Because the AA-RR mutation only partially blocks LapG cleavage of LapA in vivo, we predicted that LapG cleavage was occurring at additional sites or that the AA-RR mutation was not sufficient to completely block cleavage at residues 108 and 109. A BLASTP search performed previously with the LapG sequence revealed several LapA-like proteins encoded near genes encoding LapG homologs in other bacterial species (27). Alignment of the N termini of these LapA-like proteins revealed not only that alanines 108 and 109 are conserved residues but also that their position relative to the N terminus is also conserved (27). In addition to the pair of alanine residues at positions 108 and 109 ( Fig. 1A , double asterisks), the aligned LapA-like N termini contain many other conserved features relative to the pair of alanine residues (AA) at positions 108 and 109, including additional pairs of AA residues ( Fig. 1A , arrows), conserved residues flanking the double alanine residues at positions 108 and 109 ( Fig. 1A , highlighted in black), and a conserved predicted α-helical secondary structure found in 8 of 9 proteins with the closest homologs ( Fig. 1A , underlined). We predicted that these conserved elements might function in LapG-dependent cleavage of LapA.
Data were analyzed by one-way analysis of variance followed by Tukey’s posttest comparison. Significance was set at the traditional 95% confidence interval.
Interestingly, while deletion of the vWA domain was sufficient to eliminate biofilm formation on glass, loss of this domain or the entire C-terminal domain of LapA only moderately reduced biofilm formation on the hydrophobic plastic surface. Thus, the vWA domain appears to play a specific role in attachment to hydrophilic surfaces. These data suggest that perhaps different subdomains of the protein play differential roles in the ability of LapA to mediate attachment across a wide range of surfaces, a finding consistent with previous studies from our group (15, 16).
Our studies reported here have identified additional critical regions of the N terminus of LapA required for LapG proteolysis and release of LapA from the cell surface, namely, the predicted α-helix and the amino acids between residues 108 and 120. Our data are consistent with the model that LapG preferentially targets the AA motif at positions 108 and 109 in the WT LapA protein; in the absence of this primary cleavage site, LapG can apparently target alternative cleavage sites in vivo. Thus, amino acids in these regions of LapA are required for LapG cleavage and/or LapG recognition of LapA and thus likely participate in the LapG-dependent release of LapA from the cell surface under conditions unfavorable for biofilm formation.
Constructs for overexpression of NTerm-LapA variants.
Taken together, our data suggest that the AA residues at positions 108 and 109 and the predicted helical region encompassed by residues A81 to 95A are critical for LapG-dependent cleavage of LapA; however, residues immediately flanking the LapG cleavage site are dispensable for LapA cleavage. Furthermore, at least for the phenotypes described here, our findings are consistent with the conclusion that the only target for LapG is the LapA adhesin.
Tsuji H, Aya K, Ueguchi-Tanaka M, Shimada Y, Nakazono M, Watanabe R, et al. GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers. Plant J. 2006;47(3):427–44. https://doi.org/10.1111/j.1365-313X.2006.02795.x.
Taylor NG, Laurie S, Turner SR. Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis. Plant Cell. 2000;12(12):2529–39. https://doi.org/10.1105/tpc.12.12.2529.
Plackett ARG, Thomas SG, Wilson ZA, Hedden P. Gibberellin control of stamen development: a fertile field. Trends Plant Sci. 2011;16(10):568–78. https://doi.org/10.1016/j.tplants.2011.06.007.
Promotion of cell survival 1
The multifunctionality of GAs in the early stages of anther development, mainly tapetum and pollen, is well documented through analyses of A. thaliana and rice mutants. However, the late stages of anther development related to their final split are not well understood due to the earlier GA-deficiency mutant disturbances that prevent further anther development . In yellow lupine, expression profile studies have shown that fluctuating transcript levels of LlDELLA1, which encodes the main repressor of GA signalling, promote proper flower and pod development. The LlDELLA1 mRNA level is lowest when the anthers are opened and then increases during the fertilization and early pod developmental stages . This encouraged us to investigate the localization of GA3 in selected stages of late anther development in yellow lupine. Our results obtained in this work closely correlate with the expression pattern of the LlDELLA1 gene, in the opposite manner. The highest accumulation of GA3 occurs in the first LAD phase, which is before anther opening. The GA3 signal was observed mainly in degenerating septum cells, near the vascular bundle, in the middle layer, endothecium, and less frequently in the epidermis. In the second LAD phase, where the stomium cells were disrupted and the pollen chambers were opened, the fluorescence signal was almost unnoticeable, and no signal was detected in the third and fourth LAD stages. In the case of rice, GA4 accumulation was found to be highest in anthers just before anthesis. The level of active hormone molecules decreased, and was completely undetectable in the young seeds a week after anthesis . These results suggest that GAs play an important role in specific organs (place) at a specific stage of the life cycle (time) and that they may comprise the strict regulation of reproductive growth and development in different species.
Multiple Sequence Alignment by CLUSTALW. https://www.genome.jp/tools-bin/clustalw. Accessed 28 Oct 2020.
Taylor NG, Scheible WR, Cutler S, Somerville CR, Turner SR. The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis. Plant Cell. 1999;11(5):769–80. https://doi.org/10.1105/tpc.11.5.769.
Fernandez J, Wilson ZA. Non-destructive staging of barley reproductive development for molecular analysis based upon external morphology. J Exp Bot. 2012;63:4085–94.
Gibberellic acid (GA3) immunolocalization in selected stages (1–4) of late anther development (LAD) in yellow lupine. Green fluorescence corresponds to GA3 accumulation, and blue fluorescence indicates cell nuclei stained with DAPI. Subfigures A1/B1 are an enlargement of subfigures A/B, respectively, in the places marked with the red squares. The red arrows indicate GA3 signal in the selected magnified cells. The yellow squares marked with a dashed line in subfigures B and B′ indicate the same area of cells. Autofluorescence of the cell walls and pollen grains was visible. VB – vascular bundle, P – pollen grain, E – epidermis, En – endothecium, T – tapetum, C – connective, Se – septum, St – stomium. Scale bars: 25 μm (A, B, B′, C, D), 10 μm (A1) and 5 μm (B1)