WRKYs work to limit root growth in response to shade

Plants compete for light when growing at close proximity and detect shading from neighboring vegetation through decreases in light availability and in the red: far-red light ratio (R:FR). Changes in R:FR are sensed by specialized photoreceptors called phytochromes that trigger morphological changes known as the shade avoidance syndrome (SAS). SAS is characterized by rapid stem and petiole elongation accompanied by a change in leaf orientation (hyponasty; Casal, 2012). Shade also promotes plant susceptibility towards pathogens via disruption of jasmonic acid and ethylene-mediated defense responses (de Wit et al., 2013; Courbier et al., 2020). Shade signals are typically perceived in the shoot but limit belowground root growth as well. Indeed, shade perceived in the shoot leads to reduced lateral root formation in Arabidopsis (Arabidopsis thaliana; van Gelderen et al., 2018), but the exact mechanisms behind shade-induced responses in plant roots are still relatively unclear.

In this issue of Plant Physiology, Rosado et al. (2021) shed light on the role of WRKY transcription factors (WRKYs) and ethylene signaling in root growth inhibition and lateral root formation during shade. To understand the molecular events leading to the inhibition of root growth by shade, the authors performed a transcriptome study on Arabidopsis and tomato (Solanum lycopersicum) roots sampled from seedlings exposed to either control or FR-enriched light. A gene ontology (GO) analysis on the FR-induced genes revealed that overrepresented GO categories in both species were associated with ethylene signaling and stress responses, including low oxygen and biotic stress responses, suggesting that shade-induced transcriptional dynamics closely resemble (a)biotic stress-induced transcriptional responses.

Interestingly, a third of the shade-induced genes displayed W-box motifs in their promoter; this motif is known as a WRKY transcription factor binding site. WRKYs are among the largest family of transcriptional regulators in plants and typically control biotic and abiotic stress responses (Pandey and Somssich, 2009). Accordingly, the authors found many WRKYs transcriptionally upregulated by shade, suggesting that the observed transcriptional abiotic and biotic stress responses could be driven by these WRKYs. Whether the WRKYs and (a)biotic-related transcriptional responses contribute to the observed root growth phenotype remains unclear, but both low oxygen and biotic stress signaling have previously been linked to root development (Shukla et al., 2019).

To further elucidate the role of WRKYs in root growth inhibition in response to shade, the authors generated Arabidopsis fluorescent protein-fusion reporter lines that over-express 10 of the detected WRKYs. They observed WRKYs were mostly localized in both shared and distinct root cell types in response to shade. Next, the authors compared shoot and root growth phenotypes of the 10 WRKY overexpression lines (WRKYox) in both shaded and unshaded conditions. Interestingly, none of the WRKYox lines showed differences in shoot elongation compared to the wild type in response to shade. However, WRKY26ox and WRKY45ox displayed a reduction in primary root length and lateral root density compared to wild-type plants after shade and control treatments, suggesting that WRKY26 and WRKY45 may play a role in shade-induced root growth inhibition. In contrast, wrky26 and wrky45 single mutants still responded to shade similarly to wild type, ruling out a prominent role in root growth regulation. As the authors mention, several WRKYs may contribute to root growth responses in a redundant fashion, and future work could test their involvement by creating higher-order knock-out mutants that include wrky26 and wrky45.

Ethylene plays a primary role in both shade avoidance responses and root growth cessation (Das et al., 2016; Pandey et al., 2021), and “response to ethylene” was among the top GO categories enriched in shade-induced genes in both Arabidopsis and tomato. Therefore, the authors studied the effect of ethylene signaling in shade-induced root growth inhibition. Treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) indeed led to shorter primary roots and increased lateral root density in both shaded and unshaded conditions. At higher ACC concentrations, root phenotype differences between the shade and control plants disappeared, suggesting shade-induced root growth inhibition could be ethylene-regulated. Moreover, ethylene-insensitive mutants no longer reduced root growth in response to shade but showed constitutively shorter primary roots. This latter result is somewhat surprising since ethylene is a negative regulator of root growth and insensitive mutants are instead expected to have longer primary roots. Still, the work highlights that ethylene signaling is required for shade-induced root growth inhibition in Arabidopsis.

Collectively, Rosado et al. identify WRKYs, namely WRKY26 and WRKY45, as well as ethylene signaling as regulators of the shade-induced root growth inhibition in Arabidopsis (and possibly tomato). A potential connection between WRKYs and ethylene signaling in response to shade remains to be investigated. The authors provide evidence that WRKYs could exert their role in shade-induced root growth inhibition downstream of ethylene signaling. ACC treatment led to increased WRKY45 and WRKY26 transcript levels in the roots of both shaded and unshaded plants. These results correspond with the report that both WRKYs are direct targets of the principal ethylene signaling regulator ETHYLENE INSENSITIVE3 and are upregulated by ethylene (Chang et al., 2013). However, ethylene-dependent WRKY26 and WRKY46 upregulation during shade remain to be confirmed, for instance, through studying their expression levels in ethylene-insensitive mutants during shade treatments.

The work of Rosado et al. further contributes to our understanding of the complex role of WRKYs in plant responses to their environment and pinpoints them as potential regulators of root growth during the competition for light. Their findings also highlight that shade-induced transcriptional root responses could be conserved between Arabidopsis and important crop species like tomato, which could help identify common pathways by which plants reallocate energy resources in response to their environment.