Root properties such as root architecture and root function have a major impact on crop yield and production (Lynch 2007), but insights from laboratory experiments under controlled conditions can be only partly transferred to a field scale due to spatial constraints in the lab and highly variable properties of different soils, as well as fluctuating weather conditions in the field (Passioura 2006; Langstroff et al. 2022). In the current study, maize was cultivated under limiting nutrient supply. Different types of field-derived data were measured to shed light into processes impacting on water uptake, nutrition, growth, and interactions with the environment. We identified that the growth stage is a decisive driver for elemental composition of maize leaves, but also a moderate driver of root gene expression. For both element composition and gene expression, the effect of substrate is enhanced during the aging of maize, and the development of drought stress. These results provide experimental evidence how maize responds to the environment, and support the aim to increase the sustainability of maize cropping systems (Cordero et al. 2019). The presence of root hairs mattered for shoot growth and total nutrient accumulation, but had a minimal influence on element concentrations and root gene expression profiles. This suggests that the functional role of root hairs is related to efficient nutrient acquisition and maize growth propagation (Zhu et al. 2005).
We observed that the global gene expression patterns were not significantly different (PERMANOVA, P < 0.05) between rth3 and WT maize although the loss of elongated root hairs resulted in lower shoot biomass and lower total uptake of nutrients. This observation opposed our fourth hypothesis, which stated that effects of root hair formation on root traits and root gene expression should be higher in the field than laboratory settings. In fact, the minor extent of changes in gene expression was highly consistent with earlier data on maize root architecture(Lippold et al. 2021) and root gene expression during BBCH14 in soil columns (Ganther et al. 2021), but also regarding the transcript abundances in young primary roots (Rüger, MG, in preparation). The fact that the total uptake of nutrients was higher in WT, but not root biomass, might indicate the nutrient uptake and/or transport rates per root volume are higher in WT than rth3. Since transporter gene expression was not affected, it is possible that transporter activity is regulated at the protein level. For instance, post-translational modifications such as ubiquitination and phosphorylation are widespread among the members of phosphate transporter 1 family, affecting the localization, abundance and activity of the transporters (Wang et al. 2017). Our data for total nutrient uptake were also in accordance with observations made in a greenhouse experiment assaying the rth2 maize mutant which forms extremely short root hairs (Klamer et al. 2019): While the concentration of P did not differ in rth2 compared to the wild type, the total P content of the juvenile maize plants was reduced by 50% under combined water stress and P deficiency. Furthermore, WT plants did not show higher P concentrations, but higher total shoot P contents, a product of P uptake and biomass generation. Lower biomass gain by rth3 could thus be related to a more inefficient use of the incorporated carbon by the root hair mutant, caused for instance by higher rate respiration rate (Earl et al. 2012). When cumulative water extraction was normalized to shoot dry weight, Jorda et al. (submitted) detected higher water use efficiency of WT than rth3 maize. Lower water use efficiency of rth3 could be explained by a more effective regulation of stomata in response to drought (Benešová et al. 2012), which could also affect respiration rates.
Elemental levels and related gene expression are growth stage-dependent
Plant development depends on a balanced supply of macro- and micronutrients, and an equilibrium between individual elements is maintained by homeostatic mechanisms. The age of a plant or plant organ represents an important factor modulating plant nutrient concentration (Marschner 1995); for example, Peng et al. (2012) showed that relative NPK uptake from the soil differs throughout the maize growth season. This relevance of growth stage was also reflected by strong regulation of nutrient concentration and gene expression data in our study.
We found that maize plants did not receive adequate P, K and Zn from soil during the three growth stages, and were supplied with too little N and Mn during BBCH59 (Bergmann 1986). For BBCH59, the limitation of nutrients was particularly strong, most likely due to the fact that the topsoil dried out completely, rendering fertilizer-applied nutrients unavailable for plant uptake (Bista et al. 2018). Low water availability and high temperatures could have also limited shoot growth (He and Dijkstra 2014; Dodig et al. 2021) and thus decreased nutrient demand.
Leaf (Baxter et al. 2008; Stich et al. 2020) and root (Courbet et al. 2021) ionome studies from hydroponics and from the field (Watanabe et al. 2015) have shown that this homeostasis is mediated by strong interactions between individual nutrients. In line with this, our field study found significant correlations between the concentration profiles; the macronutrients N, Ca, Mg and S, and micronutrients Fe and Mn followed a similar trend (Fig. 1b). The reports on interactions between multiple nutrient elements suggest that their uptake, transport, and/or assimilation levels are correlated, and supports chemical element stoichiometry in plant tissues (Kumar et al. 2021). For instance, Briat et al. (2015) presented a molecular framework in Arabidopsis thaliana for interactions between P, S, Fe and Zn homeostasis. In our study, these nutrient elements showed in part contrasting patterns according to sampling stage and substrate (Figs. 4–6).
During the transition between vegetative and reproductive growth (Chen et al. 2016), but also when root mineral uptake is limited and cannot fully support plant growth (Maillard et al. 2015), nutrient reallocation has been identified as a strategy by higher plants to improve mineral resource availability of sink tissues. Whereas N can be readily remobilized from leaves, K, S, P, Mg, Cu, Mo, Fe and Zn are predominantly mobilized during nutrient deficiency (Courbet et al. 2021). Our data showed lower N and Mn levels in remainder shoot (RS) than younger leaves at all stages, and lower K levels during BBCH59 (Supplementary Fig. 7, ESM1). Leaf mineral nutrient remobilization has been described in maize, but maize was among the least efficient plants in nutrient reallocation from senescent to young leaves (Maillard et al. 2015). Our data thus indicates that maize did not efficiently compensate for the loss of nutrients in young leaves by nutrient reallocation from the rest of the shoot.
Nutrient starvation cues are immediately transmitted from the shoots to the roots via signaling molecules to maintain nutrient homeostasis. This signaling leads to the activation of transporter gene expression, secretion of organic acids, siderophores or enzymes, associations with mycorrhizal fungi and alterations in root architecture and growth to enhance mineral uptake (Bouain et al. 2014). For some of the surveyed transport genes, we also saw complementary expression profiles in dependence of the developmental stage or substrate. These included high-affinity transporters NRT1 and NRT2 which are induced by N starvation (Trevisan et al. 2008) and accordingly induced in our study on sand with lower N concentration in leaves. A similar compensatory pattern was found for P transporters PHT1 and PHT2, as well as several purple acid phosphatases (Supplementary Table 8, ESM5) that are known to be induced during P starvation (Yun and Kaeppler 2001). The stronger expression in sand than loam suggests that the topsoil in sand was dry and nutrient availability decreased, leading to higher mineral transporter gene expression. The characterization of these gene “responders” in the field could be applied for future studies to target and possibly elicit certain plant responses to environmental stresses.
However, we found that leaf elemental concentrations declined from higher levels during the first two stages of growth to the last stage BBCH59 (Figs. 4–6). Apparently, maize could not compensate for acquisition of these nutrients by faster root growth or enhanced nutrient transporter or siderophore biosynthesis gene expression (Figs. 1, 6). Still, one of the most highly enriched GO terms by developmental stage was nicotianamine biosynthesis. Nicotianamine represents a critical metabolite in the biosynthetic pathway to mugineic acid family phytosiderophores, natural Fe and Zn chelators (Takahashi et al. 2003), that graminaceous plants secrete from their roots to mobilize and take up Fe and Zn from the rhizosphere (Wiren et al. 1996; Nozoye et al. 2011; Hanikenne et al. 2021). As Fe concentration in the maize leaf remained low during BBCH59, the elevated expression of genes involved in nicotianamine biosynthesis could be interpreted as a response to that.
Root resource allocation and biomass were also affected by plant age, with a higher level of carbon (atomic % of 13C) and nitrogen (15N) uptake during BBCH14, and higher root-shoot ratio during BBCH14 than BBCH59, in both substrates (Fig. 1). This suggests a relative increase in shoot sink activity at the cost of the root system, which would be normal during maize development in soils under no water limitation. Since the precipitation was low during BBCH19 and continued to be so during BBCH59 (Supplementary Fig. 1, ESM1), a stronger resource and biomass investment to the roots than during sufficient soil moisture was expected (Gargallo-Garriga et al. 2014). Root sink limitation by environmental factors, like water or nutrient availability, may thus be more constraining for growth and development than C availability (Körner 2015). The temporal pattern of root resource allocation was also reflected in related gene expression. When root C allocation decreased over time, this was accompanied by a decrease in cell wall-related gene expression (e.g. beta-glucosidase 40, xyloglucan endotransglycosylase). Comparing substrates, C allocation was consistently higher in sand than loam. In terms of gene expression, this was supported by a higher gene expression in sand for transport and cell wall-related genes (Fig. 1). These findings were also in line with higher root length densities of plants grown on sand (Vetterlein et al. 2022). Root length density (root length per volume soil) describes root architecture in respect to the size of the soil volume that can be explored by roots, and it is thus related to the efficiency of water and nutrient uptake (Ren et al. 2017). By contrast, for plants on loam we found an enrichment of genes supporting GO categories related to microbial defenses and stress (for example chitin, resp. to herbivory; Supplementary Table 6, ESM3). These findings support our first hypothesis, that the balancing of resources in order to favor either growth or defense (Schultz et al. 2013) is reflected on a transcriptomic level.
Water stress towards BBCH59 leads to differences between substrates in root-shoot ratios and aquaporin root gene expression
Within the same field experiment, water transpiration data from Jorda et al. (submitted) demonstrated that severe water stress occurred just before tassel emergence (BBCH59 stage). Water stress is especially detrimental during the reproductive stages when water requirement is the highest (Pandey et al. 2000; Çakir 2004; Araus et al. 2012). According to the water transpiration data, drought stress occurred earlier in loam than sand substrate and earlier in the wild type than the rth3 mutant. This could possibly attributed to the higher root-shoot ratios observed in plants grown on sand, as an adaptation to water stress (Gargallo-Garriga et al. 2014). In wheat, it was shown that increased root-shoot ratios supported growth under drought conditions, as they served to limit inefficient transpiration (Bacher et al. 2021; Collins et al. 2021). For maize, transpiration-limiting variants were also estimated to produce to higher yields under extreme conditions (Messina et al. 2015).
Water transport in plants is conveyed by intrinsic membrane proteins of the aquaporin family (Siefritz et al. 2002), and the role of aquaporins in root water uptake has been abundantly documented (Aroca et al. 2012). Plant aquaporin genes are regulated in a complex manner, for instance some up- and others down-regulated by drought, suggesting that the maintenance of water transport requires spatial regulation of aquaporin gene expression (Galmés et al. 2007; Šurbanovski et al. 2013). Elevated aquaporin expression, with higher values for sand than loam was observed at BBCH59. This would fit a compensatory pattern in aquaporin expression, taking into account measurements of plant available water by (Vetterlein et al. 2022) that demonstrate a sharp decline of plant available water during the transition from BBCH19 to BBCH59. At BBCH59, plants on loam (both root hair genotypes) had already experienced drought stress for 1–2 weeks, but plants on sand (wild type) only for 4–5 days. The fact that aquaporin expression is higher in sand even though they experienced later onset of drought than loam could perhaps indicate a differential stress response in relation to immediate or prolonged water stress exposition. Plants grown on sand were sampled during the recent onset of drought stress which led to elevated aquaporin gene expression. Another possibility could be that plants on sand generally expressed higher levels of aquaporin genes and thus were better equipped to face drought stress. Elevated aquaporin expression in sandy substrate was also found for a companion soil column experiment conducted under finely controlled growth and watering conditions (Ganther et al. 2021), suggesting that the induction could also in part be related to the properties of the root system. Interestingly, both field and lab grown plants developed thicker root diameters in sand (Lippold et al. 2021; Vetterlein et al. 2022), and the thicker roots might compensate for potential increase in radial resistance by increased aquaporin expression (Del Carmen Martinez-Ballesta et al. 2011).
Depth profiles are more pronounced at BBCH59 and affect selected N, P and water transport genes
At BBCH59 compared to BBCH19, more depth-related gene expression among the surveyed genes has been detected, which is in line with our third hypothesis, stating that depth profiles become more pronounced towards later growth stages. At BBCH19 no gene expression differences between depths 0–20 and 20–40 cm were observed, but at BBCH59 a stronger induction of N, P and water transporters in the upper layer, in particular for the loam substrate, was shown. Root length density was higher in the top 20 cm than the lower depth at BBCH59 (Vetterlein et al. 2022), suggesting that the roots in the upper layer may have exhausted some of the available nutrients. This possibly was exacerbated by the drying out of the topsoil. For maize under field conditions, Wiesler and Horst (1994) observed that nitrate uptake rate per unit root length generally increased with soil depth, most likely due to increased root growth. That aquaporin genes PIP1-5, 2–3 and 2–6 were higher expressed in the upper soil, is probably related to lower soil moisture, and higher expression levels of root exudation-related genes in the topsoil suggests that root exudation is also enhanced by low soil moisture (Zhang et al. 1995). Of the analyzed maize phosphate transporters, PHT2 (Nagy et al. 2006) and PHT7 (Liu et al. 2016) are up-regulated by arbuscular mycorrhizal fungi, implying that these transporters might participate with other P transporters in mediating inorganic P absorption and/or transport by the mycorrhizal pathway. Mycorrhiza formation in this experiment was higher in the topsoil than in the lower depth (Vetterlein et al. 2022), and this matched the higher expression PHT2 and PHT12 in the topsoil layer, but only for loam-grown plants.