Arabidopsis
monogalactosyldiacylglycerol desaturase
(palmitate-specific) (FAD5) is
possibly similar to animal
and fungal acyl-CoA desaturases
Genes or clones have previously been identified for 6 of the
8 acyl desaturase reactions shown in Figure 2.
However, FAD4 and FAD5 are so far identified only by the existence of
putative mutations in these reactions (reactions #15 and #20 in Figure 2). One of the most intriguing differences in the
abundance of ESTs between rice and Arabidopsis concerns a gene family that is
most similar to animal and fungal acyl-CoA desaturases. There are 10
independent ESTs in Arabidopsis, 4 in tomato and 2 in Brassica, but this
sequence class is completely missing from rice. At this time the precise function of these desaturase-like genes
remains unknown. However, further
analysis suggests that members of this family encode acyl-lipid desaturases
which likely include palmitate-specific monogalactosyldiacylglycerol desaturase
(FAD5).
In animals
and yeast this class of desaturase genes encodes palmitoyl and stearoyl delta 9
desaturases, which use acyl-CoA (or possibly acyl-lipids) as substrate (Stukey et
al., 1989; Stukey et al., 1990; Thiede et al., 1986). However, desaturation at the delta 9
position in higher plants is catalyzed by stearoyl-ACP desaturases and the
currently established pathway of higher plant glycerolipid biosynthesis does
not involve any other steps of delta 9 desaturation. Thus, it is possible that
enzymes encoded by this gene family are responsible for another reaction(s)
which is similar to desaturation of palmitoyl and stearoyl at the delta 9
position.
The most obvious
candidates for these reactions would be desaturation of palmitoyl at the delta
7 position on monogalactosyldiacylglycerol or at the delta 3 (trans)
position on phosphatidylglycerol, the reactions that have been associated with
Arabidopsis mutations fad4 (delta 3) and fad5 (delta 7) (Browse,
1985; Kunst, 1989). Because
desaturation of palmitate at delta 3 and delta 7 in higher plants occurs in the
chloroplasts the corresponding enzymes should be synthesized as precursors with
chloroplast transit peptides. We did multiple alignment analysis of amino acid
sequences for all cDNA, genomic and EST clones of higher plants similar to
delta 9 acyl-CoA desaturases (see the alignment below). Surveying genomic
Arabidopsis sequences that have not been annotated we identified a tandem of
genes in chromosome 3 that encodes two proteins similar to animal and fungal
acyl-CoA and acyl-lipid desaturases (AB017071).
When aligned with similar proteins these sequences which we name putative
FAD5_1 and FAD5_2 have N-terminal extensions which are recognized as
chloroplast transit peptides by ChloroP software. The fad5 mutation has been mapped in chromosome 3, at 28±6
cM from locus gl1 (Hugly, 1991). Clone MSJ11 (AB017071) with the
putative gene FAD5 is located at the distance 27.9 cM from the clone
with GL1 and in the same relative position to locus tt5
(http://genome‑www3.stanford.edu/cgi‑bin/AtDB/SEQmap?chr=3&beg=19&end=24æ).Thus,
it is likely that we have identified genomic sequences which are strong
candidates for plastidial palmitate-specific monogalactosyldiacylglycerol
desaturase. Putative gene FAD5_1
does not have ESTs in Arabidopsis. However, searching dbEST with the amino acid
sequence of the protein encoded by FAD5_1 we found 2 similar EST
sequences in Brassica which overlap with the predicted transit peptide (L38104
and H07631). Thus, in other cruciferous
plants protein products of similar genes are
likely to be imported into chloroplasts. Gene FAD5_2 in
Arabidopsis is represented by EST clones 145O4 (AI099992 and T76134) and 122A14
(R87006). Both clones appear to be full length and have the sequence
corresponding to the predicted transit peptide. We also identified ESTs similar
to animal and fungal acyl-CoA and acyl-lipid desaturases with potential
chloroplast transit peptides from tomato and Chlamydomonas.
Finally, we
note that occurrence of the transcripts similar to delta 9 acyl desaturases in
Arabidopsis, Brassica, tomato and Chlamydomonas and their
apparent absence in rice correlate with our biochemical understanding of desaturation
patterns of acyl residues in the sn-2 position of DGG in these species. All species which contain these sequences
are considered "16:3 plants" and carry out delta 7 desaturation of
palmitate, as opposed to many other angiosperms, including rice (Mongrand et
al., 1998), which are “18:3" plants and lack this reaction. The ESTs
similar to delta 9 acyl desaturases are clearly associated with the 16:3
phenotype. Thus, based on four criteria: sequence similarity to acyl-CoA
desaturases, presence of transit peptides, chromosomal location, and
association with the 16:3 phenotype, our survey of public databases has
provided strong evidence for the function of one of the previously unidentified
members of the desaturase gene family in plants.
Potential transit peptides encoded by plant ESTs and
Arabidopsis genomic sequences
similar to animal and fungal acyl-CoA desaturases are shown by green arrows.
Accession numbers for ESTs are highlighted with
yellow.
At
T46147 : ------------------------------------------------------------------------------------------------
At
AA041026 :
------------------------------------------------------------------------------------------------
At
Z30751 : ------------------------------------------------------------------------------------------------
At ADS1 :
------------------------------------------------------------------------------------------------
At
AA586250 :
------------------------------------------------------------------------------------------------
At Z35340 :
------------------------------------------------------------------------------------------------
At T20690 :
------------------------------------------------------------------------------------------------
At N97264 :
------------------------------------------------------------------------------------------------
At R90185 :
------------------------------------------------------------------------------------------------
At T42777 : ------------------------------------------------------------------------------------------------
At R65180 :
------------------------------------------------------------------------------------------------
At T88304 : ------------------------------------------------------------------------------------------------
At Z35339 :
------------------------------------------------------------------------------------------------
At Z32595 :
------------------------------------------------------------------------------------------------
At N96488 :
------------------------------------------------------------------------------------------------
Tomato : ---------------------------------RIRHELLSPPPSKSKPYPFSPLPRAVHRQGRSTQTTKNVNSLSCTSMTTKIGVVNKWNCYGLE
At ADS2 :
------------------------------------------------------------------------------------------------
At AI099992 :
------------------------------------------------------------------------------------------------
At
T76134 :
----------------------------------------------------------------MASXLTKPKPVFLCXPSLSPRTLNTATPSLNX
At
R87006 :
----------------------------------------------------------------MASXLTKPKPVFLCXPSXSPRTLNTATPSLNL
At FAD5_1 :
----------------------------------------------------------------MASLLTKPKPVFLCSPSLSPRTLNTATPSLNF
Brassica :
----------------------------------------------------------------MVSLCTTLKPLSPFXPFV--KQGNLNYTSALL
At
FAD5_2 :
----------------------------------------------------------------MMSLSTTLKPLSHFSPFV--KRHNPKTNNTLF
Chlamidomonas : QVERRKCLGCGTSTCAGLSAACAVRTXPPLHQLRPASQPCCSSGQPWACARRRAAGSGRRGAAASKYMRPLRLGSRVLGLPRLAGGQRGGRVPPPG
ROSE : ------------------------------------------------------------------------------------------------
At
T46147 :
------------------------------------------------------------------------------------------------
At AA041026 : ---------------------------------------------------GAVSKEKR--PYIHREWSWADIIRALTVINVHFLCLLA--PFNYK
At
Z30751 :
-------------------------------------------------------------AMWERKWKRLDIVKAFASLFVHFLCLLA--PFNFT
At ADS1 : ----------------MSLSASEKE----------------ENNKKMAADKAEMGRKKR--AMWERKWKRLDIVKAFASLFVHFLCLLA--PFNFT
At
AA586250 :
------------------------------------------------------------------------------------------------
At
Z35340 :
------------------------------------------------------------------------------------------------
At
T20690 :
------------------------------------------------------------------------------------------------
At
N97264 : ----------------MSVTSTVEE----------------NHQKNPSTPAGWEEKKKRRWVFWDRRWRRLDYVKXSASFTVHSLALLA--PFYFT
At
R90185 : -----------------SVTSTVEE----------------NHQKNPSTPAGWEEKKKRRWVFWXRRWRRLDYXKXSASXTVHSLALLA--PFXXT
At
T42777 : -----------------SVTSTVEE----------------NHQKNPSTPAGWXEKKKRRWVFWDRRWRRLDYVKFSASFTVHSLALLA--PFYFT
At
R65180 :
----------------------------------------------------------------------LDYVKFSASFTVHSLALLA--PFYFT
At
T88304 : -----------------SVTSTVEE----------------NHQKNPSTPAGWEEKKKRRWVFWDRRWRRLDYXKFSXSXTVHXLALLA--PFXFT
At
Z35339 : ----------------MSVTSTVEE----------------NHQKNPSTPAAVEEKKKRRWVFWDRRWRRLDYVKFSASFTVHSLALLA--PFYFT
At
Z32595 : ----------------MSVTSTVEE----------------NHQKNPSTPAAVEEKKKRRWVLWYRRWRRLDYVKFSASHTAHSLALLA--PFYHT
At
N96488 : ---------------------TVEE----------------NHQKNPSTPAGWXEKKKRRWVFWDRRWRRLDYVKFSASFTVHSLALLA--PFYFT
Tomato : GKVRFSRRVMLMPIVNAAVTQPVSENERESNFKRILLSDVVVTRPRDVLWG-------RKW-------NSMDMASAAVVVSMHLLCLFA--PFTFN
At ADS2 : ----------------MSVTSTVEE----------------NHQKNPSTPAAVEEKKKRRWVFWDRRWRRLDYVKFSASFTVHSLALLA--PFYFT
At
AI099992 :
------------------------------------------------------------------------------------------------
At T76134 : TRISFTHHQKLAPXKPPSL--VVAFSEKGLKRDVTTAAAATEGDYRRXMLSXVLVKKKEKVVWWEXXWESYGLWGCXXXVLXM-------------
At
R87006 : TRIXFTHHQKLAPXKPPSL--VVAFXEKGLKRDVTTAAAATEGDYRRXMLSXVLVKEEGKSSLV--------------------------------
At FAD5_1 : TRISFTHHQKLAPFKPPSL--VVAFSEKGLKRDVTTAAAATEGDYRRIMLSDVLVKKKEKVVWWEREWKAMDFGAVAVVLSMHLLSLLA--PFQFN
Brassica : TYRTSNSANSFLPKRGGTV-AVHDAP------------DQVESSCRIPLTEVVAVRKK--SAFWERSWSSRDVRNVVVLGGVHLLSLFG--PLYFS
At FAD5_2 : TLDTHNFTNSFWSKRGGSVSAVYEAP------------DHVESSWRRLLSEVVVVRTKR--SFWERSWTSWDVSKLVIFVGTHLLSLLA--PFYFS
Chlamidomonas : GGRLGLHVLXSAVRQALHQASRAIGAGDAQGDQQAGXGCAEQRGGCFQGLCVGCACSGKRPLWFGRKWSITDISYGVYLGGMHLLACLA--PFTFS
ROSE :
------------------------------------------------MPSRLINWQVQ---FFGREWDFMDLYHLTLFLGVPFVCLLA--PFQFT