TTG2-regulated development is related to expression of putative AUXIN RESPONSE FACTOR genes in tobacco

Abstract

Background: The phytohormone auxin mediates a stunning array of plant development through the functions of AUXIN RESPONSE FACTORs (ARFs), which belong to transcription factors and are present as a protein family comprising 10-43 members so far identified in different plant species. Plant development is also subject to regulation by TRANSPARENT TESTA GLABRA (TTG) proteins, such as NtTTG2 that we recently characterized in tobacco Nicotiana tabacum. To find the functional linkage between TTG and auxin in the regulation of plant development, we performed de novo assembly of the tobacco transcriptome to identify candidates of NtTTG2-regulated ARF genes.

Results: The role of NtTTG2 in tobacco growth and development was studied by analyzing the biological effects of gene silencing and overexpression. The NtTTG2 gene silencing causes repressive effects on vegetative growth, floral anthocyanin synthesis, flower colorization, and seed production. By contrast, the plant growth and development processes are promoted by NtTTG2 overexpression. The growth/developmental function of NtTTG2 associates with differential expression of putative ARF genes identified by de novo assembly of the tobacco transcriptome. The transcriptome contains a total of 54,906 unigenes, including 30,124 unigenes (54.86%) with annotated functions and at least 8,024 unigenes (14.61%) assigned to plant growth and development. The transcriptome also contains 455 unigenes (0.83%) related to auxin responses, including 40 putative ARF genes. Based on quantitative analyses, the expression of the putative genes is either promoted or inhibited by NtTTG2.

Conclusions: The biological effects of the NtTTG2 gene silencing and overexpression suggest that NtTTG2 is an essential regulator of growth and development in tobacco. The effects of the altered NtTTG2 expression on expression levels of putative ARF genes identified in the transcriptome suggest that NtTTG2 functions in relation to ARF transcription factors.

Figure 1

The effect of NtTTG2 silencing on tobacco growth. The NtTTG2-silenced tobacco line TTG2RNAi4 was compared to the control WT RFP plant in terms of the vegetative growth and the expression of NtTTG2 and EXP genes. (a, b) Root monitoring after 10-day growth on the medium. (c) Plants grown in pots. Floral development stages S1–S5 are indicated. (d, e) The biomass of plants from (c). (f) Real-time RT-PCR analyses of the indicated genes expressed in roots of plants from (b). (g) Real-time RT-PCR analyses of gene expression in leaves of 45-day-old plants grown in pots and equivalent to those in (c). In (b, d-g), data shown are mean values ± standard deviation (SD) bas of results from five experimental repeats (50 plants/repeat). Data were analyzed by the ANOVA method along with one-way Fisher’s Least Significant Difference (LSD) test. Significant differences between data pairs at the corresponding time points are indicated by single asterisks (P < 0.05) or double asterisks (P < 0.01) on the graphs.

Figure 2

The effects of NtTTG2 silencing on flower and seed development. (a) Flowers at five development stages (S1-S5) from 120-d-old WT/RFP and 160-d-old TTG2RNAi4 plants. (b, c) Flower anthocyanin extracts and spectrophotometric analyses. The floral anthocyanin content was quantified as relative unit (r.u.). (d) Real-time RT-PCR analyses of floral transcripts of the NtTTG2 gene as well as the DFR and ANS genes, which are involved in the late stage of the anthocyanin biosynthesis pathway. (e) Growing fruits (left) from 140-d-old WT/RFP and 170-d-old TTG2RNAi4, and mature fruits (right) from 170-d-old WT/RFP and 210-d-old TTG2RNAi4 plants. (f) The number of seeds per mature fruit. Data were given as mean values ± SD bars of results from three (c, d) and five (f) experimental repeats (30 fruits/repeat). Significant levels (*P < 0.05; **P < 0.01) in differences between corresponding data pairs were analyzed by one-way ANOVA and LSD test.

Figure 3

The effects of NtTTG2 overexpression on main characters of tobacco development. (a) Forty-day-old plants. (b) Real-time RT-PCR analyses of EXP1 and EXP2 expression in leaves of plants from (a). (c) The biomass of plants from (a). (d) S5 flowers and mature seeds. (e) S5 flower anthocyanin content given as relative unit (r.u.). (f) The number of seeds per mature fruit. Quantitative data are presented as mean values ± SD bars of results from five experimental repeats each involving 15 plants (b, c), 30 flowers (e), or 30 fruits (f). Different letters on curve and bar graphs indicate significant (P < 0.01) differences by one-way ANOVA and LSD test.

Figure 4

Assessment of the transcriptome assembly quality. (a) Components of raw reads. The character N in the “Containing N” type indicate that nucleotides were not recognized. Different colors represent components. (b) Unigene length distribution.

Figure 5

Distributions of unigenes in seven public databases.

Figure 6

Characteristics of similarity search against Nr databases. (a) E-value distribution of BLAST hits for unigenes with a cutoff E-value of 1.0E-5. (b) Similarity distribution of the top BLAST hits for unigenes. (c, d) Organism species distribution of the top BLAST hits for unigenes in Nr database. The numbers of unigenes are indicated together with percentages placed in parentheses.

Figure 7

GO classifications of assembled unigenes.

Figure 8

KOG/COG classification of unigenes.

Figure 9

KEGG classification of unigenes. Capital letters back to the colored bars indicate five main categories: A, Cellular Processes; B, Environmental Information Processing; C, Genetic Information Processing; D, Metabolism; and E, Organism Systems.

Figure 10

Phylogenic protein families involved in auxin responses and identified in the transcriptome. The number of genes in the corresponding protein family group (bottom character panel) is shown on top of the bar graph. The “Others” group mainly contains auxin-repressed and auxin-induced proteins that do not belong to any of the designated groups.

Figure 11

The effects of NtTTG2 on the expression of putative ARF genes. The effects were determined by analyzing the expression of putative ARF genes in WT RFP, P35S:TTG2:RFP#1, and TTG2RNAi4 plants. Putative ARF genes are shown as identification code in the transcriptome inventory. Gene expression levels in the control plant were regarded as 1 to qualify relative amounts of the same gene transcripts in the other phenotypes of plant. Data shown are mean values ± SD bars of results from three experimental repeats (15 plants/repeat). Different letters on bar graphs indicate significant (P < 0.01) differences by one-way ANOVA and LSD test.