AUCSIA

Abstract

Aucsia is a green plant gene family. In Angiosperms, Aucsia genes control several aspects of auxin biology, including polar auxin transport. AUCSIA miniproteins are produced via splicing of three exons. The first two exons span the conserved AUCSIA motif, while the third exon(s) encodes the more variable carboxyterminal end. AUCSIA presence in green algae indicates that the Aucsia gene family predated the emergence of land plants and the complex auxin biology of Angiosperms. In algae, however, AUCSIA might have been involved in a primitive auxin biology, when auxin was just a simple metabolite, probably noxious at high concentrations, and consequently pump out via the ancestral auxin exporters, i.e., ABCB1/19 homologs. This speculative scenario implies that in green algae AUCSIA is involved in controlling the ABCB-dependent efflux of noxious metabolites, including auxin. Such speculative hypothesis might be tested in living green algae.

AUCSIA and Auxin Biology

Two recent papers have reported the identification of a novel green plant gene family, named Aucsia.^1,2 Both in Solanum lycopersicum and Arabidopsis thaliana, suppression of Aucsia genes causes phenotypes ascribable to perturbed auxin biology. In tomato, silencing of Aucsia gene family causes parthenocarpy (i.e., the development of the fruit in the absence of fertilization), alterations of leaf morphogenesis, reduced auxin-induced rhizogenesis, and reduced polar auxin transport (PAT).¹ In Arabidopsis, ataucsia-1 mutants and AtAucsia-1 overexpressing lines have demonstrated that AtAucsia-1, by itself, is required for PAT and is involved in root auxin biology.² A kinesin-related protein has been identified as an interacting partner of AtAUCSIA-1 indicating a novel intracellular connection between components of auxin biology and the cytoskeleton.²

AUCSIA Phylogenetic Ranking

Aucsia genes are widespread in the green plant lineage (Fig. 1) and encode miniproteins ranging from 40 to 56 amino acids.^3-7 The high degree of conservation of AUCSIA proteins both in land plants, in chlorophyte and streptophyte algae, i.e., the closest relatives of extant land plants, reveals that Aucsia is a gene already present before the divergence of Chlorophyta and Streptophyta, and yet it controls fruit initiation, a biological innovation likely not older than 160 MY.

Figure 1. Phylogenetic analysis of Aucsia orthologs in green plant lineages. The amino acid sequences for Aucsia orthologs, predicted from EST clones, were aligned using the ClustalW program with default alignment parameters using BLOSUM as protein matrix.^3,4 The evolutionary history was inferred using the UPGMA method.⁵ The optimal tree with the sum of branch length = 209.37865453 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the number of differences method and are in the units of the number of amino acid differences per sequence.⁶ Evolutionary analyses were conducted in MEGA5.⁷ Taxonomy abbreviations: Am (Antirrhinum majus); Amt (Amborella trichopoda); At (Arabidopsis thaliana); Bb (Botryococcus braunii); Bp (Bathycoccus prasinos); Cg (Chaetosphaeridium globosum); Cos (Coccomyxa subellipsoidea); Cr (Cycas rumphii); Cs (Citrus sinensis); Cv (Chlorella variabilis); Eo (Elaeis oleifera); Fa (Festuca arundinacea); Gb (Ginkgo biloba); Gm (Glycine max); He (Helicosporidium sp ex Simulium jonesi); Hv (Hordeum vulgare); Lt (Liriodendron tulipifera); Mxd (Malus x domestica); Mp (Marchantia polymorpha); Mt (Medicago truncatula); Nt (Nicotiana tabacum); Os (Oryza sativa); Ol (Ostreococcus lucimarinus); Pa (Prunus armeniaca); Pem (Penium margaritaceum); Pg (Picea glauca); Php (Physcomitrella patens); Pit (Pinus taeda); Pot (Populus trichocarpa); Pp (Pinus pinaster); Psm (Pseudotsuga menziesii); Pw (Prototheca wickerhamii); Sah (Saruma henryi); Sb (Sorghum bicolor); Sh (Saccharum hybrid); Sl (Solanum lycopersicum); Sm (Selaginella moellendorffii); Ta (Triticum aestivum); Tr (Tortula ruralis); Vv (Vitis vinifera); Wm (Welwitschia mirabilis); Zm (Zea mays).

Besides land plants, auxin (IAA) is synthesized in algae and prokaryotes.⁸ The presence/absence of genes deputed to auxin biology at different levels of phylogenetic ranking within green plants, have allowed to predict the time of emergence of molecular components of auxin biology either predating (e.g., ABCB, YUC, ABP1) or appearing during land plants evolution (e.g., PIN, PID/D6PK, TIR1-AUX/IAA-ARF).^8,9 In the land plant lineage, mosses represent the first group where PAT has been experimentally ascertained, although auxin can move directionally in the gametophyte of a streptophyte alga.^10,11 Genome and transcriptome data have revealed that chlorophyte green algae possess systems for auxin import and export based on AUX1-like, ABCB/PGP-like proteins and the ER-localized transporters PILS, whereas putative orthologs of PIN transporters have not been detected.^8,12 A putative PIN protein was recently identified in an alga (Spirogyra pratensis) of the Streptophyta clade, but it appears to be localized at the ER.⁸ Thus, an ancestral auxin transport was probably deputed to regulate auxin intracellular homeostasis, while PAT emerged later, during the evolution of land plants.¹⁰ ABCB tranporters were probably the first ancestral IAA efflux transporter, predating land plants emergence. This interpretation is consistent with the finding that Ectocarpus silicosus, a brown alga, has no genetic information for auxin transporters similar either to AUX1/LAX or PIN, but it codes for two ABCB transporters with significant similarity to the ABCB19 IAA-exporter of Arabidopsis.¹³ The function of AUCSIA in unicellular algae is unknown, and yet during the conquest of land by green plants, AUCSIA proteins have been recruited in the bauplan of land plants, as indicated by their role in the auxin biology of Angiosperms.

The comparison of AUCSIA proteins of green algae, bryophytes, lycophytes and spermatophytes indicates that the central part, containing the 16 amino acids-long AUCSIA motif (PYSGXSTLALVARXSA), has been well conserved during evolution, whereas both N-terminal and C-terminal portions have undergone several modifications (Fig. 2A).^1,3,4 For instance, Aucsia ortologues in Ostreococcus lucimarinus and Bathycoccus prasinos within the Prasinophyceae, an early branch of the Chlorophyta, are 44 and 40 amino acids-long, respectively, whereas in streptophytes AUCSIA proteins are longer than 50 amino acids. The C-terminal portion of AUCSIA proteins is generally characterized by a high content of lysine, and commonly by two terminal histidine residues (Fig. 2A) except for some of green algal Aucsia orthologs.^3,4 In addition, the N-terminal region has been expanded in bryophytes and spermatophytes as compared with green algae (Fig. 2A).^3,4 All the modifications observed in the aminoacid sequence of land plants AUCSIA proteins might be further elaborated via post-translational modifications.¹ Consequently, some AUCSIA features, for instance cellular localization, stability and interaction with protein partners, might have been diversified during plant evolution.

Figure 2. (A) Sequence comparison of putative AUCSIA peptides among green plants. The amino acid sequences for Aucsia orthologs were aligned using the ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) program with default alignment parameters selecting BLOSUM for the protein matrix, and choosing the input order of the sequences for output option.^3,4 Consensus symbols: “*” identical residues; “.” residues that are somewhat similar. Black arrows indicate the position of the two introns deduced from the analysis of AUCSIA-1 and AUCSIA-2 from Arabidopsis thaliana genome. Representative members of chlorophytes (B.prasinos, O.lucimarinus, C.subellipsoidea, B.braunii, C.variabilis, P.wickerhamii, H. ex Simulium jonesi), charophytes (P.margaritaceum, C.globosum) and land plants (M.polymorpha, P.patens, S.moellendorffii, G.biloba) are reported in the alignment. (B) Comparison of Aucsia ortologous gene structures in different Angiosperms. The structure of Aucsia genes appears quite stable among different species, with two introns that divide the coding sequences into three exons. The second exon encodes for 22–23 amino acids and is flanked by 5′ and 3′ exons encoding 16 and 13–17 aminoacids, respectively.

AUCSIA Gene Family

The structure of Aucsia genes is conserved in Angiosperms (Fig. 2B).^3,4 AUCSIA coding region comprises three exons, the length of the first two exons is rather conserved, whereas the size of the third exon is variable (Fig. 2B).^3,4 The Aucsia genes of O. lucimarinus and B. prasinos do not contain introns. The EST databases of several angiosperms have revealed the existence, in most of the analyzed cases, of two different Aucsia transcripts. At the genomic level, we unequivocally identified two Aucsia genes in Arabidopsis thaliana, Vitis vinifera, Zea mays, Glycine max and three genes in Popolus trichocarpa (Fig. 2B).^3,4 In A. thaliana, we have shown that the two Aucsia genes have different patterns of expression, suggesting that after duplication the two genes were subjected to sequence divergence in their promoter regions and likely to sub-functionalization via differential expression.² A further layer of complexity within AtAucsia genes is the presence of alternative transcripts. The AtAucsia-1 splicing variant (At3g01130.2) encodes a putative 50 amino acids-long protein. The first and the second exons are identical to those observed in the original protein, while the third is shorter and displays a quite different amino acidic composition. The AtAucsia-2 splicing variant (At5g15320.2) encodes a protein isoform of 50 amino acids, that differs from the original protein for the length of the second exon that lacks the three terminal aminoacids. The presence of Aucsia splicing variants encoding putative shorter proteins is not limited to A. thaliana (e.g., V. vinifera, data not shown).

The molecular function(s) of AUCSIA proteins is currently unknown. This evolutionary analysis and the occurrence of Aucsia homologs in unicellular chlorophyte algae found in marine and freshwater habitats and even in algae with saprophytic feeding habits, suggest an ancestral function for Aucsia genes, perhaps in the cellular homeostasis of IAA and other metabolites noxious at high concentrations. The modifications emerged in land plants Aucsia orthologs at the level of gene structure, coding sequence and transcript processing, are consistent with the cooptation of AUCSIA miniproteins in the bauplan of land plants and the subsequent acquisition of novel roles, likely via subfunctionalization(s), in auxin biology and in the development of new organs, such as roots, flowers, fruits.

Glossary

Abbreviations:

IAA

indole-3-acetic acid

PAT

polar auxin transport

MY

million year

bauplan

body plan

PIN

PIN-formed

AUX1/LAX

AUXIN1/ LIKE AUX1

PILS

PIN-LIKES

YUC

YUCCA

ABCB/PGP

ATP Binding Cassette subfamily B /P-glycoprotein

ABP1

AUXIN BINDING PROTEIN1

TIR1-AUX/IAA-ARF

TRANSPORT INHIBITOR RESPONSE1/ indole-3-acetic acid-auxin response factor

PID/D6PK

PINOID/D6 PROTEIN KINASE

ER

endoplasmic reticulum

Molesini B, Pandolfini T, Pii Y, Korte A, Spena A. Arabidopsis thaliana AUCSIA-1 regulates auxin biology and physically interacts with a kinesin-related protein. PLoS One. 2012;7:e41327. doi: 10.1371/journal.pone.0041327.