Liquid Phase Fluorescence in situ RT-PCR Analysis for Gene Expression Analysis in Woody Stems

Abstract

We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen (Populus tremula L. × tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.

Key words

Confocal laser scanning microscopy - in situ RT-PCR - Populus - vascular cambium - wood - xylem

References

• 1 Allona I., Quinn M., Shoop E., Swope K., St. Cyr S., Carlis J., Riedl J., Retzel E., Campbel M. M., Sederoff R. R., Whetten R. W.. Analysis of xylem formation in pine by cDNA sequencing.  Proceedings of the National Academy of Sciences. (1998);  95 9693-9698
• 2 Burlat V., Kwon M., Davin L. B., Lewis N. G.. Dirigent proteins and dirigent sites in lignifying tissues.  Phytochemistry. (2001);  57 883-897
• 3 Catesson A. M.. Cambial ultrastructure and biochemistry: changes in relation to vascular tissue differentiation and the seasonal cycle.  International Journal of Plant Sciences. (1994);  155 251-261
• 4 Chang S., Puryear J., Cairney J.. A simple and efficient method for isolating RNA from pine trees.  Plant Molecular Biology Reporter. (1993);  11 113-116
• 5 Choi S.-B., Wang C., Muench D. G., Ozawa K., Franceschi V. R., Wu Y., Okita T. W.. Messenger RNA targeting of rice seed storage proteins to specific ER subdomains.  Nature. (2000);  407 765-767
• 6 Church G., Gilbert W.. Genomic sequencing.  Proc. Natl. Acad. Sci. USA. (1984);  81 1991-1995
• 7 Harding S. A., Leshkevich J., Chiang V. L., Tsai C. J.. Differential substrate inhibition couples kinetically distinct 4-coumarate: coenzyme A ligases with spatially distinct metabolic roles in quaking aspen.  Plant Physiology. (2002);  128 428-438
• 8 Hawkins S., Boudet A., Grima-Pettenati J.. Characterisation of caffeic acid O-methyltransferase and cinnamyl alcohol dehydrogenase expression patterns by in situ hybridisation in Eucalyptus gunnii Hook. plantlets.  Plant Science. (2003);  164 165-173
• 9 Hawkins S., Samaj J., Lauvergeat V., Boudet A., Grima-Pettenati J.. Cinnamyl alcohol dehydrogenase: identification of new sites of promoter activity in transgenic poplar.  Plant Physiology. (1997);  113 321-325
• 10 Hertzberg M., Aspeborg H., Schrader J., Andersson A., Erlandsson R., Blomqvist K., . Bhalerao R., Uhlén M., Teeri T. T., Lundeberg J., Sundberg B., Nilsson P., Sandberg G.. A transcriptional roadmap to wood formation.  Proceedings of the National Academy of Sciences, USA. (2001);  98 14732-14737
• 11 Johansen B.. In situ PCR on plant material with sub-cellular resolution.  Annals of Botany. (1997);  80 697-700
• 12 Koltai H., Bird D. M.. High throughput cellular localization of specific plant mRNAs by liquid-phase in situ reverse transcription-polymerase chain reaction of tissue sections.  Plant Physiology. (2000);  123 1203-1212
• 13 Kwon M., Davin L. B., Lewis N. G.. In situ hybridization and immunolocalization of lignan reductases in woody tissues: implications for heartwood formation and other forms of vascular tissue preservation.  Phytochemistry. (2001);  57 899-914
• 14 Milioni D., Sado P. E., Stacey N. J., Domingo C., Roberts K., McCann M. C.. Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system.  Plant Molecular Biology. (2001);  47 221-238
• 15 Oelmüller R.. Photooxidative destruction of chloroplasts and its effect on nuclear gene expression and extraplastidic enzyme levels.  Photochemistry and Photobiology. (1989);  49 229-239
• 16 Panchuk-Voloshima N., Haugland R. P., Bishop-Stewart J., Bhalgat M. K., Millard P. J., Mao F., Leung W.-Y., Haugland R. P.. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugate.  The Journal of Histochemistry & Cytochemistry. (1999);  47 1179-1188
• 17 Regan S., Bourquin V., Tuominen H., Sundberg B.. Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues.  Plant Journal. (1999);  19 363-369
• 18 Riding R. T., Little C. H. A.. Histochemistry of the dormant cascular cambium of Abies balsamea - Changes associated with tree age and crown position.  Canadian Journals of Botany. (1986);  64 2082-2087
• 19 Ruiz-Medrano R., Xoconostle-Cázares B., Lucas W. J.. Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants.  Development. (1999);  126 4405-4419
• 20 Sterky F., Regan S., Karlsson J., Hertzberg M., Rohde A., Holmberg A., Amini B., Bhalerao R., Larsson M., Villarroel R., VanMontagu M., Sandberg G., Olsson O., Teeri T. T., Boerjan W., Gustafsson P., Uhlén M., Sundberg B., Lundeberg J.. Gene discovery in the wood-forming tissues of poplar: analysis of 5692 expressed sequence tags.  Proceedings of the National Academy of Sciences, USA. (1998);  95 13330-13335
• 21 Thompson G. A., Schulz A.. Macromolecular trafficking in the phloem.  Trends in Plant Science. (1999);  4 354-360
• 22 Taylor W. C.. Regulatory interactions between nuclear and plastid genomes.  Annual Review of Plant Physiology and Plant Molecular Biology. (1989);  40 211-233
• 23 Wu L. G., Joshi S. P., Chiang V. L.. A xylem-specific cellulose synthase gene from aspen (Populus tremuloides) is responsive to mechanical stress.  Plant Journal. (2000);  22 495-502

Section Editor: H. Rennenberg