| L-Serine (L-Ser) is involved in protein biosynthesis, and essential for synthesis of many important biomolecules in the cell. Hence, L-Ser deficiency was proved to cause arrested root development, pollen shrunken and altered, and insensitivity to ABA. It is also proposed as a possible signal molecule, as the previous work in our lab has shown that exogenous L-Ser prompted the senescence of intact plant and excised half-fronds in Spirodela polyrrhiza P143.To further investigate the effects and the mechanism of L-Ser to plants, Arabidopsis (a dicot) and Lemna minor (a monocot) were employed in this study. Based on our new findings about the phenotype changes caused by exogenous L-Ser treatment, we first investigated the mechanism underlying L-Ser effect which leads to an arrested primary root development and a promoted lateral root formation. Then, the serine:glyoxylate aminotransferase(AtAGTl), an important enzyme for L-Ser metabolism during photorespiration, was studied by both overexpression and knock-down, in order to change the expression level of SGAT, and to change the endogenous L-Ser level. From these studies, we wanted to confirm the function of L-Ser to plants founded by L-Ser treatment and at the same time to reveal new functions of SGAT. Additionally, a plant transformation vector of PAtAGT1:GUS was constructed and the histological expression of AtAGTl was studied. To further investigate the function of L-Ser in roots, overexpression vectors for3enzymes of the main L-Ser biosynthesis pathway in the root,3-phosphoglycerate dehydrogenase (PGDH),3-phosphoserine aminotransferase (PS AT) and3-phosphoserine phosphatase (PSP), respectively, were constructed and some of the transgenic plants were obtained. The main results of this study are summarized as follows:1. Foliar-spraying of1mM L-Ser (once per day.for10days) caused appearance of chlorosis and necrosis of Arabidopsis leaves,’Which were not found in the controlled treatment with1mM L-Trp, indicating that high level of L-Ser enhanced senescence in leaves of Arabidopsis. When L-Ser was applied into the culture medium of Arabidopsis germinating seedlings, it was found that treatment with more than1mM L-Ser inhibited the primary root development, but promoted the formation of lateral roots. Treated with2mM L-Ser for7d, the primary root length was found to be only1/4of the control plants. The effect was more obvious along with the increase of L-Ser concentration. The endogenous serine content was measured, and the results showed its alteration with the treatment of exogenous L-Ser. For example, the L-Ser content in roots of1.5mM L-Ser treated seedlings was4.5-fold of the control. Treatment of Arabidopsis DR5:GUS reporter plants by addition of L-Ser into the culture medium caused the enhancement of GUS activity in the primary root and the root tip, and also the alteration of GUS activity in the lateral root tips, which happened already from the2d and was very obvious after6d of the treatment. Gene expression analysis results showed that L-Ser treatment caused the up-regulation of the auxin influx carrier gene AUX1and the senescence-related transcription factor SAG12, and the down-regulation of the SGAT encoding gene AtAGT1.2. Treatment of L. minor callus with L-Ser improved the frond regeneration. Except for L-Ser, the other19amino acids were also tested and the results showed that L-Gly and L-Cys promoted regeneration, too. However, the regeneration promotion effects of these two amino acids were smaller than that of L-Ser. L-Gly and L-Ser can be converted to each other in plants. It was observed that with L-Gly treatment the endogenous level of L-Gly did not increase very much and that the increase was much less than the increase in endogenous level of L-Ser, indicating that much of the L-Gly absorbed was converted into L-Ser, which in turn promoted the regeneration. As the precursor of L-Cys, L-Ser content might be maintained on a certain higher level by decreasing its conversion to L-Cys when the callus was cultured on the medium added with L-Cys. With the addition of1mM L-Ser, the callus regeneration frequency reached46%and87%, on the11th and14th day, respectively. And this fast speed has never been reported. The best concentration for L-Ser and L-Gly to promote callus regeneration was1.0mM and1.5mM, respectively.3. Overexpression (OE) of AtAGT1in L. minor showed the increase of SGAT activity and decrease of L-Ser. The OE transgenic duckweed displayed no significant difference with wide type (WT). However, when cultured with salinity stress, the salt resistance of transgenic OE duckweed was enhanced. The transgenic OE plants showed an enhanced protection against root abscission (the abscission root number was only1/3of the WT), a higher photosystem Ⅱ maximum quantum yield (Fv/Fm), a reduction in MDA, a reduction of reactive oxygen species (ROS) accumulation, and an improved antioxidant system. The SGAT activity in both transgenic OE plants and WT was induced in moderate salt stress (100mM) and inhibited in severe salt stress (300mM), and the activity of SGAT in transgenic OE plant was significantly higher than the one of WT; the L-Ser content in the WT declined with the enhancement of salt stress, however, the L-Ser content in transgenic plants increased in moderate salt stress and reduced in severe salt stress. These results showed that the enhancement of photorespiration pathway increased the tolerance to salinity due to the interaction with ROS and antioxidant system.4. The AtAGT1expression has been knocked down by RNAi. The L-Ser content was increased in AtAGT1-RNAi transgenic lines. And experiment results showed that the down-expression of AtAGT1caused dwarfism, decreased in root length, chlorosis and necrosis. Further study showed that the net photosynthetic rate in the AtAGT1-RNAi transgenic lines decreased. And the absorption spectrum of photosynthetic pigments showed that the photosynthetic pigments declined in the AtAGT1-RNAi transgenic plants, especially the chlorophyll a and chlorophyll b. The lutein content in the AtAGT1-RNAi transgenic plants was increased.5. The promoter region of the Arabidopsis AtAGT1was analyzed by bioinformatics and showed cis-acting element of abscisic acid (ABA), methyl jasmonate (MeJA), gibberellin (GA), and salicylic acid (SA), and the consensus sequences of root. These results indicated the responding of AtAGT1to those plant hormones, and the root location of AtAGT1protein. The vector of PAtAGT1:GUS was constructed and the PAtAGT1:GUS transgenic Arabidopsis was obtained. The GUS assay was taken and the GUS activity can be detected in cotyledons, leaves, hypocotyls, and roots. This result proved that AtAGT1was expressed in both aerial part and root. Moreover, the GUS activity was stronger in young leaves than in old leaves. Salinity and drought stress enhanced the activity of GUS. Finally, PAtAGT1: GUS might also respond to the hormone treatment, and therefore, the mechanism underlying needs a detailed investigation in the future. |
PDF Full Text Request