Exogenous carbon source supplementation counteracts root and hypocotyl growth limitations under increased cotyledon shading, with glucose and sucrose differentially modulating growth curves

ABSTRACT

Plant growth is continuously modulated by endogenous and exogenous stimuli. By no means the only, but well described, signaling molecules produced in plants and distributed through the plant body to orchestrate efficient growth are photosynthates. Light is a potent exogenous stimulus that determines, first, the rate of photosynthesis, but also the rate of plant growth. Root meristem activity is reduced with direct illumination but enhanced with increased sugar levels. With reduced cotyledon illumination, the seedling increases hypocotyl elongation until adequate light exposure is again provided. If endogenous carbon sources are limited, this leads to a temporary inhibition of root growth. Experimental growth conditions include exogenous supplementation of sucrose or glucose in addition to culturing seedlings under light exposure in Petri dishes. We compared total root length and hypocotyl elongation of Arabidopsis thaliana wild type Col-0 in response to illumination status and carbon source in the growth medium. Overall, sucrose supplementation promoted hypocotyl and root length to a greater extent than glucose supplementation. Glucose promoted root length compared to non-supplemented seedlings especially when cotyledon illumination was greatly reduced.

Plants are exposed to a constantly changing environment and have therefore evolved a finely tuned network of intracellular processes to adapt their growth rate and architecture.^1–11 Previously published studies have shown that established plant cultivation protocols that include direct root illumination and often sucrose supplementation either mask or alter plant growth responses.^2,12–16 Direct root illumination is considered a stress-inducing growth escape mechanism and is known to interfere with signaling events in the root tip, including suppressing root meristem activity.^2,17–20 Dark-grown roots (DGR) form longer roots compared to light-grown roots (LGR) due to a more active meristem and respond differently to additive stress treatment or exogenous phytohormone addition.^2,16 Sugar increases meristem activity and auxin biosynthesis in the root tip, resulting in highly divergent root expression compared with non-sucrose-supplemented seedlings^13,14,21. Light and sugar signaling pathways are closely intertwined and play a major role in shaping plant growth direction and architecture.^{2,4,13,20,22,23} In particular, the hypocotyl, which consists of approximately 20 cells that can elongate even up to 10-fold, is very sensitive to light and sugar signals.^24–26 In this study, we compared the effect of growth conditions on plant development by measuring total root length and hypocotyl length of fully illuminated seedlings, seedlings with roots grown in shade before direct illumination (DGR) using the so-called D-root system,² seedlings shaded from light by growing at a defined position below the edge of the D-root system, and of etiolated seedlings (Figures 1 and Figures 2, A-F; Suppl. Figure 1 B: Suppl. Figure 2 showing experimental setup). In addition, we investigated the influence of exogenous supplementation of the growth medium with a carbon source (Figures 1 and 2, G-I; Suppl. Figure 1 A and C).

Figure 1.

Impact of carbon source supplementation of the growth medium on root length

Figure 2.

Impact of carbon source supplementation of the growth medium on hypocotyl length

We measured the growth of seven-day-old seedlings of wild-type Arabidopsis thaliana line Col-0 cultured at a temperature of 21°C ± 2°C and a light intensity of 100 µmol/sec/m². Seedlings were grown vertically on ½ Murashige and Skoog medium supplemented with 1% agar and either 1% sucrose,1% glucose or no sugar. Plates were scanned to obtain images of the seedlings, and changes in growth characteristics were quantified using the Image J program from NIH (raw data are summarized in Suppl. Table 1). Statistical evaluation was performed using GraphPad prism and R. Normally distributed data was analyzed using One-Way ANOVA with Tukey HSD post-hoc comparisons while not normally distributed data was tested using the Kruskal-Wallis method with Dunn’s multiple comparison (Raw Data and Statistic Analysis added available as Data Set).

Comparison of root length arranged according to the root light source (A-F) and carbon supplementation used (G-I). Statistical analysis within groups was performed through One-Way ANOVA with Tukey HSD post-hoc test or Kruskal-Wallis with Dunn’s multiple comparison test (not normally distributed data, denoted by □ at the right corner of the plot). Different letters between groups denote statistical significance (p < 0,05; n = 17–49 plants, Raw Data and Statistical Analysis available as Supplement Files, Example Pictures available as Supplement Figure 1, scalebar is 5 mm).

Total root length gradually decreases when cotyledons are not fully illuminated and even more depending on carbohydrate supplementation (Figure 1). Previous studies showed that root growth is negatively influenced by direct root illumination,^2,22 including on one side reduced meristematic activity, but on the other side enhancing escaping mechanism, negative phototropism, by triggering cell elongation.^2,22,27,28 Additionally, direct root illumination results in differential distribution of siganling molecules, including phytohormones and reactive oxygen species (ROS), and reduced ability of nutrient uptake, all factors are crucial for proper root development.² In the study of Silva-Navas et al., 2015, it is further shown that root length differences depending on the root illumination status accumulate with age of the seedling, with striking differences twelve days after germination,² whereby all experiments were done on medium supplemented with 1% sucrose. In García-González et al., 2021a,²² we showed that differences in root length and gravitropic index are already measurable at seven days after germination. Whereby, sucrose enhances root length in dark and light grown roots, but without sucrose light grown roots are stronger impaired comparing them with dark grown roots due to the inhibitory effect of direct root illumination on meristem activity.^2,18,22

In this study, we wanted to examine furthermore to which extend reduced illumination of the cotyledons, which is affecting photosynthetic activity, additively modulates plant growth and if exogenous supplementation of one kind of carbon source is enough to stimulate root growth. When cotyledons are exposed to light, total root length differs due to root illumination status but not because sucrose was supplemented or not (Figure 1. A-B and G-H). With increased shading and especially in the case of etiolated seedlings, the total root length on cultivation medium without sucrose supplementation is significantly shorter compared to seedlings cultured with sucrose supplementation, but sucrose supplementation alone doesn’t promote root growth to the same extent compared to growth conditions when cotyledons are exposed to light (Figure 1. C-F and G-H). It will be necessary to investigate to which extend other photosynthetic products are crucial for efficient root growth modulation, how far they are coupled to the synthesis of signaling molecules like phytohormones, or if root growth is inhibited because to long dark periods indicate that investment in primary root growth is counterproductive.

Furthermore, when we exchanged sucrose with glucose root growth changed dramatically depending on the illumination status of the cotyledons, implicating that not only changes in photosynthetic activity but also the distribution of individual sugars has a crucial impact on root growth (Figure 1. A-F and I). When the cultivation medium is supplemented with glucose, the total root length is shorter compared with roots cultivated with sucrose supplementation or on medium without exogenous sugar addition as long as the cotyledons are still exposed to a certain amount of light (Figure 1. A-D). Only upon increased shading and especially under etiolated growth conditions, glucose increases root length compared with roots grown on medium without exogenous sucrose supplementation, but significantly less compared with sucrose (Figure 1. E-F and G-I).

Altogether, light grown roots deviate on tissue and cell level strongly from dark grown roots,^2,22 and it is still unknown to which extend availability and distribution of endogenous sugars, between shoot and root, and further through the root tip modulate root growth. Experiments with differential carbon source supplementation show that not all sugars equally orchestrate root growth.^29,30 Impact of individual sugars is among others dependent on applied concentration and experimental design, which sometimes includes pre-cultivation on sucrose containing medium followed by transfer of seedlings on medium with other carbon sources, which may result in deviating results in-between individual studies, and therefore, we suggest careful evaluation of applied setups.^29,30

Comparison of hypocotyl length arranged according to the root light source (A-F) and carbon supplementation used (G-I). Statistical analysis within groups was performed through One-Way ANOVA with Tukey HSD post-hoc test or Kruskal-Wallis with Dunn’s multiple comparison test (not normally distributed data, denoted by □ at the right corner of the plot). Different letters between groups denote statistical significance (p < 0,05; n = 16–51 plants, Raw Data and Statistical Analysis available as Supplement Files, Example Pictures available as Supplement Figure 1, scalebar is 5 mm).

After exogenous sugar supplementation, both hypocotyl elongation and root growth are enhanced (Figure 2. A-I and Figure 1. A-I). Unlike root length, the increase in hypocotyl length under sucrose and glucose supplementation shows the same growth curve under changing light conditions, probably because hypocotyl length is defined by elongation, whereas root length results of elongation and proliferation events (Figure 1. G-I and Figure 2. G-I). When cotyledons are well exposed, significant difference in hypocotyl length as a function of growth medium supplementation is measurable (Figure 2. A), and gets even more obvious with increased shading, gradually proportional to increasing darkness, while sucrose supplementation accelerates hypocotyl elongation more compared with no sugar and glucose supplementation (Figure 2. B-F), which also shows that sucrose and glucose differently modulate length of roots and hypocotyls, maybe already indicating their differential role in orchestrating elongation versus proliferation events.

With reduced cotyledon illumination status, endogenous energy resources produced via photosynthesis in the form of sugars are less available to maintain continuous growth of all plant organs. Seedlings with cotyledons covered when growing in soil or shaded by other plants, for example, inhibit root growth and promote hypocotyl elongation until the cotyledons are illuminated again.⁴ Treatment of seedlings in the laboratory by exposure to artificial light conditions or supplementation with carbon sources in the growth medium obscures the interplay between the different plant organs and also masks the phenotypes of the mutants.^2,22 Therefore, our work encourages other colleagues to consider the critical role of individual sugars in modulating plant growth not only as energy sources or building blocks, but also as signaling molecules that orchestrate various growth processes. After decades of research, we now know how specialized the role of sugar signaling is at the tissue level and at different developmental steps, and beyond that in concert with changing environmental stimuli, but we still design experiments the same way we did decades ago. The same is true for the choice of lighting conditions for plants. It is time to rethink standard laboratory growth methods in the context of individual scientific questions.

Funding Statement

This work was supported by the grantová agentura české republiky [19-13375Y]; ministerstvo školství, mládeže a tělovýchovy [LM2018129]; ministerstvo školství, mládeže a tělovýchovy [CZ.02.1.01/0.0/0.0/16_019/0000738].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website