ABSTRACT
Sugar signaling forms the basis of metabolic activities crucial for an organism to perform essential life activities. In plants, sugars like glucose, mediate a wide range of physiological responses ranging from seed germination to cell senescence. This has led to the elucidation of cell signaling pathways involving glucose and its counterparts and the mechanism of how these sugars take control over major hormonal pathways such as auxin, ethylene, abscisic acid and cytokinin in Arabidopsis. Plants use HXK1(Hexokinase) as a glucose sensor to modulate changes in photosynthetic gene expression in response to high glucose levels. Other proteins such as SIZ1, a major SUMO E3 ligase have recently been implicated in controlling sugar responses via transcriptional and translational regulation of a wide array of sugar metabolic genes. Here, we show that these two genes work antagonistically and are epistatic in controlling responsiveness toward high glucose conditions.
Results and discussion
Sugars are key molecules governing major aspects of plant growth and development. Sugar signals interconnect with major phytohormonal networks to induce profound changes in plants physiology throughout their life cycles.Citation1,Citation2 In photosynthetic organisms, sugars control these aspects of growth and development through the major sugar sensor, HXK1 that exhibits both catalytic and regulatory/signaling attributes.Citation3 However, high exogenous glucose supplementation causes post-germination growth arrest (PGGA), delayed cotyledon expansion and greening of seedlings.Citation1,Citation4 Moreover, hypersensitivity to glucose also predisposes the seedlings to ABA, suggesting a link between phytohormonal crosstalk with sugar-mediated signaling.Citation5
Earlier, HXK1 mutants named gin2 (glucose insensitive 2) had been characterized in Arabidopsis thaliana as a glucose sensor which functions independently of its metabolism-related activityCitation1 and mediates glucose-dependent gene repression of key photosynthetic genes viz. chlorophyll a/b binding protein (CAB), ribulose-1,5-bisphosphate carboxylase (RBCS) and carbonic anhydrase (CAA).Citation4 Furthermore, glucose also regulates expression of other genes independently of HXK1, suggesting the presence of multiple parallel sugar responsive pathways.Citation4
Like many other sugar response mutants, the siz1 (for SAP AND MIZ1 DOMAIN-CONTAINING LIGASE1) mutant shows hypersensitivity toward high exogenous glucose concentrations.Citation6 In particular, the siz1 mutant exhibits enhanced sensitivity toward glucose and sucrose in terms of inhibition of root growth, seed germination and cotyledon greening.Citation6 These data point toward the implication of sumoylation of essential proteins at early life stages of a plant in response to sugars since sugars have been implicated in the control of early seed development and seedling establishment.Citation5
To investigate for the role of HXK1 and SIZ1 in controlling plant growth and development, T-DNA mutants of both HXK1 (hxk1–3, CS69135) and SIZ1 (siz1–5, SALK_111280) (independent mutant allele less characterized than siz1–2 and siz1–3) mutants were obtained from the Arabidopsis Biological Resource Centre (ABRC). Literature mining of these two loss-of-function mutants indicated that for hxk1–3, the insertion of T-DNA was in the 1st intronCitation7 while for siz1–5, 15th exon was disrupted by T-DNA,Citation8 in their respective genomes (). The T-DNA insertions in the single mutants and the double mutant was then confirmed by routine genotyping PCR. The results showed that all mutants were homozygous T-DNA insertion lines (). Phenotypic analyses of WT, hxk1–3, siz1–5 and the homozygous double mutant, hxk1-3siz1–5, revealed that knockout of either allele or in combination did not affect the normal growth and development of the double mutant (). In particular, hxk1-3siz1–5 plants flowered early under short days (8 h light/16 h dark) and had rosette and leaf size similar to the siz1–5 mutants when compared to the WT (), suggesting that the hxk1–3 mutation lies upstream of siz1–5 mutation.
Since the siz1 mutation has been shown to be hypersensitive to high exogenous glucose concentrations,Citation6 we therefore asked if the disruption of the evolutionary conserved HXK1 gene in the siz1 background would confer insensitivity toward glucose. Glucose has been shown to delay seed germination in Arabidopsis,Citation5 we therefore first examined percentage seed germination of different genotypes under increasing concentration of glucose. In the absence of glucose (no sugar), there were no significant differences in terms of seed germination between WT (Col-0) and the hxk1–3, siz1–5 and hxk1–3 siz1–5 mutants (). However, at higher glucose concentrations (3% and 5%), PGGA of the siz1–5 was significantly higher when compared to the other genotypes. In the presence of glucose, the cotyledon greening of the siz1–5 mutant was also visibly impaired (). Most importantly, this inhibition of seed germination and cotyledon greening of the siz1–5 was nearly reversed in the hxk1–3 siz1–5 double mutant (). This suggests that the disruption of the HXK1 gene in siz1–5 confers insensitivity toward inhibition of seed germination under high glucose concentrations. Alternatively, the hxk1 mutation is epistatic to siz1 mutation in mediating response to inhibition of high glucose concentrations.
Next, we studied hypocotyl growth in dark under glucose supplemented conditions of the above mentioned genotypes. As expected, glucose-mediated inhibition of hypocotyl growth inhibition was more in the siz1–5 mutant than the WT, hxk1–3 and the double mutant grown in 3% glucose supplemented media (). Interestingly, the introduction of the hxk1–3 mutation in the siz1–5 background resulted in an intermediate phenotype of the hxk1-3siz1–5 double mutant, suggesting that SIZ1 and H×K1 have distinct and opposite role in sugar sensing and signaling during hypocotyl growth in Arabidopsis ().
In conclusion, this report suggests the involvement of HXK1 in SIZ1-modulated sugar signaling is conditionally epistatic across normal overall development (ontogeny) and growth conditions (sugars). Overall, the disruption of HXK1 in siz1–5 background markedly attenuated the glucose-oversensitive phenotype of the latter in terms of seed germination and hypocotyl elongation under high glucose conditions, suggesting that the function of SIZ1 in sugar signaling might be dependent upon HXK1 activity. Overall, the interaction between these two proteins might govern key developmental aspects of this organism during different phases of the life cycle. In particular, the significance of this report lies in unraveling the molecular machinery behind how plants perceive and react to glucose signals, influencing fundamental processes of seed germination and hypocotyl growth (). Moreover, as SIZ1 is involved in salicylic acid signaling, therefore an interconnection between HXK1 and the hormone can be explored.
This finding, however, is in contrast to what had been mentioned earlier in the literature.Citation9 This discrepancy might have arisen due to the hxk1 allele being used in Castro et al., 2020,Citation9 which differs from the one used in the present study. The hxk1–3 is a null mutant which has been characterized previouslyCitation10 to be a knockout mutant, the transcript levels being lower than the other mutant alleles, hxk1–1 and hxk1–2.Citation10,Citation11 The residual HXK1mRNA expression in the above two lines might be the reason for the obscured genetic analysis.
Acknowledgements
SSR acknowledges Council of Scientific and Industrial Research (CSIR) and Fellowship from NIPGR. SS acknowledges Department of Biotechnology (DBT). AL acknowledges NIPGR core grant, DBT: NWBA-2015 (BT/HRD/NWBA/37/01/2015(xii)) and JC Bose fellowship 2021 (JCB/2021/000012). The authors are grateful to the DBT-eLibrary Consortium (DeLCON) for providing access to e-resources.
Disclosure statement
No potential conflict of interest was reported by the author(s).
References
- Moore B, Zhou L, Rolland F, Hall Q, Cheng W-H, Liu Y-X, Hwang I, Jones T, Sheen J. Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science. 2003;300(5617):332–5. doi:10.1126/science.1080585.
- Sami F, Siddiqui H, Hayat S. Interaction of glucose and phytohormone signaling in plants. Plant Physiol Biochem PPB. 2019;135:119–126. doi:10.1016/j.plaphy.2018.11.005.
- Jang JC, Sheen J. Sugar sensing in higher plants. Plant Cell. 1994;6(11):1665–1679. doi:10.1105/tpc.6.11.1665.
- Sheen J, Zhou L, Jang JC. Sugars as signaling molecules. Curr Opin Plant Biol. 1999;2(5):410–418. doi:10.1016/S1369-5266(99)00014-X.
- Dekkers BJW, Schuurmans JAMJ, Smeekens SCM. Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis. Plant Mol Biol. 2008;67(1–2):151–167. doi:10.1007/s11103-008-9308-6.
- Castro PH, Verde N, Lourenço T, Magalhães AP, Tavares RM, Bejarano ER, Azevedo H. SIZ1-Dependent post-translational modification by SUMO modulates sugar signaling and metabolism in Arabidopsis thaliana. Plant Cell Physiol. 2015;56(12):2297–2311. doi:10.1093/pcp/pcv149.
- Huang J-P, Tunc-Ozdemir M, Chang Y, Jones AM. Cooperative control between AtRGS1 and AtHXK1 in a WD40-repeat protein pathway in Arabidopsis thaliana. Front Plant Sci. 2015;6:851. doi:10.3389/fpls.2015.00851.
- Watson SJ, Li N, Ye Y, Wu F, Ling Q, Jarvis RP. Crosstalk between the chloroplast protein import and SUMO systems revealed through genetic and molecular investigation in Arabidopsis. Elife. 2021;10. doi:10.7554/eLife.60960.
- Castro PH, Verde N, Azevedo H. Arabidopsis thaliana growth is independently controlled by the SUMO E3 ligase SIZ1 and Hexokinase 1. MicroPubl Biol. 2020;2020.
- Huang JP, Tunc-Ozdemir M, Chang Y, Jones AM. Cooperative control between AtRGS1 and AtHXK1 in a WD40-repeat protein pathway in Arabidopsis thaliana. Front Plant Sci. 2015;6:160979. doi:10.3389/fpls.2015.00851.
- Aki T, Konishi M, Kikuchi T, Fujimori T, Yoneyama T, Yanagisawa S. Distinct modulations of the Hexokinase1-mediated glucose response and Hexokinase1-independent processes by HYS1/CPR5 in Arabidopsis. J Exp Bot. 2007;58(12):3239–3248. doi:10.1093/jxb/erm169.