Physiology And Biochemistry, Gene And MicroRNA Expression Of Citrus Sinensis Seedlings In Response To Boron Deficiency

Citrus belong to subtropical fruit trees. Boron （B）-deficiency is frequently observed in citrus orchards due to leaching of B from soils, B soil fixation, uncoordinated application of N, P and Ca fertilizatizers, and unfavorable climate conditions. In this study, seedlings of’Xuegan’（Citrus sinensis） grown in pots containing fine river sand were irrigated for 15 weeks with nutrient solution at a B concentration of 0 （B-deficiency） or 10 （controls） μM every other day. Thereafter, the effects of B-deficiency on growth, physiology and biochemistry in roots and leaves, expression of genes and miRNAs expression in roots and leaves were investigated. The objectives were to understand the tolerance mechanisms of citrus plants to B-deficiency at biochemical, physiological and molecular levels and to provide scientific basis for citrus cultivation of high yield and good quality of citrus cultivation.1 Effects of B deficiency on growth of C. sinensis seedlingsUnder 10 μM B treatment, C. sinensis seedlings displayed normal growth without any visible B-deficient symptoms and leaf B concentration in leaves was within the sufficiency range of 30-100 μg g g-1 DW for citrus. However,0 μM B treatment significantly decreased leaf, stem and root DW, and leaf B concentration. The B concentration in 0μM B-treated leaves was much lower than the sufficiency range. In addition, a typical B-deficient symptom, corky split veins, was observed in 0 μM B-treated leaves. Based on these results, plants treated with 0 μM B are considered B-deficient, and those treated with10 μM B are considered B-sufficient （controls）.2 Effects of B-deficiency on physiology and biochemistry in C. sinensis roots and leavesFifteen weeks after B treatments, gas exchange, concentrations of nonstructural carbohydrates, organic acids, total phenolics, amino acids, and total soluble proteins, and activities of key enzymes involved in organic acid and amino acid metabolism in leaves and roots were investigated. B-deficient leaves displayed excessive accumulation of nonstructural carbohydrates and much lower CO2 assimilation, demonstrating feedback inhibition of photosynthesis. Dark respiration, concentrations of most organic acids [malate, citrate, oxaloacetate （OAA）, pyruvate and phosphoenolpyruvate （PEP）] and activities of enzymes [PEP carboxylase （PEPC）, NAD-malate dehydrogenase （NAD-MDH）, NAD-malic enzyme （NAD-ME）, NADP-ME, pyruvate kinase （PK）, PEP phosphatase （PEPP）, citrate synthase （CS）, aconitase （ACO）, NADP-isocitrate dehydrogenase （NADP-IDH） and hexokinase （HK）] involved in glycolysis, the tricarboxylic acid （TCA） cycle and the anapleurotic reaction were higher in B-deficient leaves than in controls. Also, total free amino acid （TFAA） concentration and related enzyme [NADH-dependent glutamate 2-oxoglutarate aminotransferase （NADH-GOGAT） and glutamate oxaloacetate transaminase （GOT） activities were enhanced in B-deficient leaves. By contrast, respiration, concentrations of nonstructural carbohydrates and three organic acids （malate, citrate and pyruvate）, and activities of most enzymes （PEPC, NADP-ME, PK, PEPP, CS, ACO, NAD-IDH, NADP-IDH and HK） involved in glycolysis, the TCA cycle and the anapleurotic reaction, as well as concentration of TFAA and activities of related enzymes [nitrate reductase, NADH-GOGAT, glutamate pyruvate transaminase （GPT） and glutamine synthetase （GS）] were lower in B-deficient roots than in controls. However, leaf and root concentration of total phenolics increased, whereas that of total soluble protein decreased, in response to B-deficiency. In conclusion, respiration, organic acid （glycolysis and the TCA cycle） metabolism, the anapleurotic pathway and amino acid biosynthesis were upregulated in B-deficient leaves with excessive accumulation of carbohydrates to’consume’the excessive carbon available, but downregulated in B-deficient roots with less accumulation of carbohydrates to maintain the net carbon balance.3 Isolation and identification of B-deficiency-responsive genes in C. sinensis roots and leavesUsing cDNA-AFLP, we isolated 54 （38） and 38 （45） up （down）-regulated genes from B-deficient leaves and roots, respectively. These genes were mainly involved in protein and amino acid metabolism, carbohydrate and energy metabolism, nucleic acid metabolism, cell transport, signal transduction, and stress response and defense. The majority of the differentially expressed genes were isolated only from B-deficient roots or leaves, only seven genes with the same GenBank ID （XP₀06484536.1, XP₀06488862.1, XP₀07042812.1, XP₀07043058.1, NP₅64354.1, XP₀06492455.1 and BAF01964.1） were isolated from the both. Among the seven genes shared by B-deficient roots and leaves, only three genes （XP₀06484536.1, NP₅64354.1 and XP₀06492455.1） displayed similar expression trend in response to B-deficiency. Obviously, many differences existed in B-deficiency-induced changes in gene expression between roots and leaves. For example, UDP-glycosyltransferases were induced in B-deficient roots, but lowered in B-deficient leaves; however, genes involved in ATP biosynthesis were up-regulated in the former, but unaffected in the latter. All B-deficiency-responsive genes associated with signal transduction were down-regulated in roots, but up-regulated in leaves except for one gene. Genes related to protein ubiquitination and proteolysis were induced in B-deficient leaves except for one gene, while only two down-regulated genes involved in ubiquitination were detected in B-deficient roots. Genes involved in stress defense were down-regulated in B-deficient roots, but up-regulated in B-deficient leaves except for one gene.4 Identification of boron-deficiency-responsive microRN As in C. sinensis rootsWe isolated 52 （40 known and 12 novel） up-regulated and 82 （72 known and 10 novel） down-regulated miRNAs from B-deficient roots, demonstrating remarkable metabolic flexibility of roots by Illumina sequencing, which might contribute to the tolerance of plants to B-deficiency. MiRNAs might regulate the adaptations of roots to B-deficiency through following several aspects:（a） inactivating reactive oxygen species （ROS） signaling and scavenging through up-regulating miR474 and down-regulating miR782 and miR843; （b） increasing lateral root number by lowering miR5023 expression and maintaining a certain phenotype favorable for B-deficiency-tolerance by increasing miR394 expression; （c） enhancing cell transport by decreasing the transcripts of miR830, miR5266 and miR3465; （d） improving osmoprotection （miR474） and regulating other metabolic reactions （miR5023 and miR821）; （e） upregulating the transcript of miR472 and miR211, and decreasing the expression of their target genes, which are involved in disease resistance, and hence, the disease resistance of roots.5 Isolation and identification of B-deficiency-responsive microRNAs in C. sinensis leavesBy Illumina sequencing, we identified 91 （83 known and 8 novel） up-regulated and 81 （75 known and 6 novel） down-regulated miRNAs, demonstrating that miRNA expression was greatly altered by B-deficiency, which might contribute to the tolerance of citrus to B-deficiency. The adaptive responses of leaf miRNAs to B-deficiency might mainly include following several aspects:（a） attenuation of plant growth and development by repressing auxin signaling due to decreased TIR1 level and ARF-mediated gene expression by altering the expression of miR393, miR160 and miR3946; （b） maintaining leaf phenotype and enhancing the stress tolerance by up-regulating MYBs targeted by miR159, miR782, miR3946 and miR7539; （c） activation of the stress responses and antioxidant system through down-regulating the expression of miR164, miR6260, miR5929, miR6214, miR3946 and miR3446; （d） decreasing the expression of major facilitator superfamily protein genes targeted by miR5037, thus lowering B export from plants. Also, B-deficiency-induced down-regulation of miR408 might play a role in plant tolerance to B-deficiency by regulating Cu homeostasis and enhancing superoxide dismutase activity.