Profiling Gene Expression in Germinating Brassica Roots

Abstract

Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

Keywords: Brassica; Gene expression profiling; Solid-phase gene extraction; mRNA quality.

Fig. 1

LabChip® 90 electropherograms of DEPC-treated water (a), total RNA extracted from roots of Brasscia seedlings (b), messenger RNA (mRNA) extracted from total RNA (c), and mRNA extracted from total RNA by SPGE (d). Peaks represent the lead marker (50 nt, arrow head) or ribosomal RNAs

Fig. 2

Amounts of SPGE-extracted mRNA with probes coated with different concentrations of the NH₂-oligo (dT)₁₅ nucleotides; average ± SE, n = 9. Least significance difference for SPGE-extracted mRNA is 0.41 ng (p < 0.05)

Fig. 3

SPGE-extracted mRNA (a) and relative amount of ACT7 per SPGE probe (b). SPGE probes were either incubated in total RNA of Brassica root and directly reverse transcribed or washed with buffer before reverse transcription and quantification; average ± SE, n = 9. Least significant difference (p < 0.05) for SPGE-extracted mRNA is 0.40 ng and for ACT7 copies is 1,164 copies

Fig. 4

Quantity of mRNA extracted from total RNA by SPGE (—) and number of copies of ACT7 after multiple usage of SPGE probes (- - -). The probes were washed before release of mRNA (80 °C for 3 min). Average ± SE, n = 9. Least significance difference (p < 0.05) for amounts of SPGE-extracted mRNA amounts of SPGE-extracted mRNA is 0.39 ng, and for number of copies of ACT7 is 1,403 copies

Fig. 5

Amount of mRNA (squares) and copies of ACT7 per probe (circles) as a function of storage at 4 °C (filled symbols) or RT (open symbols) after storing SPGE probes prior to extraction (a) and the stability of mRNA on the SPGE probe prior to reverse transcription (b). Average ± SE, n = 9. The least significance difference (p < 0.05) for SPGE-extracted mRNA is 0.35 ng and for ACT7 copy number is 1046 (Fig. 5a). The least significance difference for SPGE-extracted mRNA is 208 copies (Fig. 5b)

Fig. 6

Relative expression of highly expressed genes (ACT7, UBQ; a) or low expressed genes (TUB, GLK; b) along 10-mm-long primary Brassica roots. Root were sampled about 36 h after germination at five positions (a, insert) and the copy number of ACT7, UBQ, TUB, and GLK determined at each sample position [1 (tip) to 5 (root–shoot junction); average ± SE, n = 9]. Least significance differences at p < 0.05 for copy numbers of ACT7, UBQ, TUB, and GLK are 61, 229, 32, and 21 copies, respectively

Fig. 7

Expression of ubiquitin (UBQ, top) and glucokinase (GLK, bottom) of Brasscia primary roots. The roots were sampled at the meristem (square), elongation zone (diamond) and root-shoot junction (circle) between 6 and 24 h after germination; average ± SE, n = 9. Least significance differences (p < 0.05) for UBQ copy numbers in MZ, EZ, and RS are 164, 683, and 666 copies, respectively; for GLK in MZ, EZ, and RS, the values are 5.7, 18.7, and 10.8 copies, respectively