The REPLUMLESS Transcription Factor Controls the Expression of the RECEPTOR-LIKE CYTOPLASMIC KINASE VI_A2 Gene Involved in Shoot and Fruit Patterning of Arabidopsis thaliana

Abstract

The promoter of the RECEPTOR-LIKE CYTOPLASMIC KINASE VI_A2 (RLCK VI_A2) gene contains nine binding sites for the REPLUMLESS (RPL) transcription factor. In agreement, the expression of the kinase gene was strongly downregulated in the rpl-4 mutant. Comparing phenotypes of loss-of-function mutants, it was revealed that both genes are involved in stem growth, phyllotaxis, organization of the vascular tissues, and the replum, highlighting potential functional interactions. The expression of the RLCKVI_A2 gene from the constitutive 35S promoter could not complement the rpl-4 phenotypes but exhibited a dominant positive effect on stem growth and affected vascular differentiation and organization. The results also indicated that the number of vascular bundles is regulated independently from stem thickness. Although our study cannot demonstrate a direct link between the RPL and RLVKVI_A2 genes, it highlights the significance of the proper developmental regulation of the RLCKVI_A2 promoter for balanced stem development.

Keywords: BELLRINGER transcription factor; ROP GTPase-activated kinase; phyllotaxis; replum; stem thickness; vascular bundles.

Figure 1

Comparison of the expression of the At2G18890 (RLCKVI_A2) promoter driving the GUS marker gene in wild-type (A,C,E,G,I,K,M) and rpl-4 mutant (B,D,F,H,J,L,N) genetic backgrounds.

Figure 2

The phyllotactic pattern of siliques of wild-type (wt) (A,D,G), rpl-4 (B,E,H), and rlckvi_a2 (C,F,I) mutants without (A–C) and with (D–I) ectopic expression (OX1 and OX2) of the RLCKVI_A2 gene under the control of the 35S promoter, respectively, is shown. The divergence angles of two successive siliques on the stem were determined for a minimum of ten T4 generation plants at the developmental stage 8 [28]. The distribution of measured angles falling into the indicated angle size categories is shown according to [29].

Figure 3

The replum of wild-type (wt), rpl-4, and rlckvi_a2 fruits. Scanning electron microscopy of the fruit surface (A–C) and cross-sections (D–F) verified a thinner visible (indicated by ] in (A–C)) and smaller inner (D–G) replum for the rpl-4 mutant compared to the other two lines. Overlaying cross-sections for three replums per line shows that the inner replum of the rlckvi_a2 mutant is larger than that of the wild-type (G), although the visible replum of these two lines is about the same size (A,C).

Figure 4

Stem thickness and vascular organization of wild-type (wt), rlckvi_a2, and rpl-4 mutant plants. Cross-sections of the inflorescence stems of the three investigated lines (A) were analyzed for stem cross-sectional area (B), the number of vascular bundles per stem (C), xylem size ((D); see dashed lines in (G)), the number of xylem vessels per xylem (E), and the size of xylem vessels (F). Stem parameters were determined for ten plants per line. Xylem parameters were measured for all xylems in four plants per line. The distribution of the measured values is represented as a box plot for each line. Significant differences from the wild-type were determined by Student’s t-test (p < 0.05 = *; p < 0.01 = **). The organization of vascular bundles is exemplified in (G). vb—vascular bundle; xv—xylem vessel; pc—arc of procambium.

Figure 5

Effect of ectopic RLCKVI_A2 expression on stem thickness and the vasculature. Stem cross-sectional area (A,D,G); the number of vascular bundles per stem (B,E,H); and the average size of xylems (C,F,I) were determined for wild-type (wt; (A–C)), rpl-4 (D–F), or rlckvi_a2 (G–I) plants without and with overexpression (OX1 and OX2) of the 35S:RLCKVI_A2 gene. Ten plants per line were measured. The distribution of the measured values is represented as a box plot for each line. The data of the overexpressor lines (OX1 and OX2) were compared to their respective control using Student’s t-test (p < 0.01 = **).

Figure 6

Stem cross-sections of wild-type (wt) and mutant plants (rpl-4 or rlckvi_a2) without and with overexpression (OX1 and OX2) of the 35S:RLCKVI_A2 gene. Red asterisks indicate closely placed/clustered vascular bundles.