Periplasmic Microtubules And ABA Were Involved In The Cell Growth Of Arabidopsis Seedlings

Microtubules(MTs) and abscisic acid(ABA) are both involved in varied stages regulation of plant growth and development. The dynamic of microtubule arrays are mainly regulated by microtubule associated proteins(MAPs). While it remains unclear whether there are some components mediated the potential interaction between microtubule and ABA and how they working on. In order to elucidate the specific mechanism of the potential link between ABA and MTs, we use Arabidopsis wild type and kinds of mutants sad2-2?aba3?abi1,2?abi4 and abi5 as donor materials to process integrated analysis including phenotype screening, cell biology observation and transcript analysis aiming to find out the key components of ABA and MTs interaction. The experimental results are shown as follows:(1) Results of root length phenotype analysis: Seven-day-old Arabidopsis seedlings on the MS medium were upside down and then analysis the average growth rate of roots. The average growth rate of sensitive to ABA and drought mutant sad2-2 is 4.40 compare to wild type 3.58 mm/d, while the average growth rate of ABA synthesis deficient mutant aba3 is 0 mm/d and other ABA insensitive mutants abi1,2, abi4 and abi5 are 3.57 mm/d, 3.71 mm/d and 3.96 mm/d. Seven-day-old seedlings on the MS medium were transfer to a new MS culture medium containing 0.3 ?mol/L oryzalin and then kept been cultured upside down and analyzed the average growth rate of roots. The average growth rate of wild type is 3.66 mm/d, mutant sad2-2 are 5.26 mm/d, and mutant aba3 is 0 mm/d and other ABA insensitive mutants abi1,2, abi4 and abi5 are 3.66 mm/d, 4.18mm/d and 4.01mm/d. Seven-day-old seedlings on the MS medium were transfer to a new MS culture medium containing 1 ?mol/L taxol and kept been cultured upside down and analyzed the average growth rate of roots., the average growth rate of wild type and sad2-2 aba3 are 3.19 mm/d, 4.57 mm/d, and 0 mm/d.According to the above data, sensitive to ABA and drought sad2-2 is insensitive to oryzalin and taxol. While ABA synthesis deficient mutant aba3 is sensitive to taxol, which indicate that the ABA level affected the growth of Arabidopsis seedlings roots. Results of ABA insensitive mutants screening suggest that ABA insensitive mutants abi4 is insensitive to oryzalin.(2) Observation of cortical microtubule array of the kinds of mutants via confocal microscope: Results showed that the cortical arrays of mutant sad2-2 are tend to more dense with relatively strong green fluorescence compare to wild type, while cortical arrays of aba3 are loose and depolymerized and oblique. After application of 0.3 ?mol/L oryzalin, specific polymerization inhibitor, microtubule arrays of wild type are depolymerized, but less in sad2-2 while more severe in aba3 than that of WT. In the group of ABA insensitive mutants, microtubule arrays of abi4 are mostly not depolymerized. The observation of hypocotyl cell microtubules arrays show that wild type and mutant abi4 are mostly in the state of parallel, and after treated with 0.3 ?mol/L oryzalin microtubule arrays of wild type are become oblique(73%), however abi4 are still mostly in the state of parallel. After treated with 1 ?mol/L taxol, microtubule arrays of wild type are mostly in the state of parallel, while mutant abi4 are become oblique(67%).According to the above data, mutant aba3 is sensitive to taxol but insensitive to oryzalin, which indicates that ABA level influence the growth of Arabidopsis roots and cortical microtubule arrays are also involved in the progress. And ABA insensitive mutants abi4 is also insensitive to oryzalin.(3) According to the data of the Arabidopsis Information Resource(TAIR) that MAP18 may be regulated by ABA, we analyze the MAP18 transcription in wild type and different mutants seedlings with and without 5 ?mol/L ABA treatment using Real-time PCR. The results show that MAP18 gene expression of mutant abi1,2, abi5, sad2-2 and aba3 were increased 2.95 fold, 1.98 fold, 2.95 fold and 3.97 fold comparing to wild type control while mutant abi4 is 0.6 fold of wild type. After ABA treatment, wt were 0.49 fold of control. Mutant abi1,2, abi5, sad2-2 and aba3 were 2.82 fold,1.89 fold,2.13 fold and 2.50 fold of control wt while mutant abi4 is 0.51 of control wt.Based on the analysis of phenotype, microtubule array observation and MAP18 transcription, we find that there is a link between ABA and MTs in regulating Arabidopsis root and hypocotyl growth. And microtubule associated proteins MAP18 and transcription factors ABI4 may mediate the interaction as the downstream factors. Our data provide clues to further investigate the mechanism and potential key factors of the interaction between MTs and ABA that co-regulate the plant growth and development.