Resolving the role of strigolactone in the early steps of rice axillary bud dormancy

The mechanisms underlying axillary bud outgrowth in apical dominance, i.e. the outgrowth of axillary buds following the removal of the apical bud, have been studied for many decades (Thimann and Skoog, 1934). This process is a major determinant of shoot branching and, as such, of aerial plant architecture (Barbier et al., 2017). Auxin has long been implicated in axillary bud outgrowth (Bennett et al., 2006). However, given that auxin itself does not enter the bud, it was postulated that either auxin flow into the main stem or the presence of a second messenger transmitting the auxin signal to the axillary bud was responsible. Although cytokinin had been proposed as a suitable candidate, just over a decade ago strigolactones were conclusively demonstrated to be the phytohormones which inhibit axillary growth (Gomez-Roldan et al., 2008; Umehara et al., 2008). In this issue, (Luo et al., 2019), used a combination of highly spatially resolved in situ hybridizations alongside transcriptomics on hydroponically grown rice. In doing so they were able to uncover that cell cycle and ribosomal genes were among the active genes in dormant buds, whilst ABA inducible genes were among the inactive genes in this tissue. Furthermore, they showed that application of ABA suppressed the growth of axillary buds of strigolactone mutants and wild type while the tiller number was decreased in transgenic lines in which ABA biosynthesis was upregulated. Taken together these results demonstrate that the main site of strigolactone function is in the lead primordia in the axillary bud and that ABA is involved in strigolactone-mediated axillary bud dormancy (Figure 1). The role of strigolactones in axillary bud outgrowth. (a) Tiller buds in WT and strigolactone-deficient mutant, d10-2. At P5.5, buds in WT and d10-2 are active, but at later stage, the buds stop growing in WT which resulted in reduced tiller number. The middle panel shows the expression of histone H4 as revealed by in situ hybridizations. The scale bar represents 100 mm (b) Relationship among genes involved in SL-regulated bud dormancy. Solid red lines indicate published relationship between genes. Solid blue lines indicate relationship demonstrated in this study. Dashed blue lines indicate the possible role of gene. Abbreviations SPL, squamosa promoter-binding protein-like; FC, FINE CULM1; GT, GRASSY TILLERS1; DRM, DORMANCY ASSOCIATED GENE; NAC, NAM (for no apical meristem), ATAF1 and −2, and CUC2 (for cup-shaped cotyledon); AWPM, AWPM-19 (ABA-induced Wheat Plasma Membrane Polypeptide-19). Professor Junko Kyozuka has held her current position at Tohoku University since 2015, previously working at the University of Tokyo between 2001 and 2014. Her research focuses on plant development with highlights being the discovery of strigolactones and further investigation of their developmental biological roles. Her co-corresponding author, Le Luo, was a PhD student at the onset of the project but is currently a lecturer at Nanjing Agricultural University. The work described in this article, which was also carried out with collaboration input from Professor Seo of the RIKEN Center for Sustainability Resource Science and Professor Itoh of Nagoya University, took a total of 8 years. Given the length of time taken, which was in part due to the wish to repeat the experiments several times to ensure their reproducibility, the work extended well beyond Le′s PhD and encompassed the moves of Le to Nanjing and Junko to Tohoku, where their works focus on the role of nutrients in shoot branching and determining the ancestral roles of strigolactones and unknown ligands perceived by strigolactone paralogs, respectively. As mentioned above the nature of the terpenoid plant hormones strigolactones was elucidated in a pair of seminal papers that identified their structure in rice and pea with Junko being one of the co-authors of the rice paper (Gomez-Roldan et al., 2008; and Umehara et al., 2008). Subsequently the pathway of their synthesis in rice was rapidly completed by the identification of the pathway to and beyond carlactone (Alder et al., 2012). Thereafter, the perception of strigolactones by DWARF14 (D14) – the strigolactone receptor triggers a D14:D3:D53 complex formation leading to ubiquitination of D53 followed by its degradation and thus de-represses proteins downstream of D53 (Jiang et al., 2013; Al-Babili and Bouwmeester, 2015). Despite the rapid advances in the understanding of the biosynthetic pathway and strigolactone signaling mechanistic insight into the function of strigolactones lags behind. Prior to the article of Luo et al. (2019) two modes of action of strigolactones had been proposed (i) that they act via controlling the subcellular location of the PIN protein, which acts as an efflux transporter of auxin; or (ii) that they activate downstream transcriptional cascades. In order to gain further insight into which of these modes of action are functional or dominant a highly controllable hydroponic system was used and strigolactone-deficient mutants grown in this system were observed to display differential axillary bud growth. Careful inspection of the emergence of this phenotype over time revealed the time point at which the wild-type bud becomes dormant by a sudden arrest of cell division. However, further analysis revealed that the buds maintain the capacity to resume growth once conditions render it favorable. Junko explained that the sampling was the most difficult and critical part of this project since they wanted to compare transcriptomes between the SL mutants and WT before the differences in their developmental patterns became obvious. Therefore, they first needed to find the critical timing when the phenotype becomes detectable, and to sample axillary buds just before this time point. Le repeatedly grew seedlings again and again to set up stable growth conditions and to determine the critical developmental stage for analysis. Having defined the time at which the switch to bud dormancy occurs laser capture microdissection was used to dissect the bud of the second leaf at the time immediately preceding and following the onset of dormancy and evaluated changes in genome-wide gene expression. This study revealed induction of dormancy-associated genes whilst genes associated with the cell cycle and cell proliferation were decreased. Furthermore, evaluation of the transcriptomic data revealed that several ABA responsive genes as well as genes associated to ABA biosynthesis were induced on the transition to dormancy. Given this observation Luo et al. (2019) next tested whether ABA suppresses bud outgrowth in rice by adding low concentrations of ABA which do not invoke major changes in plant growth. Intriguingly, while this addition of ABA did not affect tiller bud growth in wild-type plants it suppressed the growth of tiller buds in the strigolactone-deficient d10-1 mutant via an effect on cell division as well as their growth in the strigolactone signaling mutant d14-1. Overexpression of the key ABA biosynthesis associated gene NCED1 resulted in lines with higher ABA content and suppressed axillary bud outgrowth. Furthermore, measurement of the level of ABA levels in the basal part of the stem revealed that the d14-1 mutant, but not the d10-1 mutant, displayed reduced levels of ABA. When taken together these observations clearly demonstrate the involvement of ABA in strigolactone-mediated axillary bud dormancy thus opening a new window into the study of strigolactone function. However, the lack of congruence in the ABA levels of the strigolactone biosynthesis and signaling mutants, as well as how ABA and indeed strigolactone-mediated effects interact with the signals influencing axillary branching recently reported to emanate from the signal metabolite trehalose 6-phosphate (Fichtner et al., 2017), remains to be investigated. Beyond this specific question the work presented by Luo et al. (2019), provides a highly important example of how hormone function contributes to the control of normal growth. Such insight is lacking not just for strigolactones but also for most other phytohormones. In addition, given that shoot branching is one of the most important traits for agriculture the findings reported in their article provide important fundamental knowledge, which could have dramatic implications for applied agriculture.