Acidic α-galactosidase is the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum)

Abstract

Background and aims: To date, most floral nectarins (nectar proteins) are reported to function in nectar defence, particularly for insect-pollinated outcrossing species. We compared nectarin composition and abundance in selfing common tobacco (Nicotiana tobaccum) with outcrossing ornamental tobacco plants to elucidate the functional difference of nectarins in different reproductive systems.

Methods: Common tobacco (CT) nectarins were separated by SDS-PAGE and the N terminus of the most abundant nectarin was sequenced via Edman degradation. The full-length nectarin gene was amplified and cloned from genomic DNA and mRNA with hiTail-PCR and RACE (rapid amplification of cDNA ends), and expression patterns were then investigated in different tissues using semi-quantitative reverse transcriptase PCR. Additionally, high-performance liquid chromatography and enzymatic analyses of nectar sugar composition, and other biochemical traits and functions of the novel nectarin were studied.

Key results: The most abundant nectarin in CT nectar is an acidic α-galactosidase, here designated NTα-Gal. This compound has a molecular mass of 40 013 Da and a theoretical pI of 5·33. NTα-Gal has a conserved α-Gal characteristic signature, encodes a mature protein of 364 amino acids and is expressed in different organs. Compared with 27 other melliferous plant species from different families, CT floral nectar demonstrated the highest α-Gal activity, which is inhibited by d-galactose. Raffinose family oligosaccharides were not detected in CT nectar, indicating that NTα-Gal does not function in post-secretory hydrolysis. Moreover, tobacco plant fruits did not develop intact skin with galactose inhibition of NTα-Gal activity in nectar, suggesting that NTα-Gal induces cell-wall surface restructuring during the initial stages of fruit development.

Conclusions: α-Gal was the most abundant nectarin in selfing CT plants, but was not detected in the nectar of strictly outcrossing sister tobacco species. No function was demonstrated in antimicrobial defence. Therefore, floral nectarins in selfing species maintain their functional significance in reproductive organ development.

Fig. 1.

HPLC-ELSD chromatogram of tobacco floral nectar. HPLC analysis was performed using an Agilent 1100 Elite P230 series HPLC-ELSD system equipped with a Cosmosil Sugar-D column (25 × 4·6 mm, i.d. 5 µm).

Fig. 2.

SDS-PAGE of tobacco nectar proteins. Lane 1 shows different ranges of reference proteins with the molecular masses at the standards indicated; lane 2 contains tobacco nectar proteins under reducing and denaturing conditions. The six distinguishable protein bands are labelled NTB1 to NTB6. NTB5 was selected for N-terminal protein sequence analysis.

Fig. 3.

NTα-Gal gene. (A) Schematic representation of the genomic organization of NTα-Gal. The genomic structure of NTα-Gal is characterized by 15 exons (indicated by a rectangular box) and 14 introns. An initiation codon and a termination codon are shown. Binding sites and primer directions are indicated by arrows and labelled as in Table 2. All primers for NTα-Gal amplification were located on exons except primer intR1. (B) Nucleotide (GenBank accession no. HQ877671) and deduced amino acid sequences of NTα-Gal. The nucleotide sequence is numbered to the right. The deduced amino acids are shown in a one-letter code above the corresponding codons. Signal peptide sequence data are shown in italics. The N-terminal sequence determined by Edman degradation, stop codon and poly(A) addition signal are indicated in bold. Conserved amino acids acting in NTα-Gal enzymatic activity are boxed.

Fig. 4.

Semi-quantitative RT-PCR analysis of NTα-Gal expression in flower, stem and leaf. The housekeeping gene, actin, was used as the control.

Fig. 5.

α—Gal activity in nectar. (A) α—Gal activity in floral nectar of 28 plant species; (B) effects of pH on NTα—Gal activity; (C) effects of temperature on NTα—Gal activity; (D) effects of temperature on NTα—Gal stability; (E) inhibitory effects of monosaccharides and disaccharides on NTα—Gal activity.

Fig. 6.

Fresh weights of common tobacco plant fruits treated with d-galactose and d-lactose. Photos of typical fruits treated with galactose, lactose and control are shown above each bar. GAL, d-galactose; CK, control; LAC, lactose. Standard errors are given and different letters indicate significant differences by Bonferroni post-hoc test at P < 0·05.