μl) was used as the template in a PCR reaction with the oligonucleotides 5′-CCATCCTCTCCCTCTCCTCT-3′ and 5′-GGTGTCAAGACAACGACCAA-3′. A 1.5-kb fragment was obtained, cloned in a pGEMt vector (Promega), and sequenced in both strands. The OsNTRC gene sequence was deposited at the EMBL data bank with accession number AJ582621. Expression and Purification of Recombinant Proteins and the Production of Anti-OsNTRC Polyclonal Antibodies—Rice OsNTRC was expressed in E. coli as a His-tagged polypeptide. The coding sequence, excluding the putative signal peptide (36 residues at the N terminus, Fig. 1) was amplified from the full-length cDNA with the oligonucleotides 5′-GAGAGAGCTCGATCTTGGCAAGGGAG-3′ and 5′-GAGAAAGCTTTCATTTGTTTGACTCG-3′, which added a SacI site at the 5′-end and a HindIII site at the 3′-end (underlined). The PCR fragment was digested with SacI and HindIII, subcloned into the pQE-30 expression vector (Qiagen), and introduced into E. coli XL1-Blue. The NTR domain of OsNTRC (residues 37 to 359, Fig. 1), was amplified with the oligonucleotides 5′-GAGAGAGCTCGATCTTGGCAAGGGAG-3′) and (5′-GAGAAAGCTTTTCAACAAGAAGGTCG-3′). The thioredoxin domain (residues 360 to 488, Fig. 1), was amplified with oligonucleotides (5′-GAGAGAGCTCCACCAGCCTGTTCGCG-3′) and (5′-GAGAAAGCTTTCATTTGTTTGACTCG-3′). Both of these truncated proteins were produced in E. coli as described for full-length OsNTRC. Overexpressed proteins were purified by Cu2+ affinity chromatography in pre-packed Hi-Trap affinity columns (Amersham Biosciences). Anti-OsNTRC antibodies were raised by immunizing rabbits with purified His-tagged Trx-D at the Service for Animal Production (University of Seville, Spain). Relative Reverse Transcription PCR, Western Blot Analysis, and Activity Assays—Reverse transcription PCR analysis was performed as described previously (20Sánchez R. Cejudo F.J. Plant Physiol. 2003; 132: 949-957Crossref PubMed Scopus (112) Google Scholar) with pairs of gene-specific oligonucleotides for OsNTRC (5′-CATGTGGTCCTTGCAGAAC-3′ and 5′-AATAGGAGCGACCGGCTAAT-3′) and OsNTRB (5′-TCCAAGATCATGCAGGCC-3′ and 5′-ACGCTGGTGGTCGCTTCCTC-3′). Western-blot analysis was performed as described previously (16Serrato A.J. Pérez-Ruiz J.M. Cejudo F.J. Biochem. J. 2002; 217: 392-399Google Scholar). NTR activity was determined by the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) according to the method described (21Holmgren A. Björnstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (817) Google Scholar). Thioredoxin activity was determined with the insulin reduction assay (22Serrato A.J. Crespo J.L. Florencio F.J. Cejudo F.J. Plant Mol. Biol. 2001; 46: 361-371Crossref PubMed Scopus (70) Google Scholar). A Gene Encoding a Novel NTR from Rice—A search of the rice genome data base identified three putative NTR encoding genes, here called OsNTRA, OsNTRB, and OsNTRC. According to the sequence comparison shown in Fig. 1, OsNTRB is an ortholog of AtNTRB (66.7% identity), the cytosolic form of the enzyme (14Jacquot J.-P. Rivera-Madrid R. Marinho P. Kollavora M. LeMarechal P. Miginiac-Maslow M. Meyer Y. J. Mol. Biol. 1994; 235: 1357-1363Crossref PubMed Scopus (129) Google Scholar), whereas OsNTRA is the ortholog of AtNTRA (66.5% identity), the mitochondrial form of NTR (9Laloi C. Rayapuram N. Chartier Y. Grienenberger J.-N. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (223) Google Scholar). The OsNTRC and AtNTRC (72.4% identity) genes encode a novel NTR not yet described. A full OsNTRC cDNA was cloned from rice that encodes a polypeptide composed of a domain showing high similarity to other plant NTRs (Fig. 1). This domain contains the FAD binding site (motifs GXGXX(G/A) and TXXXX-VFAAGD; Fig. 1, inverted triangles), the active site (Fig. 1, asterisks), and the NADPH binding motif GXGXXA (Fig. 1, triangles). Unlike the other NTRs described to date, OsNTRC (and AtNTRC) contain an extension at the C terminus with the sequence CGPC (Fig. 1, box), a putative thioredoxin active site. These data suggest that OsNTRC encodes an NTR/thioredoxin system in a single polypeptide. This unusual type of NTR was found in cyanobacteria, and the Nostoc sp. PCC7120 gene (60.1% identity with OsNTRC) is included in Fig. 1 but not in other bacteria, fungi, or mammals. To establish the phylogeny of this novel NTR, a phylogenetic tree was constructed (Fig. 2). OsNTRC and AtNTRC genes are clustered with NTRs from cyanobacteria defining a novel group of NTRs (Fig. 2). Functional Characterization of the OsNTRC Polypeptide—To analyze the biochemical properties of the polypeptide encoded by OsNTRC gene, it was expressed in E. coli without the 36 amino acids at the N terminus (Fig. 3A). Truncated polypeptides containing the NTR- or Trx-like domains of OsNTRC were also produced (Fig. 3A) and purified by Cu2+ chromatography (Fig. 3B, lanes 2–4). For comparison, purified His-tagged NTRB (16Serrato A.J. Pérez-Ruiz J.M. Cejudo F.J. Biochem. J. 2002; 217: 392-399Google Scholar), the cytosolic form, and TRXhA (22Serrato A.J. Crespo J.L. Florencio F.J. Cejudo F.J. Plant Mol. Biol. 2001; 46: 361-371Crossref PubMed Scopus (70) Google Scholar) from wheat were also included (Fig. 3B, lanes 1 and 5, respectively). Western blots of the gel shown in Fig. 3B were probed with anti-NTRB antibodies (16Serrato A.J. Pérez-Ruiz J.M. Cejudo F.J. Biochem. J. 2002; 217: 392-399Google Scholar), which detected NTR-D (Fig. 3C, lane 3) more efficiently than the full-length OsNTRC (Fig. 3C, lane 2). To obtain polyclonal antibodies specifically cross-reacting with OsNTRC, rabbits were immunized with the purified Trx-D polypeptide found only in OsNTRC. As expected, this antibody specifically cross-reacted with Trx-D (Fig. 3D, lane 4) and OsNTRC (Fig. 3D, lane 2). Furthermore, an antibody against wheat thioredoxin h did not cross-react with either Trx-D or OsNTRC (Fig. 3E). Because OsNTRC contains both NTR and Trx-like domains, we tested whether this single polypeptide functions as an NTR/thioredoxin system. OsNTRC catalyzes the dithiothreitol-dependent reduction of insulin, but no activity was detected in the presence of NADPH (Fig. 4A). The thioredoxin activity of OsNTRC was due to the Trx-like domain as shown by the insulin reduction activity of the isolated Trx-D polypeptide (Fig. 4B). Both OsNTRC and the NTR domain showed NADPH-dependent reduction of DTNB (Fig. 4C). The Trx-like domain does not affect the NTR activity of OsNTRC (Fig. 4C) and, as expected, did not show any NTR activity. These results show that OsNTRC is a bifunctional enzyme, showing NTR and Trx activity, but it is not an NTR/thioredoxin system when assayed with insulin as the substrate. As for other NTRs, OsNTRC showed higher affinity for NADPH (Km of 10.4 μm) than for NADH (Km of 1.2 mm). We also tested whether the OsNTRC polypeptide was able to transfer electrons to the Trx-like module, to wheat TRXhA or to thioredoxins and from of were for OsNTRC not Expression of the OsNTRC reverse transcription PCR was used to analyze the expression of OsNTRC and cytosolic OsNTRB in rice OsNTRC and OsNTRB at similar in roots and shoots (Fig. A similar and of the OsNTRB polypeptide was detected in both (Fig. in with the presence of OsNTRB Surprisingly, the OsNTRC polypeptide was detected exclusively in shoots (Fig. showing that NTRC is or of low in roots the presence of OsNTRC in this The that the OsNTRC gene is to with the presence of the OsNTRC polypeptide in its chloroplast To this we performed fractionation experiments chloroplasts purified from rice leaves. Western analysis showed that NTRC was more in the chloroplast it was also detected in the (Fig. Both the and the were of or mitochondrial as shown by the of signal when Western blots were probed with the anti-NTRB antibody (Fig. Characterization of an Arabidopsis NTRC T-DNA insertion line of the Arabidopsis NTRC gene was identified in the (19Alonso J.M. Stepanova A.N. Leisse T.J. Kim C.J. Chen H. Shinn P. Stevenson D.K. Zimmerman J. Barajas P. Cheuk R. Gadrinab C. Heller C. Jeske A. Koesema E. Meyers C.C. Parker H. Prednis L. Ansari Y. Choy N. Deen H. Geralt M. Hazari N. Hom E. Karnes M. Mulholland C. Ndubaku R. Schmidt I. Guzman P. Aguilar-Henonin L. Schmid M. Weigel D. Carter D.E. Marchand T. Risseeuw E. Brogden D. Zeko A. Crosby W.L. Berry C.C. Ecker J.R. Science. 2003; 301: 653-657Crossref PubMed Scopus (4127) Google Scholar). identified both and homozygous for the T-DNA (Fig. and the homozygous line was For the antibody detected the large from Arabidopsis (Fig. the Western showed the of NTRC in the mutant (Fig. whereas it wild-type of NTRB (Fig. As in rice seedlings, NTRC was not detected in roots (Fig. showing that it is either or in very low in of the Arabidopsis NTRC knock-out mutant germinated as efficiently as wild-type but mutant plants showed leaves with a (Fig. and growth which were after (Fig. and in plants (Fig. This that growth stress to the Because NTRC polypeptide is localized in the this to chloroplast protection against oxidative To this wild-type and mutant plants were with methyl viologen, a that oxidative stress J.A. Plant Physiol. PubMed Google Scholar). plants showed a higher to methyl than did wild-type plants (Fig. as well as more of (Fig. Because other oxidative we tested the of the NTRC mutant to salt (Fig. and (Fig. stress. the of both was more in the mutant than in wild-type plants. For comparison, wild-type and mutant plants are also shown (Fig. In this paper we describe a gene from rice and Arabidopsis encoding a novel NTR that is found exclusively in In to sequence similarity of show that OsNTRC encodes an NTR. The expressed in E. coli, is a not detected by a polyclonal antibody against the cytosolic form of the NTRB enzyme from wheat (16Serrato A.J. Pérez-Ruiz J.M. Cejudo F.J. Biochem. J. 2002; 217: 392-399Google Scholar) and NADPH-dependent DTNB reduction activity (Fig. 4A). The of the enzyme for NADPH μm) is in the of reported for NTRs from different plant (14Jacquot J.-P. Rivera-Madrid R. Marinho P. Kollavora M. LeMarechal P. Miginiac-Maslow M. Meyer Y. J. Mol. Biol. 1994; 235: 1357-1363Crossref PubMed Scopus (129) Google Scholar, 16Serrato A.J. Pérez-Ruiz J.M. Cejudo F.J. Biochem. J. 2002; 217: 392-399Google Scholar) and, as for other NTRs described to date, it a higher affinity for NADPH than for OsNTRC is an unusual NTR due to the presence of a Trx-like domain at the C terminus, which the that this enzyme as an NTR/thioredoxin system. We that OsNTRC both NTR and thioredoxin activity due to the NTR and Trx-like domains of the OsNTRC was to the NADPH-dependent reduction of we that OsNTRC is a bifunctional enzyme and not an NTR/thioredoxin at these assay data show that OsNTRC or the truncated polypeptide containing the NTR domain was to transfer electrons to the thioredoxin-like domain of the Furthermore, it was not able to wheat E. coli and thioredoxins and from the that the OsNTRC gene encodes an NTR, of the plant thioredoxins are for this According to this novel NTR to to plants and phylogenetic analysis a of OsNTRC and AtNTRC with the NTR The only of a protein with NTR and thioredoxin domains was identified in D. A. R. T. Mol. 1995; PubMed Scopus Google Scholar), and, this gene is with the NTRs and not with NTRC genes (Fig. 2). This gene other contain genes for NTR and Trx D. A. R. T. Mol. 1995; PubMed Scopus Google Scholar). In with the rice OsNTRC the protein functions as an NTR/thioredoxin system J. R. A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The analysis of the expression of the OsNTRC gene an The OsNTRC gene was expressed in roots and shoots from rice (Fig. the OsNTRC polypeptide was detected in in both rice and Arabidopsis and this polypeptide was localized in the chloroplast and, to a lower in the (Fig. This that the extension of the protein at the N terminus as a to this sequence as a but is not only on sequence D. Meyer Y. 1999; PubMed Scopus Google Scholar). The or low of OsNTRC in the presence of the that either the is not efficiently or that the protein is but the for the of of protein in is not yet The of this novel NTR in the chloroplast and its high affinity for NADPH that it involved in the of from Because the chloroplast thioredoxins involved in are not for the that NTRC is involved in of enzymes is that this NTR is involved in for chloroplast protection against oxidative The of NTRC in Arabidopsis is not but growth inhibition (Fig. The of the mutant leaves and their as well as their hypersensitivity to oxidative to a of NTRC in chloroplast protection against oxidative of the system for chloroplast protection against oxidative have been as the M. Plant J. 1997; PubMed Scopus Google Scholar, M. Plant Physiol. 1999; PubMed Scopus Google Scholar, M. Trends Plant Sci. 1999; 4: Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. U. G. Schürmann P. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). This system electrons that are by the thioredoxin M. S. P. Plant 2000; Scopus Google Scholar), but to it is not electrons of the are to this system. The of the Arabidopsis NTRC knock-out mutant the of NTRC in this but the molecular of this the identification of NTRC which is The of R. was We A. for thioredoxins and m, M. for rice and (University of for of the

Evidence is accumulating to challenge the paradigm that biogenic methanogenesis, considered a strictly anaerobic process, is exclusive to archaea. We demonstrate that cyanobacteria living in marine, freshwater, and terrestrial environments produce methane at substantial rates under light, dark, oxic, and anoxic conditions, linking methane production with light-driven primary productivity in a globally relevant and ancient group of photoautotrophs. Methane production, attributed to cyanobacteria using stable isotope labeling techniques, was enhanced during oxygenic photosynthesis. We suggest that the formation of methane by cyanobacteria contributes to methane accumulation in oxygen-saturated marine and limnic surface waters. In these environments, frequent cyanobacterial blooms are predicted to further increase because of global warming potentially having a direct positive feedback on climate change. We conclude that this newly identified source contributes to the current natural methane budget and most likely has been producing methane since cyanobacteria first evolved on Earth.

The synthesis of ribosomes is one of the major cellular activities, and in eukaryotes, it takes place primarily, although not exclusively, in a specialized subnuclear compartment termed the nucleolus (125, 155). There, the rRNA genes are transcribed as precursors (pre-rRNAs), which undergo processing and covalent modification. Maturation of pre-rRNAs is intimately linked to their assembly with the ribosomal proteins (r-proteins). These processes depend on various cis-acting elements (6, 188), and they require a large number of nonribosomal protein trans-acting factors (97, 174, 193). Experimental evidence suggests that the basic outline of ribosome synthesis is conserved throughout eukaryotes. However, most of our knowledge comes from the combination of molecular genetic and biochemical approaches in the yeast Saccharomyces cerevisiae. This minireview is aimed at giving an insight into the functions of the many protein trans-acting factors involved in ribosome biogenesis in S. cerevisiae.

One of the mechanisms plants have developed for chloroplast protection against oxidative damage involves a 2-Cys peroxiredoxin, which has been proposed to be reduced by ferredoxin and plastid thioredoxins, Trx x and CDSP32, the FTR/Trx pathway. We show that rice (Oryza sativa) chloroplast NADPH THIOREDOXIN REDUCTASE (NTRC), with a thioredoxin domain, uses NADPH to reduce the chloroplast 2-Cys peroxiredoxin BAS1, which then reduces hydrogen peroxide. The presence of both NTR and Trx-like domains in a single polypeptide is absolutely required for the high catalytic efficiency of NTRC. An Arabidopsis thaliana knockout mutant for NTRC shows irregular mesophyll cell shape, abnormal chloroplast structure, and unbalanced BAS1 redox state, resulting in impaired photosynthesis rate under low light. Constitutive expression of wild-type NTRC in mutant transgenic lines rescued this phenotype. Moreover, prolonged darkness followed by light/dark incubation produced an increase in hydrogen peroxide and lipid peroxidation in leaves and accelerated senescence of NTRC-deficient plants. We propose that NTRC constitutes an alternative system for chloroplast protection against oxidative damage, using NADPH as the source of reducing power. Since no light-driven reduced ferredoxin is produced at night, the NTRC-BAS1 pathway may be a key detoxification system during darkness, with NADPH produced by the oxidative pentose phosphate pathway as the source of reducing power.

Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems.

Hydrogen sulfide-mediated signaling pathways regulate many physiological and pathophysiological processes in mammalian and plant systems. The molecular mechanism by which hydrogen sulfide exerts its action involves the post-translational modification of cysteine residues to form a persulfidated thiol motif, a process called protein persulfidation. We have developed a comparative and quantitative proteomic analysis approach for the detection of endogenous persulfidated proteins in wild-type Arabidopsis and L-CYSTEINE DESULFHYDRASE 1 mutant leaves using the tag-switch method. The 2015 identified persulfidated proteins were isolated from plants grown under controlled conditions, and therefore, at least 5% of the entire Arabidopsis proteome may undergo persulfidation under baseline conditions. Bioinformatic analysis revealed that persulfidated cysteines participate in a wide range of biological functions, regulating important processes such as carbon metabolism, plant responses to abiotic and biotic stresses, plant growth and development, and RNA translation. Quantitative analysis in both genetic backgrounds reveals that protein persulfidation is mainly involved in primary metabolic pathways such as the tricarboxylic acid cycle, glycolysis, and the Calvin cycle, suggesting that this protein modification is a new regulatory component in these pathways.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

Nitrogen sources commonly used by cyanobacteria include ammonium, nitrate, nitrite, urea and atmospheric N(2), and some cyanobacteria can also assimilate arginine or glutamine. ABC (ATP-binding cassette)-type permeases are involved in the uptake of nitrate/nitrite, urea and most amino acids, whereas secondary transporters take up ammonium and, in some strains, nitrate/nitrite. In cyanobacteria, nitrate and nitrite reductases are ferredoxin-dependent enzymes, arginine is catabolized by a combination of the urea cycle and arginase pathway, and urea is degraded by a Ni(2+)-dependent urease. These pathways provide ammonium that is incorporated into carbon skeletons through the glutamine synthetase-glutamate synthase cycle, in which 2-oxoglutarate is the final nitrogen acceptor. The expression of many nitrogen assimilation genes is subjected to regulation being activated by the nitrogen-control transcription factor NtcA, which is autoregulatory and whose activity appears to be influenced by 2-oxoglutarate and the signal transduction protein P(II). In some filamentous cyanobacteria, N(2) fixation takes place in specialized cells called heterocysts that differentiate from vegetative cells in a process strictly controlled by NtcA.

S function in the context of guard cell ABA signaling, but also demonstrate the presence of a rapid signal integration mechanism involving specific and reversible redox-based post-translational modifications that occur in response to changing environmental conditions.

Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H2S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H2S is enzymatically produced in the cytosol through the activity of l-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H2S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H2S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H2S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H2S, suggesting that H2S required a functional ABI1. Further studies to unravel the cross talk between H2S and NO indicate that (1) H2S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H2S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells.

The fixation of atmospheric N(2) by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena, this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of >10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N(2) fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify >900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N(2) fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF(+) motif, a palindrome at or close to position -35 that seems essential for heterocyst-specific expression of certain genes.

Reactive oxygen species (ROS) and autophagy have been historically associated with cell death. However, more recent evidence indicates that both ROS and autophagy play important roles in signaling and cellular adaptation to stress. As a catabolic process, autophagy allows eukaryotic cells to recycle intracellular components including entire organelles during development or under stress conditions such as nutrient limitation. Degradation and recycling of macromolecules via autophagy provides a source of building blocks (amino acids, lipids and sugars) that allow temporal adaptation of cells to adverse conditions. In addition to recycling, autophagy is required for the degradation of damaged or toxic material that can be generated as a result of ROS accumulation during oxidative stress. The mitochondrial electron-transport chain and the peroxisomes are primary sources of ROS production in most eukaryotes. The plant cell contains an additional organelle, the chloroplast, with an intense electron flow that leads to high rates of ROS production. Studies in plants and algae have demonstrated that autophagy is structurally and functionally conserved in photosynthetic organisms and plays an important role in the cellular response and adaptation to different stress conditions that involve generation of ROS such as oxidative and drought stresses, pathogen infection or photo-oxidative damage. These findings suggested a strong link between autophagy and ROS in photosynthetic eukaryotes. Here we review recent studies in plants and algae describing redox control of autophagy and discuss about conserved regulatory proteins that may transmit redox signals to the autophagic machinery.