BACKGROUND
low temperature is one of the key environmental stresses that many plants have to cope with during their life cycle, which can influence growth, development, as well as the yield, quality, postharvest life, and geographic distribution of crop plants  <cit> .
cold stress can be classified as chilling  and freezing  stresses.
generally, plants from temperate regions have the capacity to cold acclimate, that is, to develop increased freezing tolerance after being exposed to low, nonfreezing temperatures  <cit> , but many important crops, such as rice, maize, soybean, cotton, and tomato, which originated in the tropics and subtropics, lack the cold acclimation mechanism and are sensitive to chilling stress  <cit> .
moreover, different varieties of the same species can also exhibit a high degree of genetic variability for cold tolerance  <cit> .
in conventional crop cross-breeding, the cold-tolerant varieties are usually used as the main resource for increasing the cold tolerance of cultivars  <cit> .
however, the lack of detailed knowledge of molecular mechanisms responsible for cold stress limits the potential for crop improvement.
investigation of gene expression profiles in response to cold stress will shed light on the sensing and regulatory networks for cold acclimation in plants and provide an effective approach to select targeted candidate genes for manipulation and/or cross-breeding of agronomic plants  <cit> .
musa spp.
, which originated in the tropics are giant perennial herbaceous monocots.
they are vital staple food in many african countries and the most popular fruit in industrialized countries  <cit> .
musa spp.
exhibits a high degree of genetic variability for cold tolerance, with banana  being more cold sensitive than plantain .
when the temperature drops to 8°c, banana growth is arrested, injury is inflicted  <cit> , and irreversible damage often occurs with temperatures below 12°c  <cit> .
in milder cases of cold injury, the fruit can be harvested, but its ripening process is abnormal.
in contrast to the cavendish “dessert” banana, the plantain species, dajiao has superior cold tolerance, enabling it to tolerate temperatures of 0-4°c  <cit> , and has been proposed as a potential germplasm resource of cold tolerance in banana breeding  <cit> .
although musa spp.
appear to lack a cold acclimation mechanism, transgenic plantain constitutively overexpressing the arabidopsis transcription factor dreb1b/cbf <dig> becomes highly tolerant to cold by increasing sod activities, decreasing mda content and the electrolyte leakage rate.
meanwhile the growth rates of these transgenic plants are severely retarded under normal growth conditions  <cit> .
in the herbaceous monocot rice , which also originated in the tropics, a novel mybs3-dependent pathway has recently been identified as essential for cold tolerance.
mybs <dig> was found to repress the cbf-dependent cold signaling pathway.
molecular evidence indicates that the sequential expression of cbf and mybs <dig> provides two complementary mechanisms for conferring cold tolerance, with the cbf-mediated process initiating the immediate cold shock response and the mybs3-mediated system adjusting the long-term cold adaptation in rice  <cit> .
recently, a new quantitative trait locus cold <dig> was identified, which interacts with g protein to activate the ca2+ channel for low temperature sensing that confers chilling tolerance in japonica rice, compared with cold-sensitive indica rice.
overexpression of cold1jap significantly enhances chilling tolerance, whereas rice lines with deficiency or down-regulation of cold1jap are sensitive to cold stress  <cit> .
in the past decade, some physiological responses to cold stress were analyzed, comparatively, between banana and plantain  <cit> , and another cold-resistance related plantain mprci  has been identified, that enhances low temperature resistance when heterologously expressed in transgenic tobacco  <cit> .
through the traditional map-based cloning and transgenic methods, one can identify some key stress-related genes.
but it’s generally tedious and time-consuming, especially for fruit trees which have a relatively long juvenile phase.
in recent years, advances in novel high-throughput sequencing technologies, such as solexa/illumina rna-seq  and digital gene expression  has provided an opportunity to explore cold resistance and signaling-associated genes in different species by denovo assembly or mapping, facilitating rapid identification and analysis of many transcriptomes  <cit> .
transcriptome research has become an effective means to investigate the gene expression patterns of fruit trees in the growth and development process and under various stresses, which has been reported in tropical and subtropical fruit trees, such as litchi  <cit> , mango  <cit> , papaya  <cit> , citrus  <cit>  and grape  <cit>  etc.
our quantitative proteomic analysis reveals that molecular mechanisms for the higher cold resistance found in plantain are associated with increased redox potential characterized by adapted ros scavenging capability, reduced ros production, decreased lipid peroxidation, and cell wall stabilization  <cit> .
gene expression under cold stress is very sensitive and it depends on the species, the temperature and the length of exposure to low temperature  <cit> .
although several important clues are suggested from intensive proteomic research of cold-tolerance plantain and comparing those results with functional gene analysis conducted in other plants, a comprehensive differential transcriptome analysis in response to cold stress between banana and plantain has not been reported and remains unknown.
in this study, plantain collected from a subtropical region of china with high cold-tolerance was used to investigate responses to cold stress at the transcriptional level.
a cold-sensitive species, banana was examined as a control.
a well-established whole genome transcriptome analysis method, based on rna-seq, and incorporating real time-pcr, was utilized to screen the differential transcripts associated with cold tolerance between banana and plantain.
our study provides a broad picture of the banana and plantain cold-responsive transcriptomes, with a new insight to cold-tolerance molecular mechanisms of plantain under cold stress.
RESULTS
phenotypes and electrolyte leakage of banana and plantain to cold stress
to accurately evaluate the tolerance of plantain, we compared the physiological changes of plantain versus banana in parallel during cold stress.
a significant phenotypic difference is that the banana leaves drooped after 6 h of cold treatment and the second and third leaves from the top displayed severe necrosis and wilting symptoms after 48 h of cold treatment at 10°c, while plantain leaves remained normal .
the relative leakage increased from  <dig>  to  <dig> % in banana leaves, and from  <dig>  to  <dig> % in plantain leaves respectively after 6 h of cold treatment.
after 48 h of cold stress, the leakage of plantain leaves decreased to the basal level, but the leakage of banana leaves still increased and the damage was not alleviated .
these observations suggest that the seedlings of plantain leaves had adapted to the cold stress to some extent after 6 h of treatment and appeared to protect the membrane from further damage, suggesting that some important cold-tolerant genes, such as the early signal transduction genes of plantain, may work at the beginning 6 h of cold treatment.
both phenotypic response and relative leakage of plantain support the notion that plantain once again, has stronger cold tolerance than banana.figure  <dig> phenotypic and physiological responses of banana and plantain under cold stress.
six-leaf stage seedlings of banana and plantain were treated at 10°c for  <dig>   <dig> and 6 h ; and comparison of phenotypic difference of the three leaves from the top of banana and plantain following 48 h of cold treatment ; the relative electrolyte leakage was determined for the banana and plantain treated at 10°c for  <dig>   <dig>   <dig>   <dig> and 48 h. the different letters  labeled above columns indicate a significant difference at p ≤  <dig>  between the columns by duncan’s test using spss statistical software .
the columns with the same letters mean no significant difference  between each other .
rna-seq and alignment of unique reads to banana reference genome
the raw data quality assessment of banana and plantain libraries with control and cold treatment are shown in additional file 1: table s <dig>  for banana, after filtering out low quality reads, raw reads containing ‘n’ and adaptor sequences,  <dig>  m to  <dig>  m clean reads were sequenced from four biological replicates of  <dig>   <dig> and 6 h cold treated libraries, respectively .
clean reads were aligned to banana reference genome database .
about 60% cavendish clean reads mapped to the reference genome.
multiple mapped clean reads in each library were excluded from further analysis.
finally a total of  <dig>  m to  <dig>  m uniquely mapped clean reads were used for subsequent analysis.
the distribution of unique reads with chromosome ‘+/−’ chain and splice/non-splice in each library were counted and are shown in table  <dig> table  <dig>
statistic analysis of cavendish and dajiao reads mapped to
m. acuminate
reference genome
sample name
cavendish
dajiao
10°c
10°c
10°c
10°c
10°c
10°c
0 h replicates
3 h replicates
6 h replicates
0 h replicates
3 h replicates
6 h replicates
total reads
a
total mapped
multiple mapped
uniquely mapped
reads map to ‘+’
reads map to ‘-’
non-splice reads
splice reads
atotal reads: clean data obtained after filtering the reads containing adapter, poly-n and low quality reads from raw data.
.
differentially expressed genes  in cavendish and dajiao during cold treatment for  <dig> and 6 hours
cavendish
unique in cavendish
dajiao
unique in dajiao
common in both
for plantain, after filtering out low quality reads, raw reads containing ‘n’ and adaptor sequences,  <dig>  m to  <dig>  m clean reads were generated from four biological replicates of  <dig>   <dig> and 6 h cold treated libraries, respectively .
clean reads were aligning to banana reference genome database.
there are also about 60% of plantain clean reads mapped to the reference genome.
finally a total of  <dig>  m to  <dig>  m uniquely mapped clean reads were used for further analysis.
the distribution of unique reads with chromosome ‘+/−’ chain and splice/non-splice in each library were counted and are shown in table  <dig>
differential expression profiling of cold stress between banana and plantain
to comprehensively investigate the differences in gene expression between cold-sensitive banana and cold-tolerant plantain in response to cold stress, we performed comparative transcriptome analysis using the aligned reads .
after 3 h of cold stress, a total of  <dig> ,  <dig>  cold-responsive genes were identified in banana and plantain, respectively  with the threshold of  <dig>  for corrected p-value and  <dig> for log <dig> base of fold-change.
out of the  <dig> degs,  <dig> cold-responsive genes  were exclusively identified in banana, whereas  <dig> cold-responsive genes  were uniquely observed in plantain.
the remaining  <dig> genes  were commonly regulated by cold stress in both banana and plantain.
after 6 h of cold stress, a total of  <dig> ,  <dig>  cold-responsive genes were identified in banana and plantain, respectively .
out of the  <dig> degs,  <dig> cold-responsive genes  were exclusively identified in banana, whereas  <dig> cold-responsive genes  were uniquely observed in plantain and  <dig> genes  were commonly regulated by cold stress in both banana and plantain.
the small number of cold-responsive genes identified in cold-tolerant plantain suggests that some inherent adaptation and regulation mechanisms may be attributed to the cold tolerance in plantain.
the degs with at least a 2-fold change are shown in additional file 4: table s <dig> and table  <dig>  after 3 h of cold stress,  <dig> up-regulated genes commonly identified in both banana and plantain, include gsmua_achr9g07610_ <dig> , gsmua_achr6g32910_ <dig> , gsmua_ achr2g13410_ <dig> , gsmua_achr6g25670_ <dig> , gsmua_achr7g26580_ <dig>  gsmua_achr7g05900_ <dig>  and gsmua_achr3g23000_ <dig> .
meanwhile,  <dig> up-regulated genes including gsmua_achr8g21550_ <dig>  and gsmua_achr3g05220_ <dig>  and  <dig> down-regulated gene: gsmua_achr3g04360_ <dig>  were significantly changed in plantain at 3 hours.
interestingly, the above  <dig> genes with the identical trend of changes were present in banana after 6 h of cold stress, suggesting that those  <dig> genes be specifically responsive to cold stress, and their early response maybe critical in the cold resistance.
in addition, this timely delay of response to cold stress was also found in plantain.
out of  <dig> degs found in banana after 3 h of cold stress,  <dig> genes with the identical response profiling were present in plantain only after 6 h of cold stress.
those  <dig> genes include gsmua_achr5g21050_ <dig> , gsmua_achr7g06910_ <dig> , gsmua_achr3g01650_ <dig> , gsmua_achr9g04780_ <dig> , gsmua_achr7g21780_ <dig>  gsmua_achr3g <dig> _ <dig> , gsmua_achr10g22580_ <dig> , gsmua_achr 7g21070_ <dig> , gsmua_achr10g25070_ <dig> , gsmua_achr7g00180_ <dig> , gsmua_achr6g13020_ <dig> , gsmua_achr10g24780_ <dig>  with up-regulation, and gsmua_achr11g02890_ <dig> , gsmua_achr7g22100_ <dig>  and gsmua_achr 8g30530_ <dig>  with down-regulation.
these delayed, expressed genes may be of importance to the adaptation of cold stress for sustainable cold tolerance in plantain.table  <dig>
primary functional classification on differentially expressed genes of dajiao with fold changes > or < 2-fold
gene_id
rpkm dajiao 0 h
rpkm dajiao 3 h
rpkm dajiao 6 h
log2fold change 3 h
log2fold change 6 h
gene name
regulation of transcription
response to stress
protein modification
signal transduction
cell wall organization and biogenesis
transport
nucleosome assembly
iron-sulfur cluster assembly
proteolysis
oxidation reduction
rna modification
copper ion homeostasis
photosynthesis and photorespiration
response to sucrose stimulus
unknown
there are  <dig> more degs being identified in plantain for 6 h of cold stress.
of which  <dig> genes include gsmua_achr8g25220_ <dig> , gsmua_achr 6g15840_ <dig> , gsmua_achr3g25160_ <dig> , gsmua_achr11g04410_ <dig> , gsmua_achr6g14750_ <dig> , gsmua_ achr5g07340_ <dig> , gsmua_achr4g33270_ <dig> , gsmua_achr8g06870_ <dig> , gsmua_achr7g11200_ <dig> , gsmua_achr7g01430_ <dig> , gsmua_achr 2g13700_ <dig>  gsmua_achr4g11450_ <dig> , gsmua_ achr6g16830_ <dig> , gsmua_ achr6g <dig> _ <dig> , gsmua_achr7g27650_ <dig> , gsmua_achr1g22700_ <dig> , gsmua_ achr7g12120_ <dig> , gsmua_achr3g03820_ <dig>  gsmua_achr2g22400_ <dig> , gsmua_achr6g10300_ <dig> , gsmua_ achr2g22290_ <dig> , gsmua_ achr5g01760_ <dig> , gsmua_achr 6g35120_ <dig> .
a similar differential profile for those  <dig> genes was also found in banana for 6 h of cold stress, suggesting they share the similar adaptation mechanism to cold stress.
the remaining  <dig> genes were differentially expressed only in plantain .
these genes include gsmua_achr4g16550_ <dig> , gsmua_achr1g07160_ <dig> , gsmua_ achr <dig> g05390_ <dig>  gsmua_achr7g16570_ <dig> , gsmua_achr3g16210_ <dig> , gsmua_achr3g28910_ <dig> , gsmua_achr8g05250_ <dig>  with up-regulation, and gsmua_achr8g20530_ <dig> , gsmua_ achr7g20770_ <dig> , gsmua_achr4g24680_ <dig> , gsmua_achr10g19930_ <dig> , gsmua_ achr3g26220_ <dig> , gsmua_achr1g17220_ <dig> , gsmua_achr 11g11670_ <dig> , gsmua_achr6g01490_ <dig> , gsmua_achr9g12290_ <dig>  and gsmua_achr9g26820_ <dig>  with down regulation.
these  <dig> genes and their regulations appear likely attributed to the cold tolerance for plantain.
besides aforementioned  <dig> differential genes,  <dig> more degs were found only in banana for 6 h of cold stress, which are expected to be involved in cold stress for banana only.
functional classification of cold stress-related degs
the functional classification of degs in banana and plantain was further examined to explore the pattern of transcriptome regulation that occurs during cold stress.
genes matching well-characterized proteins or proteins with putative functions were grouped using the gene ontology  and summarized in additional file 4: table s <dig> and table  <dig>  the majority of degs identified in this study are in the categories regulation of transcription, response to stress, transport, protein modification, nucleosome assembly, cell wall organization and biogenesis, signal transduction, oxidation reduction, rna modification, cell redox homeostasis, etc.
no obvious difference of go classification was observed for those degs from either banana  or plantain , despite the fact that few degs in each go category were identified in plantain than in banana.
validation of the degs by quantitative rt-pcr analysis
plantain leaves showed significant phenotypic difference from banana after 3 h cold treatment at 10°c, we suspect that early response genes of plantain are closely associated with its cold resistance, thus  <dig> degs of plantain identified in this study after 3 h cold treatment and  <dig> critical cold-response homologous genes in arabidopsis ice <dig> and rice mybs <dig> were selected for quantitative rt-pcr analysis.
two additional extended time points for  <dig> and 48 h of cold stress were also measured in parallel with initial  <dig>   <dig> and 6 h cold stressed samples in both banana and plantain.
the primers of selected genes are listed in additional file 5: table s <dig>  25s ribosomal rna gene was used as reference gene for data normalization according to van den berg et al.
<cit> .
the quantitative rt-pcr results from banana and plantain are shown in figure  <dig>  the expression profiles of all  <dig> detected genes show the same trend and consistent results between the rt-pcr and the solexa-sequencing methods.
for  <dig> and 48 h of cold stress,  <dig> out of  <dig> degs displayed the same down regulation in both banana and plantain, including gsmua_achr7g05900_ <dig> , gsmua_achr9g07610_ <dig> , gsmua_achr6g32910_ <dig> , gsmua_achr7g26580_ <dig> , gsmua_ achr3g <dig> _ <dig> , gsmua_achr3g23000_ <dig> .
however,  <dig> of them showed opposite changes between banana and plantain, including gsmua_achr6g25670_ <dig> , gsmua_achr8g21550_ <dig> , gsmua_ achr2g13410_ <dig>  and gsmua_achr 3g05220_ <dig> .
strikingly, there is a remarkable difference of ice <dig> and mybs <dig> expression profile between plantain and banana in response to cold stress.
the ice <dig> was significantly down-regulated  under cold stress at early stage  in banana, but its decrease  didn’t appear until at 24 h in plantain .
although the mybs <dig> was down-regulated similarly at early stage by 9-fold and 4-fold in banana and plantain respectively, at 6 h of cold stress , it can be recovered much quickly in plantain than banana.
with the extended cold stress, the plantain mybs <dig> was rapidly recovered to the level of 41%  and 75% , while the banana mybs <dig> was further decreased to 4% at 24 h and then recovered to 24% at 48 h of cold stress.
the considerably different expression profiles of the two important transcriptional factors with a remarkable time delayed response in banana versus plantain suggest that the specific time course-based expression of ice <dig> and mybs <dig> in plantain might be related to its cold tolerance.figure  <dig> relative mrna levels of  <dig> degs in banana and plantain seedlings were determined by quantitative rt-pcr analyses.
six-leaf stage seedlings were incubated at 10°c for the indicated time.
transcript abundances of genes encoding dehydration-responsive element-binding protein 1d , ethylene insensitive 3-like  <dig> protein , probable cytosolic iron-sulfur protein assembly protein  <dig> , zinc finger protein  <dig> , u-box domain- containing protein  <dig> , zinc finger ccch domain-containing protein  <dig> , udp-glucose 6-dehydrogenase , probable xyloglucan endotransglucosylase/hydrolase protein  <dig> , ethylene-responsive transcription factor rap2- <dig> , calcium-binding protein kic , transcription factor ice <dig>  and mybs <dig>  from both banana and plantain were determined and compared across the time course of cold stress.
data represent means ± sd in four replicates .
the different lowercase letters labeled above columns indicate a significant difference at p ≤  <dig>  between the columns by duncan’s test using spss statistical software .
the columns with the same letters mean no significant difference  between each other.
discussion and
CONCLUSIONS
in our previous study on the temporal responses of plantain to cold stress using quantitative proteomics analysis, we have revealed that antioxidation mechanisms contribute to cold tolerance in plantain at the global proteome level  <cit> .
however, due to the limitation of current technologies in overcoming the issue of the wide dynamic range in proteomics samples, quantitative proteomics analysis often misses the detection of many low abundance proteins, yielding only 10-30% proteome coverage for any given non-model species.
obviously this will reduce the number of low abundance proteins identified during global functional studies.
given the above consideration and to focus on the molecular mechanisms involving low abundance genes on the cold-tolerance of plantain, a comparative transcriptomics analysis of cold-sensitive banana and cold-tolerant plantain was conducted by rna-seq and real time rt-pcr with time course of cold treatment.
after 48 h of cold stress at 10°c for banana and plantain seedlings, both phenotype and electrolyte leakage analyses clearly indicate that plantain seedlings exhibited a more robust cold tolerance than banana seedlings .
it is this phenotypic difference in the cold response between banana and plantain that provides an excellent model system allowing us to study differential gene expression in response to cold stress, as well as to elucidate the potential different cold responsive mechanisms between banana and plantain.
rna-seq analysis shows that  <dig> and  <dig> degs are identified for 3 h and 6 h of cold stress respectively in plantain, while the equivalent degs are  <dig> and  <dig> being identified in banana , indicating banana is much more sensitive to cold stress than plantain.
go classification analysis shows the majority of degs identified in this study belong to the following  <dig> categories: regulation of transcription, response to stress, transport, protein modification, nucleosome assembly, cell wall organization and biogenesis, signal transduction, oxidation reduction, rna modification, cell redox homeostasis, etc.
since there is no difference of go classification found between banana  and plantain , it suggests that cold stress appears to have a broad range of impacts on cellular activities.
however, when we further performed the pathway analysis for some specific degs either found uniquely in plantain particularly or detected at variable cold stress time points in both banana and plantain, we found that many degs and their associated pathways are likely to be involved in cold-tolerance of plantain.
below we discuss intensively some of the important pathways and their possible mechanisms associated with the observed cold tolerance in plantain.
ice <dig> and mybs <dig> pathways
ice gene was initially identified and isolated from arabidopsis thaliana.
the function of atice <dig> is to activate gene expression of c-repeat binding factor  to enhance the cold resistance of arabidopsis  <cit> .
in oryza sativa, three novel myb proteins  containing one highly conserved dna-binding domain mediate sugar and hormone regulation by promoting gene expression of alpha-amylase  <cit> .
recent studies reveal that mybs <dig> is also involved in adaptation of cold stress in rice.
compared to the ice <dig>  mybs <dig> functions particularly in late stage of plant for adaptation of cold stress.
for example, transgenic rice overexpressing mybs <dig> can resist cold stress at 4°c for a week  <cit> .
in the early stage of cold response, the transient activation of αamy <dig> expression by cbf allows hydrolysis of reserved starch to meet the immediate need for a carbon source and energy to combat the cold shock, while the subsequent suppression of αamy <dig> expression by mybs <dig> allows rice to conserve carbohydrates until regrowth is allowed at elevated temperatures  <cit> .
in this study using rna-seq analysis for  <dig>   <dig>  and 6 h of cold stressed samples, although both ice <dig> and mybs <dig> genes were detected, neither was identified as deg.
this is not surprising, because ice <dig> requires activation, which is regulated by post-translational modifications via phosphorylation and sumoylation of the siz <dig> gene  <cit> .
thus, ice <dig> is likely not regulated at the transcriptional level.
since mybs <dig> plays a role in cold adaptation at late stage of plant growth, mybs <dig> is expected not present in early time points from cold stressed samples.
in fact, we did identify both genes by rna-seq analysis at a low level with relatively large variation across the  <dig> replicate samples.
given the well-known functions of both ice <dig> and mybs <dig> genes in plant resistance to cold stress, we decided to use quantitative rt-pcr for assessing their changes with the extended time points at  <dig> and 48 h of cold stress in this study.
the results indicate that although there is no significant change of ice <dig> in plantain at  <dig> and 6 h cold stress, both an upstream gene: gsmua_achr9p04380_ <dig> and a downstream gene: gsmua_ achr <dig> g05900_ <dig>  in an ice1-cbf-cor pathway, were up-regulated .
since the ice1-cbf-cor is a well-known pathway involved in plant cold-tolerance  <cit> , the up-regulation of cbf is therefore, expected to facilitate the cold-tolerant pathway.
a  <dig> -fold increase of cor <dig> protein found in our proteomics analysis of plantain for 6 h under cold stress at 8°c  <cit>  supports this observation with the ice1-cbf-cor pathway being activated in response to the cold stress in this study.
interestingly, at 24 h of cold stress, both plantain ice <dig> and cbf were down regulated.
meanwhile, the mybs <dig> gene had almost completely recovered , which may offset the down-regulated ice1-cbf-cor pathway as a means of sustaining the cold resistance.
in banana, however both ice <dig> and mybs <dig> were significantly decreased under cold stress.
the cbf response to cold stress in banana is significantly slower than that in plantain, until 6 h of cold stress, when the banana cbf gene was up-regulated, only to go down again at 24 h of cold stress.
consistently, the time delayed response of mybs <dig> gene to the cold stress was also observed in banana.
however, at 48 h of cold stress, the banana mybs <dig> gene appeared recover from its initial cold suppression.
based on the observation of slow response to the cold stress in banana for both ice <dig> and mybs <dig> as the two critical transcriptional factors responsible for early and late stage respectively against cold stress, we suspect that the relatively low expression of these two key genes along with their delayed response to the cold stress are among the main reasons for cold sensitive banana.
signals transduction
as an important second messenger, ca2+ plays a vital role in the plant cold-stress response.
the concentration of ca2+ inside the cell increases rapidly during cold stress, followed by a number of signals mediated by a series of protein phosphorylation cascades  <cit> .
during rice domestication, an amino acid mutant of cold <dig> from met187/thr <dig> to lys <dig> in japonica cultivars was found to enhance cold tolerance partly because it facilitates the formation of an appropriate ca2+ signal by increased ca2+ concentration  <cit> .
interestingly, our study shows that most of the degs involving in signal transduction are indeed related to the calcium-dependent signal pathway, such as orthologs of cipk , kic  and cml .
all three genes in banana and plantain were up-regulated in response to cold stress.
in addition, overexpression of osmsr <dig>  a novel rice calmodulin-like gene, improves resistance of drought and salt and increases aba sensitivity in arabidopsis  <cit> .
in this work, we found the dramatically increased expression of cml <dig> , an ortholog of osmsr <dig>  in response to cold stress in plantain but not in banana, suggesting that there is a more efficient response of the ca2+ signal transduction pathway in plantain so that it can quickly and effectively regulate downstream signaling and gene expression in response to cold stress.
as a signal molecule, auxin plays an important function under various abiotic stress conditions  <cit> .
intriguingly, compared to the control group , the expression of gsmua_achr4g16550_ <dig>  was increased by  <dig> fold in 6 h cold treatment in plantain, while no differential expression of the gene was detected in banana.
on the plasma membrane, air <dig> is able to receive auxin signal to promote lateral root morphogenesis and participates in the decomposition of glucose  <cit> , while it has been proven that the content of soluble sugar and acid can affect the plant’s cold tolerance  <cit> .
our results indicate that the increased expression of air <dig> in plantain could facilitate the growth of plantain root and the content of sugar and acid, which might be attributed to the better resistance to cold.
transcription regulation
in this study, we have identified  <dig> differentially expressed transcription factors in response to cold stress in banana and plantain, including nac, erf , dreb , myb,wrky, c3h <dig>  eil <dig> , ado <dig> , zfp  and rpoa .
nac transcription factors were up-regulated in banana and plantain after 6 h cold stress, and over-expression of snac <dig> was reported to increase cold and salt resistance in rice  <cit> , suggesting that nacs are likely to participate in cold response in banana and plantain.
effects of ethylene on plants under cold stress have recently been recognized, the erf transcription factors which related to ethylene, were up-regulated in banana and plantain after 6 h cold stress.
overexpression of transcription factor terf2/leerf <dig> in tobacco and tomato was reported to result in cold tolerance by facilitating ethylene biosynthesis  <cit> , which supports the important role of erf in plant cold tolerance.
dreb transcription factors, with a conservative ap2/erebp domain, are involved in regulation of stress-related gene in response to the external environment, through binding dre cis-acting elements  <cit> .
our data shows that overexpression of dreb <dig> occurs at 3 h of cold stress in plantain versus 6 h in banana, suggesting that the early response of dreb <dig> may be critical in cold-tolerant plantain.
due to a large number of myb transcription factor found in plants such as arabidopsis thaliana and zea mays, an increased amount of research has been focused on its role in transcriptional regulation and its impact on a broad range of physiological functions, e.g., overexpression of rice myb genes  enabled to significantly enhanced the cold tolerance of transgenic arabidopsis thaliana  <cit> .
in our work,we found gsmua_achr8g25220_001-a myb transcription factor increased after 6 h cold stress while it was undetectable at 0 h in both banana and plantain, indicating that this gene is a cold-induced gene.
the change of this myb gene could affect downstream related genes.
in arabidopsis thaliana, the wrky gene family contains  <dig> genes induced by cold stress, most of which are activated in its early stages  <cit> .
after 6 h cold stress, the wrky genes showed up-regulation, among them, gsmua_achr7g05200_ <dig> was overexpressed whereas it was undetectable at 0 h, indicating it is also a cold-induced gene.
in plantain, only one cold-induced wrky gene-gsmua_achr6g15840_ <dig> was detected after 6 h of cold stress.
however, its expression was much higher than that in banana, suggesting that this wrky gene may contribute to the cold-resistance of plantain.
it has been reported that zinc-finger proteins are involved in resistance to adversity in plant  <cit> .
for example, gmzf <dig> from soybean was found to enhance the tolerance of arabidopsis to cold stress by expression of cold-regulation genes in the transgenic arabidopsis  <cit> .
strikingly, banana zfp <dig> is not up-regulated until 24 h of cold stress and starts to decrease at 48 h, while plantain zfp <dig> is constantly up-regulated beyond 48 h of cold stress, suggesting zfp <dig> may play a role in late stages of cold stress .
expression of eil <dig> in plantain reached ~11-fold higher induction at 3 h cold treatment and then decreased back to initial levels at 48 h. in banana, the eil <dig> was up-regulated by ~9-fold at 6 h and further increased to ~13-fold at 48 h of cold treatment .
as a primary transcription factor in ethylene signal transduction, eil regulates transcription of downstream genes to complete the ethylene response  <cit> .
given the higher expression of eil at very early stages and its rapid decrease in response to cold stress in plantain, we suspect that rapid and early activation of the ethylene signal transduction pathway appears to play a critical role in the rapid response to cold stress allowing early protection in plantain.
genes associated with stress response
plants often activate similar pathways in response to different types of abiotic stress.
in this cold stress study, we found quite a few genes related to stress response in both banana and plantain, such as sthy , p2c <dig> , ubiquitin-protein ligase genes, thil- <dig> , grp and drt <dig> , etc.
those genes showed similar patterns of expression in response to cold treatment in banana and plantain, suggesting the stress response genes share some common response pathways in both species.
however, it should be noted that some of the degs are located in the chloroplast of plantain only, such as elip, ctl <dig>  sca and mks <dig>  in rhododendron catawbiens, expression of rcelip  resulted in adaptive responses to cold and high light in winter-adapted rhododendron leaves, suggesting a critical role of elip in protection of photosynthetic apparatus from these stresses  <cit> .
in arabidopsis thaliana, ctl <dig>  is involved in altering the architecture of the root system in response to multiple environmental conditions  <cit> .
as an map kinase substrate, arabidopsis mks <dig> may be involved in mpk4-regulated activation of plant defense by coupling the kinase to specific wrky transcription factors  <cit> .
due to the expression of elip, ctl <dig>  sca and mks <dig> induced by cold treatment exclusively in plantain , we believe that plantain possesses the highly superior aspects of photosynthesis, root architecture and plant defense regulated by mpk, which makes plantain broadly resist to abiotic stress resulting in a better cold tolerance.
other degs involved in functional regulations
in addition to the above four categories, we found some degs in banana and plantain are also involved in protein/rna modification, cell wall composition and occurrence, transit, nucleosome assembly, cellular redox balance, iron-sulfur cluster assembly, protein hydrolysis, oxidation and reduction, copper ion balance, photosynthesis, respiration and sugar stimulus response.
but again some of the cold responsive degs are found exclusively in plantain, including mtp <dig>  involved in copper ion balance, rbcs  functioning in photosynthesis and respiration in chloroplast, cp <dig>  involved in sugar stimulus response in chloroplast, and ubq <dig>  functioning in protein modification.
in yeast, putmt <dig>  has been verified to play a critical role in improving tolerance to metal and reactive oxygen species  <cit> .
thus the up-regulation of mtp <dig> would allow plantain for improved tolerance to the increased reactive oxygen species  due to the cold stress  <cit> .
in plant, rbsc encodes the small subunit of rubisco  involved in the first major step of carbon fixation and the rbsc gene family functions to yield sufficient rubisco content for leaf photosynthetic capacity  <cit> .
as the expression of rbcs was highly repressed in plantain, we speculate that the protection mechanism is activated in response to cold treatment in chloroplast in order to decrease the production of ros through the weakened photosynthesis.
arabidopsis cp <dig> forms a complex with gapdh  and prk in regulating tca cycle  <cit> .
a dramatic repression of cp <dig> suggests that plantain must reduce the photosynthesis in response to cold stress.
this is consistent with the decreased rbcs but as another pathway for the self-protection mechanism.
ubq  is involved in protein hydrolysis in the ubiquitin-proteasome pathway.
in order to compensate cells for ubiquitin molecules in signal transmission, expression of poly-ubiquitin gene is increased in response to stress  <cit> .
given the increased expression of ubq in response to cold treatment, we suspect that ubq might be involved in stress response in plantain.
in summary,a global, comparative transcriptomic profile of banana and plantain in response to cold treatment has been intensively investigated by transcriptome sequencing  and quantitative rt-pcr .
at the transcriptomic level, both plantain and banana share similar profiles for the majority of degs in time course response to cold treatment.
many of the degs have been reported and involved in the development of cold resistance or other stress resistance in plant, such as: cipk, kic, cml , nac, erf, dreb, myb, wrky, c3h <dig>  ado <dig>  rpoa , sthy, p2c <dig>  ubiquitin-protein ligase genes, thil- <dig>  gr and drt <dig> , etc.
despite the similar profiles of degs found in banana and plantain, several dozen degs involved in several different pathways were successfully identified and validated only in cold-tolerant plantain.
for example, the cold-tolerant ice <dig> and mybs <dig> pathways were rapidly activated and switched in early  and late  stages respectively, of response to cold treatment in plantain.
while in cold-sensitive banana, no such equivalent regulation for ice1-cbf-cor pathway was found and slow response to mybs <dig> pathway responsible for the cold stress was observed.
thus, the low expression of the two key genes along with their delayed response to the cold stress may be one of the main reasons for cold sensitivity of banana.
the cold-tolerant plantain also appears closely related to expressions of several specific genes such as cml <dig>  air <dig> , zfp <dig>  eil <dig> , elip, ctl <dig>  sca, mks <dig> , mtp <dig> , rbcs , cp <dig>  and ubq <dig> .
combined with previous research, we rationalize that in early stage of cold stress response, changes in cell membrane phase trigger modification of actin cytoskeleton, which activates ice1-cbf-cor metabolic pathways by ca2+ and phosphorylation signaling pathways.
also it triggers an early response via the ethylene signal transduction pathway.
in late stage of cold stress, expression of mybs <dig> begins to recover.
mybs <dig> is involved in coordination with the expression of multiple genes for regulation of oxidation/reduction, oxylipin biosynthetic process, photosynthesis, photorespiration, glycolysis, tricarboxylic acid cycle, carbohydrate metabolic process, fatty acid biosynthetic process and beta-oxidation, enhanced adoption to cold tress .
the next questions need to be answered are what causes the observed different expression profiles between ice <dig> and mybs <dig> pathways in plantain?
how do both coordinate together for effectively regulating cold-resistance?
addressing these questions will further enhance the understanding on signaling and metabolic pathways of cold tolerance in plantain, but also conducive to nurturing new varieties of cold tolerant banana cultivars.figure  <dig> a schematic diagram of cold-tolerance transcriptional network in plantain, adapted initially from v. chinnusamy et al.
<cit>  and c.f.
su et al.
<cit>  and revised based on this study.
at the early stage of cold stress, plantain cells probably sense low temperatures through membrane rigidification and/or other cellular changes, which might induce a calcium signature and activate protein kinases necessary for cold tolerance.
constitutively expressed ice <dig> is activated by cold stress through sumoylation and phosphorylation.
sumoylation of ice <dig> is critical for ice1-activation of transcription of cbfs and repression of myb <dig>  cbfs regulate the expression of cor genes that confer cold tolerance.
the expression of cbfs is negatively regulated by myb <dig>  hos <dig> mediates the ubiquitination and proteosomal degradation of ice <dig> and, thus, negatively regulates cbf regulons.
cbfs can constitutively regulate the expression of downstream cold-responsive transcription factor genes raps, which might control sub-regulons of the cbf regulon.
cbfs also activate αamy <dig> expression to hydrolyse reserved starch.
at the late stage of cold stress,mybs <dig> inhibits cbfs and αamy <dig> expression.
the effective coordination across the early and late stages of cold stress by at least two different regulatory pathways appears to efficiently regulate the following metabolic pathways including oxidation reduction, oxylipin biosynthetic process, photosynthesis, photorespiration, glycolysis, tricarboxylic acid cycle, carbohydrate metabolic process, fatty acid biosynthetic process and beta-oxidation.
the rapid activation and selective induction of ice <dig> and mybs <dig> cold tolerance pathways in plantain, along with expression of other cold-specific genes, may be one of the main reasons that plantain has higher cold resistance than banana .
broken arrows indicate post-translational regulation; solid arrows indicate activation, whereas lines ending with a bar show negative regulation; the two stars  indicate unknown cis-elements.
p, phosphorylation; s, sumo; u, ubiquitin.

 low temperature is one of the key environmental stresses that many plants have to cope with during their life cycle, which can influence growth, development, as well as the yield, quality, postharvest life, and geographic distribution of crop plants. cold stress can be classified as chilling and freezing stresses. generally, plants from temperate regions have the capacity to cold acclimate, that is, to develop increased freezing tolerance after being exposed to low, nonfreezing temperatures, but many important crops, such as rice, maize, soybean, cotton, and tomato, which originated in the tropics and subtropics, lack the cold acclimation mechanism and are sensitive to chilling stress. moreover, different varieties of the same species can also exhibit a high degree of genetic variability for cold tolerance. in conventional crop cross-breeding, the cold-tolerant varieties are usually used as the main resource for increasing the cold tolerance of cultivars. however, the lack of detailed knowledge of molecular mechanisms responsible for cold stress limits the potential for crop improvement. investigation of gene expression profiles in response to cold stress will shed light on the sensing and regulatory networks for cold acclimation in plants and provide an effective approach to select targeted candidate genes for manipulation and/or cross-breeding of agronomic plants. musa spp., which originated in the tropics are giant perennial herbaceous monocots. they are vital staple food in many african countries and the most popular fruit in industrialized countries. musa spp. exhibits a high degree of genetic variability for cold tolerance, with banana being more cold sensitive than plantain. when the temperature drops to 8°c, banana growth is arrested, injury is inflicted, and irreversible damage often occurs with temperatures below 12°c. in milder cases of cold injury, the fruit can be harvested, but its ripening process is abnormal. in contrast to the cavendish “dessert” banana, the plantain species, dajiao has superior cold tolerance, enabling it to tolerate temperatures of 0-4°c, and has been proposed as a potential germplasm resource of cold tolerance in banana breeding. although musa spp. appear to lack a cold acclimation mechanism, transgenic plantain constitutively overexpressing the arabidopsis transcription factor dreb1b/cbf becomes highly tolerant to cold by increasing sod activities, decreasing mda content and the electrolyte leakage rate. meanwhile the growth rates of these transgenic plants are severely retarded under normal growth conditions. in the herbaceous monocot rice, which also originated in the tropics, a novel mybs3-dependent pathway has recently been identified as essential for cold tolerance. mybs was found to repress the cbf-dependent cold signaling pathway. molecular evidence indicates that the sequential expression of cbf and mybs provides two complementary mechanisms for conferring cold tolerance, with the cbf-mediated process initiating the immediate cold shock response and the mybs3-mediated system adjusting the long-term cold adaptation in rice. recently, a new quantitative trait locus cold was identified, which interacts with g protein to activate the ca2+ channel for low temperature sensing that confers chilling tolerance in japonica rice, compared with cold-sensitive indica rice. overexpression of cold1jap significantly enhances chilling tolerance, whereas rice lines with deficiency or down-regulation of cold1jap are sensitive to cold stress. in the past decade, some physiological responses to cold stress were analyzed, comparatively, between banana and plantain, and another cold-resistance related plantain mprci has been identified, that enhances low temperature resistance when heterologously expressed in transgenic tobacco. through the traditional map-based cloning and transgenic methods, one can identify some key stress-related genes. but it’s generally tedious and time-consuming, especially for fruit trees which have a relatively long juvenile phase. in recent years, advances in novel high-throughput sequencing technologies, such as solexa/illumina rna-seq and digital gene expression has provided an opportunity to explore cold resistance and signaling-associated genes in different species by denovo assembly or mapping, facilitating rapid identification and analysis of many transcriptomes. transcriptome research has become an effective means to investigate the gene expression patterns of fruit trees in the growth and development process and under various stresses, which has been reported in tropical and subtropical fruit trees, such as litchi, mango, papaya, citrus and grape etc. our quantitative proteomic analysis reveals that molecular mechanisms for the higher cold resistance found in plantain are associated with increased redox potential characterized by adapted ros scavenging capability, reduced ros production, decreased lipid peroxidation, and cell wall stabilization. gene expression under cold stress is very sensitive and it depends on the species, the temperature and the length of exposure to low temperature. although several important clues are suggested from intensive proteomic research of cold-tolerance plantain and comparing those results with functional gene analysis conducted in other plants, a comprehensive differential transcriptome analysis in response to cold stress between banana and plantain has not been reported and remains unknown. in this study, plantain collected from a subtropical region of china with high cold-tolerance was used to investigate responses to cold stress at the transcriptional level. a cold-sensitive species, banana was examined as a control. a well-established whole genome transcriptome analysis method, based on rna-seq, and incorporating real time-pcr, was utilized to screen the differential transcripts associated with cold tolerance between banana and plantain. our study provides a broad picture of the banana and plantain cold-responsive transcriptomes, with a new insight to cold-tolerance molecular mechanisms of plantain under cold stress.  phenotypes and electrolyte leakage of banana and plantain to cold stress to accurately evaluate the tolerance of plantain, we compared the physiological changes of plantain versus banana in parallel during cold stress. a significant phenotypic difference is that the banana leaves drooped after 6 h of cold treatment and the second and third leaves from the top displayed severe necrosis and wilting symptoms after 48 h of cold treatment at 10°c, while plantain leaves remained normal. the relative leakage increased from to % in banana leaves, and from to % in plantain leaves respectively after 6 h of cold treatment. after 48 h of cold stress, the leakage of plantain leaves decreased to the basal level, but the leakage of banana leaves still increased and the damage was not alleviated. these observations suggest that the seedlings of plantain leaves had adapted to the cold stress to some extent after 6 h of treatment and appeared to protect the membrane from further damage, suggesting that some important cold-tolerant genes, such as the early signal transduction genes of plantain, may work at the beginning 6 h of cold treatment. both phenotypic response and relative leakage of plantain support the notion that plantain once again, has stronger cold tolerance than banana.figure phenotypic and physiological responses of banana and plantain under cold stress. six-leaf stage seedlings of banana and plantain were treated at 10°c for and 6 h; and comparison of phenotypic difference of the three leaves from the top of banana and plantain following 48 h of cold treatment; the relative electrolyte leakage was determined for the banana and plantain treated at 10°c for and 48 h. the different letters labeled above columns indicate a significant difference at p ≤  between the columns by duncan’s test using spss statistical software. the columns with the same letters mean no significant difference between each other. rna-seq and alignment of unique reads to banana reference genome the raw data quality assessment of banana and plantain libraries with control and cold treatment are shown in additional file 1: table s for banana, after filtering out low quality reads, raw reads containing ‘n’ and adaptor sequences,  m to  m clean reads were sequenced from four biological replicates of and 6 h cold treated libraries, respectively. clean reads were aligned to banana reference genome database. about 60% cavendish clean reads mapped to the reference genome. multiple mapped clean reads in each library were excluded from further analysis. finally a total of  m to  m uniquely mapped clean reads were used for subsequent analysis. the distribution of unique reads with chromosome ‘+/−’ chain and splice/non-splice in each library were counted and are shown in table  table statistic analysis of cavendish and dajiao reads mapped to m. acuminate reference genome sample name cavendish dajiao 10°c 10°c 10°c 10°c 10°c 10°c 0 h replicates 3 h replicates 6 h replicates 0 h replicates 3 h replicates 6 h replicates total reads a total mapped multiple mapped uniquely mapped reads map to ‘+’ reads map to ‘-’ non-splice reads splice reads atotal reads: clean data obtained after filtering the reads containing adapter, poly-n and low quality reads from raw data.. differentially expressed genes in cavendish and dajiao during cold treatment for and 6 hours cavendish unique in cavendish dajiao unique in dajiao common in both for plantain, after filtering out low quality reads, raw reads containing ‘n’ and adaptor sequences,  m to  m clean reads were generated from four biological replicates of and 6 h cold treated libraries, respectively. clean reads were aligning to banana reference genome database. there are also about 60% of plantain clean reads mapped to the reference genome. finally a total of  m to  m uniquely mapped clean reads were used for further analysis. the distribution of unique reads with chromosome ‘+/−’ chain and splice/non-splice in each library were counted and are shown in table  differential expression profiling of cold stress between banana and plantain to comprehensively investigate the differences in gene expression between cold-sensitive banana and cold-tolerant plantain in response to cold stress, we performed comparative transcriptome analysis using the aligned reads. after 3 h of cold stress, a total of, cold-responsive genes were identified in banana and plantain, respectively with the threshold of for corrected p-value and for log base of fold-change. out of the degs, cold-responsive genes were exclusively identified in banana, whereas cold-responsive genes were uniquely observed in plantain. the remaining genes were commonly regulated by cold stress in both banana and plantain. after 6 h of cold stress, a total of, cold-responsive genes were identified in banana and plantain, respectively. out of the degs, cold-responsive genes were exclusively identified in banana, whereas cold-responsive genes were uniquely observed in plantain and genes were commonly regulated by cold stress in both banana and plantain. the small number of cold-responsive genes identified in cold-tolerant plantain suggests that some inherent adaptation and regulation mechanisms may be attributed to the cold tolerance in plantain. the degs with at least a 2-fold change are shown in additional file 4: table s and table  after 3 h of cold stress, up-regulated genes commonly identified in both banana and plantain, include gsmua_achr9g07610_, gsmua_achr6g32910_, gsmua_ achr2g13410_, gsmua_achr6g25670_, gsmua_achr7g26580_ gsmua_achr7g05900_ and gsmua_achr3g23000_. meanwhile, up-regulated genes including gsmua_achr8g21550_ and gsmua_achr3g05220_ and down-regulated gene: gsmua_achr3g04360_ were significantly changed in plantain at 3 hours. interestingly, the above genes with the identical trend of changes were present in banana after 6 h of cold stress, suggesting that those genes be specifically responsive to cold stress, and their early response maybe critical in the cold resistance. in addition, this timely delay of response to cold stress was also found in plantain. out of degs found in banana after 3 h of cold stress, genes with the identical response profiling were present in plantain only after 6 h of cold stress. those genes include gsmua_achr5g21050_, gsmua_achr7g06910_, gsmua_achr3g01650_, gsmua_achr9g04780_, gsmua_achr7g21780_ gsmua_achr3g _, gsmua_achr10g22580_, gsmua_achr 7g21070_, gsmua_achr10g25070_, gsmua_achr7g00180_, gsmua_achr6g13020_, gsmua_achr10g24780_ with up-regulation, and gsmua_achr11g02890_, gsmua_achr7g22100_ and gsmua_achr 8g30530_ with down-regulation. these delayed, expressed genes may be of importance to the adaptation of cold stress for sustainable cold tolerance in plantain.table primary functional classification on differentially expressed genes of dajiao with fold changes > or < 2-fold gene_id rpkm dajiao 0 h rpkm dajiao 3 h rpkm dajiao 6 h log2fold change 3 h log2fold change 6 h gene name regulation of transcription response to stress protein modification signal transduction cell wall organization and biogenesis transport nucleosome assembly iron-sulfur cluster assembly proteolysis oxidation reduction rna modification copper ion homeostasis photosynthesis and photorespiration response to sucrose stimulus unknown there are more degs being identified in plantain for 6 h of cold stress. of which genes include gsmua_achr8g25220_, gsmua_achr 6g15840_, gsmua_achr3g25160_, gsmua_achr11g04410_, gsmua_achr6g14750_, gsmua_ achr5g07340_, gsmua_achr4g33270_, gsmua_achr8g06870_, gsmua_achr7g11200_, gsmua_achr7g01430_, gsmua_achr 2g13700_ gsmua_achr4g11450_, gsmua_ achr6g16830_, gsmua_ achr6g _, gsmua_achr7g27650_, gsmua_achr1g22700_, gsmua_ achr7g12120_, gsmua_achr3g03820_ gsmua_achr2g22400_, gsmua_achr6g10300_, gsmua_ achr2g22290_, gsmua_ achr5g01760_, gsmua_achr 6g35120_. a similar differential profile for those genes was also found in banana for 6 h of cold stress, suggesting they share the similar adaptation mechanism to cold stress. the remaining genes were differentially expressed only in plantain. these genes include gsmua_achr4g16550_, gsmua_achr1g07160_, gsmua_ achr g05390_ gsmua_achr7g16570_, gsmua_achr3g16210_, gsmua_achr3g28910_, gsmua_achr8g05250_ with up-regulation, and gsmua_achr8g20530_, gsmua_ achr7g20770_, gsmua_achr4g24680_, gsmua_achr10g19930_, gsmua_ achr3g26220_, gsmua_achr1g17220_, gsmua_achr 11g11670_, gsmua_achr6g01490_, gsmua_achr9g12290_ and gsmua_achr9g26820_ with down regulation. these genes and their regulations appear likely attributed to the cold tolerance for plantain. besides aforementioned differential genes, more degs were found only in banana for 6 h of cold stress, which are expected to be involved in cold stress for banana only. functional classification of cold stress-related degs the functional classification of degs in banana and plantain was further examined to explore the pattern of transcriptome regulation that occurs during cold stress. genes matching well-characterized proteins or proteins with putative functions were grouped using the gene ontology and summarized in additional file 4: table s and table  the majority of degs identified in this study are in the categories regulation of transcription, response to stress, transport, protein modification, nucleosome assembly, cell wall organization and biogenesis, signal transduction, oxidation reduction, rna modification, cell redox homeostasis, etc. no obvious difference of go classification was observed for those degs from either banana or plantain, despite the fact that few degs in each go category were identified in plantain than in banana. validation of the degs by quantitative rt-pcr analysis plantain leaves showed significant phenotypic difference from banana after 3 h cold treatment at 10°c, we suspect that early response genes of plantain are closely associated with its cold resistance, thus degs of plantain identified in this study after 3 h cold treatment and critical cold-response homologous genes in arabidopsis ice and rice mybs were selected for quantitative rt-pcr analysis. two additional extended time points for and 48 h of cold stress were also measured in parallel with initial and 6 h cold stressed samples in both banana and plantain. the primers of selected genes are listed in additional file 5: table s 25s ribosomal rna gene was used as reference gene for data normalization according to van den berg et al.. the quantitative rt-pcr results from banana and plantain are shown in figure  the expression profiles of all detected genes show the same trend and consistent results between the rt-pcr and the solexa-sequencing methods. for and 48 h of cold stress, out of degs displayed the same down regulation in both banana and plantain, including gsmua_achr7g05900_, gsmua_achr9g07610_, gsmua_achr6g32910_, gsmua_achr7g26580_, gsmua_ achr3g _, gsmua_achr3g23000_. however, of them showed opposite changes between banana and plantain, including gsmua_achr6g25670_, gsmua_achr8g21550_, gsmua_ achr2g13410_ and gsmua_achr 3g05220_. strikingly, there is a remarkable difference of ice and mybs expression profile between plantain and banana in response to cold stress. the ice was significantly down-regulated under cold stress at early stage in banana, but its decrease didn’t appear until at 24 h in plantain. although the mybs was down-regulated similarly at early stage by 9-fold and 4-fold in banana and plantain respectively, at 6 h of cold stress, it can be recovered much quickly in plantain than banana. with the extended cold stress, the plantain mybs was rapidly recovered to the level of 41% and 75%, while the banana mybs was further decreased to 4% at 24 h and then recovered to 24% at 48 h of cold stress. the considerably different expression profiles of the two important transcriptional factors with a remarkable time delayed response in banana versus plantain suggest that the specific time course-based expression of ice and mybs in plantain might be related to its cold tolerance.figure relative mrna levels of degs in banana and plantain seedlings were determined by quantitative rt-pcr analyses. six-leaf stage seedlings were incubated at 10°c for the indicated time. transcript abundances of genes encoding dehydration-responsive element-binding protein 1d, ethylene insensitive 3-like protein, probable cytosolic iron-sulfur protein assembly protein, zinc finger protein, u-box domain- containing protein, zinc finger ccch domain-containing protein, udp-glucose 6-dehydrogenase, probable xyloglucan endotransglucosylase/hydrolase protein, ethylene-responsive transcription factor rap2-, calcium-binding protein kic, transcription factor ice and mybs from both banana and plantain were determined and compared across the time course of cold stress. data represent means ± sd in four replicates. the different lowercase letters labeled above columns indicate a significant difference at p ≤  between the columns by duncan’s test using spss statistical software. the columns with the same letters mean no significant difference between each other. discussion and  in our previous study on the temporal responses of plantain to cold stress using quantitative proteomics analysis, we have revealed that antioxidation mechanisms contribute to cold tolerance in plantain at the global proteome level. however, due to the limitation of current technologies in overcoming the issue of the wide dynamic range in proteomics samples, quantitative proteomics analysis often misses the detection of many low abundance proteins, yielding only 10-30% proteome coverage for any given non-model species. obviously this will reduce the number of low abundance proteins identified during global functional studies. given the above consideration and to focus on the molecular mechanisms involving low abundance genes on the cold-tolerance of plantain, a comparative transcriptomics analysis of cold-sensitive banana and cold-tolerant plantain was conducted by rna-seq and real time rt-pcr with time course of cold treatment. after 48 h of cold stress at 10°c for banana and plantain seedlings, both phenotype and electrolyte leakage analyses clearly indicate that plantain seedlings exhibited a more robust cold tolerance than banana seedlings. it is this phenotypic difference in the cold response between banana and plantain that provides an excellent model system allowing us to study differential gene expression in response to cold stress, as well as to elucidate the potential different cold responsive mechanisms between banana and plantain. rna-seq analysis shows that and degs are identified for 3 h and 6 h of cold stress respectively in plantain, while the equivalent degs are and being identified in banana, indicating banana is much more sensitive to cold stress than plantain. go classification analysis shows the majority of degs identified in this study belong to the following categories: regulation of transcription, response to stress, transport, protein modification, nucleosome assembly, cell wall organization and biogenesis, signal transduction, oxidation reduction, rna modification, cell redox homeostasis, etc. since there is no difference of go classification found between banana and plantain, it suggests that cold stress appears to have a broad range of impacts on cellular activities. however, when we further performed the pathway analysis for some specific degs either found uniquely in plantain particularly or detected at variable cold stress time points in both banana and plantain, we found that many degs and their associated pathways are likely to be involved in cold-tolerance of plantain. below we discuss intensively some of the important pathways and their possible mechanisms associated with the observed cold tolerance in plantain. ice and mybs pathways ice gene was initially identified and isolated from arabidopsis thaliana. the function of atice is to activate gene expression of c-repeat binding factor to enhance the cold resistance of arabidopsis. in oryza sativa, three novel myb proteins containing one highly conserved dna-binding domain mediate sugar and hormone regulation by promoting gene expression of alpha-amylase. recent studies reveal that mybs is also involved in adaptation of cold stress in rice. compared to the ice mybs functions particularly in late stage of plant for adaptation of cold stress. for example, transgenic rice overexpressing mybs can resist cold stress at 4°c for a week. in the early stage of cold response, the transient activation of αamy expression by cbf allows hydrolysis of reserved starch to meet the immediate need for a carbon source and energy to combat the cold shock, while the subsequent suppression of αamy expression by mybs allows rice to conserve carbohydrates until regrowth is allowed at elevated temperatures. in this study using rna-seq analysis for and 6 h of cold stressed samples, although both ice and mybs genes were detected, neither was identified as deg. this is not surprising, because ice requires activation, which is regulated by post-translational modifications via phosphorylation and sumoylation of the siz gene. thus, ice is likely not regulated at the transcriptional level. since mybs plays a role in cold adaptation at late stage of plant growth, mybs is expected not present in early time points from cold stressed samples. in fact, we did identify both genes by rna-seq analysis at a low level with relatively large variation across the replicate samples. given the well-known functions of both ice and mybs genes in plant resistance to cold stress, we decided to use quantitative rt-pcr for assessing their changes with the extended time points at and 48 h of cold stress in this study. the results indicate that although there is no significant change of ice in plantain at and 6 h cold stress, both an upstream gene: gsmua_achr9p04380_ and a downstream gene: gsmua_ achr g05900_ in an ice1-cbf-cor pathway, were up-regulated. since the ice1-cbf-cor is a well-known pathway involved in plant cold-tolerance, the up-regulation of cbf is therefore, expected to facilitate the cold-tolerant pathway. a -fold increase of cor protein found in our proteomics analysis of plantain for 6 h under cold stress at 8°c supports this observation with the ice1-cbf-cor pathway being activated in response to the cold stress in this study. interestingly, at 24 h of cold stress, both plantain ice and cbf were down regulated. meanwhile, the mybs gene had almost completely recovered, which may offset the down-regulated ice1-cbf-cor pathway as a means of sustaining the cold resistance. in banana, however both ice and mybs were significantly decreased under cold stress. the cbf response to cold stress in banana is significantly slower than that in plantain, until 6 h of cold stress, when the banana cbf gene was up-regulated, only to go down again at 24 h of cold stress. consistently, the time delayed response of mybs gene to the cold stress was also observed in banana. however, at 48 h of cold stress, the banana mybs gene appeared recover from its initial cold suppression. based on the observation of slow response to the cold stress in banana for both ice and mybs as the two critical transcriptional factors responsible for early and late stage respectively against cold stress, we suspect that the relatively low expression of these two key genes along with their delayed response to the cold stress are among the main reasons for cold sensitive banana. signals transduction as an important second messenger, ca2+ plays a vital role in the plant cold-stress response. the concentration of ca2+ inside the cell increases rapidly during cold stress, followed by a number of signals mediated by a series of protein phosphorylation cascades. during rice domestication, an amino acid mutant of cold from met187/thr to lys in japonica cultivars was found to enhance cold tolerance partly because it facilitates the formation of an appropriate ca2+ signal by increased ca2+ concentration. interestingly, our study shows that most of the degs involving in signal transduction are indeed related to the calcium-dependent signal pathway, such as orthologs of cipk, kic and cml. all three genes in banana and plantain were up-regulated in response to cold stress. in addition, overexpression of osmsr a novel rice calmodulin-like gene, improves resistance of drought and salt and increases aba sensitivity in arabidopsis. in this work, we found the dramatically increased expression of cml, an ortholog of osmsr in response to cold stress in plantain but not in banana, suggesting that there is a more efficient response of the ca2+ signal transduction pathway in plantain so that it can quickly and effectively regulate downstream signaling and gene expression in response to cold stress. as a signal molecule, auxin plays an important function under various abiotic stress conditions. intriguingly, compared to the control group, the expression of gsmua_achr4g16550_ was increased by fold in 6 h cold treatment in plantain, while no differential expression of the gene was detected in banana. on the plasma membrane, air is able to receive auxin signal to promote lateral root morphogenesis and participates in the decomposition of glucose, while it has been proven that the content of soluble sugar and acid can affect the plant’s cold tolerance. our results indicate that the increased expression of air in plantain could facilitate the growth of plantain root and the content of sugar and acid, which might be attributed to the better resistance to cold. transcription regulation in this study, we have identified differentially expressed transcription factors in response to cold stress in banana and plantain, including nac, erf, dreb, myb,wrky, c3h eil, ado, zfp and rpoa. nac transcription factors were up-regulated in banana and plantain after 6 h cold stress, and over-expression of snac was reported to increase cold and salt resistance in rice, suggesting that nacs are likely to participate in cold response in banana and plantain. effects of ethylene on plants under cold stress have recently been recognized, the erf transcription factors which related to ethylene, were up-regulated in banana and plantain after 6 h cold stress. overexpression of transcription factor terf2/leerf in tobacco and tomato was reported to result in cold tolerance by facilitating ethylene biosynthesis, which supports the important role of erf in plant cold tolerance. dreb transcription factors, with a conservative ap2/erebp domain, are involved in regulation of stress-related gene in response to the external environment, through binding dre cis-acting elements. our data shows that overexpression of dreb occurs at 3 h of cold stress in plantain versus 6 h in banana, suggesting that the early response of dreb may be critical in cold-tolerant plantain. due to a large number of myb transcription factor found in plants such as arabidopsis thaliana and zea mays, an increased amount of research has been focused on its role in transcriptional regulation and its impact on a broad range of physiological functions, e.g., overexpression of rice myb genes enabled to significantly enhanced the cold tolerance of transgenic arabidopsis thaliana. in our work,we found gsmua_achr8g25220_001-a myb transcription factor increased after 6 h cold stress while it was undetectable at 0 h in both banana and plantain, indicating that this gene is a cold-induced gene. the change of this myb gene could affect downstream related genes. in arabidopsis thaliana, the wrky gene family contains genes induced by cold stress, most of which are activated in its early stages. after 6 h cold stress, the wrky genes showed up-regulation, among them, gsmua_achr7g05200_ was overexpressed whereas it was undetectable at 0 h, indicating it is also a cold-induced gene. in plantain, only one cold-induced wrky gene-gsmua_achr6g15840_ was detected after 6 h of cold stress. however, its expression was much higher than that in banana, suggesting that this wrky gene may contribute to the cold-resistance of plantain. it has been reported that zinc-finger proteins are involved in resistance to adversity in plant. for example, gmzf from soybean was found to enhance the tolerance of arabidopsis to cold stress by expression of cold-regulation genes in the transgenic arabidopsis. strikingly, banana zfp is not up-regulated until 24 h of cold stress and starts to decrease at 48 h, while plantain zfp is constantly up-regulated beyond 48 h of cold stress, suggesting zfp may play a role in late stages of cold stress. expression of eil in plantain reached ~11-fold higher induction at 3 h cold treatment and then decreased back to initial levels at 48 h. in banana, the eil was up-regulated by ~9-fold at 6 h and further increased to ~13-fold at 48 h of cold treatment. as a primary transcription factor in ethylene signal transduction, eil regulates transcription of downstream genes to complete the ethylene response. given the higher expression of eil at very early stages and its rapid decrease in response to cold stress in plantain, we suspect that rapid and early activation of the ethylene signal transduction pathway appears to play a critical role in the rapid response to cold stress allowing early protection in plantain. genes associated with stress response plants often activate similar pathways in response to different types of abiotic stress. in this cold stress study, we found quite a few genes related to stress response in both banana and plantain, such as sthy, p2c, ubiquitin-protein ligase genes, thil-, grp and drt, etc. those genes showed similar patterns of expression in response to cold treatment in banana and plantain, suggesting the stress response genes share some common response pathways in both species. however, it should be noted that some of the degs are located in the chloroplast of plantain only, such as elip, ctl sca and mks in rhododendron catawbiens, expression of rcelip resulted in adaptive responses to cold and high light in winter-adapted rhododendron leaves, suggesting a critical role of elip in protection of photosynthetic apparatus from these stresses. in arabidopsis thaliana, ctl is involved in altering the architecture of the root system in response to multiple environmental conditions. as an map kinase substrate, arabidopsis mks may be involved in mpk4-regulated activation of plant defense by coupling the kinase to specific wrky transcription factors. due to the expression of elip, ctl sca and mks induced by cold treatment exclusively in plantain, we believe that plantain possesses the highly superior aspects of photosynthesis, root architecture and plant defense regulated by mpk, which makes plantain broadly resist to abiotic stress resulting in a better cold tolerance. other degs involved in functional regulations in addition to the above four categories, we found some degs in banana and plantain are also involved in protein/rna modification, cell wall composition and occurrence, transit, nucleosome assembly, cellular redox balance, iron-sulfur cluster assembly, protein hydrolysis, oxidation and reduction, copper ion balance, photosynthesis, respiration and sugar stimulus response. but again some of the cold responsive degs are found exclusively in plantain, including mtp involved in copper ion balance, rbcs functioning in photosynthesis and respiration in chloroplast, cp involved in sugar stimulus response in chloroplast, and ubq functioning in protein modification. in yeast, putmt has been verified to play a critical role in improving tolerance to metal and reactive oxygen species. thus the up-regulation of mtp would allow plantain for improved tolerance to the increased reactive oxygen species due to the cold stress. in plant, rbsc encodes the small subunit of rubisco involved in the first major step of carbon fixation and the rbsc gene family functions to yield sufficient rubisco content for leaf photosynthetic capacity. as the expression of rbcs was highly repressed in plantain, we speculate that the protection mechanism is activated in response to cold treatment in chloroplast in order to decrease the production of ros through the weakened photosynthesis. arabidopsis cp forms a complex with gapdh and prk in regulating tca cycle. a dramatic repression of cp suggests that plantain must reduce the photosynthesis in response to cold stress. this is consistent with the decreased rbcs but as another pathway for the self-protection mechanism. ubq is involved in protein hydrolysis in the ubiquitin-proteasome pathway. in order to compensate cells for ubiquitin molecules in signal transmission, expression of poly-ubiquitin gene is increased in response to stress. given the increased expression of ubq in response to cold treatment, we suspect that ubq might be involved in stress response in plantain. in summary,a global, comparative transcriptomic profile of banana and plantain in response to cold treatment has been intensively investigated by transcriptome sequencing and quantitative rt-pcr. at the transcriptomic level, both plantain and banana share similar profiles for the majority of degs in time course response to cold treatment. many of the degs have been reported and involved in the development of cold resistance or other stress resistance in plant, such as: cipk, kic, cml, nac, erf, dreb, myb, wrky, c3h ado rpoa, sthy, p2c ubiquitin-protein ligase genes, thil- gr and drt, etc. despite the similar profiles of degs found in banana and plantain, several dozen degs involved in several different pathways were successfully identified and validated only in cold-tolerant plantain. for example, the cold-tolerant ice and mybs pathways were rapidly activated and switched in early and late stages respectively, of response to cold treatment in plantain. while in cold-sensitive banana, no such equivalent regulation for ice1-cbf-cor pathway was found and slow response to mybs pathway responsible for the cold stress was observed. thus, the low expression of the two key genes along with their delayed response to the cold stress may be one of the main reasons for cold sensitivity of banana. the cold-tolerant plantain also appears closely related to expressions of several specific genes such as cml air, zfp eil, elip, ctl sca, mks, mtp, rbcs, cp and ubq. combined with previous research, we rationalize that in early stage of cold stress response, changes in cell membrane phase trigger modification of actin cytoskeleton, which activates ice1-cbf-cor metabolic pathways by ca2+ and phosphorylation signaling pathways. also it triggers an early response via the ethylene signal transduction pathway. in late stage of cold stress, expression of mybs begins to recover. mybs is involved in coordination with the expression of multiple genes for regulation of oxidation/reduction, oxylipin biosynthetic process, photosynthesis, photorespiration, glycolysis, tricarboxylic acid cycle, carbohydrate metabolic process, fatty acid biosynthetic process and beta-oxidation, enhanced adoption to cold tress. the next questions need to be answered are what causes the observed different expression profiles between ice and mybs pathways in plantain? how do both coordinate together for effectively regulating cold-resistance? addressing these questions will further enhance the understanding on signaling and metabolic pathways of cold tolerance in plantain, but also conducive to nurturing new varieties of cold tolerant banana cultivars.figure a schematic diagram of cold-tolerance transcriptional network in plantain, adapted initially from v. chinnusamy et al. and c.f. su et al. and revised based on this study. at the early stage of cold stress, plantain cells probably sense low temperatures through membrane rigidification and/or other cellular changes, which might induce a calcium signature and activate protein kinases necessary for cold tolerance. constitutively expressed ice is activated by cold stress through sumoylation and phosphorylation. sumoylation of ice is critical for ice1-activation of transcription of cbfs and repression of myb cbfs regulate the expression of cor genes that confer cold tolerance. the expression of cbfs is negatively regulated by myb hos mediates the ubiquitination and proteosomal degradation of ice and, thus, negatively regulates cbf regulons. cbfs can constitutively regulate the expression of downstream cold-responsive transcription factor genes raps, which might control sub-regulons of the cbf regulon. cbfs also activate αamy expression to hydrolyse reserved starch. at the late stage of cold stress,mybs inhibits cbfs and αamy expression. the effective coordination across the early and late stages of cold stress by at least two different regulatory pathways appears to efficiently regulate the following metabolic pathways including oxidation reduction, oxylipin biosynthetic process, photosynthesis, photorespiration, glycolysis, tricarboxylic acid cycle, carbohydrate metabolic process, fatty acid biosynthetic process and beta-oxidation. the rapid activation and selective induction of ice and mybs cold tolerance pathways in plantain, along with expression of other cold-specific genes, may be one of the main reasons that plantain has higher cold resistance than banana. broken arrows indicate post-translational regulation; solid arrows indicate activation, whereas lines ending with a bar show negative regulation; the two stars indicate unknown cis-elements. p, phosphorylation; s, sumo; u, ubiquitin. 
we conclude that the rapid activation and selective induction of ice and mybs cold tolerance pathways in plantain, along with expression of other cold-specific genes, may be one of the main reasons that plantain has higher cold resistance than banana. the results show that and differentially expressed genes are identified for and 6 h of cold stress respectively in plantain, while and degs are identified respectively in banana. to understand the molecular mechanism of the cold-tolerance in plantain we used rna-seq based comparative transcriptomics analyses for both cold-sensitive banana and cold-tolerant plantain subjected to the cold stress for and 6 h. twelve early responsive genes including ice and mybs were selected and further assessed and confirmed by qpcr in the extended time course experiments, which revealed significant expression difference of key genes in response to cold stress, especially ice and mybs between cold-sensitive banana and cold-tolerant plantain. low temperature is one of the key environmental stresses which greatly affects the global banana production. there are degs found uniquely in cold-tolerance plantain, which were involved in signal transduction, abiotic stress, copper ion equilibrium, photosynthesis and photorespiration, sugar stimulation, protein modifications etc. a similar profile for degs was found in both banana and plantain for 6 h of cold stress, suggesting both share some common adaptation processes in response to cold stress. we found that the cold-tolerance pathway appears selectively activated by regulation of ice and mybs expression in plantain under different stages of cold stress. the cold-response genes at early stage are identified and grouped in both species by go analysis. go classification analyses show that the majority of degs identified in both banana and plantain belong to categories including regulation of transcription, response to stress signal transduction, etc.
 banana and plantain comprise an important part of diets for millions of people around the globe. low temperature is one of the key environmental stresses which greatly affects the global banana production. to understand the molecular mechanism of the cold-tolerance in plantain we used rna-seq based comparative transcriptomics analyses for both cold-sensitive banana and cold-tolerant plantain subjected to the cold stress for and 6 h.  the cold-response genes at early stage are identified and grouped in both species by go analysis. the results show that and differentially expressed genes are identified for and 6 h of cold stress respectively in plantain, while and degs are identified respectively in banana. go classification analyses show that the majority of degs identified in both banana and plantain belong to categories including regulation of transcription, response to stress signal transduction, etc. a similar profile for degs was found in both banana and plantain for 6 h of cold stress, suggesting both share some common adaptation processes in response to cold stress. there are degs found uniquely in cold-tolerance plantain, which were involved in signal transduction, abiotic stress, copper ion equilibrium, photosynthesis and photorespiration, sugar stimulation, protein modifications etc. twelve early responsive genes including ice and mybs were selected and further assessed and confirmed by qpcr in the extended time course experiments, which revealed significant expression difference of key genes in response to cold stress, especially ice and mybs between cold-sensitive banana and cold-tolerant plantain.  we found that the cold-tolerance pathway appears selectively activated by regulation of ice and mybs expression in plantain under different stages of cold stress. we conclude that the rapid activation and selective induction of ice and mybs cold tolerance pathways in plantain, along with expression of other cold-specific genes, may be one of the main reasons that plantain has higher cold resistance than banana. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords comparative transcriptome analysiscold tolerancebananaplantainice1mybs pathwaysissue-copyright-statement© the author 2015 