Phosphorus uptake in eucalypts plants under split root system

doi:10.21203/rs.3.rs-5038051/v1

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Phosphorus uptake in eucalypts plants under split root system

https://doi.org/10.21203/rs.3.rs-5038051/v1

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Phosphorus (P) often limits plant growth and development because its availability in most soils is low, mainly in tropical soils. Various phosphate transporters and regulatory elements, including transcription factors, are involved in the uptake and transport of P from the soil into root cells and to other plant organs. The split-root technique was applied to three eucalypt species to understand better the mechanisms of the root-leaf signaling and remobilization response to P supply. Two-month-old seedlings of Eucalypts grandis, E. globulus, and E. tereticornis were used, with each half of the split root system placed in pots. The P treatments were: +P/+P, +P/-P, and -P/-P (+ P = P supplementation and –P = P depleted). P was supplied as 440 µM nutrient solution. Eucalypts plants were grown for six weeks and RT-qPCR evaluated the expression of genes related to P uptake, transport, and utilization in roots and leaves. P supply on one side of the root seemed to compensate for the lack of P on the other side in the + P/-P treatment, so the plant did not show P stress responses, and root-to-root signaling and remobilization in this treatment differed depending on the species. The genes analyzed were mostly induced when plants were under P deprivation, and the expression response was species-dependent. Therefore, this indicates that different mechanisms may be involved in plant response to low P and that signaling control 1may also be linked to the adaptation of eucalypts species to low soil P.

PSR

phosphate transporters

phosphorus stress

plant nutrition

split-root

Eucalypt forests occupy an area of about 18 million hectares global and have superior silvicultural characteristics compared to other forestal genera, such as rapid growth, the ability to adapt to a wide variety of climates and soils, and good quality wood for pulp production (Grattapaglia and Kirst 2008; Pérez-Cruzado et al. 2011). Competition for scarce terrestrial resources between food crops and trees has intensified and agroforestry systems with eucalyptus are an alternative to balance this competition (Nadir et al. 2018). Eucalyptus is a fast-growing tree widely planted throughout the world. Different varieties and hybrids spread across the 625 species and subspecies are planted on various agricultural lands, both as monocultures and as components of agroforestry programs due to their ease of cultivation and ability to grow in adverse conditions (Raj et al. 2018).

Several studies report the performance of agricultural crops under Eucalyptus tree plantations, in order to evaluate the trees' potential for agroforestry (Nadir et al. 2018; Pezzopane et al. 2021; Chavan et al. 2023). Crops of common bean, Irish potato and nightshade (Solanum villosum) were planted along rows of E. grandis (3 and 6 years old). From the results, germination of crops under trees was higher than that in open field, and crops grown under eucalyptus trees gave higher yields compared to crops grown in open fields but not fertilized (Nadir et al. 2018).

Eucalypts plantations occupy more than 50% of the planted forest area in Brazil. They are often established in soils with low fertility, including a low phosphorus (P) availability, demanding phosphate fertilization, mainly in the initial stages of crop growth (Stape et al. 2010; Da Silva et al. 2016).

P is essential for plant growth and development, as it participates in many cellular and biochemical processes and is a component of essential molecules, such as ATP. In the medium and long term, P fertilizers may become a global problem because of the limited number of mines and the continuous increase of P-fertilizers use in agriculture. Thus, there is an enormous concern in the scientific community to ensure a sustainable food supply for the growing global population (Prathap et al. 2022).

Plants may have different adaptation mechanisms to cope with low soil P to optimize its absorption and maintain cellular P homeostasis in all organs (Zhang et al. 2014). The array of molecular responses involved in those processes is called PSR (Phosphorus Starvation Response) and includes the secretion of organic acids, acid phosphatases, protons, and ribonuclease to solubilize inaccessible forms of P. Other changes include increased expression of high-affinity P-transporters, the increase in root-to-shoot ratio, the establishment of symbiotic relationships with mycorrhizal fungi (Tang et al. 2020; Rawat et al. 2021; Li et al. 2022), and strategies that increase soil exploration, such as foraging, promoting changes in architecture and root growth or changes in the amount and length of root hairs (Trindade and Araújo 2014). The set of these molecular strategies and structural and physiological changes in PSR involves not only cellular perception of Pi in different organs, but also interorgan communication of Pi (inorganic phosphorus) levels via systemic signaling (Doerner 2008; Rouached et al. 2010; Puga et al. 2017). Many discoveries and advances in recent years have taken place and allowed the understanding of the complex mechanisms and the knowledge of several genes that control PSR (Gonçalves et al. 2020).

The physical, chemical and biological characteristics of the soil when in a state different from normal undergo changes, and these are detected by the roots (Shabala et al. 2015). Changes in external P concentrations arouse signaling molecules such as orthophosphate, sugars, hormones, small peptides and microRNAs, and these signals can act locally and/or serve as systemic signals to elicit responses at a distance (Chiou and Lin 2011). Plant performance is optimized under specific conditions through the coordinated regulation of metabolic mechanisms and pathways, all done by a network of signals propagated both within the root itself and between root and shoot. (Shabala et al. 2015).

The perception of roots to low P triggers the expression of several genes related to different functions, including metabolic re-organization at the cell level. Most responses to Pi deprivation are modulated by PHR1 (PHOSPHATE STARVATION RESPONSE 1) transcription factors, which adjust Pi uptake and mobilization mainly by regulating the expression of genes. These genes regulate the absorption, assimilation and relocation of P (synthesis of nucleic acids, galactolipids, sulfolipids, RNAses and phosphatases). In addition, they regulate protein kinases and small RNAs, involved in local and systemic regulatory pathways (Li et al. 2022). PHR1 is involved in the activation of several genes related to Pi deficiency, such as PHT1, PHO1 and miR399. PHR1 binds to the PHR1-binding site motif (P1BS; GNATATNC) in the promoter region of the low Pi stress-responsive genes (Secco et al. 2012). When P is available to the plant, PHR1 is inactively bound to proteins with SPX domain, and in the opposite situation, when P is scarce for the plant, the SPX-PHR1 complex is dissociated, and PHR1 binds to the P1BS region of the responsive gene (PSR gene), thus activating its expression, for example, inducing the expression of PHT1 phosphate transporters and miR827(Wang et al. 2017; Gonçalves et al. 2020).

The imbalance of essential nutrients in many soils is seen as one of the main challenges to which plants are exposed. Many of the PSR gene responses are involved in the mechanisms adopted by plants to improve P uptake and internal recycling, modifying the root-shoot partition and expanding the absorption surface of the root system. Long-distance signaling (signaling root-to-shoot-to-root) transmits PSR information from the roots to the entire plant, allowing the perception of the absence of P in the soil, while the signals emitted by the shoots guarantee that the P obtained by the roots is used efficiently to meet the nutritional demand (Lough and Lucas 2006; Hermans et al. 2006). Several molecules signaling for nutrient deficiency have been found for P, but also for N and S, and, to a lesser extent K and Mg. Information is also available about the direction of their movement and the genes involved in signaling cascades (Kondhare et al. 2021). Long-distance signals are expected to support this cross-talk triggered by nutritional cues partially, but the mechanisms are complex and still largely unknown (Ruffel 2018).

Split-root systems make it possible to understand how the root system adjusts nutrient acquisition to meet variations in leaf demand or external nutrient availability, thus maintaining its efficiency (Coppa et al. 2018). It can also help to understand nutrient remobilization in the plants. In this regard, a split-root system can be used as a tool to analyze transcriptional, biochemical, and physiological changes in roots in response to nutritional challenges and root-shoot-root signaling (Ferreira Torres et al. 2021). This approach has been used mostly with fast-growing plants, most herbaceous, where quick changes in the plant metabolism can be tracked.

A broad study with several eucalyptus species was carried out with the aim of classifying these species in terms of their efficiency and responsiveness to P. Several species were classified as Efficient Non-Responsive (ENR) and Not Efficient Responsive (NER), with few being classified as Not Efficient Non-Responsive (NENR). No species was clearly positioned in the Efficient Responsive (ER) quadrant. This study raised new questions about the distinct capacity of eucalyptus species to use available P in the soil (Bulgarelli et al. 2019). Here, we used the split-root technique to understand the response of eucalyptus plants to low P availability and its remobilization in the plant. We used three eucalyptus species known to differ regarding their growth to low P.

2.1 Experimental design and treatments

The experiment was performed in a greenhouse in Campinas, São Paulo, Brazil (22°49′10.38" S 47°04′12.88" W). A 3 × 2 factorial design was used, with the factors being: three eucalypt species and combinations of two P levels (absent and sufficient) at the sides of the split-root system, with three replicates per treatment for biomass and gene expression and five replicates for nutrient contents determination. Seeds of eucalypt species were obtained from the Caiçara Sementes company (Brejo Alegre, São Paulo, Brazil) or the Forestry Science and Research Institute (IPEF, Piracicaba, SP), all originating from clonal garden plants. The species used were Eucalypts grandis W. Hill ex Maiden, E. globulus Labill., and E. tereticornis Sm. The choice of these three species was based on previous results of our research group, which evaluated the efficiency and responsiveness of 24 eucalypt species to P availability (Bulgarelli et al. 2019).

Seeds were germinated in vermiculite and maintained in a growth chamber at 25-28 °C. After two months, seedlings were transferred to a greenhouse and maintained under micro-sprinkler irrigation. After four weeks of acclimatization, each seedling was transferred into a split-root system consisting of two plastic cups of 290 mL with vermiculite. The seedlings were selected according to the size and development of the root system to guarantee the splitting of the root into two similar parts. After placing the seedlings in the cups, they received half-strength modified Hoagland solution (Hoagland and Arnon 1950), with the absence of P (0 μM, -P) and a sufficient level of P (440 μM, +P) (Bahar et al. 2018). The P-treatments in the split-root systems were the following: +P/+P, +P/-P, -P/-P. Plants were collected after six weeks under these conditions.

The collection of biological material (leaf and root) was carried out in a single and quick way, in order to avoid possible stresses for the plant that would result in the results obtained, mainly in the gene expression part. The collection of leaves and roots for gene expression analysis was conducted on the same day as the harvest, and they were promptly frozen in liquid nitrogen and stored at -80 C. After removing the plants from the tube, they had the root separated from the aerial part so that the root could be cleaned in running water. Subsequently, 1-2 cm of secondary and tertiary root tips were removed for gene expression analyses. Regarding the aerial part, leaves (from the first to the fourth, from the tip of the main branch and branches) were collected for gene expression. The rest of the leaves were separated from the stem, and dried in an oven at 65 °C for five days along with the rest of the roots, for analysis of biomass and nutrients. P and gene expression analyses were performed on both sides of roots of the split-root the system.

2.2 Plant biomass and quantification of macro and micronutrients

The dried material was ground and 100 mg were weighed for nutrient analysis. P, K, S, Ca, Mg, Fe, Mn, Cu, Zn, Ni, and B concentrations were determined in leaves and roots by inductively coupled plasma optical emission spectrometry (ICP-OES) following nitro-perchloric acid digestion of the samples (Malavolta et al. 1997). We validated the ICP analysis with two certified standard reference materials from the National Institute of Standards and Technology (NIST: SRM 1515 Apple leaves and SRM 1547 Peach leaves).

2.3 Analysis of gene expression in leaves and roots

At harvest, leaves and roots were collected for the evaluation of the expression of the genes. The genes were from three functional groups: P transport (PHT1.1, PHT1.3, PHT1.5, PHT1.6, PHT1.10, PHT1.11), P uptake (PHO1.8) and sulfolipid synthesis (SQD). Four replicates were used per treatment. Leaf and root samples were macerated in a Geno/Grinder® (SPEX Europe). For RNA extraction, the CTAB/SSTE protocol was followed (Chang et al. 1993), with adaptations. The quality of the extracted RNA was checked on 1% agarose gel and quantified in the NanoDrop® 2000 spectrophotometer (Thermo Scientific). Contamination by genomic DNA was eliminated with the DNase I, RNase-free Kit (Thermo Scientific), and cDNA synthesis was performed with the Platus Transcriber RNase H kit (Sinapse Biotecnologia).

The primers were designed using the Primer3Plus online software and the analysis of secondary structures (hairpins and dimers) was performed using the Primer Quest Tool – IDT (Supplementary Table 1). Data were analyzed using StepOnePlus Real-Time PCR System (Applied Biosystems) to determine sample amplification and detection cycle threshold values (Ct). The specificity of the PCR products generated for each set of primers was verified by analyzing the Tm (dissociation curve) of the amplified products. The expression of Ef1 gene was used as a constitutive reference (Moura et al. 2012). To calibrate the relative value between samples, we considered the mean values of both sides of the treatment +P/+P as reference treatment. The relative expression was calculated using the 2^-^ΔΔ^Ct method (Livak and Schmittgen 2001).

2.4 Statistical analysis

The effects of the heterogeneous availability of P and the eucalypt species on the variables analyzed (biomass and nutrient concentrations) and the expression levels of genes of the PSR were evaluated using analysis of variance (two way ANOVA). The ANOVA was carried out to detect the influence of the factors +P/-P, -P/-P and +P/+P for each dependent variable. Averages were compared using the Tukey’s test at p ≤ 0.05, using the SISVAR software (Ferreira 2011). When necessary, the data were transformed to obtain a normal distribution. Additionally, principal component analysis (PCA) was performed for the nutrients dataset to reduce the dimensionality of the data and identify the variables that best explained the highest proportion of total variance, using XLStat^® software.

3.1 Plant biomass and macro and micronutrients contents

The low availability of nutrients in the soil has been considered a major limitation for plant growth and productivity. The lack of one or more nutrients causes physiological, morphological, and biochemical disturbs, reducing plant growth and inducing biomass reductions. To avoid the low availability of P, plants were provided with several strategies to maximize uptake by the roots through long-distance and local signaling, and improve P utilization in the several physiological processes (Shabala et al. 2015; Bulgarelli et al. 2019). The integration between endogenous signals and environmental factors is responsible for the regulation of plant growth and development. So plants are able to sense the internal and external P-status, triggering appropriate responses. External Pi concentration sensing involves local signaling and plant tissue Pi status involves systemic or long-distance signaling (Chiou and Lin 2011).

Here we used a split root system to study P uptake and allocation and the systemic response of the genes involved in P-use efficiency to P heterogeneity in the root environment. The split root system set-up is shown in Figure 1.

Fig.1 Eucalypt plants after the split-root experiments. Treatments (+P/+P, +P/-P, -P/-P) and the three repetitions of each of the treatments illustrate the biomass production of the studied species: (a) E. tereticornis, (b) E. globulus and (c) E. grandis

In our study, the presence or privation of P did not significantly influence foliar biomass production in E. globulus and E. tereticornis plants (Figure 2a). However, the lack of P on one or both sides of the split root system affected the leaf biomass of E. grandis. Considering the biomass of the whole root system, the lack of P decreased root biomass of -P/-P and +P/-P in E. globulus plants and -P/-P of E. grandis plants (Figure 2b). In E. globulus the roots of the -P side of the +P/-P treatment grew more than roots in the +P side. E. tereticornis plants showed a large variation of the root biomass production between the two sides of the +P/+P treatment. The +P side of the +P/-P treatments showed the highest root biomass accumulation (Figure 1b). In our experiment plants were maintained for six weeks at the split root system and irrigated with the -P and +P solutions and because P is mobile within the plant (Hinsinger et al. 2011) we were not expecting too many differences regarding biomass accumulation in +P/-P plants. In these plants, P might have been redistributed from the +P side to the -P side and supported growth. Also, the +P side might transfer P to the shoots, supporting growth. This is corroborated by the lower average root biomass in -P/-P plants of E. globulus and the clear trend of a lower leaf mass accumulation. The same may be applied to E. grandis, but in this species, leaf and root growth was significantly affected even when just one side was under -P conditions, suggesting that is more sensitive to low P availability than the other two species. A recent work reported that root biomass of E. grandis was higher in plants grown with sufficient P than with low P (Silva et al. 2022). It was observed by (Bulgarelli et al. 2019) that plants of E. tereticornis and E. globulus growing in soils with low and sufficient P did not differ regarding shoot biomass accumulation.

Fig.2 Leaf (a) and root (b) biomass of eucalypt species cultivated in a split-root system, with P-repleted (+P) and P-deprived (-P) conditions. Bars represent the means of five replicates and the lines are the standard error. The average value of the two root sides was calculated to compare the treatment in the same species and statistical differences are indicated by different small letters. Asterisk indicates a significant difference between the sides of each treatment in each species (Tukey's test, p < 0.05)

In plants of E. grandis and E. tereticornis, the P concentration in the leaves did not show significant differences among plants receiving P-repleted or P-depleted solutions, although a trend of a higher P concentration was observed in +P/+P and +P/-P plants if compared with -P/-P plants (Figure 3a). The average root P concentrations at both root sides were lower in E. globulus and E. grandis at the -P/-P treatment, but no difference was observed between the sides of +P/-P treatment. E. tereticornis showed higher P concentration in the leaves and roots than the other species. E. tereticornis had a large P concentration variation in roots but did not show a significant difference between -P/-P treatments and the other two treatments (Figure 3b). The total P content per plant was calculated (P concentration multiplied by biomass) to verify if the -P/-P treatment were effective in decreasing P accumulation. Because of the large variation of P content among plants, we did not observe significant differences for E. globulus and E. tereticornis, as observed in E. grandis, but the values found were lower than the other treatments (Figure 3c). This shows that P was supplied to the plants from the +P side of the +P/-P treatment.

Fig.3 Phosphorus concentration (g kg^-1) in leaves (a) and roots (b) and total P content (mg plant^-1) in plants (c) of eucalypt species cultivated under split root condition, with P-repleted (+P) and P-deprived (-P) conditions. Bars represent the means of five replicates, and the lines are the standard error. The average value of the two root sides was calculated to compare the treatment in the same species, and different small letters indicate statistical differences (Tukey's test, p < 0.05)

Supplementary Tables 2 and 3 show the concentrations of macro- and micronutrients in the leaves and roots, respectively. In general, plants grown under split root conditions with -P/-P and +P/+P showed varying concentrations of the nutrients, and the foliar concentration of most of the nutrients varied depending on the species. In leaves, P availability significantly affected foliar B and Cu concentrations. B was highest in leaves of +P/+P plants of E. tereticornis. In E. grandis, +P/-P plants had higher foliar B and Cu concentrations. No significant effect was observed for the concentration of other nutrients. In the roots, the concentration of nutrients had a significant effect that varied among species and treatments. Interestingly, although only P was varied in the nutrient solutions, other nutrients, like K, Mg, Cu, Fe, and B, showed significantly different concentrations, influenced by P presence or absence. Among the studied species, higher K concentrations were found in roots of E. tereticornis in +P/-P and -P/-P treatments and was highest in the -P side of +P/-P split root. For E. grandis, comparing the three treatments, K concentration was highest when one side of the split system was +P, either the +P/+P or +P/-P.

Similar to K, in E. grandis, the highest concentrations of Mg occurred when one side of the split root system was +P, either +P/+P or +P/-P. In E. tereticornis, the lowest concentrations of Mg were observed in the +P/+P treatment, either among or within the species. Regarding the Cu concentration, significant differences occurred only between species. Cu had the highest concentration when one side of the roots was -P, either in the -P/-P or the +P/-P treatments. For Fe, the highest concentration was observed in E. grandis in the +P/+P treatment. For B, the differences were only significant when comparing the sides of each split root, with the highest concentration when one side of the split system was -P, either the -P/-P or +P/-P.

Copper toxicity due to the deliberate use of fungicides induces nutritional imbalances and restrictions on plant growth, which is related to P (Feil et al. 2020). In eucalyptus plants, higher concentrations of Cu occurred in treatments that had P deficiency on at least one side of the root, either in leaf (E. grandis) or root (E. tereticornis). Interestingly, it was the root sides, or foliar treatment, with the lowest biomass (Figure 2). Hydroponically grown cucumber plants were used to investigate the influence of exposure to different Cu concentrations. High concentrations of Cu (above 25 μM) resulted in lower shoot and root growth, and the rate of P influx decreased. The data shown by the authors clearly show that high concentrations of Cu can disrupt the uptake of P by the root, probably through a direct action on gene transporters (Feil et al. 2020).

Responses related to Fe over accumulation and toxicity has been observed in plants under Pi starvation, as P may negatively and positively regulate genes associated with Fe metabolism (Gonçalves et al. 2020). Using available transcriptomic data, (Gonçalves et al. 2020) devised new functions for already known genes in Pi metabolism and identified responsive genes with unknown functions, although upregulated under Pi starvation. This includes the downregulation of genes involved in activating Fe starvation responses, such as Fe transporters, and genes involved in cellular Fe homeostasis. Although the regulation of these genes under Pi deprivation is likely a consequence of Fe over accumulation and the following responses to avoid Fe toxicity, some of these genes are likely also mediators of P-Fe interaction (Gonçalves et al. 2020), which possibly explains the fact that the Fe concentration was not higher in the roots under P-starvation conditions (-P/-P), or perhaps also because the +P/-P treatment allowed signaling between the sides of the roots, and the +P side has maintained a suitable P supply for the plant. Another hypothesis is that genes related to Fe transport and uptake were downregulated. In a split-root system with tomato plants, it was analyzed whether the sulfur (S) concentration could modify the plant's ability to absorb and accumulate Fe (Coppa et al. 2018). The authors concluded that the S supply, even if located only on one side of the root system, contributed to the Fe status in the shoot. In rice, expression levels of Fe-regulated marker genes on the non-Fe side of the root were increased, but not as much as when the two sides of the root did not contain Fe, indicating signaling processes between the two sides of the split roots (Chen et al. 2018).

P deficiency has been reported to influence K uptake in rice (Li and Rengel 2012). The interaction between these two nutrients can influence their absorption and utilization in tomatoes, and P and K deficiency (either individually or both) significantly reduced root biomass density, and the ratios of P, and K contents in shoots and roots were positively correlated with root volume (Naciri et al. 2022).

3.2 Principal component analysis

PCA was performed separately for variables related to the nutrient concentrations of leaves (Figure 4a) and roots (Figure 4b). For leaves, the first (PC1) and the second (PC2) components explained 48.7% and 28.8% of the total variation, respectively. For roots, PC1 and PC2 explained 58.1% and 18.3% of the total variation, respectively. In leaves, the treatments -P/-P and +P/+P of E. grandis and +P/+P of E. globulus were separated from all other variables. E. globulus (+P/-P and -P/-P) showed the closest correlation with Fe and K, Ca, and Mg. E. tereticornis (+P/+P and -P/-P) did not show a close correlation with any nutrient, and it was slightly correlated with B, while E. tereticornis and E. grandis (+P/-P) were closely correlated with Mn. The most correlated nutrients were Cu, P, and B. Fe, K, Ca, and Mg were positioned in another quadrant.

P separated from all other variables in the roots but close to E. tereticornis in the treatment +P/+P. The closest nutrients were Fe, Cu, and Mg, which correlated with E. globulus (+P in the +P/+P and +P/-P), E. grandis (+P in the +P/-P treatments), and E. tereticornis (-P in the treatments +P/-P and -P/-P treatments). B did not have a positive correlation with the other nutrients and was closest to E. globulus in the -P/-P treatment. The same occurred with K, which was more correlated with E. tereticornis (-P/-P treatment), and Ca, which was more correlated with E. tereticornis in the -P/-P treatment.

A negative correlation between Fe and P in the leaves was reported in Citrus grandis (Meng et al. 2021) and E. grandis (De Oliveira et al. 2022), species that accumulated more Fe in stems and roots under P deficiency. Opposite observations were obtained in the present study for E. grandis, which showed higher Fe levels in the roots of the +P/+P treatment compared to the low P treatment. Low Fe availability promotes P uptake, while P-starvation promotes Fe accumulation in tissues because of ROS activity, causing callose deposition and preventing plasmodesmal communication (Müller et al. 2015) by regulating genes involved in Fe homeostasis. In our study, interestingly, the lack of P did not promote the accumulation of Fe, with the highest concentration in the +P/+P treatment, which is possibly explained by the negative and positive regulation of genes involved in cellular Fe homeostasis and those related to the synthesis of ferritin, a protein that can bind over one thousands of Fe atoms, regulating the concentration of free Fe inside the cell (Stein et al. 2009).

Fig.4 Principal component analysis (PCA) of nutrient concentrations of leaves (a) and the split roots (b) of the eucalypt species. Different colors were used to indicate species and treatments in the leaves and on both sides of the split roots. The side of the split root is indicated using bold letters. The analysis was performed on the correlation matrix of least square means of averaged accessions. Numbers in parentheses indicate the variation percentage explained by PC1 (F1) and PC2 (F2)

3.3 PSR gene expression in leaves and roots

The split-root system is a relevant tool to address the long-distance signaling and remobilization effects of nutrient heterogeneity at the root environment, as it allows separating local and systemic responses to understand better their respective molecular mechanisms (Ferreira Torres et al. 2021). We observed a large variation in gene expression levels, but in general, the expression of the analyzed genes was highest in the leaves and roots of plants grown under -P. Genes from three functional groups were analyzed: P transport (PHT1), P uptake (PHO1), and sulfolipid synthesis (SQD).

3.3.1 Genes related to transport and uptake P (PHT1 and PHO1)

Most members of the PHT1 membrane protein family are involved in the direct uptake of Pi from the soil, its transport through root cortical cells, in arbuscular mycorrhizal symbiosis and in the transport and remobilization of this nutrient within the plant (Gu et al. 2016; Wang et al. 2017). The analyzed PHT1 genes responded differently among species and treatments. In the leaves of E. globulus, the expression level of the genes of the PHT1.1 and PHT1.6 transporters was higher in the -P/-P treated plants than in the +P/+P (Figures 5a, 5b). PHT1.6 expression was also higher in in leaves of E. tereticornis under -P/-P conditions. On the contrary, the expression of PHT1.5 and PHT1.6 transporters were higher in the leaves of E. grandis plants under +P/+P conditions (Figures 5b, 5c), which may be related to P transport and remobilization (Wang et al. 2022). In Medicago truncatula the PHT1 transporter MtPT5 was proved to play an important role in leaf growth and Pi accumulation (Wang et al. 2022).

Fig.5 Expression analysis of genes included in the "P transport (PHT1)" group in leaves of eucalypts species cultivated under split root condition, with P-repleted (+P) and P-deprived (-P) conditions. A= PHT1.1, B= PHT1.5, C= PHT1.6, D= PHT1.10, E= PHT1.11. The relative quantification of each transcript in each treatment was normalized against the constitutive Ef1 gene. Bars represent the means of three replicates, and the lines represent the standard error. Different letters indicate statistical significance among treatments in the same species (Tukey's test, p < 0.05)

In the roots PHT1.3 expression in the three species showed the highest levels in plants from the treatments +P/+P in E. globulus and -P/-P in E. tereticornis. The comparison of the root sides in each species showed statistical significance only in the -P side of the -P/-P treatment in E. grandis and E. tereticornis (-P/-P). The expression of the PHT1.3 in E. globulus roots clearly showed what might be expected regarding signaling between the two roots in the +P/-P treatment. On the -P side of the +P/-P treatment, the expression was slightly lower than on the -P/-P treatment, reflecting the better P status of the plant which probably triggered a signal to the -P side, indicating to the plant the non-total absence of P (Figure 6a).

The induction of the PHT1 transporters group may be related to the increase in P uptake. Studies with plants deficient in PHT1 expression showed that these multigene families are composed of transporters that may have high or low affinity for Pi, corroborating with kinetics studies of absorption performed with plants, which showed that the high-affinity system works in the range of 2,5 to 12 μM and the low-affinity system between 50 and 100 μM (Poirier and Jung 2015). The passage of Pi through the membrane through these transporters takes place via proton-Pi co-transport (Baker et al. 2015). It is not known what defines the greater or lesser affinity for Pi transport, but it was recently discovered that it is not related to the Pi binding site of the protein, suggesting that these proteins could have dual affinity and that post-translational changes would be involved in defining the high or low-affinity transporter function (Ceasar et al. 2016).

The PHO1 family genes mainly express in stellar cells of roots and hypocotyls (Zhang et al. 2023). In Arabidopsis, PHO1 is responsible for the efflux of Pi out of the cells towards the xylem vessel, that is, with an essential role in the transport of Pi from the roots to the leaves (Arpat et al. 2012). The PHO1 gene may be weakly upregulated under P-deprivation (Hamburger et al. 2002; Zhang et al. 2023). Here, PHO1.8 expression was species-dependent, it was induced only in E. grandis in -P (average root values in the +P/-P and -P/-P treatments) and in the -P side of the +P/-P treatment. This same side had higher expression in E. tereticornis (Figure 6b). Under low P, SPX and PHO1 were induced in several crop plants, including rice, chickpea, switchgrass, and alfalfa (Ding et al. 2021; Li et al. 2022).

Fig.6 Expression analysis of genes included in the “P transport”: PHT1.3 (a) and “P uptake”: PHO1.8 (b) group in roots of eucalypts species cultivated under split root condition, with P-repleted (+P) and P-deprived (-P) conditions. The relative quantification of each transcript in each treatment was normalized against the constitutive Ef1 gene. Bars represent the means of three replicates, and the lines represent the standard error. The average value of the two root sides was calculated to compare the treatment in the same species, and statistical differences are indicated by different small letters. Asterisk indicates a significant difference between the sides of each treatment in each species (Tukey's test, p < 0.05)

Plant cells generally have a higher P concentration, between 5 and 20 mM, than the soil solution, between1 and 10 μM (Hinsinger 2001). Thus, the uptake of P from the soil to the root is an energy-dependent process (Prathap et al. 2022) and that needs membrane transporters, including those codified by PHT1 and PHO1 genes. So far, 36 members of the PHT1 transporter family have been identified in wheat (TaPHT1;1-TaPHT1;36), 14 in soybean (GmPHT1;1-GmPHT1;14), 13 in corn (ZmPHT1;1-ZmPHT1;13), 13 the genome of rice (OsPT1;1-OsPT1;13), 11 in barley (HvPHT1;1-HvPHT1;11) and nine members in Arabidopsis (AtPHT1;1-AtPHT1;9) (Prathap et al. 2022). In E. grandis 22 transporters belonging to the PHT1 family have been identified (Che et al. 2022).

As a large family of transporters, PHT1 transporters show differences in expression sites and affinities for P (Nussaume et al. 2011; Teng et al. 2013). The function and location of these transporters are very broad, whether in Arabidopsis or rice, the two well-characterized species for this family of transporters. Studies carried out with PHT1 transporters in Arabidopsis showed variation in the expression of transporters in different plant organs. Mobilization of P from the root to the leaves was observed for PHT1;8 and PHT1;9, while mobilization from the leaves to the root by PHT1;5 (Liu et al. 2012). In Arabidopsis and rice, expression of PHT1;7, and PHT1;12 transporters are confined to the anthers (Mudge et al. 2002). AtPHT1.1 and AtPHT1.4 are responsible for P acquisition by roots in both high and low P sources (Catarecha et al. 2007), and have high activity at the root-soil interface, including the epidermis, root hair cells, and the root cap under low-P conditions (Mudge et al. 2002). AtPHT1.8 and AtPHT1.9 act during the translocation of Pi from root to shoot (Lapis-Gaza et al. 2014). In rice, transporters also have distinct roles; OsPHT1.6 is classified as a high-affinity transporter responsible for Pi uptake and translocation throughout the plant, with an active role in the epidermal and cortical cells of primary and young lateral roots. OsPHT1.2 is a low affinity transporter with a role in Pi translocation. It has a specific location in the stele of primary and lateral roots (Ai et al. 2009).

Some studies have been carried out with eucalypts and putative P transporter genes to assess expression in response to P deprivation. PHT1 and PHT2 were evaluated in E. grandis, and the expression of EgPHT1;4 and EgPHT2;1 was induced in P deficiency, mainly in roots (Pereira et al. 2019). Also under P deprivation, PHT1 was induced in C. citriodora and E. dunni, and in the latter, the high expression level was possibly related to the greater demand for P because of the high growth rate of this species (Niu et al. 2015). Most PHT1 transporters are found in various tissues and organs, often overlapping with other PHT1 members, evidencing their functional complexity. It is important to mention that this location is often influenced by environmental or development factors (Mudge et al. 2002). This would explain the different expression patterns obtained between the analyzed PHT1 transporters between roots and leaves. Most PHT1 transporters are preferentially expressed in roots, where their expression is activated by P-starvation-induced PHR1 transcription factor and directly mediates P acquisition from the soil (Bayle et al. 2011).

In the present study, the PHT1 transporters that showed the highest level of expression under +P condition may be involved in P uptake under conditions of greater P availability. Although the PHT1 genes expression is usually involved with P scarcity and partially repressed under high P concentrations to prevent its excessive accumulation, some members of the PHT1 family have basal expression levels that are important for maintaining P absorption even under adequate availability P (Zhang et al. 2021). Additionally, the repression of four PHT1 genes by P deficiency in wheat has been reported for transporters. Under low-P conditions, expression of TaPHT1.1, 1.2, 1.9 and 1.10 in roots was lower than under high-P conditions (Teng et al. 2013). This down regulation by P deficiency could be partially explained by the colonization of arbuscular mycorrhizal fungi, which are upregulated under P deficiency (not analyzed in this experiment). High concentrations of Cu may also disrupt P uptake by the root, probably through a direct action on gene transporters (Feil et al. 2020).

3.3.2 Sulfolipid synthesis (SQD)

The lipid composition of plants is responsive to environmental conditions, including changes in nutrient availability (Bolik et al. 2022). P-deficient plants synthesize the non-P glycolipid sulfoquinovosyldiacylglycerol (SQD). This sulfolipid may replace membrane phospholipids, releasing P for remobilization (Dissanayaka et al. 2018) and improving the efficiency of P utilization (Watanabe et al. 2020; Luo et al. 2022). Studies developed mainly on rice (Jeong et al. 2017; Gho et al. 2018; Gonçalves et al. 2020) show that OsSQD2 is highly induced under P starvation, as also shown in Arabidopsis (Yu et al. 2002). This mechanism seems to be widely exploited in leaves but much less in roots (Murakawa et al. 2019). The role of SQD in rice is poorly understood, which makes it interesting for further investigation, especially in tree species.

In our study, SQD was induced in the leaves of plants of E. globulus and E. tereticornis grown on -P/-P, with a similar trend in E. grandis, despite not statically significant (Figure 7a). In roots SQD expression was only higher for - E. globulus under the P/-P conditions (Figure 7c).

Fig.7 Expression analysis of SQDs genes in the leaves (a) and (b) roots of eucalypt species cultivated under split root condition, with P-repleted (+P) and P-deprived (-P) conditions. The relative quantification of each transcript in each treatment was normalized against the constitutive Ef1 gene. Bars represent the means of three replicates, and the lines represent the standard error. The average value of the two root sides was calculated to compare the treatment in the same species, and statistical differences are indicated by different small letters (Tukey's test, p < 0.05)

Lipid remodeling occurs more frequently in young photosynthetic tissues, as photosynthesis demands P (Pfaff et al. 2020). In leaves, E. tereticornis that showed an increase in biomass under P deprivation, had a greater expression of SQD under -P/-P conditions suggesting the presence of the strategy to replace P-lipids as an attempt to optimize P utilization. In addition, the low concentration of P in the leaves of -P/-P E. tereticornis may have contributed to membrane remodeling to overcome P-deficiency.

In a split root system experiment, transcriptomic data revealed that genes involved in Pi transport as well as Pi uptake and recycling are generally systemically regulated, and genes linked to stress- or hormone-related responses are locally regulated (Thibaud et al. 2010). The growth of shoots and roots is reciprocally dependent, reinforcing the importance of signaling pathway networks (Chiou and Lin 2011). A recent study showed sulfolipid and phospholipid remodeling in eucalyptus plants at low P (Silva et al. 2022).

Our results show a complex response of eucalypt species to uneven P distribution in the root environment. In addition to P, the concentrations of Fe, B and Cu were affected by P availability in the root environment, as was the expression of genes related to functions as P uptake, transport and metabolism. P remobilization was likely to be the reason of high P contents at the -P root side when the other root side was supplied with P, and also the reason in the lack of a clear response for some of the PSR genes under P-depleted conditions. In general, the PSR genes were more expressed when plants were P- deprived, and the response to P was species-dependent. It is important to point out that eucalypt plants under split root systems take months to stabilize (with roots of adequate size to provide a sufficient amount of material for the analyses), which may have exacerbated the effects of P remobilization. Understanding how a specific nutrient affects plant growth is critical for understanding their mobilization and how signaling pathways are integrated to result in diverse and dynamic plant responses.

Competing Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

LFT, PM and SALA projected the experimental design, LFT set up and developed the experiment. LFT wrote the manuscript and PM and SALA reviewed it. All authors read and approved the final version of the manuscript.

Funding

The authors thank the financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo - Fapesp (grant 2016/25498-0). LFT thanks Fapesp for a post-doctoral fellowship (grant 2020/02867-5) and PM thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq for a research fellowship (grant 306002/2020-5).

Acknowledgments

We thank Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP and University of Campinas - UNICAMP.

Ai P, Sun S, Zhao J et al (2009) Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. Plant J 57:798–809. https://doi.org/10.1111/j.1365-313X.2008.03726.x
Arpat AB, Magliano P, Wege S et al (2012) Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate. Plant J 71:479–491. https://doi.org/10.1111/j.1365-313X.2012.05004.x
Bahar NHA, Gauthier PPG, O’Sullivan OS et al (2018) Phosphorus deficiency alters scaling relationships between leaf gas exchange and associated traits in a wide range of contrasting Eucalyptus species. Funct Plant Biol 45:813–826. https://doi.org/10.1071/FP17134
Baker A, Ceasar SA, Palmer AJ et al (2015) Replace, reuse, recycle: improving the sustainable use of phosphorus by plants. J Exp Bot 66:3523–3540. https://doi.org/10.1093/jxb/erv210
Bayle V, Arrighi J-F, Creff A et al (2011) Arabidopsis thaliana High-Affinity Phosphate Transporters Exhibit Multiple Levels of Posttranslational Regulation. Plant Cell 23:1523–1535. https://doi.org/10.1105/tpc.110.081067
Bolik S, Albrieux C, Schneck E et al (2022) Sulfoquinovosyldiacylglycerol and phosphatidylglycerol bilayers share biophysical properties and are good mutual substitutes in photosynthetic membranes. Biochim et Biophys Acta (BBA) - Biomembr 1864:184037. https://doi.org/10.1016/j.bbamem.2022.184037
Bulgarelli RG, de Oliveira Silva FM, Bichara S et al (2019) Eucalypts and low phosphorus availability: between responsiveness and efficiency. Plant Soil 445:349–368. https://doi.org/10.1007/s11104-019-04316-2
Catarecha P, Segura MD, Franco-Zorrilla JM et al (2007) A Mutant of the Arabidopsis Phosphate Transporter PHT1;1 Displays Enhanced Arsenic Accumulation. Plant Cell 19:1123–1133. https://doi.org/10.1105/tpc.106.041871
Ceasar SA, Baker A, Muench SP et al (2016) The conservation of phosphate-binding residues among PHT1 transporters suggests that distinct transport affinities are unlikely to result from differences in the phosphate-binding site. Biochem Soc Trans 44:1541–1548. https://doi.org/10.1042/BST20160016
Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116. https://doi.org/10.1007/BF02670468
Chavan SB, Dhillon RS, Sirohi C et al (2023) Carbon Sequestration Potential of Commercial Agroforestry Systems in Indo-Gangetic Plains of India: Poplar and Eucalyptus-Based Agroforestry Systems. Forests 14:559. https://doi.org/10.3390/f14030559
Che X, Lai W, Wang S et al (2022) Multiple PHT1 family phosphate transporters are recruited for mycorrhizal symbiosis in Eucalyptus grandis and conserved PHT1;4 is a requirement for the arbuscular mycorrhizal symbiosis. Tree Physiol 42:2020–2039. https://doi.org/10.1093/treephys/tpac050
Chen L, Wang G, Chen P et al (2018) Shoot-Root Communication Plays a Key Role in Physiological Alterations of Rice (Oryza sativa) Under Iron Deficiency. Front Plant Sci 9
Chiou T-J, Lin S-I (2011) Signaling Network in Sensing Phosphate Availability in Plants. Annu Rev Plant Biol 62:185–206. https://doi.org/10.1146/annurev-arplant-042110-103849
Coppa E, Celletti S, Pii Y et al (2018) Revisiting Fe/S interplay in tomato: A split-root approach to study the systemic and local responses. Plant Sci 276:134–142. https://doi.org/10.1016/j.plantsci.2018.08.015
Da Silva RML, Hakamada RE, Bazani JH et al (2016) Fertilization Response, Light Use, and Growth Efficiency in Eucalyptus Plantations across Soil and Climate Gradients in Brazil. Forests 7:117. https://doi.org/10.3390/f7060117
De Oliveira VH, Mazzafera P, de Andrade SAL (2022) Alleviation of low phosphorus stress in Eucalyptus grandis by arbuscular mycorrhizal symbiosis and excess Mn. Plant Stress 5:100104. https://doi.org/10.1016/j.stress.2022.100104
Ding N, Huertas R, Torres-Jerez I et al (2021) Transcriptional, metabolic, physiological and developmental responses of switchgrass to phosphorus limitation. Plant Cell Environ 44:186–202. https://doi.org/10.1111/pce.13872
Dissanayaka Dm. s. b., Plaxton WC, Lambers H et al (2018) Molecular mechanisms underpinning phosphorus-use efficiency in rice. Plant, Cell & Environment 41:1483–1496. https://doi.org/10.1111/pce.13191
Doerner P (2008) Phosphate starvation signaling: a threesome controls systemic Pi homeostasis. Curr Opin Plant Biol 11:536–540. https://doi.org/10.1016/j.pbi.2008.05.006
Feil SB, Pii Y, Valentinuzzi F et al (2020) Copper toxicity affects phosphorus uptake mechanisms at molecular and physiological levels in Cucumis sativus plants. Plant Physiol Biochem 157:138–147. https://doi.org/10.1016/j.plaphy.2020.10.023
Ferreira DF (2011) Sisvar: a computer statistical analysis system. Ciênc agrotec 35:1039–1042. https://doi.org/10.1590/S1413-70542011000600001
Ferreira Torres L, López de Andrade SA, Mazzafera P (2021) Split-root, grafting and girdling as experimental tools to study root-to shoot-to root signaling. Environ Exp Bot 191:104631. https://doi.org/10.1016/j.envexpbot.2021.104631
Gho Y-S, An G, Park H-M, Jung K-H (2018) A systemic view of phosphate starvation-responsive genes in rice roots to enhance phosphate use efficiency in rice. Plant Biotechnol Rep 12:249–264. https://doi.org/10.1007/s11816-018-0490-y
Gonçalves BX, Lima-Melo Y, Maraschin F, dos Margis-Pinheiro S M (2020) Phosphate starvation responses in crop roots: from well-known players to novel candidates. Environ Exp Bot 178:104162. https://doi.org/10.1016/j.envexpbot.2020.104162
Grattapaglia D, Kirst M (2008) Eucalyptus applied genomics: from gene sequences to breeding tools. New Phytol 179:911–929. https://doi.org/10.1111/j.1469-8137.2008.02503.x
Gu M, Chen A, Sun S, Xu G (2016) Complex Regulation of Plant Phosphate Transporters and the Gap between Molecular Mechanisms and Practical Application: What Is Missing? Mol Plant 9:396–416. https://doi.org/10.1016/j.molp.2015.12.012
Hamburger D, Rezzonico E, MacDonald-Comber Petétot J et al (2002) Identification and characterization of the Arabidopsis PHO1 gene involved in phosphate loading to the xylem. Plant Cell 14:889–902. https://doi.org/10.1105/tpc.000745
Hermans C, Hammond JP, White PJ, Verbruggen N (2006) How do plants respond to nutrient shortage by biomass allocation? Trends Plant Sci 11:610–617. https://doi.org/10.1016/j.tplants.2006.10.007
Hinsinger P (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 237:173–195. https://doi.org/10.1023/A:1013351617532
Hinsinger P, Brauman A, Devau N et al (2011) Acquisition of phosphorus and other poorly mobile nutrients by roots. Where do plant nutrition models fail? Plant Soil 348:29–61. https://doi.org/10.1007/s11104-011-0903-y
Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Circular Calif Agricultural Exp Stn 347
Jeong K, Baten A, Waters DLE et al (2017) Phosphorus remobilization from rice flag leaves during grain filling: an RNA-seq study. Plant Biotechnol J 15:15–26. https://doi.org/10.1111/pbi.12586
Kondhare KR, Patil NS, Banerjee AK (2021) A historical overview of long-distance signalling in plants. J Exp Bot 72:4218–4236. https://doi.org/10.1093/jxb/erab048
Lapis-Gaza HR, Jost R, Finnegan PM (2014) Arabidopsis PHOSPHATE TRANSPORTER1 genes PHT1;8 and PHT1;9 are involved in root-to-shoot translocation of orthophosphate. BMC Plant Biol 14:334. https://doi.org/10.1186/s12870-014-0334-z
Li L, Rengel Z (2012) Soil Acidification as Affected by Phosphorus Sources and Interspecific Root Interactions between Wheat and Chickpea. Commun Soil Sci Plant Anal 43:1749–1756. https://doi.org/10.1080/00103624.2012.684821
Li Z, Hu J, Wu Y et al (2022) Integrative analysis of the metabolome and transcriptome reveal the phosphate deficiency response pathways of alfalfa. Plant Physiol Biochem 170:49–63. https://doi.org/10.1016/j.plaphy.2021.11.039
Liu T-Y, Huang T-K, Tseng C-Y et al (2012) PHO2-Dependent Degradation of PHO1 Modulates Phosphate Homeostasis in Arabidopsis. Plant Cell 24:2168–2183. https://doi.org/10.1105/tpc.112.096636
Livak KJ, Schmittgen TD (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 – ∆∆CT Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lough TJ, Lucas WJ (2006) INTEGRATIVE PLANT BIOLOGY: Role of Phloem Long-Distance Macromolecular Trafficking. Annu Rev Plant Biol 57:203–232. https://doi.org/10.1146/annurev.arplant.56.032604.144145
Luo J, Cai Z, Huang R et al (2022) Integrated multi-omics reveals the molecular mechanisms underlying efficient phosphorus use under phosphate deficiency in elephant grass (Pennisetum purpureum). Front Plant Sci 13:1069191. https://doi.org/10.3389/fpls.2022.1069191
Malavolta E, Vitti GC, de Oliveira SA (1997) Avaliação do estado nutricional das plantas. princípios e aplicações
Meng X, Chen W-W, Wang Y-Y et al (2021) Effects of phosphorus deficiency on the absorption of mineral nutrients, photosynthetic system performance and antioxidant metabolism in Citrus grandis. PLoS ONE 16:e0246944. https://doi.org/10.1371/journal.pone.0246944
Moura JCMS, Araújo P, Brito M dos S, et al (2012) Validation of reference genes from Eucalyptus spp. under different stress conditions. BMC Res Notes 5:634. https://doi.org/10.1186/1756-0500-5-634
Mudge SR, Rae AL, Diatloff E, Smith FW (2002) Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis. Plant J 31:341–353. https://doi.org/10.1046/j.1365-313X.2002.01356.x
Müller J, Toev T, Heisters M et al (2015) Iron-Dependent Callose Deposition Adjusts Root Meristem Maintenance to Phosphate Availability. Dev Cell 33:216–230. https://doi.org/10.1016/j.devcel.2015.02.007
Murakawa M, Ohta H, Shimojima M (2019) Lipid remodeling under acidic conditions and its interplay with low Pi stress in Arabidopsis. Plant Mol Biol 101:81–93. https://doi.org/10.1007/s11103-019-00891-1
Naciri R, Rajib W, Chtouki M et al (2022) Potassium and phosphorus content ratio in hydroponic culture affects tomato plant growth and nutrient uptake. Physiol Mol Biol Plants 28:763–774. https://doi.org/10.1007/s12298-022-01178-4
Nadir SW, Ng’etich WK, Kebeney SJ (2018) Performance of crops under Eucalyptus tree-crop mixtures and its potential for adoption in agroforestry systems. Australian Journal of Crop Science
Niu F-H, Li Z-H, Malladi A et al (2015) Expression Dynamics of Two Pht1 Genes in Eucalyptus dunnii and Corymbia citriodora in Relation to Phosphate Supply. Medicine and Biopharmaceutical. WORLD SCIENTIFIC, pp 1374–1385
Nussaume L, Kanno S, Javot H et al (2011) Phosphate Import in Plants: Focus on the PHT1 Transporters. Front Plant Sci 2
Pereira FCM, Tayengwa R, Alves PLDCA, Peer WA (2019) Phosphate Status Affects Phosphate Transporter Expression and Glyphosate Uptake and Transport in Grand Eucalyptus (Eucalyptus grandis). Weed Sci 67:29–40. https://doi.org/10.1017/wsc.2018.58
Pérez-Cruzado C, Merino A, Rodríguez-Soalleiro R (2011) A management tool for estimating bioenergy production and carbon sequestration in Eucalyptus globulus and Eucalyptus nitens grown as short rotation woody crops in north-west Spain. Biomass Bioenergy 35:2839–2851. https://doi.org/10.1016/j.biombioe.2011.03.020
Pezzopane JRM, Bosi C, de Campos Bernardi AC et al (2021) Managing eucalyptus trees in agroforestry systems: Productivity parameters and PAR transmittance. Agric Ecosyst Environ 312:107350. https://doi.org/10.1016/j.agee.2021.107350
Pfaff J, Denton AK, Usadel B, Pfaff C (2020) Phosphate starvation causes different stress responses in the lipid metabolism of tomato leaves and roots. Biochimica et Biophysica Acta (BBA) -. Mol Cell Biology Lipids 1865:158763. https://doi.org/10.1016/j.bbalip.2020.158763
Poirier Y, Jung J-Y (2015) Phosphate Transporters. Annual Plant Reviews Volume 48. Wiley, Ltd, pp 125–158
Prathap V, Kumar A, Maheshwari C, Tyagi A (2022) Phosphorus homeostasis: acquisition, sensing, and long-distance signaling in plants. Mol Biol Rep 49:8071–8086. https://doi.org/10.1007/s11033-022-07354-9
Puga MI, Rojas-Triana M, de Lorenzo L et al (2017) Novel signals in the regulation of Pi starvation responses in plants: facts and promises. Curr Opin Plant Biol 39:40–49. https://doi.org/10.1016/j.pbi.2017.05.007
Rawat P, Das S, Shankhdhar D, Shankhdhar SC (2021) Phosphate-Solubilizing Microorganisms: Mechanism and Their Role in Phosphate Solubilization and Uptake. J Soil Sci Plant Nutr 21:49–68. https://doi.org/10.1007/s42729-020-00342-7
Rouached H, Arpat AB, Poirier Y (2010) Regulation of Phosphate Starvation Responses in Plants: Signaling Players and Cross-Talks. Mol Plant 3:288–299. https://doi.org/10.1093/mp/ssp120
Ruffel S (2018) Nutrient-Related Long-Distance Signals: Common Players and Possible Cross-Talk. Plant Cell Physiol 59:1723–1732. https://doi.org/10.1093/pcp/pcy152
Secco D, Wang C, Arpat BA et al (2012) The emerging importance of the SPX domain-containing proteins in phosphate homeostasis. New Phytol 193:842–851. https://doi.org/10.1111/j.1469-8137.2011.04002.x
Shabala S, White RG, Djordjevic MA et al (2015) Root-to-shoot signalling: integration of diverse molecules, pathways and functions. Funct Plant Biol 43:87–104. https://doi.org/10.1071/FP15252
Silva FM, de O, Bulgarelli RG, Mubeen U et al (2022) Low phosphorus induces differential metabolic responses in eucalyptus species improving nutrient use efficiency. Front Plant Sci 13
Stape JL, Binkley D, Ryan MG et al (2010) The Brazil Eucalyptus Potential Productivity Project: Influence of water, nutrients and stand uniformity on wood production. For Ecol Manag 259:1684–1694. https://doi.org/10.1016/j.foreco.2010.01.012
Stein RJ, Ricachenevsky FK, Fett JP (2009) Differential regulation of the two rice ferritin genes (OsFER1 and OsFER2). Plant Sci 177:563–569. https://doi.org/10.1016/j.plantsci.2009.08.001
Tang H, Chen X, Gao Y et al (2020) Alteration in root morphological and physiological traits of two maize cultivars in response to phosphorus deficiency. Rhizosphere 14:100201. https://doi.org/10.1016/j.rhisph.2020.100201
Teng W, Deng Y, Chen X-P et al (2013) Characterization of root response to phosphorus supply from morphology to gene analysis in field-grown wheat. J Exp Bot 64:1403–1411. https://doi.org/10.1093/jxb/ert023
Thibaud M-C, Arrighi J-F, Bayle V et al (2010) Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis. Plant J 64:775–789. https://doi.org/10.1111/j.1365-313X.2010.04375.x
Trindade R, dos Araújo S AP (2014) Variability of root traits in common bean genotypes at different levels of phosphorus supply and ontogenetic stages. Rev Bras Ciênc Solo 38:1170–1180. https://doi.org/10.1590/S0100-06832014000400013
Wang D, Lv S, Jiang P, Li Y (2017) Roles, Regulation, and Agricultural Application of Plant Phosphate Transporters. Front Plant Sci 8
Wang X, Wei C, He F, Yang Q (2022) MtPT5 phosphate transporter is involved in leaf growth and phosphate accumulation of Medicago truncatula. Front Plant Sci 13
Watanabe M, Walther D, Ueda Y et al (2020) Metabolomic markers and physiological adaptations for high phosphate utilization efficiency in rice. Plant Cell Environ 43:2066–2079. https://doi.org/10.1111/pce.13777
Yu B, Xu C, Benning C (2002) Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth. Proceedings of the National Academy of Sciences 99:5732–5737. https://doi.org/10.1073/pnas.082696499
Zhang J, Gu M, Liang R et al (2021) OsWRKY21 and OsWRKY108 function redundantly to promote phosphate accumulation through maintaining the constitutive expression of OsPHT1;1 under phosphate-replete conditions. New Phytol 229:1598–1614. https://doi.org/10.1111/nph.16931
Zhang J-F, Chu H-H, Liao D et al (2023) Comprehensive Evolution and Expression anaLysis of PHOSPHATE 1 Gene Family in Allotetraploid Brassica napus and Its Diploid Ancestors. Biochem Genet. https://doi.org/10.1007/s10528-023-10375-z
Zhang Z, Liao H, Lucas WJ (2014) Molecular mechanisms underlying phosphate sensing, signaling, and adaptation in plants. J Integr Plant Biol 56:192–220. https://doi.org/10.1111/jipb.12163
Supplementary Table 1 Sequences of primers used for amplification of constitutive and target genes in leaves and roots of each eucalyptus species by RT-qPCR

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Phosphorus uptake in eucalypts plants under split root system

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Abstract

Figures

1 Introduction

2 Material and Methods

2.1 Experimental design and treatments

2.2 Plant biomass and quantification of macro and micronutrients

2.3 Analysis of gene expression in leaves and roots

2.4 Statistical analysis

3 Results and discussion

3.1 Plant biomass and macro and micronutrients contents

3.2 Principal component analysis

3.3 PSR gene expression in leaves and roots

3.3.1 Genes related to transport and uptake P (PHT1 and PHO1)

3.3.2 Sulfolipid synthesis (SQD)

4 Conclusions

Declarations

Competing Interests

Author Contributions

Funding

Acknowledgments

References

Additional Declarations

Supplementary Files

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