| Somatic embryos (SEs) have been defined as structures that arise from somatic cells butresemble zygotic embryos. This system is recognized as a model to study the totipotency ofplant cells and the cellular and molecular mechanisms of early embryo development. DuringSE induction in Arabidopsis, removal of exogenous auxin from the medium triggers bothauxin polar transport and auxin gradient establishment in the embryonic callus that is essentialfor the SE initiation, but whether the auxin biosynthesis is also essential for the formation ofSE is not yet clear. Ethylene has been shown to be one of the important hormones which canregulate SE initiation and development. However, the molecular mechanisms underlying thefunction of ethylene on SE initiation are not well understood. In our research, we firstlyfocused on examining the role of auxin biosynthesis during SE induction in Arabidopsis. Andthen, the effect of ethylene on auxin biosynthesis and auxin response signal gradientestablishment was also studied. Our results will provide new information on the mechanismsof hormone-regulated SE initiation in Arabidopsis. The main results are as follows:(1) YUCCA-mediated local auxin biosynthesis is required for SE inductionDuring SE initiation, the expression level of YUC1, YUC2, YUC4, YUC6, etc. inembryonic calli was dramatically increased following removal of exogenous auxin (2,4-D)from the medium. Consistent with the expression patterns of the YUC genes, the freeauxin/IAA levels in embryonic calli were also increased. By using a construct containing aGUS gene driven by the YUC promoter, we evaluated the spatial expression pattern of YUCgenes. During the early phases of the SE induction, the GUS signals were observed to beregionally distributed to the future SE initiation sites of the embryonic callus. Genetic analysisconfirmed that the production of SEs was severely inhibited in the quadruple mutants yuc1yuc4yuc10yuc11. These results suggest that the regional expression of YUCs which mediatedauxin biosynthesis are essential for SE induction(2) Ethylene biosynthesis is decreased during SE initiationWe chose two time points to analyze the transcriptomes of embryonic calli using microarray. First, the mature embryonic calli. Second, the embryonic calli were cultured inSEIM for48hours. Our data showed that during SE induction, the expression levels of someauxin response genes were down-regulated. However, the expression level of YUC wasup-regulated, implying that endogenous auxin biosynthesis was promoted following SEinduction. Moreover, genes (ACS2, ACS6and ACS8) encoding the key enzymes of ethylenebiosynthesis were down-regulated, and the expression of genes involved in ethylene responsewas also reduced. Then, we analyzed the transcript levels of ACS genes by qRT-PCR.Consistent with the microarray results, the expression level of ACS genes dramaticallydown-regulated. The rate of ethylene release detected by gas chromatography progressivelydecreased from0h to48h during SE induction. These results suggest that the removal ofauxin from the medium causes the down-regulation of ACS mediated ethylene biosynthesis.(3) Ethylene inhibits SE formationTo further examine the function of ethylene during SE induction, we increased the levelsof endogenous ethylene in the embryonic callus by adding the ethylene biosynthesis precursor,1-aminocyclopropane-1-carboxylic acid (ACC), to the medium, and the SE induction wasinhibited. The inhibition effects of ACC on SE initiation aggravated with the elevatedconcentration of ACC applied.(4) Excessive ethylene affect SE initiation by reducing YUC mediated local auxinbiosynthesisThe transcript levels of YUC were decreased in embryonic calli following treatment with200μM ACC for from0h to48h. Meanwhile, the regional distribution of YUC expressionwas not observed in the calli, instead, dispersed and weak signals were identified along theedges of the callus. The distribution of DR5rev::GFP changed correspondingly. Thus, theseresults imply that ACC treatment that induces ethylene evolution inhibits local auxinbiosynthesis mediated by YUC that might be involved in local auxin distribution required forSE induction.Similar with the ACC treatment, in the ethylene overproduction mutant eto1-1, thedecreased regeneration frequency of SEs was observed. The transcript levels of YUC4weredecreased in the eto1-1mutant compared with those in the wild type. Further, in situhybridization revealed the disrupted regional distribution of YUC4transcript signals in the eto1-1mutant compared with that in the wild type. The signals were distributed along theedges of the eto1-1callus. Thus, the results suggest that excessive ethylene production causedby mutation of ETO1inhibits SE induction through disturbing local auxin biosynthesismediated by YUC.To determine the function of ethylene signaling during SE induction, we used the ctr1-1mutant to induce SEs. Compared with the wild type, the ctr1-1mutant generated the severelyabnormal embryonic callus following culture in ECIM, which did not give rise to SEs inSEIM. The expression levels of YUC were decreased in the ctr1-1calli. In situ hybridizationresults showed that YUC4and YUC1transcript signals were restricted to the SE initiation sites.However, mutation of CTR1caused the reduced transcript signals in the callus. Moreover, theregional distribution of DR5:GUS was also disrupted by mutation of CTR1. These resultssuggest that the constitutive ethylene response inhibits local auxin biosynthesis mediated byYUC that might be involved in local auxin distribution required for SE induction.Taken together, our results reveal that YUC mediated auxin biosynthesis was alsorequired for SE initiation in Arabidopsis. Removing exogenous auxin triggered decreasedethylene production and ethylene response, which is in turn required for local auxinbiosynthesis mediated by YUC genes. We further showed that excessive ethylene disturbedSE initiation through inhibiting YUC expression that might be involved in local auxinbiosynthesis and subsequent auxin distribution. |
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