The WTM Genes in Budding Yeast Amplify Expression of the Stress-Inducible Gene RNR3

Cellular responses to DNA damage and inhibited replication are evolutionarily conserved sets of pathways that are critical to preserving genome stability. To identify new participants in these responses, we undertook a screen for regulators that, when present on a high-copy vector, alter expression of a DNA damage-inducible RNR3-lacZ reporter construct in Saccharomyces cerevisiae. From this screen we isolated a plasmid encoding two closely related paralogs, WTM1 and WTM2, that greatly increases constitutive expression of RNR3-lacZ. Moderate overexpression of both genes together, or high-level expression of WTM2 alone from a constitutive promoter, upregulates RNR3-lacZ in the absence of DNA damage. Overexpressed, tagged Wtm2p is associated with the RNR3 promoter, indicating that this effect is likely direct. Further investigation reveals that Wtm2p and Wtm1p, previously described as regulators of meiotic gene expression and transcriptional silencing, amplify transcriptional induction of RNR3 in response to replication stress and modulate expression of genes encoding other RNR subunits

Precocious shoot termination in determinate tomatoes is partially suppressed by <i>sft/</i>

<p>+<b> mutant heterozygosity.</b> (A) Schematic diagrams showing shoot architecture of a wild type (WT) indeterminate tomato plant (left) and an <i>sp</i> 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 <i>sp</i> 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 <i>sp</i> mutants alone, <i>sft/+ sp</i> 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 <i>sp</i> alone, <i>sft/+ sp</i> 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&lt;0.05, **P&lt;0.01, ***P&lt;0.001).</p

Reducing <i>SFT</i> transcripts with artificial microRNAs mimics the dosage effects of <i>sft/</i>+ heterozygosity.

<p>(A) Artificial microRNAs targeting tomato <i>SFT</i> and <i>Arabidopsis FT</i>. Shown are alignments of amiR-SFT/FT<i>^At</i>^164b and amiR-SFT/FT<i>^At</i>^319a with the complementary region of <i>SFT</i> and <i>FT</i>. 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 <i>SFT</i> transcript levels in <i>amirSFT</i> plants showing knock down. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004043#s2" target="_blank">Results</a> shown are from using primers targeting <i>SFT</i> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004043#pgen.1004043-Allen1" target="_blank">[80]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004043#pgen.1004043-Moissiard1" target="_blank">[81]</a> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004043#pgen.1004043.s005" target="_blank">Figure S2</a>). Bars indicate relative expression level and error bars indicate standard deviation among replicates. (C) Depending on the strength of suppression, <i>amirSFT</i> plants produce at least one additional sympodial unit and inflorescence compared to <i>sp</i> alone, indicating that reducing <i>SFT</i> transcript levels by artificial microRNA partially suppresses <i>sp</i> sympodial termination, mimicking the dosage effect of <i>sft/+</i> heterozygosity. Note that some <i>amirSFTc</i> progeny plants showed indeterminacy, whereas <i>amirSFTb</i> progeny plants were always indeterminate, indicating that a stronger suppression of <i>SFT</i> completely suppresses the <i>sp</i> phenotype and reverts the plants to normal sympodial cycling. Differences in sympodial unit and inflorescence numbers between <i>amirSFT</i> and <i>sp</i> plants were tested by Wilcoxon rank sum test and significance levels are marked by asterisks (* P&lt;0.05, ** P&lt;0.01, *** P&lt;0.001). (D) <i>amirSFT</i> plants have delayed primary shoot flowering time compared to <i>sp</i> and WT controls, similar to <i>sft/+</i> heterozygosity. Bars indicate average leaf numbers with standard deviations. Genotypes and sample sizes are shown below. Differences in leaf numbers between <i>amirSFT</i> and <i>sp</i> plants were tested by Wilcoxon rank sum test and significance levels are marked by asterisks (* P&lt;0.05, ** P&lt;0.01, *** P&lt;0.001).</p

<i>sft/</i>+ heterozygosity induces weak semi-dominant delays in both primary and sympodial flowering transitions.

<p>(A) <i>sft/+ sp</i> plants show slightly delayed primary shoot flowering time compared to <i>sp</i> as measured by leaf production before formation of the first inflorescence. Note the extremely delayed flowering of <i>sft sp</i> double mutants, indicating a weak semi-dominant effect for <i>sft/+</i> 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&lt;0.001). (B–G) Representative images and quantification of developmental progression (ontogeny) of meristems in the first inflorescence and sympodial shoot meristems (SYM) of <i>sp</i> (left images) and <i>sft/+ sp</i> plants (right images) at 20^th DAG. Both <i>sp</i> (B) and <i>sft/+ sp</i> (C) PSMs have completed the primary flowering transition and generated a series of floral meristems (FM) and sympodial inflorescence meristems (SIM) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004043#pgen.1004043-Pnueli1" target="_blank">[26]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004043#pgen.1004043-Lippman2" target="_blank">[30]</a>. <i>sft/+ sp</i> plants are consistently one SIM behind ontogenically, consistent with a weak delay in flowering from <i>sft/+</i> heterozygosity (D). Developmental progression of the first SYM in <i>sp</i> (E) and <i>sft/ + sp</i> (F) plants at the same time point as in B–C. While the SYM of <i>sp</i> mutants has already completed the flowering transition and differentiated into the first or second FM and initiated the next SIM, the SYM of <i>sft/+ sp</i> plants is still transitioning or initiating the first SIM, indicating a developmental delay parallel to the PSM of <i>sft/+ sp</i> 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&lt;0.001). Scale bar: 100 um.</p

Transcriptome profiling reveals an early semi-dominant delay on seedling development from <i>sft/</i>+ heterozygosity.

<p>(A) Representative 6^th expanding leaf from <i>sp</i> mutants. The same leaf and stage (3 cm long) was profiled by RNA-Seq for <i>sft/+ sp</i> and <i>sft sp</i> genotypes. (B) Molecular quantification of leaf maturation using the DDI algorithm <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004043#pgen.1004043-Efroni1" target="_blank">[31]</a>. Given that seedling development of <i>sft sp</i> is delayed compared to <i>sp</i> based on extreme late flowering, the <i>sft sp</i> 6^th expanding leaf was designated an early leaf calibration point. Dark and light green curves indicate <i>sft sp</i> and <i>sp</i> maturation score distributions based on 124 DDI-defined marker genes. The black curve for the <i>sft/+ sp</i> 6^th leaf indicates an intermediate maturation state. Numbers above indicate average maturation scores.</p

Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene

Selection for inflorescence architecture with improved flower production and yield is common to many domesticated crops. However, tomato inflorescences resemble wild ancestors, and breeders avoided excessive branching because of low fertility. We found branched variants carry mutations in two related transcription factors that were selected independently. One founder mutation enlarged the leaf-like organs on fruits and was selected as fruit size increased during domestication. The other mutation eliminated the flower abscission zone, providing ?jointless? fruit stems that reduced fruit dropping and facilitated mechanical harvesting. Stacking both beneficial traits caused undesirable branching and sterility due to epistasis, which breeders overcame with suppressors. However, this suppression restricted the opportunity for productivity gains from weak branching. Exploiting natural and engineered alleles for multiple family members, we achieved a continuum of inflorescence complexity that allowed breeding of higher-yielding hybrids. Characterizing and neutralizing similar cases of negative epistasis could improve productivity in many agricultural organisms

A cascade of arabinosyltransferases controls shoot meristem size in tomato

Shoot meristems of plants are composed of stem cells that are continuously replenished through a classical feedback circuit involving the homeobox WUSCHEL (WUS) gene and the CLAVATA (CLV) gene signaling pathway. In CLV signaling, the CLV1 receptor complex is bound by CLV3, a secreted peptide modified with sugars. However, the pathway responsible for modifying CLV3 and its relevance for CLV signaling are unknown. Here we show that tomato inflorescence branching mutants with extra flower and fruit organs due to enlarged meristems are defective in arabinosyltransferase genes. The most extreme mutant is disrupted in a hydroxyproline O-arabinosyltransferase and can be rescued with arabinosylated CLV3. Weaker mutants are defective in arabinosyltransferases that extend arabinose chains, indicating that CLV3 must be fully arabinosylated to maintain meristem size. Finally, we show that a mutation in CLV3 increased fruit size during domestication. Our findings uncover a new layer of complexity in the control of plant stem cell proliferation