BACKGROUND
f-box proteins represent one of the largest and most diverse protein families in plants, as most species have several hundred representatives  <cit> .
they are named after their highly conserved n-terminal protein-protein interaction motif of approximately  <dig> amino acid residues, known as the f-box domain.
the majority of the f-box proteins function as part of skp1-cullin1-f-box -type ubiquitin e <dig> ligases  <cit>  in which the f-box protein comprises the substrate-binding module.
f-box proteins are assembled into active scf complexes through a direct binding of the f-box motif with the scf core s-phase kinase-related protein  <dig> .
through their variable c-terminal substrate-binding domain, f-box proteins specifically bind to and deliver appropriate substrates to the scf complex for ubiquitin-mediated proteolysis by the ubiquitin-proteasome system  <cit> .
the impressive number of f-box proteins in plants and the extensive diversity of their c-terminal target-binding domains contribute to the ability of scf complexes to target a wide variety of substrates.
therefore, it is not surprising that f-box proteins are involved in numerous cellular processes within plant development and stress signaling  <cit> .
genetic approaches already revealed an essential role for f-box proteins in plant hormone perception and signaling  <cit> .
f-box proteins were also reported to be involved in circadian clock control, photomorphogenesis and flowering  <cit> , leaf senescence  <cit> , self-incompatibility  <cit>  and responses to various biotic stresses .
multiple newly discovered f-box proteins  combine various recognition mechanisms which enable tight regulation of substrate selection by the f-box protein and each complementary scf complex  <cit> .
amongst these new fbxo proteins, glycan-binding f-box  proteins were first discovered in mammals  <cit> , including fbg <dig>  fbg <dig> and fbg <dig> which bind to high-mannose n-glycosylated proteins  <cit> .
the c-terminal substrate-binding domain of both fbg <dig> and fbg <dig> specifically interacts with the inner n,n’-diacetylchitobiose  core of high-mannose n-glycans present on incompletely folded or denatured glycoproteins.
since fbg <dig> and fbg <dig> do not target free mannose structures or non-glycosylated proteins, it was concluded that both fbg proteins function as glycan-binding f-box proteins involved in the endoplasmic reticulum associated degradation pathway  <cit> .
in this pathway, proteins which fail to fold correctly or assemble into oligomeric complexes in the lumen of the endoplasmic reticulum are retro-translocated to the cytosol, where they are captured by an scffbg1/ <dig> complex before degradation by the ubiquitin-proteasome system  <cit> .
recently, fbg-like proteins were also reported in plants.
the a. thaliana genome contains multiple genes encoding f-box proteins with a putative glycan-binding or lectin-like domain  <cit> .
these proteins are referred to as the f-box-nictaba family, since their c-terminal domain highly resembles nictaba, an n-glycan-binding jasmonate-inducible lectin from tobacco  <cit> .
this f-box protein family in a. thaliana groups approximately  <dig> members that share over  <dig> and 40–64 % sequence similarity in the f-box domain and the nictaba domain, respectively  <cit> .
due to the presence of the f-box domain linked to a lectin-like c-terminal substrate-binding domain, it is tempting to speculate that these f-box-nictaba proteins function as substrate adaptors in an endoplasmic reticulum associated degradation pathway in plants similar to the mammalian fbg proteins.
furthermore, both nictaba and fbg <dig> show comparable glycan-binding properties towards the inner core structure of n-glycans  <cit> .
however, detailed analysis of the sugar specificities for the a. thaliana f-box-nictaba protein  revealed binding of its nictaba domain to n- and o-glycans containing n-acetyllactosamine  structures, lewis a, lewis x and lewis y motifs  <cit> .
at present, it is not clear if and how the  f-box-nictaba protein can bind to these structures in planta.
n-glycan modification with lewis a motifs takes place in the golgi apparatus and plant-specific lewis epitopes were only shown on some extracellular glycoproteins and on membrane-bound glycoproteins present at the plant cell surface .
moreover, in contrast to most plant families, the occurrence of lewis a motifs in a. thaliana was reported to be generally low and tissue specific with highest levels in pedicels, stems and nodes and with moderate levels in siliques and the shoot apex.
lewis a structures are not detectable in the leaves of a. thaliana plants  <cit> .
in this study, we show that the transcripts for the a. thaliana f-box-nictaba protein encoded by at2g <dig> are up-regulated after plant treatment with salicylic acid , heat stress and upon infection with pseudomonas syringae pv.
tomato strain dc <dig>  with preferential activity of its promoter sequence in leaf trichomes.
stress experiments on transgenic a. thaliana plants with altered f-box-nictaba expression suggested a role for f-box-nictaba in plant defense-related signaling pathways.
RESULTS
f-box-nictaba gene expression is up-regulated after sa application and heat shock treatment
the expression pattern of f-box-nictaba was first analysed in wild-type  a. thaliana plants grown under laboratory growth conditions.
qrt-pcr analyses demonstrated that at2g <dig> was expressed during all developmental stages and in every tissue tested .
a slightly lower expression of at2g <dig> was observed in roots from young plants and in flowers compared to the youngest stage tested .
in silico expression analysis indicated that at2g <dig> is a stress-responsive gene .
indeed, the uniform expression profile of at2g <dig> considerably changed when a. thaliana plants were subjected to treatments with various plant hormones and several abiotic stress factors  .
after 1 h  of sa treatment, at2g <dig> expression was slightly down-regulated compared to the mock treatment, but at2g <dig> mrna levels were significantly higher after 3 h with a maximal increase of almost 4-fold after 10 h of sa application .
transcript levels in treated plants slightly dropped after 24 h but were still two-fold higher than in the untreated plants.
transcript levels for the sa-inducible transcription factor wrky <dig> were up-regulated faster and reached much higher levels compared to f-box-nictaba expression levels .fig.
<dig> relative transcript levels of at2g <dig> in wt a. thaliana col- <dig> plants determined by qrt-pcr analyses of two independent biological experiments, each with multiple technical replicates as pointed out in methods section; error bars ± se.
asterisks indicate statistically significant differential expression compared to control samples .
a, at2g <dig> expression levels measured in a. thaliana plants grown under standard conditions.
b-c, at2g <dig> expression levels in 16-day-old a. thaliana seedlings after sa treatment  or after heat shock .
d-e, relative expression of at2g <dig> after infection of 5-week-old a. thaliana plants with pst dc <dig>  or with b. cinerea strain b <dig>  .
expression levels are compared  to the at2g <dig> expression level in the cotyledons stage or  to gene expression levels in the mock-treated plants
also heat stress enhanced f-box-nictaba gene expression with a maximal up-regulation of almost 3-fold after 10 h of heat stress compared to control plants .
however, compared to the at2g <dig> gene the expression of the hsp70b gene, encoding a heat shock-responsive chaperone protein, was enhanced much faster and reached much higher mrna levels .
other hormones and abiotic stress treatments either slightly down-regulated or did not affect f-box-nictaba gene expression at all .
f-box-nictaba expression is up-regulated in wt a. thaliana plants after pseudomonas infection but is slightly down-regulated by fungal infection
five-week-old wt a. thaliana col- <dig> plants were infected either with the virulent hemibiotrophic bacterium p. syringae pv.
tomato strain dc <dig>  or the necrotrophic fungus botrytis cinerea strain b <dig> .
pst dc <dig> infection strongly enhanced the expression of both control genes wrky <dig> and pr <dig> in rosette leaves .
f-box-nictaba mrna levels were significantly up-regulated approximately  <dig> -fold at  <dig>   <dig> and  <dig> dpi compared to mock-sprayed plants.
f-box-nictaba expression dropped at  <dig> and  <dig> dpi but was still higher than in the mock-treated plants .
in contrast, b. cinerea infection mildly affected f-box-nictaba mrna levels compared to mock-treated plants .
at  <dig> and  <dig> dpi, at2g <dig> expression levels were reduced almost two-fold.
the expression of the control gene pr <dig> was not altered significantly .
in contrast, the expression of the control gene pdf <dig>  was highly up-regulated reaching a 50-fold increase in infected tissues at  <dig> dpi .
f-box-nictaba promoter is particularly active in leaf trichomes
to test the activity of the 5′-upstream region of the at2g <dig> gene, a promoter at2g02360:gus reporter construct was introduced into a. thaliana col- <dig> plants and used for histochemical assays.
first data revealed a distinct and localized gus staining in the leaf trichomes of 14-day-old pat2g02360:gus a. thaliana seedlings .
subsequently, plants grown under standard conditions were also analyzed at different developmental stages .
overall, plants of different transgenic lines showed comparable gus staining patterns, but the intensity of the gus staining differed amongst plants.
in the very young seedlings  no gus staining was detected .
in the cotyledons and 2-leaves stage , plantlets showed a weak gus activity spread over mesophyll cells.
when plants developed , a very intense gus activity was observed all over the petioles and in the majority of the trichomes present on the shoot meristem and first leaves.
gus staining in the trichomes present on newly developing leaves remained throughout further development of the plants.
in plantlets of stage  <dig>  and older , gus staining was prominently visible in trichomes present on new leaves and in the petioles, the major leaf vein and some parts with mesophyll cells of older leaves.
trichomes present on older leaves also showed gus staining, especially in those located in close proximity of the petiole, but their gus staining was less intense compared to the staining detected in trichomes residing on young leaves.
in flowering plants , gus staining was mainly visible in the flowers, some siliques and in some major veins and trichomes located in close proximity of these veins on rosette and cauline leaves.
the camv35s:gus control plants showed an intense blue, homogeneous gus staining pattern in all organs and tissues of the plants throughout development .fig.
<dig> histochemical analysis of pat2g02360:gus a. thaliana lines.
a, gus staining data for 14-day-old plantlets.
b, different developmental plant stages analyzed  <cit> .
c, gus staining data for selected developmental stages with a closer look at trichomes present on new leaves  and on flower buds ; panel  <dig> b represents a close-up of rosette leaves.
scale bars represent 1 mm
f-box-nictaba is predominantly expressed in the trichomes of a. thaliana plants
given the apparent activity of the pat2g02360:gus reporter construct in leaf trichomes and the occurrence of putative cis-regulatory elements reported to be responsible for trichome-specific gene regulation in the f-box-nictaba promoter sequence including eight myb-like recognition sites and five t/g-box elements , the expression of the f-box-nictaba gene was quantified by qrt-pcr in trichomes purified from wt a. thaliana col- <dig> rosette leaves.
at2g <dig> mrna levels measured in trichomes were slightly higher compared to those measured in unprocessed rosette leaves  with increased expression in trichomes up to approximately  <dig> -fold in 4-week-old plants and 2-fold in 3- and 5-week-old plants .fig.
<dig> transcript levels of at2g <dig> in the trichomes isolated from rosette leaves of wt a. thaliana plants of col- <dig> or ler- <dig> ecotype compared to corresponding gene expression levels in complete rosette leaves from wt a. thaliana plants of col- <dig> or ler- <dig> ecotype or of glabra <dig> a. thaliana plants.
relative transcript levels were determined in 3-, 4- and 5-week-old plants by qrt-pcr analyses of two independent biological experiments, each with two technical replicates; error bars ± se.
asterisks indicate statistically significant differential expression compared to control samples .
a, gene expression levels in the trichomes of wt a. thaliana col- <dig> plants compared to complete rosette leaves from wt a. thaliana col- <dig> plants.
b, gene expression levels in the trichomes of wt a. thaliana ler- <dig> plants compared to complete rosette leaves of wt a. thaliana ler- <dig> plants or compared to complete rosette leaves of glabra <dig> a. thaliana plants as well as in complete rosette leaves of wt a. thaliana ler- <dig> plants compared to complete rosette leaves of glabra <dig> plants.
c, comparison of gene expression levels for f-box-nictaba in the trichomes and in complete rosette leaves of wt a. thaliana col- <dig> compared to wt a. thaliana ler- <dig> plants
f-box-nictaba gene expression was also analyzed in rosette leaves of mutant a. thaliana plants completely lacking trichomes.
these glabra <dig> plants are impaired in the gl <dig> gene  which encodes a myb-like transcription factor required for trichome development  <cit> .
since the glabra <dig> mutants are made in a landsberg erecta- <dig>  background, f-box-nictaba expression was also examined in wt a. thaliana ler- <dig> plants.
trichome purification from rosette leaves of a. thaliana ler- <dig> plants yielded similar amounts of trichomes as for a. thaliana col- <dig> plants.
however, the relative expression of the f-box-nictaba gene was much more pronounced in trichomes of a. thaliana ler- <dig> plants with an up-regulation of  <dig> -fold , 150-fold and 25-fold compared to total leaf tissue from 3-, 4- and 5-week-old a. thaliana ler- <dig> plants, respectively .
in comparison to glabra <dig> a. thaliana plants, at2g <dig> transcript levels were 20-, 160- and 175-fold higher in trichomes from a. thaliana ler- <dig> plants when compared to total leaf tissue from 3-, 4- and 5-week-old glabra <dig> a. thaliana plants, respectively .
the transcript levels for the f-box-nictaba gene were higher in wt a. thaliana leaves of plants with a col- <dig> background compared to leaves from wt plants with a ler- <dig> background, at all developmental stages investigated, irrespective whether the purified trichomes or complete rosette leaves of both ecotypes were assessed .
the differential expression was most striking when unprocessed rosette leaves were compared : at2g <dig> mrna levels were approx.
<dig>   <dig> and  <dig> times higher in leaves of 3-, 4- and 5-week-old plants of col- <dig> ecotype, respectively, compared to leaves of the same age from plants with a ler- <dig> background.
in the trichomes isolated from 3-, 4- and 5-week-old wt a. thaliana col- <dig> plants f-box-nictaba gene expression was 90-, 7- and 10-fold higher than in the trichomes of 3-, 4- and 5-week-old plants from wt a. thaliana ler- <dig>  respectively .
galt <dig> and fut <dig> genes are up-regulated in the trichomes of wt a. thaliana ler- <dig> plants
f-box-nictaba was identified as a functional lectin that can bind n- and o-glycans containing lactosamine structures, lewis a, lewis x and lewis y motifs  <cit> .
arabidopsis plants were only reported to contain lewis a-modified n-glycans.
to our knowledge, the presence of lewis a has not been studied yet in trichomes of a. thaliana plants.
since f-box-nictaba expression is prominent in the trichomes, we addressed whether lewis a motifs could be specifically synthesized in the trichomes.
therefore, qrt-pcr analysis was performed to analyse gene expression for the enzymes indispensable for lewis a epitope synthesis in a. thaliana: i.e., at1g <dig> encoding β <dig> -galactosyltransferase  and at1g <dig> encoding α <dig> -fucosyltransferase    <cit> .
according to fig.
<dig>  both galt <dig> and fut <dig> are expressed in rosette leaves as well as in the leaf trichomes of a. thaliana col- <dig> plants.
the transcript levels for galt <dig> were significantly lower in the trichomes of wt a. thaliana col- <dig> plants compared to those measured in the unprocessed rosette leaves of the same plants at all investigated developmental stages, the lowest value being a 5-fold decrease of galt <dig> expression in trichomes of 4-week-old plants .
in 3- and 5-week-old plants galt <dig> mrna levels were approximately two and three times lower in trichomes compared to rosette leaves, respectively .
in contrast to galt <dig>  fut <dig> did not show a significant differential expression between trichomes and unprocessed rosette leaves .fig.
<dig> transcript levels of galt <dig>  and fut <dig>  in trichomes isolated from rosette leaves of wt a. thaliana plants of col- <dig> or ler- <dig> ecotype compared to corresponding gene expression levels in complete rosette leaves of wt a. thaliana plants of col- <dig> or ler- <dig> ecotype or of glabra <dig> a. thaliana plants.
transcript levels were determined in 3-, 4- and 5-week-old plants by qrt-pcr analyses of two independent biological experiments, each with two technical replicates; error bars ± se.
asterisks indicate statistically significant differential expression compared to control samples .
a-b, relative gene expression levels of galt <dig>  and fut <dig>  in the trichomes of wt a. thaliana col- <dig> plants compared to complete rosette leaves from wt a. thaliana col- <dig> plants.
c-d, relative gene expression levels of galt <dig>  and fut <dig>  in the trichomes of wt a. thaliana ler- <dig> plants compared to complete rosette leaves of wt a. thaliana ler- <dig> plants or compared to complete rosette leaves of glabra <dig> a. thaliana plants as well as in complete rosette leaves of wt a. thaliana ler- <dig> plants compared to complete rosette leaves of glabra <dig> plants.
e-f, comparison of gene expression levels of galt <dig>  and fut <dig>  in the trichomes and in complete rosette leaves of wt a. thaliana col- <dig> plants compared to wt a. thaliana ler- <dig> plants
conversely, in trichomes of 3- and 4-week-old wt a. thaliana ler- <dig> plants, the expression of the galt <dig> gene was markedly up-regulated by 7- and 29-fold, respectively, when compared to levels in unprocessed rosette leaves originating from these plants .
comparison of the galt <dig> expression levels in trichomes of wt a. thaliana ler- <dig> plants to those in the trichomeless glabra <dig> leaves revealed that galt <dig> mrna levels are 5-, 20- and 7-fold higher in the trichomes, respectively, for the three plant stages tested .
the differences in fut <dig> expression levels between trichomes and unprocessed rosette leaves from wt a. thaliana ler- <dig> plants were smaller than for the galt <dig> gene expression.
fut <dig> expression in trichomes of 4-week-old plants was only  <dig> times higher, whereas in 3- and 5-week-old plants there was no apparent difference in fut <dig> expression in trichome rna and total rna of ler- <dig> leaf material .
however, fut <dig> expression was  <dig> -fold up-regulated in trichomes of a. thaliana ler- <dig> plants compared to glabra <dig> leaves of 4-week-old plants .
similarly to the f-box-nictaba expression pattern, galt <dig> and fut <dig> genes are more significantly up-regulated in the trichomes of 4-week-old plants compared to those of 3- and 5-week-old plants.
the galt <dig> gene was  <dig> - <dig> times more expressed in the leaves of a. thaliana col- <dig> plants compared to the corresponding leaves of a. thaliana ler- <dig> plants , with the highest differential expression in 4-week-old plants.
in contrast, galt <dig> gene expression was 3-fold and 30-fold higher in the trichomes of 3- and 4-week-old a. thaliana ler- <dig> plants compared to a. thaliana col- <dig> plants .
in general, wt a. thaliana ler- <dig> plants had slightly higher transcript levels for fut <dig> than the wt a. thaliana col- <dig> plants in unprocessed rosette leaves .
more important differences were observed in the trichomes of 3- and 4-week-old plants, where the fut <dig> transcript level was 2- and 5-fold higher in the a. thaliana plants with a ler- <dig> background .
over-expression of f-box-nictaba abrogates leaf damage and suppresses pst dc <dig> colonization at  <dig> and  <dig> dpi
since f-box-nictaba gene expression was significantly up-regulated after sa treatment  and infection with pst dc <dig> , it was suggested that f-box-nictaba plays a role in plant defense responses.
to investigate the relevance of f-box-nictaba for plant resistance towards stress, transgenic a. thaliana col- <dig> plants impaired in f-box-nictaba gene expression  as well as plants with increased f-box-nictaba gene expression   were subjected to infection with the virulent pst dc <dig> strain and assessed for disease symptoms and bacterial growth.
the level of colonization 24 h post infection reflects the initial growth of pst dc <dig> within the apoplast, whereas growth of the pathogen in the next days after infection is accompanied by host programmed cell death and necrosis.
beforehand, transgenic f-box-nictaba plants were proven to be morphologically indistinguishable from wt plants throughout growth and development .
as demonstrated in fig.
5a and in additional file 1: figure s <dig>  ko <dig> plants developed similar disease symptoms to wt plants, characterized by leaf lesions constituting up to  <dig> and 35 % of total leaf area at  <dig> and  <dig> dpi, respectively.
plants of both oe lines clearly exhibited less disease symptoms than the wt plants at  <dig> and  <dig> dpi, with lesions covering  <dig> to 12 % of total leaf area.
also at  <dig> dpi, oe <dig> plants demonstrated reduced disease symptoms compared to the wt plants.
consistent with the reduced levels of leaf damage, there was no significant difference in colonization of wt and ko <dig> plants whereas both oe lines yielded significantly lower bacterial counts compared to wt samples at  <dig> dpi and at  <dig> dpi .fig.
<dig> development of disease symptoms and pathogen growth on the leaves of wt and transgenic ko <dig>  oe <dig> and oe <dig> a. thaliana plants infected with pst dc <dig>  a, disease symptoms on leaves of infected a. thaliana plants measured as percent of lesion area.
b, pathogen growth in infected leaves.
levels of colonization are scored as cfu cm− <dig>  the analysis was performed for two independent biological experiments, each with two technical replicates; error bars ± se; *p <  <dig> ; **p <  <dig>
qrt-pcr analysis demonstrated no differential expression for any of the two positive controls wrky <dig> and pr <dig>  neither in infected and mock-treated ko <dig> plants  nor in oe <dig> and oe <dig> plants .
f-box-nictaba expression remained stable in all transgenic plants over the time course of the infection experiment, with very low transcript levels in the ko <dig> line and significant 100-200-fold over-expression in the two oe lines compared to wt plants .fig.
<dig> transcript levels of at2g <dig> and positive control genes determined by qrt-pcr in 5-week-old transgenic a. thaliana plants after pst dc <dig> infection.
a and d, expression of at3g <dig>  in the ko <dig> line  and in lines oe <dig> and oe <dig> .
b and e, expression of at2g <dig>  in the ko <dig> line , and in lines oe <dig> and oe <dig> .
c and f, expression of at2g <dig> in the ko <dig> line , and in lines oe <dig> and oe <dig> .
gene expression levels were calculated relative to the expression in mock-treated wt plants at  <dig> dpi.
for each time point different letters indicate statistically significant differential expression between different lines and compared to mock-treated plants .
values were obtained from three independent biological experiments, each with three technical replicates; error bars ± se
discussion
the qrt-pcr data and the meta-analysis database search show that, although a basal expression of at2g <dig> is present throughout plant development, this f-box-nictaba is a sa- and pathogen-inducible gene.
supported by the expression profile of the wrky <dig> gene and the promoter analysis of at2g <dig>  it is likely that the sa-inducible expression of at2g <dig> is under the control of  sa-dependent wrky transcription regulator.
judging from the delay in maximal expression for the f-box-nictaba gene in comparison to one of the key sa-dependent wrky transcription factors it is suggested that f-box-nictaba is a late sa-responsive gene.
as demonstrated by the pseudomonas infection assays, at2g <dig> expression does not play an essential role in pathogen defense.
it was reported previously that the a. thaliana genome contains more than  <dig> homologous genes encoding at2g02360-like genes for which evidence of expression is available  <cit> .
all these genes show considerable amino acid sequence similarity, reaching levels of >  <dig> and > 40 % sequence similarity for the f-box domain and the nictaba domain, respectively.
it is likely that some of these genes show functional redundancy and could fulfill the same or at least a similar role, as also demonstrated during male gametogenesis  <cit> .
therefore, it is suggested that at2g <dig> will rather act as a partner in a complex network of defense-related proteins.
since transcript levels for f-box-nictaba were up-regulated in wt a. thaliana plants after sa treatment and pst dc <dig> infection but expression was not affected by meja/ethephon treatment or infection with b. cinerea, it is reasonable to conclude that f-box-nictaba plays a role in sa-mediated plant defense reactions to pathogen infection.
f-box-nictaba expression is not only responsive towards biotic stresses, it is also up-regulated up to 3-fold after heat stress.
cross-talk between biotic and abiotic stress signaling pathways is quite common  <cit> .
f-box-nictaba up-regulation upon heat stress might be driven by heat shock-activated transcription factors which can bind to the heat shock-responsive cis-elements present in the at2g <dig> promoter sequence, but also by heat stress-induced wrky transcription factors activating f-box-nictaba expression through the multiple w-box motifs present in its promoter sequence.
expression analysis of the f-box-nictaba gene in wrky-specific mutant lines could clarify the mode of transcriptional regulation of f-box-nictaba.
in line with the plant defense-related expression of at2g <dig>  the histochemical assays showed preferential activity of the at2g <dig> promoter throughout plant development in non-glandular leaf trichomes of transgenic a. thaliana plants, especially in the trichomes present on young leaves.
similarly the qrt-pcr assays demonstrated a dominant presence of at2g <dig> mrna in arabidopsis leaf trichomes.
non-glandular trichomes are epidermal hairs on the surface of most plant aerial organs, characterized by a unicellular dendritic structure with stalk and three to four branches.
they are implicated in transpiration control, thermotolerance and protection against insects, diseases and uv irradiation  <cit> .
although non-glandular trichomes are presumably non-secreting, they do express genes involved in the biosynthesis of secondary compounds suggesting their putative role in plant defense against pathogens  <cit> .
in line with the presumed wrky-dependent regulation of f-box-nictaba, wrky transcription factors are highly expressed in trichome tissues  <cit> .
alternatively, f-box-nictaba gene expression in the trichomes may be under control of myb transcription factors  <cit> , which could bind to the multiple myb-like cis-regulatory elements present in the f-box-nictaba promoter and are reported to be responsible for trichome-specific gene regulation  <cit> .
interestingly, two other f-box-nictaba homologs are also predominantly expressed in trichomes  <cit> .
one of these homologs, encoded by at1g <dig>  is up-regulated both in the trichomes and after infection with pst dc <dig>
the higher transcript levels for both galt <dig> and fut <dig> in the trichomes of wt a. thaliana with ler- <dig> background strongly resembled the trichome-specific expression of the f-box-nictaba gene under study.
in general, all three genes are up-regulated more significantly in the trichomes of 4-week-old a. thaliana ler- <dig> plants compared to those of 3- and 5-week-old plants.
it is not entirely clear why the highest trichome-specific expression was reached at this particular time point, but the most probable explanation is that 4-week old a. thaliana plants are undergoing transition from the vegetative to the reproductive phase, characterized by stem formation and progression into bolting  <cit> .
this is the time when major physiological changes are likely to occur and trichome production was shown to be affected significantly during this process .
irrespective of the cause of the observed time-dependent differences, these data indicate that the two enzymes galt <dig> and fut <dig>  required for biosynthesis of lewis a structures, and the carbohydrate-binding protein f-box-nictaba are co-expressed in the trichomes of a. thaliana ler- <dig> plants.
the fact that lewis a containing glycans can be synthesized in the trichomes does not indicate that they are available to f-box-nictaba within the nucleocytoplasmic compartment of the cell.
future studies will include interaction studies to clarify  the availability of lewis a modified proteins in leaf trichomes and  the binding of the sa-induced f-box-nictaba protein to these glycan structures.
CONCLUSIONS
detailed analysis of the transcript levels for the carbohydrate-binding f-box-nictaba protein from a. thaliana demonstrated up-regulation after plant treatment with stress stimuli of both biotic and abiotic origin, including heat stress, the defense related plant hormone salicylic acid and after plant infection with the virulent pseudomonas syringae pv.
tomato strain dc <dig>  furthermore, the histochemical assays show that the f-box-nictaba gene is abundantly expressed in leaf trichomes, appendages involved in plant defense responses.
experiments with transgenic a. thaliana plants overexpressing the f-box-nictaba protein demonstrated reduced disease symptoms after pst dc <dig> infection compared to the wild-type plants or plants in which f-box-nictaba expression is reduced.
altogether the data strongly suggest that f-box-nictaba is a defense protein involved in the plant response against pseudomonas infection.
furthermore, we show that both f-box-nictaba as well as the glycosyltransferases required for the synthesis of glycan motifs specifically recognized by f-box-nictaba are co-expressed in the trichomes.
although the putative targets of f-box-nictaba and the underlying mechanism of action remain to be elucidated, our research sheds a new light on the putative function of the non-glandular trichomes in plant responses to pathogen attack through glycan-based stress signaling.

 f-box proteins represent one of the largest and most diverse protein families in plants, as most species have several hundred representatives. they are named after their highly conserved n-terminal protein-protein interaction motif of approximately amino acid residues, known as the f-box domain. the majority of the f-box proteins function as part of skp1-cullin1-f-box -type ubiquitin e ligases in which the f-box protein comprises the substrate-binding module. f-box proteins are assembled into active scf complexes through a direct binding of the f-box motif with the scf core s-phase kinase-related protein. through their variable c-terminal substrate-binding domain, f-box proteins specifically bind to and deliver appropriate substrates to the scf complex for ubiquitin-mediated proteolysis by the ubiquitin-proteasome system. the impressive number of f-box proteins in plants and the extensive diversity of their c-terminal target-binding domains contribute to the ability of scf complexes to target a wide variety of substrates. therefore, it is not surprising that f-box proteins are involved in numerous cellular processes within plant development and stress signaling. genetic approaches already revealed an essential role for f-box proteins in plant hormone perception and signaling. f-box proteins were also reported to be involved in circadian clock control, photomorphogenesis and flowering, leaf senescence, self-incompatibility and responses to various biotic stresses. multiple newly discovered f-box proteins combine various recognition mechanisms which enable tight regulation of substrate selection by the f-box protein and each complementary scf complex. amongst these new fbxo proteins, glycan-binding f-box proteins were first discovered in mammals, including fbg fbg and fbg which bind to high-mannose n-glycosylated proteins. the c-terminal substrate-binding domain of both fbg and fbg specifically interacts with the inner n,n’-diacetylchitobiose core of high-mannose n-glycans present on incompletely folded or denatured glycoproteins. since fbg and fbg do not target free mannose structures or non-glycosylated proteins, it was concluded that both fbg proteins function as glycan-binding f-box proteins involved in the endoplasmic reticulum associated degradation pathway. in this pathway, proteins which fail to fold correctly or assemble into oligomeric complexes in the lumen of the endoplasmic reticulum are retro-translocated to the cytosol, where they are captured by an scffbg1/ complex before degradation by the ubiquitin-proteasome system. recently, fbg-like proteins were also reported in plants. the a. thaliana genome contains multiple genes encoding f-box proteins with a putative glycan-binding or lectin-like domain. these proteins are referred to as the f-box-nictaba family, since their c-terminal domain highly resembles nictaba, an n-glycan-binding jasmonate-inducible lectin from tobacco. this f-box protein family in a. thaliana groups approximately members that share over and 40–64 % sequence similarity in the f-box domain and the nictaba domain, respectively. due to the presence of the f-box domain linked to a lectin-like c-terminal substrate-binding domain, it is tempting to speculate that these f-box-nictaba proteins function as substrate adaptors in an endoplasmic reticulum associated degradation pathway in plants similar to the mammalian fbg proteins. furthermore, both nictaba and fbg show comparable glycan-binding properties towards the inner core structure of n-glycans. however, detailed analysis of the sugar specificities for the a. thaliana f-box-nictaba protein revealed binding of its nictaba domain to n- and o-glycans containing n-acetyllactosamine structures, lewis a, lewis x and lewis y motifs. at present, it is not clear if and how the f-box-nictaba protein can bind to these structures in planta. n-glycan modification with lewis a motifs takes place in the golgi apparatus and plant-specific lewis epitopes were only shown on some extracellular glycoproteins and on membrane-bound glycoproteins present at the plant cell surface. moreover, in contrast to most plant families, the occurrence of lewis a motifs in a. thaliana was reported to be generally low and tissue specific with highest levels in pedicels, stems and nodes and with moderate levels in siliques and the shoot apex. lewis a structures are not detectable in the leaves of a. thaliana plants. in this study, we show that the transcripts for the a. thaliana f-box-nictaba protein encoded by at2g are up-regulated after plant treatment with salicylic acid, heat stress and upon infection with pseudomonas syringae pv. tomato strain dc with preferential activity of its promoter sequence in leaf trichomes. stress experiments on transgenic a. thaliana plants with altered f-box-nictaba expression suggested a role for f-box-nictaba in plant defense-related signaling pathways.  f-box-nictaba gene expression is up-regulated after sa application and heat shock treatment the expression pattern of f-box-nictaba was first analysed in wild-type a. thaliana plants grown under laboratory growth conditions. qrt-pcr analyses demonstrated that at2g was expressed during all developmental stages and in every tissue tested. a slightly lower expression of at2g was observed in roots from young plants and in flowers compared to the youngest stage tested. in silico expression analysis indicated that at2g is a stress-responsive gene. indeed, the uniform expression profile of at2g considerably changed when a. thaliana plants were subjected to treatments with various plant hormones and several abiotic stress factors. after 1 h of sa treatment, at2g expression was slightly down-regulated compared to the mock treatment, but at2g mrna levels were significantly higher after 3 h with a maximal increase of almost 4-fold after 10 h of sa application. transcript levels in treated plants slightly dropped after 24 h but were still two-fold higher than in the untreated plants. transcript levels for the sa-inducible transcription factor wrky were up-regulated faster and reached much higher levels compared to f-box-nictaba expression levels.fig. relative transcript levels of at2g in wt a. thaliana col- plants determined by qrt-pcr analyses of two independent biological experiments, each with multiple technical replicates as pointed out in methods section; error bars ± se. asterisks indicate statistically significant differential expression compared to control samples. a, at2g expression levels measured in a. thaliana plants grown under standard conditions. b-c, at2g expression levels in 16-day-old a. thaliana seedlings after sa treatment or after heat shock. d-e, relative expression of at2g after infection of 5-week-old a. thaliana plants with pst dc or with b. cinerea strain b. expression levels are compared to the at2g expression level in the cotyledons stage or to gene expression levels in the mock-treated plants also heat stress enhanced f-box-nictaba gene expression with a maximal up-regulation of almost 3-fold after 10 h of heat stress compared to control plants. however, compared to the at2g gene the expression of the hsp70b gene, encoding a heat shock-responsive chaperone protein, was enhanced much faster and reached much higher mrna levels. other hormones and abiotic stress treatments either slightly down-regulated or did not affect f-box-nictaba gene expression at all. f-box-nictaba expression is up-regulated in wt a. thaliana plants after pseudomonas infection but is slightly down-regulated by fungal infection five-week-old wt a. thaliana col- plants were infected either with the virulent hemibiotrophic bacterium p. syringae pv. tomato strain dc or the necrotrophic fungus botrytis cinerea strain b. pst dc infection strongly enhanced the expression of both control genes wrky and pr in rosette leaves. f-box-nictaba mrna levels were significantly up-regulated approximately -fold at and dpi compared to mock-sprayed plants. f-box-nictaba expression dropped at and dpi but was still higher than in the mock-treated plants. in contrast, b. cinerea infection mildly affected f-box-nictaba mrna levels compared to mock-treated plants. at and dpi, at2g expression levels were reduced almost two-fold. the expression of the control gene pr was not altered significantly. in contrast, the expression of the control gene pdf was highly up-regulated reaching a 50-fold increase in infected tissues at dpi. f-box-nictaba promoter is particularly active in leaf trichomes to test the activity of the 5′-upstream region of the at2g gene, a promoter at2g02360:gus reporter construct was introduced into a. thaliana col- plants and used for histochemical assays. first data revealed a distinct and localized gus staining in the leaf trichomes of 14-day-old pat2g02360:gus a. thaliana seedlings. subsequently, plants grown under standard conditions were also analyzed at different developmental stages. overall, plants of different transgenic lines showed comparable gus staining patterns, but the intensity of the gus staining differed amongst plants. in the very young seedlings no gus staining was detected. in the cotyledons and 2-leaves stage, plantlets showed a weak gus activity spread over mesophyll cells. when plants developed, a very intense gus activity was observed all over the petioles and in the majority of the trichomes present on the shoot meristem and first leaves. gus staining in the trichomes present on newly developing leaves remained throughout further development of the plants. in plantlets of stage and older, gus staining was prominently visible in trichomes present on new leaves and in the petioles, the major leaf vein and some parts with mesophyll cells of older leaves. trichomes present on older leaves also showed gus staining, especially in those located in close proximity of the petiole, but their gus staining was less intense compared to the staining detected in trichomes residing on young leaves. in flowering plants, gus staining was mainly visible in the flowers, some siliques and in some major veins and trichomes located in close proximity of these veins on rosette and cauline leaves. the camv35s:gus control plants showed an intense blue, homogeneous gus staining pattern in all organs and tissues of the plants throughout development.fig. histochemical analysis of pat2g02360:gus a. thaliana lines. a, gus staining data for 14-day-old plantlets. b, different developmental plant stages analyzed. c, gus staining data for selected developmental stages with a closer look at trichomes present on new leaves and on flower buds; panel b represents a close-up of rosette leaves. scale bars represent 1 mm f-box-nictaba is predominantly expressed in the trichomes of a. thaliana plants given the apparent activity of the pat2g02360:gus reporter construct in leaf trichomes and the occurrence of putative cis-regulatory elements reported to be responsible for trichome-specific gene regulation in the f-box-nictaba promoter sequence including eight myb-like recognition sites and five t/g-box elements, the expression of the f-box-nictaba gene was quantified by qrt-pcr in trichomes purified from wt a. thaliana col- rosette leaves. at2g mrna levels measured in trichomes were slightly higher compared to those measured in unprocessed rosette leaves with increased expression in trichomes up to approximately -fold in 4-week-old plants and 2-fold in 3- and 5-week-old plants.fig. transcript levels of at2g in the trichomes isolated from rosette leaves of wt a. thaliana plants of col- or ler- ecotype compared to corresponding gene expression levels in complete rosette leaves from wt a. thaliana plants of col- or ler- ecotype or of glabra a. thaliana plants. relative transcript levels were determined in 3-, 4- and 5-week-old plants by qrt-pcr analyses of two independent biological experiments, each with two technical replicates; error bars ± se. asterisks indicate statistically significant differential expression compared to control samples. a, gene expression levels in the trichomes of wt a. thaliana col- plants compared to complete rosette leaves from wt a. thaliana col- plants. b, gene expression levels in the trichomes of wt a. thaliana ler- plants compared to complete rosette leaves of wt a. thaliana ler- plants or compared to complete rosette leaves of glabra a. thaliana plants as well as in complete rosette leaves of wt a. thaliana ler- plants compared to complete rosette leaves of glabra plants. c, comparison of gene expression levels for f-box-nictaba in the trichomes and in complete rosette leaves of wt a. thaliana col- compared to wt a. thaliana ler- plants f-box-nictaba gene expression was also analyzed in rosette leaves of mutant a. thaliana plants completely lacking trichomes. these glabra plants are impaired in the gl gene which encodes a myb-like transcription factor required for trichome development. since the glabra mutants are made in a landsberg erecta- background, f-box-nictaba expression was also examined in wt a. thaliana ler- plants. trichome purification from rosette leaves of a. thaliana ler- plants yielded similar amounts of trichomes as for a. thaliana col- plants. however, the relative expression of the f-box-nictaba gene was much more pronounced in trichomes of a. thaliana ler- plants with an up-regulation of -fold, 150-fold and 25-fold compared to total leaf tissue from 3-, 4- and 5-week-old a. thaliana ler- plants, respectively. in comparison to glabra a. thaliana plants, at2g transcript levels were 20-, 160- and 175-fold higher in trichomes from a. thaliana ler- plants when compared to total leaf tissue from 3-, 4- and 5-week-old glabra a. thaliana plants, respectively. the transcript levels for the f-box-nictaba gene were higher in wt a. thaliana leaves of plants with a col- background compared to leaves from wt plants with a ler- background, at all developmental stages investigated, irrespective whether the purified trichomes or complete rosette leaves of both ecotypes were assessed. the differential expression was most striking when unprocessed rosette leaves were compared: at2g mrna levels were approx. and times higher in leaves of 3-, 4- and 5-week-old plants of col- ecotype, respectively, compared to leaves of the same age from plants with a ler- background. in the trichomes isolated from 3-, 4- and 5-week-old wt a. thaliana col- plants f-box-nictaba gene expression was 90-, 7- and 10-fold higher than in the trichomes of 3-, 4- and 5-week-old plants from wt a. thaliana ler- respectively. galt and fut genes are up-regulated in the trichomes of wt a. thaliana ler- plants f-box-nictaba was identified as a functional lectin that can bind n- and o-glycans containing lactosamine structures, lewis a, lewis x and lewis y motifs. arabidopsis plants were only reported to contain lewis a-modified n-glycans. to our knowledge, the presence of lewis a has not been studied yet in trichomes of a. thaliana plants. since f-box-nictaba expression is prominent in the trichomes, we addressed whether lewis a motifs could be specifically synthesized in the trichomes. therefore, qrt-pcr analysis was performed to analyse gene expression for the enzymes indispensable for lewis a epitope synthesis in a. thaliana: i.e., at1g encoding β -galactosyltransferase and at1g encoding α -fucosyltransferase. according to fig.  both galt and fut are expressed in rosette leaves as well as in the leaf trichomes of a. thaliana col- plants. the transcript levels for galt were significantly lower in the trichomes of wt a. thaliana col- plants compared to those measured in the unprocessed rosette leaves of the same plants at all investigated developmental stages, the lowest value being a 5-fold decrease of galt expression in trichomes of 4-week-old plants. in 3- and 5-week-old plants galt mrna levels were approximately two and three times lower in trichomes compared to rosette leaves, respectively. in contrast to galt fut did not show a significant differential expression between trichomes and unprocessed rosette leaves.fig. transcript levels of galt and fut in trichomes isolated from rosette leaves of wt a. thaliana plants of col- or ler- ecotype compared to corresponding gene expression levels in complete rosette leaves of wt a. thaliana plants of col- or ler- ecotype or of glabra a. thaliana plants. transcript levels were determined in 3-, 4- and 5-week-old plants by qrt-pcr analyses of two independent biological experiments, each with two technical replicates; error bars ± se. asterisks indicate statistically significant differential expression compared to control samples. a-b, relative gene expression levels of galt and fut in the trichomes of wt a. thaliana col- plants compared to complete rosette leaves from wt a. thaliana col- plants. c-d, relative gene expression levels of galt and fut in the trichomes of wt a. thaliana ler- plants compared to complete rosette leaves of wt a. thaliana ler- plants or compared to complete rosette leaves of glabra a. thaliana plants as well as in complete rosette leaves of wt a. thaliana ler- plants compared to complete rosette leaves of glabra plants. e-f, comparison of gene expression levels of galt and fut in the trichomes and in complete rosette leaves of wt a. thaliana col- plants compared to wt a. thaliana ler- plants conversely, in trichomes of 3- and 4-week-old wt a. thaliana ler- plants, the expression of the galt gene was markedly up-regulated by 7- and 29-fold, respectively, when compared to levels in unprocessed rosette leaves originating from these plants. comparison of the galt expression levels in trichomes of wt a. thaliana ler- plants to those in the trichomeless glabra leaves revealed that galt mrna levels are 5-, 20- and 7-fold higher in the trichomes, respectively, for the three plant stages tested. the differences in fut expression levels between trichomes and unprocessed rosette leaves from wt a. thaliana ler- plants were smaller than for the galt gene expression. fut expression in trichomes of 4-week-old plants was only times higher, whereas in 3- and 5-week-old plants there was no apparent difference in fut expression in trichome rna and total rna of ler- leaf material. however, fut expression was -fold up-regulated in trichomes of a. thaliana ler- plants compared to glabra leaves of 4-week-old plants. similarly to the f-box-nictaba expression pattern, galt and fut genes are more significantly up-regulated in the trichomes of 4-week-old plants compared to those of 3- and 5-week-old plants. the galt gene was - times more expressed in the leaves of a. thaliana col- plants compared to the corresponding leaves of a. thaliana ler- plants, with the highest differential expression in 4-week-old plants. in contrast, galt gene expression was 3-fold and 30-fold higher in the trichomes of 3- and 4-week-old a. thaliana ler- plants compared to a. thaliana col- plants. in general, wt a. thaliana ler- plants had slightly higher transcript levels for fut than the wt a. thaliana col- plants in unprocessed rosette leaves. more important differences were observed in the trichomes of 3- and 4-week-old plants, where the fut transcript level was 2- and 5-fold higher in the a. thaliana plants with a ler- background. over-expression of f-box-nictaba abrogates leaf damage and suppresses pst dc colonization at and dpi since f-box-nictaba gene expression was significantly up-regulated after sa treatment and infection with pst dc, it was suggested that f-box-nictaba plays a role in plant defense responses. to investigate the relevance of f-box-nictaba for plant resistance towards stress, transgenic a. thaliana col- plants impaired in f-box-nictaba gene expression as well as plants with increased f-box-nictaba gene expression were subjected to infection with the virulent pst dc strain and assessed for disease symptoms and bacterial growth. the level of colonization 24 h post infection reflects the initial growth of pst dc within the apoplast, whereas growth of the pathogen in the next days after infection is accompanied by host programmed cell death and necrosis. beforehand, transgenic f-box-nictaba plants were proven to be morphologically indistinguishable from wt plants throughout growth and development. as demonstrated in fig. 5a and in additional file 1: figure s ko plants developed similar disease symptoms to wt plants, characterized by leaf lesions constituting up to and 35 % of total leaf area at and dpi, respectively. plants of both oe lines clearly exhibited less disease symptoms than the wt plants at and dpi, with lesions covering to 12 % of total leaf area. also at dpi, oe plants demonstrated reduced disease symptoms compared to the wt plants. consistent with the reduced levels of leaf damage, there was no significant difference in colonization of wt and ko plants whereas both oe lines yielded significantly lower bacterial counts compared to wt samples at dpi and at dpi.fig. development of disease symptoms and pathogen growth on the leaves of wt and transgenic ko oe and oe a. thaliana plants infected with pst dc a, disease symptoms on leaves of infected a. thaliana plants measured as percent of lesion area. b, pathogen growth in infected leaves. levels of colonization are scored as cfu cm− the analysis was performed for two independent biological experiments, each with two technical replicates; error bars ± se; *p < ; **p <  qrt-pcr analysis demonstrated no differential expression for any of the two positive controls wrky and pr neither in infected and mock-treated ko plants nor in oe and oe plants. f-box-nictaba expression remained stable in all transgenic plants over the time course of the infection experiment, with very low transcript levels in the ko line and significant 100-200-fold over-expression in the two oe lines compared to wt plants.fig. transcript levels of at2g and positive control genes determined by qrt-pcr in 5-week-old transgenic a. thaliana plants after pst dc infection. a and d, expression of at3g in the ko line and in lines oe and oe. b and e, expression of at2g in the ko line, and in lines oe and oe. c and f, expression of at2g in the ko line, and in lines oe and oe. gene expression levels were calculated relative to the expression in mock-treated wt plants at dpi. for each time point different letters indicate statistically significant differential expression between different lines and compared to mock-treated plants. values were obtained from three independent biological experiments, each with three technical replicates; error bars ± se discussion the qrt-pcr data and the meta-analysis database search show that, although a basal expression of at2g is present throughout plant development, this f-box-nictaba is a sa- and pathogen-inducible gene. supported by the expression profile of the wrky gene and the promoter analysis of at2g it is likely that the sa-inducible expression of at2g is under the control of sa-dependent wrky transcription regulator. judging from the delay in maximal expression for the f-box-nictaba gene in comparison to one of the key sa-dependent wrky transcription factors it is suggested that f-box-nictaba is a late sa-responsive gene. as demonstrated by the pseudomonas infection assays, at2g expression does not play an essential role in pathogen defense. it was reported previously that the a. thaliana genome contains more than homologous genes encoding at2g02360-like genes for which evidence of expression is available. all these genes show considerable amino acid sequence similarity, reaching levels of >  and > 40 % sequence similarity for the f-box domain and the nictaba domain, respectively. it is likely that some of these genes show functional redundancy and could fulfill the same or at least a similar role, as also demonstrated during male gametogenesis. therefore, it is suggested that at2g will rather act as a partner in a complex network of defense-related proteins. since transcript levels for f-box-nictaba were up-regulated in wt a. thaliana plants after sa treatment and pst dc infection but expression was not affected by meja/ethephon treatment or infection with b. cinerea, it is reasonable to conclude that f-box-nictaba plays a role in sa-mediated plant defense reactions to pathogen infection. f-box-nictaba expression is not only responsive towards biotic stresses, it is also up-regulated up to 3-fold after heat stress. cross-talk between biotic and abiotic stress signaling pathways is quite common. f-box-nictaba up-regulation upon heat stress might be driven by heat shock-activated transcription factors which can bind to the heat shock-responsive cis-elements present in the at2g promoter sequence, but also by heat stress-induced wrky transcription factors activating f-box-nictaba expression through the multiple w-box motifs present in its promoter sequence. expression analysis of the f-box-nictaba gene in wrky-specific mutant lines could clarify the mode of transcriptional regulation of f-box-nictaba. in line with the plant defense-related expression of at2g the histochemical assays showed preferential activity of the at2g promoter throughout plant development in non-glandular leaf trichomes of transgenic a. thaliana plants, especially in the trichomes present on young leaves. similarly the qrt-pcr assays demonstrated a dominant presence of at2g mrna in arabidopsis leaf trichomes. non-glandular trichomes are epidermal hairs on the surface of most plant aerial organs, characterized by a unicellular dendritic structure with stalk and three to four branches. they are implicated in transpiration control, thermotolerance and protection against insects, diseases and uv irradiation. although non-glandular trichomes are presumably non-secreting, they do express genes involved in the biosynthesis of secondary compounds suggesting their putative role in plant defense against pathogens. in line with the presumed wrky-dependent regulation of f-box-nictaba, wrky transcription factors are highly expressed in trichome tissues. alternatively, f-box-nictaba gene expression in the trichomes may be under control of myb transcription factors, which could bind to the multiple myb-like cis-regulatory elements present in the f-box-nictaba promoter and are reported to be responsible for trichome-specific gene regulation. interestingly, two other f-box-nictaba homologs are also predominantly expressed in trichomes. one of these homologs, encoded by at1g is up-regulated both in the trichomes and after infection with pst dc the higher transcript levels for both galt and fut in the trichomes of wt a. thaliana with ler- background strongly resembled the trichome-specific expression of the f-box-nictaba gene under study. in general, all three genes are up-regulated more significantly in the trichomes of 4-week-old a. thaliana ler- plants compared to those of 3- and 5-week-old plants. it is not entirely clear why the highest trichome-specific expression was reached at this particular time point, but the most probable explanation is that 4-week old a. thaliana plants are undergoing transition from the vegetative to the reproductive phase, characterized by stem formation and progression into bolting. this is the time when major physiological changes are likely to occur and trichome production was shown to be affected significantly during this process. irrespective of the cause of the observed time-dependent differences, these data indicate that the two enzymes galt and fut required for biosynthesis of lewis a structures, and the carbohydrate-binding protein f-box-nictaba are co-expressed in the trichomes of a. thaliana ler- plants. the fact that lewis a containing glycans can be synthesized in the trichomes does not indicate that they are available to f-box-nictaba within the nucleocytoplasmic compartment of the cell. future studies will include interaction studies to clarify the availability of lewis a modified proteins in leaf trichomes and the binding of the sa-induced f-box-nictaba protein to these glycan structures.  detailed analysis of the transcript levels for the carbohydrate-binding f-box-nictaba protein from a. thaliana demonstrated up-regulation after plant treatment with stress stimuli of both biotic and abiotic origin, including heat stress, the defense related plant hormone salicylic acid and after plant infection with the virulent pseudomonas syringae pv. tomato strain dc furthermore, the histochemical assays show that the f-box-nictaba gene is abundantly expressed in leaf trichomes, appendages involved in plant defense responses. experiments with transgenic a. thaliana plants overexpressing the f-box-nictaba protein demonstrated reduced disease symptoms after pst dc infection compared to the wild-type plants or plants in which f-box-nictaba expression is reduced. altogether the data strongly suggest that f-box-nictaba is a defense protein involved in the plant response against pseudomonas infection. furthermore, we show that both f-box-nictaba as well as the glycosyltransferases required for the synthesis of glycan motifs specifically recognized by f-box-nictaba are co-expressed in the trichomes. although the putative targets of f-box-nictaba and the underlying mechanism of action remain to be elucidated, our research sheds a new light on the putative function of the non-glandular trichomes in plant responses to pathogen attack through glycan-based stress signaling. 
a. thaliana plants overexpressing the gene showed less disease symptoms after pst dc infection with reduced bacterial colonization compared to infected wild type and f-box-nictaba knock-out plants. qrt-pcr analyses confirmed high expression of f-box-nictaba in leaf trichomes. here, we present a detailed qrt-pcr expression analysis of f-box-nictaba in arabidopsis plants upon different stresses and hormone treatments. β-glucuronidase histochemical staining of transgenic arabidopsis plants displayed preferential activity of the at2g promoter in trichomes present on young rosette leaves. previously, the so-called f-box-nictaba protein, encoded by the gene at2g was shown to be a functional lectin which binds n-acetyllactosamine structures. expression of the f-box-nictaba gene was enhanced after plant treatment with salicylic acid and after plant infection with the virulent pseudomonas syringae pv. a small group of f-box proteins consisting of a conserved f-box domain linked to a domain homologous to the glycan-binding protein has been identified within the genome of arabidopsis thaliana. our data show that the arabidopsis f-box-nictaba gene is a stress-inducible gene responsive to sa, bacterial infection and heat stress, and is involved in salicylic acid related plant defense responses. f-box-nictabaglycanlectinpseudomonassalicylic acidstressfwog006114n and g002416nvan damme els j. m. universiteit gent bof2010/goa/003van damme els j. m. issue-copyright-statement© the author 2016
 a small group of f-box proteins consisting of a conserved f-box domain linked to a domain homologous to the glycan-binding protein has been identified within the genome of arabidopsis thaliana. previously, the so-called f-box-nictaba protein, encoded by the gene at2g was shown to be a functional lectin which binds n-acetyllactosamine structures. here, we present a detailed qrt-pcr expression analysis of f-box-nictaba in arabidopsis plants upon different stresses and hormone treatments.  expression of the f-box-nictaba gene was enhanced after plant treatment with salicylic acid and after plant infection with the virulent pseudomonas syringae pv. tomato strain dc. β-glucuronidase histochemical staining of transgenic arabidopsis plants displayed preferential activity of the at2g promoter in trichomes present on young rosette leaves. qrt-pcr analyses confirmed high expression of f-box-nictaba in leaf trichomes. a. thaliana plants overexpressing the gene showed less disease symptoms after pst dc infection with reduced bacterial colonization compared to infected wild type and f-box-nictaba knock-out plants.  our data show that the arabidopsis f-box-nictaba gene is a stress-inducible gene responsive to sa, bacterial infection and heat stress, and is involved in salicylic acid related plant defense responses. this knowledge enriched our understanding of the physiological importance of f-box-nictaba, and can be used to create plants with better performance in changing environmental conditions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords f-box-nictabaglycanlectinpseudomonassalicylic acidstressfwog006114n and g002416nvan damme els j. m. universiteit gent bof2010/goa/003van damme els j. m. issue-copyright-statement© the author 2016 