The Identification And Expression Analysis Of The Transcription Factor ZmPTF1Target Genes

ZmPTF1, A bHLH (basic helix-loop-helix domain) transcription factor involved in response to Pi starvation, was cloned from maize with an RT-PCR and RACE approach in our previous work by Gao Qiang. Real-time RT-PCR revealed that ZmPTF1was quickly and significantly up-regulated in the root under phosphate starvation conditions. The full-length ZmPTFl cDNA was transferred into the maize genome under the control of the ubiquitin promoter by Li et al. Overexpression of ZmPTF1in maize improved root development in different mediums and the sense lines displayed better developed roots compared to WT. When cultured in low phosphate soil, the sense plants developed more tassel branches and larger kernels and were less affected by low phosphate stress. Some low phosphate stress response genes have higher expression levels in the overexpression lines. Compared with wild type, overexpressing ZmPTF1altered the concentrations of soluble sugars in transgenic plants, in which soluble sugars (Glu+Fru+Suc) levels were lower in the leaves and higher in the roots. Overexpression of ZmPTF1enhanced the expression of fructose-1,6-bisphosphatase and sucrose phosphate synthasel participated in sucrose synthesis in the leaves but decreased them in the root, and reduced the expression of genes involved in sucrose catabolism in the roots. Overexpression of ZmPTF1resulted in the modifications on the physiology and root morphology of transgenic plants, which may contribute to more rapid adaption ability of plants to low phosphate stress and further reduce the influence of stress on plant growth, development and yields. These results are deserved by Li in our lab.In this work, seedlings of transgenic maize plants with integrated ZmPTF1and maize inbred line DH4866, were chosen as the material. Digital Gene Expression Profiling was applied to find the differentially expressed genes (DEGs) in maize roots. We use "FDR≤0.001and the absolute value of log2Ratio≥1" as the threshold to judge the significance of gene expression difference. More stringent criteria with smaller FDR and bigger fold-change value can be used to identify DEGs. The results showed that755genes were differently expressed in transgenic maize and maize inbred line,526genes were up-regulated and229genes were down-regulated. On the basis of GO enrichment analysis and pathway enrichment analysis, we identified that DEGs were significantly enriched in several metabolic pathways or signal transduction pathways comparing with the whole genome background, including PAMPs-triggered immunity, plant hormone biosynthesis and signal transduction, carbohydrate metabolism and secondary metabolism et al.Overexpression of ZmPTF1in maize induced changes in expression levels of a serial of genes involved in flagellin signaling,, including coding a LRR receptor kinase FLS2gene, MEKK1and MKK4/5of mitogen-activated protein kinase (MAPKs), WRKY22/29and WRKY25/33of WRKY transcription factor genes. Compared with WT, a lot of other genes involved in PAMPs-triggered immunity were up-regulated in ZmPTFl overexpression transgenic plants, for example, RPM1, RPS2, PBS1, etc. Besides, some genes involved in JA and SA signal transduction pathways were differently expressed. These data showed that the resistance to pathogen of maize may be strengthened by overexpression of ZmPTF1.The biosynthesis and signal transduction of some hormone, such as auxin, cytokinin, abscisic acid and gibberellin, are influenced by overexpression of ZmPTF1. In ZmPTF1overexpression plants,, auxin influx transporter A UX1and auxin response factor ARF1were down-regulated, while auxin responsive protein gene SAUR25, SAUR56and SAUR20were remarkably up-regulated. Compared with WT, the cytokinin receptor gene CRE1was down-regulated. GID1and DELLA protein gene involved in GA signal transduction pathway were up-and down-regulated respectively. The expression level of ERF1/2and other ERFs were remarkably up-regulated due to overexpression of ZmPTF1. These results suggested that overexpression of ZmPTF1induced changes in expression levels of genes involved in multiple plant hormone signaling pathways, which most likely led to expression levels changes of down-stream gene regulated by hormone signaling, further contributing to better developed roots.Overexpression of ZmPTF1enhanced the expression of sucrose phosphate synthasel and beta-fructofuranosidase participated in sucrose synthesis, and reduced the expression of genes involved in starch synthesis, such as alpha,alpha-frehalase and beta-amylase3. These data were in accordance with the results of Li.Furthermore, some genes involved in protein degradation were up-regulared to some extent in transgenic maize. These data suggested that protein degration, especially ubiqutin-mediated protein degradation pathway, were strengthened.