Aluminum-tolerance Mechanisms Of Citrus And Regulation Of Aluminum-toxicity By Phosphorus And Nitric Oxide

Citrus belong to evergreen subtropical fruit trees and are cultivated in humid and subhumid of tropical, subtropical, and temperate region of the world mainly on acid soils. High aluminum （Al） and/or low phosphorus （P） are frequently observed in citrus plantations. In this study, we investigated the effects of Al-P interactions on seedling growth, root organic acid （OA） metabolism and Al-induced-secretion of OA anions from roots, and expression of root genes involved in alternative glycolytic pathways, P acquisition and utilization using‘Xuegan’（Citrus sinensis） and‘Sour pummelo’（Citrus grandis） seedlings having different tolerance to Al and P-deficiency; and effects of sodium nitroprusside （SNP, an NO donor）-Al interactions on seedling growth and photosynthesis in leaves, and carbohydrates, antioxidant system and OA metabolism in roots and leaves of C. grandis. The objectives were to understand the mechanisms of citrus to Al-tolerance and the regulation of Al-toxicity by P and NO.1 Al-tolerance mechanisms of citrus: secretion of OA anions and immobilization of Al by phosphorus in rootsC. grandis and C. sinensis seedlings were irrigated daily for 18 weeks with nutrient solution containing 0 （-Al） and 1.2 mM AlCl₃·6H₂O （+Al）×0, 50 and 200μM KH₂PO₄. Al significantly decreased root, stem and leaf DW of C. grandis seedlings, but had little on C. sinensis ones. Al decreased P content in roots, stems and leaves. Under Al stress, root Al content increased and stem and leaf Al content decreased with increasing P supply. C. sinensis accumulated more Al in roots and less Al in shoots than C. grandis. P content was higher in C. sinensis roots, stems and leaves than in C. grandis ones. C. sinensis roots secreted more malate and citrate than C. grandis ones in response to Al. Al-induced-secretion of malate and citrate from +Al excised roots was higher than from -Al ones, while Al-preculture did not increase root malate and citrate. Al-induced-secretion of malate and citrate from +Al excised roots decreased with increasing P supply. Al-induced-secretion of malate and citrate was inhibited by 5μM anion-channel inhibitors phenylglyoxal （PG）, anthracene-9-carboxylic acid （A-9-C） and 4,4’-diisothiocyanatostilbene-2,2’-disulfonate （DIDS） and 5μM protein-synthesis inhibitor cycloheximide （CHM）, but not by 5μM anion-channel inhibitor niflumic acid （NIF）. When 25μM inhibitors were used, similar results were obtained except that A-9-C stimulated the secretion of malate. Low temperature inhibited Al-induced-secretion of malate and citrate. To conclude, P can alleviate Al-toxicity through increasing immobilization of Al in roots and P level in seedlings rather than through increasing OA anion secretion. The higher Al-tolerance of C. sinensis may involve higher secretion of OA anions and precipitation of Al by P in roots.2 Effects of Al-P interactionson expression of root genes involved in alternative glycolytic pathways, P acquisition and utilizationC. grandis and C. sinensis seedlings were irrigated daily for 18 weeks with nutrient solution containing 0 （–Al） and 1.2 mM AlCl3·6H2O （+Al）×0, 50 and 200μM KH2PO4. C. sinensis was more tolerant to Al and P-deficiency than C. grandis. qRT-PCR analysis showed that Al-activated malate transporter （ALMT1）, ATP-dependent phosphofructokinase （ATP-PFK）, pyrophosphate-dependent phosphofructokinase （PPi-PFK）, tonoplast adenosine-triphosphatase subunit A （V-ATPase A）, tonoplast pyrophosphatase （V-PPiase）, pyruvate kinase （PK）, acid phosphatase （APase）, phosphoenolpyruvate carboxylase （PEPC）, malic enzyme （ME） and malate dehydrogenase （MDH） might be involved in citrus tolerance to Al and/or P-deficiency, but any single gene could not account for the differences between C. sinensis and C. grandis. Citrus tolerance to Al and/or P-deficiency might be a result of a complex coordinated regulation of gene expression involved in alternative glycolytic pathways, P acquisition and utilization.3 NO alleviates the toxic effects of Al on citrus seedlingsC. grandis seedlings were irrigated daily for 18 weeks with nutrient solution containing 0 containing 0 （–Al） and 1.2 mM （+Al） AlCl3·6H2O×0, 10 and 500μM SNP. Under Al stress, SNP increased root P and Al, but decreased shoot Al. SNP at 10μM alleviated Al-induced-inhibition of root, stem and leaf growth, but at 500μM only alleviated leaf growth. Al decreased photosynthesis, maximum fluorescence （Fm）, maximum quantum yield of primary photochemistry （Fv/Fm） and total performance index （PItot,abs）, but increased minimum fluorescence （Fo）, K-band, relative variable fluorescence at J- and I-steps （VJ and VI）. SNP alleviated Al-induced-changes for all these parameters. SNP at 500μM had a detrimental effect on growth, which might explain why the ameliorative effect of 10μM SNP was better. SNP stimulated Al-induced-secretion of malate and citrate from +Al excised roots, while Al did not increase their contents in roots. SNP and Al interactions affected malate and citrate more in leaves than in roots. Generally speaking, antioxidant system in leaves and roots was up-regulated and down-regulated by Al, respectively. SNP prevented Al-induced-accumulation of malondialdehyde （MDA） in roots and leaves. Non-structural carbohydrates in roots and leaves were differently affected by SNP and Al interactions. In conclusion, NO alleviates Al-induced-inhibition of growth and impairment of the whole photosynthetic electron transport chain from Photosystem II （PSII） donor side up to the reduction of end acceptors of Photosystem I （PSI） through increasing Al immobilization and P level in roots and Al-induced-secretion of malate and citrate from roots, and decreasing Al accumulation in shoots, thus preventing the decrease of photosynthesis. NO is involved in the amelioration of Al-induced oxidative stress in roots and leaves. The change in carbohydrates, antioxidant system and OA metabolism differ between roots and leaves in response to NO and Al interactions.