Comparative transcriptional survey between laser-microdissected cells from laminar abscission zone and petiolar cortical tissue during ethylene-promoted abscission in citrus leaves

Abstract

Background: Abscission is the cell separation process by which plants are able to shed organs. It has a great impact on the yield of most crop plants. At the same time, the process itself also constitutes an excellent model to study cell separation processes, since it occurs in concrete areas known as abscission zones (AZs) which are composed of a specific cell type. However, molecular approaches are generally hampered by the limited area and cell number constituting the AZ. Therefore, detailed studies at the resolution of cell type are of great relevance in order to accurately describe the process and to identify potential candidate genes for biotechnological applications.

Results: Efficient protocols for the isolation of specific citrus cell types, namely laminar abscission zone (LAZ) and petiolar cortical (Pet) cells based on laser capture microdissection (LCM) and for RNA microextraction and amplification have been developed. A comparative transcriptome analysis between LAZ and Pet from citrus leaf explants subjected to an in-vitro 24 h ethylene treatment was performed utilising microarray hybridization and analysis. Our analyses of gene functional classes differentially represented in ethylene-treated LAZ revealed an activation program dominated by the expression of genes associated with protein synthesis, protein fate, cell type differentiation, development and transcription. The extensive repertoire of genes associated with cell wall biosynthesis and metabolism strongly suggests that LAZ layers activate both catabolic and anabolic wall modification pathways during the abscission program. In addition, over-representation of particular members of different transcription factor families suggests important roles for these genes in the differentiation of the effective cell separation layer within the many layers contained in the citrus LAZ. Preferential expression of stress-related and defensive genes in Pet reveals that this tissue is likely to be reprogrammed to prevent pathogen attacks and general abiotic stresses after organ shedding.

Conclusion: The LCM-based data generated in this survey represent the most accurate description of the main biological processes and genes involved in organ abscission in citrus. This study provides novel molecular insight into ethylene-promoted leaf abscission and identifies new putative target genes for characterization and manipulation of organ abscission in citrus.

Figure 1

Cellular morphology of fracture planes at the laminar abscission zone. Scanning electron micrographs of the distal (A, C and E; leaf-blade side) and the proximal (B, D and F; petiole side) fracture planes of the citrus laminar abscission zone from Citrus clementina mature leaf explants non-treated (A and B) and treated for 24 h (C and D) and 48 h (E and F) with ethylene. High magnification pictures show cells of the cortical portion of the laminar abscission zone. Bars: 1 mm and 100 μm.

Figure 2

Anatomy of laminar abscission zone. Laser microdissected-mediated isolation of laminar abscission zone (LAZ) cells and petiolar cortical (Pet) cells from 10 μm-thick longitudinal sections of Citrus clementina mature leaf explants (A). Gel analysis of LAZ and Pet amplified mRNA after two rounds of amplification (B). LAZ = laminar abscission zone; OG = oil gland; Pet = petiolar cortex; VB = vascular bundles.

Figure 3

Distribution of functional cathegories between the laminar abscission zone cells and the petiolar cortical cells. Ratio and number of ethylene-regulated ESTs in laminar abscission zone cells (open box) or petiolar cortical cells (filled box) of Citrus clementina leaves assigned to MIPS (Munich Information Center for Protein Sequences, ) categories. Positive and negative values indicate the EST fraction preferentially expressed in laminar abscission zone cells and petiolar cortical cells, respectively. The total number of ESTs included in each of the MIPS categories is shown in the vertical axis. Data are based on microarray analyses.

Figure 4

Expression of genes encoding cell-wall modifying enzymes. Expression ratio (log₂) between laminar abscission zone cells and petiolar cortical cells (LAZ/Pet) of genes encoding cell-wall modifying enzymes (exopolysaccharidases, endopolysaccharidases, other hydrolases and transferases and lyases) with significant changes after 24 h of ethylene treatment to Citrus clementina leaf explants based on microarray analyses. Positive values show transcripts preferentially expressed in LAZ and negative values those preferentially expressed in Pet. Each bar represents the expression ratio of a singleton or of different ESTs assembled in the same contig. Data are the average of two dye-swap comparisons and error bars show SE.

Figure 5

qRT-PCR analysis of genes related to cell-wall modification. Expression ratio (log₂) between laminar abscission zone cells and petiolar cortical cells (LAZ/Pet) of CitίGAL1, CitCEL1, CitPG1, CitGMP, CitLAC1 based on microarray results and quantitative real-time PCR. The qRT-PCR results confirm the tendency of expression observed in the microarray data.

Figure 6

Expression of genes encoding components of the ubiquitin/proteasome system. Expression ratio (log₂) between laminar abscission zone cells and petiolar cortical cells (LAZ/Pet) of genes encoding components of the ubiquitin/proteasome system with significant changes after 24 h of ethylene treatment to Citrus clementina leaf explants based on microarray analyses. Positive values show transcripts preferentially expressed in LAZ and negative values those preferentially expressed in the Pet. Each bar represents the expression ratio of a singleton or of different ESTs assembled in the same contig. Data are the average of two dye-swap comparisons and error bars show SE.

Figure 7

Expression of genes encoding stress-related proteins. Expression ratio (log₂) between laminar abscission zone cells and petiolar cortical cells (LAZ/Pet) of genes encoding stress-related proteins with significant changes after 24 h of ethylene treatment to Citrus clementina leaf explants based on microarray analyses. Positive values show transcripts preferentially expressed in LAZ and negative values those preferentially expressed in the Pet. Each bar represents the expression ratio of a singleton or of different ESTs assembled in the same contig. Data are the average of two dye-swap comparisons and error bars show SE.

Figure 8

Expression of genes encoding pathogenesis-related proteins. Expression ratio (log₂) between laminar abscission zone cells and petiolar cortical cells (LAZ/Pet) of genes encoding pathogenesis-related proteins with significant changes after 24 h of ethylene treatment to Citrus clementina leaf explants based on microarray analyses. Positive values show transcripts preferentially expressed in LAZ and negative values those preferentially expressed in the Pet. Each bar represents the expression ratio of a singleton or of different ESTs assembled in the same contig. Data are the average of two dye-swap comparisons and error bars show SE.

Figure 9

Expression of genes encoding protein kinases. Expression ratio (log₂) between laminar abscission zone cells and petiolar cortical cells (LAZ/Pet) of genes encoding protein kinases with significant changes after 24 h of ethylene treatment to Citrus clementina leaf explants based on microarray analyses. Positive values show transcripts preferentially expressed in LAZ and negative values those preferentially expressed in Pet. Each bar represents the expression ratio of a singleton or of different ESTs assembled in the same contig. Data are the average of two dye-swap comparisons and error bars show SE.

Figure 10

Expression of genes encoding transcription factors. Expression ratio (log₂) between laminar abscission zone cells and petiolar cortical cells (LAZ/Pet) of genes encoding transcription factors with significant changes after 24 h of ethylene treatment to Citrus clementina leaf explants based on microarray analyses. Positive values show transcripts preferentially expressed in LAZ and negative values those preferentially expressed in Pet. Each bar represents the expression ratio of a singleton or of different ESTs assembled in the same contig. Data are the average of two dye-swap comparisons and error bars show SE.

Figure 11

sqRT-PCR analysis of transcription factors. Semi-quantitative RT-PCR-based differential expression analyses of CitERF1, CitZip1, CitHDZip1, CitMADS1, CitBLH1, CitbHLH1 and CitABP between laminar abscission zone (LAZ) and petiolar cortical (Pet) cells. Numbers on the right hand side represent the cycles performed during the PCR.