Figure 1.
Precocious shoot termination in determinate tomatoes is partially suppressed by sft/
+ mutant heterozygosity. (A) Schematic diagrams showing shoot architecture of a wild type (WT) indeterminate tomato plant (left) and an sp determinate mutant (right). In WT M82 plants the primary shoot meristem (PSM) from the embryo gives rise to 7–9 leaves before terminating in the first flower of the first multi-flowered inflorescence (boxed). A specialized axillary meristem called a sympodial meristem (SYM) in the axil of the last leaf on primary shoot then generates three leaves before terminating in the first flower of the next inflorescence. In indeterminate tomatoes, this process continues indefinitely (left). In sp mutants (right), sympodial cycling accelerates progressively on all shoots causing leaf production to decrease in successive units until growth ends in two juxtaposed inflorescences (asterisks). Alternating colored groups of three ovals represent leaves within successive sympodial units numbered at right. Colored circles represent fruits and flowers within each inflorescence (red: fully ripe fruit; orange: ripening fruit; green: unripe fruit; yellow: flowers) and arrows represent canonical axillary shoots. (B) Compared to sp mutants alone, sft/+ sp plants produce more inflorescences (left) and sympodial units (right) before sympodial cycling terminates on the main shoot. Genotypes and sample sizes are shown below, and standard deviations of averages are presented. (C) Compared to sp alone, sft/+ sp plants produce more leaves in the first three sympodial units, indicating a delay in precocious termination. Colored bars indicate average leaf numbers within sympodial units with standard deviations. Statistical significance in B and C was tested by Wilcoxon rank sum test, and significance levels are indicated by asterisks (*P<0.05, **P<0.01, ***P<0.001).
Figure 2.
sft/+ heterozygosity induces weak semi-dominant delays in both primary and sympodial flowering transitions.
(A) sft/+ sp plants show slightly delayed primary shoot flowering time compared to sp as measured by leaf production before formation of the first inflorescence. Note the extremely delayed flowering of sft sp double mutants, indicating a weak semi-dominant effect for sft/+ heterozygosity. Bars indicate average leaf numbers with standard deviations. Genotypes and sample sizes are shown below. Statistical differences were tested by Wilcoxon rank sum tests and significance levels are marked by asterisks (***P<0.001). (B–G) Representative images and quantification of developmental progression (ontogeny) of meristems in the first inflorescence and sympodial shoot meristems (SYM) of sp (left images) and sft/+ sp plants (right images) at 20th DAG. Both sp (B) and sft/+ sp (C) PSMs have completed the primary flowering transition and generated a series of floral meristems (FM) and sympodial inflorescence meristems (SIM) [26], [30]. sft/+ sp plants are consistently one SIM behind ontogenically, consistent with a weak delay in flowering from sft/+ heterozygosity (D). Developmental progression of the first SYM in sp (E) and sft/ + sp (F) plants at the same time point as in B–C. While the SYM of sp mutants has already completed the flowering transition and differentiated into the first or second FM and initiated the next SIM, the SYM of sft/+ sp plants is still transitioning or initiating the first SIM, indicating a developmental delay parallel to the PSM of sft/+ sp plants (G). In D and G, bars indicate average numbers of initiated FMs with standard deviations. Genotypes and sample sizes are shown below. Statistical differences were tested by Wilcoxon rank sum tests and significance levels are marked by asterisks (***P<0.001). Scale bar: 100 um.
Figure 3.
Transcriptome profiling reveals an early semi-dominant delay on seedling development from sft/+ heterozygosity.
(A) Representative 6th expanding leaf from sp mutants. The same leaf and stage (3 cm long) was profiled by RNA-Seq for sft/+ sp and sft sp genotypes. (B) Molecular quantification of leaf maturation using the DDI algorithm [31]. Given that seedling development of sft sp is delayed compared to sp based on extreme late flowering, the sft sp 6th expanding leaf was designated an early leaf calibration point. Dark and light green curves indicate sft sp and sp maturation score distributions based on 124 DDI-defined marker genes. The black curve for the sft/+ sp 6th leaf indicates an intermediate maturation state. Numbers above indicate average maturation scores.
Figure 4.
Transcriptome profiling reveals a semi-dominant delay in meristem maturation from sft/+ heterozygosity.
(A–C) Stereoscope images showing morphology and dissection (white dashed line) of the TM stage used for mRNA-Seq from sp (A), sft/+ sp (B) and sft sp (C) genotypes. Scale bar: 100 um. Red arrows highlight identical TM morphologies. L: leaf primordium number. The additional leaf primordium at the sft/+ sp TM is consistent with the one leaf delay in primary shoot flowering time (Figure 2). (D) DDI quantification of maturation scores for sp, sft sp, and sft/+ sp predicted from the WT PSM meristem maturation atlas [34]. Colored dashed curves indicate maturation stages for the 5 PSM stages used for calibration EVM, MVM, LVM, TM and FM [the Early, Middle, and Late Vegetative Meristems, Transition Meristem and the Flower Meristem]. Colored areas define boundaries of these stages estimated from the curves. Maturation scores are derived from 637 DDI-selected marker genes (Dataset S3). Student's t-tests are presented as heat-maps of scaled 1/(−log10P) values below each graph, and associated numbers to the right indicate average maturation scores for the predicted meristems. Darker color indicates greater similarity in maturation state. Note the statistically intermediate TM maturation state of sft/+ sp relative to sft sp and sp, indicating sft/+ heterozygosity causes a semi-dominant delay in the primary flowering transition. The presence of more than one peak along the curves of the sft sp and sft/+ sp genotypes reflect mixed maturation states for these TMs, as different subsets of marker genes are driving different maturation stage estimates that translate to less uniform maturation patterns. (E–F) Stereoscope images showing morphology and dissection of the first sympodial shoot meristem (SYM) used for mRNA-Seq profiling in sp (E) and sft/+ sp genotypes (F). Meristems and leaf primordia are marked as in Figure 2. (G) DDI quantification of SYM maturation scores from sp, sft/+ sp, and WT using the PSM stages as calibrations. Maturation scores for sft/+ sp, sp and WT indicate an intermediate maturation state for the SYM of sft/+ sp plants, mirroring the delay in the PSM. P-value heat maps are shown below along with average maturation scores to the right.
Figure 5.
Reducing SFT transcripts with artificial microRNAs mimics the dosage effects of sft/+ heterozygosity.
(A) Artificial microRNAs targeting tomato SFT and Arabidopsis FT. Shown are alignments of amiR-SFT/FTAt164b and amiR-SFT/FTAt319a with the complementary region of SFT and FT. G–U wobbles and mismatches between the two amiR-SFT/FTs and the target are highlighted in the target sequence with bold blue and red, respectively. (B) Quantitative RT-PCR measurements of tomato SFT transcript levels in amirSFT plants showing knock down. Results shown are from using primers targeting SFT transcripts 5′ to the amiRNA binding site, consistent with reports of primer-dependent transitivity occurring at the 3′ to 5′ direction upon the initial target cleavage, resulting in degradation of the 5′ cleaved product of the target but not the 3′ product [80], [81] (Figure S2). Bars indicate relative expression level and error bars indicate standard deviation among replicates. (C) Depending on the strength of suppression, amirSFT plants produce at least one additional sympodial unit and inflorescence compared to sp alone, indicating that reducing SFT transcript levels by artificial microRNA partially suppresses sp sympodial termination, mimicking the dosage effect of sft/+ heterozygosity. Note that some amirSFTc progeny plants showed indeterminacy, whereas amirSFTb progeny plants were always indeterminate, indicating that a stronger suppression of SFT completely suppresses the sp phenotype and reverts the plants to normal sympodial cycling. Differences in sympodial unit and inflorescence numbers between amirSFT and sp plants were tested by Wilcoxon rank sum test and significance levels are marked by asterisks (* P<0.05, ** P<0.01, *** P<0.001). (D) amirSFT plants have delayed primary shoot flowering time compared to sp and WT controls, similar to sft/+ heterozygosity. Bars indicate average leaf numbers with standard deviations. Genotypes and sample sizes are shown below. Differences in leaf numbers between amirSFT and sp plants were tested by Wilcoxon rank sum test and significance levels are marked by asterisks (* P<0.05, ** P<0.01, *** P<0.001).
Figure 6.
Dose-dependent suppression of tfl1 (sp) by ft/+ (sft/+) heterozygosity is conserved in Arabidopsis thaliana.
(A) Representative plants from left to right of: tfl1-2 single mutants, ft-2/+ tfl1-2, ft-2 tfl1-2 double mutants, ft-2 single mutants and wild type Ler-0 (WT) showing the intermediate height of ft-2/+ tfl1-2 plants compared to tfl1-2 and ft-2 tfl1-2 genotypes. (B–C) Statistical comparisons among all genotypes for plant height and flower/fruit yield showing semi-dominant effects from ft-2/+heterozygosity in the tfl1-2 background. Bars indicate average values with standard deviation. Genotypes and sample size are shown below. Differences between genotypes were tested by a Wilcoxon rank sum test and significance levels are marked by asterisks (*P<0.05, **P<0.01, ***P<0.001). (B) ft-2 heterozygosity in a tfl1-2 mutant background partially suppresses the early flowering and early termination phenotype of the tfl1-2 mutation in a semi-dominant manner, resulting in plant height in between tfl1-2 and ft-2 tfl1-2 mutant parental lines. (C) Unlike tomato, ft/+ heterozygosity in a tfl1-2 mutant background does not drive heterosis for yield (number of total siliques and floral buds) in Arabidopsis. Rather, yield in the ft-2/+ tfl1-2 plants is intermediate to tfl1-2 and ft-2 tfl1-2 double mutants.