DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells

Abstract

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridization analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed in non-proliferating tissue such as the mature leaves and elongation zone of root. The removal of DNA damage, cyclobutane pyrimidine dimers and (6-4) photoproducts, in both mature leaves and the root apical meristem (RAM) was observed after UV irradiation under light. In the dark, DNA damage in mature leaves was not repaired efficiently, but that in the RAM was removed rapidly. Using a rice 22K custom oligo DNA microarray, we compared global gene expression patterns in the shoot apical meristem (SAM) and mature leaves. Most of the excision repair genes were more strongly expressed in SAM. These results suggested that photoreactivation is the major DNA repair pathway for the major UV-induced damage in non-proliferating cells, while both photoreactivation and excision repair are active in proliferating cells.

Figure 1

Expression of DNA repair genes in various plant tissues. Each lane was loaded with 20 µg of total RNA isolated from SAM (lane 1), mature leaves (lane 2), young leaves (lane 3), flag leaves (lane 4), panicles (lane 5) or roots (lane 6). The blot was probed with ³²P-labeled cDNA. Similar amounts of RNA were loaded in each lane as confirmed by ethidium bromide staining (lowest panel).

Figure 2

Spatial expression pattern of DNA repair genes in the shoot apex region. In situ hybridization analysis was performed using longitudinal sections from the shoot apex region of 10-day-old rice seedlings. The plant tissues were probed with antisense riboprobe labeled with digoxigenin-UTP. The result using the sense probe for CPD photolyase is shown as a control. No hybridization signals were detected when sense probes for the other genes were used (data not shown).

Figure 3

Spatial expression pattern of DNA repair genes in the root tip region. In situ hybridization analysis was performed using longtitudinal sections from the root tip region of 10-day-old rice seedlings. The plant tissues were probed with antisense riboprobe labeled with digoxigenin-UTP. The result using the sense probe for CPD photolyase is shown as a control. No hybridization signals were detected when sense probes for the other genes were used (data not shown).

Figure 4

DNA repair activity of meristematic (root tips) and non-meristematic (mature leaves) tissues. The removal of DNA damage induced by UV-B irradiation was measured under light and in total darkness. (A) Removal of CPDs in the root tips. (B) Removal of CPDs in the mature leaves. (C) Removal of (6–4) photoproducts in the root tips. (D) Removal of (6–4) photoproducts in the mature leaves. Points represent an average of at least three independent experimental values (error bars are 1 SD), expressed as the percentage of the original amount of damage.

Figure 5

Scatter plots comparing gene expression levels in SAM and mature leaves. An Agilent rice 22K custom oligo DNA microarray was used. Each spot on the array is represented by a dot in the scatter plot. The results of color-swap experiments (left, mature leaf-Cy 3/SAM-Cy 5; right, SAM-Cy 3/mature leaf-Cy 5) are shown. The red line represents the diagonal (equal expression in the two tissues). The yellow line above represents a Cy-3/Cy-5 ratio of 5:1, while that below represents a Cy-3/Cy-5 ratio of 1:5.

Figure 6

Expression data for rice DNA repair genes. Scatter plots were drawn based on expression data for 90 DNA repair genes spotted on the rice 22K custom oligo DNA microarray slide. Red and green dots represent spots for NER genes and the CPD photolyase gene, respectively. Blue dots represent spots for other dark repair genes (BER, MMR, HR, etc.). The red line represents the diagonal line.

Figure 7

Comparison of expression levels of DNA repair genes between SAM and mature leaves. The expression levels of 90 DNA repair genes are shown in a bar chart. Figures on the bars represent the fold expression.

Figure 8

Overview of DNA repair in plants.