Figure 1.
The biosynthetic pathways of nicotinamide adenine dinucleotide (NAD+).
Enzymes that are present in Chlamydomonas are indicated in blue and enzymes that are absent are indicated in red. Green arrows indicate steps specific to the aspartate pathway; dark purple arrows indicate steps specific to the tryptophan pathway; gray arrows indicate steps specific to the 4-step pathway; orange arrows indicate steps specific to the 2-step pathway; black arrows indicate steps that are commonly shared by multiple pathways. Abbreviations: ASO, L-Aspartate oxidase; QS, Quinolinate synthetase; QPT, Quinolinate phosphoribosyltransferase; NMNAT, Nicotinate/nicotinamide mononucleotide adenylyltransferase; NS, NAD+ synthetase; TDO, Tryptophan 2,3-dioxygenase; IDO: Indoleamine 2,3-dioxygenase; AFMID, Arylformamidase; KMO, Kynurenine 3-monooxygenase; KYNU, Kynureninase; 3HAO, 3-Hydroxy- anthranilate 3,4-dioxygenase; NAMase, nicotinamidase; NAPRT, nicotinate phosphoribosyltransferase; NAMPT, nicotinamide phosphoribosyltransferase; SIRT, silent information regulator two. Chlamydomonas genes identified in this study are indicated in parentheses.
Figure 2.
Chlamydomonas nicotinamide-requiring nic mutants show 3-AP sensitivity.
Chlamydomonas cells were spotted on solid rich medium (R) or medium supplemented with 10 µM NAM (nicotinamide), 10 µM NMN (nicotinamide mononucleotide), 10 µM NA (nicotinic acid), 10 µM NaAD (nicotinate adenine dinucleotide), or 10 µM 3-HA (3-hydroxyanthranilate). For 3-AP (3-acetylpyridine) sensitivity assay, cells were spotted on R medium containing 16.5 mg/l 3-AP with or without the addition of various chemical substances as indicated.
Table 1.
List of Chlamydomonas enzymes involved in de novo NAD+ biosynthesis from L-aspartate.
Figure 3.
Chlamydomonas nic mutants carry various point mutations in NIC genes.
(A) Schematic diagrams show the gene structures of NIC1, NIC2, NIC7, NIC13, NIC15 and the positions of mutations are indicated by asterisks. Blue solid box, exon; solid line, intron; dashed line, undefined length of intron. Each magenta box below the gene structure indicates changes in nucleotide/amino acid in the mutants when compared to the wild-type strain. Changes in codon that results in amino acid changes are highlighted in black. (B) PCR and enzymatic digestion to identify nic13-1 I740S reversion from 18 nic13-1 UV revertants. Top, nucleotide and amino acid sequences around the mutation points in various strains. The SfcI restriction enzyme recognition site is underlined in the nic13-1 revertant sequence. Bottom, SfcI digestion products in various strains.
Figure 4.
Chlamydomonas strain CC-125 contains an insertion in the NAMPT gene.
(A) Schematic diagram shows gene structure of the Chlamydomonas NAMPT gene. An insertion in exon 2 in the npt1-1 mutant strain is indicted by an orange arrow. Blue solid box, exon; black solid line, intron; green solid box, 5′ UTR; gray solid box, 3′ UTR; magenta and red solid boxes, sequences from the putative transposon(s); hatched boxes, incomplete regions of exon 2. (B) PCR amplification of NPT1 genomic DNA around the insertional region. Upper panel, primers spanning the insertional point can amplify the wild-type NPT1 but not NPT1 with an insert. Lower panel, one primer binds to the inserted sequence while the other binds to NPT1. Thus only NPT1 with the specific inserted sequence can be amplified. (C) RT-PCR amplification of the whole coding region of NPT1. L27a, a ribosomal protein gene serves as a control [84]. Control, no DNA template was added in PCR.
Table 2.
Progeny analysis from crosses between npt1-1 and nic mutant strains.
Figure 5.
Gene expression in wild-type, npt1-1, and nic mutant cells.
Relative real-time RT-PCR was used to detect transcripts. Results represent 2 biological replicates and standard errors are indicated by error bars.
Figure 6.
Chlamydomonas SRT2 and life span extension in Chlamydomonas nic13-1 mutant strain.
(A) Schematic diagram shows gene structures of Chlamydomonas SRT1 and SRT2. (B) Pedigree of the uni3-1 mutant strain regarding flagellar numbers, redrawn from Dutcher and Trabuco [43]. (C) Schematic diagram shows how the aging experiment was performed with Chlamydomonas cells. (D) Life span in wild-type and nic13-1 cells. Results from 2 biological replicates of each strain are shown.
Figure 7.
NAD+ biosynthesis in Chlamydomonas reveals evolutionary aspects.
Evolutionary distances between different organisms are indicated on the left. Branch lengths are not to scale. Chlamydomonas reinhardtii and Volvox carteri are indicated in bold letters. Filled dots indicate the presence of a trait in organisms and open dots indicate absence. Traits found in photosynthetic organisms are indicated in green and traits associated primarily with the animal lineage are indicated in magenta. The distinct traits of NAD+ biosynthesis in Chlamydomonas and Volvox are highlighted in the orange box. 2-step, 2-step salvage pathway of NAD+ biosynthesis; 4-step, 4-step salvage pathway of NAD+ biosynthesis; Asp, de novo biosynthesis of NAD+ from aspartic acid; Trp, de novo biosynthesis of NAD+ from tryptophan.