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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>E. coli </it>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.</p> <p>Conclusions</p> <p>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.</p

Chlorogenic acid synthesis in coffee: An analysis of CGA content and real-time RT-PCR expression of HCT, HQT, C3H1, and CCoAOMT1 genes during grain development in C. canephora

The mature coffee grain contains a high level of chlorogenic acids (CGA). We have quantified the main caffeoylquinic acids (CQA) and dicaffeoylquinic acids (diCQA) in Coffea canephora (robusta) grain during late development. This analysis indicates the CQA levels remain relatively steady during the final stages of grain development, but the levels of diCQA fall significantly. Analysis of quinic acid, a key CGA precursor in coffee, shows it is present at a high level in the early grain, but then drops to a low level as development progresses. To better understand coffee CGA synthesis, and to learn why diCQA and quinic acid levels fall late in grain development, we have cloned cDNA encoding four key enzymes for CGA synthesis in coffee; HCT, HQT, C3H1 and CCoAOMT1. The characterization of recombinant HCT, HQT and CCoAOMT1 proteins is also described. Quantitative real-time RT-PCR data is presented for different stages of grain and pericarp development, as well as several other C. canephora tissues. Elevated HCT and CCoAOMT1 expression in branch tissues strongly suggests the products of these genes are associated with increased lignin synthesis, while higher HQT expression appears to be more closely correlated with CGA accumulation. The data presented forms an important base for designing new experiments aimed at improving our understanding of CGA synthesis in coffee and other plants, and could facilitate the development of new strategies to increase the CGA content of plant foods