Localization of tomato (Solanum lycopersicum) R3 MYB protein encoded by SlTRY in Arabidopsis roots

ABSTRACT

CAPRICE (CPC), an R3-type MYB transcription factor, is known to promote root hair differentiation in the root epidermis of Arabidopsis. CPC moves from non-hair cells to adjacent hair-forming cells. In contrast, we have previously shown that there is no movement of the CPC homologs, ENHANCER OF TRY AND CPC 1, 2, and 3 (ETC1, 2, and 3), and TRYPTICHON (TRY) between root epidermal cells. We also identified a tomato homolog of CPC, named Solanum lycopersicumTRYPTICHON (SlTRY).SlTRY-introduced transgenic Arabidopsis produced many root hairs, like CPC-introduced transgenic Arabidopsis. To clarify the cell-to-cell movement ability of the SlTRY protein, in this study, we observed the distribution of GFP fluorescence in CPCp:SlTRY:GFP transgenic Arabidopsis. Unexpectedly, SlTRY moved from non-hair cells to adjacent root hair cells, like CPC, in Arabidopsis root epidermis. SlTRY does not have the cell-to-cell movement sequence (S1) defined in CPC, and the mechanism of movement is still unknown. Further investigation is necessary to elucidate the mechanism of cell-to-cell movement of SlTRY. Our results will help in the further unraveling of the functions of these MYB transcription factors in determining cell fate.

KEYWORDS:

• Arabidopsis
• MYB
• SlTRY
• tomato

CAPRICE (CPC) was identified as a gene that induces the root hairs of Arabidopsis thaliana.¹ CPC encodes an R3-type MYB transcription factor.¹ Homologous genes in Arabidopsis with sequences similar to CPC include TRYPTICHON (TRY), ENHANCER OF TRY AND CPC 1, 2, and 3 (ETC1, ETC2, and ETC3), and TRICHOMELESS 1 and 2 (TCL1 and TCL2).^2–10 These six CPC homologs induce root hairs similar to CPC in Arabidopsis. Arabidopsis roothair cells are over two underlying cortical cells, whereas non-hair cells are over a single cortical cell.^11,12 This position-determined developmental manner results in lines of roothair cells along the longitudinal root axis and has been found in Brassicaceae and other eudicot families.^13–16 This stripy root hair pattern (type 3) is one of the three types of root hair cell distribution patterns.^{13–15,17–20} Previously, we reported that CPC is expressed in non-hair cells of the root epidermis, and the translated CPC protein then migrates from non-hair cells to hair-forming cells in Arabidopsis.²¹ This ability of CPC may determine the type 3 root hair pattern. The cell-to-cell movement of transcription factors, through plasmodesmata, is important for plant cell differentiation.^22,23 On the contrary, four CPC-like MYB proteins, including TRY, ETC1, ETC2, and ETC3, do not seem to move from non-hair cells to root hair cells.^24–26 Although TRY has been shown to move between leaf epidermal cells,²⁷ the cell-to-cell movement ability is likely to be specific to CPC among R3-type MYB transcription factors in Arabidopsis.

Previously, we identified tomato (Solanum lycopersicum) homolog of Arabidopsis CPC and named it Solanum lycopersicumTRYPTICHON (SlTRY).²⁸ SlTRYwas so named because its encoded protein is more closely related to TRY than to CPC.²⁸ Tomato shows a root hair pattern different from Arabidopsis (type 3). Tomato belongs to the most prevalent type 1 root hair pattern group in which all root epidermal cells can generate root hairs.²⁹ SlTRY-introduced transgenic Arabidopsis plants produce many root hairs, a phenotype similar to CPC or CPC-like MYB-introduced transgenic Arabidopsis plants.²⁹ In this study, we explored protein localization and cell-to-cell movement of SlTRY in Arabidopsis.

As previously reported, the SlTRY-encoded protein is more closely related to TRY, than CPC or ETC1 (Figure 1).²⁸ However, the total number of amino acids in SlTRY protein is 94, which is the same as that in CPC (Figure 1). CPCp:SlTRY:GFP in Col-0 transgenic plants produce about 1.6-times more root hairs than are present in the wild-type Col-0.²⁸ CPCp:SlTRY:GFP in cpc-2 transgenic plants also produce a greater number of root hairs and completely complement the cpc-2 mutant phenotype with decreased number of root hairs.²⁸ We observed the GFP distribution of CPCp:SlTRY:GFP in Col-0 and CPCp:SlTRY:GFP in cpc-2 transgenic Arabidopsis roots (Figure 2).GFP fluorescence was observed in the nuclei of nearly all cells of the root epidermis of CPCp:SlTRY:GFP in Col-0 transgenic plants (Figure 2a), suggesting that tomato SlTRY is a transcription factor localized in the nucleus. Furthermore, it was revealed that SlTRY moved from non-hair cells, where SlTRY was expressed under the CPC promoter, to root hair cells (Figure 2a). GFP fluorescence was also observed in the nuclei of almost all the root epidermis cells of CPCp:SlTRY:GFP in cpc-2 transgenic plants (Figure 2a). Even in the cpc-2 mutant background, SlTRY showed nuclear localization and cell-to-cell movement.

Figure 1. The amino acid sequence of R3 MYB proteins. Sequence alignment of SlTRY (Solyc01g095640.1.1), CPC (FJ268773), TRY (AC007288), and ETC1 (NM100020). Black shaded letters indicate identical residues. Gray shaded letters pointed by arrows indicate identical residues where only SlTRY and CPC are identical. The R3 MYB domain is indicated by a line above the sequences. The amino acids comprising the S1 and S2 domains are indicated with boxes outlined in gray. CPC, CAPRICE; SlTRY, Solanum lycopersicum TRYPTICHON; TRY, TRYPTICHON.

Figure 2. Distribution of GFP fluorescence in CPCp:SlTRY:GFP in Col-0 and CPCp:SlTRY:GFP in cpc-2 transgenic plants. Confocal laser scanning microscopy images showing GFP (green) and PI (red) fluorescence in the root epidermis of 10-d-old seedlings. The CPCp:SlTRY:GFP in Col-0 (a) and CPCp:SlTRY:GFP in cpc-2 (b) transgenic plants were observed. CPC, CAPRICE; SlTRY, Solanum lycopersicum TRYPTICHON.

When CPC-like MYBof Arabidopsis (TRY, ETC1, ETC2, and ETC3) was expressed under the CPC promoter, the translated proteins were mainly localized in non-hair cells.^25,30-32 In contrast, CPC localized in both non-hair cells and migrated root hair cells, evenly, that is, in all the root epidermal cells.³⁰ Both CPC and CPC-like MYBs induce root hairs, but only CPC shows an obvious movement between root epidermal cells. We confirmed SlTRY:GFP fusion protein nuclear localization in all root epidermal cells for five CPCp:SlTRY:GFP in Col-0 independent homozygous transgenic lines and six CPCp:SlTRY:GFP in cpc-2 independent homozygous transgenic lines. Our results suggest that tomato SlTRY retains cell-to-cell movement ability from non-hair cells to root hair cells.

Kurata et al. identified the S1 (Sequence 1) and S2 regions in CPC, which are active sites for cell-to-cell movement of CPCin Arabidopsis epidermal cells (Figure 1).³³ The conservation of S2 (WxM) in all R3 MYBs (Figure 1) suggests that S2 is necessary but not sufficient for cell-to-cell movement ability in root epidermal cells. Surprisingly, SlTRY did not have S1, as in the case of non-mobile proteins TRY and ETC1 (Figure 1). Previously, we reported that the addition of S1 sequence to ETC1 does not allow cell-to-cell movement of ETC1.³⁴ Besides S1 and S2, there may be amino acids crucial for cell-to-cell movement. Comparing the migrating group (SlTRY and CPC) with the non-migrating group (TRY and ETC1), S, H, and K (indicated by gray arrows in Figure 1) were amino acids specific to the migrating group. These amino acids might be involved in the cell-to-cell movement of SlTRY and CPC.SlTRY and CPC, which belong to the migrating group, both have 94amino acids, so their size may be important for cell-cell movement (Figure 1). The mechanism of CPCp:SlTRY:GFPin cpc-2 mutant might be more complicated than that of Col-0.The CPC promoter might work in all epidermal cells in the cpc-2 mutant. Eventually, however, SlTRY protein moved from neighboring cells and was localized in all epidermal cells (Figure 2b).

Tomato and Arabidopsis have different root hair patterns, type1 and type 3, respectively. The function of SlTRY in tomato is still unclear. This study revealed that the unique property of cell-to-cell movement, such as in CPC, is maintained in SlTRY. Further investigation is necessary to elucidate the mechanism of cell-to-cell movement of CPC and SlTRY.

Disclosure statement

No potential conflict of interest was reported by the authors.