Physiological And Molecular Mechanisms Of Stylosanthes Adaptation To Aluminum Toxicity/Low Phosphorus Availability

Aluminum (Al) toxicity and low phosphorus (P) availability are two major limiting factors for crop production on acid soils. The content of active Al form (Al3+) in acid soils is very high, which could inhibit root growth and result in yield reduction. Phosphorus is one of the essential macronutrients for plant growth, whereas the available P in acid soils is limited. Stylosanthes (Stylo), a dominating leguminous forage in tropics and subtropics, well adapts to acid soils. Researches on the mechanisms of stylo adaptation to Al toxicity and low P availability might be beneficial for breeding new cultivars with tolerance to both Al toxicity and low P stress. The present studies employed two stylo genotypes contrasting in Al tolerance and P efficiency. Among them, TPRC2001-1(Stylosanthes guianensis) showed superior Al tolerance and P efficiency, while Fine-stem (Stylosanthes hippoeampoides) showed Al sensitivity and P inefficiency. For Al tolerance studies, the effects of Al on root growth, gene and protein expression, the roles of synthesis and exudation of organic acids to detoxify Al, as well as the protection of border cells in ameliorating Al toxicity were investigated. In the study of adaptation to low P availability, we established the full length cDNA library under Pi (Phosphate) starvation conditions, and homologously cloned and analyzed SgPT1(encoding Pi transporter) and SgSPXl (encoding the protein only containing SPX domain), and thus preliminarily evaluated the mechanisms of stylo in adaptation to low P availability. The main results were shown as follows:1) Genotypic variations for Al tolerance in stylo and their putative physiological mechanisms. Under slight Al treatment (50μM Al), the relative root length of TPRC2001-1was almost the same as XN1, the most Al tolerant rice variety, but significantly longer than Fine-stem. At72h after Al treatment, the relative root length of TPRC2001-1was well-matched with XN1and much longer than Fine-stem, indicating that TPRC2001-1has comparably superior Al tolerance to XN1, and Fine-stem is Al sensitive. Furthermore, malate accumulation and exudation in the roots of TPRC2001-1were highly stimulated by Al treatment, while Al content in the roots of TPRC2001-1was also higher than that in Fine-stem after72h of Al treatment, implying that the superior Al tolerance of TPRC2001-1might be mainly attributed by enhancing internal malate accumulation to chelate Al3+in roots, and together with the supplementary help of malate exudation to exclude Al, and thus coordinately detoxify Al.2) Establishment and analysis of suppression subtractive hybridization (SSH) library using stylo roots under different Al treatments. The roots treated by100μM Al for24h (Short-term library) and72h (Long-term library) were separately used as tester, while roots without Al treatment were as driver.200clones selected from short-term library and long-term library were sequenced, respectively.In total37nonredundant expressed sequence tag (EST) with11from short-term library and26from long-term library were obtained. The ratio of genes associated with metabolism was the largest, and the genes like MDH related to malate metabolism were up-regulated, suggesting that malate synthesis and/or exudation might be closely related to Al tolerance of TPRC2001-1.3) Two dimensional electrophoresis (2-D) and mass spectrum analysis of Al responsive proteins in-TPRC2001-1roots. The results showed that there were no notable differentially expressed proteins stimulated by Al under short-term (24h) treatment, while23proteins including13up-regulated and10down-regulated were enhanced and suppressed by72h Al stress, respectively. Furthermore, quantitative real time PCR (q-PCR) results showed that the gene transcripts correlated with the accumulations of about50%up-regulated, and40%down-regulated proteins, indicating that Al responsive proteins were controlled by both transcriptional and posttranscriptional levels in TPRC2001-1roots. Among the proteins, the proteins in carbon metabolism group had the highest proportion, which containing malate metabolism related proteins. Among them, protein accumulation and gene expression of a NADP dependent malic enzyme (ME), synthesis and exudation of malate, and stylo Al tolerance were closely correlated, indicating that ME might play important roles in the Al tolerance of TPRC2001-1.4) Functional analysis of SgME1. We homologously cloned the full length of targeting gene encoding the above NADP dependent ME and named it as SgME1(accession number:JX164253). The full length of SgME1was2173bp and the corresponding protein had the highest similarity to AtME2(AtNP196728) in Arabidopsis through phylogenetic analysis. Heterogenous transformation in onion epidermal cells found that SgME1was localized in cytosol. Furthermore, overexpressing SgME1improved Al tolerance of either yeast cells or bean transgenic hairy roots by increasing the intracellular malate concentration and also attenuated Al toxicity, suggesting that SgME1directly functioned in Al detoxification. Taken together, our results provide the first evidence that the superior Al tolerance of stylo genotype TPRC2001-1is conferred mainly by Al3+-enhanced malate accumulation in roots, together with the supplementary help of malate exudation, which functionally controlled by a novel malic enzyme, SgME1.5) Contribution of root tip exfoliates in Al tolerance of TPRC2001-1. Under normal growth conditions, the root tip of stylo could form exfoliates consisting of border cells, border-like cells and mucilage. Our results found that the seedlings of TPRC2001-1with superior Al tolerance formed more root tip exfoliates under Al treatment and thus absorbed more Al to reduce the Al uptaken by root tips. The root tip exfoliates formed under Al treatment closely encapsulated cell division zone so as to reduce the damage of Al toxicity on cell division; while the root tip exfoliates did not cover the transition zone, and thus would not block the Al signaling transduction and the activation of other Al tolerance pathways. This suggested that the formation of root tip exfoliates might be one of the important mechanisms conferred TPRC2001-1seedlings superior Al tolerance.6) Homologous cloning and gene expression analysis of SgPTl and SgSPX1. We established a full length cDNA library under Pi starvation conditions in TPRC2001-1roots, and homologously cloned SgPT1(encoding Pi transporter) and SgSPX1(encoding the protein containing SPX domain). The full length of SgPT1was1994bp, which encoded538amino acids. The protein molecular weight of SgPT1was59kD. The full length of SgSPXl was1324bp, predicted to encode292amino acids. The protein molecular weight of SgSPX1was33kD. Furthermore, gene expressions of SgPTl and SgSPX1were strongly enhanced by low P in TPRC2001-1roots, indicating that SgPTl and SgSPXl might be involved in stylo adaptation to low P stress.Taken together, we comprehensively studied the underlying mechanisms of Al tolerance in stylo from three aspects, including physiology, transcriptomics and proteomics. Combined with the physiological and molecular studies of low P tolerance in stylo, we overall analyzed the adaptive mechanisms of stylo to acid soils so as to provide theoretical basis for breeding more adaptive crop varieties to acid soils through the approaches of genetic improvements.