Ethylene-induced differential gene expression during abscission of citrus leaves

Abstract

The main objective of this work was to identify and classify genes involved in the process of leaf abscission in Clementina de Nules (Citrus clementina Hort. Ex Tan.). A 7 K unigene citrus cDNA microarray containing 12 K spots was used to characterize the transcriptome of the ethylene-induced abscission process in laminar abscission zone-enriched tissues and the petiole of debladed leaf explants. In these conditions, ethylene induced 100% leaf explant abscission in 72 h while, in air-treated samples, the abscission period started later and took 240 h. Gene expression monitored during the first 36 h of ethylene treatment showed that out of the 12 672 cDNA microarray probes, ethylene differentially induced 725 probes distributed as follows: 216 (29.8%) probes in the laminar abscission zone and 509 (70.2%) in the petiole. Functional MIPS classification and manual annotation of differentially expressed genes highlighted key processes regulating the activation and progress of the cell separation that brings about abscission. These included cell-wall modification, lipid transport, protein biosynthesis and degradation, and differential activation of signal transduction and transcription control pathways. Expression data associated with the petiole indicated the occurrence of a double defensive strategy mediated by the activation of a biochemical programme including scavenging ROS, defence and PR genes, and a physical response mostly based on lignin biosynthesis and deposition. This work identifies new genes probably involved in the onset and development of the leaf abscission process and suggests a different but co-ordinated and complementary role for the laminar abscission zone and the petiole during the process of abscission.

Keywords: Citrus clementina; cDNA microarray; expression profiling; laminar abscission zone; mandarin; petiole.

Fig. 1.

Abscission kinetics of Citrus clementina leaves under ethylene or air treatments. Data are the average of three independent experiments and error bars show SE.

Fig. 2.

Venn diagrams including the number of ethylene regulated ESTs in both the laminar abscission zone (LAZ) and the petiole of Citrus clementina leaves after 6, 12, 24, and 36 h of ethylene treatment. The total EST number is also shown in boxes. Data are based on microarray analyses.

Fig. 3.

Ratio and number of ethylene regulated ESTs in laminar abscisión zone (LAZ) (open box) or petiole (Pet) (filled box) of Citrus clementina leaves assigned to MIPS (Munich Information Center for Protein Sequences, http://www.mips.gsf.de) categories. Positive and negative values indicate the ESTs fraction preferentially expressed in LAZ and Pet, respectively. The total number of ESTs included in each one of the MIPS category is shown in the vertical axis. Data are based on microarray analyses.

Fig. 4.

Expression ratio between laminar abscission zone and petiole (log₂ LAZ/Pet) of ESTs representing cell-wall modifying enzymes with significant changes during the ethylene treatment of Citrus clementina leaves based on microarray analyses. Positive values, preferentially expressed in LAZ. Negative values, preferentially expressed in Pet. Data are the average of three independent comparisons and error bars show SE. Coloured lines represent the expression profiles of different ESTs printed in the cDNA microarray and assembled in the same contig.

Fig. 5.

Expression ratio between laminar abscission zone and petiole (log₂ LAZ/Pet) of ESTs representing cell-wall biosynthesis proteins with significant changes during the ethylene treatment of Citrus clementina leaves based on microarray analyses. Positive values, preferentially expressed in LAZ. Negative values, preferentially expressed in Pet. Data are the average of three independent comparisons and error bars show SE.

Fig. 6.

Expression ratio between laminar abscission zone and petiole (log₂ LAZ/Pet) of ESTs representing laccases with significant changes during the ethylene treatment of Citrus clementina leaves based on microarray analyses. Positive values, preferentially expressed in LAZ. Negative values, preferentially expressed in Pet. Data are the average of three independent comparisons and error bars show SE. Coloured lines represent the expression profiles of different ESTs printed in the cDNA microarray and assembled in the same contig.

Fig. 7.

Expression ratio between laminar abscission zone and petiole (log₂ LAZ/Pet) of ESTs representing lipid transfer proteins with significant changes during the ethylene treatment of Citrus clementina leaves based on microarray analyses. Positive values, preferentially expressed in LAZ. Negative values, preferentially expressed in Pet. Data are the average of three independent comparisons and error bars show SE. Coloured lines represent the expression profiles of different ESTs printed in the cDNA microarray and assembled in the same contig.

Fig. 8.

Expression ratio between laminar abscission zone and petiole (log₂ LAZ/Pet) of ESTs representing pathogenesis-related proteins with significant changes during the ethylene treatment of Citrus clementina leaves based on microarray analyses. Positive values, preferentially expressed in LAZ. Negative values, preferentially expressed in Pet. Data are the average of three independent comparisons and error bars show SE. Coloured lines represent the expression profiles of different ESTs printed in the cDNA microarray and assembled in the same contig.

Fig. 9.

Expression ratio between laminar abscission zone and petiole (log₂ LAZ/Pet) of ESTs representing oxidative stress-related proteins with significant changes during the ethylene treatment of Citrus clementina leaves based on microarray analyses. Positive values, preferentially expressed in LAZ. Negative values, preferentially expressed in Pet. Data are the average of three independent comparisons and error bars show SE. Coloured lines represent the expression profiles of different ESTs printed in the cDNA microarray and assembled in the same contig.

Fig. 10.

Phloroglucinol staining for lignin (A) and ruthenium-red staining for pectins (B) in laminar abscission zone (LAZ) and petiole (Pet) before and after ethylene treatment of Citrus clementina leaves. Note that leaf blade was completely removed and petiole was cut longitudinally.