BLADE-ON-PETIOLE1 and 2 regulate Arabidopsis inflorescence architecture in conjunction with homeobox genes KNAT6 and ATH1

Abstract

Inflorescence architecture varies widely among flowering plants, serving to optimize the display of flowers for reproductive success. In Arabidopsis thaliana, internode elongation begins at the floral transition, generating a regular spiral arrangement of upwardly-oriented flowers on the primary stem. Post-elongation, differentiation of lignified interfascicular fibers in the stem provides mechanical support. Correct inflorescence patterning requires two interacting homeodomain transcription factors: the KNOTTED1-like protein BREVIPEDICELLUS (BP) and its BEL1-like interaction partner PENNYWISE (PNY). Mutations in BP and PNY cause short internodes, irregular spacing and/or orientation of lateral organs, and altered lignin deposition in stems. Recently, we showed that these defects are caused by the misexpression of lateral organ boundary genes, BLADE-ON-PETIOLE1 (BOP1) and BOP2, which function downstream of BP-PNY in an antagonistic fashion. BOP1/2 gain-of-function in stems promotes expression of the boundary gene KNOTTED1-LIKE FROM ARABIDOPSIS THALIANA6 (KNAT6) and shown here, ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1), providing KNAT6 with a BEL1-like co-factor. Our further analyses show that defects caused by BOP1/2 gain-of-function require both KNAT6 and ATH1. These data reveal how BOP1/2-dependent activation of a boundary module in stems exerts changes in inflorescence architecture.

Figure 1. qRT-PCR analysis of relative ATH1 transcript levels in internodes of WT, bp-2, pny, and BOP1/2 gain-of-function lines: 35S:BOP2 and bop1-6D. Plant lines, growth conditions, and experimental conditions were as previously described. WT was the Columbia-0 ecotype of Arabidopsis. Gene-specific primers for ATH1 were: ATH1-qPCR-F1 (5′-ATACTCGCTCGA-TTATTCATCTCGA) and ATH-R1 (5′-ATCGATCATCCAACCATTTGAAGAAG). Asterisks, significantly different from WT (Student’s t-tests, p < 0.0001 for all). Error bars, s.e.m.

Figure 2. Phenotypic suppression of pny-40126 and bp-2 inflorescence defects by ath1-3 loss-of-function. The ath1-3 allele (see ref. 18) was obtained from the Arabidopsis Biological Resource Center. Mutant combinations were constructed by crossing and confirmed by PCR genotyping as described.^, Inflorescences of five-week-old representative plants are shown for: (A) WT. (B) ath1-3. (C) bp-2. (D) bp-2 ath1-3. (E) pny; arrow denotes cluster of siliques. (F) pny ath1-3. (G--I) Quantitative analyses of pny phenotypic rescue by ath1-3. Seven-week-old plants were analyzed as previously described. (G) Average plant height. (H) Average number of paraclades. (I) Distribution of internode lengths; internodes were measured between the first and 11th siliques on the primary stem (counting acropetally). (J) Pedicel orientation; angles were measured with a protractor (n = 55). Scale bars, 2 cm.

Figure 3. Inactivation of ATH1 rescues compact internodes in BOP2 gain-of-function plants. Plants homozygous for a 35S:BOP2 transgene were crossed to WT control plants or ath1-3 homozygous mutants. Representative F1 plants are shown. qRT-PCR analysis confirmed that BOP2 transcripts were expressed at similar levels in both genotypes. (A) 35S:BOP2/+ Col/+. (B) 35S:BOP2/+ ath1-3/+. Scale bars, 1 cm.

Figure 4. Effect of KNAT6 and ATH1 inactivation on vascular patterning in bp-2 stems. Cross sections from the base of fully elongated primary stems were stained with phloroglucinol-HCl to detect lignin as described. At least 50 sections from 4 or 5 plants per genotype were examined. Representative sections are shown. (A) WT. (B) bp-2. (C) bp-2 knat2. (D) bp-2 knat6. (E) bp-2 ath1-3. (F) bp-2 knat2 knat6. Arrowheads denote gaps in the vascular ring. Scale bars, 100 µm.

Figure 5. Summary of genetic interactions between BP-PNY, BOP1/2, and KNAT6-ATH1 in inflorescence patterning. BP and PNY restrict BOP1/2 expression to the pedicel axil at nodes. BOP1/2 in stems induces KNAT6 and ATH1 expression, permitting the formation of a KNOX-BELL complex whose potential activity is antagonistic to BP and PNY. Arrows represent transcriptional activation. Black T-bar represents transcriptional repression. Grey T-bar represents an opposing activity.