BACKGROUND
chloroplasts are derived from cyanobacteria through endosymbiosis, and massive gene transfer from the plastid to the nucleus occurred during evolution
<cit> .
chloroplasts possess approximately 100- <dig> genes in their own circular genome that encodes messenger rnas, ribosome rnas, and transfer rnas.
in addition to its own gene products, functional communication between chloroplasts and nucleus is required, and many nuclear-encoded proteins are targeted to chloroplasts and play fundamental roles in the regulation of chloroplast gene expression.
expression of chloroplast genes is commonly regulated at posttranscriptional level, including mrna processing, splicing, editing, decay, and translational control
<cit> .
a sophisticated regulatory process between chloroplasts and nuclei is required to fine-tune chloroplast gene expression, and many nuclear-encoded rna-binding proteins  have been recently regarded as the primary elements that modulate posttranscriptional steps in chloroplasts
<cit> .
although chloroplasts share some features of rna metabolism with their bacterial ancestors, chloroplasts require a more complicated mechanism of rna metabolism compared to their ancestor, which have both prokaryotic and eukaryotic characteristics
<cit> .
one particular example is the splicing of group i and group ii introns.
in contrast to self-splicing of prokaryotic introns, chloroplasts have lost their capacity to self-splice, and the splicing of group i and group ii introns in chloroplasts requires many nuclear-encoded proteins that form protein complexes similar to spliceosomal complexes found in eukaryotes
<cit> .
therefore, involvement of nuclear-encoded rbps is indispensible for posttranscriptional regulation of rna metabolism and gene expression in chloroplasts.
crm  domain-containing proteins were first found in archaea and bacteria.
based on their structural data and predicted domain structures, crm domain proteins were suggested to have rna-binding activity
<cit> .
prokaryotes contain proteins harboring only a single crm domain, whereas land plants contain proteins harboring multiple crm domains.
splicing of group i and group ii introns and trnas and processing of rrnas in chloroplasts require different complexes of crss , cafs , and cfms  that harbor multiple crm domains
<cit> .
it has also been demonstrated that mutation in crm domain-containing protein genes results in pale-green phenotypes, delayed development, and aborted seed production in plants, indicating the important roles of crm domain proteins in plant growth and development
<cit> .
despite an increased understanding of the roles of crm domain-containing proteins in chloroplast rna metabolism and plant growth and development, the functional roles of most of the crm domain-containing proteins have not been demonstrated experimentally.
the arabidopsis  and rice  genomes harbor the genes encoding  <dig> and  <dig> crm domain-containing proteins, respectively
<cit> .
among the  <dig> arabidopsis crm domain-containing protein genes, two genes  encode the smallest proteins harboring a single crm domain
<cit> .
however, the role of single crm domain-containing proteins has not been demonstrated in plants.
here, we determined the developmental and stress response roles of a single crm domain-containing protein .
because this protein belongs to subfamily group  <dig> among crm domain proteins
<cit> , we designated it as crm family member subfamily <dig> .
we show that cfm <dig> possesses rna chaperone activity and is involved in rrna processing, which is important for normal growth, development, and the stress response in plants.
RESULTS
structural features and characterization of cfm <dig> in arabidopsis
sixteen predicted crm domain family members occur in the arabidopsis genome, and they are classified into four groups, such as crs <dig> subfamily, caf subfamily, subfamily  <dig>  and subfamily  <dig>  among the  <dig> crm domain-containing protein genes, two genes  encode proteins harboring a single crm domain and are classified into subfamily group 4
<cit> .
we thus named at4g <dig> as cfm <dig>  the cfm <dig> protein contains a highly conserved gxxg sequence in the c-terminal half of the protein .
the two single crm domain-containing proteins  share approximately 56% amino acid sequence homology with each other.
to examine whether the single crm domain proteins are conserved in dicotyledonous and monocotyledonous plants, the amino acid sequences of single crm domain proteins in diverse plant species, including arabidopsis, zea mays, medicago truncatula, vitis vinifera, hordeum vulgare, sorghum bicolor, and oryza sativa, were compared.
the results showed that cfm <dig> family proteins share 35-50% amino acid sequence homology among dicot and monocot plants and share > 70% amino acid sequence homology among monocot plants , suggesting that the single crm domain-containing proteins are functionally conserved in dicots and monocots.
the crm proteins in arabidopsis and rice have been predicted to be targeted mainly to chloroplasts or mitochondria.
to determine the subcellular localization of cfm <dig>  the cdna encoding cfm <dig> was ligated in front of the green fluorescence protein  gene, and expression of the cfm4-gfp fusion protein was investigated in transgenic arabidopsis plants.
strong gfp signals were observed in chloroplasts .
to examine whether cfm <dig> is also localized to mitochondria, arabidopsis mitochondria were stained with mito-tracker that is a red-fluorescent dye and stains mitochondria in live cells, and the signals from plastids in roots and chloroplasts in leaves were examined.
the results showed that the signals from mitochondria did not overlap with the signals from chloroplasts, and gfp signals were observed exclusively in chloroplasts .
these results clearly indicate that cfm <dig> is localized to chloroplasts.
cfm <dig> plays a role in arabidopsis growth and senescence
to determine the role of cfm <dig> during plant growth and development, the t-dna insertion mutant lines in cfm <dig>  were obtained, and their phenotypes were analyzed under normal and stress conditions.
the absence of cfm <dig> expression in the knockout mutant lines was confirmed by rt-pcr analysis .
the wild-type and cfm <dig> mutant plants were grown in ms medium or soil, and their phenotypes were observed during the entire life cycle  of the plants.
growth of the wild-type and mutant plants was not significantly different at 7 days after germination  .
however, growth of the plants was markedly different at later stages in that the size of the cfm <dig> mutants was much smaller than that of the wild-type plants at  <dig> or  <dig> dag .
the difference in flowering time between the wild-type and mutant plants was evident; cfm <dig> mutants flowered approximately 7 days later than the wild-type plants .
although the cfm <dig> mutants flowered much later than the wild-type plants, the size and number of leaves at the time of bolting were not different between the wild-type and mutant plants , suggesting that cfm <dig> does not affect control of flowering time.
no significant difference in plant height was observed between the wild-type and mutant plants at the time of maturity.
the root growth of the cfm <dig> mutants was also retarded compared with that of the wild-type plants .
to determine whether the retarded root growth was due to the decreases in cell size or cell number, the plasma membranes in the roots of the wild-type and cfm <dig> mutant plants were stained with fm 4- <dig> dye and observed under confocal microscope.
the results showed that the size of cells in cfm <dig> mutants was much smaller than that in the wild-type plants .
the retarded growth phenotypes of the mutant plants recovered to normal phenotypes in the complementation lines .
all of these observations clearly demonstrate that cfm <dig> plays a role for normal growth of arabidopsis plants.
because a recent study demonstrated that the growth retardation phenotypes of an arabidopsis mutant are closely related to abscisic acid  biosynthesis and chloroplast rna metabolism
<cit> , we wanted to determine aba levels in the wild-type plants, cfm <dig> mutants, and complementation lines using an immunoassay method.
the results showed that levels of aba in cfm <dig> mutants were approximately 70-80% of those in the wild-type plants .
we also analyzed transcript levels of the genes, including aba <dig>  aba <dig>  aba <dig>  and nced <dig> that are involved in aba biosynthesis, and found that the levels of nced <dig> were significantly lower in cfm <dig> mutants than in the wild-type plants .
these results suggest that the retarded growth phenotypes of cfm <dig> mutants are related to impaired aba biosynthesis.
with the observation that cfm <dig> plays a role in the growth of arabidopsis, we subsequently examined whether cfm <dig> is involved in senescence.
in the dark-induced senescence assay, it was evident that greening of the leaves of cfm <dig> mutant plants was maintained for much longer compared with that of the wild-type plants when they were incubated under dark conditions .
total chlorophyll  contents in cfm <dig> mutant plants were much higher than those in the wild-type plants .
greening of the leaves of the complementation lines was similar to that of the wild-type plants, and the chlorophyll contents in the complementation lines were comparable with those in the wild-type plants .
these results suggest that cfm <dig> plays a positive role in senescence.
cfm <dig> is involved in arabidopsis response to abiotic stresses
to determine whether cfm <dig> plays a role in the plant response to environmental stresses, the wild-type, cfm <dig> mutants, and complementation lines were grown in ms medium supplemented with nacl for salt stress or with mannitol for dehydration stress, or the plants were grown at 10°c for cold stress treatment.
we first compared seed germination rates of the plants under normal and stress conditions.
no differences were observed in seed germination rates between the wild-type and mutant plants under normal conditions .
however, seed germination of cfm <dig> mutants was retarded compared with that of the wild-type and complementation lines under salt or cold stress conditions , whereas seed germination rates of all three genotypes were similar to each other under dehydration stress conditions .
seedling growth of cfm <dig> mutants was also retarded compared with that of the wild-type and complementation lines under salt or cold stress .
these results show that cfm <dig> affects seed germination and subsequent seedling growth of arabidopsis under salt or cold stress conditions.
cfm <dig> affects rrna processing in chloroplasts
the crm domain-containing proteins have been demonstrated to be involved in the splicing of group ii introns of chloroplast mrnas and trnas
<cit> .
therefore, we first examined whether cfm <dig> is involved in the splicing of intron-containing genes in chloroplasts.
splicing patterns of all intron-containing chloroplast transcripts, including  <dig> mrnas and  <dig> trnas, were analyzed by rt-pcr.
the results showed that the splicing patterns of all intron-containing chloroplast transcripts were not altered in cfm <dig> mutants compared with those in the wild-type plants , suggesting that cfm <dig> is not involved in intron splicing in chloroplasts.
we next examined whether cfm <dig> is involved in rrna processing.
the levels of rrnas, including 23s, 16s, 5s, and  <dig> s rrnas, in the wild type, cfm <dig> mutant, and complementation line were determined by northern blot analysis.
to accurately determine relative levels of rrna transcripts, northern blot analysis was repeated four times, and the relative intensities of rrna bands in cfm <dig> mutants and complementation lines compared with those in wild type were calculated.
the results showed that the transcript levels of the 23s and 5s rrnas in cfm <dig> mutants were comparable with those in the wild-type plants.
however, the mature-16s rrna transcript levels in cfm <dig> mutant decreased compared with those in the wild-type plants .
in addition, the levels of mature- <dig> s rrna decreased, whereas the levels of precursor- <dig> s rrna increased in cfm <dig> mutant.
these abnormal levels of 16s and  <dig> s rrnas observed in cfm <dig> mutants were restored to wild-type levels in the complementation line .
these results indicate that cfm <dig> affects the processing of 16s and  <dig> s rrnas.
cfm <dig> possesses rna chaperone activity
rna processing and intron splicing require proper folding of rna substrates, and many rbps that harbor rna chaperone activity play an important role during these cellular processes
<cit> .
because cfm <dig> harbors a crm domain that is known as an rna binding module
<cit> , we aimed to determine whether cfm <dig> possesses rna chaperone activity.
we first analyzed the complementation ability of cfm <dig> in the cold-sensitive e. coli bx <dig> mutant in which four cold shock proteins known as rna chaperones are deficient and is high sensitivity to lower temperatures
<cit> .
when the bx <dig> cells harboring each construct were incubated at 37°c, all cells grew well with no difference.
however, when the bx <dig> cells were exposed to cold shock at 20°c, the cells expressing cfm <dig> or cspa as a positive control grew well at low temperatures, whereas the cells harboring the piniii vector did not grow at low temperatures .
this result demonstrates that cfm <dig> has the ability to complement rna chaperone-deficient e. coli mutant cells, suggesting that cfm <dig> functions as an rna chaperone in e. coli under cold shock.
to further confirm whether cfm <dig> possesses rna chaperone activity, the in vitro and in vivo nucleic acid-melting abilities of cfm <dig> were assessed via dna-melting and transcription anti-termination assays.
for the analysis of in vitro dna-melting ability of cfm <dig>  the recombinant gst-cfm <dig> fusion proteins were purified from e. coli  and gst-cfm <dig> fusion protein was tested for its ability to destabilize base pairs in the synthetic dna molecules labeled with a fluorophore  and quencher .
fluorescence of the molecular beacon increased after adding the gst-cfm <dig> proteins or the gst-cspa fusion proteins , confirming the dna-melting activity of cfm <dig> .
by contrast, adding gst alone  did not increase fluorescence .
to determine whether cfm <dig> has the ability to destabilize base pairs in rna, the in vivo rna-melting ability of cfm <dig> was assessed via transcription anti-termination assays using e. coli rl <dig> cells that harbor a chloramphenicol resistance gene downstream from a trpl terminator with stem-loop structure
<cit> .
when the rl <dig> cells were grown in the ms medium containing chloramphenicol, the cells expressing cfm <dig> or cspa grew well, suggesting that cfm <dig> and cspa destabilize the stem-loop structures present in the transcription termination signal.
by contrast, the cells harboring the piniii vector did not grow in chloramphenicol-containing ms medium .
taken together, these results indicate that cfm <dig> possesses rna chaperone activity.
discussion
the results presented here demonstrate that cfm <dig>  one of two arabidopsis genes encoding single crm domain-containing protein, plays a role in the arabidopsis growth and stress response by affecting rrna processing in chloroplasts.
analysis of the t-dna knockout lines indicated that cfm <dig> exhibits important function during plant growth and senescence , which is in agreement with its postulated roles in chloroplast rna metabolism.
our current results point to the important roles of crm domain-containing proteins in plant growth and development.
it has been demonstrated that mutation in the genes encoding multiple crm domain-containing proteins causes pale-green phenotypes, delayed development, and aborted seed production in plants
<cit> .
in addition to its roles in the growth and senescence of arabidopsis under normal growth conditions, cfm <dig> also affected the plant response to abiotic stresses.
seed germination and seedling growth of cfm <dig> mutants were retarded compared with those of the wild-type plants under salt or cold stress conditions , suggesting that cfm <dig> plays a positive role in seed germination and seedling growth of arabidopsis under salt or cold stress conditions.
interestingly, the effect of cfm <dig> on seed germination and seedling growth of plants was confined to salt or cold stress, as no differences in seed germination and seedling growth were observed between the wild type and cfm <dig> mutants under dehydration stress conditions.
notably, the genes encoding single crm domain-containing proteins as well as the genes encoding multiple crm domain-containing proteins play important roles in plant growth, development, and stress response.
all multiple crm domain-containing proteins whose functions have been characterized in plants are involved in the splicing of chloroplast group ii and i introns
<cit> .
caf <dig>  caf <dig>  and crs <dig>  the three maize proteins harboring multiple copies of the domain, are required for the splicing of group ii introns in chloroplasts
<cit> .
in addition, it has been demonstrated that cfm <dig> and cfm <dig> together with crs2/caf complexes promote the splicing of chloroplast introns
<cit> .
in contrast to the well-characterized roles of multiple crm domain-containing proteins in the splicing of chloroplast introns, cfm <dig> plays no role in the splicing of chloroplast introns .
however, our current analysis shows that cfm <dig> is involved in the processing and maturation of 16s and  <dig> s rrnas .
this observation is in line with previous findings that prokaryotic proteins harboring a single crm domain participate in ribosome maturation
<cit> .
the yhby, the crm domain protein in escherichia coli, is a small protein with molecular mass of ~10 kda and is associated with pre-50s ribosomal subunits, which function in ribosome assembly
<cit> .
among the  <dig> crm domain proteins found in arabidopsis, crm <dig> is the most homologous to yhby in that it harbors little else except a single crm domain .
we propose that the delayed growth of cfm <dig> mutants is due to, at least in part, improper processing of 16s and  <dig> s rrnas in chloroplasts.
interestingly, improper rrna processing in cfm <dig> mutants resulted in impaired aba biosynthesis , which supports a notion that proper chloroplast function is required not only for active photosynthesis but also for aba biosynthesis, both of which are required for normal plant growth
<cit> .
the proposed mechanistic role of cfm <dig> as an rna chaperone in chloroplast rrna metabolism is intriguing.
rna processing as well as intron splicing requires proper folding of rna substrates, and many rbps play an important role in rna-rna and rna-protein interactions during rna metabolism
<cit> .
rna chaperones are nonspecific rbps that bind diverse rna substrates and help rnas fold by inducing structural rearrangement of misfolded rnas
<cit> .
it has recently been demonstrated that one of the minor spliceosomal proteins u11/u12-31 k possesses rna chaperone activity and is indispensible for correct intron splicing and normal growth and development in arabidopsis and rice
<cit> , which emphasizes the important role of rna chaperones in maintaining rna substrates in splicing-competent structures for correct processing to occur.
involvement of rna chaperone activity in the splicing of group i and group ii introns has been demonstrated in yeast mitochondria.
the yeast dead-box protein mss <dig> promotes the splicing of both group i and group ii introns in mitochondria via functioning as an rna chaperone
<cit> .
our current analysis clearly indicates that cfm <dig> harbors rna chaperone activity .
it is likely that rna chaperone activity of cfm <dig> is needed to maintain precursor-rrna in processing-competent structures for subsequence rrna processing.
although it is not clear at present how cfm <dig> affects processing of only two  rrnas out of the four rrnas in the chloroplast, it is possible that cfm <dig> recognizes specific sequence or structural elements in 16s and  <dig> s rrnas and the rna chaperone activity of cfm <dig> is involved in the formation of processing-competent structures of these rrnas.
CONCLUSIONS
the present study shows that cfm <dig> harboring rna chaperone activity is involved in rrna processing in chloroplasts, which is important for growth and the stress response of plants.
although much progress has been made in the characterization of the roles of crm domain-containing proteins in intron splicing and rrna processing during chloroplast gene expression, the roles of many crm proteins in plant growth and development as well as chloroplast rna metabolism remain largely unknown.
in particular, understanding the biological function of single crm domain-containing proteins is far behind compared with that of multiple crm domain-containing proteins.
exploring the importance of the number of crm domains and involvement of each crm domain in substrate recognition during intron splicing and rrna processing should provide a much deeper insight into the mechanistic role of crm family members in chloroplast rna metabolism.

 chloroplasts are derived from cyanobacteria through endosymbiosis, and massive gene transfer from the plastid to the nucleus occurred during evolution. chloroplasts possess approximately 100- genes in their own circular genome that encodes messenger rnas, ribosome rnas, and transfer rnas. in addition to its own gene products, functional communication between chloroplasts and nucleus is required, and many nuclear-encoded proteins are targeted to chloroplasts and play fundamental roles in the regulation of chloroplast gene expression. expression of chloroplast genes is commonly regulated at posttranscriptional level, including mrna processing, splicing, editing, decay, and translational control. a sophisticated regulatory process between chloroplasts and nuclei is required to fine-tune chloroplast gene expression, and many nuclear-encoded rna-binding proteins have been recently regarded as the primary elements that modulate posttranscriptional steps in chloroplasts. although chloroplasts share some features of rna metabolism with their bacterial ancestors, chloroplasts require a more complicated mechanism of rna metabolism compared to their ancestor, which have both prokaryotic and eukaryotic characteristics. one particular example is the splicing of group i and group ii introns. in contrast to self-splicing of prokaryotic introns, chloroplasts have lost their capacity to self-splice, and the splicing of group i and group ii introns in chloroplasts requires many nuclear-encoded proteins that form protein complexes similar to spliceosomal complexes found in eukaryotes. therefore, involvement of nuclear-encoded rbps is indispensible for posttranscriptional regulation of rna metabolism and gene expression in chloroplasts. crm domain-containing proteins were first found in archaea and bacteria. based on their structural data and predicted domain structures, crm domain proteins were suggested to have rna-binding activity. prokaryotes contain proteins harboring only a single crm domain, whereas land plants contain proteins harboring multiple crm domains. splicing of group i and group ii introns and trnas and processing of rrnas in chloroplasts require different complexes of crss, cafs, and cfms that harbor multiple crm domains. it has also been demonstrated that mutation in crm domain-containing protein genes results in pale-green phenotypes, delayed development, and aborted seed production in plants, indicating the important roles of crm domain proteins in plant growth and development. despite an increased understanding of the roles of crm domain-containing proteins in chloroplast rna metabolism and plant growth and development, the functional roles of most of the crm domain-containing proteins have not been demonstrated experimentally. the arabidopsis and rice genomes harbor the genes encoding and crm domain-containing proteins, respectively. among the arabidopsis crm domain-containing protein genes, two genes encode the smallest proteins harboring a single crm domain. however, the role of single crm domain-containing proteins has not been demonstrated in plants. here, we determined the developmental and stress response roles of a single crm domain-containing protein. because this protein belongs to subfamily group among crm domain proteins, we designated it as crm family member subfamily. we show that cfm possesses rna chaperone activity and is involved in rrna processing, which is important for normal growth, development, and the stress response in plants.  structural features and characterization of cfm in arabidopsis sixteen predicted crm domain family members occur in the arabidopsis genome, and they are classified into four groups, such as crs subfamily, caf subfamily, subfamily and subfamily among the crm domain-containing protein genes, two genes encode proteins harboring a single crm domain and are classified into subfamily group 4. we thus named at4g as cfm the cfm protein contains a highly conserved gxxg sequence in the c-terminal half of the protein. the two single crm domain-containing proteins share approximately 56% amino acid sequence homology with each other. to examine whether the single crm domain proteins are conserved in dicotyledonous and monocotyledonous plants, the amino acid sequences of single crm domain proteins in diverse plant species, including arabidopsis, zea mays, medicago truncatula, vitis vinifera, hordeum vulgare, sorghum bicolor, and oryza sativa, were compared. the results showed that cfm family proteins share 35-50% amino acid sequence homology among dicot and monocot plants and share > 70% amino acid sequence homology among monocot plants, suggesting that the single crm domain-containing proteins are functionally conserved in dicots and monocots. the crm proteins in arabidopsis and rice have been predicted to be targeted mainly to chloroplasts or mitochondria. to determine the subcellular localization of cfm the cdna encoding cfm was ligated in front of the green fluorescence protein gene, and expression of the cfm4-gfp fusion protein was investigated in transgenic arabidopsis plants. strong gfp signals were observed in chloroplasts. to examine whether cfm is also localized to mitochondria, arabidopsis mitochondria were stained with mito-tracker that is a red-fluorescent dye and stains mitochondria in live cells, and the signals from plastids in roots and chloroplasts in leaves were examined. the results showed that the signals from mitochondria did not overlap with the signals from chloroplasts, and gfp signals were observed exclusively in chloroplasts. these results clearly indicate that cfm is localized to chloroplasts. cfm plays a role in arabidopsis growth and senescence to determine the role of cfm during plant growth and development, the t-dna insertion mutant lines in cfm were obtained, and their phenotypes were analyzed under normal and stress conditions. the absence of cfm expression in the knockout mutant lines was confirmed by rt-pcr analysis. the wild-type and cfm mutant plants were grown in ms medium or soil, and their phenotypes were observed during the entire life cycle of the plants. growth of the wild-type and mutant plants was not significantly different at 7 days after germination. however, growth of the plants was markedly different at later stages in that the size of the cfm mutants was much smaller than that of the wild-type plants at or dag. the difference in flowering time between the wild-type and mutant plants was evident; cfm mutants flowered approximately 7 days later than the wild-type plants. although the cfm mutants flowered much later than the wild-type plants, the size and number of leaves at the time of bolting were not different between the wild-type and mutant plants, suggesting that cfm does not affect control of flowering time. no significant difference in plant height was observed between the wild-type and mutant plants at the time of maturity. the root growth of the cfm mutants was also retarded compared with that of the wild-type plants. to determine whether the retarded root growth was due to the decreases in cell size or cell number, the plasma membranes in the roots of the wild-type and cfm mutant plants were stained with fm 4- dye and observed under confocal microscope. the results showed that the size of cells in cfm mutants was much smaller than that in the wild-type plants. the retarded growth phenotypes of the mutant plants recovered to normal phenotypes in the complementation lines. all of these observations clearly demonstrate that cfm plays a role for normal growth of arabidopsis plants. because a recent study demonstrated that the growth retardation phenotypes of an arabidopsis mutant are closely related to abscisic acid biosynthesis and chloroplast rna metabolism, we wanted to determine aba levels in the wild-type plants, cfm mutants, and complementation lines using an immunoassay method. the results showed that levels of aba in cfm mutants were approximately 70-80% of those in the wild-type plants. we also analyzed transcript levels of the genes, including aba aba aba and nced that are involved in aba biosynthesis, and found that the levels of nced were significantly lower in cfm mutants than in the wild-type plants. these results suggest that the retarded growth phenotypes of cfm mutants are related to impaired aba biosynthesis. with the observation that cfm plays a role in the growth of arabidopsis, we subsequently examined whether cfm is involved in senescence. in the dark-induced senescence assay, it was evident that greening of the leaves of cfm mutant plants was maintained for much longer compared with that of the wild-type plants when they were incubated under dark conditions. total chlorophyll contents in cfm mutant plants were much higher than those in the wild-type plants. greening of the leaves of the complementation lines was similar to that of the wild-type plants, and the chlorophyll contents in the complementation lines were comparable with those in the wild-type plants. these results suggest that cfm plays a positive role in senescence. cfm is involved in arabidopsis response to abiotic stresses to determine whether cfm plays a role in the plant response to environmental stresses, the wild-type, cfm mutants, and complementation lines were grown in ms medium supplemented with nacl for salt stress or with mannitol for dehydration stress, or the plants were grown at 10°c for cold stress treatment. we first compared seed germination rates of the plants under normal and stress conditions. no differences were observed in seed germination rates between the wild-type and mutant plants under normal conditions. however, seed germination of cfm mutants was retarded compared with that of the wild-type and complementation lines under salt or cold stress conditions, whereas seed germination rates of all three genotypes were similar to each other under dehydration stress conditions. seedling growth of cfm mutants was also retarded compared with that of the wild-type and complementation lines under salt or cold stress. these results show that cfm affects seed germination and subsequent seedling growth of arabidopsis under salt or cold stress conditions. cfm affects rrna processing in chloroplasts the crm domain-containing proteins have been demonstrated to be involved in the splicing of group ii introns of chloroplast mrnas and trnas. therefore, we first examined whether cfm is involved in the splicing of intron-containing genes in chloroplasts. splicing patterns of all intron-containing chloroplast transcripts, including mrnas and trnas, were analyzed by rt-pcr. the results showed that the splicing patterns of all intron-containing chloroplast transcripts were not altered in cfm mutants compared with those in the wild-type plants, suggesting that cfm is not involved in intron splicing in chloroplasts. we next examined whether cfm is involved in rrna processing. the levels of rrnas, including 23s, 16s, 5s, and s rrnas, in the wild type, cfm mutant, and complementation line were determined by northern blot analysis. to accurately determine relative levels of rrna transcripts, northern blot analysis was repeated four times, and the relative intensities of rrna bands in cfm mutants and complementation lines compared with those in wild type were calculated. the results showed that the transcript levels of the 23s and 5s rrnas in cfm mutants were comparable with those in the wild-type plants. however, the mature-16s rrna transcript levels in cfm mutant decreased compared with those in the wild-type plants. in addition, the levels of mature- s rrna decreased, whereas the levels of precursor- s rrna increased in cfm mutant. these abnormal levels of 16s and s rrnas observed in cfm mutants were restored to wild-type levels in the complementation line. these results indicate that cfm affects the processing of 16s and s rrnas. cfm possesses rna chaperone activity rna processing and intron splicing require proper folding of rna substrates, and many rbps that harbor rna chaperone activity play an important role during these cellular processes. because cfm harbors a crm domain that is known as an rna binding module, we aimed to determine whether cfm possesses rna chaperone activity. we first analyzed the complementation ability of cfm in the cold-sensitive e. coli bx mutant in which four cold shock proteins known as rna chaperones are deficient and is high sensitivity to lower temperatures. when the bx cells harboring each construct were incubated at 37°c, all cells grew well with no difference. however, when the bx cells were exposed to cold shock at 20°c, the cells expressing cfm or cspa as a positive control grew well at low temperatures, whereas the cells harboring the piniii vector did not grow at low temperatures. this result demonstrates that cfm has the ability to complement rna chaperone-deficient e. coli mutant cells, suggesting that cfm functions as an rna chaperone in e. coli under cold shock. to further confirm whether cfm possesses rna chaperone activity, the in vitro and in vivo nucleic acid-melting abilities of cfm were assessed via dna-melting and transcription anti-termination assays. for the analysis of in vitro dna-melting ability of cfm the recombinant gst-cfm fusion proteins were purified from e. coli and gst-cfm fusion protein was tested for its ability to destabilize base pairs in the synthetic dna molecules labeled with a fluorophore and quencher. fluorescence of the molecular beacon increased after adding the gst-cfm proteins or the gst-cspa fusion proteins, confirming the dna-melting activity of cfm. by contrast, adding gst alone did not increase fluorescence. to determine whether cfm has the ability to destabilize base pairs in rna, the in vivo rna-melting ability of cfm was assessed via transcription anti-termination assays using e. coli rl cells that harbor a chloramphenicol resistance gene downstream from a trpl terminator with stem-loop structure. when the rl cells were grown in the ms medium containing chloramphenicol, the cells expressing cfm or cspa grew well, suggesting that cfm and cspa destabilize the stem-loop structures present in the transcription termination signal. by contrast, the cells harboring the piniii vector did not grow in chloramphenicol-containing ms medium. taken together, these results indicate that cfm possesses rna chaperone activity. discussion the results presented here demonstrate that cfm one of two arabidopsis genes encoding single crm domain-containing protein, plays a role in the arabidopsis growth and stress response by affecting rrna processing in chloroplasts. analysis of the t-dna knockout lines indicated that cfm exhibits important function during plant growth and senescence, which is in agreement with its postulated roles in chloroplast rna metabolism. our current results point to the important roles of crm domain-containing proteins in plant growth and development. it has been demonstrated that mutation in the genes encoding multiple crm domain-containing proteins causes pale-green phenotypes, delayed development, and aborted seed production in plants. in addition to its roles in the growth and senescence of arabidopsis under normal growth conditions, cfm also affected the plant response to abiotic stresses. seed germination and seedling growth of cfm mutants were retarded compared with those of the wild-type plants under salt or cold stress conditions, suggesting that cfm plays a positive role in seed germination and seedling growth of arabidopsis under salt or cold stress conditions. interestingly, the effect of cfm on seed germination and seedling growth of plants was confined to salt or cold stress, as no differences in seed germination and seedling growth were observed between the wild type and cfm mutants under dehydration stress conditions. notably, the genes encoding single crm domain-containing proteins as well as the genes encoding multiple crm domain-containing proteins play important roles in plant growth, development, and stress response. all multiple crm domain-containing proteins whose functions have been characterized in plants are involved in the splicing of chloroplast group ii and i introns. caf caf and crs the three maize proteins harboring multiple copies of the domain, are required for the splicing of group ii introns in chloroplasts. in addition, it has been demonstrated that cfm and cfm together with crs2/caf complexes promote the splicing of chloroplast introns. in contrast to the well-characterized roles of multiple crm domain-containing proteins in the splicing of chloroplast introns, cfm plays no role in the splicing of chloroplast introns. however, our current analysis shows that cfm is involved in the processing and maturation of 16s and s rrnas. this observation is in line with previous findings that prokaryotic proteins harboring a single crm domain participate in ribosome maturation. the yhby, the crm domain protein in escherichia coli, is a small protein with molecular mass of ~10 kda and is associated with pre-50s ribosomal subunits, which function in ribosome assembly. among the crm domain proteins found in arabidopsis, crm is the most homologous to yhby in that it harbors little else except a single crm domain. we propose that the delayed growth of cfm mutants is due to, at least in part, improper processing of 16s and s rrnas in chloroplasts. interestingly, improper rrna processing in cfm mutants resulted in impaired aba biosynthesis, which supports a notion that proper chloroplast function is required not only for active photosynthesis but also for aba biosynthesis, both of which are required for normal plant growth. the proposed mechanistic role of cfm as an rna chaperone in chloroplast rrna metabolism is intriguing. rna processing as well as intron splicing requires proper folding of rna substrates, and many rbps play an important role in rna-rna and rna-protein interactions during rna metabolism. rna chaperones are nonspecific rbps that bind diverse rna substrates and help rnas fold by inducing structural rearrangement of misfolded rnas. it has recently been demonstrated that one of the minor spliceosomal proteins u11/u12-31 k possesses rna chaperone activity and is indispensible for correct intron splicing and normal growth and development in arabidopsis and rice, which emphasizes the important role of rna chaperones in maintaining rna substrates in splicing-competent structures for correct processing to occur. involvement of rna chaperone activity in the splicing of group i and group ii introns has been demonstrated in yeast mitochondria. the yeast dead-box protein mss promotes the splicing of both group i and group ii introns in mitochondria via functioning as an rna chaperone. our current analysis clearly indicates that cfm harbors rna chaperone activity. it is likely that rna chaperone activity of cfm is needed to maintain precursor-rrna in processing-competent structures for subsequence rrna processing. although it is not clear at present how cfm affects processing of only two rrnas out of the four rrnas in the chloroplast, it is possible that cfm recognizes specific sequence or structural elements in 16s and s rrnas and the rna chaperone activity of cfm is involved in the formation of processing-competent structures of these rrnas.  the present study shows that cfm harboring rna chaperone activity is involved in rrna processing in chloroplasts, which is important for growth and the stress response of plants. although much progress has been made in the characterization of the roles of crm domain-containing proteins in intron splicing and rrna processing during chloroplast gene expression, the roles of many crm proteins in plant growth and development as well as chloroplast rna metabolism remain largely unknown. in particular, understanding the biological function of single crm domain-containing proteins is far behind compared with that of multiple crm domain-containing proteins. exploring the importance of the number of crm domains and involvement of each crm domain in substrate recognition during intron splicing and rrna processing should provide a much deeper insight into the mechanistic role of crm family members in chloroplast rna metabolism. 
although several chloroplast rna splicing and ribosome maturation domain-containing proteins have been characterized for intron splicing and rrna processing during chloroplast gene expression, the functional role of a majority of crm domain proteins in plant growth and development as well as chloroplast rna metabolism remains largely unknown. these results suggest that the chloroplast-targeted cfm one of two arabidopsis genes encoding a single crm domain-containing protein, harbors rna chaperone activity and plays a role in the arabidopsis growth and stress response by affecting rrna processing in chloroplasts. in addition, cfm mutants showed retarded seed germination and seedling growth under stress conditions. here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single crm domain, designated cfm in arabidopsis thaliana. no alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16s and s rrnas was abnormal in the mutant plants. importantly, cfm was determined to possess rna chaperone activity. the loss-of-function t-dna insertion mutants for cfm displayed retarded growth and delayed senescence, suggesting that cfm plays a role in growth and development of plants under normal growth conditions.
 although several chloroplast rna splicing and ribosome maturation domain-containing proteins have been characterized for intron splicing and rrna processing during chloroplast gene expression, the functional role of a majority of crm domain proteins in plant growth and development as well as chloroplast rna metabolism remains largely unknown. here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single crm domain, designated cfm in arabidopsis thaliana.  analysis of cfm4-gfp fusion proteins revealed that cfm is localized to chloroplasts. the loss-of-function t-dna insertion mutants for cfm displayed retarded growth and delayed senescence, suggesting that cfm plays a role in growth and development of plants under normal growth conditions. in addition, cfm mutants showed retarded seed germination and seedling growth under stress conditions. no alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16s and s rrnas was abnormal in the mutant plants. importantly, cfm was determined to possess rna chaperone activity.  these results suggest that the chloroplast-targeted cfm one of two arabidopsis genes encoding a single crm domain-containing protein, harbors rna chaperone activity and plays a role in the arabidopsis growth and stress response by affecting rrna processing in chloroplasts. arabidopsis thalianachloroplastcrm domainrna-binding proteinrna metabolism 