Nitrogen (N) and nitrate (NO 32 ) per se regulate many aspects of plant metabolism, growth, and development. N/NO 3 2 also suppresses parts of secondary metabolism, including anthocyanin synthesis. Molecular components for this repression are unknown. We report that three N/NO 3 2 -induced members of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family of transcription factors (LBD37, LBD38, and LBD39) act as negative regulators of anthocyanin biosynthesis in Arabidopsis thaliana. Overexpression of each of the three genes in the absence of N/NO 3 2 strongly suppresses the key regulators of anthocyanin synthesis PAP1 and PAP2, genes in the anthocyanin-specific part of flavonoid synthesis, as well as cyanidinbut not quercetin-or kaempferol-glycoside production. Conversely, lbd37, lbd38, or lbd39 mutants accumulate anthocyanins when grown in N/NO 3 2 -sufficient conditions and show constitutive expression of anthocyanin biosynthetic genes. The LBD genes also repress many other known N-responsive genes, including key genes required for NO 3 2 uptake and assimilation, resulting in altered NO 3 2 content, nitrate reductase activity/activation, protein, amino acid, and starch levels, and N-related growth phenotypes. The results identify LBD37 and its two close homologs as novel repressors of anthocyanin biosynthesis and N availability signals in general. They also show that, besides being developmental regulators, LBD genes fulfill roles in metabolic regulation.
INTRODUCTION
Nitrogen (N) and nitrate (NO 32 ) itself regulate many aspects of plant metabolism, growth, and development. These include NO 3 2 uptake and assimilation (Crawford, 1995;Lejay et al., 1999), starch and organic acid metabolism (Scheible et al., 1997a), secondary metabolism (e.g., anthocyanin production) Fritz et al., 2006), germination (Alboresi et al., 2005), root architecture (Zhang et al., 1999), root and shoot development (Scheible et al., 1997b), leaf expansion (Walch-Liu et al., 2000), stomatal opening (Guo et al., 2003), flowering (Bernier et al., 1993), seed set, and senescence (Crawford, 1995;Stitt, 1999;Crawford and Forde, 2002). Some molecular components involved in regulation of these N/NO 3 2 responses in Arabidopsis thaliana have been described. The MADS box transcription factor (TF) ARABIDOPSIS NITRATE REGULATED1 (ANR1) was found to act as regulator of systemic NO 3 2 repression and localized NO 3 2 stimulation of lateral root growth (Zhang and Forde, 1998). NITRATE TRANSPORTER1.1 (NRT1.1) was subsequently shown to act upstream of ANR1 in the signaling pathway triggering lateral root growth and root colonization of NO 3 2 -rich patches (Remans et al., 2006) and to regulate expression of the high-affinity NO 3 2 transporter NRT2.1 (Muñ os et al., 2004). Also, NRT2.1 is suspected to repress lateral root initiation in response to nutritional cues by acting either as a NO 3 2 sensor or signal transducer (Little et al., 2005). Recently, a CBL-interacting protein kinase, CIPK8, was shown to regulate parts of the primary NO 3 2 response, incl...