03生物技术基地班王伟

复旦大学 论文集 2007 373 A A StudyStudy onon thethe ShortShort andand LongLong TermTerm EffectsEffects AluminumAluminum ExertsExerts onon OryzaOryza RufipogonRufipogon 03 生物技术基地班王伟 生命科学学院明凤副教授 AbstractAbstractOryza rufipogon or common wild rice is a close relative of Oryza sativa the widely cultivated rice In the short term study by measuring the relative root elongation of seedlings treated with different aluminum Al concentrations we found that O rufipogon showed considerable tolerance or resistance to Al toxicity and experienced a dose dependent stimulated root growth The subsequential aniline blue and morin staining indicated that Al did have detrimental effects on the growth of O rufipogon and accumulated in it The quantification of the Al content via ICP AES has also confirmed its accumulation Therefore O rufipogon is tolerant rather than resistant to the Al toxicity The DAPI staining suggested that the recorded stimulated root growth may be owing to the dominance of the enhanced cell division over other detrimental effects at an early stage In the long term study through suppression subtractive hybridization SSH and the Northern blot the enhanced expression of the small subunit of Rubisco and a photosystem I PSI P700 apoprotein A1 the two important components in photosynthesis in leaves were observed The further investigations via the assessment of the content of chlorophyll a b and SDS PAGE of Rubisco also suggested that the aluminum also had a regulatory effect on the photosynthesis in the leaves besides the devastating effects on the roots KeyKey WordsWordsOryza rufipogon DAPI stimulated root growth SSH Rubisco IntroductionIntroduction Plants are continually confronted with challenges by environmental stresses like drought salinity low or high temperature flooding pathogen heavy metals etc Al is the most abundant metal in the earth s crust comprising about 7 45 of its mass As many plant species are sensitive to micromolar concentrations of Al the potential for soils to be A1 toxic is considerable While most of A1 is bound by ligands or occurs in other nonphytotoxic forms such as aluminosilicates and precipitates however in acidic soils the phytotoxic species Al3 is solubilized to levels that Al toxicity becomes significant According to the present knowledge the detrimental influence of Al on plants has several A Study on the Short and Long Term Effects Aluminum Exerts on Oryza Rufipogon 374 aspects including competitive inhibition of absorption sites on the cell membrane of Mg2 Ca2 by combining with pectin inhibiting the absorption and transportation of water and ions inducing symptoms of iron phosphate and calcium deficiency 1 leading to the disintegration of cortical microtubules 2 accumulating at nuclei of cells and interferencing its normal physiological function 3 thus inhibiting root growth and crop yield Several mechanisms of Al tolerance such as external exclusion and internal detoxification have been reported in various plants The external exclusion includes Al activated exudation of organic acids 4 8 and alkalinization of apoplast and rhizosphere 9 10 The internal detoxification refers to plant ability to tolerate Al in symplasm by sequestration 11 However the mechanisms of Al tolerance in most plant species including O sativa are still poorly understood O rufipogon is a kind of weed which grows largely in Yun Nan province of China where the loess and laterite comprise the majority of the arable land As the loess and laterite are leached of silicon but rich in Al due to their process of formation the pH of them is 4 5 5 5 12 Therefore Al exists in the form of phytotoxic species Al3 in these soils So O rufipogon may have great tolerance to Al toxicity In a pilot study we found that this plant grows normally in solution containing 3 5 mM Al3 with Al content in old leaves as high as 4 10 mg g 1 dry weight in old leaves though the roots were inhibited obviously Since O rufipogon is a close relative of widely cultivated rice the exploration of the mechanism of Al tolerance in O rufipogon may to some extent shed light on the study of the mechanism of Al tolerance in cultivated rice Though it is widely accepted that the detrimental effects of Al and the reactions of the treated plants commence within a relatively short period after the addition of Al we also conducted the relevant experiments after a long time course of Al treatment considering that long exposure to the Al toxicity represents the original situation that plants meet with in their living surroundings thus may also bring about some physiological and molecular differences among tolerant and sensitive species other than those in the roots Therefore in the studies of the long term exposure to Al of O 复旦大学 论文集 2007 375 rufipogon we selected O sativa as a reference and sampled the leaves for the research Since scanty knowledge has been achieved about the leaves after the treatment of Al we started our work via the suppression subtractive hybridization ResultsResults ObservationObservation ofof a a dose dependantdose dependant relativerelative rootroot elongationelongation RRE RRE stimulationstimulation While most plants are sensitive to micromolar Al O rufipogon experiences a dose dependant RRE stimulation Fig 1 When treated with 0 7 mmol L Al for 12 hours the root growth was stimulated rather than inhibited At the meantime the root growth of seedlings treated with higher or even lower concentration of Al was inhibited by more than 50 This is quite intriguing since Al is widely believed to have various detrimental effects on the growth especially the root growth of most plants 0 0 0 0 2 20 0 0 0 4 40 0 0 0 6 60 0 0 0 8 80 0 0 0 1 10 00 0 0 0 1 12 20 0 0 0 1 14 40 0 0 0 0 0 0 00 00 0 3 35 50 0 7 70 01 1 0 05 51 1 4 40 0 A Al l c co on nc ce en nt tr ra at ti io on n m mM M R Re el la at ti iv ve e R Ro oo ot t E El lo on ng ga at ti io on n FigureFigure 1 1Relative root elongation RRE of O rufipogon treated with Al of different concentrations 0 0 35 0 5 0 7 1 4mmol L for 12h RRE is defined as the root growth of the treated seedlings divided by that of the control ones The bar represents the standard error n 20 DetrimentalDetrimental effectseffects ofof AlAl onon thethe growthgrowth ofof wildwild ricerice The production of callose was accepted as a criterion to determine the extent of the damages Al has exerted on the treated plants Aniline blue staining can visualize the production of callose with help of the fluorescence microscopy According to Figure 2 the roots treated with 0 7 mmol L Al had more callose than those treated with 0 35 or 1 4 mmol L Al 4 hours after Al treatment This implied that 0 7 mmol L Al treatment can stimulate the production of callose faster at the early stage 8 or 12 hours after Al treatment the amount of the callose had a A Study on the Short and Long Term Effects Aluminum Exerts on Oryza Rufipogon 376 positive relationship with the Al concentration used in the treatment This suggested that in the long run more Al can cause more damages to the roots FigureFigure 2 2The aniline blue staining shows the formation of callose indicating the detrimental effect of Al on O rufipogon The seedlings were treated with different Al concentrations 0 0 35 0 7 1 4mmol L for different period of time 0 4 8 12h Representative photos of fluorescence and those merged with the same density of white light were taken at different magnifications field lens 10 and 20 However the amount of the callose in the roots does not necessarily represent the amount of the Al that attaches to the surface of the roots and enters the cells It only lent credit to the proposition that Al did cause damages to O rufipogon despite its stimulated root growth when treated with 0 7 mmol L Therefore the morin staining was adopted as a qualitative method to locate Al According to Figure 3 4 or 12 hours after Al treatment roots treated with 0 7 mmol L Al accumulated the largest amount of Al among the three treatments and control ones The photos of 8 hours however suggested no significant differences of Al accumulation among the three Al treatments These results together with those from the aniline blue staining indicated that compared with the other two Al treatments roots treated with 0 7 mmol L though tended to accumulate more Al yet did not always have the most serious damages during the 12 hour Al treatment Moreover these results also implied that Al may also have the beneficial effect on the root growth of O rufipogon besides the detrimental influence 复旦大学 论文集 2007 377 FigureFigure 3 3The morin staining locates Al in the roots The seedlings were form the same batch under the same treatment as figure 2 QuantificationQuantification ofof thethe accumulatedaccumulated AlAl inin O O rufipogonrufipogon Though the morin staining visualized the Al in the treated roots we still cannot rule out the possibility that Al just attaches to the surface of the roots rather than enters the cells Therefore the quantification of the accumulated Al is necessary The measurement via ICP AES proved that Al not only entered the cells but also was transported to other organisms like the stem and leaves Fig 4 Here we adopted the seedlings treated with 1 4 mmol L Al for 12 hours to meet the minimal limit of the ICP AES we used though later it turned out that samples treated with lower Al content also contained enough amount of Al to be detected by ICP AES Anyway figure 4 proved that Al can enter the cells of O rufipogon and be transported to upper ground organisms Al accumulation 020406080100120 Root Stem New leaf Old leaf Al concentration ug g FigureFigure 4 4Quantification of the accumulated Al in O rufipogon The seedlings were treated with 1 4 mmol L Al for 12h Al concentration refers to the amount of Al divided by the fresh weight of the samples Al accumulation was defined as the difference between the Al concentration of treated seedlings and that of the control ones A Study on the Short and Long Term Effects Aluminum Exerts on Oryza Rufipogon 378 DAPIDAPI stainingstaining According to the abovementioned results though damages did exist seedlings treated with 0 7 mmol L Al for 12 hours experienced the root growth stimulation So besides the detrimental effects Al should also have beneficial or at least the stimulative effects on the root growth of O rufipogon Two mechanisms may account for the enhanced root growth the increased cell elongation or the accelerated cell division However it is technically difficult to directly identify the increased cell elongation So here the second possible mechanism was first tested Using DAPI staining we were able to see the greatly enhanced cell division of roots treated with 0 7 mmol L Al for 12 hours Fig 5 Roots treated with 0 35 mmol L Al for 12 hours also experienced a little bit stimulated cell division However according to Figure 1 roots with this treatment met a more than half growth inhibition Therefore instead of increasing cell elongation Al inhibited it Therefore we presented evidence to indirectly support the proposition that the first mechanism did not exist in the case of wild rice FigureFigure 5 5The DAPI staining represents the division viability of the treated roots The seedlings were from the same batch under the same treatment as figure 2 ComparisonComparison ofof thethe RRERRE ofof O O rufipogonrufipogon andand O O sativasativa inin longlong termterm treatmenttreatment In the original surroundings plants meet with Al toxicity throughout the whole life Therefore long term study is necessary Since more nutrients were added to the treatment solution for the long term treatment 1 4 mmol L Al was used for the treatment Figure 6 showed that compared with the close relative O sativa O rufipogon had an enhanced root growth 1 3 5 days after treatment 复旦大学 论文集 2007 379 On day 7 O rufipogon still showed more tolerance to Al than O sativa However on day 15 no significant differences were shown This may attribute to the exhaust of the nutrients in the treatment solution These suggested that even in the long run O rufipogon grew better than O sativa when treated with 1 4 mmol L Al and also experienced a short period of root growth stimulation 5 50 0 0 00 0 0 0 0 00 0 5 50 0 0 00 0 1 10 00 0 0 00 0 1 15 50 0 0 00 0 2 20 00 0 0 00 0 2 25 50 0 0 00 0 0 0 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 1 10 01 11 11 12 21 13 31 14 41 15 51 16 6 d du ur ra at ti io on n d da ay ys s R Re el la at ti iv ve e R Ro oo ot t E El lo on ng ga at ti io on n O O s sa at ti iv va a O O r ru uf fi ip po og go on n FigureFigure 6 6Relative root elongation of O rufipogon and O sativa treated with 1 4 mmol L Al for different time period 0 1 3 5 7 15 days The bar represents the standard error n 10 SSHSSH screeningscreening andand northernnorthern blotblot analysisanalysis Thirty genes from shoots were found after two rounds of screening Further sequencing and northern blot uncovered 5 genes whose transcript abundance was significantly changed under Al toxicity Table 1 Fig 7 Two of them OrALI1 and OrALI2 were found involved in photosynthesis OrALI1 is a ribulose 1 5 bisphosphate carboxylase oxygenase Rubisco gene and OrALI2 is a photosytem I PSI P700 apoprotein A1 gene Both genes were up regulated under Al3 stress OrALI3 homology to the MADS domain transcription factors was also induced under Al3 Expression of the other two genes decreased at Al3 stress One is OrALR1 homological to dihydrofolate reductase dhfrXII and streptomycin 3 adenylyltransferase aadA2 genes which play an important role in the bacteria DNA synthesis The other gene is OrALR2 It is located at rice chromosome 6 with unknown function TableTable 1 1Genes found in O rufipogon which expression are induced or reduced by Al3 toxicity Gene brief Identity base Full name of homology genesGeneBank A Study on the Short and Long Term Effects Aluminum Exerts on Oryza Rufipogon 380 name aratio OrALI1352 396Rice rbcS gene for ribulose 1 5 bisphosphate arboxylase oxygenase OSRBCS OrALI2134 142Anthoceros punctatus chloroplast gene for photosystem I P700 apoprotein A1 partial cds AB013664 OrALI3212 212Gnetum gnemon mRNA for putative MADS domain transcription factor GGN132212 OrALR1206 208Klebsiella pneumoniae dihydrofolate reductase dhfrXII and streptomycin 3 adenylyltransferase aadA2 genes AF180731 OrALR2266 278Oryza sativa genomic DNA chromosome 6AP002842 a OrALI genes Oryza rufipogon L Al induced genes OrALR genes Oryza rufipogon L Al reduced genes FigureFigure 7 7Northern blot of the OrALI1 a OrALI2 b OrALI3 c OrALR1 d and OrALR2 e Al Al3 treatment CK control 28S and 18S rRNA as reference ChlorophyllChlorophyll a a b b contentcontent andand SDS PAGESDS PAGE ofof RubiscoRubisco Since the transcripts of two important components in the photosynthesis were found to be up regulated when treated with Al in the SSH assay Al seems to have a regulatory effect on the photosynthesis Therefore we measured the chlorophyll a b content and the amount of Rubisco the key enzyme in the photosynthesis to further identify the effect of Al on photosynthesis of O rufipogon According to Figure 8 the content of chlorophyll a b in the leaves of O rufipogon generally rose after Al treatment At the meantime the content of chlorophyll a and the overall content of chlorophyll a b in the leaves of O sativa declined or remained unchanged This together with the results from SSH implied that Al induced enhancement of the photosystem of the Al treated O rufipogon may account for wild rice s higher tolerance to Al than O sativa 复旦大学 论文集 2007 381 The SDS PAGE results of the large unit of Rubisco were in agreement with those of the small unit They suggested that though minor differences did exist between the Al treated samples and the control ones on day 7 or day 15 in the cases of O rufipogon and O sativa the amount of Rubisco largely remained unchanged Thus Al has a regulatory effect on Rubisco at the transcriptional level but not the translational level Altogether these results indicated that the Al induced enhancement of the photosynthesis may contribute to the wild rice s long term tolerance to Al O O r ru uf fi ip po og go on n 7 70 0 0 00 0 8 80 0 0 00 0 9 90 0 0 00 0 1 10 00 0 0 00 0 1 11 10 0 0 00 0 1 12 20 0 0 00 0 1 13 30 0 0 00 0 1 14 40 0 0 00 0 1 15 50 0 0 00 0 1 16 60 0 0 00 0 0 01 12 23 34 45 56 67 78 89 91 10 0 1 11 1 1 12 2 1 13 3 1 14 4 1 15 5 1 16 6 d du ur ra at ti io on n d da ay ys s C Ch hl l a a C Ch hl l b b C Ch hl l a a b b O O s sa at ti iv va a 7 70 0 0 00 0 7 75 5 0 00 0 8 80 0 0 00 0 8 85 5 0 00 0 9 90 0 0 00 0 9 95 5 0 00 0 1 10 00 0 0 00 0 1 10 05 5 0 00 0 1 11 10 0 0 00 0 1 11 15 5 0 00 0 1 12 20 0 0 00 0 0 01 12 23 34 45 56 67 78 89 9 1 10 0 1 11 1 1 12 2 1 13 3 1 14 4 1 15 5 1 16 6 d du ur ra at ti io on n d da ay ys s C Ch hl l a a C Ch hl l b b C Ch hl l a a b b FigureFigure 8 8The measurement of the relative chlorophyll content defined as the chlorophyll content of those treated with Al divided by that of control ones of O rufipogon and O sativa treated with 1 4 mmol L Al for different time period 1 3 5 7 15 days The bar represents the standard error n 3 A Study on the Short and Long Term Effects Aluminum Exerts on Oryza Rufipogon 382 FigureFigure 9 9SDS PAGE of the large and the small subunit of Rubisco extracted from the leaves of O rufipogon and O sativa treated with 1 4 mmol L Al for different period of time The seedlings were from the same batch as figure 7 CK represents for the control ones DiscussionDiscussion Al has long been one of the major factors that limiting the worldwide crop production leading to the root growth inhibition and thus poor crop yields Over 50 of the world s arable lands are acidic lands where Al toxicity is significant 13 14 And most of these lands are in developing countries Therefore Al has been a potential threat to the development of these nations However plants will not await their dooms They have developed several methods to dea