Tomato phytochrome B1 modulates N, P, and K deficiency response by root-to-shoot communication

Tomato phytochrome B1 modulates N, P, and K deficiency response by root-to-shoot communication | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tomato phytochrome B1 modulates N, P, and K deficiency response by root-to-shoot communication Mariana Bomfim Soares, Renato de Mello Prado, Dilier Olivera Viciedo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6938144/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Acta Physiologiae Plantarum → Version 1 posted 4 You are reading this latest preprint version Abstract Phytochromes are involved in the expression of nutrient transporter genes and participate in signaling responses in plants under nutritional deficiency. This study investigated the reciprocal interaction between phytochrome B1 (phyB1) and N, P, and K deficiency responses, specifically focusing on shoot-root communication. For this purpose, we used grafting combinations of the control genotype (WT) with the tomato phyB1-deficient mutant ( phyB1 ) under nutritional sufficiency and individual deficiencies of N, P, and K. In nutrient-sufficient conditions, shoot phyB1 stimulates the uptake of N and P in the roots in addition to increase stomatal conductance, transpiration, and dry weight production, whereas root phyB1 regulated the production of chlorophyll in the shoot. With N deficiency, grafted plants with loss of shoot phyB1 function showed increased transpiration and less efficient water use. However, the WT/ phyB1 combination attenuated the damage caused by N deficiency by increasing the dry weight of the entire plant. Under P deficiency, the absence of root phyB1 decreased N uptake and increased malondialdehyde (MDA) production. However, the deficiency of phyB1 impaired the water-use efficiency of P-deficient plants. In K-deficient tomato, N and K uptake was under the control of both shoot and root phytochromes and shoot phyB1 regulated MDA production and increased photosynthesis. We conclude that phyB1 is involved in shoot-root communication for the control of nutritional, physiological, and growth responses in tomato, raising new roles of this photoreceptor and perspectives on the plant nutrition studies. Solanum lycopersicum L. red light phyB1 mutant nutritional deficiency Figures Figure 1 Figure 2 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Nutritional deficiency is one of the main factors that limits agricultural production worldwide (Patel et al. 2020 ). Inadequate supplies of nitrogen (N), phosphorus (P), and potassium (K) are the most limiting factors for plant growth because these elements are considered the most responsive by plants (Marschner, 2012 ). Understanding the responses of crops to N, P, and K deficiencies is essential for improving the efficiency of the use of these elements and reducing the degree of fertilization. Nitrogen deficiency reduces its absorption and concentration in all parts of snap bean, citrus, and peanut, causing nutritional imbalance, growth inhibition, decreased concentration of photosynthetic pigments, and CO 2 assimilation (de Souza Osório et al. 2020 ; Patel et al. 2020 ; Huang et al. 2021 ). Furthermore, P is required for several metabolic processes in plants, such as the formation of the ATP molecule, which is used as a source of cellular energy (Prado 2021 ). A decrease in photosynthesis and stomatal conductance has been reported in P-deficient plants, in addition to the formation of reactive oxygen species (ROS) in chloroplasts (Hernández and Munné-Bosch 2015 ). In contrast, K deficiency damages photosynthesis, transpiration, and stomatal conductance (Pandey and Mahiwal 2015 ). Light modulates plant nutrition, and recently, the role of photoreceptors in this response has become increasingly evident (Carvalho et al. 2016 ; Sakuraba et al. 2018 ; Soares et al. 2021 ; D’Amico-Damião et al. 2022 ). The phytochrome B family (phyB) is involved in the stress response to nutritional deficiency, and in a previous study, phyB-9 and phyB-10 deficiency resulted in reduced P uptake in Arabidopsis plants (Sakuraba et al. 2018 ). Those authors also observed a decrease in the expression of high-affinity P transporter genes, indicating that red light is involved in the activation of P uptake, especially under this nutrient's deficiency (Sakuraba et al. 2018 ). We recently revealed that tomato phyB1 is a positive regulator of N uptake, pigment synthesis, and dry weight production under N deficiency (Soares et al. 2021 ). Moreover, the loss of function of this photoreceptor attenuated the damage caused by P deficiency, since the mutant plants showed increased production of dry weight compared to WT plants (Soares et al. 2021 ). Under K deficiency, phyB1 positively regulated P absorption and pigment production. This result suggests strong control by phyB1 over nutritional responses induced by N, P, and K (Soares et al. 2021 ). Therefore, in the present study, we hypothesized that the interaction between phyB1 and N, P, and K deficiencies involves reciprocal communication between the root and shoot. For the first time, we explored the role of the tomato PHYB1 gene in plant responses to N, P, and K deficiency, using the photomorphogenetic mutant phyB1 and grafting to understand the role of this photoreceptor in root-to-shoot communication. 2. MATERIAL AND METHODS 2.1 Growth conditions, plant material, and grafting technique The experiment was conducted inside a growth chamber and greenhouse at São Paulo State University, Jaboticabal, Brazil, for 65 days after sowing (DAS). We used Solanum lycopersicum L. cv. Moneymaker as the wild-type (WT) as well as the phyB1 mutant, which is defective for the gene PHYB1 that encodes PHYB1 apoproteins in the cv Moneymaker background (van Tuinen, Ageeth, Kerckhoffs, Huub J., Nagatani, Akira, Kendrick, Richard E., and Koornneef 1995). Seeds of phyB1 and WT were placed in polystyrene trays to germinate, which were filled with a mixture of the commercial substrate BioPlant® (composed of sphagnum peat, vermiculite, coconut fiber, rice husk, husk pine, and additives containing calcium) and expanded vermiculite in a volumetric proportion of 1: 1 (v: v). Seventeen DAS, the plants were transferred to 200 mL pots (one plant per pot) on the same substrate, and grafting was performed. Seventeen-day-old plants were grafted using the splice method with the aid of a scalpel blade and grafting clips to obtain the following graft combinations: WT/WT, phyB1 / phyB1 , WT/ phyB1 , and phyB1 /WT (scion/rootstock).After grafting, the basal quarter of the pots was submerged in water (a floating moist chamber of 25 ℃ ± 2 ℃ with a high relative humidity of 85% ± 10%) under a 12 h photoperiod at 70 µmol photons m -2 s -1 , until the graft union had completely healed (20 days after grafting, DAG) (Fig. S1). After the graft union had healed, the roots of the grafted plants were washed with deionized water to remove excess substrate, and the plants were transferred to 1.7 dm³ pots containing medium texture sand that was previously decontaminated with 0.1 mol L -1 hydrochloric acid solution and washed with deionized water. The pots were transferred to the greenhouse and irrigated daily with the nutrient solution described by Hoagland and Arnon (1950) to feed the plants and keep the humidity close to the maximum water retention capacity. The solution was initially diluted to 25% ionic strength and after ten days, the concentration was increased to 50%, where it remained until the end of the experiment. The nutrient solution was prepared with deionized water, and the pH value of the solution was maintained at 5.5 ± 0.5 using 1% sodium hydroxide and hydrochloric acid solutions. Air temperature and humidity data were monitored daily inside the greenhouse. Variations were observed in the maximum (85.1 ± 9%) and minimum (33 ± 12%) relative humidity, and maximum (28.7 ± 10 °C) and minimum (14.7 ± 7 °C) temperatures. 2.2 Treatments and experimental design Three experiments were performed with the treatments arranged in a 4 × 2 factorial scheme, with the first factor as the grafting combinations of the tomato plants (WT/WT, phyB1 / phyB1 , WT/ phyB1 , and phyB1 /WT), and the second factor defined as the nutrient solution, which was either complete (containing 15 mmol L -1 of N, 1 mmol L -1 of P and 6 mmol L -1 of K) , deficient in N (1 mmol L -1 of N using NH 4 NO 3 as the source), without P (0 mmol L -1 ), or with absence of K (0 mmol L -1 ) in the solution (Table 1). A completely randomized design with four replicates was adopted, with one replicate consisting of one plant. Table 1. Composition of nutrient solutions used to induce N, P, and K deficiencies in Solanum lycopersicum L. Values correspond to a 100% nutrient solution, which was diluted to 25% and 50%. Treatment Sources Complete -N -P -K mol L -1 mL L -1 KH 2 PO 4 1 1 - - KNO 3 5 - - - Ca (NO 3 ) 2 . 5H 2 O 5 - 5 5 MgSO 4 2 2 2 2 KCL - 5 6 - CaCl 2 . 2H 2 O - 5 - - NH 4 H 2 PO 4 - - - 1 NH 4 NO 3 - 0.5 2.5 2 Micronutrients* 1 1 1 1 Fe-EDDHA** 1 1 1 1 *In 1L: 2.86 g H 3 BO 3 ; 1.81 g MnCl 2 . 4H 2 O; 0.10 g ZnCl 2 ; 0.04 g CuCl 2 ; 0.02 g H 2 MoO 4 H 2 O. ** In 1L: 83.33 g Fe-EDDHA 2.3 Performed analysis 2.3.1 Leaf gas exchange Photosynthetic activity (A), stomatal conductance (Gs), and transpiration (E) was measured using a Li-6400 (LICOR, EUA). The water use efficiency (WUE) was calculated by dividing A by E. 2.3.2 Quantum efficiency of PSII (Fv/Fm) and pigment content The quantum efficiency of PSII was measured between 07:00 and 08:00 h. The third fully expanded leaf was dark-adapted for 30 min using clips, and the minimal (F0) and maximal fluorescence (Fm), as well as the Fv/Fm, were obtained using a portable fluorometer (Opti-Sciences, Os30P). The total chlorophyll (Chl) and carotenoid contents were determined according to the methodology described by Lichtenthaler (Lichtenthaler 1987). Fresh samples (0.025–0.030 g) of the leaves were collected from tomato plants for each treatment, and readings were performed using a Beckman DU 640 spectrophotometer at 663, 647, and 470 nm to acquire the respective contents of chlorophyll a , b , and carotenoids, based on fresh weight. The total chlorophyll content (total chl) was calculated as the sum of the concentrations of chlorophylls a and b . 2.3.3 Lipidic peroxidation Lipid peroxidation was determined in shoot samples (stems and leaves), which were collected, washed, immersed in liquid N 2, and stored at −80 °C, following the methodology of thiobarbituric acid described by Heath and Packer (Heath and Packer 1968). Metabolites of cell membranes, especially malondialdehyde (MDA), react with thiobarbituric acid and can be quantified by spectrophotometry. Briefly, shoot tissue (0.3 g) was homogenized with 1 mL of trichloroacetic acid and polyvinylpyrrolidone (20%). Subsequently, the samples were transferred to tubes and centrifuged at 10,000 × g for 15 min. The supernatant was blended with 1 mL of trichloroacetic acid 20% (m/v) and thiobarbituric acid 0.5% (m/v) and incubated in a water bath at 95 °C for 30 min. After this period, the samples were centrifuged at 11,000 × g for 5 min. The MDA concentration was determined using the coefficient 1.55 × 10−5 mol−1 cm−1 (Gratão et al., 2012), and results are expressed as nmol g−1 of fresh weight. The readings were performed in a spectrophotometer at 535 and 600 nm. 2.3.4 Growth parameters Plants were harvested at 65 DAS and separated into shoots and roots. The plant material was washed under running water with a detergent solution (0.1% v/v), followed by an HCl solution (0.3% v/v) and deionized water. Subsequently, the material was dried in an oven with forced air circulation (65 ± 5 °C) until a constant weight was reached to determine the shoot, root, and plant dry weights. 2.3.5 Nutrient concentration and use efficiency The N, P, and K concentrations in the shoots were determined as described by Bataglia et al. (1983). Nutrient accumulation was calculated based on N, P, and K concentrations and shoot dry weight. Nutrient use efficiency was calculated according to the equation (shoot dry weight) 2 /(accumulation of the respective nutrients) (Siddiqi and Glass 1981). It was not possible to determine the concentration of K in N and P-deficient plants because these treatments limited plant growth substantially. Therefore, we strongly recommend that future research uses a larger number of plants as an experimental unit. 2.4 Statistical analysis All data were subjected to a bidirectional analysis of variance (ANOVA) using the F test (p≤0.05) after checking for homogeneity of variances (Shapiro-Wilk W test). When significant, the Student’s multiple comparison test was applied to compare the means of the genotypes under each nutrient solution at a 5% probability level. The same test was used to compare the deficiency of each nutrient with the complete nutrient solution within each genotype using the statistical software Agroestat. 3. RESULTS 3.1 Complete nutrient solution Under nutritional sufficiency, WT/WT and WT/ phyB1 (scion/rootstock) induced greater N accumulation than that of phyB1 / phyB1 and phyB1 /WT (Fig. 1a), whereas P accumulation was similar among WT/ phyB1 , WT/WT, and phyB1 / phyB1 plants and less in phyB1 /WT (Fig. 1b). The genotypes did not differ in K accumulation under this condition (Fig. 1c). Regarding lipid peroxidation, we observed higher MDA concentrations in phyB1 / phyB1 than those in phyB1 /WT, which did not differ from WT/ phyB1 and WT/WT (Fig. 2a). Plants grafted with phyB1 or WT onto phyB1 rootstock ( phyB1 / phyB1 and WT/ phyB1 ) exhibited lower concentrations of total chlorophyll and carotenoids compared to WT/WT. Conversely, phyB1 /WT did not differ from WT/WT in chlorophyll concentration (Fig. 2b) but produced fewer carotenoids (Fig. 2c). Despite different physiological responses, the genotypes did not differ with respect to the quantum efficiency of PSII (Fig. 2d). No differences were observed between grafting combinations regarding photosynthetic rate and water use efficiency (Figs. 3a and 3d). On the other hand, phyB1 grafts induced lower stomatal conductance compared to WT grafts and only phyB1/WT showed lower transpiration compared to WT/WT (Fig. 3c). The N-use efficiency was higher in phyB1 / phyB1 than in WT/WT and the other genotypes (Fig. 4a); however, all genotypes showed similar P-and K-use efficiencies (Figs. 4b and 4c). No differences were observed between WT/ phyB1 or phyB1 / phyB1 and WT/WT in terms of shoot dry weight; however, lower dry weight production was observed in phyB1 /WT than in the control genotype (Fig. 5a). Furthermore, a similar pattern was observed for root and plant dry weight among the genotypes, in which WT/WT was superior to the other grafting combinations (Figs. 5b and 5c). 3.2 Nitrogen deficiency (-N) The comparison of means showed that there was no significant (p≤ 0.05) effect of genotype and nutrient solution factors on MDA concentration, or their interaction on P accumulation, P use efficiency, photosynthesis, or shoot dry weight (Tables S1-4). N deficiency decreased N, P, and K accumulation in the shoots (Fig. 1) compared to the complete treatment. The lower accumulation of nutrients caused by N deficiency led to a decrease in total Chl and carotenoids and the quantum efficiency of PSII (Figs. 2b-d). However, the grafting combinations showed similar N accumulation and total Chl and carotenoid concentrations. Regardless of the nutritional treatment, WT/ phyB1 plants accumulated more P than WT/WT and the other genotypes (Fig. 1b). Furthermore, phyB1 / phyB1 and WT/ phyB1 showed higher quantum efficiency of PSII than WT/WT and phyB1 /WT (Fig. 2d). Regardless of genotype, plants grown under N deficiency showed decreased photosynthesis, stomatal conductance, and transpiration compared to those of the complete treatment (Figs. 3a-c). However, only phyB1 / phyB1 and phyB1 /WT plants exhibited a reduction in WUE compared to the complete treatment (Fig. 3c). Under N deficiency, phyB1 /WT and phyB1 / phyB1 plants showed more transpiration than that of the WT/WT and WT/ phyB1 genotypes. However, higher WUE was observed in plants with WT scions (WT/ phyB1 and WT/WT) compared to those with phyB1 scions (Fig 3d). Low N availability also compromised plant growth, reducing N- and P-use efficiencies (Figs 4a and 4b) and consequently decreasing the production of shoot, root, and whole plant dry weights compared to the complete treatment (Fig 5). The N-deficient WT/ phyB1 and phyB1 / phyB1 plants showed higher N-use efficiency than that of WT/WT and phyB1 /WT; however, genotype had no significant effect on P-use efficiency (Figs. 4a and 4b). Thus, N-deficient WT/ phyB1 plants produced more roots and whole plant dry weights than WT/WT plants. However, regardless of the nutritional treatment, WT/ phyB1 and phyB1 / phyB1 did not differ from WT/WT in terms of shoot dry mass, although, WT/ phyB1 produced more dry weight in this part of the plant compared to phyB1 / phyB1 and phyB1 /WT (Fig. 5). 3.3 Phosphorus deficiency (-P) The comparison of means showed that there was no significant effect (p≤ 0.05) of genotype and nutrient solution interaction on P accumulation, MDA concentration, photosynthesis, stomatal conductance, P-use efficiency, or shoot, root, and whole plant dry weight (Tables S1-4). Phosphorus deficiency decreased the accumulation of N and P in the shoots of the plants; however, the plants of WT/ phyB1 that were deficient in P accumulated less N than WT/WT and the other genotypes, and together with phyB1 / phyB1 did not differ from the control genotype in relation to P accumulation, whereas lower accumulation of this element was observed in phyB1 /WT, irrespective of nutritional treatment (Figs. 1a and 1b). Regardless of the nutritional treatment, WT/ phyB1 and phyB1 / phyB1 had increased MDA concentration, whereas WT/WT and phyB1 /WT had lower concentrations of this molecule (Fig. 2a). Phosphorus-deficient plants had decreased concentrations of total Chl and carotenoids in relation to the complete treatment (Figs. 2b and 2c). However, WT/ phyB1 and phyB1 /WT showed higher concentrations of pigments than WT/WT. Phosphorus deficiency also decreased PSII quantum efficiency (Fig. 2d), photosynthesis, stomatal conductance, and plant transpiration compared to the complete treatment (Figs. 3a-c). However, the phyB1 / phyB1 and phyB1 /WT combinations showed lower quantum efficiency of PSII and stomatal conductance than WT/WT; however, photosynthesis and transpiration were similar between genotypes. Phosphorus-deficient WT/WT plants exhibited increased WUE, superior to the other genotypes, which showed lower efficiency compared to the complete treatment plants (Fig. 3d). P deficiency decreased N- and P-use efficiency (Figs. 4a and 4b), which resulted in lower shoot, root, and whole-plant dry weight production (Fig. 5). However, WT/ phyB1 had a more efficient N use than that of WT/WT, whereas P use efficiency was similar between genotypes. Regardless of nutritional condition, phyB1 /WT produced less shoot and whole plant dry weight than WT/WT and the other genotypes (Figs. 5a and 5c). The root dry weight was higher in WT/WT and lower in phyB1 /WT (Fig. 5b). 3.4 Potassium deficiency (-K) The comparison of means showed that there was no significant effect (p≤ 0.05) of the genotype on K accumulation and genotype and nutrient solution interaction on P and K accumulation, stomatal conductance, transpiration, N- and P-use efficiency, or shoot, root, and whole-plant dry weight (Tables S1-4). K deficiency decreased K accumulation in tomato plants compared to the complete treatment (Fig. 1c). However, K-deficient WT/WT plants accumulated more N than the other grafting combinations, and together with WT/ phyB1 , they accumulated more P than the phyB1 grafts ( phyB1 / phyB1 and phyB1 /WT), regardless of nutritional status (Figs. 1a and 1b). K-deficient phyB1 / phyB1 and phyB1 /WT plants had decreased and increased MDA concentrations in the shoot, respectively, in relation to the complete treatment; however, no differences were observed between the genotypes (Fig. 2a). Furthermore, K deficiency also decreased the concentration of total Chl, carotenoids, and the quantum efficiency of PSII (Figs. 2b-d); however, the genotypes did not differ from each other in these factors. Photosynthesis decreased in K-deficient plants, and this reduction was more pronounced in phyB1 / phyB1 , phyB1 /WT, and WT/ phyB1 than in WT/WT (Fig. 3a). K deficiency only reduced the stomatal conductance of phyB1 /WT plants (Fig. 3b), which together with phyB1 / phyB1 presented the lowest values in relation to WT/WT, irrespective of the nutritional condition. Regardless of genotype, K deficiency decreased transpiration (Fig. 3c) and WUE of plants (Fig. 3d); however, it was observed that phyB1 /WT and phyB1 / phyB1 transpired less than WT/WT and WT/ phyB1. Genotype had no effect on K-use efficiency. In contrast, K deficiency increased the use efficiency of this element and decreased N- and P-use efficiency compared to the plants in the complete treatment (Fig. 4). The lower efficiency in N, P, and K utilization resulted in decreased production of shoot, root, and whole plant dry weight (Fig. 5). Regardless of the nutritional condition, phyB1 / phyB1 and phyB1 /WT produced less dry weight in the shoot (Fig. 5a), and together with WT/ phyB1 , produced less dry weight in the root and in the whole plant compared to WT/WT (Figs. 5c and 5d). 4. DISCUSSION There is growing evidence that phytochrome B can modulate responses to different types of stress. Recently, Soares et al. (2021) demonstrated that the loss of tomato phyB1 function attenuated the damage caused by P deficiency, providing new perspectives for a better understanding of photoreceptor participation in nutritional responses. Since PHYB is expressed in shoots and roots, we studied the communication between these plant parts in the nutritional, physiological, and growth response signaling in tomato plants under nutritional deficiency. We used the phyB1-deficient tomato mutant ( phyB1 ) and its control genotype (WT) in grafting combinations under N, P, and K sufficiency and deficiency. The information obtained could be useful in understanding the benefits of phyB1 and identifying which nutritional deficiencies are expected to result in greater gains in nutritional and physiological processes and plant growth. This could contribute to strengthening the sustainability of various crops, as N, P, and K deficiencies are common worldwide, particularly in underdeveloped countries. 4.1 Complete nutrient solution Under nutrient-sufficient conditions, plants obtained from phyB1-deficient grafts ( phyB1 / phyB1 and phyB1 /MM) absorbed less N than that of WT/WT plants (Fig. 1a). Phytochrome is known to be involved in the transcription of nitrate transporter genes, and recent reports indicate that the loss of tomato phyB1 function decreased N accumulation in the shoot (Sakuraba and Yanagisawa, 2018; Soares et al., 2021). These results, together with those obtained in this study, reinforce the participation of phyB1 in N uptake, a process that appears to be coordinated by the shoot phytochrome. Furthermore, we observed that only the phyB1 /WT plants showed decreased P uptake (Fig. 1b), indicating that shoot phyB1 also upregulated the uptake of this element. This result may be related to the fact that this photoreceptor is involved in the expression of genes encoding high-affinity P transporters (Sakuraba et al. 2018). In contrast, all genotypes absorbed similar amounts of K compared to WT/WT (Fig. 1c), suggesting that neither shoot nor root phytochromes are directly involved in the absorption process of this element under sufficient conditions. All grafting combinations showed membrane lipid peroxidation that was similar to that of the control genotype (WT/WT); however, the highest production of MDA was observed in phyB1 / phyB1 (Fig. 2a), which can be explained by the role of phytochromes in suppressing lipid peroxidation of thylakoid membranes (Joshi et al. 1991). Thus, phyB1 / phyB1 and WT/ phyB1 plants had decreased concentrations of chlorophylls, and, together with phyB1 /MM, had less carotenoid production than that of the WT/WT. In a study on rice, a lower concentration of chlorophylls was observed in the phyB mutant, suggesting that this photoreceptor plays an important role in the synthesis of chlorophylls (Zhao et al. 2013). In our study, chlorophyll was likely controlled by the phyB1 of the root because the plants obtained from phyB1-deficient rootstocks had a lower concentration of this pigment, whereas carotenoids were under the control of both shoot and root phytochromes. Although phyB1 deficiency induced alterations in nutrient uptake and photosynthetic pigment production, the results suggest that shoot and root phyB1 do not control PSII quantum efficiency (Fig. 2d), photosynthesis (Fig. 3a), or WUE (Fig. 3d) in tomato. However, the stomatal conductance of phyB1 /WT and phyB1 / phyB1 and the transpiration of phyB1 /WT were lower than those of WT/WT. According to Mereb et al. (Mereb et al. 2020), the lower stomatal conductance and transpiration of the phytochrome mutant are associated with the lower density and stomatal index of this genotype, especially in phyB1 . Similarly, our results suggest the involvement of tomato shoot phyB1 in the positive regulation of these processes under nutritional sufficiency. The deficiency of both shoot and root phytochromes amplified the N-use efficiency of tomato (Fig. 4a); however, it did not change the P and K use efficiencies (Fig. 4b and 4c). The WT/WT plants produced a greater amount of dry weight in the root and whole plant than that of the other genotypes (Figs 5b and 5c). This may have occurred because the control genotype accumulated higher amounts of N and P, which resulted in higher concentrations of chlorophyll and carotenoids and higher stomatal conductance and transpiration. The lowest dry weight production in all parts of phyB1 /WT plants (Fig. 5) may have been a consequence of shoot phytochrome B1 deficiency, as our results suggest that this photoreceptor is involved in signaling for N and P uptake in the root, in addition to regulating carbon assimilation. 4.2 Nitrogen deficiency (-N) Carbon is the most demanded element by cultivated plants, followed by N, which is a structural component of amino acids, proteins, coenzymes, and nitrogenous bases (Marschner 2012; Prado 2021). Thus, the reduced accumulation of N caused by N deficiency (Fig. 1a) created an imbalance in P absorption compared to that of the complete treatment (Fig. 1b). However, the decrease in N uptake occurred similarly between the genotypes, suggesting that neither phytochrome regulates this process under N deficiency. Root phyB1 negatively regulated P uptake, as the accumulation of this element in WT/ phyB1 was greater than that in WT/WT, regardless of nutritional status. Chlorophyll concentration is directly related to the N level in the plant, as it is a constituent of this molecule (Mu and Chen 2021), which explains the decrease in photosynthetic pigments in plants under N deficiency (Fig. 2b and 2c). This deficiency also reduced the quantum efficiency of PSII by inhibiting the biosynthesis of photosynthetic pigments in all grafting combinations, because chlorophylls are responsible for the absorption and transport of energy to PSII and PSI (Mu et al. 2017; Zhang et al. 2019). In addition, WT/ phyB1 and phyB1 / phyB1 plants showed higher quantum efficiency of PSII than WT/WT (Fig. 2d), which can be attributed to the use of phyB1 as a rootstock, which amplified this response independently of N. We also observed a reduction in photosynthesis for all grafting combinations subjected to N deficiency, which was associated with a decrease in carbon assimilation and transpiration in plants (Figs. 3a-c). In addition, N deficiency decreased the WUE of all grafting combinations (Fig. 3d). Despite these results, no participation of shoot or root phytochromes was observed in the control of stomatal conductance under N deficiency (Fig. 3b). The phyB1 /WT and phyB1 / phyB1 plants had greater transpiration than that of the WT/WT, which decreased the WUE of these genotypes. These results suggest that shoot phyB1 is a positive regulator of transpiration in N-deficient tomato, and its deficiency results in higher water consumption per fixed carbon, as observed in the WUE results. It is estimated that approximately 5% of a plant's total dry weight is composed of N (Marschner, 2012), and this element is considered most related to dry weight production (Reis et al., 2009). Thus, all genotypes cultivated under N deficiency had reduced use efficiency of this nutrient as well as P-use efficiency (Figs. 4a and 4b), which, associated with the other nutritional and physiological changes, caused a decrease in the production of shoot, root, and whole plant dry weight (Fig 5). Regardless of N, WT/ phyB1 absorbed and accumulated more P in the shoot, which favored the greatest shoot dry weight. In addition, loss of root phyB1 function favored root and whole-plant dry weight production of WT/ phyB1 under N deficiency. These responses suggest that root phyB1 is a negative regulator of root and plant dry weight production in an N-dependent manner, which is associated with the higher PSII quantum, water-use, and N-use efficiencies of this genotype. Our results also suggest that the use of WT as a graft and of the defective mutant in phyB1 as a rootstock may be an alternative to attenuate the damage caused by N deficiency in tomato. 4.3 Phosphorus deficiency (-P) The omission of P decreased the absorption of this element (Fig. 1b) and caused an imbalance in the absorption of N in all genotypes (Fig. 1a), due to P being a constituent of the ATP molecule that is used in the process of nutrient absorption (Prado 2021). WT/ phyB1 plants under P deficiency showed reduced N uptake compared to WT/WT. This may be due to the photoreceptor's involvement in regulating the activity of key genes encoding nitrate transporters (Sakuraba and Yanagisawa, 2018), a process that, in this case, appears to be under the control of root phyB1 and P-dependent. Furthermore, regardless of nutritional status, phyB1 /WT plants accumulated less P compared to WT/WT. This can be attributed to shoot phyB1 deficiency, which appears to have a greater contribution to signaling responses for nutrient absorption than root phyB1, thereby positively regulating this process. P deficiency increased lipid peroxidation of plant membranes, regardless of genotype (Fig. 2a); however, the highest production of MDA occurred in plants deficient in root phyB1 (WT/ phyB1 ). Recently, it was demonstrated that phyB1 is related to the maintenance of membrane integrity in tomato (Soares et al., 2021). Despite MDA quantification in the shoot, our results indicate that its production is under the control of phytochrome in the root. Previous studies have reported that the concentrations of chlorophyll and carotenoids are lower in P-deficient plants (de Souza Osório et al. 2020; Patel et al. 2020). Our results agree with these findings; however, this reduction was less pronounced in WT/ phyB1 and phyB1 /WT, and more evident in WT/WT (Figs. 2b and 2c), indicating that in P-deficient plants, the lack of phyB1 from both the root and shoot provides an increase in the biosynthesis of chlorophylls and carotenoids. As a consequence of the loss of photosynthetic pigments, P deficiency also decreases the quantum efficiency of PSII, which may have been a strategy to reduce photoinhibition damage (Hernández and Munné-Bosch 2015). However, we verified that phyB1 / phyB1 and phyB1 /MM showed lower efficiency in energy transfer to PSII (Fv/Fm) than that of WT/WT, indicating that shoot phytochrome B1 is a positive regulator of this process (Fig. 2d). In this study, P deficiency negatively affected gas exchange reactions (Fig. 3). A decrease in photosynthesis occurred due to the loss of photosynthetic pigments (Fig. 3a), in addition to lower carbon assimilation and transpiration (Figs. 3b and 3c). However, in P-deficient plants, transpiration is not regulated by the shoot and root phyB1. Except for WT/WT, all other grafting combinations had decreased WUE compared to the complete treatment (Fig. 3d), which may be associated with the loss of phyB1 function, indicating the participation of this photoreceptor in water relations dependent on P. We found that regardless of genotype, phosphorus deficiency decreased N- and P-use efficiencies and resulted in lower nutrient conversion to dry weight (Fig. 5). These results were expected because, together with N and K, P is one of the most responsive nutrients for plants, and therefore, its low availability is a limiting factor for plant growth and development (Hernández and Munné-Bosch 2015). The lack of root phyB1 in P-deficient WT/ phyB1 plants increased P-use efficiency compared to that of WT/WT (Fig. 4a). Regardless of the nutritional treatment, phyB1 /WT plants showed less dry weight production in the shoot and whole plant than that of WT/WT. These results can be explained by the lower accumulation of P and lower stomatal conductance of this genotype in relation to WT/WT, showing that phyB1 deficiency in the shoot negatively affected nutritional and physiological processes, and consequently, the production of tomato dry weight. In contrast, the root dry weight results indicated that both shoot and root phytochromes participated in the dry weight production process in this part of the plant. 4.4 Potassium deficiency (-K) We demonstrated that potassium deficiency decreased the uptake of this element, as well as the uptake of N and P from plants, regardless of the genotype (Fig. 1). This may be because K acts in the activation of key enzymes for nutrient absorption, in addition to stimulating nitrate absorption, as it serves as an accompanying cation (Blevins et al. 1978). However, K-deficient genotypes accumulated less N and K than WT/WT, suggesting that both phyB1s are positive regulators of the absorption of these nutrients in a K-dependent manner, whereas shoot phyB1 is a positive regulator of P uptake that is independent of K. K deficiency can result in lipid peroxidation of membranes, and this response is modulated by shoot phyB1 in tomato, since phyB1 / phyB1 and phyB1 /WT increased the MDA concentration (Fig. 2a). Furthermore, the increase in MDA concentration may have inhibited biosynthesis and accelerated the biodegradation of pigments (Figs. 2b and 2c) and increased oxidative damage, as observed with the lower quantum efficiency of PSII results (Fig. 2d). However, these physiological changes occurred independently of the phytochromes. Our results showed that K deficiency caused a considerable reduction in the photosynthetic parameters (photosynthesis, stomatal conductance, and transpiration) compared to the complete treatment (Figs. 3a-c). The low availability of K in plants decreases its concentration in guard cells and consequently compromises stomatal regulation (Hasanuzzaman et al. 2018). Thus, the decrease in stomatal conductance minimized water loss through transpiration but reduced carbon assimilation. Previous studies have shown that photosynthetic performance can be increased in transgenic rice plants that overproduce phytochrome B (Kreslavski et al. 2018). Therefore, grafting combinations with the loss of phyB1 function decreased photosynthesis in relation to the WT/WT (Fig. 3a). However, this reduction was more pronounced in plants deficient in shoot phyB1, indicating that this photoreceptor is a positive regulator of photosynthesis in a K-dependent manner. The deficiency of shoot phyB1 decreased stomatal conductance and transpiration in plants, regardless of nutritional treatment (Figs. 3b and 3c). This suggests that phyB1 is a positive regulator of these processes. It was previously reported that adequate K availability allows for more efficient water use (Hasanuzzaman et al. 2018), which justifies the lower WUE results in plants deficient in this element compared to plants that received the complete treatment. K-deficient grafted plants showed decreased N- and P-use efficiencies compared to the complete treatment (Figs. 4a and 4b). However, K-use efficiency increased with the low availability of this element (Fig. 4c), which may have been an adaptive strategy for plants to maintain their metabolism. In addition to activating more than 50 enzymes, K plays a fundamental role in the transport of solutes from the shoot to the root via the phloem (Hafsi et al. 2014). Furthermore, this element regulates the biosynthesis, conversion, and allocation of metabolites (Hasanuzzaman et al. 2018). K deficiency negatively affected shoot, root, and whole-plant dry weight production (Fig. 5). Regardless of the nutritional condition, the deficiency of shoot phyB1 ( phyB1 /WT and phyB1 / phyB1 ) negatively affected the dry weight of this part of the plant. In addition, the loss of function of both shoot and root phytochromes decreased the dry weight of roots and whole plants. These results show that both shoot and root phyB1 are involved in some metabolic steps in the plant, whether nutritional or physiological, and consequently participate in the signaling of responses to dry weight production in tomato. In summary, based on the responses observed in the grafting combinations between the mutant phyB1 and WT, we can conclude that phytochrome B1 is involved in shoot-root communication for the control of nutritional, physiological, and growth responses in tomato. Furthermore, the action of both shoot and root phytochromes on these responses may occur independently or may depend on N, P, and K. The use of a tomato mutant deficient in phyB1 could be a tool to better understand the control of phytochromes in plant nutrition, physiology, and growth. Our results also provide new perspectives for future studies to explore the manipulation of the PHYB1 gene to improve crop productivity and mitigate damage caused by nutritional deficiency. Declarations Acknowledgments The support of the São Paulo State University (UNESP) is gratefully acknowledged. This research project was funded by Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil, Code 001. Author Contribution M.B.S, R.M.P and R.F.C conceived the idea. M.B.S and L.T.S.C carried out the experiments. M.B.S and E.G.R performed the chemical analysis. M.B.S. and R.O performed the physiological analysis. J.L.F.S analyzed the data. M.B.S, R.F.C and D.O.V wrote the manuscript. R.M.P and R.F.C revised the manuscript. All authors read and approved the manuscript. Funding This research project was funded by Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil, Code 001. Data availability Data sharing is not applicable to this article as all newly obtained data is already contained within this article. Competing interests The authors declare no competing interests. References Bataglia O, Furlani A, Teixeira J, et al (1983) Métodos de análise química de plantas. Bol Técnico, Inst Agronômico Campinas Blevins DG, Barnett NM, Frost WB (1978) Role of Potassium and Malate in Nitrate Uptake and Translocation by Wheat Seedlings. Plant Physiol 62:784–788. https://doi.org/10.1104/pp.62.5.784 Carvalho RF, Moda LR, Silva GP, et al (2016) Nutrition in tomato (Solanum lycopersicum L) as affected by light: Revealing a new role of phytochrome A. 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J Plant Nutr 4:289–302. https://doi.org/10.1080/01904168109362919 Soares MB, de Mello Prado R, Tenesaca LFL, et al (2021) Tomato phytochromes B1 and B2 are part of the responses to the nutritional stress induced by NPK deficiency. Physiol Plant 1–10. https://doi.org/10.1111/ppl.13574 van Tuinen, Ageeth, Kerckhoffs, Huub J., Nagatani, Akira, Kendrick, Richard E., and Koornneef M (1995) A Temporarily Red Light-lnsensitive Mutant of Tomato. 939–947 Zhang Y, Liang Y, Zhao X, et al (2019) Silicon compensates phosphorus deficit-induced growth inhibition by improving photosynthetic capacity, antioxidant potential, and nutrient homeostasis in tomato. Agronomy 9:1–16. https://doi.org/10.3390/agronomy9110733 Zhao J, Zhou J jun, Wang Y ying, et al (2013) Positive Regulation of Phytochrome B on Chlorophyll Biosynthesis and Chloroplast Development in Rice. Rice Sci 20:243–248. https://doi.org/10.1016/S1672-6308(13)60133-X Supplementary Files Supp.docx Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Acta Physiologiae Plantarum → Version 1 posted Reviewers agreed at journal 22 Jul, 2025 Reviewers invited by journal 02 Jul, 2025 Editor assigned by journal 22 Jun, 2025 First submitted to journal 20 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6938144","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":479409190,"identity":"95d46100-177f-46e2-b62e-7d0ccc4b9b99","order_by":0,"name":"Mariana Bomfim Soares","email":"","orcid":"","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"Bomfim","lastName":"Soares","suffix":""},{"id":479409191,"identity":"af349108-d588-4579-b309-82a2cc3e268a","order_by":1,"name":"Renato de Mello Prado","email":"","orcid":"","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":false,"prefix":"","firstName":"Renato","middleName":"de Mello","lastName":"Prado","suffix":""},{"id":479409192,"identity":"16a83f07-9553-4082-b181-ef63afac3103","order_by":2,"name":"Dilier Olivera Viciedo","email":"","orcid":"","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":false,"prefix":"","firstName":"Dilier","middleName":"Olivera","lastName":"Viciedo","suffix":""},{"id":479409193,"identity":"53b4437c-b734-40fa-b183-ffc7485cb89c","order_by":3,"name":"Eduarda Gonçalves Reis","email":"","orcid":"","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":false,"prefix":"","firstName":"Eduarda","middleName":"Gonçalves","lastName":"Reis","suffix":""},{"id":479409194,"identity":"67082e43-51dc-4d36-bd4a-681527608d40","order_by":4,"name":"Livia Tálita da Silva Carvalho","email":"","orcid":"","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":false,"prefix":"","firstName":"Livia","middleName":"Tálita da Silva","lastName":"Carvalho","suffix":""},{"id":479409195,"identity":"722e6dd1-e805-4507-b6f8-b32e16359b46","order_by":5,"name":"Reginaldo Oliveira","email":"","orcid":"","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":false,"prefix":"","firstName":"Reginaldo","middleName":"","lastName":"Oliveira","suffix":""},{"id":479409196,"identity":"bd5110d2-24e7-4805-98ac-43432001dab5","order_by":6,"name":"José Lucas Farias da Silva","email":"","orcid":"","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Lucas Farias da","lastName":"Silva","suffix":""},{"id":479409197,"identity":"d58d9716-fcc2-4414-92ba-b61007e12c6c","order_by":7,"name":"Rogério Carvalho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDAC5gNgSoYfyoAI4tXClgCmeCTbEkjVYnCMWC3mbcwPP1e22fEYHwMyeBgO5/Hznz3AXLgHtxaZY2zGkmfbknnMgAxpoJZiyRl5CcwznuHWIiHfwyDZcIaZx+w+kDGD4XDihhs8Bsw8B/BoYeNh/tlwpp7HuA3IAGnZf/4MQS1skg0Vh3kMgAyJDyBbGHIIaWEzs2yoOM4jcYzNzOKDQXrijBt5CYdn4NXC/Phmg0G1HH8b8+MbCRXWif39Zw8+LsCjBQ0YgAgeBuI1QAEPqRpGwSgYBaNgmAMAVl5HmMcKi14AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1270-7372","institution":"UNESP: Universidade Estadual Paulista Julio de Mesquita Filho","correspondingAuthor":true,"prefix":"","firstName":"Rogério","middleName":"","lastName":"Carvalho","suffix":""}],"badges":[],"createdAt":"2025-06-20 10:47:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6938144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6938144/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11738-026-03893-x","type":"published","date":"2026-02-27T15:58:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85948423,"identity":"45fdb2e3-1fa5-47f1-aa2a-896a74ca03b6","added_by":"auto","created_at":"2025-07-03 13:19:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":144954,"visible":true,"origin":"","legend":"\u003cp\u003eNutrient accumulation in the shoots of self- and reciprocal-grafted tomato WT and phyB1 grown under complete or deficient (-) nutrient solution. (a) N, (b) P, and (c) K. Values are the means ± SE of each treatment (n=4 replicates, containing one plant each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Student’s test (p≤0.05). It was not possible to determine the concentration of K in N and P-deficient plants because these treatments limited plant growth substantially\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6938144/v1/a54fe084bb5323526c65ca23.png"},{"id":85951538,"identity":"615a0fdf-2bec-4df9-9ac8-829a2776d9cd","added_by":"auto","created_at":"2025-07-03 13:57:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":207384,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Malondialdehyde (MDA), (b) chlorophyll a+b, (c) carotenoid content and (d) quantum efficiency of PSII (Fv/Fm) of self- and reciprocal-grafted tomato WT and \u003cem\u003ephyB1 \u003c/em\u003egrown under complete or deficient (-) nutrient solution. Values are the means ± SE of each treatment (n=4 replicates, containing one plants each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Student's test (p\u003cem\u003e≤\u003c/em\u003e0.05)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6938144/v1/cd1084c7d906710220569d58.png"},{"id":85948419,"identity":"011ad85c-c4e2-4e6f-8736-404c4aaeea7a","added_by":"auto","created_at":"2025-07-03 13:19:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":207384,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Malondialdehyde (MDA), (b) chlorophyll a+b, (c) carotenoid content and (d) quantum efficiency of PSII (Fv/Fm) of self- and reciprocal-grafted tomato WT and \u003cem\u003ephyB1 \u003c/em\u003egrown under complete or deficient (-) nutrient solution. Values are the means ± SE of each treatment (n=4 replicates, containing one plants each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Student's test (p\u003cem\u003e≤\u003c/em\u003e0.05)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6938144/v1/2c0b41a20027e651993d40c1.png"},{"id":85948421,"identity":"81d60b08-1528-4b31-b15d-a127c75702d2","added_by":"auto","created_at":"2025-07-03 13:19:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":193647,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Photosynthesis, (b) stomatal conductance, (c) transpiration, and (d) water use efficiency of self- and reciprocal-grafted tomato WT and \u003cem\u003ephyB1 \u003c/em\u003egrown under complete or deficient (-) nutrient solution. Values are the means ± SE of each treatment (n=4 replicates, containing one plants each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Student's test (p\u003cem\u003e≤\u003c/em\u003e0.05)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6938144/v1/f727debfab6667c8cf7373df.png"},{"id":85949305,"identity":"c0c52c22-3b2e-4f4d-9582-ec78d2ab6eb2","added_by":"auto","created_at":"2025-07-03 13:27:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":147497,"visible":true,"origin":"","legend":"\u003cp\u003eNutrient use efficiency of self- and reciprocal-grafted tomato WT and phyB1 grown under complete or deficient (-) nutrient solution. (a) N, (b), and (c) K. Values are the means ± SE of each treatment (n=4 replicates, containing one plant each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Student's test (p≤0.05). It was not possible to determine the concentration of K in N and P-deficient plants because these treatments limited plant growth substantially\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6938144/v1/44d9751049d528e0a712b4b8.png"},{"id":85949309,"identity":"a55d98da-a2d8-4256-a0cf-d93d3179d61e","added_by":"auto","created_at":"2025-07-03 13:27:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138673,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Shoot, (b) root, and (c) plant dry weight of self- and reciprocal-grafted tomato WT and phyB1 grown under complete or deficient (-) nutrient solution. (a) N, (b), and (c) K. Values are the means ± SE of each treatment (n=4 replicates, containing one plant each). Letters above the bars represent the differences in the means between the genotypes within each solution, and asterisks on top of bars indicate significant differences between the nutritional treatment and the complete solution; both were calculated using Student's test (p\u003cem\u003e≤\u003c/em\u003e0.05)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6938144/v1/a68646c5f3fd4a689e6ef7cf.png"},{"id":103765842,"identity":"44a61b9a-aca9-4589-8ced-588696561ccd","added_by":"auto","created_at":"2026-03-02 16:10:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1594468,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6938144/v1/7d83856a-2a67-4174-8f8e-a932334a7102.pdf"},{"id":85949558,"identity":"aeaaf2d6-709f-4292-9bc4-c9a2025e3592","added_by":"auto","created_at":"2025-07-03 13:35:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1312081,"visible":true,"origin":"","legend":"","description":"","filename":"Supp.docx","url":"https://assets-eu.researchsquare.com/files/rs-6938144/v1/2d53d74d7bf27bd6cba149d7.docx"}],"financialInterests":"","formattedTitle":"Tomato phytochrome B1 modulates N, P, and K deficiency response by root-to-shoot communication","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eNutritional deficiency is one of the main factors that limits agricultural production worldwide (Patel et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Inadequate supplies of nitrogen (N), phosphorus (P), and potassium (K) are the most limiting factors for plant growth because these elements are considered the most responsive by plants (Marschner, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Understanding the responses of crops to N, P, and K deficiencies is essential for improving the efficiency of the use of these elements and reducing the degree of fertilization.\u003c/p\u003e \u003cp\u003eNitrogen deficiency reduces its absorption and concentration in all parts of snap bean, citrus, and peanut, causing nutritional imbalance, growth inhibition, decreased concentration of photosynthetic pigments, and CO\u003csub\u003e2\u003c/sub\u003e assimilation (de Souza Os\u0026oacute;rio et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Patel et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, P is required for several metabolic processes in plants, such as the formation of the ATP molecule, which is used as a source of cellular energy (Prado \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A decrease in photosynthesis and stomatal conductance has been reported in P-deficient plants, in addition to the formation of reactive oxygen species (ROS) in chloroplasts (Hern\u0026aacute;ndez and Munn\u0026eacute;-Bosch \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In contrast, K deficiency damages photosynthesis, transpiration, and stomatal conductance (Pandey and Mahiwal \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLight modulates plant nutrition, and recently, the role of photoreceptors in this response has become increasingly evident (Carvalho et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sakuraba et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Soares et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; D\u0026rsquo;Amico-Dami\u0026atilde;o et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The phytochrome B family (phyB) is involved in the stress response to nutritional deficiency, and in a previous study, phyB-9 and phyB-10 deficiency resulted in reduced P uptake in Arabidopsis plants (Sakuraba et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Those authors also observed a decrease in the expression of high-affinity P transporter genes, indicating that red light is involved in the activation of P uptake, especially under this nutrient's deficiency (Sakuraba et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe recently revealed that tomato phyB1 is a positive regulator of N uptake, pigment synthesis, and dry weight production under N deficiency (Soares et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, the loss of function of this photoreceptor attenuated the damage caused by P deficiency, since the mutant plants showed increased production of dry weight compared to WT plants (Soares et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Under K deficiency, phyB1 positively regulated P absorption and pigment production. This result suggests strong control by phyB1 over nutritional responses induced by N, P, and K (Soares et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, in the present study, we hypothesized that the interaction between phyB1 and N, P, and K deficiencies involves reciprocal communication between the root and shoot. For the first time, we explored the role of the tomato \u003cem\u003ePHYB1\u003c/em\u003e gene in plant responses to N, P, and K deficiency, using the photomorphogenetic mutant \u003cem\u003ephyB1\u003c/em\u003e and grafting to understand the role of this photoreceptor in root-to-shoot communication.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cp\u003e2.1 Growth conditions, plant material, and grafting technique\u003c/p\u003e\n\u003cp\u003eThe experiment was conducted inside a growth chamber and greenhouse at S\u0026atilde;o Paulo State University, Jaboticabal, Brazil, for 65 days after sowing (DAS). We used \u003cem\u003eSolanum lycopersicum\u003c/em\u003e L. cv. Moneymaker as the wild-type (WT) as well as the \u003cem\u003ephyB1\u003c/em\u003e mutant, which is defective for the gene \u003cem\u003ePHYB1\u003c/em\u003e that encodes PHYB1 apoproteins in the cv Moneymaker background (van Tuinen, Ageeth, Kerckhoffs, Huub J., Nagatani, Akira, Kendrick, Richard E., and Koornneef 1995).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeeds of \u003cem\u003ephyB1\u003c/em\u003e and WT were placed in polystyrene trays to germinate, which were filled with a mixture of the commercial substrate BioPlant\u0026reg; (composed of sphagnum peat, vermiculite, coconut fiber, rice husk, husk pine, and additives containing calcium) and expanded vermiculite in a volumetric proportion of 1: 1 (v: v). Seventeen DAS, the plants were transferred to 200 mL pots (one plant per pot) on the same substrate, and grafting was performed. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeventeen-day-old plants were grafted using the splice method with the aid of a scalpel blade and grafting clips to obtain the following graft combinations: WT/WT, \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e, WT/\u003cem\u003ephyB1\u003c/em\u003e, and \u003cem\u003ephyB1\u003c/em\u003e/WT (scion/rootstock).After grafting, the basal quarter of the pots was submerged in water (a floating moist chamber of 25 ℃ \u0026plusmn; 2 ℃ with a high relative humidity of 85% \u0026plusmn; 10%) under a 12 h photoperiod at 70 \u0026micro;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, until the graft union had completely healed (20 days after grafting, DAG) (Fig. S1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter the graft union had healed, the roots of the grafted plants were washed with deionized water to remove excess substrate, and the plants were transferred to 1.7 dm\u0026sup3; pots containing medium texture sand that was previously decontaminated with 0.1 mol L\u003csup\u003e-1\u003c/sup\u003e hydrochloric acid solution and washed with deionized water. The pots were transferred to the greenhouse and irrigated daily with the nutrient solution described by Hoagland and Arnon (1950) to feed the plants and keep the humidity close to the maximum water retention capacity. The solution was initially diluted to 25% ionic strength and after ten days, the concentration was increased to 50%, where it remained until the end of the experiment. The nutrient solution was prepared with deionized water, and the pH value of the solution was maintained at 5.5 \u0026plusmn; 0.5 using 1% sodium hydroxide and hydrochloric acid solutions. Air temperature and humidity data were monitored daily inside the greenhouse. Variations were observed in the maximum (85.1 \u0026plusmn; 9%) and minimum (33 \u0026plusmn; 12%) relative humidity, and maximum (28.7 \u0026plusmn; 10 \u0026deg;C) and minimum (14.7 \u0026plusmn; 7 \u0026deg;C) temperatures.\u003c/p\u003e\n\u003cp\u003e2.2 Treatments and experimental design\u003c/p\u003e\n\u003cp\u003eThree experiments were performed with the treatments arranged in a 4 \u0026times; 2 factorial scheme, with the first factor as the grafting combinations of the tomato plants (WT/WT, \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e, WT/\u003cem\u003ephyB1\u003c/em\u003e, and \u003cem\u003ephyB1\u003c/em\u003e/WT), and the second factor defined as the nutrient solution, which was either complete (containing 15 mmol L\u003csup\u003e-1\u003c/sup\u003e of N, 1 mmol L\u003csup\u003e-1\u003c/sup\u003e of P and 6 mmol L\u003csup\u003e-1\u003c/sup\u003e of K) , deficient in N (1 mmol L\u003csup\u003e-1\u003c/sup\u003e of N using NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eas the source), without P (0 mmol L\u003csup\u003e-1\u003c/sup\u003e), or with absence of K (0 mmol L\u003csup\u003e-1\u003c/sup\u003e) in the solution (Table 1). A completely randomized design with four replicates was adopted, with one replicate consisting of one plant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Composition of nutrient solutions used to induce N, P, and K deficiencies in\u0026nbsp;Solanum lycopersicum\u0026nbsp;L. Values correspond to a 100% nutrient solution, which was diluted to 25% and 50%.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSources\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eComplete\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-K\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emol L\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003emL L\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCa (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. 5H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMgSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e. 2H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMicronutrients*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFe-EDDHA**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\"\u003e\n \u003cp\u003e*In 1L: 2.86 g H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e;\u003csub\u003e\u0026nbsp;\u003c/sub\u003e1.81 g\u003csub\u003e\u0026nbsp;\u003c/sub\u003eMnCl\u003csub\u003e2\u003c/sub\u003e. 4H\u003csub\u003e2\u003c/sub\u003eO; 0.10 g ZnCl\u003csub\u003e2\u003c/sub\u003e; 0.04 g CuCl\u003csub\u003e2\u003c/sub\u003e; 0.02 g H\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003eO.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e** In 1L: 83.33 g Fe-EDDHA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e2.3 Performed analysis\u003c/p\u003e\n\u003cp\u003e2.3.1 Leaf gas exchange\u003c/p\u003e\n\u003cp\u003ePhotosynthetic activity (A), stomatal conductance (Gs), and transpiration (E) was measured using a Li-6400 (LICOR, EUA). The water use efficiency (WUE) was calculated by dividing A by E.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.3.2 Quantum efficiency of PSII (Fv/Fm) and pigment content\u003c/p\u003e\n\u003cp\u003eThe quantum efficiency of PSII was measured between 07:00 and 08:00 h. The third fully expanded leaf was dark-adapted for 30 min using clips, and the minimal (F0) and maximal fluorescence (Fm), as well as the Fv/Fm, were obtained using a portable fluorometer (Opti-Sciences, Os30P). The total chlorophyll (Chl) and carotenoid contents were determined according to the methodology described by Lichtenthaler (Lichtenthaler 1987). Fresh samples (0.025\u0026ndash;0.030 g) of the leaves were collected from tomato plants for each treatment, and readings were performed using a Beckman DU 640 spectrophotometer at 663, 647, and 470 nm to acquire the respective contents of chlorophyll \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e, and carotenoids, based on fresh weight. The total chlorophyll content (total chl) was calculated as the sum of the concentrations of chlorophylls \u003cem\u003ea\u003c/em\u003e and \u003cem\u003eb\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e2.3.3 Lipidic peroxidation\u003c/p\u003e\n\u003cp\u003eLipid peroxidation was determined in shoot samples (stems and leaves), which were collected, washed, immersed in liquid N\u003csub\u003e2,\u003c/sub\u003e and stored at \u0026minus;80 \u0026deg;C, following the methodology of thiobarbituric acid described by Heath and Packer (Heath and Packer 1968). Metabolites of cell membranes, especially malondialdehyde (MDA), react with thiobarbituric acid and can be quantified by spectrophotometry. Briefly, shoot tissue (0.3 g) was homogenized with 1 mL of trichloroacetic acid and polyvinylpyrrolidone (20%). Subsequently, the samples were transferred to tubes and centrifuged at 10,000 \u003cem\u003e\u0026times; g\u003c/em\u003e for 15 min. The supernatant was blended with 1 mL of trichloroacetic acid 20% (m/v) and thiobarbituric acid 0.5% (m/v) and incubated in a water bath at 95 \u0026deg;C for 30 min. After this period, the samples were centrifuged at 11,000 \u0026times; g for 5 min. The MDA concentration was determined using the coefficient 1.55 \u0026times; 10\u0026minus;5 mol\u0026minus;1 cm\u0026minus;1 (Grat\u0026atilde;o et al., 2012), and results are expressed as nmol g\u0026minus;1 of fresh weight. The readings were performed in a spectrophotometer at 535 and 600 nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.3.4 Growth parameters\u003c/p\u003e\n\u003cp\u003ePlants were harvested at 65 DAS and separated into shoots and roots. The plant material was washed under running water with a detergent solution (0.1% v/v), followed by an HCl solution (0.3% v/v) and deionized water. Subsequently, the material was dried in an oven with forced air circulation (65 \u0026plusmn; 5 \u0026deg;C) until a constant weight was reached to determine the shoot, root, and plant dry weights.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.3.5 Nutrient concentration and use efficiency\u003c/p\u003e\n\u003cp\u003eThe N, P, and K concentrations in the shoots were determined as described by Bataglia et al. (1983). Nutrient accumulation was calculated based on N, P, and K concentrations and shoot dry weight. Nutrient use efficiency was calculated according to the equation (shoot dry weight)\u003csup\u003e2\u003c/sup\u003e/(accumulation of the respective nutrients) (Siddiqi and Glass 1981). It was not possible to determine the concentration of K in N and P-deficient plants because these treatments limited plant growth substantially. Therefore, we strongly recommend that future research uses a larger number of plants as an experimental unit.\u003c/p\u003e\n\u003cp\u003e2.4 Statistical analysis\u003c/p\u003e\n\u003cp\u003eAll data were subjected to a bidirectional analysis of variance (ANOVA) using the \u003cem\u003eF\u003c/em\u003e test (p\u0026le;0.05) after checking for homogeneity of variances (Shapiro-Wilk W test). When significant, the Student\u0026rsquo;s multiple comparison test was applied to compare the means of the genotypes under each nutrient solution at a 5% probability level. The same test was used to compare the deficiency of each nutrient with the complete nutrient solution within each genotype using the statistical software Agroestat.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e3.1 Complete nutrient solution\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnder nutritional sufficiency, WT/WT and WT/\u003cem\u003ephyB1\u003c/em\u003e (scion/rootstock) induced greater N accumulation than that of \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT (Fig. 1a), whereas P accumulation was similar among WT/\u003cem\u003ephyB1\u003c/em\u003e, WT/WT, and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e plants and less in \u003cem\u003ephyB1\u003c/em\u003e/WT (Fig. 1b). The genotypes did not differ in K accumulation under this condition (Fig. 1c).\u003c/p\u003e\n\u003cp\u003eRegarding lipid peroxidation, we observed higher MDA concentrations in \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e than those in \u003cem\u003ephyB1\u003c/em\u003e/WT, which did not differ from WT/\u003cem\u003ephyB1\u003c/em\u003e and WT/WT (Fig. 2a). Plants grafted with \u003cem\u003ephyB1\u003c/em\u003e or WT onto \u003cem\u003ephyB1\u003c/em\u003e rootstock (\u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and WT/\u003cem\u003ephyB1\u003c/em\u003e) exhibited lower concentrations of total chlorophyll and carotenoids compared to WT/WT. Conversely, \u003cem\u003ephyB1\u003c/em\u003e/WT did not differ from WT/WT in chlorophyll concentration (Fig. 2b) but produced fewer carotenoids (Fig. 2c). Despite different physiological responses, the genotypes did not differ with respect to the quantum efficiency of PSII (Fig. 2d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo differences were observed between grafting combinations regarding photosynthetic rate and water use efficiency (Figs. 3a and 3d). On the other hand, phyB1 grafts induced lower stomatal conductance compared to WT grafts and only phyB1/WT showed lower transpiration compared to WT/WT (Fig. 3c).\u003c/p\u003e\n\u003cp\u003eThe N-use efficiency was higher in \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e than in WT/WT and the other genotypes (Fig. 4a); however, all genotypes showed similar P-and K-use efficiencies (Figs. 4b and 4c).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;No differences were observed between WT/\u003cem\u003ephyB1\u003c/em\u003e or \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and WT/WT in terms of shoot dry weight; however, lower dry weight production was observed in \u003cem\u003ephyB1\u003c/em\u003e/WT than in the control genotype (Fig. 5a). Furthermore, a similar pattern was observed for root and plant dry weight among the genotypes, in which WT/WT was superior to the other grafting combinations (Figs. 5b and 5c).\u003c/p\u003e\n\u003cp\u003e3.2 Nitrogen deficiency (-N)\u003c/p\u003e\n\u003cp\u003eThe comparison of means showed that there was no significant (p\u0026le; 0.05) effect of genotype and nutrient solution factors on MDA concentration, or their interaction on P accumulation, P use efficiency, photosynthesis, or shoot dry weight (Tables S1-4).\u003c/p\u003e\n\u003cp\u003eN deficiency decreased N, P, and K accumulation in the shoots (Fig. 1) compared to the complete treatment. The lower accumulation of nutrients caused by N deficiency led to a decrease in total Chl and carotenoids and the quantum efficiency of PSII (Figs. 2b-d). However, the grafting combinations showed similar N accumulation and total Chl and carotenoid concentrations. Regardless of the nutritional treatment, WT/\u003cem\u003ephyB1\u003c/em\u003e plants accumulated more P than WT/WT and the other genotypes (Fig. 1b). Furthermore, \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and WT/\u003cem\u003ephyB1\u003c/em\u003e showed higher quantum efficiency of PSII than WT/WT and \u003cem\u003ephyB1\u003c/em\u003e/WT (Fig. 2d).\u003c/p\u003e\n\u003cp\u003eRegardless of genotype, plants grown under N deficiency showed decreased photosynthesis, stomatal conductance, and transpiration compared to those of the complete treatment (Figs. 3a-c). However, only \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT plants exhibited a reduction in WUE compared to the complete treatment (Fig. 3c). Under N deficiency, \u003cem\u003ephyB1\u003c/em\u003e/WT and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e plants showed more transpiration than that of the WT/WT and WT/\u003cem\u003ephyB1\u0026nbsp;\u003c/em\u003egenotypes. However, higher WUE was observed in plants with WT scions (WT/\u003cem\u003ephyB1\u003c/em\u003e and WT/WT) compared to those with \u003cem\u003ephyB1\u003c/em\u003e scions (Fig 3d).\u003c/p\u003e\n\u003cp\u003eLow N availability also compromised plant growth, reducing N- and P-use efficiencies (Figs 4a and 4b) and consequently decreasing the production of shoot, root, and whole plant dry weights compared to the complete treatment (Fig 5). The N-deficient WT/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e plants showed higher N-use efficiency than that of WT/WT and \u003cem\u003ephyB1\u003c/em\u003e/WT; however, genotype had no significant effect on P-use efficiency (Figs. 4a and 4b). Thus, N-deficient WT/\u003cem\u003ephyB1\u003c/em\u003e plants produced more roots and whole plant dry weights than WT/WT plants. However, regardless of the nutritional treatment, WT/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e did not differ from WT/WT in terms of shoot dry mass, although, WT/\u003cem\u003ephyB1\u003c/em\u003e produced more dry weight in this part of the plant compared to \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT (Fig. 5).\u003c/p\u003e\n\u003cp\u003e3.3 Phosphorus deficiency (-P)\u003c/p\u003e\n\u003cp\u003eThe comparison of means showed that there was no significant effect (p\u0026le; 0.05) of genotype and nutrient solution interaction on P accumulation, MDA concentration, photosynthesis, stomatal conductance, P-use efficiency, or shoot, root, and whole plant dry weight (Tables S1-4).\u003c/p\u003e\n\u003cp\u003ePhosphorus deficiency decreased the accumulation of N and P in the shoots of the plants; however, the plants of WT/\u003cem\u003ephyB1\u003c/em\u003e that were deficient in P accumulated less N than WT/WT and the other genotypes, and together with \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e did not differ from the control genotype in relation to P accumulation, whereas lower accumulation of this element was observed in \u003cem\u003ephyB1\u003c/em\u003e/WT, irrespective of nutritional treatment (Figs. 1a and 1b).\u003c/p\u003e\n\u003cp\u003eRegardless of the nutritional treatment, WT/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e had increased MDA concentration, whereas WT/WT and \u003cem\u003ephyB1\u003c/em\u003e/WT had lower concentrations of this molecule (Fig. 2a). Phosphorus-deficient plants had decreased concentrations of total Chl and carotenoids in relation to the complete treatment (Figs. 2b and 2c). However, WT/\u003cem\u003ephyB1\u0026nbsp;\u003c/em\u003eand \u003cem\u003ephyB1\u003c/em\u003e/WT showed higher concentrations of pigments than WT/WT. Phosphorus deficiency also decreased PSII quantum efficiency (Fig. 2d), photosynthesis, stomatal conductance, and plant transpiration compared to the complete treatment (Figs. 3a-c). However, the \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT combinations showed lower quantum efficiency of PSII and stomatal conductance than WT/WT; however, photosynthesis and transpiration were similar between genotypes. Phosphorus-deficient WT/WT plants exhibited increased WUE, superior to the other genotypes, which showed lower efficiency compared to the complete treatment plants (Fig. 3d).\u003c/p\u003e\n\u003cp\u003eP deficiency decreased N- and P-use efficiency (Figs. 4a and 4b), which resulted in lower shoot, root, and whole-plant dry weight production (Fig. 5). However, WT/\u003cem\u003ephyB1\u003c/em\u003e had a more efficient N use than that of WT/WT, whereas P use efficiency was similar between genotypes. Regardless of nutritional condition, \u003cem\u003ephyB1\u003c/em\u003e/WT produced less shoot and whole plant dry weight than WT/WT and the other genotypes (Figs. 5a and 5c). The root dry weight was higher in WT/WT and lower in \u003cem\u003ephyB1\u003c/em\u003e/WT (Fig. 5b).\u003c/p\u003e\n\u003cp\u003e3.4 Potassium deficiency (-K)\u003c/p\u003e\n\u003cp\u003eThe comparison of means showed that there was no significant effect (p\u0026le; 0.05) of the genotype on K accumulation and genotype and nutrient solution interaction on P and K accumulation, stomatal conductance, transpiration, N- and P-use efficiency, or shoot, root, and whole-plant dry weight (Tables S1-4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK deficiency decreased K accumulation in tomato plants compared to the complete treatment (Fig. 1c). However, K-deficient WT/WT plants accumulated more N than the other grafting combinations, and together with WT/\u003cem\u003ephyB1\u003c/em\u003e, they accumulated more P than the \u003cem\u003ephyB1\u003c/em\u003e grafts (\u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT), regardless of nutritional status (Figs. 1a and 1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK-deficient \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT plants had decreased and increased MDA concentrations in the shoot, respectively, in relation to the complete treatment; however, no differences were observed between the genotypes (Fig. 2a). Furthermore, K deficiency also decreased the concentration of total Chl, carotenoids, and the quantum efficiency of PSII (Figs. 2b-d); however, the genotypes did not differ from each other in these factors. Photosynthesis decreased in K-deficient plants, and this reduction was more pronounced in \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e, \u003cem\u003ephyB1\u003c/em\u003e/WT, and WT/\u003cem\u003ephyB1\u003c/em\u003e than in WT/WT (Fig. 3a). K deficiency only reduced the stomatal conductance of \u003cem\u003ephyB1\u003c/em\u003e/WT plants (Fig. 3b), which together with \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e presented the lowest values in relation to WT/WT, irrespective of the nutritional condition. Regardless of genotype, K deficiency decreased transpiration (Fig. 3c) and WUE of plants (Fig. 3d); however, it was observed that \u003cem\u003ephyB1\u003c/em\u003e/WT and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e transpired less than WT/WT and WT/\u003cem\u003ephyB1.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenotype had no effect on K-use efficiency. In contrast, K deficiency increased the use efficiency of this element and decreased N- and P-use efficiency compared to the plants in the complete treatment (Fig. 4). The lower efficiency in N, P, and K utilization resulted in decreased production of shoot, root, and whole plant dry weight (Fig. 5). Regardless of the nutritional condition, \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT produced less dry weight in the shoot (Fig. 5a), and together with WT/\u003cem\u003ephyB1\u003c/em\u003e, produced less dry weight in the root and in the whole plant compared to WT/WT (Figs. 5c and 5d).\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThere is growing evidence that phytochrome B can modulate responses to different types of stress. Recently, Soares et al. (2021) demonstrated that the loss of tomato phyB1 function attenuated the damage caused by P deficiency, providing new perspectives for a better understanding of photoreceptor participation in nutritional responses. Since \u003cem\u003ePHYB\u003c/em\u003e is expressed in shoots and roots, we studied the communication between these plant parts in the nutritional, physiological, and growth response signaling in tomato plants under nutritional deficiency. We used the phyB1-deficient tomato mutant (\u003cem\u003ephyB1\u003c/em\u003e) and its control genotype (WT) in grafting combinations under N, P, and K sufficiency and deficiency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe information obtained could be useful in understanding the benefits of phyB1 and identifying which nutritional deficiencies are expected to result in greater gains in nutritional and physiological processes and plant growth. This could contribute to strengthening the sustainability of various crops, as N, P, and K deficiencies are common worldwide, particularly in underdeveloped countries.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4.1 Complete nutrient solution\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnder nutrient-sufficient conditions, plants obtained from phyB1-deficient grafts (\u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/MM) absorbed less N than that of WT/WT plants (Fig. 1a). Phytochrome is known to be involved in the transcription of nitrate transporter genes, and recent reports indicate that the loss of tomato phyB1 function decreased N accumulation in the shoot (Sakuraba and Yanagisawa, 2018; Soares et al., 2021). These results, together with those obtained in this study, reinforce the participation of phyB1 in N uptake, a process that appears to be coordinated by the shoot phytochrome. Furthermore, we observed that only the \u003cem\u003ephyB1\u003c/em\u003e/WT plants showed decreased P uptake (Fig. 1b), indicating that shoot phyB1 also upregulated the uptake of this element. This result may be related to the fact that this photoreceptor is involved in the expression of genes encoding high-affinity P transporters (Sakuraba et al. 2018). In contrast, all genotypes absorbed similar amounts of K compared to WT/WT (Fig. 1c), suggesting that neither shoot nor root phytochromes are directly involved in the absorption process of this element under sufficient conditions.\u003c/p\u003e\n\u003cp\u003eAll grafting combinations showed membrane lipid peroxidation that was similar to that of the control genotype (WT/WT); however, the highest production of MDA was observed in \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u0026nbsp;\u003c/em\u003e(Fig. 2a), which can be explained by the role of phytochromes in suppressing lipid peroxidation of thylakoid membranes (Joshi et al. 1991). Thus, \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and WT/\u003cem\u003ephyB1\u003c/em\u003e plants had decreased concentrations of chlorophylls, and, together with \u003cem\u003ephyB1\u003c/em\u003e/MM, had less carotenoid production than that of the WT/WT. In a study on rice, a lower concentration of chlorophylls was observed in the \u003cem\u003ephyB\u003c/em\u003e mutant, suggesting that this photoreceptor plays an important role in the synthesis of chlorophylls (Zhao et al. 2013). In our study, chlorophyll was likely controlled by the phyB1 of the root because the plants obtained from phyB1-deficient rootstocks had a lower concentration of this pigment, whereas carotenoids were under the control of both shoot and root phytochromes. Although phyB1 deficiency induced alterations in nutrient uptake and photosynthetic pigment production, the results suggest that shoot and root phyB1 do not control PSII quantum efficiency (Fig. 2d), photosynthesis (Fig. 3a), or WUE (Fig. 3d) in tomato. However, the stomatal conductance of \u003cem\u003ephyB1\u003c/em\u003e/WT and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and the transpiration of \u003cem\u003ephyB1\u003c/em\u003e/WT were lower than those of WT/WT. According to Mereb et al. (Mereb et al. 2020), the lower stomatal conductance and transpiration of the phytochrome mutant are associated with the lower density and stomatal index of this genotype, especially in \u003cem\u003ephyB1\u003c/em\u003e. Similarly, our results suggest the involvement of tomato shoot phyB1 in the positive regulation of these processes under nutritional sufficiency.\u003c/p\u003e\n\u003cp\u003eThe deficiency of both shoot and root phytochromes amplified the N-use efficiency of tomato (Fig. 4a); however, it did not change the P and K use efficiencies (Fig. 4b and 4c). The WT/WT plants produced a greater amount of dry weight in the root and whole plant than that of the other genotypes (Figs 5b and 5c). This may have occurred because the control genotype accumulated higher amounts of N and P, which resulted in higher concentrations of chlorophyll and carotenoids and higher stomatal conductance and transpiration. The lowest dry weight production in all parts of \u003cem\u003ephyB1\u003c/em\u003e/WT plants (Fig. 5) may have been a consequence of shoot phytochrome B1 deficiency, as our results suggest that this photoreceptor is involved in signaling for N and P uptake in the root, in addition to regulating carbon assimilation.\u003c/p\u003e\n\u003cp\u003e4.2 Nitrogen deficiency (-N)\u003c/p\u003e\n\u003cp\u003eCarbon is the most demanded element by cultivated plants, followed by N, which is a structural component of amino acids, proteins, coenzymes, and nitrogenous bases (Marschner 2012; Prado 2021). Thus, the reduced accumulation of N caused by N deficiency (Fig. 1a) created an imbalance in P absorption compared to that of the complete treatment (Fig. 1b). However, the decrease in N uptake occurred similarly between the genotypes, suggesting that neither phytochrome regulates this process under N deficiency. Root phyB1 negatively regulated P uptake, as the accumulation of this element in WT/\u003cem\u003ephyB1\u003c/em\u003e was greater than that in WT/WT, regardless of nutritional status.\u003c/p\u003e\n\u003cp\u003eChlorophyll concentration is directly related to the N level in the plant, as it is a constituent of this molecule (Mu and Chen 2021), which explains the decrease in photosynthetic pigments in plants under N deficiency (Fig. 2b and 2c). This deficiency also reduced the quantum efficiency of PSII by inhibiting the biosynthesis of photosynthetic pigments in all grafting combinations, because chlorophylls are responsible for the absorption and transport of energy to PSII and PSI (Mu et al. 2017; Zhang et al. 2019). In addition, WT/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e plants showed higher quantum efficiency of PSII than WT/WT (Fig. 2d), which can be attributed to the use of \u003cem\u003ephyB1\u003c/em\u003e as a rootstock, which amplified this response independently of N.\u003c/p\u003e\n\u003cp\u003eWe also observed a reduction in photosynthesis for all grafting combinations subjected to N deficiency, which was associated with a decrease in carbon assimilation and transpiration in plants (Figs. 3a-c). In addition, N deficiency decreased the WUE of all grafting combinations (Fig. 3d). Despite these results, no participation of shoot or root phytochromes was observed in the control of stomatal conductance under N deficiency (Fig. 3b). The \u003cem\u003ephyB1\u003c/em\u003e/WT and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e plants had greater transpiration than that of the WT/WT, which decreased the WUE of these genotypes. These results suggest that shoot phyB1 is a positive regulator of transpiration in N-deficient tomato, and its deficiency results in higher water consumption per fixed carbon, as observed in the WUE results.\u003c/p\u003e\n\u003cp\u003eIt is estimated that approximately 5% of a plant\u0026apos;s total dry weight is composed of N (Marschner, 2012), and this element is considered most related to dry weight production (Reis et al., 2009). Thus, all genotypes cultivated under N deficiency had reduced use efficiency of this nutrient as well as P-use efficiency (Figs. 4a and 4b), which, associated with the other nutritional and physiological changes, caused a decrease in the production of shoot, root, and whole plant dry weight (Fig 5). Regardless of N, WT/\u003cem\u003ephyB1\u003c/em\u003e absorbed and accumulated more P in the shoot, which favored the greatest shoot dry weight. In addition, loss of root phyB1 function favored root and whole-plant dry weight production of WT/\u003cem\u003ephyB1\u003c/em\u003e under N deficiency. These responses suggest that root phyB1 is a negative regulator of root and plant dry weight production in an N-dependent manner, which is associated with the higher PSII quantum, water-use, and N-use efficiencies of this genotype.\u003c/p\u003e\n\u003cp\u003eOur results also suggest that the use of WT as a graft and of the defective mutant in phyB1 as a rootstock may be an alternative to attenuate the damage caused by N deficiency in tomato.\u003c/p\u003e\n\u003cp\u003e4.3 Phosphorus deficiency (-P)\u003c/p\u003e\n\u003cp\u003eThe omission of P decreased the absorption of this element (Fig. 1b) and caused an imbalance in the absorption of N in all genotypes (Fig. 1a), due to P being a constituent of the ATP molecule that is used in the process of nutrient absorption (Prado 2021). WT/\u003cem\u003ephyB1\u003c/em\u003e plants under P deficiency showed reduced N uptake compared to WT/WT. This may be due to the photoreceptor\u0026apos;s involvement in regulating the activity of key genes encoding nitrate transporters (Sakuraba and Yanagisawa, 2018), a process that, in this case, appears to be under the control of root phyB1 and P-dependent. Furthermore, regardless of nutritional status, \u003cem\u003ephyB1\u003c/em\u003e/WT plants accumulated less P compared to WT/WT. This can be attributed to shoot phyB1 deficiency, which appears to have a greater contribution to signaling responses for nutrient absorption than root phyB1, thereby positively regulating this process.\u003c/p\u003e\n\u003cp\u003eP deficiency increased lipid peroxidation of plant membranes, regardless of genotype (Fig. 2a); however, the highest production of MDA occurred in plants deficient in root phyB1 (WT/\u003cem\u003ephyB1\u003c/em\u003e). Recently, it was demonstrated that phyB1 is related to the maintenance of membrane integrity in tomato (Soares et al., 2021). Despite MDA quantification in the shoot, our results indicate that its production is under the control of phytochrome in the root. Previous studies have reported that the concentrations of chlorophyll and carotenoids are lower in P-deficient plants (de Souza Os\u0026oacute;rio et al. 2020; Patel et al. 2020). Our results agree with these findings; however, this reduction was less pronounced in WT/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT, and more evident in WT/WT (Figs. 2b and 2c), indicating that in P-deficient plants, the lack of phyB1 from both the root and shoot provides an increase in the biosynthesis of chlorophylls and carotenoids. As a consequence of the loss of photosynthetic pigments, P deficiency also decreases the quantum efficiency of PSII, which may have been a strategy to reduce photoinhibition damage (Hern\u0026aacute;ndez and Munn\u0026eacute;-Bosch 2015). However, we verified that \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/MM showed lower efficiency in energy transfer to PSII (Fv/Fm) than that of WT/WT, indicating that shoot phytochrome B1 is a positive regulator of this process (Fig. 2d).\u003c/p\u003e\n\u003cp\u003eIn this study, P deficiency negatively affected gas exchange reactions (Fig. 3). A decrease in photosynthesis occurred due to the loss of photosynthetic pigments (Fig. 3a), in addition to lower carbon assimilation and transpiration (Figs. 3b and 3c). However, in P-deficient plants, transpiration is not regulated by the shoot and root phyB1. Except for WT/WT, all other grafting combinations had decreased WUE compared to the complete treatment (Fig. 3d), which may be associated with the loss of phyB1 function, indicating the participation of this photoreceptor in water relations dependent on P.\u003c/p\u003e\n\u003cp\u003eWe found that regardless of genotype, phosphorus deficiency decreased N- and P-use efficiencies and resulted in lower nutrient conversion to dry weight (Fig. 5). These results were expected because, together with N and K, P is one of the most responsive nutrients for plants, and therefore, its low availability is a limiting factor for plant growth and development (Hern\u0026aacute;ndez and Munn\u0026eacute;-Bosch 2015). The lack of root phyB1 in P-deficient WT/\u003cem\u003ephyB1\u003c/em\u003e plants increased P-use efficiency compared to that of WT/WT (Fig. 4a). Regardless of the nutritional treatment, \u003cem\u003ephyB1\u003c/em\u003e/WT plants showed less dry weight production in the shoot and whole plant than that of WT/WT. These results can be explained by the lower accumulation of P and lower stomatal conductance of this genotype in relation to WT/WT, showing that phyB1 deficiency in the shoot negatively affected nutritional and physiological processes, and consequently, the production of tomato dry weight. In contrast, the root dry weight results indicated that both shoot and root phytochromes participated in the dry weight production process in this part of the plant.\u003c/p\u003e\n\u003cp\u003e4.4 Potassium deficiency (-K)\u003c/p\u003e\n\u003cp\u003eWe demonstrated that potassium deficiency decreased the uptake of this element, as well as the uptake of N and P from plants, regardless of the genotype (Fig. 1). This may be because K acts in the activation of key enzymes for nutrient absorption, in addition to stimulating nitrate absorption, as it serves as an accompanying cation (Blevins et al. 1978). However, K-deficient genotypes accumulated less N and K than WT/WT, suggesting that both phyB1s are positive regulators of the absorption of these nutrients in a K-dependent manner, whereas shoot phyB1 is a positive regulator of P uptake that is independent of K.\u003c/p\u003e\n\u003cp\u003eK deficiency can result in lipid peroxidation of membranes, and this response is modulated by shoot phyB1 in tomato, since \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e and \u003cem\u003ephyB1\u003c/em\u003e/WT increased the MDA concentration (Fig. 2a). Furthermore, the increase in MDA concentration may have inhibited biosynthesis and accelerated the biodegradation of pigments (Figs. 2b and 2c) and increased oxidative damage, as observed with the lower quantum efficiency of PSII results (Fig. 2d). However, these physiological changes occurred independently of the phytochromes. Our results showed that K deficiency caused a considerable reduction in the photosynthetic parameters (photosynthesis, stomatal conductance, and transpiration) compared to the complete treatment (Figs. 3a-c). The low availability of K in plants decreases its concentration in guard cells and consequently compromises stomatal regulation (Hasanuzzaman et al. 2018). Thus, the decrease in stomatal conductance minimized water loss through transpiration but reduced carbon assimilation. Previous studies have shown that photosynthetic performance can be increased in transgenic rice plants that overproduce phytochrome B (Kreslavski et al. 2018). Therefore, grafting combinations with the loss of phyB1 function decreased photosynthesis in relation to the WT/WT (Fig. 3a). However, this reduction was more pronounced in plants deficient in shoot phyB1, indicating that this photoreceptor is a positive regulator of photosynthesis in a K-dependent manner. The deficiency of shoot phyB1 decreased stomatal conductance and transpiration in plants, regardless of nutritional treatment (Figs. 3b and 3c). This suggests that phyB1 is a positive regulator of these processes. It was previously reported that adequate K availability allows for more efficient water use (Hasanuzzaman et al. 2018), which justifies the lower WUE results in plants deficient in this element compared to plants that received the complete treatment.\u003c/p\u003e\n\u003cp\u003eK-deficient grafted plants showed decreased N- and P-use efficiencies compared to the complete treatment (Figs. 4a and 4b). However, K-use efficiency increased with the low availability of this element (Fig. 4c), which may have been an adaptive strategy for plants to maintain their metabolism. In addition to activating more than 50 enzymes, K plays a fundamental role in the transport of solutes from the shoot to the root via the phloem (Hafsi et al. 2014). Furthermore, this element regulates the biosynthesis, conversion, and allocation of metabolites (Hasanuzzaman et al. 2018). K deficiency negatively affected shoot, root, and whole-plant dry weight production (Fig. 5). Regardless of the nutritional condition, the deficiency of shoot phyB1 (\u003cem\u003ephyB1\u003c/em\u003e/WT and \u003cem\u003ephyB1\u003c/em\u003e/\u003cem\u003ephyB1\u003c/em\u003e) negatively affected the dry weight of this part of the plant. In addition, the loss of function of both shoot and root phytochromes decreased the dry weight of roots and whole plants. These results show that both shoot and root phyB1 are involved in some metabolic steps in the plant, whether nutritional or physiological, and consequently participate in the signaling of responses to dry weight production in tomato.\u003c/p\u003e\n\u003cp\u003eIn summary, based on the responses observed in the grafting combinations between the mutant \u003cem\u003ephyB1\u003c/em\u003e and WT, we can conclude that phytochrome B1 is involved in shoot-root communication for the control of nutritional, physiological, and growth responses in tomato. Furthermore, the action of both shoot and root phytochromes on these responses may occur independently or may depend on N, P, and K. The use of a tomato mutant deficient in phyB1 could be a tool to better understand the control of phytochromes in plant nutrition, physiology, and growth. Our results also provide new perspectives for future studies to explore the manipulation of the \u003cem\u003ePHYB1\u003c/em\u003e gene to improve crop productivity and mitigate damage caused by nutritional deficiency.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe support of the S\u0026atilde;o Paulo State University (UNESP) is gratefully acknowledged. This research project was funded by Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil, Code 001.\u003c/p\u003e\n\u003cp\u003eAuthor Contribution\u003c/p\u003e\n\u003cp\u003eM.B.S, R.M.P and R.F.C conceived the idea. M.B.S and L.T.S.C carried out the experiments. M.B.S and E.G.R performed the chemical analysis. M.B.S. and R.O performed the physiological analysis. J.L.F.S analyzed the data. M.B.S, R.F.C and D.O.V wrote the manuscript. R.M.P and R.F.C revised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research project was funded by Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil, Code 001.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eData sharing is not applicable to this article as all newly obtained data is already contained within this article.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBataglia O, Furlani A, Teixeira J, et al (1983) M\u0026eacute;todos de an\u0026aacute;lise qu\u0026iacute;mica de plantas. Bol T\u0026eacute;cnico, Inst Agron\u0026ocirc;mico Campinas\u003c/li\u003e\n \u003cli\u003eBlevins DG, Barnett NM, Frost WB (1978) Role of Potassium and Malate in Nitrate Uptake and Translocation by Wheat Seedlings. 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Biochim Biophys Acta - Bioenerg 1859:400\u0026ndash;408. https://doi.org/10.1016/j.bbabio.2018.03.003\u003c/li\u003e\n \u003cli\u003eLichtenthaler HK (1987) Chlorophylls and Carotenoids: Pigments of Photosynthetic Biomembranes. Methods Enzymol 148:350\u0026ndash;382. https://doi.org/10.1016/0076-6879(87)48036-1\u003c/li\u003e\n \u003cli\u003eMarschner H (2012) Mineral Nutrition of Higher Plants, 3rd editio. Academic Press, London\u003c/li\u003e\n \u003cli\u003eMereb EL, Alves FRR, Rezende MH, et al (2020) Morphophysiological responses of tomato phytochrome mutants under sun and shade conditions. Rev Bras Bot 43:45\u0026ndash;54. https://doi.org/10.1007/s40415-020-00584-w\u003c/li\u003e\n \u003cli\u003eMu X, Chen Q, Chen F, et al (2017) A RNA-seq analysis of the response of photosynthetic system to low nitrogen supply in maize leaf. 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Physiol Plant 1\u0026ndash;10. https://doi.org/10.1111/ppl.13574\u003c/li\u003e\n \u003cli\u003evan Tuinen, Ageeth, Kerckhoffs, Huub J., Nagatani, Akira, Kendrick, Richard E., and Koornneef M (1995) A Temporarily Red Light-lnsensitive Mutant of Tomato. 939\u0026ndash;947\u003c/li\u003e\n \u003cli\u003eZhang Y, Liang Y, Zhao X, et al (2019) Silicon compensates phosphorus deficit-induced growth inhibition by improving photosynthetic capacity, antioxidant potential, and nutrient homeostasis in tomato. Agronomy 9:1\u0026ndash;16. https://doi.org/10.3390/agronomy9110733\u003c/li\u003e\n \u003cli\u003eZhao J, Zhou J jun, Wang Y ying, et al (2013) Positive Regulation of Phytochrome B on Chlorophyll Biosynthesis and Chloroplast Development in Rice. Rice Sci 20:243\u0026ndash;248. https://doi.org/10.1016/S1672-6308(13)60133-X\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Solanum lycopersicum L., red light, phyB1, mutant, nutritional deficiency","lastPublishedDoi":"10.21203/rs.3.rs-6938144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6938144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhytochromes are involved in the expression of nutrient transporter genes and participate in signaling responses in plants under nutritional deficiency. This study investigated the reciprocal interaction between phytochrome B1 (phyB1) and N, P, and K deficiency responses, specifically focusing on shoot-root communication. For this purpose, we used grafting combinations of the control genotype (WT) with the tomato phyB1-deficient mutant (\u003cem\u003ephyB1\u003c/em\u003e) under nutritional sufficiency and individual deficiencies of N, P, and K. In nutrient-sufficient conditions, shoot \u003cem\u003ephyB1\u003c/em\u003e stimulates the uptake of N and P in the roots in addition to increase stomatal conductance, transpiration, and dry weight production, whereas root \u003cem\u003ephyB1\u003c/em\u003e regulated the production of chlorophyll in the shoot. With N deficiency, grafted plants with loss of shoot \u003cem\u003ephyB1\u003c/em\u003e function showed increased transpiration and less efficient water use. However, the WT/\u003cem\u003ephyB1\u003c/em\u003e combination attenuated the damage caused by N deficiency by increasing the dry weight of the entire plant. Under P deficiency, the absence of root \u003cem\u003ephyB1\u003c/em\u003e decreased N uptake and increased malondialdehyde (MDA) production. However, the deficiency of phyB1 impaired the water-use efficiency of P-deficient plants. In K-deficient tomato, N and K uptake was under the control of both shoot and root phytochromes and shoot phyB1 regulated MDA production and increased photosynthesis. We conclude that phyB1 is involved in shoot-root communication for the control of nutritional, physiological, and growth responses in tomato, raising new roles of this photoreceptor and perspectives on the plant nutrition studies.\u003c/p\u003e","manuscriptTitle":"Tomato phytochrome B1 modulates N, P, and K deficiency response by root-to-shoot communication","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-03 13:19:27","doi":"10.21203/rs.3.rs-6938144/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-22T22:57:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-02T06:12:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-23T03:53:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Physiologiae Plantarum","date":"2025-06-20T06:47:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b793fd6-4c23-4d12-b200-6d06a5979ab0","owner":[],"postedDate":"July 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:07:00+00:00","versionOfRecord":{"articleIdentity":"rs-6938144","link":"https://doi.org/10.1007/s11738-026-03893-x","journal":{"identity":"acta-physiologiae-plantarum","isVorOnly":false,"title":"Acta Physiologiae Plantarum"},"publishedOn":"2026-02-27 15:58:49","publishedOnDateReadable":"February 27th, 2026"},"versionCreatedAt":"2025-07-03 13:19:27","video":"","vorDoi":"10.1007/s11738-026-03893-x","vorDoiUrl":"https://doi.org/10.1007/s11738-026-03893-x","workflowStages":[]},"version":"v1","identity":"rs-6938144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6938144","identity":"rs-6938144","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}