Nature and Nurture: Genotype-Dependent Differential Responses of Root Architecture to Agar and Soil Environments

Abstract

Root development is crucial for plant growth and therefore a key factor in plant performance and food production. Arabidopsis thaliana is the most commonly used system to study root system architecture (RSA). Growing plants on agar-based media has always been routine practice, but this approach poorly reflects the natural situation, which fact in recent years has led to a dramatic shift toward studying RSA in soil. Here, we directly compare RSA responses to agar-based medium (plates) and potting soil (rhizotrons) for a set of redundant loss-of-function plethora (plt) CRISPR mutants with variable degrees of secondary root defects. We demonstrate that plt3plt7 and plt3plt5plt7 plants, which produce only a handful of emerged secondary roots, can be distinguished from other genotypes based on both RSA shape and individual traits on plates and rhizotrons. However, in rhizotrons the secondary root density and the total contribution of the side root system to the RSA is increased in these two mutants, effectively rendering their phenotypes less distinct compared to WT. On the other hand, plt3, plt3plt5, and plt5plt7 mutants showed an opposite effect by having reduced secondary root density in rhizotrons. This leads us to believe that plate versus rhizotron responses are genotype dependent, and these differential responses were also observed in unrelated mutants short-root and scarecrow. Our study demonstrates that the type of growth system affects the RSA differently across genotypes, hence the optimal choice of growth conditions to analyze RSA phenotype is not predetermined.

Redundant PLETHORA activity promotes development of early embryonic cell lineages in Arabidopsis

“…Although PLT7 is expressed at the one-cell stage in the apical and basal cells, we did not identify embryonic abortion in plt2 plt7 and bbm plt7 double mutants nor in plt1 plt2 plt3 plt7 quadruple mutants (Table 1). Since single and higher-order mutants of plt3, plt5 and plt7 have been extensively studied for their role in root and shoot architecture; (Du and Scheres, 2017; Hofhuis et al, 2013; Kerstens et al, 2021; Pinon et al, 2013; Prasad et al, 2011), this excludes the possibility that they are mandatory in early embryo development. Moreover, embryogenesis proceeded normally in plt3 plt5 plt7-cr mutants (Kerstens et al, 2021) in which we also knocked out BBM with CRISPR/Cas9 ( plt3 bbm plt5 plt7-cr , Table 1).…”

“…plt5-2 and plt7-1 were described in Prasad et al (2011). plt3 plt5 plt7-cr triple mutant was described in Kerstens et al (2021). Growth conditions of plants are described in (Willemsen et al, 1998).…”

“…plt2 and bbm CRISPR constructs were generated as described in Kerstens et al (2021), except that we used a Zea mays codon-optimised intronised Cas9 protein (zCas9i; Grützner et al, 2021), using one sgRNA per gene (5’-CTTAGGAGTGAGCAAATCGG-3’ and 5’-AACTCGATGAATAACTGGTT-3’, respectively). The zCas9i module pRPS5A::ΩTMV:zCas9i:nosT was assembled by combining pICH41233-pRPS5A (Kerstens et al, 2021), pICH41402 (Addgene #50285; ΩTMV 5’UTR), pAGM47523 (Addgene #153221; zCas9i) and pICH41421 (Addgene #50339; nosT) in pICH47742 (Addgene #48001) using golden gate cloning (Engler et al, 2014). All constructs were electroporated into Agrobacterium tumefaciens (C58C1.pMP90) and transformed into Col-0 wild-type plants as described in (Clough and Bent, 1998).…”

“…In roots, PLTs indirectly read out an auxin gradient to establish stable tissue zonation by controlling cell division and differentiation (Mahonen et al, 2014; Roszak et al, 2021; Santuari et al, 2016) and they are required for stem cell maintenance (Aida et al, 2004; Blilou et al, 2005). Although PLTs are involved in a wide array of developmental processes including development of the primary roots (Aida et al, 2004; Galinha et al, 2007), lateral roots (Du and Scheres, 2017; Hofhuis et al, 2013; Kerstens et al, 2021), phyllotaxis (Pinon et al, 2013; Prasad et al, 2011), and pluripotency acquisition in callus (Zhai and Xu, 2021), their role during early embryogenesis has not been resolved. However, homozygous combinations of plt2 and bbm in PLT1, PLT2 seedlings were not recovered when we attempted to generate higher-order mutants of, AIL/PLT clade members (Galinha et al, 2007), suggesting that specific PLTs are required for early embryogenesis.…”

Genetic Variability of Arabidopsis thaliana Mature Root System Architecture and Genome-Wide Association Study

“…The rhizotron system consists of flat boxes comprising two plastic sheets with a space of 4 mm filled with potting soil. A detailed protocol on the rhizotron assembly and preparation can be found in the study by Kerstens et al (2021). The soil substrate in this study was a commercial mix (Aveve potting soil universal use 20L/bag).…”