The microRNA159-regulated GAMYB-like genes inhibit growth and promote programmed cell death in Arabidopsis

Abstract

The microRNA159 (miR159) family represses the conserved GAMYB-like genes that encode R2R3 MYB domain transcription factors that have been implicated in gibberellin (GA) signaling in anthers and germinating seeds. In Arabidopsis (Arabidopsis thaliana), the two major miR159 family members, miR159a and miR159b, are functionally specific for two GAMYB-like genes, MYB33 and MYB65. These transcription factors have been shown to be involved in anther development, but there are differing reports about their role in the promotion of flowering and little is known about their function in seed germination. To understand the function of this pathway, we identified the genes and processes controlled by these GAMYB-like genes. First, we demonstrate that miR159 completely represses MYB33 and MYB65 in vegetative tissues. We show that GA does not release this repression and that these transcription factors are not required for flowering or growth. By contrast, in the absence of miR159, the deregulation of MYB33 and MYB65 in vegetative tissues up-regulates genes that are highly expressed in the aleurone and GA induced during seed germination. Confirming that these genes are GAMYB-like regulated, their expression was reduced in myb33.myb65.myb101 seeds. Aleurone vacuolation, a GA-mediated programmed cell death process required for germination, was impaired in these seeds. Finally, the deregulation of MYB33 and MYB65 in vegetative tissues inhibits growth by reducing cell proliferation. Therefore, we conclude that miR159 acts as a molecular switch, only permitting the expression of GAMYB-like genes in anthers and seeds. In seeds, these transcription factors participate in GA-induced pathways required for aleurone development and death.

Figure 1.

Flowering time of myb33.myb65 and mir159ab. Flowering time under long-day (A) and short-day (B) conditions of wild-type Columbia-0 (Col-0), myb33.myb65, and mir159ab is shown. Error bars represent sd, and asterisks mark statistically significant changes.

Figure 2.

GA response of the miR159-GAMYB pathway in vegetative tissues. A, Effect of GA and control (ethanol) treatments on the flowering time of wild-type Columbia-0 (Col-0) and mutant plants under short-day conditions. B, mRNA levels of MYB33 and MYB65 and the levels of mature miR159a and miR159b in wild-type SARs. C, LFY, GA3OX1, and SCL3 mRNA levels in wild-type Columbia-0 and myb33.myb65 SARs grown under short-day conditions. D, Petiole length of 23-d-old third leaves of plants grown under long-day conditions. E, mRNA levels of MYB33 and MYB65 in 32-d-old wild-type rosettes grown under short-day conditions. Black bars represent control (ethanol-treated) plants, and gray bars represent GA-treated plants. Error bars represent sd, and asterisks mark statistically significant changes.

Figure 3.

MYB33 is strongly repressed by miR159 in vegetative tissues. A to D, Histochemical staining for GUS activity in 14-d-old seedlings of MYB33:GUS after 5 d of staining (A and B), mMYB33:GUS after 16 h of staining (C), and wild-type Columbia-0 (Col-0) after 5 d of staining (D). Bars = 100 μm (A) and 200 μm (B–D). E, Levels of MYB33:GUS (MYB) and mMYB33:GUS (mMYB) mRNA in five independent lines detected by qRT-PCR. F, GUS activity in five independent MYB33:GUS and mMYB33:GUS averaged lines. Error bars represent sd. G, Illustration depicting the positions of the primers used to quantify the mRNA of the transgenes. The primers span the miRNA target site of the MYB33:GUS construct and therefore only detect uncleaved mRNA.

Figure 4.

Many genes up-regulated in mir159ab are GA regulated in seeds and preferentially expressed in the aleurone. A, Early GA induction in ga1-3 seeds of 16 genes up-regulated in mir159ab. B, Late GA response in ga1-3 seeds of 14 up-regulated genes in mir159ab as determined by qRT-PCR. C, Normalized expression levels for MYB33, MYB65, and the GA-induced genes in the aleurone and embryo. Numbers on top of the bars are absolute expression values in the aleurone. Data for A were obtained from AtGenExpress (Schmid et al., 2005), and data for C were obtained from the Arabidopsis eFP browser (Winter et al., 2007).

Figure 5.

Identification of GAMYB-like regulated genes in the seed. A and B, Visualization of MYB33:GUS expression (pink crystals) in the seed (A) and aleurone (B) with dark-field optics. C, Fold changes in mRNA levels of the GA-responsive genes CP1, GASA1, BXL1, BXL2, and CP in myb33.myb65, myb101, and myb33.myb65.myb101 mutants as determined by qRT-PCR. D, Genomic structure of the myb101 allele (SALK_061355). The conserved R2R3 MYB domain (R2R3) is represented in the gene. E, MYB101 mRNA levels in myb101 mutant seeds. F to H, mRNA levels of MYB33, MYB65, and MYB101 in wild-type Columbia-0 (Col-0) and ga1-3 seeds treated with GA. Error bars represent sd.

Figure 6.

The Arabidopsis GAMYB-like genes promote aleurone PCD. A, Number of PSVs per aleurone cell in wild-type Columbia-0 (Col) and myb33.myb65.myb101 (triple mutant) seeds at different time points during germination. n = 50 cells, and sd ranges from 0 to 7.71. Error bars have been omitted for clarity. B and C, Typical images of aleurone cells incubated at 30°C for 5 d without (B) and with (C) GA. D, Percentage of vacuolated aleurone layers after 5 d of incubation at 30°C with or without GA (n = 20).

Figure 7.

Trypan blue staining of third leaves of 13-d-old wild-type Columbia-0 (Col-0) and mir159ab plants. Necrotic rosette leaves from 6-week-old wild-type plants were used as a positive control. Bars = 2 mm. [See online article for color version of this figure.]

Figure 8.

mir159ab displays a hypertrophic SAM. A and B, Differential interference contrast microscopy of cleared tissue from 4-d-old wild-type Columbia-0 (Col-0; A) and mir159ab (B) seedlings. SAM regions have been shaded. C and D, Median longitudinal cross-sections of 14-d-old wild-type (C) and mir159ab (D) SAMs. The red line is delimiting the L3 and subtending meristematic region. Bars = 50 μm.

Figure 9.

Alterations to leaf development in mir159ab plants. A and B, Transverse sections of 24-d-old wild-type Columbia-0 (Col-0; A) and mir159ab (B) fifth leaves. C and D, Cryo-fracture of 24-d-old wild-type (C) and mir159ab (D) fifth leaves. E and F, Differences between the wild type and mir159ab concerning mesophyll cell size (E) and cell density (F). G to J, SEM analysis of wild-type adaxial (G) and abaxial (H) as well as mir159ab adaxial (I) and abaxial (J) surfaces. K and L, Epidermal cell number (K) and cell size (L). M and N, Midvein cross-sections in the wild type (M) and mir159ab (N). P, Phloem; X, xylem. O to R, Venation pattern of 14-d-old wild-type (O) and mir159ab (Q) cotyledons as well as wild-type (P) and mir159ab (R) first leaves. Error bars represent sd, and bars = 50 μm (A–D, G–J, M, and N) and 200 μm (O–R). [See online article for color version of this figure.]

Figure 10.

Proposed model of the miR159-GAMYB regulatory pathway in Arabidopsis. GAMYB-like proteins are present in the aleurone, indicating low activity of miR159. In this tissue, we hypothesize that they transduce the GA signal for the activation of GA-induced genes, which leads to nutrient secretion and the progression of PCD. Conversely, strong miR159 activity fully represses MYB33 and MYB65 in vegetative tissues, ensuring that aleurone-related genes remain inactive to allow the progression of growth. GA appears not associated with the miR159-GAMYB regulatory module in these tissues.