Pale-Green Phenotype of atl31 atl6 Double Mutant Leaves Is Caused by Disruption of 5-Aminolevulinic Acid Biosynthesis in Arabidopsis thaliana

February Pale-Green Phenotype of atl31 atl6 Double Mutant Leaves Is Caused by Disruption of 5-Aminolevulinic Acid Biosynthesis in Arabidopsis thaliana Shugo Maekawa 0 1 2 Atsushi Takabayashi 0 1 2 Thais Huarancca Reyes 0 1 2 Hiroko Yamamoto 0 1 2 Ayumi Tanaka 0 1 2 Takeo Sato 0 1 2 Junji Yamaguchi 0 1 2 0 Current address: Biotechnology Research Center, The University of Tokyo , Bunkyo-ku, Tokyo , Japan 1 1 Faculty of Science and Graduate School of Life Science, Hokkaido University , Sapporo, Hokkaido , Japan , 2 Institute of Low Temperature Science, Hokkaido University , Sapporo, Hokkaido , Japan 2 Academic Editor: Wagner L. Araujo, Universidade Federal de Vicosa , BRAZIL Arabidopsis ubiquitin ligases ATL31 and homologue ATL6 control the carbon/nitrogen nutrient and pathogen responses. A mutant with the loss-of-function of both atl31 and atl6 developed light intensity-dependent pale-green true leaves, whereas the single knockout mutants did not. Plastid ultrastructure and Blue Native-PAGE analyses revealed that pale-green leaves contain abnormal plastid structure with highly reduced levels of thylakoid proteins. In contrast, the pale-green leaves of the atl31/atl6 mutant showed normal Fv/Fm. In the palegreen leaves of the atl31/atl6, the expression of HEMA1, which encodes the key enzyme for 5-aminolevulinic acid synthesis, the rate-limiting step in chlorophyll biosynthesis, was markedly down-regulated. The expression of key transcription factor GLK1, which directly promotes HEMA1 transcription, was also significantly decreased in atl31/atl6 mutant. Finally, application of 5-aminolevulinic acid to the atl31/atl6 mutants resulted in recovery to a green phenotype. Taken together, these findings indicate that the 5-aminolevulinic acid biosynthesis step was inhibited through the down-regulation of chlorophyll biosynthesis-related genes in the pale-green leaves of atl31/atl6 mutant. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Grants-in-Aid for Scientific Research to JY [No. 23380198, 24114701 and 25112501] and by a Grant in-Aid to SM for Scientific Research for a Plant Graduate Student from the Nara Institute of Science and Technology (2009) by The Ministry of Education, Culture, Sports, Science and Technology of Japan. SM was also supported by Research Fellowships from the Japan Society for the Promotion of Science Chlorophyll (Chl) biosynthesis is regulated primarily at the level of 5-aminolevulinic acid (ALA) synthesis. In higher plants, ALA is synthesized from glutamate in the plastid in three enzymatic steps. In the first step, the enzyme glutamyl-tRNA synthetase aminoacylates tRNAGlu with glutamate, generating the substrate for plastid translation and tetrapyrrole biosynthesis. In the following steps, tRNAGlu is reduced by glutamyl-tRNA reductase (GluTR) to glutamate-1semialdehyde, which is converted to ALA by glutamate-1-semialdehyde aminotransferase [1,2]. for Young Scientists (2010-2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Of the three enzymes responsible for ALA synthesis, GluTR is regarded as the main target of regulatory mechanisms [3]. The transcription and activity of GluTR is controlled by light, an endogenous clock, cytokinin and development [25]. In Arabidopsis, three HEMA genes encode GluTR. HEMA3 is considered a pseudogene [6], whereas HEMA1 and HEMA2 encode proteins that are 81% identical at the amino acid level [7,8]. Since HEMA1 is highly expressed in photosynthetic tissues of leaves and stems, whereas HEMA2 is constitutively expressed at low levels in all tissues, HEMA1 is considered the dominant form of GluTR for Chl biosynthesis [9,10]. RNA interference (RNAi)-induced down-regulation of HEMA1 expression in Arabidopsis and tobacco resulted in a reduction in plant Chl [5,11]. Furthermore, a hema1 mutant showed a yellowish phenotype, due to a drastically reduced level of Chl with severe growth retardation [12]. Thus, regulating HEMA1 is essential for Chl biosynthesis. Genes in the Arabidopsis Txicos en Levadura (ATL) family encode plant-specific putative RING-type ubiquitin ligases with transmembrane domains. In Arabidopsis, the ATL family is composed of 91 members [13,14]. ATL31 and its closest homolog ATL6 are membraneassociated ubiquitin ligases, shown to be involved in the carbon/nitrogen (C/N) response by regulating the stability of 1433 proteins through their ubiquitination activity [1517]. Plants overexpressing full-length ATL31 or ATL6 (35S-ATL31 and 35S-ATL6) were insensitive to high C/N stress conditions, whereas the single knockout mutants atl311 and atl61, as well as an atl311/atl61 double knockout mutant showed increased sensitivity to high C/N stress conditions [15,18]. ATL31 and ATL6 are also involve (...truncated)