Highlight An increasing number of reports question conclusions based on loss-of-function lines that have unexpected genetic backgrounds. In this opinion paper, we urge researchers to meticulously (re)investigate phenotypes retrieved from various genetic backgrounds and be critical regarding some previously drawn conclusions. As an example, we provide new evidence that acr4-2 mutant phenotypes with respect to columella stem cells are due to the lack of ACR4 and not – at least not as a major contributor – to a mutation in QRT1. In addition, we take the opportunity to alert the scientific community about the qrt1-2 background of a large number of Syngenta Arabidopsis Insertion Library (SAIL) T-DNA lines, a feature that is not commonly recognized by Arabidopsis researchers. This qrt1-2 background might have an important impact on the interpretation of the results obtained using these research tools, now and in the past. In conclusion, as a community, we should continuously assess and – if necessary – correct our conclusions based on the large number of (genetic) tools our work is built on. In addition, the positive or negative results of this self-criticism should be made available to the scientific community. ACR4, Arabidopsis thaliana, loss-of-function lines, SAIL, self-criticism, QRT1 Meticulously (re)investigating phenotypes retrieved from various genetic backgrounds is needed. As an example, it is shown that acr4-2 columella stem cell phenotypes are indeed due to the lack of ACR4. Since the dawn of Arabidopsis research, various genetic tools, including ethyl methanesulfonate mutants and T-DNA lines, have been used to functionally characterize genes and assign roles to them. However, there are several examples in the literature with specific mutant alleles, such as those for AUXIN-BINDING PROTEIN 1 (abp1-1), CORONATINE-INSENSITIVE PROTEIN 1 (coi1-16), ABSCISIC ACID-INSENSITIVE1 (abi1-3) and TRANSPARENT TESTA 4 (tt4(2YY6)) (Bennett et al., 2006; Westphal et al., 2008; Wu et al., 2015; Enders et al., 2015; Kriegel et al., 2015; Hazak et al., 2017), along with our own work (unpublished results) that illustrate that trusting published and available genetic tools and building hypotheses on them can be risky. In addition to best practice for verifying both the identity of genetic stocks and the causal relationship between a genetic variant and a phenotype (Bergelson et al., 2016), we therefore need, as a community, to assess and, if necessary, correct our current conclusions, and continuously be critical of the (genetic) tools our work is built on. In addition, the positive or negative results of this self-criticism should be made available to the scientific community (for example as has been done for AUXIN-BINDING PROTEIN 1; Michalko et al., 2015; Dai et al., 2015; Enders et al., 2015). Taken together, this will create a much stronger foundation for future experiments, hypotheses and conclusions. To illustrate this, we recently became aware that the acr4-2 allele, a T-DNA insertion line derived from the Syngenta Arabidopsis Insertion Library (SAIL) collection (see Supplementary Fig. S1 at JXB online; O’Malley et al., 2015), was generated in a qrt1-2 mutant background (Preuss et al., 1994; Copenhaver et al., 2000). QRT1 codes for a pectin methylesterase involved in post-meiotic spore release. Absence of QRT1 activity compromises the normal separation of the four products of qrt1-2−/− male sporogenesis and results in the release of pollen grains as tetrads (Francis et al., 2006), a trait reflected in the acr4-2 pollen phenotype (Fig. 1A). The non-separating tetrads allow for non-destructive visual analysis of the outcome of male meiosis and studying meiotic recombination events (Johnson-Brousseau and McCormick, 2004; Francis et al., 2007). Although the qrt1-2 background of a subset of SAIL lines had been picked up earlier in the literature (Zhang et al., 2009; Ishiguro et al., 2010; Yamaoka et al., 2013; Gehl et al., 2014), this information does not appear to be common knowledge in the scientific community. An awareness of this background is nevertheless relevant, as 48% of the SAIL lines (about 28500 lines) appear to be in the qrt1-2 mutant background, which in turn is in the Col-3 background (O’Malley and Ecker, 2010). Therefore, we urge the scientific community to check and validate results from SAIL lines, and to re-evaluate published phenotypes if necessary. Fig. 1. View largeDownload slide Analysis of acr4 columella stem cell phenotypes in view of acr4-2 background. (A) Quartet pollen phenotype in acr4-2 and qrt1-2, compared with Col-0. (B) Representative images of Col-0, acr4-2, acr4-25, and qrt1-2 root tip. Pink arrowhead indicates undifferentiated stem cells and yellow asterisk the quiescent center. Modified pseudo-Schiff propidium iodide staining for Col-0, acr4-2, and acr4-25, and propidium iodide staining for qrt1-2. (C, D) Quantification of columella stem cell phenotypes with respect to stem cell differentiation (C) and irregular columella organization (D) for Col-0 (n=356, from seven replicates), qrt1-2 (n=234, from three replicates), acr4-2 (n=120, from two replicates), acr4-2 pACR4::ACR4:GSyellow (n=306, from four replicates), acr4-10 (n=21, from one replicate) and acr4-25 (n=204, from three replicates). Statistical significance (Z test calculator for two population proportions, P<0.05) compared with Col-0 or qrt1-2 is indicated by asterisk for two layers of undifferentiated columella layers (C) and irregular columella organization (D). n.s., not significant. Fig. 1. View largeDownload slide Analysis of acr4 columella stem cell phenotypes in view of acr4-2 background. (A) Quartet pollen phenotype in acr4-2 and qrt1-2, compared with Col-0. (B) Representative images of Col-0, acr4-2, acr4-25, and qrt1-2 root tip. Pink arrowhead indicates undifferentiated stem cells and yellow asterisk the quiescent center. Modified pseudo-Schiff propidium iodide staining for Col-0, acr4-2, and acr4-25, and propidium iodide staining for qrt1-2. (C, D) Quantification of columella stem cell phenotypes with respect to stem cell differentiation (C) and irregular columella organization (D) for Col-0 (n=356, from seven replicates), qrt1-2 (n=234, from three replicates), acr4-2 (n=120, from two replicates), acr4-2 pACR4::ACR4:GSyellow (n=306, from four replicates), acr4-10 (n=21, from one replicate) and acr4-25 (n=204, from three replicates). Statistical significance (Z test calculator for two population proportions, P<0.05) compared with Col-0 or qrt1-2 is indicated by asterisk for two layers of undifferentiated columella layers (C) and irregular columella organization (D). n.s., not significant. In Arabidopsis, functional analysis of ACR4 has largely been performed based on the study of mutant alleles among which acr4-2 occupies a central position (Gifford et al., 2003; Watanabe et al., 2004; De Smet et al., 2008, 2010, Stahl et al., 2009, 2013; Roeder et al., 2012; San-Bento et al., 2014; Chang et al., 2015; Yue et al., 2016). However, the presence of the qrt1-2 mutation in the background of the acr4-2 allele might jeopardize our current views on ACR4 functioning in columella organization and stem cell division in the primary root (De Smet et al., 2008; Stahl et al., 2009, 2013; Yue et al., 2016). Therefore, to lead the way, we re-evaluated the columella phenotypes in acr4-2 in view of the qrt1-2 background information we recently became aware of. In order to explore this oversight in the genetic analyses of ACR4, we identified an additional acr4 allele (in Col-0), namely acr4-25 (SALK_043679) (see Supplementary Fig. S1; Supplementary Methods). In addition, we looked into another existing acr4 allele, namely acr4-10 (Supplementary Fig. S1; Gifford et al., 2005). Surprisingly, none of these acr4 alleles displayed a complete loss of ACR4 expression, and acr4-2 actually showed a slight increase in expression level of a fragment located C-terminally of the T-DNA (Supplementary Fig. S1; Supplementary Methods). It should be noted that this is likely an incomplete transcript, as full length ACR4 does not appear to be present in acr4-2 and acr4-25 (Supplementary Fig. S1). In this context, all the acr4 mutant alleles we analysed, namely acr4-2, acr4-10 and acr4-25, presented the earlier described rounded seeds with wrinkled seed coats instead of the normal ellipsoid shape with smooth seed coat characteristics of Col-0 and qrt1-2 (Gifford et al., 2003; Watanabe et al., 2004; Supplementary Fig. S2; Supplementary Methods). In addition, similar to acr4-2, both acr4-10 and acr4-25 seedlings often displayed two layers of columella stem cells, 19 and 33%, respectively, and had an irregularly patterned columella, 14 and 23%, respectively, compared with 10% and 4% for Col-0, respectively (Fig. 1B–D). Furthermore, qrt1-2 seedlings were not significantly different from Col-0 with respect to the columella stem cell phenotype (Fig. 1B–D; Supplementary Methods). Taken together, these results confirm that other acr4 alleles display some of the previously observed and characterized phenotypes, including columella stem cell defects, and are likely to be independent of the qrt1-2 mutant background. This is further supported by published expression analyses (Francis et al., 2006) and Arabidopsis eFP Browser transcriptome data, which indicate absence of QRT1 expression in the primary root tip columella cells (Supplementary Fig. S3). To further confirm that the described columella stem cell phenotype in acr4-2 is due to the loss of ACR4 activity, we performed complementation experiments. Indeed, expression of an ACR4 translational fusion (pACR4::ACR4:GSyellow) in acr4-2 could restore the columella defects (Fig. 1B–D; Supplementary Fig. S4; Supplementary Methods). Similarly, we could complement the abnormal seed coats of acr4-2 by introducing pACR4::ACR4:GSyellow (Supplementary Fig. S2). This further supports that these previously described phenotypes are indeed caused by loss of ACR4 function. While these various T-DNA lines appear to act as loss-of-function lines, and at least acr4-2 can be complemented by introducing ACR4:GSyellow under the ACR4 promoter, at the moment we do not know the impact of the remaining or even higher expression of ACR4 (fragments) observed in these T-DNA lines (Supplementary Fig. S1). In this Opinion Paper, we call for the scientific community to be self-critical and re-evaluate genetic tools meticulously. We further encourage that the positive or negative results of this self-criticism are made available to the scientific community. To lead the way, we used a novel and a previously reported acr4 allele and complementation of acr4-2 with pACR4::ACR4:GSyellow to confirm that the before-described acr4-2 mutant phenotypes with respect to columella stem cells are due to the lack of functional ACR4 and not – at least not as a major contributor – to a mutation in QRT1. In conclusion, the use of multiple independent mutant alleles and even whole genome sequencing to identify background single-nucleotide polymorphisms will yield valuable results, but the genetic complementation of the mutant remains the best test. Supplementary data Supplementary data are available at JXB online. Methods. Fig. S1. Detailed information on acr4 alleles used in this study. Fig. S2. Representative images for Col-0 (11/2), qrt1-2 (10/0), acr4-2 (0/15), acr4-10 (0/11), acr4-25 (0/11), acr4-26 (0/15), and acr4-2 pACR4::ACR4:GSyellow (9/3) seed coat phenotype. Fig. S3. Arabidopsis eFP Browser data (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) on QRT1 (AT5G55590) expression. Fig. S4. Expression of pACR4::ACR4:GSyellow in acr4-2. Author contributions NN, KY, TB and IDS designed the experiments, analysed data, and wrote the manuscript; NN and KY conducted all experiments. Acknowledgements We thank Geert De Jaeger, Astrid Gadeyne and Eveline Van De Slijke for generating constructs. KY was supported by a grant from the Chinese Scholarship Council (CSC) and a grant by BELSPO (Belgian Science Policy). Work in the laboratory of TB is supported by grants from the Research Foundation – Flanders (project no. G022516N and G0273.13N). References Bennett T , Sieberer T , Willett B , Booker J , Luschnig C , Leyser O . 2006 . 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Journal of Experimental Botany – Oxford University Press
Published: May 16, 2018
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