Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination

Abstract

Background: Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases.

Results: Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels.

Conclusions: Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death associated with post-germination of the coffee grain. Expression analysis of the cysteine proteinase inhibitor genes suggests that CcCPI-1 could primarily be involved in modulating the activity of grain CP activity; while CcCPI-4 may play roles modulating grain CP activity and in the protection of the young coffee seedlings from insects and pathogens. CcCPI-2 and CcCPI-3, having lower and more widespread expression, could be more general "house-keeping" CPI genes.

Figure 1

Alignment of C. canephora CP1 and CP4 sequences with highly homologous plant sequences. The alignments were done using CLUSTAL W. The key conserved amino acid characteristics and motifs are noted. Amino acids shaded in grey indicate most conserved sequences. Panel A: Database accession numbers are Coffea canephora CP1 (GeneBank sequence #AEQ54770), Arabidopsis thaliana AtCP (AAL49820) and Vicia sativa VsCPR4 (CAB16316). The catalytic triad Cys, His, and Asn and also the Gln active site residues are indicated by an asterisk. The GCXGG motif is double underlined. The cathepsin propeptide inhibitor domain (I29) is shown in a rectangular box. Note: an ERFNIN-like sequence, shown in italics, exists in the propeptide region of CP1 (ie. ERFNAQ). Panel B: Database accession numbers: Coffea canephora CP4-KDDL (GeneBank sequence #AEQ54771), Nicotiana tabacum NtCP56-KDEL (ACB70409) and Solanum lycopersicum SlCysEP-KDEL (ABV22590). Symbols for key amino acids and motifs are as above, plus the ERFNIN motif is indicated in italics and the KDEL (K358-L361 for NtCP56) motif is shown in a double lined rectangular box. Arrows indicates the site of auto-hydrolysis of NtCP56 reported by Zhang et al. (2009) [4].

Figure 2

Quantitative expression analysis of CcCP1 and CcCP4 in different tissues and at different stages for Robusta BP409. The expression of each gene was measured in the various maturing and germinating grain samples, as well as maturing pericarp samples plus in roots, branches, and leaves. For germination stages T5 and T6, only the grain (T5) or grain/emerging cotyledons were extracted for RNA. The expression levels were obtained using quantitative RT-PCR. RQ is the expression level of each gene relative to the constitutively expressed gene RPL39. GSG, small green stage grain; GLG, large green stage grain; GY, yellow stage grain; GR, red stage grain. PSG, pericarp small green stage; PLG, pericarp large green stage; PY, pericarp yellow stage; PR, pericarp red stage. T1 is the sterilized and washed material obtained just before placing on the "germination" media; T2 = 3 DAI, T3 = 5 DAI, T4 = 14 DAI (FE), T5 = FE + 1 month, T6 = FE + 2 months. DAI represents "Days After Imbibition", FE represents "First Evidence" of germination.

Figure 3

Coomassie SDS-Page gel analysis of CcCP1 et CcCP4 recombinant proteins. Protein samples were run on SDS-PAGE gels and stained with coomassie blue. Panel A: Lane M, molecular mass marker with the sizes shown on the left in kDa; Lane 1, empty BL21(DE3) whole cell lysate; Lane 2, Induced BL21(DE3) + pET-SUMO whole cell lysate; Lane 3, Not Induced BL21 (DE3) + pET-SUMO/CP4 whole cell lysate; Lane 4, Induced BL21 (DE3) + pET-SUMO/CP4 whole cell lysate. Panel B: Lane M, molecular mass markers with the sizes shown on the left in kDa; Lane 5, Not Induced BL21 (DE3) + pET-SUMO/CP1 whole cell lysate; Lane 6, Induced BL21 (DE3) + pET-SUMO/CP1 whole cell lysate.

Figure 4

Detection of CcCP4 protease activity. The assay was run for different lengths of time as described in the Materials and Methods and samples were then run on SDS-PAGE gels followed by coomassie staininig. The markers (lane M) as in Figure 3. 'Activated' indicates HIS-SUMO-CP4 which has been acid treated. T = reaction times with BSA.

Figure 5

Quantitative expression analysis of coffee cysteine proteinase inhibitor genes CPI1, CPI2, CPI3 and CPI4 in different tissues and at different grain/pericarp development stages for Robusta BP409. The expression of each gene was measured in the various samples indicated. RQ is the expression level of each gene relative to the constitutively expressed gene RPL39 in that sample. Sample symbols are as in Figure 2.

Figure 6

Quantitative expression analysis of CcCPI1, CcCPI2, CcCPI3 and CcCPI4 in leaves at different maturation stages for Arabica T2308. The expression of each gene was measured in two independent sets of leaf samples during development and senescence. The expression levels were obtained using QRT-PCR. RQ is the expression level of each gene relative to the constitutively expressed gene RPL39. Symbols: VYL, very young leaves; YL, young leaves; ML, mature leaves; OL, old leaves.