BACKGROUND
programmed cell death  is a genetically defined process associated with distinctive morphological and biochemical characteristics, and is an integral part of the life cycle of multicellular organisms  <cit> .
in plants, pcd occurs during developmental processes and in response to abiotic and biotic stresses  <cit> .
the major pcd signaling pathways involve mitochondria and plasma membrane receptors, although it has recently been shown that er stress caused by impaired er function can also induce apoptotic pathways in animals and plants  <cit> .
the er performs several important functions, including protein targeting and secretion, vesicle trafficking, and membrane biogenesis, and its proper function is essential to cell survival.
perturbations in er homeostasis disrupt folding of proteins, leading to accumulation of unfolded proteins and protein aggregates.
this condition is called er stress and is detrimental to cell survival  <cit> .
under conditions of er stress, a cell activates a signal transduction pathway termed the unfolded protein response  to limit the damage and maintain er homeostasis  <cit> .
prolonged and excessive er stress induces apoptosis, evidenced by dna fragmentation, cytochrome c release, and induction of caspase activity  <cit> .
in mammals, the er stress-induced apoptotic pathway involves cross-talk between er and mitochondria through bcl- <dig> family members bcl- <dig>  bax and bak, which are localized in both mitochondria and er  <cit> .
recent reports suggest that in animal cells, the mitochondrial apoptotic pathway mediated by apaf- <dig> is an integral part of er stress-induced apoptosis  <cit> .
in plants, treatment with tunicamycin, an inhibitor of n-linked protein glycosylation, and cyclopiazonic acid, a blocker of plant er-type iia calcium pumps, elicits er stress, followed by activation of pcd with typical apoptotic morphology  <cit> .
however, the mechanisms of upr and er stress-induced pcd in plants remain largely unknown.
one mechanism of er stress response in arabidopsis involves membrane-associated bzip transcription factors; following er stress, bzip <dig> and bzip <dig> are processed and their released n-terminal domains are translocated to the nucleus to upregulate the expression of er stress response genes including bips , bi- <dig> , pdi , and calnexin  <cit> .
genomic amplification  of n-myc oncogene in human neuroblastoma correlates with aggressive tumor growth and poor prognosis  <cit> .
neuroblastoma amplified gene  was first identified as a gene co-amplified with the n-myc gene, although nag is widely expressed in normal human tissues and its homologous sequences are found in plant databases  <cit> .
recently, it has been shown that nag is localized in er and is a component of the syntaxin  <dig> complex that is involved in golgi-to-er retrograde transport using human cell lines  <cit> .
in this study, we investigated in planta functions of nag in nicotiana benthamiana.
we showed that n. benthamiana nag  played a role in protein transport pathway, and nbnag deficiency caused er stress and cell death, suggesting its essential role in plant growth and survival.
RESULTS
identification of nbnag
functional genomics using virus-induced gene silencing  revealed that silencing of the n. benthamiana homolog of neuroblastoma amplified gene , designated nbnag, results in growth arrest and acute plant death.
the ~ <dig>  kb full-length nbnag cdna encodes a polypeptide of  <dig>  amino acids with a predicted molecular mass of  <dig> .
<dig> da .
database searches identified closely related genes in human, mouse, zebrafish, c. elegans, arabidopsis and rice.
the nag homolog encodes a protein of ~ <dig>  amino acids in arabidopsis , rice , and human , and is a single-copy gene in all three genomes.
our analyses showed that there is no null mutant of nag in arabidopsis t-dna insertion lines.
the nbnag protein contains the sec <dig> domain  that has been implicated in er-golgi trafficking in yeast  <cit> .
overall, nbnag shows 49%, 42% and 29% sequence identity to the nag homologs from arabidopsis, rice, and human, respectively.
nbnag sequence was aligned with those of the nag homologs from arabidopsis and rice .
er localization of nbnag in tobacco by- <dig> cells
to determine subcellular localization of nbnag, we performed immunolabeling experiments in tobacco by- <dig> cells using anti-nbnag antibodies .
after immunolabeling with anti-nbnag antibodies, by- <dig> cells were briefly stained with er tracker™ blue-white dpx as an er marker.
confocal laser scanning microscopy revealed that red fluorescent signals of nbnag overlapped with blue fluorescence of the er tracker near the nuclei and along the network in a typical er localization pattern in by- <dig> cells  <cit>  .
when the by- <dig> cells were examined by fluorescence microscopy, they exhibited a network pattern of red fluorescence in the cytosol and in the nuclear periphery .
these results suggest that nbnag was mainly localized in the er, consistent with the er localization of the yeast and mammalian nag  <cit> .
expression of arabidopsis nag
we transformed arabidopsis with a fusion construct between the arabidopsis nag promoter  and gus .
the  <dig>  bp promoter was the maximum size due to a closely located adjacent gene in arabidopsis genome.
gus staining of seedlings demonstrated that atnag promoter activity was mainly detected in the aerial parts, particularly in shoot apical meristem, leaf primordia, developing vasculature, and stomata .
in roots, gus activity was localized in emerging lateral roots and root vasculature, albeit weakly .
in the reproductive stage, flower buds and axillary buds showed gus staining .
in general, nag expression was concentrated in young aerial tissues containing dividing cells.
interestingly, atnagp::gus activity in a seedling increased in response to increasing concentrations of tunicamycin , an inhibitor of n-linked glycosylation .
vigs phenotypes and suppression of endogenous nbnag transcripts
for vigs, we cloned two different fragments of nbnag cdna into the trv -based vigs vector ptv <dig>  and infiltrated n. benthamiana plants with agrobacterium containing each plasmid.
trv:nag and trv:nag contain the non-overlapping  <dig> bp and  <dig> bp n-terminal regions of the cdna, respectively .
the effect of gene silencing on the level of nbnag mrna was examined using semiquantitative rt-pcr .
reduced amounts of pcr product were produced in three independent plants from the trv:nag and trv:nag lines compared with trv control, indicating that the endogenous level of the nbnag transcripts was reduced in those plants.
the transcript levels of actin, which served as the control, remained constant.
vigs with trv:nag and trv:nag constructs resulted in growth arrest and acute plant death .
necrotic lesions were evident in young leaves around the shoot apex at 15– <dig> days after infiltration  .
at  <dig> dai, the shoot apex was completely abolished with no stem growth or new leaf formation .
the lesions progressively expanded, leading to premature death of the plants.
root growth was also affected by nbnag vigs at  <dig> and  <dig> dai .
the petiole of the fourth leaf above the infiltrated leaf, and the stem where the fourth leaf above the infiltrated leaf was attached were cross-sectioned freehand and observed under light microscopy .
the localized brown pigment in the petiole  and the stem  at  <dig> dai indicates cell death in the vasculature of trv:nag lines, particularly in the cambium.
fluorescence microscopy revealed that trv:nag lines accumulated large amounts of autofluorescent secondary metabolites, which were also observed at infection sites during hypersensitive cell death  <cit> , in the stem vasculature , while trv control exhibited the fluorescence only in the xylem tracheary elements .
analysis of programmed cell death phenotypes
although morphology and sizes of abaxial epidermal cells of the trv:nag leaves remained normal compared with trv control , extension and fragmentation of nuclear chromatin was evident in trv:nag lines at  <dig> dai , but not at  <dig> dai when lesions were not formed yet .
dna laddering was also observed in genomic dna isolated from the trv:nag leaves .
since nuclear fragmentation and dna laddering are hallmark features of pcd, these results suggest that silencing of nbnag activates pcd in plants.
during apoptosis in animal cells, activation of the cell death pathway is initiated by modification of mitochondrial membrane permeability  <cit> .
mitochondrial membrane potential of leaf protoplasts from vigs lines was monitored by tmrm  fluorescent probes that accumulate in mitochondria in proportion to the mitochondrial membrane potential  <cit> .
the average tmrm fluorescence of trv:nag protoplasts was ~4-fold lower than that of trv control at  <dig> dai, indicating reduced mitochondrial membrane potential , but there was no difference in tmrm fluorescence between trv and trv:nag lines at  <dig> dai .
chlorophyll autofluorescence was not affected in trv:nag protoplasts at either  <dig> dai  or  <dig> dai .
to test whether reactive oxygen species  are involved in the cell death phenotype of trv:nag plants, leaf protoplasts prepared from vigs lines  were incubated with h2dcfda that becomes activated in the presence of h2o <dig> to produce green fluorescence.
accumulation of fluorescent h2dcfda in trv:nag protoplasts was ~ <dig> -fold higher than in trv control, indicating h2o <dig> accumulation .
ultrastructural analyses of the cell death phenotype
light  and transmission electron microscopy   of transverse leaf sections revealed degenerating spongy mesophyll cells at early and late stages in trv:nag lines, in contrast to trv control cells at  <dig> dai.
tem also showed disintegrating chloroplasts  and mitochondria , ruptured vacuoles , and abnormal nuclei  in trv:nag lines, compared with trv control .
these results suggest that the nbnag-induced cell death involves vacuole collapse leading to enzymatic degradation of organelles and cell contents, as demonstrated in pcd induced by other stimuli  <cit> .
nbnag deficiency inhibits intracellular protein transport
yeast 82-kda sec39p  is localized to the er  <cit> , and associated with the syntaxin  <dig> complex that is involved in golgi-to-er retrograde transport  <cit> .
human nag containing a sec39p-homologous region appears to be the ortholog of yeast sec39p despite the marked difference in their molecular sizes, and plays a similar role in the golgi-er retrograde trafficking  <cit> .
nbnag also contains the sec <dig> domain , indicating that nbnag may be involved in protein trafficking in a plant cell.
to test this possibility, protoplasts isolated from trv and trv:nag leaves  were transformed with sporamin:gfp  construct encoding a fusion protein between green fluorescent protein  and the n-terminal region of sporamin as a vacuolar reporter gene .
previous experiments with arabidopsis protoplasts showed that spo:gfp is targeted to the central vacuole, where it is processed from a 40-kda precursor to a 30-kda form by proteolysis  <cit> .
consistent with the results, fluorescence microscopy revealed spo:gfp in the central vacuole in both trv and trv:nag protoplasts , while gfp alone was localized in the cytosol and the nucleus in trv protoplasts .
to measure trafficking efficiency, relative amounts of the precursor and the processed form of spo:gfp were compared in transformed trv and trv:nag protoplasts by immunoblotting using anti-gfp antibody .
in trv protoplasts, ~44% of spo:gfp was in the  <dig> kda precursor form; however, in trv:nag protoplasts, the precursor form is visibly dominant, indicating that nbnag deficiency leads to reduced vacuole trafficking of spo:gfp.
we next examined the effects of nbnag depletion on the secretory pathway using invertase:gfp, a chimeric protein consisting of full-length secretory invertase and gfp  <cit> , as a reporter .
after transformation with the invertase:gfp construct, green fluorescent signals were not detectable in trv protoplasts, indicating that invertase:gfp was secreted into the medium as previously observed  <cit> .
however, invertase:gfp signal was readily detected in trv:nag protoplasts .
to confirm this finding, proteins were extracted from the protoplasts and the incubation medium, and analyzed by western blot analysis with anti-gfp antibody .
in trv controls, invertase:gfp  was mainly detected in the medium, while a minor portion of the protein remained in the protoplasts.
in contrast, in the trv:nag line the fusion protein was predominantly present in the protoplasts, suggesting that nbnag depletion blocked the secretion of invertase:gfp into the medium.
next, we examined er-golgi transport using an artificial er marker protein, gkx  <cit> .
trv protoplasts transformed with gkx exhibited a reticular network pattern of green fluorescence  as observed previously  <cit> .
however, trv:nag protoplasts expressing gkx displayed a punctate and aggregated fluorescence signal, suggesting disruption of the er network.
since gkx contains an n-glycosylation site, we examined the sensitivity of the n-glycans of gkx to endoglycosidase h  treatment.
it has been reported that the n-glycans of er proteins are sensitive to endo h, while the n-glycans of golgi proteins are resistant due to modification  <cit> .
protein extracts from trv and trv:nag protoplasts were digested by endo h prior to western blotting with anti-gfp antibody .
in the trv control, endo h digestion of gkx resulted in two bands, ~ <dig> kda endo h-sensitive gkx proteins  and ~ <dig> kda endo h-resistant proteins .
these results indicate that a large part of gkx proteins was transported from er to golgi complex through anterograde trafficking.
trv:nag lines also contained both the er and the golgi forms, but the ratio of the er form to the golgi form was higher than in trv controls .
these results provide evidence that protein transport from er to golgi complex was inhibited by nbnag deficiency.
nbnag-silencing causes er stress
the abnormal er morphology shown by gkx expression raised a possibility that nbnag-deficient plants may have been subjected to er stress.
transcriptome analysis in response to tunicamycin-induced er stress in arabidopsis revealed transcriptional up-regulation of the genes encoding bips, bi <dig> , cnk <dig>  and hsp <dig>  <cit> ; therefore, we examined transcript levels of these er stress-related genes in trv and trv:nag lines.
semiquantitative rt-pcr revealed increased transcript levels of bi <dig> and bip <dig> in trv:nag lines compared with trv control at  <dig> dai, while bi <dig>  bip <dig>  bip <dig>  cnk <dig>  and hsp <dig> genes were all induced at  <dig> dai .
to further observe er morphology, we transiently expressed a chimeric lumenal protein, bip:gfp, a fusion protein between bip  and gfp, in protoplasts from trv and trv:nag lines .
at  <dig> dai, the fluorescent signal of bip:gfp was localized in a reticular membranous network throughout the cytoplasm of both trv control and trv:nag lines.
at  <dig> dai, the signal was punctate and fragmented in trv:nag lines indicating a disrupted er network, while trv exhibited normal er morphology .
we next tested whether the chemical chaperone 4-phenyl butyric acid  alleviates nbnag-induced cell death .
pba relieves er stress by directly reducing the load of misfolded proteins retained in the er and has been shown to mitigate er stress-induced cell death in mammals and plants  <cit> .
treatment with pba  partially rescued the growth retardation and cell death phenotypes of trv:nag lines .
elevated ion leakage, an indicator of cellular membrane leakage, in trv:nag lines was also alleviated by pba treatment in the 4th leaf above the infiltrated leaf  and in the leaf near the shoot apex .
these results suggest that the defects in protein transport may cause er stress in nbnag-deficient cells.
nuclear translocation of bzip28
in arabidopsis, tunicamycin-induced er stress causes proteolytic processing and nuclear relocation of an er membrane-associated transcription factor, bzip <dig>  <cit> .
we therefore tested whether silencing of nbnag similarly induced nuclear relocation of bzip <dig> from the er membrane .
a gfp:bzip <dig> construct in which gfp was fused to arabidopsis bzip <dig> was transiently expressed in trv and trv:nag leaves  by agro-infiltration.
protoplasts prepared from the infiltrated leaves were examined by confocal laser scanning microscopy  and fluorescence microscopy  to observe gfp:bzip <dig> fluorescence in the mesophyll cells.
in addition, infiltrated leaves were directly examined to observe the fluorescence in the epidermal cells .
protoplasts and leaf epidermal cells from trv control exhibited a network pattern of green fluorescence in the cytosol and in the nuclear periphery as previously observed  <cit> .
however, protoplasts from trv:nag lines and tunicamycin -treated trv lines  frequently showed a punctuate and aggregated gfp signal in the cytosol, revealing an abnormal er network .
furthermore, some of the protoplasts and the epidermal cells exhibited strong gfp fluorescence in the nucleus with little fluorescence remaining in the cytoplasm .
since nuclear relocation of gfp:bzip <dig> was not observed in trv protoplasts or trv epidermal cells, these results suggest that nbnag deficiency activates the er stress response.
discussion
neuroblastoma-amplified gene  encodes a large protein  containing the sec <dig> domain in plants and mammals.
in human neuroblastoma, nag was found to be co-amplified with n-myc oncogene, of which genomic amplification correlates with aggressive tumor growth  <cit> .
despite the marked size difference, the nag ortholog of yeast appears to be sec39p , a cytosolic protein of ~ <dig> kda, that is peripherally associated with the er membrane  <cit> .
sec39p was first identified as an essential protein involved in er-golgi transport in a large-scale promoter shutoff analysis of essential yeast genes  <cit> .
further characterization revealed that sec39p is a component of the dsl1p complex that also includes dsl1p, tip20p, and er-localized q-snare proteins  <cit> .
the dsl1p complex is essential for retrograde traffic from the golgi to er, and consistently, sec <dig> showed strong genetic interaction with other factors required for golgi-er retrograde transport  <cit> .
interestingly, the temperature-sensitive sec <dig> mutants also exhibited defects in forward transport between the er and golgi  <cit> , consistent with the results by mnaimneh et al.
<cit> .
the phenotype can be explained by the fact that retrograde traffic plays an important role in recycling er-resident proteins that have escaped from the er  <cit> .
thus, a blocked retrograde pathway can result in failure to retrieve these proteins and lead to a concomitant block in anterograde transport.
mammals possess functional orthologs of the components of the yeast dsl1p complex, despite a low degree of sequence conservation between the yeast and mammalian counterparts  <cit> .
recently, it has been revealed that mammalian nag is a subunit of the syntaxin  <dig> complex involved in golgi-to-er retrograde trafficking and serves as a link between p <dig> and zw10-rint- <dig> in the mammalian er fusion machinery  <cit> .
interestingly, silencing of nag in human cell lines using rna interference did not substantially disrupt golgi morphology or the er-to-golgi anterograde trafficking, but caused defects in protein glycosylation  <cit> .
in this study, we characterized in vivo effects of nag deficiency in plants.
nag from n. benthamiana, arabidopsis, and rice all encodes a ~ <dig>  kda protein with the sec <dig> domain that is mainly localized in er, similar to the mammalian nag.
knockdown of nbnag using vigs led to reduced protein transport between er and golgi, and a concomitant decrease in trafficking of the marker proteins to vacuole and to plasma membrane for secretion.
furthermore, nag depletion in planta caused er stress and pcd.
our data suggest that er stress caused by disrupted protein transport contributes to pcd activation in nbnag-silenced plants.
first, expression of the upr-related genes such as bip <dig>  bip <dig>  bi <dig> , hsp <dig>  and cnk <dig> was up-regulated in nbnag-silenced vigs plants.
bips encode er-localized chaperones and have been used as marker genes for upr activation in plants  <cit> , and similarly, expression of bi <dig>  hsp <dig>  and cnk <dig> was induced during tunicamycin-induced er stress in arabidopsis <cit> .
second, nbnag deficiency caused proteolytic processing and nuclear relocation of bzip <dig> transcription factor, which is normally associated with the er membrane.
it has been shown that bzip <dig> processing is activated by er stress-inducing agents such as tunicamycin and dithiothreitol, but not by salt stress  <cit> .
third, the chemical chaperone pba alleviated the growth retardation and cell death phenotypes of nbnag vigs plants.
pba suppresses er stress and er-mediated apoptosis by chemically enhancing the capacity of the er to remove misfolded proteins in mammals and plants  <cit> .
in addition, expression analyses of the arabidopsis nag promoter-gus fusion gene showed that atnag promoter activity was stimulated in response to tunicamycin.
thus nbnag appears to play a crucial role in protein transport during plant growth and development, and its deficiency causes er stress response and subsequent activation of pcd.
nbnag-mediated cell death was first observed in young tissues containing actively dividing cells such as the shoot apex and vascular cambium before expanding to other tissues , consistent with the fact that young tissues have elevated demands for protein synthesis and transport.
interestingly, mutations that inhibit the er to golgi trafficking have not always caused er stress and pcd as observed in nbnag vigs plants.
for example, a missense mutation in the copii coat protein sec24a caused the formation of aberrant tubular clusters of er and golgi membrane in arabidopsis without inducing pcd  <cit> .
in yeast and mammals, the copii coat forms transport vesicles on the er surface for the er-golgi anterograde trafficking, and the copii coat protein sec24a is believed to have a specific role in cargo selection via site-specific recognition of cargo signals  <cit> .
in another case, overexpression of atpra <dig> b <dig>  a prenylated rab acceptor  <dig>  resulted in inhibition of copii vesicle-mediated anterograde trafficking but did not induce either er stress or pcd  <cit> .
the lack of pcd activation in the sec24a mutant may be linked to the fact that the missense mutation caused only a partial loss of sec24a function, affecting the anterograde trafficking of only a subset of cargos  <cit> .
total loss of sec24a function instead led to an embryonic lethality, suggesting that the gene function is essential in arabidopsis <cit> .
similarly, because atpra <dig> b <dig> functions as a negative regulator of the anterograde transport of only a subset of proteins at the er, the effect of its overexpression appeared to be rather specific and limited  <cit> .
in this study, the severe consequences of nbnag depletion suggest that nbnag function in the protein transport pathway may be essential and/or common so that its deficiency severely disturbs the system.
alternatively, nbnag may have additional, yet unidentified, functions, of which disruption induces pcd in a plant cell.
although the underlying mechanism remains unclear, recent evidence indicates that mitochondria-dependent and –independent cell death pathways both play a role in er stress-induced apoptosis  <cit> .
well-known regulators of mammalian apoptosis, such as the bcl- <dig> family and caspases, are activated during er stress  <cit> .
bcl- <dig>  bax and bak reside in the er membrane as well as in the mitochondrial outer membrane, regulating homeostasis and apoptosis in the er  <cit> .
overexpression of bcl- <dig> or deficiency of the proapoptotic proteins bax and bak confers protection against er stress-induced apoptosis, indicating that bcl- <dig> family members participate in the integration of apoptotic signals between the er and mitochondria  <cit> .
in this study, nbnag-induced cell death showed apoptotic hallmarks, such as nuclear dna fragmentation, decreased mitochondrial membrane potential, and excessive production of reactive oxygen species.
these apoptotic features are similar to the phenotypes of er stress-induced pcd in arabidopsis roots and soybean cell cultures caused by tunicamycin and cyclopiazonic acid, respectively  <cit> .
in particular, the decreased mitochondrial membrane potential strongly indicates involvement of mitochondria in nbnag-induced pcd, at least at the stage we examined.
it will be important to determine whether several pro-apoptotic pathways simultaneously function to commit the cell to death during er stress, and how the different signals from the er are integrated to activate pcd in plants.
CONCLUSIONS
nicotiana benthamania neuroblastoma-amplified gene  encodes an er-localized protein of  <dig>  amino acids that contains the secretory pathway sec <dig> domain.
nbnag plays a role in protein transport pathway, and nbnag deficiency resulted in er stress and programmed cell death, presumably caused by a blocked secretion pathway.
these results suggest that nag, a conserved protein from yeast to mammals, plays an essential role in plant growth and development.

 programmed cell death is a genetically defined process associated with distinctive morphological and biochemical characteristics, and is an integral part of the life cycle of multicellular organisms. in plants, pcd occurs during developmental processes and in response to abiotic and biotic stresses. the major pcd signaling pathways involve mitochondria and plasma membrane receptors, although it has recently been shown that er stress caused by impaired er function can also induce apoptotic pathways in animals and plants. the er performs several important functions, including protein targeting and secretion, vesicle trafficking, and membrane biogenesis, and its proper function is essential to cell survival. perturbations in er homeostasis disrupt folding of proteins, leading to accumulation of unfolded proteins and protein aggregates. this condition is called er stress and is detrimental to cell survival. under conditions of er stress, a cell activates a signal transduction pathway termed the unfolded protein response to limit the damage and maintain er homeostasis. prolonged and excessive er stress induces apoptosis, evidenced by dna fragmentation, cytochrome c release, and induction of caspase activity. in mammals, the er stress-induced apoptotic pathway involves cross-talk between er and mitochondria through bcl- family members bcl- bax and bak, which are localized in both mitochondria and er. recent reports suggest that in animal cells, the mitochondrial apoptotic pathway mediated by apaf- is an integral part of er stress-induced apoptosis. in plants, treatment with tunicamycin, an inhibitor of n-linked protein glycosylation, and cyclopiazonic acid, a blocker of plant er-type iia calcium pumps, elicits er stress, followed by activation of pcd with typical apoptotic morphology. however, the mechanisms of upr and er stress-induced pcd in plants remain largely unknown. one mechanism of er stress response in arabidopsis involves membrane-associated bzip transcription factors; following er stress, bzip and bzip are processed and their released n-terminal domains are translocated to the nucleus to upregulate the expression of er stress response genes including bips, bi-, pdi, and calnexin. genomic amplification of n-myc oncogene in human neuroblastoma correlates with aggressive tumor growth and poor prognosis. neuroblastoma amplified gene was first identified as a gene co-amplified with the n-myc gene, although nag is widely expressed in normal human tissues and its homologous sequences are found in plant databases. recently, it has been shown that nag is localized in er and is a component of the syntaxin complex that is involved in golgi-to-er retrograde transport using human cell lines. in this study, we investigated in planta functions of nag in nicotiana benthamiana. we showed that n. benthamiana nag played a role in protein transport pathway, and nbnag deficiency caused er stress and cell death, suggesting its essential role in plant growth and survival.  identification of nbnag functional genomics using virus-induced gene silencing revealed that silencing of the n. benthamiana homolog of neuroblastoma amplified gene, designated nbnag, results in growth arrest and acute plant death. the ~ kb full-length nbnag cdna encodes a polypeptide of amino acids with a predicted molecular mass of. da. database searches identified closely related genes in human, mouse, zebrafish, c. elegans, arabidopsis and rice. the nag homolog encodes a protein of ~ amino acids in arabidopsis, rice, and human, and is a single-copy gene in all three genomes. our analyses showed that there is no null mutant of nag in arabidopsis t-dna insertion lines. the nbnag protein contains the sec domain that has been implicated in er-golgi trafficking in yeast. overall, nbnag shows 49%, 42% and 29% sequence identity to the nag homologs from arabidopsis, rice, and human, respectively. nbnag sequence was aligned with those of the nag homologs from arabidopsis and rice. er localization of nbnag in tobacco by- cells to determine subcellular localization of nbnag, we performed immunolabeling experiments in tobacco by- cells using anti-nbnag antibodies. after immunolabeling with anti-nbnag antibodies, by- cells were briefly stained with er tracker™ blue-white dpx as an er marker. confocal laser scanning microscopy revealed that red fluorescent signals of nbnag overlapped with blue fluorescence of the er tracker near the nuclei and along the network in a typical er localization pattern in by- cells. when the by- cells were examined by fluorescence microscopy, they exhibited a network pattern of red fluorescence in the cytosol and in the nuclear periphery. these results suggest that nbnag was mainly localized in the er, consistent with the er localization of the yeast and mammalian nag. expression of arabidopsis nag we transformed arabidopsis with a fusion construct between the arabidopsis nag promoter and gus. the bp promoter was the maximum size due to a closely located adjacent gene in arabidopsis genome. gus staining of seedlings demonstrated that atnag promoter activity was mainly detected in the aerial parts, particularly in shoot apical meristem, leaf primordia, developing vasculature, and stomata. in roots, gus activity was localized in emerging lateral roots and root vasculature, albeit weakly. in the reproductive stage, flower buds and axillary buds showed gus staining. in general, nag expression was concentrated in young aerial tissues containing dividing cells. interestingly, atnagp::gus activity in a seedling increased in response to increasing concentrations of tunicamycin, an inhibitor of n-linked glycosylation. vigs phenotypes and suppression of endogenous nbnag transcripts for vigs, we cloned two different fragments of nbnag cdna into the trv -based vigs vector ptv and infiltrated n. benthamiana plants with agrobacterium containing each plasmid. trv:nag and trv:nag contain the non-overlapping bp and bp n-terminal regions of the cdna, respectively. the effect of gene silencing on the level of nbnag mrna was examined using semiquantitative rt-pcr. reduced amounts of pcr product were produced in three independent plants from the trv:nag and trv:nag lines compared with trv control, indicating that the endogenous level of the nbnag transcripts was reduced in those plants. the transcript levels of actin, which served as the control, remained constant. vigs with trv:nag and trv:nag constructs resulted in growth arrest and acute plant death. necrotic lesions were evident in young leaves around the shoot apex at 15– days after infiltration. at dai, the shoot apex was completely abolished with no stem growth or new leaf formation. the lesions progressively expanded, leading to premature death of the plants. root growth was also affected by nbnag vigs at and dai. the petiole of the fourth leaf above the infiltrated leaf, and the stem where the fourth leaf above the infiltrated leaf was attached were cross-sectioned freehand and observed under light microscopy. the localized brown pigment in the petiole and the stem at dai indicates cell death in the vasculature of trv:nag lines, particularly in the cambium. fluorescence microscopy revealed that trv:nag lines accumulated large amounts of autofluorescent secondary metabolites, which were also observed at infection sites during hypersensitive cell death, in the stem vasculature, while trv control exhibited the fluorescence only in the xylem tracheary elements. analysis of programmed cell death phenotypes although morphology and sizes of abaxial epidermal cells of the trv:nag leaves remained normal compared with trv control, extension and fragmentation of nuclear chromatin was evident in trv:nag lines at dai, but not at dai when lesions were not formed yet. dna laddering was also observed in genomic dna isolated from the trv:nag leaves. since nuclear fragmentation and dna laddering are hallmark features of pcd, these results suggest that silencing of nbnag activates pcd in plants. during apoptosis in animal cells, activation of the cell death pathway is initiated by modification of mitochondrial membrane permeability. mitochondrial membrane potential of leaf protoplasts from vigs lines was monitored by tmrm fluorescent probes that accumulate in mitochondria in proportion to the mitochondrial membrane potential. the average tmrm fluorescence of trv:nag protoplasts was ~4-fold lower than that of trv control at dai, indicating reduced mitochondrial membrane potential, but there was no difference in tmrm fluorescence between trv and trv:nag lines at dai. chlorophyll autofluorescence was not affected in trv:nag protoplasts at either dai or dai. to test whether reactive oxygen species are involved in the cell death phenotype of trv:nag plants, leaf protoplasts prepared from vigs lines were incubated with h2dcfda that becomes activated in the presence of h2o to produce green fluorescence. accumulation of fluorescent h2dcfda in trv:nag protoplasts was ~ -fold higher than in trv control, indicating h2o accumulation. ultrastructural analyses of the cell death phenotype light and transmission electron microscopy of transverse leaf sections revealed degenerating spongy mesophyll cells at early and late stages in trv:nag lines, in contrast to trv control cells at dai. tem also showed disintegrating chloroplasts and mitochondria, ruptured vacuoles, and abnormal nuclei in trv:nag lines, compared with trv control. these results suggest that the nbnag-induced cell death involves vacuole collapse leading to enzymatic degradation of organelles and cell contents, as demonstrated in pcd induced by other stimuli. nbnag deficiency inhibits intracellular protein transport yeast 82-kda sec39p is localized to the er, and associated with the syntaxin complex that is involved in golgi-to-er retrograde transport. human nag containing a sec39p-homologous region appears to be the ortholog of yeast sec39p despite the marked difference in their molecular sizes, and plays a similar role in the golgi-er retrograde trafficking. nbnag also contains the sec domain, indicating that nbnag may be involved in protein trafficking in a plant cell. to test this possibility, protoplasts isolated from trv and trv:nag leaves were transformed with sporamin:gfp construct encoding a fusion protein between green fluorescent protein and the n-terminal region of sporamin as a vacuolar reporter gene. previous experiments with arabidopsis protoplasts showed that spo:gfp is targeted to the central vacuole, where it is processed from a 40-kda precursor to a 30-kda form by proteolysis. consistent with the results, fluorescence microscopy revealed spo:gfp in the central vacuole in both trv and trv:nag protoplasts, while gfp alone was localized in the cytosol and the nucleus in trv protoplasts. to measure trafficking efficiency, relative amounts of the precursor and the processed form of spo:gfp were compared in transformed trv and trv:nag protoplasts by immunoblotting using anti-gfp antibody. in trv protoplasts, ~44% of spo:gfp was in the kda precursor form; however, in trv:nag protoplasts, the precursor form is visibly dominant, indicating that nbnag deficiency leads to reduced vacuole trafficking of spo:gfp. we next examined the effects of nbnag depletion on the secretory pathway using invertase:gfp, a chimeric protein consisting of full-length secretory invertase and gfp, as a reporter. after transformation with the invertase:gfp construct, green fluorescent signals were not detectable in trv protoplasts, indicating that invertase:gfp was secreted into the medium as previously observed. however, invertase:gfp signal was readily detected in trv:nag protoplasts. to confirm this finding, proteins were extracted from the protoplasts and the incubation medium, and analyzed by western blot analysis with anti-gfp antibody. in trv controls, invertase:gfp was mainly detected in the medium, while a minor portion of the protein remained in the protoplasts. in contrast, in the trv:nag line the fusion protein was predominantly present in the protoplasts, suggesting that nbnag depletion blocked the secretion of invertase:gfp into the medium. next, we examined er-golgi transport using an artificial er marker protein, gkx. trv protoplasts transformed with gkx exhibited a reticular network pattern of green fluorescence as observed previously. however, trv:nag protoplasts expressing gkx displayed a punctate and aggregated fluorescence signal, suggesting disruption of the er network. since gkx contains an n-glycosylation site, we examined the sensitivity of the n-glycans of gkx to endoglycosidase h treatment. it has been reported that the n-glycans of er proteins are sensitive to endo h, while the n-glycans of golgi proteins are resistant due to modification. protein extracts from trv and trv:nag protoplasts were digested by endo h prior to western blotting with anti-gfp antibody. in the trv control, endo h digestion of gkx resulted in two bands, ~ kda endo h-sensitive gkx proteins and ~ kda endo h-resistant proteins. these results indicate that a large part of gkx proteins was transported from er to golgi complex through anterograde trafficking. trv:nag lines also contained both the er and the golgi forms, but the ratio of the er form to the golgi form was higher than in trv controls. these results provide evidence that protein transport from er to golgi complex was inhibited by nbnag deficiency. nbnag-silencing causes er stress the abnormal er morphology shown by gkx expression raised a possibility that nbnag-deficient plants may have been subjected to er stress. transcriptome analysis in response to tunicamycin-induced er stress in arabidopsis revealed transcriptional up-regulation of the genes encoding bips, bi, cnk and hsp; therefore, we examined transcript levels of these er stress-related genes in trv and trv:nag lines. semiquantitative rt-pcr revealed increased transcript levels of bi and bip in trv:nag lines compared with trv control at dai, while bi bip bip cnk and hsp genes were all induced at dai. to further observe er morphology, we transiently expressed a chimeric lumenal protein, bip:gfp, a fusion protein between bip and gfp, in protoplasts from trv and trv:nag lines. at dai, the fluorescent signal of bip:gfp was localized in a reticular membranous network throughout the cytoplasm of both trv control and trv:nag lines. at dai, the signal was punctate and fragmented in trv:nag lines indicating a disrupted er network, while trv exhibited normal er morphology. we next tested whether the chemical chaperone 4-phenyl butyric acid alleviates nbnag-induced cell death. pba relieves er stress by directly reducing the load of misfolded proteins retained in the er and has been shown to mitigate er stress-induced cell death in mammals and plants. treatment with pba partially rescued the growth retardation and cell death phenotypes of trv:nag lines. elevated ion leakage, an indicator of cellular membrane leakage, in trv:nag lines was also alleviated by pba treatment in the 4th leaf above the infiltrated leaf and in the leaf near the shoot apex. these results suggest that the defects in protein transport may cause er stress in nbnag-deficient cells. nuclear translocation of bzip28 in arabidopsis, tunicamycin-induced er stress causes proteolytic processing and nuclear relocation of an er membrane-associated transcription factor, bzip. we therefore tested whether silencing of nbnag similarly induced nuclear relocation of bzip from the er membrane. a gfp:bzip construct in which gfp was fused to arabidopsis bzip was transiently expressed in trv and trv:nag leaves by agro-infiltration. protoplasts prepared from the infiltrated leaves were examined by confocal laser scanning microscopy and fluorescence microscopy to observe gfp:bzip fluorescence in the mesophyll cells. in addition, infiltrated leaves were directly examined to observe the fluorescence in the epidermal cells. protoplasts and leaf epidermal cells from trv control exhibited a network pattern of green fluorescence in the cytosol and in the nuclear periphery as previously observed. however, protoplasts from trv:nag lines and tunicamycin -treated trv lines frequently showed a punctuate and aggregated gfp signal in the cytosol, revealing an abnormal er network. furthermore, some of the protoplasts and the epidermal cells exhibited strong gfp fluorescence in the nucleus with little fluorescence remaining in the cytoplasm. since nuclear relocation of gfp:bzip was not observed in trv protoplasts or trv epidermal cells, these results suggest that nbnag deficiency activates the er stress response. discussion neuroblastoma-amplified gene encodes a large protein containing the sec domain in plants and mammals. in human neuroblastoma, nag was found to be co-amplified with n-myc oncogene, of which genomic amplification correlates with aggressive tumor growth. despite the marked size difference, the nag ortholog of yeast appears to be sec39p, a cytosolic protein of ~ kda, that is peripherally associated with the er membrane. sec39p was first identified as an essential protein involved in er-golgi transport in a large-scale promoter shutoff analysis of essential yeast genes. further characterization revealed that sec39p is a component of the dsl1p complex that also includes dsl1p, tip20p, and er-localized q-snare proteins. the dsl1p complex is essential for retrograde traffic from the golgi to er, and consistently, sec showed strong genetic interaction with other factors required for golgi-er retrograde transport. interestingly, the temperature-sensitive sec mutants also exhibited defects in forward transport between the er and golgi, consistent with the results by mnaimneh et al.. the phenotype can be explained by the fact that retrograde traffic plays an important role in recycling er-resident proteins that have escaped from the er. thus, a blocked retrograde pathway can result in failure to retrieve these proteins and lead to a concomitant block in anterograde transport. mammals possess functional orthologs of the components of the yeast dsl1p complex, despite a low degree of sequence conservation between the yeast and mammalian counterparts. recently, it has been revealed that mammalian nag is a subunit of the syntaxin complex involved in golgi-to-er retrograde trafficking and serves as a link between p and zw10-rint- in the mammalian er fusion machinery. interestingly, silencing of nag in human cell lines using rna interference did not substantially disrupt golgi morphology or the er-to-golgi anterograde trafficking, but caused defects in protein glycosylation. in this study, we characterized in vivo effects of nag deficiency in plants. nag from n. benthamiana, arabidopsis, and rice all encodes a ~ kda protein with the sec domain that is mainly localized in er, similar to the mammalian nag. knockdown of nbnag using vigs led to reduced protein transport between er and golgi, and a concomitant decrease in trafficking of the marker proteins to vacuole and to plasma membrane for secretion. furthermore, nag depletion in planta caused er stress and pcd. our data suggest that er stress caused by disrupted protein transport contributes to pcd activation in nbnag-silenced plants. first, expression of the upr-related genes such as bip bip bi, hsp and cnk was up-regulated in nbnag-silenced vigs plants. bips encode er-localized chaperones and have been used as marker genes for upr activation in plants, and similarly, expression of bi hsp and cnk was induced during tunicamycin-induced er stress in arabidopsis. second, nbnag deficiency caused proteolytic processing and nuclear relocation of bzip transcription factor, which is normally associated with the er membrane. it has been shown that bzip processing is activated by er stress-inducing agents such as tunicamycin and dithiothreitol, but not by salt stress. third, the chemical chaperone pba alleviated the growth retardation and cell death phenotypes of nbnag vigs plants. pba suppresses er stress and er-mediated apoptosis by chemically enhancing the capacity of the er to remove misfolded proteins in mammals and plants. in addition, expression analyses of the arabidopsis nag promoter-gus fusion gene showed that atnag promoter activity was stimulated in response to tunicamycin. thus nbnag appears to play a crucial role in protein transport during plant growth and development, and its deficiency causes er stress response and subsequent activation of pcd. nbnag-mediated cell death was first observed in young tissues containing actively dividing cells such as the shoot apex and vascular cambium before expanding to other tissues, consistent with the fact that young tissues have elevated demands for protein synthesis and transport. interestingly, mutations that inhibit the er to golgi trafficking have not always caused er stress and pcd as observed in nbnag vigs plants. for example, a missense mutation in the copii coat protein sec24a caused the formation of aberrant tubular clusters of er and golgi membrane in arabidopsis without inducing pcd. in yeast and mammals, the copii coat forms transport vesicles on the er surface for the er-golgi anterograde trafficking, and the copii coat protein sec24a is believed to have a specific role in cargo selection via site-specific recognition of cargo signals. in another case, overexpression of atpra b a prenylated rab acceptor resulted in inhibition of copii vesicle-mediated anterograde trafficking but did not induce either er stress or pcd. the lack of pcd activation in the sec24a mutant may be linked to the fact that the missense mutation caused only a partial loss of sec24a function, affecting the anterograde trafficking of only a subset of cargos. total loss of sec24a function instead led to an embryonic lethality, suggesting that the gene function is essential in arabidopsis. similarly, because atpra b functions as a negative regulator of the anterograde transport of only a subset of proteins at the er, the effect of its overexpression appeared to be rather specific and limited. in this study, the severe consequences of nbnag depletion suggest that nbnag function in the protein transport pathway may be essential and/or common so that its deficiency severely disturbs the system. alternatively, nbnag may have additional, yet unidentified, functions, of which disruption induces pcd in a plant cell. although the underlying mechanism remains unclear, recent evidence indicates that mitochondria-dependent and –independent cell death pathways both play a role in er stress-induced apoptosis. well-known regulators of mammalian apoptosis, such as the bcl- family and caspases, are activated during er stress. bcl- bax and bak reside in the er membrane as well as in the mitochondrial outer membrane, regulating homeostasis and apoptosis in the er. overexpression of bcl- or deficiency of the proapoptotic proteins bax and bak confers protection against er stress-induced apoptosis, indicating that bcl- family members participate in the integration of apoptotic signals between the er and mitochondria. in this study, nbnag-induced cell death showed apoptotic hallmarks, such as nuclear dna fragmentation, decreased mitochondrial membrane potential, and excessive production of reactive oxygen species. these apoptotic features are similar to the phenotypes of er stress-induced pcd in arabidopsis roots and soybean cell cultures caused by tunicamycin and cyclopiazonic acid, respectively. in particular, the decreased mitochondrial membrane potential strongly indicates involvement of mitochondria in nbnag-induced pcd, at least at the stage we examined. it will be important to determine whether several pro-apoptotic pathways simultaneously function to commit the cell to death during er stress, and how the different signals from the er are integrated to activate pcd in plants.  nicotiana benthamania neuroblastoma-amplified gene encodes an er-localized protein of amino acids that contains the secretory pathway sec domain. nbnag plays a role in protein transport pathway, and nbnag deficiency resulted in er stress and programmed cell death, presumably caused by a blocked secretion pathway. these results suggest that nag, a conserved protein from yeast to mammals, plays an essential role in plant growth and development. 
later it was found that nag is localized in endoplasmic reticulum and is a component of the syntaxin complex that is involved in golgi-to-er retrograde transport in human cells. these results suggest that nag, a conserved protein from yeast to mammals, plays an essential role in plant growth and development by modulating protein transport pathway, er stress response and pcd. silencing of nbnag by virus-induced gene silencing resulted in growth arrest and acute plant death with morphological markers of programmed cell death, which include chromatin fragmentation and modification of mitochondrial membrane potential. nbnag deficiency caused induction of er stress genes, disruption of the er network, and relocation of bzip transcription factor from the er membrane to the nucleus, similar to the phenotypes of tunicamycin-induced er stress in a plant cell. nicotiana benthamania neuroblastoma-amplified gene encodes a protein of amino acids that contains the secretory pathway sec domain and is mainly localized in the er. nbnag silencing caused defects in intracellular transport of diverse cargo proteins, suggesting that a blocked secretion pathway by nbnag deficiency causes er stress and programmed cell death. homologous sequences of nag are found in plant databases, but its function in plant cells remains unknown. neuroblastoma amplified gene was identified as a gene co-amplified with the n-myc gene, whose genomic amplification correlates with poor prognosis of neuroblastoma. bzip28er stress gene expressionpromoter-gus fusionprotein transport assayvirus-induced gene silencing
 neuroblastoma amplified gene was identified as a gene co-amplified with the n-myc gene, whose genomic amplification correlates with poor prognosis of neuroblastoma. later it was found that nag is localized in endoplasmic reticulum and is a component of the syntaxin complex that is involved in golgi-to-er retrograde transport in human cells. homologous sequences of nag are found in plant databases, but its function in plant cells remains unknown.  nicotiana benthamania neuroblastoma-amplified gene encodes a protein of amino acids that contains the secretory pathway sec domain and is mainly localized in the er. silencing of nbnag by virus-induced gene silencing resulted in growth arrest and acute plant death with morphological markers of programmed cell death, which include chromatin fragmentation and modification of mitochondrial membrane potential. nbnag deficiency caused induction of er stress genes, disruption of the er network, and relocation of bzip transcription factor from the er membrane to the nucleus, similar to the phenotypes of tunicamycin-induced er stress in a plant cell. nbnag silencing caused defects in intracellular transport of diverse cargo proteins, suggesting that a blocked secretion pathway by nbnag deficiency causes er stress and programmed cell death.  these results suggest that nag, a conserved protein from yeast to mammals, plays an essential role in plant growth and development by modulating protein transport pathway, er stress response and pcd. bzip28er stress gene expressionpromoter-gus fusionprotein transport assayvirus-induced gene silencing 