The Binding Of Aluminum By Cell Wall Hemicelluloses And Its Regulation In Arabidopsis

Aluminum (Al) toxicity is a major growth-limiting factor for crop production on acid soils worldwide. Ionic aluminum inhibits root elongation rapidly although the mechanism underlying Al-induced root growth inhibition remains unclear. Aluminum resistant plants have developed two mechanisms to cope with Al toxicity. One is based on the exclusion of Al from the root symplasm, whereas the other relies on the ability to tolerate symplastic Al. Although the well documented exclusion mechanism is to prevent Al from entering root cells by secretion of organic acid anions by the root apex, recently, increasing evidence has shown that binding of Al to cell wall appears to be closely related to Al resistance. However, the exact mechanism of how cell wall interacted with Al is still in early stages, and needs further study.In the present study, we first investigated the principle contribution of the cell wall hemicellulose to the Al adsorption in Arabidopsis roots; then on this basis, through using the mutants related to the content, O-acetylation modification and branched chain structure of hemicellulose xyloglucan:xth31, xth17, xth15, axy4, axy3, axy8and tbl7, we studied the effects of hemicellulose to Al deeply and systematically; Furthermore, the present study also using mpk6, yucca and so on to study the mechanism of hormone and kinase regulation of the Al bound to cell wall hemicellulose. The main results are followed below:1. Cell wall hemicellulose contributes to Al adsorptionPectin was considered as the major cell wall Al binding site for past years. However, in the previous study, Zheng et al (2004) found that after removal the pectin with pectinase, the adsorption of aluminum in the cell wall was declined, but there’s still a lot of aluminum accumulated in the cell wall. In this study, using the model plant Arabidopsis thaliana, we found that the hemicellulose1were increased significantly after Al treatment in Arabidopsis, which is in consistant with rice, and only20%of total Al content was decreased through using hot water to remove pectin. Furthermore, the Al adsorpted by the cell wall also decreased20%after pectin was removed, on the contrary, less than5%Al was retained in the cell wall and the amount of Al adsorbed by the cell wall was decreased to30%after hemicellulose1was removed, indicating that cell wall hemicellulose contributed significantly to the Al adsorption, the contribution that even greater than pectin. As xyloglucan endotransglucosylase (XET) action plays important roles in the regulation of hemicellulose, in vivo localization showed that Al greatly inhibited this enzyme activity, which is caused by transcriptional regulation, and among the eleven XTH genes that expressed in roots, XTH-14,15and31were significantly down regulated after Al treatment, as shown by the real-time reverse transcription-PCR. All these results demonstrated that hemicellulose is the major pool for Al accumulation. Furthermore, Al-induced reduction of XET action could play an important role in Al-induced root growth inhibition.2. XTH31regulates xyloglucan content and Arabidopsis Al sensitivityThen, how is the functions of the individual XTH gene in terms of root growth regulation in response to Al stress? We used a T-DNA insertional mutant-xth31, which is obtained from Janet Braam, a professor from Rice university (USA), as materials. The full-length of XTH31gene is882bp, which encoded a294amino acids, bioinformatics analysis showed that there’s19amino acids worked as signal peptide targeted to the plasma membrane at the N terminus of the protein. Although the mutant grows slowly under normal condition, its Al resistance was increased. For instance, after treated with50μM Al for24h, there’s almost40%inhibition of root elongation in WT (Col-0) while there’s almost no effect on xth31, however, when xth31was complemented by35S:XTH31, its Al resistance was disappeared and restored to the WT level, indicated that XTH31direct controlled its Al resistance. In the xth31mutant, the XET activity/action was decreased, which correlated well with its slower growth under normal condition. However, after heterologously expressed XTH31in yeast, enzyme kinetic analysis showed that this protein has strong XEH activity, with lower XET activitity. Al content analysis showed that there’s less Al accumulated in the root, cell wall and hemicellulose, and in accordance with this, the Al adsorption capacity of its cell wall was decreased. MALDI-TOF MS analysis indicated that xyloglucan was decreased significantly in the mutant cell wall, and root Al accumulation was decreased significantly while plant became more Al resistant when xyloglucan was applied exogenously. All these indicated that xyloglucan maybe combined with Al to form a complex. As expected, Al and xyloglucan really can form complex as suggested by27A1-NMR. Furthermore, Al accumulation and cell wall Al adsorption capacity was decreased in the xxtlxxt2double mutant, which has no detectable xyloglucan. All these results for the first time demonstrated the mechanism of XTH31, a gene related to xyloglucan content, in response to plant Al resistance.3. XTH17interacts with XTH31to encode XET action and attribute to Al sensitivityIn our previous study, we found that XTH31expressed heterologously exhibited rich XEH activity and low XET activity. However, the XET activity was significantly reduced while there’s almost no change of the XEH activity in xth31mutant. Then, what is the reason for this inconsistency? As we known, there are33members of the identified XTH genes in Arabidopsis thaliana, and one-third occurs as clusters resulting from genome duplication. XTH31is in group IIIA, and has very high XEH activity and low XET activity when expressed in pichia in vitro, which is consistent with the prediction of Baumann et al (2007). In order to interpret this inconsistence between in vitro and in vivo, we obtained a XTH17protein that can direct interact with XTH31through yeast two hybrid and co precipitation in tobacco (N. benthamiana). Moreover, the response of xth17mutant to Al was studied. xth17grown similar to xth31in normal conditions, which means that the xth17mutant also has low XET action and grown slowly, however, it showed greater resistance when in response to Al stress. Both the Al retention and adsorption capacity of Al decreased significantly in the xth17mutant root cell wall, accompanied by a reduction in cell wall hemicellulose content, which showed the same tendency to the xth31mutant. Overall, all these results indicated that XTH17can direct interact with XTH31to form a complex to promote XET action, thus modulate the cell wall Al binding capacity in Arabidopsis.4. The O-acetylation of xyloglucan modulate Al binding capacity of cell wall hemicelluloseIn our previous study, we found that xyloglucan is the major Al binding site in Arabidopsis cell wall. Then whether the modification of the xyloglucan will affect its Al binding capacity is still not known. Now, in our present study, we found that the O-acetylation modulation of xyloglucan can influence the response of Arabidopsis to Al stress. Under Al stress, the O-acetylation degree was significantly decreased in Arabidopsis cell wall, especially the xyloglucan. The Al resistance was decreased in axy4-1and axy4-3mutants, both has reduced xyloglucan O-acetylation level while the Al resistance was unchanged in the35S:AXY4lines (AXY4overexpressing lines), indicated that the O-acetylation modulation of xyloglucan can impact the plant response to Al stress, made them more sensitivity. Further study demonstrated that there was more Al accumulation in the root and cell wall although there’s almost no change in the pectin content, PME activity and hemicelluloses content compared with WT. However, although there’s more Al accumulation in the axy4mutants cell wall, there’s almost no change of the Al retention in the pectin, and the only difference between axy4mutants and the WT is the O-acetylation degree of xyloglucan, thus it indirect explained that this modulation of the xyloglucan O-acetylation is similar to the modulation of PME, both of which induced the Al accumulation in the cell wall and thus made the plants more Al sensitivity.5. Fucosylated xyloglucan contributes to the Al binding capacity of cell wall hemicelluloseIn our present study here, we found that it is the fucosylated xyloglucan that contributed greatly to the cell wall Al binding capacity in Arabidopsis. Although there’s an increment of both fucosylated and galactosylated xyloglucan when in response to Al, both axy3.2and axy3.3, with reduced fucosylated xyloglucan oligos, exhibited more Al sensitivity compared with WT. In accordance with this, there’s more Al accumulated in the mutant roots while less Al accumulated in the mutants cell wall, especially in the hemicellulose, although there’s almost no change in the pectin content, PME activity and hemicellulose content when compared with WT. Moreover, as there’s almost no change in the Al retention in the pectin, and the only difference between axy3mutants and WT is the structure of the xyloglucan, further demonstrated that the reduction of the fucosylated xyloglucan directly related to the reduced cell wall Al accumulation, and more Al was entered into the cell of axy3mutants. However, the expression of ALS1, which is localized in the vacuole membrane and involved in the Al sequestration into the vacuole, was not changed in the axy3mutants compared with WT, indicated that more Al entered to the cell of axy3mutants that attributed to their Al sensitivity. To further demonstrated our view, we observed a contrary phenotype in axy8mutants, both of which have increased fucosylated xyloglucan. There’s less Al accumulated in the axy8-5and axy8-6roots, while there’s more Al retained in the axy8-5and axy8-6cell wall, thus less Al entered into the cell compared with WT. The almost none changed expression of ALS1in the axy8-5and axy8-6mutants further suggested that this reduced Al accumulation in the cell deciding their Al resistance. All these results demonstrated that fucosylated xyloglucan contributes to the Al binding capacity of cell wall hemicellulose.6. Auxin regulates aluminum bound to cell wall hemicelluloseThen, how is the functions of other individual XTH gene besides XTH31in terms of root growth regulation in response to Al stress? Similar to XTH31, knockout of XTH15resulted in the Al resistance. It is interesting that there’s lower auxin content in xth15mutant when compared with Col-0. Then, whether auxin can regulate the plant Al resistance? To this end, we analyzed the endogenous auxin overproducing mutant yucca and WT applied with an auxin analog-NAA exogenously, and found that both of them exhibited Al sensitivity, which is contrary to the Al resistance phenotype of xth15. However, there’s no difference of the root Al cotent among xthl5, yucca, WT and WT applied with auxin exogenously, although less Al was accumulated in the cell wall of xth15, yucca and WT applied with auxin exogenously compared with WT. In company with this reduced cell wall Al content, the expression of ALS1, a gene that functions in Al redistribution between the cytoplasm and vacuole and contributes to symplastic Al detoxification, was more abundant in xthl5, less abundant in yucca and WT applied with auxin exogenously, and this was further demonstrated by morin staining, which is consistent with possible ALS1function. Thus, auxin can exacerbate Al toxicity, with the reduction of the cell wall Al content and ALS1expression, then more Al entered into the cell and can’t detoxify by sequestration into the vacuole, which finally resulted in the Al sensitivity phenotype of Arabidopsis. However, as there are no auxin response elements (AuxREs) TGTCTC in the2kb-promoter regions of ALS1genes, the effect may be indirect, needing further investigation. The Al resistance of xthl5further demonstrated the importance of the coordination of apoplastic and symplastic detoxification of Al toxicity.As auxin can negatively regulate plant Al tolerance through altering ALS1expression and Al distribution in plant cells, then, whether this effect is direct or indirect? In the present study, it is interesting that Al treatment can induce the increment of auxin and NO, and this increment of auxin content under Al stress further induced the production of NO. Moreover, we found that exogenous application of NO donor (GSNO) exacerbated the Al inhibited root elongation, induced more Al accumulation in the WT roots while less Al accumulation in its cell wall, especially the hemicellulose, which is in company with the lowered xyloglucan content. In accordance with this, the endogenous NO reduced mutants noal (NOA pathway) and nialnia2(NR pathway) are more Al resistant with the lower root Al content and higher xyloglucan content, thus more Al retained in their root cell wall, especially the hemicellulose. Furthermore, auxin applied exogenously also reduced the Al retention in the hemicellulose in company with the reduction of xyloglucan content, which is similar to GSNO applied exogenously. In yucca and auxl-7mutants, exogenous application of NO could bring its response similar to the behavior of WT while exogenous application of auxin almost has no effect on the noal mutant under Al stress, although exogenous application of auxin has similar effect on the nia1nia2mutant and WT, further indicated that auxin acts upstream of NO through a NOA pathway to regulate the Al adsorption capacity of the plant cell wall. On the other hand, irrespectively in the presence or absence of auxin, exogenous application of GSNO has no effect on the ALS1expression, a gene that functions in Al redistribution between the cytoplasm and vacuole and contributes to symplastic Al detoxification. All these results demonstrated that auxin acts upstream of NO through a NOA pathway to regulate the xyloglucan content, thus influence the Al adsorption capacity of the plant cell wall.7. MPK6regulates aluminum bound to cell wall hemicelluloses through TBL7In our previous study, we found that the structure of xyloglucan is in response to Al stress in Arabidopsis, with the increment of the fucosylated, galactosylated xyloglucan and other xyloglucan oligosaccharide. However, this increment is disappeared in the mpk6mutant when in response to Al stress. In order to investigate how MPK6influence cell wall xyloglucan content when in response to Al, a series of xyloglucan modification and structure related proteins were selected for the bioinformatics analysis. It is interesting that "TBL7" harbored a cluster of four potential MAPK phosphorylation sites (Ser-22, Ser-33, Ser-41and Ser-50) in the N terminus, thus it may acted as the substrate of the MAPK6. As expected, the in vitro yeast two hybrid and in vitro phosphorylation analysis showed that MPK6can phosphorylated the TBL7, thus can interact with each other. Furthermore, there’s less cell wall and hemicellulose Al content in mpk6and tbl7mutant, this similar phenotype further demonstrated the biological significances of the interaction of MPK6and TBL7to regulate the cell wall Al binding capacity.