SUPPRESSOR OF MORE AXILLARY GROWTH2 1 controls seed germination and seedling development in Arabidopsis

Abstract

Abiotic chemical signals discovered in smoke that are known as karrikins (KARs) and the endogenous hormone strigolactone (SL) control plant growth through a shared MORE AXILLARY GROWTH2 (MAX2)-dependent pathway. A SL biosynthetic pathway and candidate KAR/SL receptors have been characterized, but signaling downstream of MAX2 is poorly defined. A screen for genetic suppressors of the enhanced seed dormancy phenotype of max2 in Arabidopsis (Arabidopsis thaliana) led to identification of a suppressor of max2 1 (smax1) mutant. smax1 restores the seed germination and seedling photomorphogenesis phenotypes of max2 but does not affect the lateral root formation, axillary shoot growth, or senescence phenotypes of max2. Expression of three transcriptional markers of KAR/SL signaling, D14-LIKE2, KAR-UP F-BOX1, and INDOLE-3-ACETIC ACID INDUCIBLE1, is rescued in smax1 max2 seedlings. SMAX1 is a member of an eight-gene family in Arabidopsis that has weak similarity to HEAT SHOCK PROTEIN 101, which encodes a caseinolytic peptidase B chaperonin required for thermotolerance. SMAX1 and the SMAX1-like (SMXL) homologs are differentially expressed in Arabidopsis tissues. SMAX1 transcripts are most abundant in dry seed, consistent with its function in seed germination control. Several SMXL genes are up-regulated in seedlings treated with the synthetic SL GR24. SMAX1 and SMXL2 transcripts are reduced in max2 seedlings, which could indicate negative feedback regulation by KAR/SL signaling. smax1 seed and seedling growth mimics the wild type treated with KAR/SL, but smax1 seedlings are still responsive to 2H-furo[2,3-c]pyran-2-one (KAR2) or GR24. We conclude that SMAX1 is an important component of KAR/SL signaling during seed germination and seedling growth but is not necessary for all MAX2-dependent responses. We hypothesize that one or more SMXL proteins may also act downstream of MAX2 to control the diverse developmental responses to KARs and SLs.

Figure 1.

Identification of smax1 alleles. A, The smax1-1 mutation (dark triangle) causes a premature stop at codon 292 of At5g57710. Four exons shown; large triangles indicate T-DNA alleles (smax1-2: SALK_128579; smax1-3: SALK_097346). Small arrows indicate primer pair loci for the expression analysis shown in B. B, Abundance of SMAX1 transcript at three loci detected by real-time reverse transcription-PCR using complementary DNA derived from 7-d-old light-grown seedlings. Expression values are relative to the CLATHRIN ADAPTOR COMPLEX SUBUNIT (CACS) reference gene and normalized to Col-0 = 1. Mean ± se (n = three independent samples, >50 seedlings per sample). Significance assessed by Student’s t test (*P < 0.05, **P < 0.01).

Figure 2.

smax1 suppresses seed and seedling stage phenotypes of max2 mutants. The figure key applies to panels A, B, and C. A, The germination phenotype of max2 is suppressed in smax1 max2 mutants. Primary dormant seeds were grown on 0.8% agar plates containing 1 µm KAR₁, 1 µm KAR₂, or 10 µm GR24 for 5 d at 24°C. Mean ± sd (n = three experimental trials of 75–100 seeds per sample). B, smax1 suppresses the elongated hypocotyl phenotype of 4-d-old max2 seedlings grown in red light on 1 µm KAR₁, 1 µm KAR₂, or 1 µm GR24. Mean ± se (n = two experimental trials of 20–50 hypocotyls per sample). Statistical groupings were determined by ANOVA with Tukey-Kramer HSD (P < 0.01). C, smax1 suppresses the small cotyledon phenotype of 4-d-old max2 seedlings grown in red light on 1 µm KAR₁, 1 µm KAR₂, or 1 µm GR24. Mean ± sd (n = 26–50 cotyledons per sample). Statistical groupings were determined by ANOVA with Tukey-Kramer HSD (P < 0.01). D, Enhanced contrast image of 7-d-old seedlings grown on soil in high humidity in 16-h white light/8-h dark. smax1 suppresses cotyledonary petiole angle (E) and cotyledonary petiole length (F) of 7-d-old seedlings. Mean ± se (n = three experimental trials of 26–40 petioles per sample). Statistical groupings were determined by ANOVA with Tukey-Kramer HSD (P < 0.001).

Figure 3.

smax1 restores expression of KAR/SL transcriptional markers in max2 seedlings. Transcripts detected by real-time reverse transcription-PCR using complementary DNA derived from 4-d-old red-light-grown seedlings. Expression values are relative to the CACS reference gene and scaled to Col-0 = 1. Mean ± se (n = three independent samples, >50 seedlings per sample). Significant difference from Col-0 was assessed by Student’s t test (*P < 0.025, **P < 0.001).

Figure 4.

smax1 does not suppress max2 lateral root branching, axillary branching, primary inflorescence height, or dark-induced senescence phenotypes. A, Lateral roots per centimeter of primary root in 8-d-old seedlings. Mean ± se (n = two experimental trials of 19–36 roots per sample). Significant differences compared with Col-0 were assessed by Student’s t test (**P < 1×10⁻⁵). B, Thirty-nine-day-old plants. Axillary branching (C) and primary inflorescence height (D) phenotypes of 8-week-old max2 plants. Mean ± sd (n = nine 8-week-old plants per genotype). Significant difference from Col-0 was assessed by Student’s t test (*P < 0.001). E, Short-day-grown plants. Arrowheads indicate leaves kept in dark for 6 d to induce senescence.

Figure 5.

Maximum-likelihood phylogram of SMAX1 and SMAX1-like proteins in Arabidopsis. SMAX1, SMXL2 (At4g30350), SMXL3 (At3g52490), SMXL4 (At4g29920), SMXL5 (At5g57130), SMXL6 (At1g07200), SMXL7 (At2g29970), SMXL8 (At2g40130), and HSP101 were assigned to the tree by PhyML based on protein sequence similarity. Numbers above the branches represent bootstrap support derived from 100 bootstrap replicates. Scale bar (branch length) represents substitutions per site.

Figure 6.

Relative abundance of SMAX1 and SMXL transcripts in Arabidopsis tissues. Transcripts detected by real-time reverse transcription-PCR using complementary DNA derived from the following tissues from wild-type plants: dry seed (n = four samples, approximately 40 mg per sample), 4-d-old red-light-grown seedlings (n = five samples, >50 seedlings per sample), 13-d-old roots (n = four samples, >50 roots per sample), 5-cm-long sections from axillary branches on 6-week-old plants, each section containing three axillary buds and cauline leaves (n = four samples, one section per sample), 2-cm-long rosette leaves from 6-week-old plants (n = four samples, two leaves per sample), and senescent rosette leaves with approximately 50% remaining green from 7-week-old plants (n = four samples, one leaf per sample). Abundance values are relative to the CACS reference gene. Mean ± se. Statistical groupings for each tissue type were determined by ANOVA with Tukey-Kramer HSD (P < 0.01).

Figure 7.

Transcriptional responses to KAR/SL in the SMXL gene family. A, SMAX1 and SMXL transcriptional responses to growth on 1 µm KAR2 or 1 µm GR24 in 4-d-old red-light-grown seedlings. DLK2 included as a positive control for KAR₂ and GR24 response. Transcripts detected by real-time reverse transcription-PCR. Expression values are relative to the CACS reference gene. Mean ± se (n = five independent samples, >50 seedlings per sample). Significant differences compared with the control treatment were assessed by Student’s t test (*P < 0.05, **P < 0.01). B, SMAX1 and SMXL2 transcripts in 4-d-old red-light-grown max2 seedlings. Transcripts detected by real-time reverse transcription-PCR, and values are relative to CACS and scaled to Col-0 = 1. Mean ± se (n = three independent samples, >50 seedlings per sample). Significant differences to Col-0 were assessed by Student’s t test (*P < 0.05).

Figure 8.

smax1 single mutant phenotypes. The figure key applies to B, C, and D. A, Relative abundance of KAR/SL-responsive transcripts detected by real-time reverse transcription-PCR using complementary DNA derived from 4-d-old red-light-grown seedlings. Values are relative to the CACS reference gene and scaled to Col-0 = 1. Mean ± se (n = three independent samples, >50 seedlings per sample). Significant difference from Col-0 was assessed by Student’s t test (*P < 0.05). B, Hypocotyl lengths of 4-d-old seedlings grown in red light on 1 µm KAR₁, 1 µm KAR₂, or 1 µm GR24. Mean ± se (n = two experimental trials of 20–50 hypocotyls per sample). Statistical groupings were determined by ANOVA with Tukey-Kramer HSD (P < 0.01). C, Cotyledon area of 4-d-old seedlings grown in red light on 1 µm KAR₁, 1 µm KAR₂, or 1 µm GR24. Mean ± sd (n = 32–50 cotyledons per sample). Statistical groupings were determined by ANOVA with Tukey-Kramer HSD (P < 0.01). D, Primary dormant seeds were grown on 0.8% agar plates containing 1 µm KAR₁, 1 µm KAR₂, or 10 µm GR24 for 5 d at 24°C. Mean ± sd (n = three experimental trials of 75–100 seeds per sample).