Citrus sinensis seedlings were irrigated with nutrient solution at a concentration of 0 (Mg-deficiency) or 2 (Mg-sufficiency) mM Mg (NO₃)₂ for 16 weeks. Mg-deficiency-induced interveinal chlorosis, vein enlargement and corkiness, and alterations of gas exchange, pigments, chlorophyll a fluorescence (OJIP) transients and related parameters were observed in middle and lower leaves, especially in the latter, but not in upper leaves. Mg-deficiency might impair the whole photosynthetic electron transport, including structural damage to thylakoids, ungrouping of photosystem II (PSII), inactivation of oxygen-evolving complex (OEC) and reaction centers (RCs), increased reduction of primary quinone electron acceptor (Q_A) and plastoquinone pool at PSII acceptor side and oxidation of PSI end-electron acceptors, thus lowering energy transfer and absorption efficiency and the transfer of electrons to the dark reactions, hence, the rate of CO₂ assimilation in Mg-deficiency middle and lower leaves. Although potassium, Mg, manganese and zinc concentration in blades displayed a significant and positive relationship with the corresponding element concentration in veins, respectively, great differences existed in Mg-deficiency-induced alterations of nutrient concentrations between leaf blades and veins. For example, Mg-deficiency increased boron level in the blades of upper leaves, decreased boron level in the blades of lower leaves, but did not affect boron level in the blades of middle leaves and veins of upper, middle and lower leaves. To conclude, Mg-deficiency-induced interveinal chlorosis, vein enlargement, and corkiness, and alterations to photosynthesis and related parameters increased with increasing leaf age. Mg-deficiency-induced enlargement and corkiness of veins were not caused by Mg-deficiency-induced boron-starvation.

Limited data are available on the physiological responses of Citrus to nitrogen (N) deficiency. ‘Xuegan’ (Citrus sinensis (L.) Osbeck) and ‘Shantian pummelo’ (Citrus grandis (L.) Osbeck) seedlings were fertilized with nutrient solution at a N concentration of 0, 5, 10, 15 or 20 mM for 10 weeks. N deficiency decreased N uptake and N concentration in leaves, stems and roots and disturbed nutrient balance and homeostasis in plants, thus inhibiting plant growth, as well as reducing photosynthetic pigment levels and impairing thylakoid structure and photosynthetic electron transport chain (PETC) in leaves, hence lowering CO₂ assimilation. The imbalance of nutrients intensified N deficiency’s adverse impacts on biomass, PETC, CO₂ assimilation and biosynthesis of photosynthetic pigments. Citrus displayed adaptive responses to N deficiency, including (a) elevating the distributions of N and other elements in roots, as well as root dry weight (DW)/shoot DW ratio and root-surface-per-unit volume and (b) improving photosynthetic N use efficiency (PNUE). In general, N deficiency had less impact on biomass and photosynthetic pigment levels in C. grandis than in C. sinensis seedlings, demonstrating that the tolerance of C. grandis seedlings to N deficiency was slightly higher than that of C. sinensis seedlings, which might be related to the higher PNUE of the former.

Limited data are available on the sulfur (S)-mediated-alleviation of aluminum (Al)-toxicity in higher plants. Citrus grandis seedlings were irrigated for 18 weeks with 0.5 mM MgSO₄ or 0.5 mM MgSO₄ + 0.5 mM Na₂SO₄, and 0 (−Al) or 1 mM AlCl₃·6H₂O (+Al, Al-toxicity). Under Al-toxicity, S decreased the level of Al in leaves; increased the relative water content (RWC) of roots and leaves, the contents of phosphorus (P), calcium (Ca) and magnesium (Mg) per plant, the dry weights (DW) of roots and shoots, the ratios of root DW/shoot DW, and the Al-induced secretion of citrate from root; and alleviated the Al-induced inhibition of photosynthesis via mitigating the Al-induced decrease of electron transport capacity resulting from the impaired photosynthetic electron transport chain. In addition to decreasing the Al-stimulated H₂O₂ production, the S-induced upregulation of both S metabolism-related enzymes and antioxidant enzymes also contributed to the S-mediated-alleviation of oxidative damage in Al-treated roots and leaves. Decreased transport of Al from roots to shoots and relatively little accumulation of Al in leaves, and increased leaf and root RWC and P, Ca, and Mg contents per plant might also play a role in the S-mediated-alleviation of Al-toxicity.

Citrus sinensis seedlings were supplied with a nutrient solution containing 15 (control) or 0 (nitrogen (N) deficiency) mM N for 10 weeks. Extensive metabolic and gene reprogramming occurred in 0 mM N-treated roots (RN0) to cope with N deficiency, including: (a) enhancing the ability to keep phosphate homeostasis by elevating the abundances of metabolites containing phosphorus and the compartmentation of phosphate in plastids, and/or downregulating low-phosphate-inducible genes; (b) improving the ability to keep N homeostasis by lowering the levels of metabolites containing N but not phosphorus, upregulating N compound degradation, the root/shoot ratio, and the expression of genes involved in N uptake, and resulting in transitions from N-rich alkaloids to carbon (C)-rich phenylpropanoids and phenolic compounds (excluding indole alkaloids) and from N-rich amino acids to C-rich carbohydrates and organic acids; (c) upregulating the ability to maintain energy homeostasis by increasing energy production (tricarboxylic acid cycle, glycolysis/gluconeogenesis, oxidative phosphorylation, and ATP biosynthetic process) and decreasing energy utilization for amino acid and protein biosynthesis and new root building; (d) elevating the transmembrane transport of metabolites, thus enhancing the remobilization and recycling of useful compounds; and (e) activating protein processing in the endoplasmic reticulum. RN0 had a higher ability to detoxify reactive oxygen species and aldehydes, thus protecting RN0 against oxidative injury and delaying root senescence.

Bordeaux mixture (Bm) is a copper (Cu)-based pesticide that has been widely used for controlling citrus scab and citrus canker. However, frequent spraying of Bm is toxic to citrus. To our knowledge, few studies are available that discuss how the photosynthetic characteristics and chloroplast ultrastructure of citrus leaves are affected by Cu toxicity induced by excessive Bm. In the study, two-year-old seedlings of Citrus grandis (C. grandis) and Citrus sinensis (C. sinensis), which were precultured in pots, were foliar-sprayed with deionized water (as control) or Bm diluted 500-fold at intervals of 7 days for 6 times (4 times as recommended by the manufacturer) to investigate the leaf Cu absorption, photosynthesis, chloroplast ultrastructure and antioxidant enzymatic activities. Bm foliar-sprayed 6 times on citrus seedlings increased the leaf Cu content, decreased the photosynthetic pigments content and destroyed the chloroplast ultrastructure, which induced leaf chlorosis and photosynthetic inhibition. A lower Cu absorption, a higher light photon-electron transfer efficiency, a relative integrity of chloroplast ultrastructure and a promoted antioxidant protection contributed to a higher photosynthetic activity of C. grandis than C. sinensis under excessive spraying of Bm. The present study provides crucial references for screening and selecting citrus species with a higher tolerance to Cu toxicity induced by excessive Bm.

Manganese (Mn) excess and low pH often coexist in some citrus orchard soils. Little information is known about the underlying mechanism by which raising pH reduces Mn toxicity in citrus plants. ‘Sour pummelo’ (Citrus grandis (L.) Osbeck) seedlings were treated with 2 (Mn2) or 500 (Mn500) μM Mn at a pH of 3 (P3) or 5 (P5) for 25 weeks. Raising pH mitigated Mn500-induced increases in Mn, iron, copper, and zinc concentrations in roots, stems, and leaves, as well as nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, copper, iron, and zinc distributions in roots, but it mitigated Mn500-induced decreases in nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, and boron concentrations in roots, stems, and leaves, as well as nutrient imbalance. Raising pH mitigated Mn500-induced necrotic spots on old leaves, yellowing of young leaves, decreases in seedling growth, leaf chlorophyll concentration, and CO₂ assimilation (A_CO2), increase in root dry weight (DW)/shoot DW, and alterations of leaf chlorophyll a fluorescence (OJIP) transients and related indexes. Further analysis indicated that raising pH ameliorated Mn500-induced impairment of nutrient homeostasis, leaf thylakoid structure by iron deficiency and competition of Mn with magnesium, and photosynthetic electron transport chain (PETC), thereby reducing Mn500-induced declines in A_CO2 and subsequent seedling growth. These results validated the hypothesis that raising pH reduced Mn toxicity in ‘Sour pummelo’ seedlings by (a) reducing Mn uptake, (b) efficient maintenance of nutrient homeostasis under Mn stress, (c) reducing Mn excess-induced impairment of thylakoid structure and PEPC and inhibition of chlorophyll biosynthesis, and (d) increasing A_CO2 and subsequent seedling growth under Mn excess.