EPFL Signals in the Boundary Region of the SAM Restrict Its Size and Promote Leaf Initiation

Abstract

The shoot apical meristem (SAM) enables the formation of new organs throughout the life of a plant. ERECTA family (ERf) receptors restrict SAM size and promote initiation of leaves while simultaneously supporting establishment of correct phyllotaxy. In the epidermis and during organ elongation ERf activity is regulated by a family of Epidermal Patterning Factor-Like (EPFL) secreted Cys-rich small proteins. Here we show that ERfs play a critical role in communication between the SAM leaf boundary and the central zone in Arabidopsis (Arabidopsis thaliana). Ectopic expression of ERECTA in the central zone using the CLAVATA3 promoter is sufficient to restrict meristem size and promote leaf initiation. Genetic analysis demonstrated that four putative ligands: EPFL1, EPFL2, EPFL4, and EPFL6 function redundantly in the SAM. These genes are expressed at the SAM-leaf boundary and in the peripheral zone. Previously EPFL4 and EPFL6 have been linked with elongation of aboveground organs. Here we demonstrate that EPFL1 and EPFL2 promote organ elongation as well. In addition, we show that expression of ERECTA in the central zone of the SAM has a strong impact on elongation of internodes and pedicels and growth of leaves. These results suggest that ERfs can stimulate organ growth cell nonautonomously.

Figure 1.

Ectopic expression of ERECTA (ER) in the SAM using heterologous promoters. A, Representative DIC images of in situ hybridization with a sense and an antisense probe for ER using 3-DPG T3 or T4 transgenic seedlings. B, RT-qPCR analysis of ER in 5-DPG seedlings of wild-type (WT) and transgenic plants. The average of three biological replicates is presented. Error bars represent se. All images are under the same magnification.

Figure 2.

Expression of ERECTA in the central zone (pCLV3:ER) or broadly in the meristem (pSTM:ER) rescues SAM size defects the most effectively. SAM width measurements were performed by DIC microscopy using 3-DPG (A) and 5-DPG (B) seedlings. The width of SAM was measured as indicated by an arrow in Figure 8C. Three genetically independent transgenic lines were analyzed (line #1 is blue; line #2 is red; line #3 is yellow; n for each transgenic line = 7 to 11). The mean is indicated as a thick horizontal line. In all cases presented, the sd of the mean was less than 1.5 mm and can be considered insignificant. Different letters indicate significant difference at P < 0.05, as determined by one-way ANOVA with Tukey’s post test.

Figure 3.

Expression of ERECTA in the central zone (pCLV3:ER) or broadly in the meristem (pSTM:ER) rescues leaf initiation most efficiently. The number of leaf primordia formed was measured by DIC microscopy using 3-DPG (A) and 5-DPG (B) seedlings. Three genetically independent transgenic lines were analyzed for each construct and the data were combined to determine the mean (n for each transgenic line = 7 to 11; total n for each construct = 27 to 65). Error bars represent se. Different letters indicate significant difference at P < 0.05, as determined by one-way ANOVA with Tukey’s post test.

Figure 4.

Expression of ERECTA using the CLV3 or STM promoters most efficiently rescues leaf shape defects of the er erl1 erl2 mutant. A, 20-DPG plants, bar = 1 cm. B, RT-qPCR analysis of ER in leaves of wild-type (wt) and T3 to T6 transgenic plants. The average of three biological replicates is presented. Error bars represent se. All images are under the same magnification.

Figure 5.

Expression of ERECTA under a variety of promoters can fully or partially rescue elongation of stem and pedicels in the er erl1 erl2 mutant. Plant height (A) and pedicel length (B) were measured in mature 2-month-old plants. Two independent transgenic lines were analyzed; n = 10 to 30 for heights and n = 64 for pedicel length. Error bars represent sd. Different letters indicate significant difference at P < 0.01, as determined by one-way ANOVA with Tukey’s post test.

Figure 6.

A reporter gene assay of the EPF/EPFL gene family in the SAM demonstrates distinct patterns of expression. A, Longitudinal sections of shoot apices of T2 7-DPG or 10-DPG wild-type seedlings expressing indicated pEPFL:EGFP-GUS constructs. The dotted line in the EPFL6 insert emphasizes the L1 layer of the SAM. B, Epi-fluorescence microscopy of plants expressing pEPFL1:EGFP-GUS and pEPFL2:EGFP-GUS in torpedo embryos and pEPFL6:EGFP-GUS in bend cotyledons embryos. For the first two constructs, the same embryo is represented from two different perspectives. All images are under the same magnification in (A) and in (B).

Figure 7.

EPFL1, EPFL2, EPFL4, and EPFL6 synergistically regulate stem and pedicel elongation with EPFL4 and EPFL6 playing the key role. A, Height of fully grown plants (n = 27 to 46 except er erl1 erl2, n = 12). B, Lengths of mature pedicels on the main stem (n = 100 to 120). C, Number of siliques on the main stem (n = 10). A to C, Bars represent the average; error bars represent sd. Values significantly different from er-105 are indicated by asterisks (based on Student t test; P < 0.001). D, 6-week-old plants of er-105, epfl1-1 epfl2-1 epfl4 epfl6, and er-105 erl1-2 erl2-1. Scale bar: 1 cm. E, Influorescence apices from the wild type, er, er erl1 erl2, and various combinations of epfl mutants. Bar = 25 mm. All images are under the same magnification in (D) and in (E). wt, wild type.

Figure 8.

EPFL1, EPFL2, EPFL4, and EPFL6 redundantly regulate the size of the SAM and the rate of leaf initiation. Comparison of the SAM width (A) and the number of formed leaf primordia (B) in the wild type, er erl1 erl2, and epfl family mutants determined by DIC microscopy at 3 DPG (solid bars) and 5 DPG (dotted bars). Bars represent the average; error bars represent sd. n = 10 to 11. Values significantly different from the wild type are indicated by asterisks (based on Student t test; P < 0.006). C, DIC images of meristematic regions in the wild type, er erl1 erl2, and epfl1,2,4,6 at 3 DPG. The meristem width is displayed with an arrow. All images are under the same magnification in (C). wt, wild type.

Figure 9.

The meristematic phenotype of epfl1,2,4,6 can be fully rescued by expression of EPFL1 or EPFL2 under endogenous promoters or by expression of EPFL1 under KAN promoter but not CLV3. Comparison of the SAM width (A) and the number of formed leaf primordia (B) in the wild type (WT), selected mutants as indicated and in independent transgenic lines expressing indicated constructs in epfl1,2,4,6 background as determined by DIC microscopy in 5-DPG seedlings. Bars represent the average; error bars represent sd. n = 7 to 14. Different letters indicate significant difference at P < 0.01, as determined by one-way ANOVA with Tukey’s post test.