Gene Expression And Metabolism Of Reactive Oxygen Species Of Physiological Male-Sterile Anther In Common Wheat (Triticum Aestivum L.)

Physiological male sterility (PMS) induced by chemical hybrid agents (CHA) and its utilization are more successful system in wheat heterosis today. Utilization of CHA hybrids has many merits, the latest released varieties can be directly used for producing hybrid specially; SQ-1 is a new type of wheat CHAs, also the best wheat CHAs at home and abroad. However, understanding of its sterile mechanism is still poor, specially on the levels of gene and protein expression. In the paper, the sterile effects and absorbency of different leaves and spikes to CHA SQ-1 were studied, the cytological observation of pollen and ROS (reactive oxygen species) metabolism of anthers at different developmental stages were performed, then the differential gene expression was also compared using cDNA-AFLP technique, the genes related to male sterility induced by SQ-1 were identified and cloned, the relationship between genes expression and sterility was studied, the main results were as follows:1 At Feeke's 8.5～9.0 stage, daubing sufficient CHA SQ-1 on flag leaf, penultimate leaf, the third reciprocal leaf separately and two or three leaves simultaneous (application concentration: 1.5kg/300kg water ) can cause complete male sterility of wheat spikes; for the ability of absorbency and transfer, flag leaf is the best, the third reciprocal leaf is bad, the adaxial surface is better than that of abaxial surface of same leaf; daubing directly the spikelet with 5.01ug or 8.3ug SQ-1 at late mononuclear pollen stage will cause higher male sterility of daubed spikelet, but at late binuclear pollen stage only caused partial male sterility of daubed spikelet. Further, SQ-1 might be transported at regular direction in wheat spikes from male sterility of treatments D7～D10 and CK3. Daubing sufficient SQ-1 on the caulis leaf caused complete male sterility of caulis spike and partial male sterility of tillering spikes. The pollens abortion can be happen at any developmental stages from mononuclear to trinucleated cell, but the majority of pollens aborted stayed at late mononuclear to initial stage of binuclear cell, and the ration of aborted pollen at late mononuclear pollens was the most. Aborted pollen showed abnormal shape, without starch grains. Abortion-occurring stage was associated with spraying or daubing dose and developmental stage of pollen.2 The study on ROS metabolism of PMS showed:①During the young spike stage, o ?2. production rate and contents of H2O2 and malondialdehyde (MDA) were higher than those of corresponding controls, the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were also higher than those of corresponding controls, but the activities of peroxidase (POD)and catalase (CAT) were lower or significantly lower than those of their corresponding controls, so the susceptibility of different antioxidase to CHA stress before pollen abortion was different②From early mononuclear pollen stage to initial stage of binuclear pollen, on the one hand, o ?2. production rate and contents of H2O2 and MDA were significantly higher than those of their corresponding controls, but then the activities of SOD, POD, CAT and APX (except for early mononuclear pollen) were significantly lower than those of the corresponding controls, hence the metabolism balance of ROS in treated anthers was seriously destroyed, violent membrane lipid peroxidation was caused, subsequently the microspore developing was inhibited. At trinucleated pollen stage, the gaps of the o ?2. production rate and content of H2O2 were reduced, but that of MDA was still increased, at the same time the activities of four antioxidases between treatments and controls remained significant different. Therefore, ROS of aborted anthers still caused damage to themselves. During the key abortion stage, the increment of ROS in 1376-CHA anther were higher, but the decrement of activities of antioxidase was lower than those of 2611-CHA, which may be the reasons why the male sterility rate of xinong 1376-CHA was higher than that of xinong 2611-CHA. Excessive accumulation of o ?2.,H2O2 and MDA and significant reduction of SOD, POD and CAT antioxidase activities happened ahead of pollen abortion and went on during pollen abortion stage in the anthers of wheat PMS, the imbalance of ROS metabolism was the main physiological reason of pollen abortion in PMS.3 cDNA-AFLP analysis was conducted in PMS and normal anthers at key abortion stage. Total 144 non-redundant transcript-derived fragments (TDFs) were cloned and identified, the majority of these cloned genes were complete presence or absence (qualitative variants), and the others were differentially expressed (quantitative variants). The biological role of these TDFs mainly involved in defense system against oxidation stress(5.6%), signal transduction and transcriptional regulation(15.3%), nucleic acid metabolism(2.8%), energy and protein metabolism(12.5% and 13.2%), intracellular transporters (5.6%), cell wall and cytoskeleton components(2.8%) , whereas 35.2% of them either had lower similarity with known function gene (or protein) or their function were unknown.The results showed that CHA SQ-1 triggered dynamic changes at transcriptional regulation levels when uninucleate pollen developed to bi-nuclear pollen. The course of pollen abortion involved in intricate physiological and biochemical metabolism. The majority of genes were involved transcriptional regulation, energy and protein metabolism. But the relationship between transcriptional factors and other genes remained to be studied. 4 Seven interested TDFs were extended by electronic cloning, six of them were obtained cDNA sequences with entire ORF, their encoded amino acid had typical conserved domains, they were Urm1(ubiquitin related modifier 1), ubiquitin-S27a, U-box domain protein, pyruvate dehydrogenase E1 alpha subunit, Arf GTPase activating protein, Cytochrome P450 family protein and aconitase, respectively. The cDNA and amino acid sequences of these genes were compared with some homology genes. The partial cDNA sequences of Urm1 and ubiqutin-S27a were amplified and cloned.5 Two genes related to oxidation stress, four key genes related to ubiquitin-26S proteasome pathway (UPP) and three genes related to energy metabolism were selected to conduct RT-PCR analysis for revealing the relationship between them and male sterility induced by CHA SQ-1, the results indicated that cytoplasmic APX and Pyridine nucleotide-disulphide oxidoreductase were down-regulated in PMS anthers at key abortion period, and caused the imbalance of ROS production and scavenging, some key enzymes were oxidized and resulted in pollen abortion. The result was accordant with ROS determination. Urm1, ubiquitin-S27a and F-box domain protein (TDF360) showed up-regulated expression at key abortion stage, but the other F-box domain protein (TDF362) showed significantly down-regulated in PMS anther, the two F-box domain proteins were obviously expressed during instantaneous period. So the degradation of some key enzyme or protein related to anther development was accurately controlled by ubiquitin-26S proteasome pathway, CHA SQ-1 leaded to PMS in wheat through affecting expression of regulating proteins of the key enzymes.6 GAPDH, a key enzyme of glycolysis pathway, aconitase, a key enzyme of tricarboxylic acid cycle (TCA cycle), they all showed down-regulated expression in PMS anther at different developmental stage, especially at key stage of pollen abortion, so ATP production was impeded; NFU domain protein4 mainly provides molecule scaffolds of Fe-S cluster biogenesis located in mitochondria such as aconitase and succinate dehydrogenase, and involved in electron transfer, its down-regulated expression in sterile anther maybe caused male sterility by repressing the efficiency of electron transfer and affecting energy supply. Further study about the gene is underway.