Isolation of New Gravitropic Mutants under Hypergravity Conditions

Abstract

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis.

The gravistimulation-induced very slow Ca2+ increase in Arabidopsis seedlings requires MCA1, a Ca2+-permeable mechanosensitive channel

“…Enhanced gravitropism by hypergravity (hyper-gravitropism) is reported in Arabidopsis roots 32 , stems 33 and hypocotyls 34 . The analysis of the plant growth under the sequential hypergravity conditions (30–500 g for 1 day) shows that hypergravity suppressed elongation growth of hypocotyls, but this effect was reduced in hypocotyls of mca -null mutants compared with the wild type 28 , suggesting that MCAs are involved in the sensing of gravity signals in plants.…”

Micromanipulation of amyloplasts with optical tweezers in <i>Arabidopsis</i> stems

“…This makes centrifugation a useful technique to investigate the relationship between amyloplast dynamics and gravity sensing (Toyota et al 2014). Indeed, the reduced gravitropic sensitivities in pgm and sgr mutants were restored by centrifugal hypergravity (Fitzelle and Kiss 2001;Mori et al 2016;, which correlated with the resumption of amyloplast sedimentation (Fitzelle and Kiss 2001;Toyota et al 2013b). These studies suggested that intracellular distribution of amyloplasts toward the gravity vector is critical for gravity perception in plants.…”

Micromanipulation of amyloplasts with optical tweezers inArabidopsisstems

“…This makes centrifugation a useful technique to investigate the relationship between amyloplast dynamics and gravity sensing (Toyota et al 2014). Indeed, the reduced gravitropic sensitivities in pgm and sgr mutants were restored by centrifugal hypergravity (Fitzelle and Kiss 2001; Toyota et al 2014; Mori et al 2016), which correlated with the resumption of amyloplast sedimentation (Fitzelle and Kiss 2001; Toyota et al 2013b). These studies suggested that intracellular distribution of amyloplasts toward the gravity vector is critical for gravity perception in plants.…”