Accession-specific modifiers act with ZWILLE/ARGONAUTE10 to maintain shoot meristem stem cells during embryogenesis in Arabidopsis

Abstract

Background: Stem cells located in the centre of the shoot apical meristem are required for the repetitive formation of new organs such as leaves, branches and flowers. In Arabidopsis thaliana, the ZWILLE/PINHEAD/AGO10 (ZLL) gene encodes a member of the ARGONAUTE (AGO) protein family and is required to maintain shoot meristem stem cells during embryogenesis. In the Landsberg erecta (Ler) acession, ZLL is essential for stem cell maintenance, whereas in the Columbia (Col) accession its requirement appears masked by genetic modifiers. The genetic basis for this variation has remained elusive.

Results: To understand the impact of natural variation on shoot stem cell maintenance, we analysed 28 wild-type Arabidopsis accessions from around the world and show that ZLL function is essential for stem cell maintenance in accessions mainly originating from Germany, but is dispensable for accessions from other regions. Quantitative Trait Loci (QTL) mapping using Ler/Col recombinant inbred lines indicated that at least five genomic regions, referred to as FLETSCHE (FHE) 1-5, modify ZLL function in stem cell maintenance. Characterisation of Col zll near isogenic lines confirmed that the major QTL, FHE2, is preferentially maintained as a Ler allele in seedlings lacking stem cells, suggesting that this region harbours an important modifier of ZLL function. Comparison of torpedo-stage embryo expression profiles to QTL map data revealed candidate FHE genes, including the Arabidopsis Cyclophilin-40 homologue SQUINT (SQN), and functional studies revealed a previously uncharacterised role for SQN in stem cell regulation.

Conclusions: Multiple genetic modifiers from different Arabidopsis accessions influence the role of ZLL in embryonic stem cell maintenance. Of the five FHE loci modifying stem cell maintenance in Ler-0 and Col-0, FHE2 was the most prominent and was tightly linked to the SQN gene, which encodes a cofactor that supports AGO1 activity. SQN shows variable embryonic expression levels between accessions and altered ZLL-dependency in transgenic assays, confirming a key role in stem cell maintenance. Reduced SQN expression levels in Col-0 correlate with transposon insertions adjoining the transcriptional start site, which may contribute to stem cell maintenance in other ZLL-independent accessions.

Figure 1

Shoot meristem development in Arabidopsis wild-type and zll mutants. A. Stem cell termination phenotypes in zll-1 Landsberg erecta (Ler) mutants. Seedlings show either a wild-type-like meristem (WT-like), stem cell termination after the formation of two leaves (2L), a single central leaf-like organ (SL), a single central filament (FIL) or a flat apex without any organ formation (NM). Bar = 2.5 mm. B. Most zll-1 Columbia-0 (Col) seedlings cannot be phenotypically discerned from Col WT. Similarly, the zll ^ago10-1 Col mutant appears WT-like in the vast majority of seedlings and stem cell termination is not enhanced by mutations in the Col ERECTA (ER) gene, such as er-103. Bar = 2.5 mm. C. Frequency of stem cell termination phenotypes in the F₂ progeny of crosses between Ler zll-1, Ler-0 and Col-0. Abbreviations as per A. D. Mature embryos stained with propidium iodide show subtle differences in the number of L1 cells (red dots) between Ler-0 and Col-0. The width of the meristem is SAM, shoot apical meristem, C, cotyledon indicated with arrows. Bar = 25 μm.

Figure 2

Shoot meristem development in F ₂ progeny from 28 accessions of Arabidopsis crossed to Ler zll-1. Columns present the proportion of zll-1 homozygous seedlings showing a stem cell termination phenotype. Error bars show standard deviation after four independent seedling counts. The country of origin for each accession is indicated. US, United States of America, CA, Canada, CV, Cape Verde Islands, PT, Portugal, ES, Spain, GB, United Kingdom, BE, Belgium, NL, Netherlands, FR, France, IT, Italy, DE, Germany, CZ, Czech Republic, RU, Russia, TJ, Tajikistan.

Figure 3

Molecular phylogenetic relationships between 28 Arabidopsis accessions based on 59 single nucleotide polymorphisms. Genotypes were extracted from http://www.naturalvariation.org/. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The tree with the highest log likelihood (-1257.96) is shown and was generated in MEGA5.2. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (above the branches). All positions containing gaps and missing data were eliminated. Red shading indicates a capacity for a high frequency of zll-dependent stem cell termination, yellow indicates a medium capacity and green indicates a low capacity.

Figure 4

QTL analysis of zll-1 x Ler/Col Recombinant Inbred Lines. A. Frequency distribution plot of stem cell termination phenotypes in the F₂ progeny of 101 crosses between Ler zll-1 and 99 RILs, Ler-0 and Col-0. B. Chromosome-wide Logarithm of the Odds (LOD) scores of QTL influencing stem cell termination. The horizontal line indicates the significance threshold and marker positions are shown below each plot. LOD values and significance thresholds were determined using PlabMQTL software.

Figure 5

Shoot meristem development in F3 progeny from 18 zll-1 Col introgression lines. The frequency of seedlings showing stem cell termination phenotypes in each zll-1 near isogenic line (NIL) is shown. Error bars show standard deviation after three replicate seedling counts.