Fluorescent imaging of GUS activity and RT-PCR analysis of gene expression in the shoot apical meristem.
The use of promoter-reporter gene constructs in transgenic plants is a powerful tool in the analysis of gene expression which can, however, be limited in the resolution of small structures, such as the apical meristem. This paper reports on a fluorescent imaging technique for the analysis of GUS reporter gene expression to cellular resolution in the apical meristem of tomato. Using this technique in combination with an RT-PCR analysis of RBS gene-specific transcript levels, it is shown that: 5' upstream sequences of RBCS genes are sufficient to mimic the pattern of transcripts revealed by in situ hybridisation (no expression in the apical meristem, high expression in the initiated leaf primordia); the genes RBCS2, RBCS3A and RBCS3B are transcriptionally activated upon primordium initiation with transcripts for RBCS1 and RBCS3C accumulating later in leaf development; and that RBCS promoter activity cannot be induced in the apical meristem by light, an environmental signal which elevates RBCS transcript level in other aerial parts of the plant. These data provide a detailed picture of the complex transcriptional events occurring on leaf initiation and the establishment of the photosynthetic machinery; they describe two complementary techniques which allow the analysis of such complex events at the tissue and cellular level; and they characterize an in vivo assay system which can be used to analyse the factors involved in the initiation and maintenance of gene expression patterns in the apical meristem.
Institute of Plant Physiology, University of Berne, Switzerland.
A J
AJ
Fleming
T
T
Manzara
W
W
Gruissem
C
C
Kuhlemeier
D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics / Q000302:isolation & purification; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018551:Solanum lycopersicum / Q000235:genetics; D018519:Meristem / Q000033:anatomy & histology / Q000201:enzymology / Q000254:growth & development*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009024:Morphogenesis; D005810:Multigene Family; D018515:Plant Leaves / Q000166:cytology; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification; D018749:RNA, Plant / Q000302:isolation & purification; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic
D012333:RNA, Messenger; D018749:RNA, Plant; D005966:Glucuronidase; D012273:Ribulose-Bisphosphate Carboxylase