Effects Of Arabinogalactan-proteins On Early Embryogenic Pattern Formation Of Microspore-derived Embryo In Brassica

Arabinogalactan proteins(AGPs)widely exist in plant organs like roots,stems,leaves,flowers and seeds which can be secreted into the extracellular matrix,and involved in all aspects of plant growth and development,such as nutrition,reproduction,cell division and growth,organs formation,embryonic development and programmed cell death.In this paper,Brassica microspores were used as experimental materials to study the effect of AGPs on early embryogenic pattern formation by using microspore culture technology,cell biology tools,and statistical analysis,the main results are as follows:1.In the conditional rape microspore culture,mcirospores could carry on emberogenesis and formed the embryos without suspensor.Comparing to embryogenesis of intact-exine microspore,the micro spore-derived embryos with suspensor were found in exine-dehisced microspore culture.The cell polarity was generated by the exine rupture,resulting in the different cell fate of the daughter cells and forming the embryo with suspensor.This process was similar to zygotic embryogeneis.On the other hand,the de-exined microspores could not embryogenesis and the de-exined microspore just developed into the irregular cell clusters.Those results showed that the exine may regulate microspore embryogenesis.2.?Glc Y,an artificial synthetic components which can bind AGPs and inhibit the factor of AGPs,can inhibit microspore initial division and can not result in embryogenesis.Compared with the control culture,no embryos or very little embryos were observed when micro spores were treated with 50?M ?GlcY for 5 days.The further studies showed that?GlcY mainly inhibit the expression of GRP,an relative embryonic gene.Treated with ?GlcY for 1 day,3 days and 5 days,respectively,the number of microspores expressed GRP was less than 1%,while the control group can reach to 10%in 5th day.To further examine whether the effect of ?GlcY inhibition is reversible,microspores were moved to fresh NLN-13 medium after treated with ?GlcY for 1 day,2days and 3days,respectively.The results suggested that?GlcY inhibition could be reversible.The microspores can continue to divide and develop into a normal embryo.There was no significant difference compared with control group.3.The interaction of ?GlcY/AGPs can affect embryonic cell differentiation and cell fate.Cytological evidence showed that the embryo epidermal cells swelled,tissues loosed,cells arranged irregularly,vascular tissue construction changed,many root-hair-like structures emerged and cotyledon developed abnormally when microspore-derived embryos were treated with ?GlcY.These changes suggested that AGPs/?GlcY interaction play an important role in pattern formation during the development of microspore-derived embryo.The expression of the auxin transport vector gene PIN1-GFP and auxin response reporter gene DR5-GFP were studied in the transition from the globular stage to heart stage of micro spore embryo genesis.The results indicated that PGlcY can have an effect on auxin polar transport.In the control group,PIN1 expressed mainly in the apical epidermal cells and vascular tissue of the early globular embryo showing no polarity,while in heart-stage embryo PIN1 expressed in the apical epidermal cells and vascular tissue showing polarity distribution.When the globular embryos were treated with ?GlcY for 2 days,PIN1 expression decreased significantly in the epidermal cells and the vascular tissue of embryos.In the control group,DR5 expressed in the proper and the base cells of the globular stage embryos,while in heart stage embryos,DR5 was only expressed in a little layers of basal pole of embryos.The expression of DR5 in globular embryos was inhibited after treated with ?GlcY.A diffuse pattern was found in the globular stage embryo.The abnormal embryos with one cotyledon were found during the culture and DR5 was expressed in the site of the abnormal cotyledons.These results indicate that ?GlcY/AGPs interaction changed the auxin transport level in microspore embryogenesis,resulting in the abnormal development of embryos.4.?GlcY affect embryonic development of epidermal cells in micropsore-derived embryos.Embryonic gene LEC1 and GRP can only expressed in the epidermal cells of microspores-derived embryos.LEC1 hardly expressed in the globular stage embryos after treated with ?GlcY for 2 days.If the globular stage embryo develop into the heart-shaped embryo after treated,the expression of LEC1 were only found in a few epidermal cells of embryos.The expression pattern of GRP was the similar to that of LEC1 when the globular stage embryos were stunted after treated.If the globular embryos developed into the heart-shaped embryos after treated,the expression of GRP was scattered in a few cells weakly.The results showed that ?GlcY/AGPs interaction regulate cell fate of the epidermal cells in embryos.