Cytokinin And Auxin-regulated Root Meristem Establishment During Arabidopsis Somatic Embryogenesis

Somatic embryos (SE) have been defined as structures that arise from somatic cells but resemble zygotic embryos. Somatic embryogenesis is an important way to generate SE and whole plants. Since the initial description of SE formation from carrot callus cells, this unique developmental process has been recognized both as an important way for plants regeneration from cell culture systems and as a potential model for studying early regulatory and morphogenetic events in plant embryogenesis. Thus, somatic embryogenesis has been used for studying the plant cell totipotency and the developmental events in zygotic embryogenesis. However, molecular mechanisms of somatic embryogenesis remain largely unknown. Previous studies in our laboratory have demonstrated that auxin-induced WUS expression is essential for shoot apical meristem (SAM) formation during Arabidopsis somatic embryogenesis. Herein, we focus on studying the mechanism of root apical meristem (RAM) establishment and effects of microRNA (miRNA) in Arabidopsis somatic embryogenesis. Our results shed new light on molecular mechanisms regulating somatic embryogenesis in Arabidopsis. The main results are as follows:(1) RAM establishment was regulated by cytokinin during early somatic embryogenesisIn order to study the initiation of RAM stem cells during somatic embryogenesis in Arabidopsis, we analyzed the expression pattern of genes which were reported to express in the RAM during embryogenesis using laser scanning confocal microscope. The localization of root quiescent center-specific gene WUSCHEL RELATED HOMEOBOX 5 (WOX5) was analyzed by pWOX5::GFP expression. WOX5 gene expression was detected in embryonic calli within 16 hours after SE induction. Then, signals of other genes involved in root stem cell specification during early embryogenesis such as PLETHORA 2 (PLT2), SHORT ROOT (SHR) and SCARECROW (SCR), were detected sequentially within two days after SE induction. More importantly, repressing the expression of WOX5 or PLT resulted in abnormal somatic embryogenesis. Embryonic callus of pER8-WOX5-anti almost lost the ability to reproduce SE after induction by 17β-estradiol. Primary SE of the plt1-1 plt2-1 double mutant could form normal shoot apex with cotyledons, but defected in root apex and SE elongation. These results indicated that RAM-specific genes play important roles in SE root development during early somatic embryogenesis.Auxin that is important in SAM renewal only concentrated around shoot apex during early somatic embryogenesis, but not in RAM region, because the genes related to auxin biosynthesis and transport only expressed in shoot apex. Fluorescence signal of DR5rev::YFP or DR5rev::GFP, which mark auxin distribution, did not overlap with the signal of pWOX5::GFP or pPLT2::RFP during early somatic embryogenesis. Only after 5 days induction, auxin response signal could be detected in the root apex.Application of cytokinin synthesis inhibitor lovastatin led to complete loss of embryonic ability in the callus, and failure to induce SE. Further experiments showed that the expression region of ARABIDOPSIS RESPONSE REGULATOR 7 (ARR7), which is used to mark cytokinin response, could overlap the expression region of PLT2 during early somatic embryogenesis, and the expression of cytokinin biosynthetic gene IPT5 was detected in the root apex. Furthermore, repressing the cytokinin signal transduction resulted in defective somatic embryogenesis. The double mutants of cytokinin receptor genes ARABIDOPSIS HISTIDINE KINASE (AHK), ahk2 ahk4 and ahk3 ahk4, showed similar phenotype to that of the double mutant plt1-1 plt2-1 in the root apex. Overexpression of ARR7 and ARR15 both resulted in abnormal primary and secondary SE. These results indicated that cytokinin specifically distributed in root apex, and regulated the establishment of RAM through its signal transduction.Our results suggest that although the interaction between auxin and cytokinin plays an important role in the establishment of apical-basal polarity in zygotic embryo, there is a different pattern in the SE shoot-root apex polarity. Auxin regulates WUS expression and SAM renewal in shoot apex while cytokinin controls WOX5 expression and RAM formation in root apex.(2) MiR167 regulated somatic embryogenesis through its target genes ARF6 and ARF8 MiRNAs are small, endogenous RNAs that regulate gene expression in plants and animals. In plants, these ~21 nucleotide RNAs are processed from stem-loop regions of long primary transcripts and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs of their target genes. In recent years, more and more functions of miRNA were revealed at various stages of plant growth and development. We detected a large number of miRNA differentially expressed during SE induction by microarray technology, and found that miR167 played an important role in Arabidopsis somatic embryogenesis.First, we showed that overexpression of miR167 family members decreased somatic embryogenesis frequency. Moreover, the expression of WUS and embryo-specific genes, such as LEAFY COTYLEDON 1 (LEC1), LEC2 and FUSCA 3 (FUS3), were repressed. Further experiments indicated that the polar localization of PIN-FORMED 1 (PIN1) and normal expression pattern of WUS were not detected in 35S::miR167c, resulting in disrupted pattern of SE shoot stem cell renewal.Both miR167c and its target genes AUXIN RESPONSE FACTOR 6 (ARF6)/ARF8 expressed specifically in embryonic callus where SE was produced. In addition, mutants of ARF6 and ARF8 also showed defects in somatic embryogenesis. Primary SE of arf6-2, arf8-3 and arf6-2 arf8-3/+ were morphologically normal, but the frequency of secondary SE induction decreased obviously.Our results suggested that miR167 regulated auxin polar transport and gradient distribution through auxin response factor ARF6 and ARF8, thereby affecting RAM establishment in somatic embryogenesis.