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Systematic Analysis of Alkaline/Neutral Invertase Genes Reveals the Involvement of Smi-miR399 in Regulation of SmNINV3 and SmNINV4 in Salvia miltiorrhiza - PubMed

  • ️Tue Jan 01 2019

Systematic Analysis of Alkaline/Neutral Invertase Genes Reveals the Involvement of Smi-miR399 in Regulation of SmNINV3 and SmNINV4 in Salvia miltiorrhiza

Hong Zhou et al. Plants (Basel). 2019.

Abstract

Alkaline/neutral invertases (NINVs), which irreversibly catalyze the hydrolysis of sucrose into fructose and glucose, play crucial roles in carbohydrate metabolism and plant development. Comprehensive insights into NINV genes are lacking in Salvia miltiorrhiza, a well-known traditional Chinese medicinal (TCM) plant with significant medicinal and economic value. Through genome-wide prediction, nine putative SmNINV genes, termed SmNINV1-SmNINV9, were identified. Integrated analysis of gene structures, sequence features, conserved domains, conserved motifs and phylogenetic trees revealed the conservation and divergence of SmNINVs. The identified SmNINVs were differentially expressed in roots, stems, leaves, flowers, and different root tissues. They also responded to drought, salicylic acid, yeast extract, and methyl jasmonate treatments. More importantly, computational prediction and experimental validation showed that SmNINV3 and SmNINV4 were targets of Smi-miR399, a conserved miRNA previously shown to affect Pi uptake and translocation through the cleavage of PHOSPHATE2 (PHO2). Consistently, analysis of 43 NINV genes and 26 miR399 sequences from Arabidopsis thaliana, Populus trichocarpa, Manihot esculenta, and Solanum lycopersicum showed that various AtNINV, PtNINV, MeNINV, and SlNINV genes were regulated by miR399. It indicates that the miR399-NINV module exists widely in plants. Furthermore, Smi-miR399 also cleaved SmPHO2 transcripts in S. miltiorrhiza, suggesting the complexity of NINVs, PHO2, and miR399 networks.

Keywords: PHO2; Salvia milltiorrhiza; Smi-miR399; alkaline/neutral invertase.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Figure 1
Figure 1

Amino acid sequence alignment of eighteen neutral/alkaline invertases in S. miltiorrhiza and Arabidopsis. Dark-blue, violet and light blue shadings reflect 100%, 75%, and 50% amino acid conservation, respectively. Catalytic residues and substrate-binding residues are indicated by red and blue arrows, respectively. Signal peptide sequences predicted by Target P 1.1 were marked by red boxes.

Figure 2
Figure 2

Sequence feature of NINVs in S. miltiorrhiza. (a) Exon–intron structure of nine SmNINVs. Exons and introns are shown. (b) Conserved domains in SmNINVs. Conserved domains were predicated by searching Pfam and shown in blue boxes. Names of conserved domains are indicated.

Figure 3
Figure 3

Phylogenetic analysis of 77 alkaline/neutral invertase (NINV) proteins from eight plant species. The phylogenetic tree was constructed by the neighbor-Joining method (1000 bootstrap replicates) using MEGA version 7.0 [45]. Red dots indicate NINVs from S. miltiorrhiza. Groups and subgroups are indicated by α/β and α1/α2/β1/β2, respectively.

Figure 4
Figure 4

Distribution of conserved motifs of NINV proteins from S. miltiorrhiza and A. thaliana. (a) Sequence logos of 15 conserved motifs in NINVs. The sequences were identified and the logos were created using MEME. (b) Architecture of conserved motifs in SmNINVs and AtNINVs. The neighbor-joining (NJ) tree was constructed with full-length amino acid sequences of SmNINVs and AtNINVs using MEGA7 software [45] with 1000 bootstraps. Each motif is indicated by a colored box. Box size indicates the length of motifs. Phylogenetic groups and the number of motifs are shown.

Figure 5
Figure 5

Expression of SmNINVs in S. miltiorrhiza. (a) Number of reads per kilobase per million mapped reads (RPKM) of SmNINVs in RNA-seq data from roots (Rt), stems (St), leaves (Le), and flowers (Fl). (b) Number of reads (RPKM) of SmNINVs in RNA-seq data from root periderm (Rpe), root phloem (Rph) and root xylem (Rxy). (c) Relative expression of SmNINVs in roots (Rt), stems (St), leaves (Le) and flowers (Fl) of S. miltiorrhiza. Expression level in leaves was arbitrarily set to 1 and the levels in other organs were given relative to this. One-way ANOVA was calculated for qRT-PCR data using IBM SPSS 20 software. P < 0.05 was considered statistically significant and was represented by different letters. The bars represent standard errors. Fold changes of SmNINV expression in RNA-seq are shown by the red lines.

Figure 6
Figure 6

Expression of SmNINV genes in S. miltiorrhiza under various stresses. (a) Responses of SmNINVs to drought stress. (b) Responses of SmNINVs in S. miltiorrhiza cell cultures treated with salicylic acid (SA) for 0 (control), 2 (SA-2h), and 8 h (SA-8h). (c) Responses of SmNINVs in S. miltiorrhiza hair roots treated with methyl jasmonate (MeJA) for 0 (control), 1 (MeJA-1h) and 6 h (MeJA-6h) and yeast extract (YE) for 1 (YE-1h) and 2 h (YE-2h). P < 0.05 was considered statistically significant. * represents significant differential transcript abundance compared with control.

Figure 7
Figure 7

Smi-miR399 in S. miltiorrhiza. (a) Hairpin structures of Smi-MIR399 precursors. Mature miRNA sequences are indicated in red. miRNA* sequences are indicated in blue. (b) Number of reads per million mapped reads (RPM) of Smi-miR399s in the libraries of small RNAs from mature roots (MR), young roots (YR), stems (St), mature leaves (ML), young leaves (YL), and flowers (Fl).

Figure 8
Figure 8

Degradome analysis of Smi-miR399-directed cleavage. X-axis shows the nucleotide (nt) position of gene and Y-axis shows the number of reads obtained by degradome sequencing. Each black line represents a degradome fragment mapped to the genes. The red spots indicate that the products are resulted from Smi-miR399-directed cleavage.

Figure 9
Figure 9

Experimental validation of Smi-miR399-directed cleavage using the 5′ RLM-RACE method. Black lines represent ORFs. Grey lines represent the 5’ and 3’ UTRs. The nucleotide positions of the Smi-miR399 complementary sites (red) of genes are indicated. The mRNA sequence of each complementary site from 5′ to 3′ and the mautre Smi-miR399 sequences from 3′ to 5′ are shown. The expectations predicted using psRNATarget are shown in parentheses. Watson–Crick pairings are indicated by vertical dashes. G:U wobble pairings are indicated by circles. Vertical red arrows indicate the 5′ termini of miR399-directed cleavage products, as identified by 5′ RLM-RACE, with the frequency of clones shown.

Figure 10
Figure 10

The miR399-NINV regulatory module in other plant species. (a) Degradome analysis of Ath-miR399b/c-directed cleavage of AT1G56560 and AT3G06500. X-axis shows the nucleotide (nt) position of NINV ORFs and Y-axis shows the number of reads obtained by degradome sequencing. Each black line represents a degradome fragment mapped to the ORFs. The red spots indicate that the products are resulted from Ath-miR399b/c-directed cleavage. (b) Computational prediction of miR399-directed cleavage of NINVs in A. thaliana (At), P. trichocarpa (Pt), M. esculenta (Me), and S. lycopersicum (Sl). Heavy black lines represent ORFs. The miR399 complementary sites (red) with the nucleotide positions of SmNINVs are indicated. The mRNA sequence of each complementary site from 5′ to 3′ and the mautre miR399 sequences from 3′ to 5′ are shown in the expanded regions. The expectations predicted using psRNATarget are shown in parentheses. Watson–Crick pairings are indicated by vertical dashes. G:U wobble pairings are indicated by circles.

Figure 11
Figure 11

Relative expression levels of miR399a-miR399e, SmNINV3, and SmNINV4 in leaves of S. miltiorrhiza subjected to exogenous sucrose treatments. Fold changes of miR399s (a), SmNINV3 and SmNINV4 (b) in leaves of S. miltiorrhiza plantlets treated with 3% sucrose for 3, 12, and 24 h are shown. The level of transcripts in leaves treated with Hoagland’s medium (CK) was arbitrarily set to 1 and the levels in leaves treated with Hoagland’s medium containing 3% sucrose were given relative to this. Mean values and standard deviations were obtained from three biological and three technical replicates. ANOVA (analysis of variance) was calculated using SPSS. P < 0.05 (*) and P < 0.01 (**) were considered statistically significant and extremely significant, respectively.

Figure 12
Figure 12

Integration of the miR399-PHO2 pathway and the miR399-NINV pathway in plants. In the miR399-PHO2 pathway (blue), Pi starvation and endogenous sucrose induce the expression of miR399, which further improves Pi uptake and translocation through down-regulation of PHO2. In the miR399-NINV pathway (brown), exogenous sucrose inhibits the expression of miR399, resulting in up-regulation of NINVs and sucrose hydrolysis. MiR399 acts as an integrator of the two pathways.

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