Functional Characterization of Invertase Inhibitors PtC/VIF1 and 2 Revealed Their Involvements in the Defense Response to Fungal Pathogen in Populus trichocarpa

Abstract

In higher plants, cell wall invertase (CWI) and vacuolar invertase (VI) were considered to be essential coordinators in carbohydrate partitioning, sink strength determination, and stress responses. An increasing body of evidence revealed that the tight regulation of CWI and VI substantially depends on the post-translational mechanisms, which were mediated by small proteinaceous inhibitors (C/VIFs, Inhibitor of β-Fructosidases). As yet, the extensive survey of the molecular basis and biochemical property of C/VIFs remains largely unknown in black cottonwood (Populus trichocarpa Torr. & A. Gray), a model species of woody plants. In the present work, we have initiated a systematic review of the genomic structures, phylogenies, cis-regulatory elements, and conserved motifs as well as the tissue-specific expression, resulting in the identification of 39 genes encoding C/VIF in poplar genome. We characterized two putative invertase inhibitors PtC/VIF1 and 2, showing predominant transcript levels in the roots and highly divergent responses to the selected stress cues including fusarium wilt, drought, ABA, wound, and senescence. In silico prediction of the signal peptide hinted us that they both likely had the apoplastic targets. Based on the experimental visualization via the transient and stable transformation assays, we confirmed that PtC/VIF1 and 2 indeed secreted to the extracellular compartments. Further validation of their recombinant enzymes revealed that they displayed the potent inhibitory affinities on the extracted CWI, supporting the patterns that act as the typical apoplastic invertase inhibitors. To our knowledge, it is the first report on molecular characterization of the functional C/VIF proteins in poplar. Our results indicate that PtC/VIF1 and 2 may exert essential roles in defense- and stress-related responses. Moreover, novel findings of the up- and downregulated C/VIF genes and functional enzyme activities enable us to further unravel the molecular mechanisms in the promotion of woody plant performance and adapted-biotic stress, underlying the homeostatic control of sugar in the apoplast.

Keywords: apoplast; defense response; drought; invertase inhibitor; pathogen; poplar; sucrose.

Figure 1

Genomic structures and the chromosomal distribution of PtC/VIF candidate genes. (A) Gene structures showing the exon/intron organization that was analyzed by the online tool GSDS. The full-length sequences of mRNA were aligned by ClustalW Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) to generate the Neighbour-joining tree with branched length by a cladogram, and on the left, the gene classification was indicated. The lengths of exons are displayed proportionally to the scale on the bottom (B) Thirty-nine PtC/VIF/PMEIs were anchored on 16 chromosomes. Pairs of gene speculated to have undergone segmental/tandem duplication are lined and labeled in the same color.

Figure 2

The in silico prediction of the cis-regulatory elements in the promoters. Upstream 1.5 kb sequences of each gene promoter were analyzed in the PlantCARE server. The stress- and phytohormone-related cis-regulatory elements are boxed in different colors.

Figure 3

The deduction of conserved motifs and amino acid residues. The distribution of signal peptides, conserved domain, and 15 motifs of 39 PtC/VIF/PMEIs was programmed by Pfam (32.0) and MEME. The motif-1 contains the first pair of Cys residues that were characterized to be involved in the formation of the disulfide bridge.

Figure 4

Phylogenetic relationships of PMEI and C/VIF homologs between poplar and other plant species. Multiple protein sequences of PMEI/C/VIF were aligned with the other nine plant species by ClustalW. The unrooted phylogenetic tree was constructed by MEGA7 (https://www.megasoftware.net/) using the neighbor-joining method (Kumar et al., 2016). The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in the 1000 bootstrap test is shown next to the branches. The experimentally verified PMEI and C/VIF were reported in N. tabacum (Nt), A. thaliana (At), B. vulgaris (Bv), I. batatas (Ib), C. intybus (Ci), G. max (Gm), S. lycopersicum (Sl), S. tuberosum (St), and A. deliciosa (Ad). The accession numbers in Genbank are adjacent to the corresponding genes.

Figure 5

Expression profiles of PtC/VIF/PMEIs in various tissues and effects on transcripts of PtC/VIF1 and 2 upon stress factors. (A) Transcriptomic analyses in a heat map showing the transcript abundance of PtC/VIF/PMEIs in vegetative tissues of P. trichocarpa. (B, C) qRT-PCR analyses showing the tissue-specific expression and (D, E) the transcript effects of PtC/VIF1 and PtC/VIF2 upon the pathogenic F. solani, drought, ABA, wound, and seasonal senescence. The RNA-seq results were given in fragments per kilobase per million reads expression values. The heat map presented for RNA-seq was generated by the online program CIMminer (http://discover.nci.nih.gov/cimminer/home.do). Expression data represent mean values standard error (± SE) of at least three independent biological replicates for qRT-PCR. PtActin, PtUBIC, and PtEFα1 were used as reference genes. The asterisks indicate significant differences in comparison with the control using Student’s t-test: ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 6

Apoplastic localizations of PtC/VIF1 and 2 in tobacco and Arabidopsis. (A-F) Tobacco leaves were co-infiltrated with A. tumefaciens (C58C1) culture harboring the florescent fusion constructs of 35S: PtC/VIF1: YFP and 35S:AtCIF1: RFP or 35S: PtC/VIF2: YFP and 35S:AtCIF1: RFP. (A, D) Epidermal cells of tobacco leaf depicting YFP (green) fluorescence. (B, E) The red fluorescent signals of a cell wall marker AtCIF1. (C, F) The yellow fluorescent signals captured from the overlap of YFP and RFP fusion. (G) Images of CLSM in transgenic Arabidopsis showed the yellow fluorescent (green) signal of PtC/VIF1. Fluorescent images showing (H) YFP (green) signals, (I) the contracted vacuoles, (J) PI staining (red), and (K) the overlapping signals (yellow) from YFP and PI after plasmolysis (200 mM mannitol). PI (propidium iodide) was used as a marker to track the cell wall for fresh cells. The Arabidopsis seedlings grew for five days under short-day conditions and were harvested for the CLSM analysis.

Figure 7

The inhibitory functions in vitro of the recombinant PtC/VIF1 and 2. (A) Multiple sequence alignment of PtC/VIF homologs showed the presence of targeting sequences (underlined in green), the four Cys residues (boxed in red), and the reported small motif PKF (boxed in blue). (B, C) SDS-PAGE analyses showed the induction and purification of the recombinant proteins. (D, E) The functional activities in vitro of recombinant proteins were determined by the inhibition of CWI and VI, which were extracted from the roots. The minimum dose input caused maximum inhibitory activities of CWI and VI was 100 ng for the recombinant PtC/VIF1, and 800 ng for the recombinant PtC/VIF2. Determination of the functional enzyme activities represents means ± SE of at least four independent biological replicates.