A novel Filamentous Flower mutant suppresses brevipedicellus developmental defects and modulates glucosinolate and auxin levels

Abstract

BREVIPEDICELLUS (BP) encodes a class-I KNOTTED1-like homeobox (KNOX) transcription factor that plays a critical role in conditioning a replication competent state in the apical meristem, and it also governs growth and cellular differentiation in internodes and pedicels. To search for factors that modify BP signaling, we conducted a suppressor screen on bp er (erecta) plants and identified a mutant that ameliorates many of the pleiotropic defects of the parent line. Map based cloning and complementation studies revealed that the defect lies in the FILAMENTOUS FLOWER (FIL) gene, a member of the YABBY family of transcriptional regulators that contribute to meristem organization and function, phyllotaxy, leaf and floral organ growth and polarity, and are also known to repress KNOX gene expression. Genetic and cytological analyses of the fil-10 suppressor line indicate that the role of FIL in promoting growth is independent of its previously characterized influences on meristem identity and lateral organ polarity, and likely occurs non-cell-autonomously from superior floral organs. Transcription profiling of inflorescences revealed that FIL downregulates numerous transcription factors which in turn may subordinately regulate inflorescence architecture. In addition, FIL, directly or indirectly, activates over a dozen genes involved in glucosinolate production in part by activating MYB28, a known activator of many aliphatic glucosinolate biosynthesis genes. In the bp er fil-10 suppressor mutant background, enhanced expression of CYP71A13, AMIDASE1 (AMI) and NITRILASE genes suggest that auxin levels can be modulated by shunting glucosinolate metabolites into the IAA biosynthetic pathway, and increased IAA levels in the bp er fil-10 suppressor accompany enhanced internode and pedicel elongation. We propose that FIL acts to oppose KNOX1 gene function through a complex regulatory network that involves changes in secondary metabolites and auxin.

Fig 1. Suppression of bp er pedicel phenotypes by the fil-10 mutation.

A bp er plant showing short pedicels that bend downwards. (B) bp er fil-10 plant exhibiting enhanced internode growth and elongated pedicels perpendicular to the stem axis. The acute pedicel angle defect is partially ameliorated. (C) bp er inflorescence cluster with closed floral buds. (D) Young bp er fil-10 flowers with visible inner whorl organs due to aberrant sepal development. (E) Hand section of a bp er pedicel. Note the lack of chlorenchyma development on the abaxial side (arrow). (F) Hand section of a bp er fil-10 pedicel, revealing a continuous ring of chlorenchyma tissue. (G) The bp er pedicels display files of short cells on their abaxial sides and differentiation of guard cells is repressed. (H) In bp er fil-10, the pedicel stripe is confined to a narrow band of stomata free tissue on lateral sides, but abaxial cells are larger and assume the irregular shapes found in wild type. Differentiation of stomata is also observed (arrows) (I-K) Receptacles of bp er fil-10 (I), fil-10 er (J) and Ler (K). Note expansion in fil-10 er and Ler but lack of enlargement in bp er fil-10 (arrow). Bars in panels G and H are 50 μM.

Fig 2. Morphometric analyses and differential effects of mutations in bp, er, and/or fil.

Crosses were used to generate all combinations of single, double and triple mutants in a Landsberg (Lan) background. (A) Plant height was measured from the rosette to the inflorescence tip in six week old plants. (B-C) Mature, senescing plants were used to measure pedicel length (B) and angle (C). The error bars represent standard error of the mean. Data were compared by one way ANOVA using Tukey’s Honest Significant Differences method. Letters above the bars indicate significance categories where p< 0.01. For all measurements, n = 15–150. Similar trends were observed in two independent experiments.

Fig 3. fil-10 conditions floral organ abnormalities.

(A-G) fil-10 er flowers. (A) Early inflorescences showing symmetrically located sepal primordia. (B) An early bud with a gap (arrow) between two sepals. (C) A flower formed late in development with stigmatic tissue (arrow) on the tip of a sepal. (D) A flower with a third whorl filament lacking an anther. (E) A gynoecium with a bend. (F) A gynoecium in the midst of bending due to sustained contact with the inner face of a lateral sepal. (G) Medial region of a gynoecium showing a bulge of style tissue (arrow) under the stigma. (H) fil-10 ap1-1 er flower showing transformation of medial sepals (arrows) into carpelloid organs.

Fig 4. Mutations in FIL and LAS affect inflorescence architecture in a similar fashion.

(A-C) bp er fil-4 plants showing elongated pedicels (A), upward-oriented floral buds with gaps between sepals (arrow; B) and bends in pedicels at filamentous organs (C). (D) Locations of characterized mutations in the FIL gene. The nature of each mutation is shown in parentheses: I = insertional mutant, S = splice junction mutant; the asterisk represents a stop codon. (E-F) In situ hybridization with a FIL probe showing expression in sepal primordia (central bud) and in floral organs of older, peripheral buds (E), and gynoecium valve expression in a stage 9 pedicel (F). Note the absence of FIL expression in pedicel tissue (arrows) at stages that precede the period of pedicel elongation [59]. (G-I) A collage of a stage 9 bud from a transgenic plant expressing a FILpro::FIL::GFP transgene. The left panel shows FIL::GFP expression on the abaxial side of floral organs; the middle panel is the chlorophyll autofluorescence (red channel) and the right panel is the merged image. (J) Mature flower illustrating FIL::GFP in floral organs only. (K) The bp er las-11 triple mutant exhibits a phenotype nearly identical to that of bp er fil-10.

Fig 5. Suppressive effects of mutations in leunig and yabby3.

(A) bp er lug plant showing suppressed pedicel angles. (B) bp er lug abaxial pedicel showing enlarged cells and stomata (arrows). (C) bp er yab-3 plant. In rare cases, we observed pedicel suppression effects (arrow) of some axillary branches on plants which otherwise exhibited the bp er-like habit.

Fig 6. Aliphatic glucosinolate biosynthesis genes are down-regulated by fil10.

(A) Schematic representation of the aliphatic glucosinolate biosynthetic pathway showing genes involved in various steps. The numbers beside the AGI identifiers indicate the change in expression of these genes in bp er fil-10 suppressor vs. the parent bp er line as gauged by microarray analysis. Question marks indicate uncertainly about the involvement of these genes in the indicated steps. The green text identifies specific glucosinolate metabolites that are products of the enzymatic steps and for which quantitative analysis was performed (see Table 3). (B) QRT-PCR analyses of selected GSL biosynthetic genes, confirming down regulation of these genes in bp er fil-10 verses the bp er parent. The GSTF11 gene (At3g03190) was selected for analysis as its expression pattern is very similar to that of FIL (eFP browser data) and the gene has been implicated in GSL biosynthesis. The relative expression ratio of the bp er fil-10 mutant is shown and error bars are the standard error of the mean. Pair-wise t-tests on linear transformed ΔCT values revealed that all differences are statistically significant (p<0.034).

Fig 7. Auxin levels are altered in bp and fil mutants.

(A) Auxin levels in Ler, bp er and bp er fil-10. Wildtype FIL is required for the bp er phenotype and is associated with lower auxin levels. Pairwise T-tests revealed significant differences between Ler and bp er (p < 0.001), and between bp er and bp er fil-10 (p = 0.01). (B) Multiplex PCR on four independent transformants of both bp er or bp er fil-10 harboring the auxin reporter DR5::GUS. The lower band represents a single copy control gene (AMI) while the upper band assesses the presence/level of the DR5::GUS reporter gene. The bp lane is a non-transformed control, (-) is no DNA template. Lower left panels: X-gluc stained seedlings of four independent bp er transformants. Lower right panels: X-gluc stained seedlings of four independent bp er fil-10 transformants. In all cases, the bp er fil-10 suppressor lines exhibited broader and more intense staining than the bp er lines, despite the fact that the copy number of the auxin reporter gene was similar or even lower in the bp er fil-10 lines (panel B).

Fig 8. Changes in the expression of indolic glucosinolate and auxin biosynthesis genes in bp er fil-10.

(A) Inferred and speculative intersections of auxin and glucosinolate biosynthetic pathways. Some pathway steps are embellished with gene designations where empirical data implicate specific associations (red text). The green text identifies specific glucosinolate metabolites that are products of the enzymatic steps and for which quantitative analysis was performed (see Table 3). (B) QRT-PCR data on selected genes implicated in indolic glucosinolate and auxin metabolism. The relative level of transcripts in bp er fil-10 vs bp er is shown. Error bars represent standard error of the mean. Pairwise t-tests on linear transformed ΔCt revealed all differences to be statistically significant (p<0.02) except YUC1 (p = 0.069) and YUC6 (p = 0.55).