A Novel Mutant In Arabidopsis With Alterated Responses To Cytokinins And Defective In Root Gravitropism

This work, started with screening of T-DNA insert Arabidopsis thaliana stocks, a gravitropic response extremely reduced mutant, ckrc1 (Cytokinin induced Root Curling 1), were isolated. Segregation and phenotype of the back-cross progeny (F_1BC1 and F_1BC2) revealed that ckrc1 is a recessive homozygous mutant. And the flanking sequence cloned by inverse-PCR revealed that At1g70560, which encode an alliinase-like protein, was interrupted. Alliinase is the most abundant protein in Allium plant. With its C-S lyase activity, alliinase is able to cleave the C-S bond of alliin to produce allicin. Because many of Allium plants are important economic vegetables, allinase structure, function, in vivo localization has been carried out. However, functions of alliinase in Arabidopsis thaliana were not clear till now.Incubation on TDZ containing MS medium for 2 weeks, ckrc1 has a longer roots than the wild type (wt). However, callus induced from ckrc1 explants by TDZ were not as vigorous as that from wt, with a less growth rate and frequency of callus induction/explant, ckrc1 is insensitive to cytokinin, the possible reasons might be: (i) cytokinin signal transduction in ckrc1 was interrupted; (ii) exogenous cytokinin decreased the endogenous IAA concentration, with a less decrease in ckrc1 than in wt. On IAA or TIBA containing medium, ckrc1 has the same phenotypes as wt.Incubation on MS basal medium for 7 days followed by incubation on cytokinin or TIBA containing medium for 4 days, roots oickrc1 curled while wt did not so any altered response. In a gravitropic response experiment, gravitropism of ckrc1 disappeared. ckrc2 and ckrc3, the two allelic mutants, donated by different laboratory, have the same phenotype as ckrc1. An auxin gradient across root tip caused by IAA polar transport determines root growth direction. In case of ckrc1, we thought, IAA polar transport was diminished and its redistribution across root tip was interrupted that led to a reduced gravitropism to ckrc1. The disappeared gravitropism can be rescued by NAA, an auxin analog that efficiently diffuses through the plasma membrane, but not by IAA, as the auxin is known to require the auxin influx carrier to penetrate the cells. Taken together, a primary conclusion can be made here: functions of auxin influx carriers were interrupted and caused reduced response to gravity and exogenous cytokinin stimulation in ckrc1 roots. However, the negative gravitropism in ckrc1 fluorescence stem was not affected, this may reflects the different gravity detector between root and shoot. Further more. CKRC expression in roots, revealed by semi-quantity PCR, was found much higher than in fluorescence stem, so CKRC may not be as important as in root.On MS basal medium wt grow faster than ckrc1, while when planted in soil no difference was found except the flowering time. The cortex and endodermis of ckrc1 root tip were not well differentiated and characterized by atactic cell shape, disorder delimitation and shorter root tip length, however, root cap structure was normal.Auxin and cytokinin mediated genes (IAA1, IAA2, ARR5 and ARR15) expression were elevated by IAA/ZT, respectively, but no difference were found between ckrc1 and wt. Further more, three plasmids (cDNA_CKRC-pBI121, full length CKRC-pCAMBIA1300, P_CKRC-pBI121) were constructed and the transformed seeds had been harvested. Rescued gravitropism was found in the transformed seedlings, and about 1/4 cDNA_CKRC-pBI121 transformed T2 seeling roots grown faster than the wild type...