Origin of plant auxin biosynthesis in charophyte algae Chunyang Wang 1, 2* , Yang Liu 2* , Si-Shen Li 2 , and Guan-Zhu Han 1, 3 1 Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, China 2 State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’an, Shandong 271018, China 3 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721, USA The TRYPTOPHAN AMINOTRANSFERASE OF ARABI- DOPSIS (TAA)/YUCCA flavin monooxygenase (YUC) pathway is the main auxin biosynthesis pathway in plants [1]. The origin of plant auxin biosynthesis remains unclear and controversial. Recently Yue et al. addressed this issue by performing phylogenetic analysis of TAA and YUC proteins [2]. They found that land plant TAA proteins are most closely related to proteins from non-plant eukaryotes (such as Choanoflagellida and Haptophyta). Moreover, by searching against expressed sequence tag (EST) database, Yue et al. claimed that no TAA and YUC protein homologs were found in Charophyta, a group of freshwater green algae from which land plants originated [3,4]. Therefore, they concluded that there is ‘little phylo- genetic evidence for the presence of the TAA/YUC pathway in any algal group’ and thus suggest the TAA/YUC path- way originated in the most recent common ancestor (MRCA) of land plants [2]. However, EST data only repre- sent a fraction of the genomic information of an organism. When one searches against EST data to identify protein homologs, the presence of significant hits indicates the presence of homologs, but the absence of significant hits does not necessarily indicate the absence of homologs. Recently the genome sequence of one charophyte (Kleb- sormidium flaccidum) and transcriptome data of four charophytes (Nitella hyalina, Nitella mirabilis, Penium margaritaceum, and Spirogyra pratensis) have been pub- lished [3,5]; we have made use of these data to revisit the question of the origin of the TAA/YUC pathway in plants. To obtain a more complete picture, the genome sequences of three representative chlorophytes (Coccomyxa subellip- soidea, Ostreococcus lucimarinus, and Ostreococcus tauri) were also included in our study. We found significant homologs (using the BLAST algorithm with Arabidopsis thaliana proteins as queries and an e value threshold of 10 À5 ) of both TAA and YUC proteins in the K. flaccidum genome, and significant homologs of YUC proteins in the N. hyaline and N. mirabilis transcriptome data and in the three chlorophyte genomes. It is worth noting that, similarly to using EST data, the absence of significant hits in transcriptome data (especially when the coverage is low) does not necessarily indicate the absence of homologs. One might argue that these algal proteins are distantly related homologs of TAA and YUC proteins in land plants. To assess this possibility, we conducted phylogenetic anal- ysis of TAA and YUC homologs of bacteria, archaea, and eukaryotes (including land plants, charophytes, and other eukaryotes). Our phylogenetic analysis (Figure 1) shows that the K. flaccidum TAA homolog (kfl00051_0080) groups with land plant TAA proteins, and the K. flaccidum YUC homolog (kfl00109_0340) and the N. hyaline YUC homolog (JO301227) group with land plant YUC proteins. There- fore, our results provide clear evidence that the TAA/YUC pathway is already present in Charophyta. We found that plant TAA proteins nest within the diversity of TAA homologs of non-plant eukaryotic origin; the most parsimonious explanation is a horizontal gene transfer (HGT) event of TAA proteins from non-plant eukaryotes to plants. Yue et al. suggest that YUCs in land plants are derived from an HGT event from bacteria to the MRCA of land plants [2]. However, we believe that the evolutionary scenario might be complex and involve multiple HGT events. Three equally probable explanations for the phylogenetic topology of YUC-related proteins could be conceived: (i) an HGT event from bacteria to plants, (ii) an HGT from bacteria to plants and a subsequent HGT from plants to bacteria, and (iii) independent HGT events from bacteria to land plants and Charophyta. Sequencing of additional charophyte and basal land plant genomes might reveal which is the most likely explanation. Never- theless, our results show that the TAA/YUC pathway is present in Charophyta, and this is consistent with the finding that endogenous auxins have been detected in K. flaccidum [6]. Our results illustrate the limitations of proving the absence of particular proteins in an organism from EST data alone. Based on the limited information available, we are not sure whether Charophyta acquired TAA and YUC proteins synchronously or asynchronously. The possibility that the earlier-branching charophytes (such as Mesostigma and Chlorokybus) only have one of the proteins cannot be formally excluded. Given that our analyses pinpoint the presence of the TAA/YUC pathway in Charophyta, Yue et al. seem to overstate the role of the origin of TAA/YUC auxin biosyn- thesis pathway in the emergence of land plants [2]. Because microbes exist everywhere, regulating and counteracting the microbial activity proposed by Yu et al. [2] might be still a potential mechanism for auxin biosynthesis initially evolving in algae. However, the microbes may not be those encountered in the terrestrial environment as proposed by Yu et al. [2], and are more likely to be those encountered in the aquatic environment. Interestingly, the presence Letters 1360-1385/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2014.10.004 Corresponding authors: Li, S.-S. (ssli@sdau.edu.cn); Han, G.-Z. (guanzhu@email.arizona.edu) * These authors contributed equally to this work. Trends in Plant Science, December 2014, Vol. 19, No. 12 741