Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica

Abstract

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3-5 pmol/mg protein with an apparent dissociation constant (K (d)) between 3 and 5 microM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22-24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31-33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-(3)H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.

Fig. 1

Microsomal membranes from the moss Funaria hygrometrica were incubated on ice with 83 nM ³H IAA for 30 min in dark followed by the addition of increasing concentrations of non-radioactive IAA (0, 6.25, 12.5, 25, 50 and 100 μM) in six different reaction tubes. The reaction mixture was filtered through cellulose acetate filters, washed with binding buffer and the radioactivity is determined. The standard deviation of triplicate filters was plotted as a function of concentration of IAA. The concentration corresponding to ~K _m is shown by an arrow. The variation between samples by the filter assay was observed to be <5%

Fig. 2

Sodium cholate solubilised total proteins from moss (lane 1) and corn (lane 2) were resolved in 12% SDS-PAGE, transferred to nitrocellulose filters and probed with anti-ABP1 antibodies or pre-immune sera at a dilution of 1/1,000

Fig. 3

Cell free extracts from moss (M) and corn (C) were fractionated by differential centrifugation to obtain various pellets (P1–P3) and post-microsomal supernatant (Sup) as described in “Materials and methods”. Proteins from the pellets were solubilised with 1% sodium cholate and 15 μg of solubilised proteins from each sample were separated using 12% SDS-PAGE, transferred to nitrocellulose filters and probed with anti-ABP1 antibodies. A 28 kDa putative ABP1 specific to P3 sub-cellular fraction of moss is marked by an arrow

Fig. 4

The P2 (a, c) and P3 (b, d) sub-cellular fractions of moss (a, b) and corn (c, d) were processed for transmission microscopy as described in “Materials and methods”. The ER membrane vesicles and ribosomal dots are marked by arrows

Fig. 5

Protein samples from the membrane enriched fractions of moss, Funaria hygrometrica (M) and corn, Zea mays (C) were immuno-precipitated using anti-KDEL monoclonal antibodies. The polypeptides were separated on a SDS gel and either stained by silver (left side of the marker) or probed on a Western blot using anti-ABP1 antiserum as described in “Materials and methods”

Fig. 6

Proteins from the acetone-washed microsomal vesicles of moss were solubilised in buffer as described in “Materials and methods”. Equal amount of proteins from both buffer-soluble fraction and insoluble pellet were incubated with 0.33 μm azido-IAA in the presence of increasing amount of non-radioactive IAA for 20 min under red safe light (lanes 1–5 with 0 nM, 100 nM, 500 nM, 2.5 μM and 1 μM, respectively). The contents were exposed to UV light for 30 s, denatured in SDS sample buffer, separated using 14% SDS-PAGE. The gels were washed, fluorographed and autoradiographed for 25 days at −70°C. Note the highest labelling of the 28 kDa polypeptide in the particulate fraction

Fig. 7

Phylogenetic tree of ABP1 proteins among 13 land plants. The ABP1 sequences for each organism are indicated by a five letter code, as follows: Arath: Arabidopsis thaliana, Avesa: Avena sativa, Cerpu: Ceratodon purpureus, Cerri: Ceratopteris richardii, Glyma: Glycine max, Medtr: Medicago truncatula, Orysa: Oryza sativa, Phypa: Physcomitrella patens, Poptr: Populus tremula, Selmo: Selaginella moellendorffii, Sorbi: Sorghum bicolor, Vitvi: Vitis vinifera, Zeama: Zea mays. The phylogenetic tree was constructed using Neighbour Joining as implemented in quicktree (Howe et al. 2002) using the ScoreDist distance matrix (Sonnhammer and Hollich 2005), 1,000 bootstrap replicates and rooted at the longest internal branch

Fig. 8

Multiple amino acid sequence alignment of the auxin-binding protein 1 (ABP1) in land plants. Sequences were aligned using MAFFT L-INSI (Katoh et al. 2005). Black shaded boxes highlight identical residues. Grey shaded boxes mark similar residues. Gaps are marked by dashes in the alignment. Within the consensus line conserved amino acids are depicted in capital letters. The amino acids belonging to the KDEL retention sequence are highlighted by red squares