BIG regulates sugar response and C/N balance in Arabidopsis

ABSTRACT

Mutations in BIG gene not only produce pleiotropic phenotypes of plant development but also impair plant adaptive responses under various stresses. However, the role of BIG gene in sugar signaling is not known. In this study, we first found that BIG deficiency significantly sensitized the sugar-induced anthocyanin accumulation and the sugar-inhibited primary root growth, suggesting BIG is an important component of the sugar signaling pathway. Then we found that big mutant plants had higher sugar levels compared with the wild type, indicating the involvement of BIG gene in regulating plant sugar homeostasis. Importantly, we also found that the relative ratio of carbon to nitrogen (C/N) was greatly enhanced by BIG deficiency. Overall, our work expands the known functionality of BIG and reveals its role in regulating sugar response and C/N balance. It is likely that BIG connects nutrient, light, and hormone signaling networks for regulating plant development and adaptive responses.

KEYWORDS:

• BIG gene
• sugar response
• C/N balance
• plant development

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Sugars in particular sucrose (Suc) and glucose (Glc) which serve as the main sources of carbon-skeleton and energy are increasingly recognized as signaling molecules involved in various developmental processes such as seed germination, cotyledons expansion, root growth, flowering, senescence, and stress responses in plants.¹BIG gene encoding a putative calossin-like protein with a molecular weight of 560 KD, was identified originally as a regulator of light signaling,² auxin transport, apical dominance,³ and later was shown to control multiple hormone signaling pathways,⁴ vesicle trafficking and endocytosis ,^5,6 and recently was implicated in participating in the dynamic adjustment of circadian period which is profoundly affected by the availability of sucrose.^7,8 Mutations in BIG as well as the homologous Pushover produce pleiotropic phenotypes.^9,10 More recently, works by our lab have suggested that BIG is a component required for elevated CO₂-induced stomatal closure and plant defense,^11,12 two processes that are both modulated by sugars,^13,14 prompting us to investigate whether BIG deficiency affects cellular sugar homeostasis and sugar signaling.

High concentration of sugars arrests plant primary root growth.¹⁵ To examine whether BIG participates in sugar response, we first assayed big mutant and wild-type Col-0 (WT) seedlings in parallel and found that when higher concentration (4%) of sugars was presented in the half-strength Murashige and Skoog (MS) media, big mutants showed enhanced sensitivity to sucrose- and glucose-mediated inhibition of primary root growth (Figure 1a,b). We then examined whether the enhanced sensitivity to sugars was due to altered sugar contents by monitoring the glucose and sucrose levels of big mutant and WT plants, we observed a deviation of the dynamics of sugar accumulations. For example, as shown in Figure 1c,d, at all three test time points during the artificial day (when the light is on) mutant leaves consistently accumulated more glucose than WT, in particular, at 12:30 pm the difference was statistically significant, and the elevation of sucrose levels in big mutant was more pronounced as even at 9:00am (one hour after the onset of the artificial day) we observed a significant difference between mutant and WT plants. Intriguingly, the elevated sugar levels in big mutants dropped in the afternoon and even returned to the WT level at 5 pm (one hour before the onset of night). Whether the enhanced soluble sugars were a consequence of the enhanced photosynthesis and/or of the disturbed distribution/utilization of the photosynthates awaits further investigation. Notably, big mutant seedlings produced significantly more anthocyanin than those of WT when grown with 4% glucose or sucrose (Figure 1e,f), in accordance with the reported stimulatory effect of sugar on anthocyanin biosynthesis.^16,17 These results strongly indicate that BIG is involved in regulating sugar accumulation and responses in Arabidopsis.

Figure 1. BIG negatively regulates sugar responses.

(a) Representative images showing BIG gene mutants are more sensitive to sugar-mediated inhibition of primary root elongation than that wild-type Col-0 (WT). Seedlings of indicated genotypes were grown on half-strength MS medium for 4 days, then transferred to half-strength MS media with 1% sucrose (control) or with 4% sucrose or 4% glucose and grown for another 5 days before measurement. Scale bar, 1 cm. (b) Relative inhibition of primary root length was analyzed from (a). (c) Glucose levels of leaves from four-week-old WT and big mutant plants were measured. (d) Leaf sucrose content was measured in four-week-old WT and big mutants. (e) Representative images showing that big mutants are more sensitive to glucose-induced anthocyanin accumulation than WT. Seedlings were grown on half-strength medium with 1% sucrose (control) or with 4% sucrose or 4% glucose for 11 days. Scale bar, 1 cm. (f) Anthocyanin contents were measured in (e). For (b), (c), (d) and (f), asterisks indicate significant differences detected using Student’s t-test (*P < .05, **P < .01) when compared to WT.

To sustain optimal growth and development for plants, cellular carbon (C) and nitrogen (N) metabolism must be tightly coordinated.¹⁸ For example, when the C/N ratio is too high, the number and density of lateral root would decrease dramatically in Arabidopsis,¹⁹ whereas when the C/N ratio is too low, the whole plant growth and photosynthesis are both inhibited in maize.²⁰ Having shown that mutations in BIG gene perturbed the accumulations of sucrose and glucose, we next investigated whether BIG impacts on the concentrations of C and N as well as the C/N ratio. We first determined the carbon content and found that the carbon level in the leaves of the mutants was significantly higher than that of WT plants (Figure 2a). In contrast to the increased C levels, the nitrogen concentration in mutant plants was markedly reduced (Figure 2b). Accordingly, the C/N ratio in big mutants was greatly elevated (Figure 2c), in agreement with the phenotype of much reduced lateral roots (LR) of big mutants as a high C/N ratio inhibits LR initiation in Arabidopsis.^4,19,21 Given plant growth at elevated CO₂ stimulates carbohydrate production that will activate sugar sensing systems but lead to lower N concentration ,²² and BIG is required for stomatal responses to CO₂ which is an indispensable substrate that plants need to carry out photosynthesis ,¹¹ we wondered whether BIG has a role to play in adjusting C and N metabolism under elevated CO₂ condition. Therefore, the C and N contents of both WT and big mutant plants grown under elevated CO₂ (1000 ppm) conditions were determined. Results presented in Figure 2d,e show that in WT elevated CO₂ caused a significant increase of C level but decrease of N level, resulting in an increase of C/N ratio, whereas big mutant plants exhibited reduced sensitivity to elevated CO₂-induced elevation of C/N ratio (Figure 2f).

Figure 2. BIG is important for regulating carbon/nitrogen balance.

(a) and (b) The percentage of C content was significantly increased in big mutants compared to WT, whereas the percentage of N content was significantly decreased in big mutants than WT. The C/N contents of rosette leaves from five-week-old plants were determined by elemental analyzer. (c) The C/N ratio was significantly increased in big mutants. (d) BIG gene mutants are less sensitive to elevated CO₂-induced C content increase. (e) BIG gene mutants are less sensitive to elevated CO₂-induced N content decrease. (f) BIG gene mutants are less sensitive to elevated CO₂-induced increase of C/N ratio. For (a), (b) and (c), asterisks indicate significant differences detected using Student’s t-test (*P < .05, **P < .01) when compared to WT.

Rising CO₂ levels profoundly impact not only plant physiology but also defense responses.^23,24 For example, CO₂ enrichment experiments have consistently shown that when fed on elevated CO₂-grown plants, most leaf-chewing insects exhibit reduced growth and survival rates because of increased C/N ratios and reduced N contents.^25,26 In a recent study, we found that BIG deficiency allowed more growth of pathogens when challenged with both avirulent and virulent bacteria, and interestingly big mutant plants retained significantly fewer larvae of pest S. exigua when larvae were given the choices between big mutant and WT plants, suggesting that BIG is required for plant defense.¹² Our observation that N concentration in big mutant plants was markedly lower than that in WT (Figure 2b) which is suggestive of lacking proteins and nutritional values might partly account for the feeding deterrent effects of big mutants. Whether BIG plays essential roles in plant defense against insect feeding on elevated CO₂-grown plants merits further investigation, as the findings might provide new insights into the feeding habit of phytophagous insects and the future of agriculture in a high-CO₂ world.

Many research efforts have been made to understand the links between BIG gene and auxin pathway, as big mutants show typical defects in lateral root growth, organ elongation and apical dominance that have long been associated with auxin action.^3,6,27,28 Two recent reports have suggested that sucrose itself, rather than auxin, is acting in regulating root growth and apical dominance.^29,30 If this is the case, the decrease in apical dominance and some growth arrests observed in big mutants could be at least partly attributed to the perturbed sugar homeostasis/partitioning. Of note, mutations in BIG gene could greatly alter JA responses including JA-induced anthocyanin biosynthesis,¹² which is also modulated by sugars.^17,3133 Taking our findings and the well-documented roles of BIG in conveying light information and hormonal signaling into consideration, it is tempting for us to speculate that BIG connects light, nutrient and hormone signaling networks to regulate plant development and defense response, of which the mechanistic basis merits further investigation.

Disclosure of potential conflicts of interest

The authors declare that they have no conflicts of interest in the research.

Acknowledgments

This work was supported by the National Natural Science Foundation of China under grant numbers 31171356 and 31470360.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [31171356 and 31470360].