Preferential expression of atTOC159 and atTOC90 in leaves
To determine whether the expression of atTOC159 homologous genes was influenced by tissue-specific signals, promoter-GUS fusions were constructed for atToc159 family members and analyzed in transgenic Arabidopsis plants. Approximately 1-kb upstream sequences of atTOC159 homologs were individually placed into a binary vector pCAMBIA1391Z containing a GUS coding sequence as a reporter. The resulting constructs p159PUI, p132PUI, p120PUI and p90PUI contained a 1.4-kb, 0.82-kb, 0.89-kb and 1.14-kb upstream sequences of atTOC159, atTOC132, atTOC120 and atTOC90 genes, respectively (Figure 1). These plasmids were further transformed into wild type Arabidopsis by the floral dipping method. Twenty one, nine, nine and fourteen independent transgenic lines containing p159PUI, p132PUI, p120PUI and p90PUI, respectively, were obtained. These transgenic plants were named 159PUI-1 ~ 21, 132PUI-1 ~ 9, 120PUI-1 ~ 9, and 90PUI-1 ~ 14. Three homozygous T3 transformants representing each construct were used for further GUS analyses.
The GUS activity in 1-to 2-week-old leaves of various transgenic plants was compared first. Among these transgenic plants, the GUS activity of 159PUI and 120 PUI transformants had the highest and lowest expression levels in 1-week-old leaves, respectively (Figure 2). Their GUS expression levels in 2-week-old leaves were not different from those in 1-week-old leaves. Both 132PUI and 90PUI plants had a moderate expression level in 1-week-old leaves. However, the expression was reduced to 75% of that for 132PUI plants in 2-week-old leaves, but remained steady for 90PUI plants. These results suggest that atTOC159, atTOC120 and atTOC90 are expressed at a constant level through the developmental stages of vegetative growth. In contrast, atTOC132 expression was related to plant age, with highest promoter activity in the young leaves and a settled yield in the mature leaves. From above, it is obviously that atTOC159 expression is predominantly and constantly expressed whereas atTOC132 is modulated in an age-dependent manner in leaves.
Next, the expression differences of atTOC159 homologous genes in 3-week-old roots were determined. The expression levels of atTOC159 and atTOC132 were similar and both were approximately three times higher than those of atTOC120 and atTOC90 (Figure 2). This reveals that atTOC159 and atTOC132 play a more significant role than others in roots. In fact, atTOC120 and atTOC90 seemed expressing at a basal level when compared to control construct p1391Z. This implies that atTOC120 and atTOC90 might not important for the root function. However, atTOC90 expression in both 1-week-old and 2-week-old leaves was about 2.5 times higher than in roots, suggesting that atTOC90 is preferentially expressed in leaves. Similar to the expression pattern of atTOC90, atTOC159 expression in leaves was approximately 2 times higher than in roots, indicating that atTOC159 is also preferentially expressed in leaves. Altogether, among the members of atTOC159 gene family in Arabidopsis, the expression level of atTOC159 is the highest in all vegetative tissues and atTOC159 preferentially expressed in leaves. The atTOC132 is moderately expressed in all tissues and is regulated in an age-specific manner in leaves. The atTOC120 is poorly expressed in all tissues. Finally, atTOC90 is modestly and preferentially expressed in leaves, but barely expressed in roots.
Differential expression of atTOC159 homologous genes in reproductive tissues
In order to understand if the expression of atTOC159 homologous genes was modulated by developmental signals in reproductive tissues, GUS activity of flower buds and different stages of siliques collected from various transgenic plants was determined. In flower buds and 0.5-cm siliques, the GUS activity of 159PUI, 132PUI, 120PUI and 90PUI was alike and about 2 to 3 times higher than that of control plant 1391Z (Figure 3). In 1.0-cm siliques, the GUS activity of 132PUI, 120PUI and 90PUI remained about 2 times higher than that of 1391Z, but the GUS activity of 159PUI was 4.6 times higher than that of 1391Z (Figure 3). These data indicate that atTOC159, atTOC132, atTOC120 and atTOC90 all participate in the regulation of the flower and fruit development. In addition, atTOC159 appears to have a greater role during late fruit development.
Up-regulation of atTOC120 and atTOC90 by their endogenous leader intron
Leader intron in 5′ UTR is to moderate the level and pattern of gene expression in Arabidopsis (Norris et al., 1993;Jeong et al., 2006;Chen and Sun, 2010). The mechanism of intron-mediated enhancement (IME) is largely unknown, but IME might increase transcription efficiency or mRNA stability (Rose et al., 2008). As atTOC132, atTOC120 and atTOC90 have similar gene structures and include intron in 5′ UTR, we want to examine whether the leader intron sequences play a role in regulating the atTOC132, atTOC120 and atTOC90 expression in stable transgenic plants. With similar strategies, the pCAMBIA1391Z-based transgenes containing upstream sequence and 5′ UTR prior to endogenous leader intron sequence were transformed into wild type Arabidopsis to create 132P, 120P and 90P transgenic plants (Figure 1). The p132P plasmid contained 383-bp promoter and 74-bp 5′ UTR sequences of atTOC132. The p120P plasmid contained 673-bp promoter and 23-bp 5′ UTR sequences of atTOC120. The p90P plasmid included 575-bp promoter and 56-bp 5′ UTR sequences of atTOC90. The precise position of 5′ UTR and intron of atTOC159 is undetermined, so its intron effect is not able to verify in this study. Twenty three, fifty and eleven independent transgenic lines containing construct p132P, p120P and p90P, respectively, were identified. They were named 132P-1 ~ 23, 120P-1 ~ 50 and 90-1 ~ 11. Three homozygous 132P (132P-1, 132P-13, 132P-19), three homozygous 120P (120P-3, 120P-12, 120P-28) and three homozygous 90P (90P-1, 90P-3, 90P-9) T3 transformants were used for further GUS analyses.
The GUS activity of three transgenic lines contained the same construct from 5-day-old seedlings, 2-week-old leaves, and mature flowers was examined histochemically. Plant 132PUI-7, 132P-13, 120PUI-3, 120P-28, 90PUI-3 and 90P-9 were representatives in Figure 4. The 132PUI and 132P plants had similar GUS activity in all tissues examined. However, compared to 120P plant, 120PUI plant had obviously higher GUS activity in root tips, rosette leaves, and mature flowers. In comparison with 90P plant, 90PUI had higher GUS activity in cotyledons, rosette leaves and flowers, significantly notable in anthers. These data reveal that the endogenous intron sequence indeed up-regulates atTOC132 and atTOC90 expression in a tissue-specific manner, but it has no effect in moderating atTOC132 expression.