Research articleArabidopsis glucose-sensitive mutant 3 affects ABA biosynthesis and sensitivity during early seedling development
Introduction
As one of the primary sugars, Glc is a key regulator and signaling molecule of many crucial processes in plants, for example, seed germination, seedling establish, vegetative growth, photosynthesis, carbon and nitrogen metabolism, flowering, stress responses, and senescence (Rolland et al., 2002). Although the mechanisms how plants produce, transport, metabolize, store and sense Glc signal have been elucidated (Granot et al., 2014; Yu et al., 2015), it is difficult to separate Glc metabolism from Glc sensing and signaling events, and create powerful tools to investigate the potential roles of regulators whose essential functions are covered up by mutant lethality or redundancy of gene function. Therefore, lots of problems still need to be addressed to better understand the mechanisms in details.
Hexokinases (HXKs), which are the first demonstrated intracellular Glc sensors in plants, appear to serve overlapping and distinct functions in signaling and metabolism. In Arabidopsis, HXK1 plays dual roles in glucose signaling and metabolism (Moore et al., 2003; Cho et al., 2006). Besides, extensive studies have shown that cross-talk exists between Glc and ABA signaling pathways (Zhang et al., 2008). High Glc increases ABA accumulation by regulating the expression of genes involved in ABA synthesis or metabolism during seedling development (Carvalho et al., 2010). A large number of Glc-sensitive mutants also exhibit defects in ABA synthesis or response. Among them, ABA INSENSITIVE4 (ABI4) that plays a central role in ABA signaling, has been isolated on the basis of its strong sugar-resistant phenotype (Bossi et al., 2009). ABI4 is regulated by Glc and ABA at the transcript level (Shkolnik-Inbar and Bar-Zvi, 2010), and acts as either an activator or repressor to control the expression of many specific ABA- or Glc-responsive genes by recognizing CE1 cis-elements (CACCG and CCAC motif) in their promoters (Niu et al., 2002; Carvalho et al., 2010). For example, ABI4 directly regulates the transcripts of ABI5 and SBE2.2 to mediate sugar signaling (Carvalho et al., 2010), and also participates in regulating seed dormancy by directly repressing the transcripts of CYP707A1 and CYP707A2 to promote ABA biosynthesis (Shu et al., 2013).
Reactive oxygen species (ROS) has been believed to be connected with biotic and abiotic stress, such as oxidative, drought, and salt stresses (Torres and Dangl, 2005; Miller et al., 2008). Disruption of the ROS homeostasis causes the failure of seed germination (Bailly et al., 2008). Thus, the balance between ROS production and elimination should be under elaborate and strict control. In plant cells, chloroplasts are the main source of ROS, which are produced mainly in the photosynthetic electron transport chain.
Previous study reported that At1g56200 (EMB1303) loci encodes a chloroplast-localized protein and loss-of-function of At1g56200 led to defects in chloroplast development and growth (Huang et al., 2009). In this study, a T-DNA insertion line of At1g56200 locus termed gsm3 was isolated for its sensitivity to Glc, ABA, and sodium chloride (NaCl). Compared to the wild type, gsm3 exhibited reduced germination and cotyledon expansion under Glc, ABA, or NaCl. Further analyses suggested that increased ABA synthesis is responsible for the sensitivity of gsm3 to Glc and NaCl, as indicated by the result that addition of fluridone could rescue the cotyledon expansion defects of gsm3 under Glc or NaCl. In addition, disruption of ABI4 or AAO3 in gsm3 background led to decreased sensitivity to Glc and NaCl. We measured the levels of H2O2 and O2•- and found gsm3 accumulated more ROS than the wild type, which may result in high sensitivity of gsm3 to H2O2 during cotyledon expansion and root growth. Taken together, these results indicated that Arabidopsis GSM3 participates in the Glc-ABA signaling during seed germination and early seedling growth.
Section snippets
Materials and growth conditions
Arabidopsis thaliana wild type (WT, Col-0), gsm3 (SALK_202427), abi4-1 (CS8104), and aao3-4 (SALK_072361) mutants used in this study were obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University, Columbus, OH, USA). To gain the gsm3 abi4-1 and gsm3 aao3-4 double mutants, gsm3 was crossed with abi4-1 and aao3-4 mutants respectively and then screened via PCR until the double homozygous mutants were obtained. Plants were grown aseptically or on soil in a growth chamber
gsm3 is sensitive to Glc during seed germination and early seedling development
To identify novel components essential for Glc response or signaling, an efficient method was used to screen transfer-DNA (T-DNA) insertion lines on 1/2 Murashige and Skoog (MS) medium supplemented with 1% agar and Glc (3% or 4.5%), Mtl (3% or 4.5%) was used as a control. In the presence of high Glc conditions, wild-type seeds germinated and grew normally, whereas Glc-sensitive seeds showed cotyledon expansion defects and yielded bleached seedlings at subsequent growth stages (Zheng et al., 2019
Discussion
A previous study reported that loss of At1g56200 activity led to a complete arrest of plant growth and seedling lethality when emb1303-1 was grown in soil (Huang et al., 2009), indicating an important role of At1g56200 in chloroplast and embryogenesis. In this study, gsm3 exhibited sensitivity to Glc and NaCl (Fig. 1, Fig. 4, and Supplementary Fig. S2), which was proved to be caused by Glc- or NaCl-induced ABA synthesis and signaling pathways (Fig. 4, Fig. 5, Fig. 6). Sugars and ABA have been
Author contributions
JQ and TFS performed most of the experiments and all of the data analyses; JWY assisted in plasmid construction and Arabidopsis transformation; JQ, YFH, and MZ participated in the preparation of the manuscript; MZ conceived and designed the studies; All authors have read and approved the final manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31600987) and the Program for Innovation Team Building at Institution of Higher Education in Chongqing (CXTDX201601009).
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