Multiple Interactions between Glucose and Brassinosteroid Signal Transduction Pathways in Arabidopsis Are Uncovered by Whole-Genome Transcriptional Profiling

Abstract

Brassinosteroid (BR) and glucose (Glc) regulate many common responses in plants. Here, we demonstrate that under etiolated growth conditions, extensive interdependence/overlap occurs between BR- and Glc-regulated gene expression as well as physiological responses. Glc could regulate the transcript level of 72% of BR-regulated genes at the whole-genome level, of which 58% of genes were affected synergistically and 42% of genes were regulated antagonistically. Presence of Glc along with BR in medium could affect BR induction/repression of 85% of BR-regulated genes. Glc could also regulate several genes involved in BR metabolism and signaling. Both BR and Glc coregulate a large number of genes involved in abiotic/biotic stress responses and growth and development. Physiologically, Glc and BR interact to regulate hypocotyl elongation growth of etiolated Arabidopsis (Arabidopsis thaliana) seedlings in a dose-dependent manner. Glc may interact with BR via a HEXOKINASE1 (HXK1)-mediated pathway to regulate etiolated hypocotyl elongation. BRASSINOSTEROID INSENSITIVE1 (BRI1) is epistatic to HXK1, as the Glc insensitive2bri1-6 double mutant displayed severe defects in hypocotyl elongation growth similar to its bri1-6 parent. Analysis of Glc and BR sensitivity in mutants defective in auxin response/signaling further suggested that Glc and BR signals may converge at S-phase kinase-associated protein1-Cullin-F-box-TRANSPORT INHIBITOR RESPONSE1/AUXIN-RELATED F-BOX-AUXIN/INDOLE-3-ACETIC ACID-mediated auxin-signaling machinery to regulate etiolated hypocotyl elongation growth in Arabidopsis. Plants constantly sense the changes in their environment and transmit these signals as part of normal development. For optimal growth and development, plants need to coordinate complex developmental processes and, at the same time, sense and respond to endogenous physiological factors and external environmental stimuli. Many factors such as light, nutrients, and phytohormones are known to regulate these developmental processes. All these factors probably form a complex signal response network to bring about optimum growth changes to enable better fitness in plants. Among the phytohormones, brassinosteroids (BRs) are very important for plant growth and development. BRs are a class of polyhydroxylated sterol derivatives that are small growth-promoting molecules found at low concentrations throughout the plant kingdom. The BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) heterodimerizes with BRI1-ASSOCIATED KINASE1 (BAK1) after binding to BR. BRI1 and BAK1 subsequently act together to inhibit a Glycogen synthase kinase3-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2; Li et al., 2001). In absence of BR, BIN2 phosphorylates BRASSINAZOLE RESISTANT1 (BZR1). BIN2-mediated phosphorylation inhibits DNA binding capacity of BZR1 and promotes its binding to 14-3-3 proteins, which ultimately leads to its cytoplasmic retention or degradation (He et al., 2002; Gampala et al., 2007; Ryu et al., 2007). Signaling by BRI1/BAK1 removes this inhibition, and unphosphorylated BZR1 translocates to the nucleus, where it acts together with the transcription factor BRI1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) to regulate expression of BR-inducible genes (Wang et al., 2002; Yin et al., 2002, 2005). BZR1 not only activates BR-induced genes and promotes cell elongation, but also suppresses BR biosynthetic genes such as CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM (CPD), leading to feedback inhibition of BR biosynthesis (He et al., 2002). BRs govern many processes involved in morphogenetic changes. BRs stimulate cell elongation by increasing cell wall plasticity and can also affect cell shape and expansion via regulation of microtubule dynamics. BRs control a vast number of responses in plants, such as seed germination, hypocotyl elongation, and senescence. BRs are also involved in controlling stomatal development and physiology. They have also been implicated to play a role in both biotic and abiotic stresses (Dhaubhadel et al., 1999, 2002; Kagale et al., 2007; Koh et al., 2007; Xia et al., 2009). BRs also interact with auxin to regulate both root elongation and tropic responses in plants (Bao et al., 2004; Li et al., 2005; Kim et al., 2007). In plants, the availability of nutrient resources regulates many physiological and developmental processes profoundly. Apart from their metabolic functions, sugars such as Suc and Glc have also been reported to act as signals that can trigger changes in gene expression in plants (Price et al., 2004). Glc as a signaling molecule can also influence almost every aspect of plant growth and development such as cell proliferation and death, cell expansion and elongation, seed germination, seedling growth and development, primary root length, shoot and root gravitropism, lateral roots, root hairs, shoot meristem maintenance, reproduction, senescence, photosynthetic gene expression, crop yield and product quality, carbon and nitrogen metabolism, and stress responses (Rolland et al., 2002, 2006; Rolland and Sheen, 2005; Chen et al., 2006; Ramon et al., 2008; Mishra et al., 2009; Smeekens et al., 2010; Eveland and Jackson, 2012; Gupta et al., 2012; Singh et al., 2014). There are three distinct Glc-signaling pathways in plants: the Arabidopsis (Arabidopsis thaliana) HEXOKINASE1 (AtHXK1) signaling function-dependent pathway, the AtHXK1 catalytic function-dependent pathway, and the AtHXK1-independent pathway (Xiao et al., 2000). In the AtHXK1-dependent pathway, AtHXK1 acts as a Glc sensor (Moore et al., 2003). The AtHXK1-independent pathway involves G-protein signaling (Chen and Jones, 2004). The G-protein complex is represented by single Gα (G-PROTEIN α SUBUNIT1 [GPA1]), Gβ (G-PROTEIN β SUBUNIT1), and Gγ (G-PROTEIN γ SUBUNIT1 [AGG1] and AGG2) subunits. The REGULATOR OF G-PROTEIN SIGNALING1 (AtRGS1) contained a seven-transmembrane domain and RGS box and accelerates intrinsic guanosine triphosphatase activity of G-protein. The THYLAKOID FORMATION1 (THF1) protein is located on the outer membrane and stroma of plastids. The heterotrimeric G-protein complex via GPA1 interacts with the AtRGS1 and THF1 (Huang et al., 2006). Sugar signaling has been reported to exhibit cross talk with several different response pathways, such as those involved in nitrogen responses, environmental responses, light responses, phytohormones, and stress responses. However, not much is known about Glc and BR signaling interaction. High level of sugar causes repression of CPD, an important gene involved in brassinolide biosynthesis (Szekeres et al., 1996; Smeekens, 1998). The CPD-antisense lines as well as cabbage1 (dwarf1-6 [dwf1-6]) mutant Arabidopsis plants display a clear reduction in starch content and assimilatory capacity (Schlüter et al., 2002). BRs are reported to regulate the process of sugar uptake in tomato (Lycopersicon peruvianum; Goetz et al., 2000). The dx mutation leads to production of a nonfunctional DWARF enzyme in tomato. In dx fruits, levels of starch and various sugars are reduced, which could be partially normalized by BR application (Lisso et al., 2006). The sugar hypersensitivity of a brassinosteroid, light, sugar mutant could be rescued by exogenous BR application, suggesting an interaction of sugar with BR (Laxmi et al., 2004). In rice (Oryza sativa), overexpression of a sterol C-22 hydroxylase, which controls BR levels, increased seed weight, which was associated with more allocation of sugars to seeds (Wu et al., 2008). These results suggest a role of BR in sugar allocation during grain filling in rice. In Saccharum spp., the LEUCINE-RICH REPEAT RECEPTOR-LIKE PROTEIN KINASE ScBAK1 is found to be expressed predominantly in bundle sheath cells of the mature leaf and potentially involved in cellular signaling cascades mediated by high levels of sugar in this organ (Vicentini et al., 2009). UDP-glycosyltransferases UGT73C5 and UGT73C6 function in conjugation of Glc to BR, which ultimately leads to reduced BR activity (Poppenberger et al., 2005; Husar et al., 2011). Recently, our group has also shown a dual role for BR-Glc cross talk in modulating shoot and root gravitropic responses of Arabidopsis seedlings (Gupta et al., 2012; Singh et al., 2014). Although a number of developmental responses of early seedlings are simultaneously controlled by BR and sugar, no systematic study has been performed to explore the interaction between Glc and BR signaling on global gene expression profiles and, in turn, on plant growth and development in the dark. In this study, whole-genome transcript profiling along with physiological analysis has been performed to find out the interdependence/overlap between Glc and BR response pathways in the etiolated seedlings of the model plant system Arabidopsis.