The dehydrins are a group of specialized plant proteins that are involved in protecting cells from dehydration-related stresses (Close, 1996
; Allagulova et al., 2003
). Dehydrins also participate in two important and rather specific aspects of seeds. (1) They are widely perceived to participate, with other LEA proteins, in the dehydration process that occurs during the late stages of seed maturation by assisting the acclimatization of seed tissues to the lower water content found in mature seeds (Close, 1996
; Nylander et al., 2001
). (2) It is presumed that the dehydrins synthesized in seeds during maturation continue to stabilize the associated cellular structures during seed quiescence. In this latter context, it should also be noted that it has recently been proposed that dehydrins may also possess a radical-scavenging capability (Hara et al., 2003
) and have metal-binding properties (Alsheikh et al., 2003
), both characteristics that are likely to be useful during long periods of seed storage.
Despite the involvement of dehydrin proteins in plant resistance to osmotic stresses such as drought stress, and the probable importance of the dehydrins during grain development, little information is available on these genes in coffee. Thus it has been decided to take advantage of the new coffee EST collection to isolate cDNAs encoding highly expressed coffee dehydrins, and to establish the expression patterns of the dehydrins in different tissues, as well as during coffee grain and pericarp maturation. Five cDNAs from C. canephora
, which represent three distinct dehydrin genes (CcDH1, CcDH2 and CcDH3) are presented here, as well as a cDNA representing an LEA gene (CcLEA1) with an extremely restricted expression pattern. The gene CcDH1 is represented by two cDNAs that differ by 21 bases and have over 95
% identity. The small sequence differences translate into six amino acid changes, three of which result from a 9
bp deletion in the ORF of CcDH1a. Because these two cDNAs show such a high level of identity, and the fact that the protein sequence changes fall outside the highly conserved regions in this protein sequence family (), it is considered that these two cDNAs represent different alleles of one gene.
Two distinct unigene sequences were found to encode an identical dehydrin protein (CcDH2a and CcDH2b). However, closer examination of full-length cDNA clones representing these two unigenes indicated that unigene #123405, which consisted of two ESTs, actually contained an intron within the predicted ORF sequence. This intron containing cDNA (CcCH2b; pcccs4630p1) also contained a poly (A) tail sequence indicating that the corresponding mRNA was polyadenylated but not spliced. Interestingly, the 3′ splice site of the intron in CcDH2b was slightly different from the equivalent genomic sequence of CcDH2a [; ttatgg/TCG versus genomic sequence ttatag/T(or A)CG]. The only other differences between the intron sequence of the cDNA and the intron sequences in three independently obtained genomic sequences were a few punctual base changes. None of these changes in the intron were conserved in each of the three genomic sequences. The fact that the two ESTs of unigene #123405 (CcDH2b) had the same splice site mutations and were found in two different grain specific libraries (30-week and 46-week libraries), strongly suggests that this intron-containing transcript is real and not a cloning artefact or sequencing error. Therefore, the available data strongly suggest that the absence of splicing in the two cDNAs of unigene #123405 were due to the single base pair change in the splice site (gg versus ag). As the 30-week and 46-week grain libraries were made from more than one variety, it is currently not known which varieties harbours this mutation. It is also not known if this mutation has any biological consequences. Interestingly, it has been been found recently that approx. 5
% of the cDNAs in a large set of full-length arabidopsis cDNAs harbour unspliced introns, which the authors have called ‘retained introns’ (Iida et al., 2004
). This result is consistent with the observation that cDNA with poly (A) tails and containing unspliced introns can be found relatively easily in the new coffee EST libraries (J. McCarthy et al
., unpubl. res.). Much further work is needed to determine to what extent such ‘retained introns’ are the result of problems such as slow primary transcript processing, or base changes causing the reduced/loss of splicing, or alternatively, whether more complex mechanisms/functions are involved.
Although their primary amino acid sequences are relatively different (CcDH1a shares 47.3
% identity with CcDH2a), both CcDH1 and CcDH2 can be classified as Y3
dehydrins based on the distribution of the Y, S, and K motifs in the respective sequences. In addition to being relatively close in motif composition, CcDH1 and CcDH2 also exhibit some similarities in their patterns of expression. Both genes are strongly expressed during the later stages of grain development (). However, it appears that CcDH1 is also weakly expressed in several other tissues, albeit at different levels in arabica and robusta. In contrast, the expression of CcDH2 is limited to the grain in both species. It is noted that one CcDH2 EST was found in the leaf EST library, suggesting that this gene may, under some conditions, be expressed in the leaf. Further experiments are necessary to determine whether CcDH2 expression is inducible, for example, by dehydration stress or by exposure to cold temperatures. Given that the expression patterns for CcDH1 and CcDH2 are different, and the fact that the proteins vary slightly, even in the highly conserved Y, S and K motifs, could imply that these proteins may have related, but not identical roles. For example, it is possible that the CcDH1 and CcDH2 dehydrins are involved in protecting different functional targets. Alternatively, they could have similar functional targets, but their expression is controlled differently to enable these genes to be induced differentially in particular tissues by specific developmental and/or environmental cues. The coffee dehydrin CcDH3 has the structure SK3
, and is also expressed during the late stages of grain development (). In addition, CcDH3 expression is detected in several other tissues, as was CcDH1 expression. This latter point suggests that the expression of CcDH1 and CcDH3 could be controlled by relatively similar signals, although differences in the absolute levels of transcripts indicates transcription induction and/or transcript stabilities are not identical for both genes. As for the majority of the dehydrins previously described from other plants, the precise functions of the coffee dehydrins described here are not yet known. Nonetheless, the significant expression of the CcDH1, CcDH2 and CcDH3 genes during the late stages of grain maturation clearly suggests that the three dehydrins play a significant role(s) in conditioning the coffee grain for maturation-induced dehydration, and probably are also important in protecting the mature grain tissues before germination. Future experiments will be aimed at determining whether one or more of these dehydrins are induced in coffee under conditions of water and low temperature stress.
The LEA gene described here (CcLEA1) has an unusual expression pattern, with transcripts being detected only during one relatively short period of grain development, and not in any other tissues. In robusta, CcLEA1 expression was only detected at the large green grain stage, and not in either the small green or yellow stages of grain development. This stage of grain development spans the period when the perisperm tissue undergoes a substantial size reduction and the endosperm expands significantly (unpubl. res.), thus suggesting that the CcLEA probably plays a specific role during the perisperm/endosperm transition. A Blast analysis of the protein database with the CcLEA1 protein uncovered three potentially related protein sequences from arabidopsis, Picia
(white spruce), and maize. Although the highest level of identity was only 47.9
% for the Picia
sequence, the protein alignment seen in shows that the three related proteins did share several highly conserved blocks of homology, especially after the more variable N-terminal end of the protein. None of the homologous proteins have been assigned a function. However, the maize cDNA sequence, which is called ‘root cap protein 2’, was isolated with another highly similar cDNA called ‘root cap protein 1’ from maize root caps using a differential screen (Matsuyama et al., 1999
). Transcription of both genes was restricted to the outermost cells of the maize root cap. These cells are believed to be first associated with the production of mucilage and then, later, are sloughed off (Moore and McClelen, 1983
). It also appears that, as they sloughed off, the root cap cells undergo cell death (Matsuyama et al., 1999
). Although expression of CcLEA1 was not detected in roots, it is quite possible that the root cap fraction of the total root samples tested here were very small and thus genes specifically expressed in root caps were below the level of detection. Further expression analysis using particular regions of the root should clarify whether CcLEA1 is expressed in the root cap region of coffee. It is interesting to speculate that this class of protein may play a similar role in both the root cap and the perisperm region as it develops into endosperm during grain maturation. Although no data concerning the putative arabidopsis LEA homologue (accession number NP200248) have been found in the literature, an examination of the arabidopsis MPSS expression database (http://mpss.udel.edu
) shows that this arabidopsis gene is expressed in both the root and in germinating seeds. No expression was detected for this gene in any of the other tissues described in the arabidopsis MPSS database. The detection of transcripts for the arabidopsis homologue in root supports the idea that the arabidopsis protein is a homologue to the maize root cap protein and possibly CcLEA1. It will be interesting in the future to investigate whether the putative homologue expressed during the germination of arabidopsis seeds is associated with tissues undergoing cell death, and similarly, if the root cap proteins of maize are expressed during maize seed development and/or germination. Finally, one striking similarity between CcLEA1 and its putative homologues is the near absolute conservation of the cysteines, a feature which was first observed in the maize root cap proteins (Matsuyama et al., 1999
). However, the functional significance of these conserved cysteines is currently unknown.
Because the CcDH2 gene appears to be grain specific, the promoter of this gene was isolated for further study. The promoter sequence obtained () has several previously recognized regulatory sequence motifs that are expected to be involved in the temporal and spatial control of CcDH2 dehydrin expression. Considering that the detailed testing of coffee grain-specific promoters can take more than 3–4 years due to the slow development and flowering of coffee, the possibility of testing the capacity of this promoter sequence is being examined to direct grain-specific expression in model plants such as arabidopsis or tomato. It is now well established that dehydrin proteins somehow participate in protecting plants from water-related environmental stresses (Allagulova et al., 2003
). Therefore the detailed information on the coffee dehydrins presented here, and the identification of other LEA cDNA in the coffee EST bank (), should open some new avenues for research on stress tolerance in coffee. For example, it is now possible to study the induction of these genes in different tissues in response to various stresses. It is also possible to examine whether there are variations in the expression of these genes in coffee varieties exhibiting dramatically different tolerances to drought and low temperatures. The information on the coffee dehydrin and LEA sequences presented here also opens up the possibility of investigating the potential relationship(s) between the poor storage capability of coffee grain and the expression levels of various dehydrin and LEA genes.