Description

The ability of developing seedlings to respond and adapt to diverse environmental conditions including light is critical for their emergence and establishment (Benvenuti et al., 2001; Forcella et al., 2000; Salter et al., 2003; Yu and Huang, 2017). Cell expansion within the hypocotyl optimizes light and energy capture by the cotyledons, and enables the transition to autotrophic status (Botterweg-Paredes et al., 2020; Dowson‐Day and Millar, 1999; Oh et al., 2013). Hypocotyl elongation is regulated by multiple factors including temperature, phytohormones, circadian clock and light (Dowson‐Day and Millar, 1999; Ma et al., 2016; Procko et al., 2014; Reed et al., 2018; Yu and Huang, 2017). Endogenous and exogenous sugars are also important regulators of hypocotyl cell expansion (Lilley et al., 2012; Liu et al., 2011; Pfeiffer and Kutschera, 1995; Simon et al., 2018b; Singh et al., 2017; Zhang et al., 2010, 2015). Under constant darkness, the interaction between plant hormones such as brassinosteroid and gibberellin and sugar signalling is proposed to stimulate increase in hypocotyl length (Simon et al., 2018b; Zhang et al., 2010, 2015). Hypocotyl phenotypes are important tools in plant biology as they have been used to screen for mutants with altered responses to light and sugar signalling and some of the identified genes have revolutionized plant research (Nakano, 2019). In multicellular organisms, actomyosin-dependent transport constitutes an essential component of the cellular structure and dynamics (Duan and Tominaga, 2018; Kurth et al., 2017; Peremyslov et al., 2010). The Arabidopsis genome encodes 17 myosins classified into two distantly-related groups comprising 4 class VIII myosins and 13 class XI plant myosins (Haraguchi et al., 2019; Reddy and Day, 2001; Ryan and Nebenführ, 2018). The plant-specific-myosin XI has been implicated in diverse developmental processes (Cai et al., 2014), but the contribution of members of class VIII myosins including myosin1/ATM1 to plant development stills remains elusive.

Here, we analyzed the role of plant myosins in sucrose-induced hypocotyl elongation under continuous darkness conditions. To start the molecular characterization of ATM1 (AT3G19960), we analyzed four T-DNA insertional alleles (Fig. 1A), and mutations were confirmed via PCR-based genotyping. We also performed RT-qPCR to quantify ATM1 transcripts in the four alleles, of which atm1-1 (SAIL_405_BO8 previously described by Haraguchi et al., 2014) showed reduced ATM1 expression among these alleles compared to the wild type (Fig. 1B). Notably, atm1-3 exhibits increased mRNA levels and may be a overexpression allele. Since atm1-1 had the lowest ATM1 transcript level, and was previously described as a null (Haraguchi et al., 2014) we decided to use this allele for further phenotypic analysis. To understand the contribution of plant myosins in sugar-regulated hypocotyl cell expansion we performed growth assays using Col-0 (WT: wild-type), atm1-1, a class XI myosin single gene mutant (xik) and the triple knockout line (3KO; xi1 xi2 xik). This triple mutant was previously generated using SALK insertion lines in XI-K (SALK_067972; At5g20490), XI-1 (SALK_019031; At1g17580) and XI-2 (SALK_055785; At5g43900) (Prokhnevsky et. al., 2008; Valera et. al., 2010). All genotypes were grown on 0.5X Murashige and Skoog (MS) medium without or with 15 mM sucrose under constant darkness for 5 days. Under the sucrose-free condition (control), all the three myosin mutants showed reduced hypocotyl length compared to the WT plants (Fig. 1C and D), suggesting the role of myosins in cell elongation. Specifically, the 3KO mutant was severely impaired in hypocotyl cell expansion in the absence of sucrose when compared to the single mutants, hinting that this gene family may redundantly regulate plant development. In the presence of sucrose under constant darkness, the short hypocotyl phenotype was not fully rescued when compared to the WT plants except for atm1-1.

Altogether our findings suggest that plant-specific myosins may be involved in sugar-regulated hypocotyl elongation under continuous darkness. These myosins have diverse subcellular localization patterns (Haraguchi et al., 2014; Peremyslov et al., 2012, 2008) and thus may represent a new downstream signaling output following sucrose signals within the developing shoot. Sucrose-induced hypocotyl elongation has been linked to brassinosteroid, gibberellins, phytochrome interacting factors (PIFs) and SnRK1 (Sucrose Nonfermenting Kinase 1) signaling (Laxmi et al., 2004; Liu et al., 2011; Simon et al., 2018a; Zhang et al., 2015, 2010; Zhang and He, 2015). Other proteins that are important for this process include TANG1 (Zheng et al., 2015), a WD40 protein AtGHS40 (Hsiao et al., 2016), a plant-specific protein SR45 (Carvalho et al., 2010), and HIGH SUGAR RESPONSE 8 (HSR8) (Li et al., 2007). Further studies will be needed to determine how these molecular factors work in concert or parallel to regulate skotomorphogenesis.