Gene Expression Analysis Of Citrus Roots And Leaves In Response To Boron Toxicity Revealed By CDNA-AFLP

Seedlings of ‘Sour pummelo’(Citrus grandis) and ‘Xuegan’(Citrus sinensis) with adifferent tolerance to boron (B)-toxicity were used in this study. Potted seedlings were fertilizedfor15weeks with nutrient solution at a boron (B) concentration of10or400μM every other day.Thereafter, effects of B-toxicity on growth and the content of B in roots and leaves wereinvestigated; at the sam time, root tips and leaves for total RNA extraction and subsequentcDNA-AFLP analysis were collected. The main results are as follows:1. In ‘Sour pummelo’, symptoms of B-toxicity were first appeared in old leaves as tipyellowing, followed by marginal and interveinal chlorosis. In the later stages, these chloroticleaves became necrotic and shed premature. In contrast, almost no visible symptoms occurred in‘Xuegan’ leaves except for very few plants. Boron-toxicity deseased the dry weights (DWs) ofcitrus roots, shoots and the whole plants and the ratio of root DW to shoot DW and increased thecontent of B in roots and leaves, especially in leaves.2. Two hundreds and fifty-six pairs of primers were used for amplification of citrus root andleaf cDNA. Fifteen to forty bands were amplified by each pair of primers. In roots,332differentially expressed TDFs were detected, among these TDFs,245TDFs were sequencedsuccessfully and192TDFs were identified. In leaves,268differentially expressed TDFs weredetected, among these TDFs,230TDFs were sequenced successfully and182TDFs wereidentified. All these identified TDFs were analysed by BLASTX and were associated withcarbohydrate and energy metabolism, cell wall and cytoskeleton, lipid metabolism, stressresponses, nucleic acid metabolism, protein metabolism, biological regulation and signaltransduction, transport, etc.3. In ‘Sour pummelo’ roots, the expression of138TDFs were altered by B-toxicity,52ofwhich displayed increased and86displayed decreased expression levels in response to B-toxicity;while in ‘Xuegan’ roots, only76TDFs expression were affected by B toxicity,20of whichwere regulated and56were down-regulated by B-toxicity. This suggests that B toxicityaffected gene expression in ‘Sour pummelo’ roots more than in ‘Xuegan’ ones. Boron-toxicityincreased the expression levels of genes involved in signal transduction (calcium-bindingEF-hand-containing protein gene) and stress resonses [WD repeat domainphosphoinositide-interacting protein, regulatory factor (14-3-3protein), homogentisatephytyltransferase1, alternative oxidase and Hsp70gens] in ‘Sour pummelo’ roots, whereas in ‘Xuegan’ roots B-toxicity only increased the expression levels of genes encoding aspartic protease,dioxygenase, H+-ATPase4. This suggests that the mechanisms of B-toxicity tolerance differbetween ‘Sour pummelo’ and ‘Xuegan’ roots.4.‘Xuegan’ leaves was more tolerant to B-toxicity than ‘Sour pummelo’ ones, which mightbe related to the findings that compared with ‘Sour pummelo’, B-toxicity ‘Xuegan’ leaves hadhigher expression levels of genes involved in photosynthesis, which might contribute the higherphotosynthetic rate and light utilization and less excess light energy in ‘Xuegan’ leaves than ‘Sourpummelo” ones and prevent them from photo-oxidative damage; and the results that that theB-toxicity-induced alteration in the expression levels of PSY, Nudix hydrolases, CESA, F-box andSAMS2genes differed between ‘Sour pummelo’ and ‘Xuegan’ leaves.