Integrative analysis of the late maturation programme and desiccation tolerance mechanisms in intermediate coffee seeds

Abstract

The 'intermediate seed' category was defined in the early 1990s using coffee (Coffea arabica) as a model. In contrast to orthodox seeds, intermediate seeds cannot survive complete drying, which is a major constraint for seed storage and has implications for both biodiversity conservation and agricultural purposes. However, intermediate seeds are considerably more tolerant to drying than recalcitrant seeds, which are highly sensitive to desiccation. To gain insight into the mechanisms governing such differences, changes in desiccation tolerance (DT), hormone contents, and the transcriptome were analysed in developing coffee seeds. Acquisition of DT coincided with a dramatic transcriptional switch characterised by the repression of primary metabolism, photosynthesis, and respiration, and the up-regulation of genes coding for late-embryogenesis abundant (LEA) proteins, heat-shock proteins (HSPs), and antioxidant enzymes. Analysis of the heat-stable proteome in mature coffee seeds confirmed the accumulation of LEA proteins identified at the transcript level. Transcriptome analysis also suggested a major role for ABA and for the transcription factors CaHSFA9, CaDREB2G, CaANAC029, CaPLATZ, and CaDOG-like in DT acquisition. The ability of CaHSFA9 and CaDREB2G to trigger HSP gene transcription was validated by Agrobacterium-mediated transformation of coffee somatic embryos.

Fig. 1.

Acquisition of desiccation tolerance (DT) during coffee seed development. (A) Acquisition of germinative capacity and changes in moisture content (on a fresh weight basis) during coffee seed maturation. (B) Changes in viability (%) after equilibration drying at 81%, 62%, 45%, and 23% relative humidity (RH) during seed development. (C) Viability of zygotic embryos at 150 d after flowering (DAF, stage 5) and 200 DAF (stage 6) after equilibration drying at 9%, 23%, 32%, 45% and 62% RH. (D) Schematic representation of the seven developmental stages studied and transcript profile of three genes chosen to illustrate accumulation of storage proteins, triacylglycerols, and cell wall polysaccharides: 11S globulin (SSP1, Cc03_g05570), oleosin (OLE-1, Cc02_g04750), and mannan synthase (ManS1, Cc06_g04240), respectively. (E) Changes in ABA, cytokinins (isopentenyladenine, trans-Zeatin), and auxin (IAA) during seed maturation. The DT acquisition timeframe, i.e. the stage 5–6 transition, is indicated by the dashed vertical lines. (F) Numbers of up- and down-regulated genes at each of the six developmental transitions.

Fig. 2.

Functional analysis of genes differentially expressed during acquisition of desiccation tolerance (DT) in developing coffee seeds. Pie-charts of functions assigned to the top 100 up-regulated (A) and top 100 down-regulated (B) genes. Functional enrichment analysis (normalised frequencies of Mapman categories and bootstrap standard deviations) of the 1521 up-regulated (C) and the 677 down-regulated genes (D).

Fig. 3.

Transcript profiles of desiccation tolerance (DT)-associated reactive oxygen species (ROS) scavenging and detoxification enzymes. Gene expression levels at the seven developmental stages are indicated by the different levels of shading, as indicated in the key, and are presented as percentages of the maximum normalised transcript abundance of the gene. ALDH, aldehyde dehydrogenase; APX, ascorbate peroxidase; AOX, alternative oxidase; CAT, catalase; DHAR, dehydroascorbate reductase; GPX, glutathione peroxidase; GR, glutathione reductase; GRX, glutaredoxin; GST, glutathione-S-transferase; LAC, laccase (diphenol oxidase); LOX, lipoxygenase; MDAR, monodehydroascorbate reductase; MSR, methionine sulfoxide reductase; NDH2, type-2 NAD(P)H dehydrogenase; OXO, oxalate oxidase; PDI, protein disulphide isomerise; PPO, polyphenol oxidase; PTOX, plastid terminal oxidase; SOD, superoxide dismutase; THRX, thioredoxin.

Fig. 4.

Genome-wide analysis of late-embryogenesis abundant (LEA) proteins in coffee. LEA genes were classified according to hierarchical clustering analysis of transcript abundance in C. canephora roots, stamens, pistils, leaves, and perisperm at 180 d after flowering (DAF) and in endosperm at 260 DAF. Expression levels in C. canephora tissues and in C. arabica zygotic embryos and endosperm at 150 and 200 DAF are indicated by the blue dots, the size and intensity of which represent the number of reads per kilobase and million reads (rpkm; see key). LEA peptides detected in the heat-stable proteome of mature seeds are labelled with an orange star. LEA genes differentially expressed during seed development as shown by microarray data analysis are labelled with a diamond (closed diamonds indicate LEA genes differentially expressed during the stage 5–6 transition).

Fig. 5.

Functional validation of HSFA9 and DREB2G. Small heat-shock protein (sHSP) gene expression and desiccation tolerance. (A) Real-time RT-PCR analysis of HSFA9 and DREB2G ectopic expression in coffee somatic embryos overexpressing HSFA9 or DREB2G (HSFA9+ and DREB2+, respectively). Values are means (±SD) of four independent transgenic lines. (B) Real-time RT-PCR analysis of sHSP gene expression in HSFA9+ and DREB2+ transgenic somatic embryos. (C) Viability of HSFA9+ (circles), DREB2+ (squares), and control (diamonds) somatic coffee embryos after drying to equilibrium at 81%, 85%, 92%, and 97% relative humidity (RH).