BACKGROUND
height of cultivated banana generally exceeds 2 m, and its false stem is easily broken in typhoon-frequented areas.
the stocky build of dwarf banana varieties can resist typhoon damage to a certain extent and offers the advantages of cultivation convenience, field management, labor savings, close planting, and so on.
the dwarf mutant is a useful material for excavating and researching dwarf-related genes.
identification and utilization of banana dwarf-related genes are of considerable significance in breeding dwarf banana varieties.
we obtained the dwarf mutant ‘8818-1’ through ems mutagenesis of williams banana  <dig>  the stature of the 8818- <dig> false stem is approximately  <dig>  m. williams 8818- <dig> is stronger, bears shorter fruits, and presents dwarf characteristics in comparison with its parent,  <dig>  previous studies reveal that hormone-deficient dwarf mutants can be restored by application of the corresponding exogenous active hormones in which the active hormone biosynthesis pathway is inhibited or blocked .
while dwarf mutants may become hormone-insensitive because of problems in hormone signal absorption, transfer, metabolic regulation genes, application of the corresponding exogenous active hormone can‘t restore the dwarf type  <cit> .
total gas contents in the false stem of williams banana dwarf mutant 8818- <dig> are significantly lower than those in its parent  <dig>  and the plant stature of 8818- <dig> can be restored by application of exogenous active gibberellin ga <dig>  we thus speculate that 8818- <dig> may be a hormone-deficient dwarf mutant.
gas perform fundamental functions in plant growth and development, participating in the regulation of numerous developmental processes, such as seed germination  <cit> , stem elongation  <cit> , leaf stretching  <cit> , flower induction  <cit> , and fruit-setting  <cit> .
reduction of active gas content causes plants to exhibit the dwarf phenotype.
ga biosynthesis pathway is well elucidated in model plants, and their related mutants have been isolated  <cit> .
gas are biosynthesized from geranyl diphosphate, a common c <dig> precursor for diterpenoids.
biosynthesis enzymes, including ent-copalyl diphosphate synthase , ent-kaurene synthase , ent-kaurene oxidase , ent-kaurenoic acid oxidase , ga 20-oxidase, ga 3-oxidase, and ga 2-oxidase  <cit> , may be classified as terpene synthases , including cps and ks, cytochrome p <dig> monooxygenases , including ko and kao, and 2-oxoglutarate–dependent dioxygenases , including ga20ox, ga3ox, and ga2ox.
cps, ks, ko, and kao enzymes involved in the early steps of the ga metabolism pathway are usually encoded by a single or few genes  <cit> .
their mutants display severe dwarfism and loss of fertility, which can be recovered after spraying with exogenous active gas .
although multiple homologous genes are present in numerous plants, only one of these genes participates in the ga metabolism pathway.
for instance, the rice oscps and osks-like gene families consist of  <dig> and  <dig> members, respectively, but only oscps <dig> and osks <dig> are responsible for ent-kaurene biosynthesis  <cit> .
ga20ox, ga3ox and ga2ox are three enzymes that catalyze later reactions in the ga biosynthesis pathway and belong to the 2og-fe  oxygenase superfamily.
in numerous plant species, the enzymes are independently encoded by different gene families  <cit> , thus accounting for certain functional redundancy, as well as tissue specificity  <cit> .
the loss of function of these ga oxidase genes  in plants can generate a dwarf phenotype, which is restored by the application of exogenous ga .
for instance, the well-known green revolution gene, sd- <dig>  is generated from loss of function in osga20ox <dig> of rice  <cit> .
by contrast, ga2ox decreases levels of active gas in plants, and overexpression of ga2ox genes can lead to dwarf types  <cit> .
ga metabolism genes have been identified in fungi, bacteria  <cit> , arabidopsis , rice  <cit> , maize  <cit> , soybean  <cit> , pumpkin  <cit> , pea  <cit> , cucumber  <cit> , grapevine  <cit> , brachypodium  <cit> , bread wheat  <cit> , and salvia miltiorrhiza  <cit> , among others.
most publications focus on the systematic evolutionary analysis of the ga oxidase gene family in these plants, and gene functional research on individual pathway member from several plants has been conducted.
previous results have shown that rice  possesses  <dig> ga20ox,  <dig> ga3ox, and  <dig> ga2ox genes; arabidopsis possesses  <dig> ga20ox,  <dig> ga3ox, and  <dig> ga2ox genes; and soybean  contains  <dig> ga20ox,  <dig> ga3ox, and  <dig> ga2ox genes  <cit> .
these ga oxidase genes exhibit a unique expression pattern and perform distinct developmental functions in different organs, tissues, and developmental stages of plants  <cit> .
for instance, atga3ox <dig> and atga3ox <dig> are responsible for bioactive ga biosynthesis during vegetative growth, while atga3ox <dig>  atga3ox <dig>  and atga3ox <dig> are important for the development of reproductive organs  <cit> .
among the  <dig> atga20ox genes, atga20ox <dig>  atga20ox <dig>  and atga20ox <dig> are the dominant paralogs  <cit> .
atga20ox <dig> is functionally redundant with atga20ox <dig> and atga20ox <dig>  whereas atga20ox <dig> and atga20ox <dig> perform minor roles in most developmental stages  <cit> .
differential expression and distinct developmental functions have also been observed in rice  <cit> .
moreover, the transcription levels of several, but not all, ga metabolism genes are under feedback control .
control includes inhibition of the expression levels of several ga20ox and ga3ox genes, as well as activation of several ga2ox genes  <cit> .
banana a genome sequencing was completed in  <dig>  <cit> , but related information on ga metabolism in banana is limited.
the numbers of ga metabolism genes in the banana a genome and their phylogenetic evolution, function, tissue specificity, and timing of expression have neither been verified nor explored.
to understand the distribution and system evolution of ga metabolism genes in banana a genome, we searched all ga metabolism genes in the banana genome hub and the national center for biotechnology information .
preliminary analyses of the system evolution of these genes have laid the foundation for research on banana ga metabolism genes.
the expression levels of ga metabolism genes in williams banana  <dig> and 8818- <dig> and the principal genes regulating gas content remain unknown.
to elucidate possible causes of the 8818- <dig> dwarf phenotype, we analyzed tissue specificity and compared the gene expression differences in seven kinds of genes encoding early ga biosynthesis genes and ga oxidase genes between  <dig> and 8818- <dig>  these results improve our current understanding of the ga metabolism pathway in banana and contribute to research in other closely related species with significant agricultural importance.
RESULTS
gas content analysis and exogenous ga <dig> application treatment
in the field, the adult 8818- <dig> plant presented stronger, shorter false stems and shorter fruits in comparison with the parent  <dig> .
total gas content was determined in different tissues of williams  <dig> and its mutant, 8818- <dig>  the results are shown in fig.
1b.
in addition to that in leaves, the total gas contents in most tissues of 8818- <dig> were lower than those in  <dig> during different developmental stages.
total gas contents of false stems during the young and adult development stages in  <dig> were 113 % and 145 % higher than those in 8818- <dig>  respectively.
total gas contents of young fruits and roots in  <dig> were also significantly higher than those in 8818- <dig>  either during adulthood or the seedling stage, the total gas content of 8818- <dig> false stems was significantly lower than that of  <dig>  gas have several forms and many of them are inactive and intermediates, and only few are active forms, namely ga <dig>  ga <dig> and ga <dig>  so contents of ga <dig>  ga <dig> and ga <dig> were determined in false stems of  <dig> and 8818- <dig> .
the results showed that ga <dig> was the highest content active ga and the three kinds of active gas content of false stems in 8818- <dig> were all lower than those in  <dig>  among them the difference of ga <dig> content between  <dig> and 8818- <dig> was significant.
false stems are closely related to plant stature; therefore, 8818- <dig> is significantly shorter than  <dig>  which may be due to a decline in gas content in the former, especially ga <dig> content.fig.
<dig> phenotypes and gibberellins levels of banana mutant 8818- <dig> and its wide type.
a comparison of the plant height between  <dig> and 8881- <dig> in the harvest period.
b total gas contents between  <dig> and 8818- <dig> in different tissues at different ages.
c active gas  contents in false stems of  <dig> and 8818- <dig>  significant difference of total gas contents for each tissue and active ga contents for each ga between  <dig> and 8818- <dig> estimated by t-test was reported on the graphics .
stars  indicate significant differences of total gas content between the same organ of  <dig> and 8818- <dig>  or between the same active ga of  <dig> and 8818- <dig>
exogenous ga <dig>  application was conducted on 8818-1; in this experiment, water was used as a control.
results suggested that treatment with all three concentrations could restore the plant height of 8818- <dig> to  <dig> levels or even higher .
ga <dig> exerted a dose-dependent effect on 8818-1; the higher the concentration, the more rapidly the false stems elongated within the scope of 50–200 mg/l ga <dig> fig.
<dig> effect of exogenous ga <dig> treatments on plant height of 8818- <dig> with different concentrations.
each value was the mean of ten biological replicates with the standard error indicated and evaluated by duncan’s test .
means labeled by the same letter are not significantly different
considering the results of gas content determination and plant height recovery, we can speculate that the dwarfism of 8818- <dig> may be caused by reduction of gas content in false stems.
isolation of putative ga metabolism genes in banana
to identify the genes encoding seven kinds of ga metabolism enzymes in the banana a genome, we screened all available banana amino acid sequences in the banana genome hub and ncbi.
the banana a genome was sequenced and published in  <dig>  the sequenced genotype is a doubled-haploid  from the musa acuminata  subsp.
malaccencis dh-pahang  <cit> .
three cps-like genes ,  <dig> ks-like genes ,  <dig> kao-like genes ,  <dig> ko-like gene ,  <dig> ga20ox-like genes ,  <dig> ga3ox-like genes , and  <dig> ga2ox-like genes  were searched.
in the banana a genome,  <dig> candidate genes were distributed across all  <dig> banana chromosomes and  <dig> random chromosome .
we named the genes according to their position in the chromosome.table  <dig> gibberellin metabolism genes and their homologs in banana a genome
macps1
macps2
macps3
maks1
maks2
mako
makao1
makao2
maga20ox1
maga20ox2
maga20ox3
maga20ox4
maga20ox5
maga20ox6
maga20ox7
maga20ox8
maga20ox9
maga20ox10
maga3ox1
maga3ox2
maga3ox3
maga3ox4
maga3ox5
maga2ox1
maga2ox2
maga2ox3
maga2ox4
maga2ox5
maga2ox6
maga2ox7
maga2ox8
maga2ox9
maga2ox10
maga2ox11
maga2ox12
maga2ox13
maga2ox14
maga2ox15
early ga biosynthesis genes
we searched two cps-like complete cdna sequences  and one cps-like  fragment sequence in the banana genome hub and then searched the complete cdna sequence of macps <dig> in ncbi.
the three genes were all located in chromosome  <dig>  macps <dig> presented  <dig>  and  <dig>  % identities with macps <dig> and macps <dig>  respectively, and macps  <dig>   <dig>  and  <dig> showed  <dig> ,  <dig> , and  <dig>  % identities with oscps .
in ncbi, blast analysis revealed that macps  <dig>   <dig>  and  <dig> showed the highest similarity to the cps of phoenix dactylifera, as well as  <dig>   <dig>  and 76 % identities with pdcps, respectively.
two maks-like complete cdna sequences were searched in ncbi.
both sequences were located on chromosome  <dig> and shared  <dig>  % identity.
in ncbi, maks-like revealed the highest similarity to ks of elaeis guineensis  but shared only  <dig>  and  <dig>  % identity with osks .
in ncbi and the banana genome hub, we found only one mako-like gene, which was located in chromosome  <dig>  sharing the highest similarity to ko of phoenix dactylifera  and  <dig>  % identity with osko/cyp701a .
two makao-like genes were located in chromosomes  <dig> and  <dig> shared  <dig>  % identity with each other, maximum similarities to kao of phoenix dactylifera , and  <dig>  and  <dig>  % identity with oskao/cyp88a <dig> , respectively.
ga oxidase genes
ga20ox, ga3ox, and ga2ox are three enzymes that catalyze later reactions in the ga biosynthesis pathway.
these enzymes belong to the 2og-fe  oxygenase superfamily and are encoded by a multigene family  <cit> .
ten ga20ox-like genes were found in the banana a genome; in comparison,  <dig> and  <dig> copies of ga20ox genes have been reported in arabidopsis and rice, respectively  <cit> .
ten ga20ox-like genes were located on chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> .
in rice, osga20ox <dig> is reported as the rice green revolution gene and is previously known as semi-dwarf <dig>   <cit> ; loss of function of osga20ox <dig> can generate the dwarf phenotype.
the deduced amino acid sequence of banana maga20ox <dig>  showed the highest homology with osga20ox2/sd <dig> ; by comparison, maga20ox <dig>  revealed only  <dig>  % identity with the gene.
five ga3ox-like genes were searched in the banana genome hub.
however, four ga3ox genes were searched in ncbi.
four ga3ox-like genes in the banana genome hub respectively matched four ga3ox genes searched by blast in ncbi.
meanwhile, maga3ox <dig>  showed 100 % identity with banana ga20ox genes by blast x in ncbi.
phylogenetic analysis also revealed that maga3ox <dig> was grouped as a single clade and possessed a distant genetic relationship with the ga3ox genes of rice, maize, and arabidopsis.
therefore, the annotation of gsmua achr1p <dig> in the banana genome hub should be revised.
in comparison, two and four copies of ga3ox genes have been reported in arabidopsis and rice, respectively  <cit> .
genetic evidence from the d <dig> mutant  proves that osga3ox <dig> is essential and that loss of function of osga3ox2/d <dig> can generate the dwarf phenotype.
four ga3ox-like genes  showed  <dig> ,  <dig> ,  <dig> , and  <dig>  % identities with this gene.
fifteen ga2ox-like genes were searched in the banana a genome.
by comparison,  <dig> and  <dig> copies of ga2ox genes have been reported in arabidopsis and rice, respectively  <cit> .
fifteen ga2ox-like genes were distributed to the rest of the chromosomes, except for chromosomes  <dig>   <dig>   <dig>  however, blast x in ncbi revealed that maga2ox <dig>  shared 100 % identity with the musa acuminata probable 2-oxoglutarate-dependent dioxygenase gene.
phylogenetic analysis of ga oxidase genes showed that maga2ox <dig> presented a distant genetic relationship with other ga2ox genes.
thus, we speculate that maga2ox <dig> belongs to the 2og-fe  oxygenase superfamily and not the ga2ox family.
analyses of phylogenetic tree and conserved protein domains of ga metabolism genes in banana and other plants
early ga biosynthesis genes
phylogenetic analysis of diterpene cyclases  and cyt p <dig> monooxygenases   amino acid sequences from banana, rice, maize, soybean, and arabidopsis  revealed that cps, ks, ko, and kao proteins could be divided into monocot and dicot groups.
this finding is consistent with banana, rice, and maize which are monocot plants.
the monocot group was subdivided into two subgroups; rice and maize were grouped in the same clade, whereas banana presented a distant genetic relationship with rice and maize among monocot plants.
in ncbi, blast analysis showed that elaeis guineensis and phoenix dactylifera shared the highest similarity to banana.
phylogenetic analysis revealed that three cps-like proteins were highly similar and grouped in the same clade; moreover, two ks-like and kao-like which belonged to cyt p <dig> monooxygenases were grouped in the same clade .fig.
<dig> analysis of phylogenetic relationships and conserved protein motifs among ga metabolism genes.
a early ga biosynthesis genes .
b ga oxidase genes .
ma, musa acuminata; at, arabidopsis thaliana; os, oryza sativa; gm, glycine max; zm, zea mays.
the accession numbers of protein sequences cited in this study are in additional file 2
analysis of conserved domains  revealed that all cps possessed motifs  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> in common, whereas ks owned motifs  <dig>   <dig>   <dig>  and  <dig>  we thus speculate that protein domains  <dig>   <dig>   <dig>  and  <dig> are specific to the diterpene cyclases.
cps differed from ks by possessing conserved motifs  <dig> and  <dig>  kao only contained conserved motifs  <dig>   <dig>   <dig>  and  <dig>  which suggested evolutionary conservation.
ko only possessed motif  <dig>  which could be common in all cyt p450-dependent monooxygenases.
ga oxidase genes
to identify the evolutionary relationships of the ga oxidase genes in banana, arabidopsis, and rice, we constructed multiple sequence alignments based on the ga20ox, ga3ox, and ga2ox protein sequences of banana, arabidopsis, and rice .
an evolutionary tree was established according to the alignment results by using the neighbor joining  method .
phylogenetic analysis showed that most ga oxidase genes could be mainly separated into four subgroups .
subgroups i, ii, and iii clearly corresponded to differences among the functions of ga20ox, ga3ox, and ga2ox.
ga20ox and ga3ox can promote the production of active ga, whereas ga2ox inactivates ga, thereby regulating ga content in plants  <cit> .
the phylogenetic tree revealed that the ga oxidases of rice, arabidopsis, and banana were more similar to their respective homologs within each subgroup than to each other.
this finding indicated that expansion of ga oxidase genes occurred early in the evolution of this protein family.
ga3ox belonged to a smaller gene family than ga20ox and ga2ox.
four, two, and four copies of ga3ox genes were discovered in arabidopsis, rice, and banana, respectively.
by contrast,  <dig>   <dig>  and  <dig> copies of ga20ox genes and  <dig>   <dig>  and  <dig> copies of ga2ox genes were discovered in arabidopsis, rice, and banana, respectively.
this finding indicated that the ga3ox gene family was more conserved than the ga20ox and ga2ox families.
moreover ga20ox and ga3ox were separated by a relatively small distance , whereas ga2ox was located farther from these genes.
several homologous sequences of ga20ox and ga2ox showed low sequence identity, and certain branches disclosed a pronounced divergence and did not cluster together.
six maga2ox genes  didn’t appear in subgroups i, ii, and iii.
these genes constituted a separate branch with osga2ox <dig>  osga2ox <dig>  osga2ox <dig>  osga2ox <dig>  atga2ox <dig>  and atga2ox <dig>  showing less similarity to other ga2ox proteins.
previous results have verified that osga2ox <dig>  osga2ox <dig>  osga2ox <dig>  osga2ox <dig>  atga2ox <dig>  and atga2ox <dig> belong to c <dig> ga2ox  <cit> .
thus, six maga2ox genes may also belong to c <dig> ga2ox.
c <dig> ga2ox was found to hydroxylate c20-ga precursors  but not c19-gas, thus decreasing active ga levels  <cit> .
for instance, osga2ox <dig> have been verified to inactivate bioactive ga <dig>  thereby repressing cell growth  <cit> , similar to members in subgroup iii.
overexpression of wild-type or modified c <dig> ga2ox in rice can produce a semi-dwarf type, increase root systems, and higher tiller numbers  <cit> .
c <dig> ga2ox split from c <dig> ga2ox in the phylogenetic tree , but the key functional regions of coding sequences in ga oxidase were less variable .
c <dig> ga2ox exists not only in rice, arabidopsis, and banana but also in other plants, such as soga2ox <dig> from spinach  <cit>  and gmga2ox <dig> from soybean  <cit> .
in banana, six c <dig> ga2oxs are found, which suggests that c <dig> ga2ox may be widespread in plant ga metabolism.
moreover, several ga oxidases, such as osga20ox <dig>  osga20ox <dig>  osga20ox <dig>  osga20ox <dig>  maga20ox <dig>  and maga20ox <dig>  didn’t appeared in the four subgroups and were not clustered together with ga20ox, which implies ga20ox genes may have more complicated evolution.
protein domains  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> were in common in most ga20ox, ga2ox, and ga3ox genes.
we found that protein domain  <dig> was unique to subgroup i and subgroup iii exclusively possessed protein domains  <dig> and  <dig>  protein domain  <dig> was exclusively contained by c20-ga2ox, and subgroup ii possessed no special protein domain, suggesting greater conservation in evolution.
protein domain  <dig> only existed in subgroups i and ii; this domain was lacking in subgroup iii and c20-ga2ox.
c20-ga2ox didn’t possess protein domain  <dig> which existed in subgroups i, ii, and iii.
these special motifs may account for the function difference.
in three kinds of ga oxidase genes, the numbers of genes of ga20ox and ga2ox were greater than that of ga3ox and these genes possessed considerably longer branches in the phylogenetic trees.
these findings indicated that ga20ox and ga2ox evolved more rapidly than ga3ox.
ga20ox and ga2ox demonstrated more dynamic evolutionary routes, thereby resulting in greater functional redundancy.
in addition, more copies of ga20ox and ga2ox could cause relaxed selective pressure or loosened constraints in the evolution process.
subgroups i, ii, and iii contained both monocot and dicot proteins.
this evolutionary relationship suggests that every subgroup of ga20ox/ga3ox/ga2ox proteins may perform homologous functions crossing between monocot and dicot plants  <cit> .
tissue specificity analysis of ga metabolism genes in williams banana
quantitative real-time polymerase chain reaction  analysis revealed that the isolated ga metabolism genes were expressed at different levels in various tissues of williams banana 8818- <dig> .fig.
<dig> tissue-specific expression of the ga metabolism genes in various tissues of banana dwarf mutant 8818- <dig>  total rnas were isolated from leaves , bracts , false stems , roots , young fruits , and approximately mature fruits  in growth and development period, and qrt-pcr was conducted.
relative expression value was calculated using ‘actin’ as a reference by 2-ΔΔct method.
expression level of macps <dig>  maga3ox <dig>  maga20ox <dig> and maga2ox <dig> in the root were respectively defined as  <dig> in a, b, c and d. fold change of the target gene in every tissue, normalized to actin and relative to the expression of macps <dig>  maga3ox <dig>  maga20ox <dig>  maga2ox <dig> in the root respectively in a, b, c and d, was calculated for each sample using the 2-ΔΔct method.
the ct value of each gene was the average of three technical replicates with the standard error indicated.
significant difference of relative expression value in different tissues of the same target gene estimated by duncan’s test was reported on the graphics .
means labeled by the same letter are not significantly different
macps <dig>  maks <dig>  mako <dig>  and makao <dig> were broadly expressed at different levels in all tested tissues of williams banana 8818- <dig>  including leaves, roots, false stems, bracts, young fruits, and approximately mature fruits .
the expression level of makao <dig> gene in different tissues was generally higher than those of the three other genes in the corresponding tissues.
the expression level of makao <dig> was the highest in the bract, followed by leaves, false stems, and young fruits.
the highest gene expression levels of macps <dig> and maks <dig> were observed in bracts, whereas the highest level of mako <dig> expression was found in young fruits.
as a whole, expression level of makao <dig> in all tissues was the highest among the early ga biosynthesis genes tested, while difference among other three genes expression levels in all tissues was small, thus suggesting that makao <dig> might play an important regulating role in transcription level in ga biosynthesis of the banana.
analysis of four ga3ox-like genes  revealed that they were expressed at different levels in six tissues .
maga3ox <dig> expression levels were higher in young fruits and bracts but lower in approximately mature fruits.
compared with maga3ox <dig> and maga3ox <dig>  maga3ox <dig> and maga3ox <dig> were present at lower expression levels.
the relative expression level of maga3ox <dig> in young fruits was the highest among six tissues, but the relative expression value remained below  <dig> , similar to the relative expression value of maga3ox <dig>  in all tissues not including roots.
maga3ox <dig> was strongly expressed in young fruits, bracts, and leaves by 22-fold, 18-fold, and 16-fold, respectively, compared with that of maga3ox <dig> in roots.
while maga3ox <dig> was weakly expressed in the roots, false stems, and approximately mature fruits.
the maga3ox <dig> and maga3ox <dig> of four ga3ox-like genes may be the key genes regulating ga content in the normal development of banana.
however, different genes perform different functions in various tissues.
maga20ox <dig>   <dig>  and  <dig> showed relatively low expression and revealed less obvious tissue specificity in vegetative tissues.
by contrast, other genes exhibited high expression in several tissues at least .
maga20ox <dig> showed relatively high expression in leaves, bracts, and young fruits but low expression in roots, false stems, and approximately mature fruits.
the expression level of maga20ox <dig> was relatively high in all tested plant tissues, presenting the highest expression in young fruits and the lowest expression in leaves.
maga20ox <dig> was prominently expressed in leaves, false stems, and approximately mature fruits and lowly expressed in roots.
maga20ox <dig> was also expressed in all tissues, and showed extremely high levels in young fruits and extremely low levels in roots.
these results reveal obvious tissue specificity.
maga20ox <dig> and maga20ox <dig> expression levels were relatively similar, showing evident tissue specificity, particularly high expression in young fruits and low expression in roots, false stems, and approximately mature fruits.
maga20ox <dig> expression was higher than those of maga20ox <dig>   <dig> and  <dig> but lower than those of abundant genes, such as maga20ox <dig>  maga20ox <dig>  and so on.
tissue specificity among these genes was evident.
in general, young fruits contained abundant genes, except maga20ox <dig> and maga20ox <dig>  and other maga20ox genes all demonstrated maximum expression levels in young fruits.
fourteen maga2ox genes could generally be divided into two categories.
genes included in the first category were strongly expressed in most tissues and expressed differently in most tissues.
this group included maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox <dig>  genes in the second category were weakly expressed and slightly high expression in individual tissues.
this group included maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox <dig>  maga2ox <dig> demonstrated the highest expression level in roots, followed maga2ox14; other maga2ox genes were weakly expressed.
in false stems, maga2ox <dig> was the most abundant gene, although maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox <dig> were also strongly expressed.
other maga2ox genes were weakly expressed.
maga2ox <dig> showed the highest expression in leaves, followed by maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox8; by contrast, maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox <dig> were lowly expressed in this tissue.
in bracts, the top three most abundantly expressed genes included maga2ox <dig>  maga2ox <dig>  and maga2ox <dig>  numbers of high-expression genes in young fruits exceeded those in other tissues.
maga2ox <dig> was the most high- expression gene, followed by maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox <dig>
in approximately mature fruits, highly expressed maga2ox genes were few.
maga2ox <dig> was the most strongly expressed gene, followed by maga2ox14; all other genes were weakly expressed.
the analysis described above reveals that genes expressed strongly in most tissues, namely, maga2ox <dig>  maga2ox <dig>  and maga2ox <dig>  and so on, may be the key genes regulating ga content.
higher expression levels of genes were found in false stems, bracts, leaves, especially in young fruits, and such expression was of considerable significance for regulating ga contents in these tissues.
maga2ox gene family expression patterns in different tissues in 8818- <dig> may explain changes in the morphological characteristics of 8818- <dig>  to a certain extent, such as dwarf false stems and shorter fruits, in comparison with those of  <dig>
differential expression analysis of ga metabolism genes in the false stem of williams banana  <dig> and 8818-1
the differential expression of macps <dig>  maks <dig>  mako <dig>  and makao <dig> in false stems of williams banana  <dig> and its mutant 8818- <dig> was analyzed .
makao <dig> was the most highly expressed gene among the four early ga biosynthesis genes in the false stems of  <dig> and 8818- <dig>  compared with  <dig>  makao <dig> was more highly expressed in 8818- <dig>  by contrast, 8818- <dig> showed lower expression levels of macps <dig> than  <dig>  and the difference observed was significant.
maks <dig> and mako <dig> did’t show significantly different expression levels between  <dig> and 8818- <dig> fig.
<dig> expression analysis of ga metabolism genes in false stems of williams banana  <dig> and its mutant 8818- <dig>  a early ga biosynthesis genes .
b
maga3ox.
c
maga20ox.
d
maga2ox.
total rnas were isolated from  <dig> to 8818- <dig> false stems when the plant had grown to eight leaves and qrt-pcr was conducted.
relative expression value was calculated using ‘actin’ by 2-ΔΔct method.
expression level of maks <dig>  maga20ox <dig>  maga3ox <dig>  maga2ox <dig> in  <dig> were respectively defined as  <dig> in a, b, c and d. the ct value of each gene was the average of three technical replicates with the standard error indicated.
significant difference of relative expression value in the same target gene estimated by duncan’s test was reported on the graphics .
means labeled by the same letter are not significantly different
maga3ox <dig> and maga3ox <dig> of the four ga3ox-like genes showed higher expression levels than maga3ox <dig> and maga3ox <dig> in false stems .
the expression levels of the four genes in  <dig> and 8818- <dig> showed differences that were not significant.
after exogenous ga <dig> application on 8818- <dig>  expression levels of four genes were all more or less higher than those in 8818- <dig> treated with water, had no significant difference between them.
ga3ox gene expression may not be the main cause of total gas and active ga contents differences in the false stems of  <dig> and 8818- <dig>
analysis of the expression levels of ten maga20ox-like genes in the false stems of williams banana  <dig> and its mutant 8818- <dig> revealed that maga20ox <dig> was the most prominently expressed ga20ox family member, being expressed 18-fold higher in  <dig> than maga20ox <dig> in  <dig> and 3-fold higher than maga20ox <dig> in  <dig>  .
maga20ox <dig> expression in 8818- <dig> was significantly lower than that in  <dig>  and only 65 % of the expression value in  <dig>  moreover, the expression levels of maga20ox <dig>  maga20ox <dig>  and maga20ox <dig> were higher than those of other maga20ox genes.
the maga20ox <dig> and maga20ox <dig> expression levels in  <dig> was  <dig> -fold and 3-fold higher than those in 8818- <dig>  respectively.
the expression level of maga20ox <dig> in  <dig> was higher than that in 8818- <dig>  but no significant difference between them.
other maga20ox genes, such as maga20ox <dig>  maga20ox <dig>  maga20ox <dig>  maga20ox <dig>  maga20ox <dig>  and maga20ox <dig>  showed similar expression levels in  <dig> and 8818- <dig> with no significant difference.
after exogenous ga <dig> application on 8818- <dig>  the results revealed the expression levels of maga20ox <dig>  maga20ox <dig>  and maga20ox <dig> increased significantly than those in 8818- <dig> treated with water.
although expression levels of other maga20ox genes were more or less increased after exogenous ga <dig> application, they had no significant difference.
these results suggested that maga20ox <dig>  maga20ox <dig>  and maga20ox <dig> in maga20ox gene family were the main genes induced by exogenous active ga in false stems of banana.
overall, expression levels of maga20ox <dig>  maga20ox <dig>  and maga20ox <dig> in 8818- <dig> were significantly lower than those in  <dig>  meanwhile, these three genes were dramatically induced in 8818- <dig> false stems.
therefore we speculate that maga20ox <dig>  maga20ox <dig>  and maga20ox <dig>  may play important regulating roles in ga synthesis of banana false stems, and their expression differences may cause differences of total gas and active ga contents between  <dig> and 8818- <dig>
as shown in fig.
5d, maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox <dig> exhibited higher expression levels among  <dig> maga20ox-like gene family members.
the expression levels of maga2ox <dig>  maga2ox <dig>  and maga2ox <dig> in 8818- <dig> were prominently lower than those in  <dig>  respectively only 15 %, 25 %, and 63 % of the expression levels in 8818; maga2ox <dig> expression level was not significantly different between  <dig> and 8818- <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox <dig> demonstrated lower expression levels than the above four genes  in false stems with significant difference between  <dig> and 8818- <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  and maga2ox <dig> also showed lower expression levels than the above four genes  in false stems, but no significant differences between  <dig> and 8818- <dig> were observed.
in 8818- <dig> false stems, most genes of maga2ox gene family were inhibited by exogenous ga <dig> treatment.
expression of maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga2ox <dig> and maga2ox <dig> were inhibited and had significant difference in 8818- <dig> false stems between ga <dig> and water treatment.
maga2ox <dig> and maga2ox <dig> were significantly inhibited by ga <dig> treatment, but they showed no significant difference between  <dig> and 8818- <dig>  so they may be not the key regulating genes.
maga2ox <dig>  maga2ox <dig> and maga2ox <dig> were significantly inhibited by ga <dig> treatment and their expression levels had significant differences between  <dig> and 8818- <dig>  but they showed lower expression levels, perhaps not the main regulating genes.
maga2ox <dig>  maga2ox <dig>  and maga2ox <dig> not only had higher expression levels, but also their expression levels in 8818- <dig> were prominently lower than those in  <dig>  probably play important regulating roles in ga synthesis in banana false stems.
discussion
phylogenetic analysis of ga metabolism enzyme genes in banana
early ga biosynthesis genes
in banana a genome, genes corresponding to enzymes involved in the early stages of ga biosynthesis pathway have several homologous sequences, which is also reported in other plants .
although multiple cps-like, ks-like, kao-like, and ko-like genes are present, only one of these genes is a ga metabolism enzyme in most plants  <cit> .
in arabidopsis, the cps, ks, and ko enzymes involved in ga biosynthesis are encoded by single genes  <cit> .
the rice genome contains two genes that encode a functional cps, but only oscps <dig> has been proven to participate in ga biosynthesis by mutant studies; oscps <dig> is involved in the biosynthesis of diterpene phytoalexins  <cit> .
prisic and xu  <cit>  compared oscps1ent, oscps2ent, and oscpssyn sequences with an1/zmcps1ent from maize  and found that oscps1ent was more similar to an1/zmcps1ent  than to either one of its paralogs .
three cps-like genes were screened in banana a genome; macps <dig> and macps <dig> possess particularly high homology, whereas compared with macps <dig> and macps <dig>  macps <dig> shares higher identity with oscps <dig>  macps <dig> probably involves in ga synthesis, whereas macps <dig> and macps <dig> may involve in the biosynthesis of phytoalexins in banana.
however, this hypothesis needs further investigation.
although ks and ko each possess two homologous genes with low identity, both enzymes probably perform different functions, similar to multiple ks-like and ko-like sequences existing in the rice genome.
however, ks and ko in ga biosynthesis are also encoded by single genes  <cit> .
late ga biosynthesis and inactivation genes
we studied the evolutionary properties of ga oxidase gene family in rice, arabidopsis, and banana to elucidate the expansion patterns of banana ga oxidase genes after species split.
according to their phylogeny, most of ga oxidase genes were mainly divided into four subgroups , thus reflecting functional classifications corresponding to ga20ox, ga3ox, ga2ox, and c <dig> ga2ox.
these results are consistent with a study on rice, arabidopsis, and soybean by han and zhu  <cit> .
previous results suggest that  <dig>   <dig>   <dig> and  <dig> ga oxidase genes are discovered in arabidopsis, rice, grapevine, and soybean, respectively  <cit> .
in comparison, banana a genome contains up to  <dig> ga oxidase genes, thereby suggesting that ga oxidases in banana are more divergent than those in arabidopsis and rice.
given the common conservative protein domain observed, these four subgroups perform similar functions.
few sequence changes occur in ga3ox in arabidopsis, rice, and banana, thus implying that ga3ox may be under greater pressure and more conserved in comparison with other ga oxidases.
however, more copies of ga20ox and ga2ox exist, and the sequences of these genes are more divergent.
ga2ox gene family is divided into two subgroups, namely, subgroups iii and c <dig> ga2ox, and several ga oxidase genes are not included in the four subgroups.
these genes, including osga20ox <dig>  osga20ox <dig>  osga20ox <dig>  osga20ox <dig>  maga20ox <dig>  and maga20ox <dig>  evolved from ga20ox.
these results suggest that ga20ox and ga2ox likely undergo a more dynamic evolutionary route, resulting in greater functional redundancy, especially in banana.
more copies of ga20ox and ga2ox probably cause relaxed selective pressure or fewer constraints during evolution.
on the other hand, ga20ox and ga3ox are separated by a relatively small distance.
together with the positive regulation of ga20ox and ga3ox in ga biosynthesis, the results indicate that genes essential for survival are preferentially produced.
although the ga2ox gene family possesses more divergent sequences, subgroups iii and c <dig> ga2ox may share similar functions.
overexpression of c <dig> ga2ox in wild-type is found to cause semi-dwarfism by inactivating c <dig> ga precursors  <cit> .
these findings imply that ga2ox genes perform important functions in a variety of approaches.
differential expression of ga metabolism genes in dwarf and normal banana cultivars
the tissue-specific expression patterns of ga metabolism genes suggest that each member of them may perform different physiological functions.
precise regulation of ga content appears to be critical in plant normal development.
early ga biosynthesis genes  are often single-copy genes in most plants.
although several homologous genes exist, only one gene is proven by mutant studies to participate in the ga metabolic pathway  <cit> .
as these genes are expressed in the early ga synthesis phase, extreme dwarf plants easily occur with mutation.
however, if only the expression level of one gene is changed, dwarfism will probably not occur, because control of the ga biosynthesis pathway is mainly exerted by ga oxidases through feedback loop mechanisms  <cit>  and localization of their expression to limited tissues.
early ga biosynthesis genes  are broadly expressed in all tested tissues of williams banana 8818- <dig>  macps <dig> is the first gene in ga synthesis pathway and the expression level of macps <dig> is lower in 8818- <dig> in comparison with  <dig>  while, makao <dig> shows higher expression in 8818- <dig>  therefore, the expression difference of macps <dig> may not be enough to decrease gas and active ga contents in 8818- <dig>
in ga3ox gene family, maga3ox <dig> and maga3ox <dig> present higher expression levels and evident tissue specificity.
thus, the two genes likely perform important functions in different tissues during certain development stages in banana.
the expression levels of the four genes in  <dig> and 8818- <dig> show statistically insignificant differences.
therefore, changes in the expression levels of these genes may not lead to ga content reduction in false stems of 8881- <dig>
the gene families of ga20ox and ga2ox include more family members than ga3ox gene family.
previous research suggests that functional diversification of surviving duplicated genes is an important feature of long-term evolution of polyploids  <cit> .
this finding implies that the ga20ox and ga2ox gene families probably perform diverse and important functions in banana.
most of the evidences point to the dynamic regulation of ga biosynthesis through regulation of 2-odd genes in contrast to terpene cyclase and cytochrome p <dig> genes in earlier stages of the pathway  <cit> .
alteration of the expression levels of ga oxidases has been proven to be successful in controlling plant stature in most plants and often affects several traits  <cit> .
loss of function of osga20ox <dig> can generate dwarf phenotypes  <cit> .
overexpression of ptaga2ox <dig> in poplar produce a short and stout phenotype  <cit> , whereas a dwarf plum hybrid shows enhanced expression of pslga2ox  <cit> .
except for maga20ox <dig>   <dig>  and  <dig>  other maga20ox genes show high expression levels in several tissues, thus revealing evident tissue specificity.
maga20ox genes present the highest expression levels in young fruits, possibly because the young fruit is in a critical period of growth and development and needs more active ga to promote fruit elongation.
by contrast, high-expression genes in roots are few, indicating that the root in detected period is not the main area of ga synthesis.
maga20ox <dig> and maga20ox <dig> were the most highly expressed genes among the detected tissues, meanwhile, they show significantly different expression levels between  <dig> and 8818- <dig> false stems.
these observations suggest that the two ga20ox genes not only perform important functions in the development of whole plant organs but also may be the core genes regulating ga content in banana false stems.
it is noteworthy that maga20ox <dig> and maga20ox <dig> are excluded from the four subgroups, along with osga20ox <dig>  osga20ox <dig>  and osga20ox <dig>  and don’t cluster together with other ga20ox.
recent research has also demonstrated that osga20ox <dig> and osga20ox <dig> are expressed in callus, root, leaf, and flower in rice  <cit> .
these results imply that this type of genes have important functions in the plant development.
moreover, some researchers have postulated that multiple functionalization of ga20ox can lead to sequence divergence  <cit> .
on the basis of the distances in the phylogenetic tree, maga2ox genes are divided into subgroups iii  and c <dig> ga2ox.
according to the expression levels in detected tissues, maga2ox genes are divided into high-expression and low-expression genes.
subgroups iii and c <dig> ga2ox both include high-expression and low-expression genes with similar numbers, maybe the two subgroups both play important roles in banana growth and development.
in rice, improvements in plant architecture, such as semi-dwarfism, increased root systems, and higher tiller numbers, can be induced by overexpression of c <dig> ga2ox or modified c <dig> ga2ox  <cit> .
c <dig> ga2ox provides an alternative mechanism for controlling endogenous ga levels.
tissue specificity analysis reveals that more high-expression ga oxidase genes are present in young fruits and false stems.
in young fruits in particular, the genes are of considerable significance for regulating activity of active ga content.
multiple genes regulation may greatly aid in maintaining ga contents at the appropriate levels and satisfying the needs of cells and organs at different developmental stages.
ga concentrations can be regulated precisely at this stage and transported to tissues requiring further elongation.
bioactive hormone concentrations are regulated at the level of hormone synthesis through controlled inactivation  <cit> .
down-regulation of certain ga20ox and ga3ox genes  and up-regulation of several ga2ox genes  can control bioactive ga content.
maga2ox <dig>  maga2ox <dig>  and maga2ox <dig> of the maga2ox gene family present significantly different expressions with high-expression levels in  <dig> and 8818- <dig> false stems, and are markedly inhibited by exogenous ga <dig>  maga20ox <dig>  maga20ox <dig>  and maga20ox <dig> also present significantly different expressions with high- expression levels in  <dig> and 8818- <dig> false stems, and are markedly induced by exogenous ga <dig>  the low expression of maga20ox genes  in 8818- <dig> and the high expression of maga2ox genes  in 8818- <dig> can both lead to reducing active ga content.
to a certain extent, the different expression between  <dig> and 8818- <dig> and the expression patterns of maga20ox and maga2ox genes in different tissues of 8818- <dig> can explain the changes in morphological characteristics of 8818- <dig>  such as dwarf false stems and shorter fruits, in comparison with  <dig>  these genes may be the key genes regulating ga content in williams banana false stems.
CONCLUSIONS
in this research, we screened all banana ga metabolism genes in a genome, as well as analyzed their homologous evolutionary relationship and identified the tissue specificity of these genes.
these analyses may help elucidate metabolism genes in other monocotyledonous herbs.
we found  <dig> ga metabolism genes and gene features and phylogenetic analysis showed their conservation and divergence in williams banana.
moreover, our study found that the most high-expression ga oxidase genes existed in young fruits.
this finding demonstrates that young fruits are the most active areas of ga metabolism, which contributes to fruit length regulation.
we investigated the key genes regulating ga content in banana false stem by expression difference analysis not only between  <dig> and its dwarf mutant 8818- <dig>  but also in 8818- <dig> treated with water and ga <dig>  ga oxidase genes, including maga2ox <dig>  maga2ox <dig>  maga2ox <dig>  maga20ox <dig>  maga20ox <dig>  and maga20ox <dig>  may perform key regulating functions.
differences in the expression levels of these genes in terms of ga content in 8818- <dig> false stems are lower than those in  <dig>  thus explaining the semi-dwarf phenotype of the 8818- <dig> mutant.
based on tissue specificity and expression level differences, identifying important ga metabolism enzyme genes can help us obtain new banana cultivars with stocky features but comparable fruit lengths through genetic engineering.
the present results will also benefit research on other closely related species with significant agricultural importance.

 height of cultivated banana generally exceeds 2 m, and its false stem is easily broken in typhoon-frequented areas. the stocky build of dwarf banana varieties can resist typhoon damage to a certain extent and offers the advantages of cultivation convenience, field management, labor savings, close planting, and so on. the dwarf mutant is a useful material for excavating and researching dwarf-related genes. identification and utilization of banana dwarf-related genes are of considerable significance in breeding dwarf banana varieties. we obtained the dwarf mutant ‘8818-1’ through ems mutagenesis of williams banana the stature of the 8818- false stem is approximately  m. williams 8818- is stronger, bears shorter fruits, and presents dwarf characteristics in comparison with its parent, previous studies reveal that hormone-deficient dwarf mutants can be restored by application of the corresponding exogenous active hormones in which the active hormone biosynthesis pathway is inhibited or blocked. while dwarf mutants may become hormone-insensitive because of problems in hormone signal absorption, transfer, metabolic regulation genes, application of the corresponding exogenous active hormone can‘t restore the dwarf type. total gas contents in the false stem of williams banana dwarf mutant 8818- are significantly lower than those in its parent and the plant stature of 8818- can be restored by application of exogenous active gibberellin ga we thus speculate that 8818- may be a hormone-deficient dwarf mutant. gas perform fundamental functions in plant growth and development, participating in the regulation of numerous developmental processes, such as seed germination, stem elongation, leaf stretching, flower induction, and fruit-setting. reduction of active gas content causes plants to exhibit the dwarf phenotype. ga biosynthesis pathway is well elucidated in model plants, and their related mutants have been isolated. gas are biosynthesized from geranyl diphosphate, a common c precursor for diterpenoids. biosynthesis enzymes, including ent-copalyl diphosphate synthase, ent-kaurene synthase, ent-kaurene oxidase, ent-kaurenoic acid oxidase, ga 20-oxidase, ga 3-oxidase, and ga 2-oxidase, may be classified as terpene synthases, including cps and ks, cytochrome p monooxygenases, including ko and kao, and 2-oxoglutarate–dependent dioxygenases, including ga20ox, ga3ox, and ga2ox. cps, ks, ko, and kao enzymes involved in the early steps of the ga metabolism pathway are usually encoded by a single or few genes. their mutants display severe dwarfism and loss of fertility, which can be recovered after spraying with exogenous active gas. although multiple homologous genes are present in numerous plants, only one of these genes participates in the ga metabolism pathway. for instance, the rice oscps and osks-like gene families consist of and members, respectively, but only oscps and osks are responsible for ent-kaurene biosynthesis. ga20ox, ga3ox and ga2ox are three enzymes that catalyze later reactions in the ga biosynthesis pathway and belong to the 2og-fe oxygenase superfamily. in numerous plant species, the enzymes are independently encoded by different gene families, thus accounting for certain functional redundancy, as well as tissue specificity. the loss of function of these ga oxidase genes in plants can generate a dwarf phenotype, which is restored by the application of exogenous ga. for instance, the well-known green revolution gene, sd- is generated from loss of function in osga20ox of rice. by contrast, ga2ox decreases levels of active gas in plants, and overexpression of ga2ox genes can lead to dwarf types. ga metabolism genes have been identified in fungi, bacteria, arabidopsis, rice, maize, soybean, pumpkin, pea, cucumber, grapevine, brachypodium, bread wheat, and salvia miltiorrhiza, among others. most publications focus on the systematic evolutionary analysis of the ga oxidase gene family in these plants, and gene functional research on individual pathway member from several plants has been conducted. previous results have shown that rice possesses ga20ox, ga3ox, and ga2ox genes; arabidopsis possesses ga20ox, ga3ox, and ga2ox genes; and soybean contains ga20ox, ga3ox, and ga2ox genes. these ga oxidase genes exhibit a unique expression pattern and perform distinct developmental functions in different organs, tissues, and developmental stages of plants. for instance, atga3ox and atga3ox are responsible for bioactive ga biosynthesis during vegetative growth, while atga3ox atga3ox and atga3ox are important for the development of reproductive organs. among the atga20ox genes, atga20ox atga20ox and atga20ox are the dominant paralogs. atga20ox is functionally redundant with atga20ox and atga20ox whereas atga20ox and atga20ox perform minor roles in most developmental stages. differential expression and distinct developmental functions have also been observed in rice. moreover, the transcription levels of several, but not all, ga metabolism genes are under feedback control. control includes inhibition of the expression levels of several ga20ox and ga3ox genes, as well as activation of several ga2ox genes. banana a genome sequencing was completed in, but related information on ga metabolism in banana is limited. the numbers of ga metabolism genes in the banana a genome and their phylogenetic evolution, function, tissue specificity, and timing of expression have neither been verified nor explored. to understand the distribution and system evolution of ga metabolism genes in banana a genome, we searched all ga metabolism genes in the banana genome hub and the national center for biotechnology information. preliminary analyses of the system evolution of these genes have laid the foundation for research on banana ga metabolism genes. the expression levels of ga metabolism genes in williams banana and 8818- and the principal genes regulating gas content remain unknown. to elucidate possible causes of the 8818- dwarf phenotype, we analyzed tissue specificity and compared the gene expression differences in seven kinds of genes encoding early ga biosynthesis genes and ga oxidase genes between and 8818- these results improve our current understanding of the ga metabolism pathway in banana and contribute to research in other closely related species with significant agricultural importance.  gas content analysis and exogenous ga application treatment in the field, the adult 8818- plant presented stronger, shorter false stems and shorter fruits in comparison with the parent. total gas content was determined in different tissues of williams and its mutant, 8818- the results are shown in fig. 1b. in addition to that in leaves, the total gas contents in most tissues of 8818- were lower than those in during different developmental stages. total gas contents of false stems during the young and adult development stages in were 113 % and 145 % higher than those in 8818- respectively. total gas contents of young fruits and roots in were also significantly higher than those in 8818- either during adulthood or the seedling stage, the total gas content of 8818- false stems was significantly lower than that of gas have several forms and many of them are inactive and intermediates, and only few are active forms, namely ga ga and ga so contents of ga ga and ga were determined in false stems of and 8818-. the results showed that ga was the highest content active ga and the three kinds of active gas content of false stems in 8818- were all lower than those in among them the difference of ga content between and 8818- was significant. false stems are closely related to plant stature; therefore, 8818- is significantly shorter than which may be due to a decline in gas content in the former, especially ga content.fig. phenotypes and gibberellins levels of banana mutant 8818- and its wide type. a comparison of the plant height between and 8881- in the harvest period. b total gas contents between and 8818- in different tissues at different ages. c active gas contents in false stems of and 8818- significant difference of total gas contents for each tissue and active ga contents for each ga between and 8818- estimated by t-test was reported on the graphics. stars indicate significant differences of total gas content between the same organ of and 8818- or between the same active ga of and 8818- exogenous ga application was conducted on 8818-1; in this experiment, water was used as a control. results suggested that treatment with all three concentrations could restore the plant height of 8818- to levels or even higher. ga exerted a dose-dependent effect on 8818-1; the higher the concentration, the more rapidly the false stems elongated within the scope of 50–200 mg/l ga fig. effect of exogenous ga treatments on plant height of 8818- with different concentrations. each value was the mean of ten biological replicates with the standard error indicated and evaluated by duncan’s test. means labeled by the same letter are not significantly different considering the results of gas content determination and plant height recovery, we can speculate that the dwarfism of 8818- may be caused by reduction of gas content in false stems. isolation of putative ga metabolism genes in banana to identify the genes encoding seven kinds of ga metabolism enzymes in the banana a genome, we screened all available banana amino acid sequences in the banana genome hub and ncbi. the banana a genome was sequenced and published in the sequenced genotype is a doubled-haploid from the musa acuminata subsp. malaccencis dh-pahang. three cps-like genes, ks-like genes, kao-like genes, ko-like gene, ga20ox-like genes, ga3ox-like genes, and ga2ox-like genes were searched. in the banana a genome, candidate genes were distributed across all banana chromosomes and random chromosome. we named the genes according to their position in the chromosome.table gibberellin metabolism genes and their homologs in banana a genome macps1 macps2 macps3 maks1 maks2 mako makao1 makao2 maga20ox1 maga20ox2 maga20ox3 maga20ox4 maga20ox5 maga20ox6 maga20ox7 maga20ox8 maga20ox9 maga20ox10 maga3ox1 maga3ox2 maga3ox3 maga3ox4 maga3ox5 maga2ox1 maga2ox2 maga2ox3 maga2ox4 maga2ox5 maga2ox6 maga2ox7 maga2ox8 maga2ox9 maga2ox10 maga2ox11 maga2ox12 maga2ox13 maga2ox14 maga2ox15 early ga biosynthesis genes we searched two cps-like complete cdna sequences and one cps-like fragment sequence in the banana genome hub and then searched the complete cdna sequence of macps in ncbi. the three genes were all located in chromosome macps presented and  % identities with macps and macps respectively, and macps and showed,, and  % identities with oscps. in ncbi, blast analysis revealed that macps and showed the highest similarity to the cps of phoenix dactylifera, as well as and 76 % identities with pdcps, respectively. two maks-like complete cdna sequences were searched in ncbi. both sequences were located on chromosome and shared  % identity. in ncbi, maks-like revealed the highest similarity to ks of elaeis guineensis but shared only and  % identity with osks. in ncbi and the banana genome hub, we found only one mako-like gene, which was located in chromosome sharing the highest similarity to ko of phoenix dactylifera and  % identity with osko/cyp701a. two makao-like genes were located in chromosomes and shared  % identity with each other, maximum similarities to kao of phoenix dactylifera, and and  % identity with oskao/cyp88a, respectively. ga oxidase genes ga20ox, ga3ox, and ga2ox are three enzymes that catalyze later reactions in the ga biosynthesis pathway. these enzymes belong to the 2og-fe oxygenase superfamily and are encoded by a multigene family. ten ga20ox-like genes were found in the banana a genome; in comparison, and copies of ga20ox genes have been reported in arabidopsis and rice, respectively. ten ga20ox-like genes were located on chromosomes and. in rice, osga20ox is reported as the rice green revolution gene and is previously known as semi-dwarf; loss of function of osga20ox can generate the dwarf phenotype. the deduced amino acid sequence of banana maga20ox showed the highest homology with osga20ox2/sd; by comparison, maga20ox revealed only  % identity with the gene. five ga3ox-like genes were searched in the banana genome hub. however, four ga3ox genes were searched in ncbi. four ga3ox-like genes in the banana genome hub respectively matched four ga3ox genes searched by blast in ncbi. meanwhile, maga3ox showed 100 % identity with banana ga20ox genes by blast x in ncbi. phylogenetic analysis also revealed that maga3ox was grouped as a single clade and possessed a distant genetic relationship with the ga3ox genes of rice, maize, and arabidopsis. therefore, the annotation of gsmua achr1p in the banana genome hub should be revised. in comparison, two and four copies of ga3ox genes have been reported in arabidopsis and rice, respectively. genetic evidence from the d mutant proves that osga3ox is essential and that loss of function of osga3ox2/d can generate the dwarf phenotype. four ga3ox-like genes showed,,, and  % identities with this gene. fifteen ga2ox-like genes were searched in the banana a genome. by comparison, and copies of ga2ox genes have been reported in arabidopsis and rice, respectively. fifteen ga2ox-like genes were distributed to the rest of the chromosomes, except for chromosomes however, blast x in ncbi revealed that maga2ox shared 100 % identity with the musa acuminata probable 2-oxoglutarate-dependent dioxygenase gene. phylogenetic analysis of ga oxidase genes showed that maga2ox presented a distant genetic relationship with other ga2ox genes. thus, we speculate that maga2ox belongs to the 2og-fe oxygenase superfamily and not the ga2ox family. analyses of phylogenetic tree and conserved protein domains of ga metabolism genes in banana and other plants early ga biosynthesis genes phylogenetic analysis of diterpene cyclases and cyt p monooxygenases amino acid sequences from banana, rice, maize, soybean, and arabidopsis revealed that cps, ks, ko, and kao proteins could be divided into monocot and dicot groups. this finding is consistent with banana, rice, and maize which are monocot plants. the monocot group was subdivided into two subgroups; rice and maize were grouped in the same clade, whereas banana presented a distant genetic relationship with rice and maize among monocot plants. in ncbi, blast analysis showed that elaeis guineensis and phoenix dactylifera shared the highest similarity to banana. phylogenetic analysis revealed that three cps-like proteins were highly similar and grouped in the same clade; moreover, two ks-like and kao-like which belonged to cyt p monooxygenases were grouped in the same clade.fig. analysis of phylogenetic relationships and conserved protein motifs among ga metabolism genes. a early ga biosynthesis genes. b ga oxidase genes. ma, musa acuminata; at, arabidopsis thaliana; os, oryza sativa; gm, glycine max; zm, zea mays. the accession numbers of protein sequences cited in this study are in additional file 2 analysis of conserved domains revealed that all cps possessed motifs and in common, whereas ks owned motifs and we thus speculate that protein domains and are specific to the diterpene cyclases. cps differed from ks by possessing conserved motifs and kao only contained conserved motifs and which suggested evolutionary conservation. ko only possessed motif which could be common in all cyt p450-dependent monooxygenases. ga oxidase genes to identify the evolutionary relationships of the ga oxidase genes in banana, arabidopsis, and rice, we constructed multiple sequence alignments based on the ga20ox, ga3ox, and ga2ox protein sequences of banana, arabidopsis, and rice. an evolutionary tree was established according to the alignment results by using the neighbor joining method. phylogenetic analysis showed that most ga oxidase genes could be mainly separated into four subgroups. subgroups i, ii, and iii clearly corresponded to differences among the functions of ga20ox, ga3ox, and ga2ox. ga20ox and ga3ox can promote the production of active ga, whereas ga2ox inactivates ga, thereby regulating ga content in plants. the phylogenetic tree revealed that the ga oxidases of rice, arabidopsis, and banana were more similar to their respective homologs within each subgroup than to each other. this finding indicated that expansion of ga oxidase genes occurred early in the evolution of this protein family. ga3ox belonged to a smaller gene family than ga20ox and ga2ox. four, two, and four copies of ga3ox genes were discovered in arabidopsis, rice, and banana, respectively. by contrast, and copies of ga20ox genes and and copies of ga2ox genes were discovered in arabidopsis, rice, and banana, respectively. this finding indicated that the ga3ox gene family was more conserved than the ga20ox and ga2ox families. moreover ga20ox and ga3ox were separated by a relatively small distance, whereas ga2ox was located farther from these genes. several homologous sequences of ga20ox and ga2ox showed low sequence identity, and certain branches disclosed a pronounced divergence and did not cluster together. six maga2ox genes didn’t appear in subgroups i, ii, and iii. these genes constituted a separate branch with osga2ox osga2ox osga2ox osga2ox atga2ox and atga2ox showing less similarity to other ga2ox proteins. previous results have verified that osga2ox osga2ox osga2ox osga2ox atga2ox and atga2ox belong to c ga2ox. thus, six maga2ox genes may also belong to c ga2ox. c ga2ox was found to hydroxylate c20-ga precursors but not c19-gas, thus decreasing active ga levels. for instance, osga2ox have been verified to inactivate bioactive ga thereby repressing cell growth, similar to members in subgroup iii. overexpression of wild-type or modified c ga2ox in rice can produce a semi-dwarf type, increase root systems, and higher tiller numbers. c ga2ox split from c ga2ox in the phylogenetic tree, but the key functional regions of coding sequences in ga oxidase were less variable. c ga2ox exists not only in rice, arabidopsis, and banana but also in other plants, such as soga2ox from spinach and gmga2ox from soybean. in banana, six c ga2oxs are found, which suggests that c ga2ox may be widespread in plant ga metabolism. moreover, several ga oxidases, such as osga20ox osga20ox osga20ox osga20ox maga20ox and maga20ox didn’t appeared in the four subgroups and were not clustered together with ga20ox, which implies ga20ox genes may have more complicated evolution. protein domains and were in common in most ga20ox, ga2ox, and ga3ox genes. we found that protein domain was unique to subgroup i and subgroup iii exclusively possessed protein domains and protein domain was exclusively contained by c20-ga2ox, and subgroup ii possessed no special protein domain, suggesting greater conservation in evolution. protein domain only existed in subgroups i and ii; this domain was lacking in subgroup iii and c20-ga2ox. c20-ga2ox didn’t possess protein domain which existed in subgroups i, ii, and iii. these special motifs may account for the function difference. in three kinds of ga oxidase genes, the numbers of genes of ga20ox and ga2ox were greater than that of ga3ox and these genes possessed considerably longer branches in the phylogenetic trees. these findings indicated that ga20ox and ga2ox evolved more rapidly than ga3ox. ga20ox and ga2ox demonstrated more dynamic evolutionary routes, thereby resulting in greater functional redundancy. in addition, more copies of ga20ox and ga2ox could cause relaxed selective pressure or loosened constraints in the evolution process. subgroups i, ii, and iii contained both monocot and dicot proteins. this evolutionary relationship suggests that every subgroup of ga20ox/ga3ox/ga2ox proteins may perform homologous functions crossing between monocot and dicot plants. tissue specificity analysis of ga metabolism genes in williams banana quantitative real-time polymerase chain reaction analysis revealed that the isolated ga metabolism genes were expressed at different levels in various tissues of williams banana 8818-.fig. tissue-specific expression of the ga metabolism genes in various tissues of banana dwarf mutant 8818- total rnas were isolated from leaves, bracts, false stems, roots, young fruits, and approximately mature fruits in growth and development period, and qrt-pcr was conducted. relative expression value was calculated using ‘actin’ as a reference by 2-ΔΔct method. expression level of macps maga3ox maga20ox and maga2ox in the root were respectively defined as in a, b, c and d. fold change of the target gene in every tissue, normalized to actin and relative to the expression of macps maga3ox maga20ox maga2ox in the root respectively in a, b, c and d, was calculated for each sample using the 2-ΔΔct method. the ct value of each gene was the average of three technical replicates with the standard error indicated. significant difference of relative expression value in different tissues of the same target gene estimated by duncan’s test was reported on the graphics. means labeled by the same letter are not significantly different macps maks mako and makao were broadly expressed at different levels in all tested tissues of williams banana 8818- including leaves, roots, false stems, bracts, young fruits, and approximately mature fruits. the expression level of makao gene in different tissues was generally higher than those of the three other genes in the corresponding tissues. the expression level of makao was the highest in the bract, followed by leaves, false stems, and young fruits. the highest gene expression levels of macps and maks were observed in bracts, whereas the highest level of mako expression was found in young fruits. as a whole, expression level of makao in all tissues was the highest among the early ga biosynthesis genes tested, while difference among other three genes expression levels in all tissues was small, thus suggesting that makao might play an important regulating role in transcription level in ga biosynthesis of the banana. analysis of four ga3ox-like genes revealed that they were expressed at different levels in six tissues. maga3ox expression levels were higher in young fruits and bracts but lower in approximately mature fruits. compared with maga3ox and maga3ox maga3ox and maga3ox were present at lower expression levels. the relative expression level of maga3ox in young fruits was the highest among six tissues, but the relative expression value remained below, similar to the relative expression value of maga3ox in all tissues not including roots. maga3ox was strongly expressed in young fruits, bracts, and leaves by 22-fold, 18-fold, and 16-fold, respectively, compared with that of maga3ox in roots. while maga3ox was weakly expressed in the roots, false stems, and approximately mature fruits. the maga3ox and maga3ox of four ga3ox-like genes may be the key genes regulating ga content in the normal development of banana. however, different genes perform different functions in various tissues. maga20ox and showed relatively low expression and revealed less obvious tissue specificity in vegetative tissues. by contrast, other genes exhibited high expression in several tissues at least. maga20ox showed relatively high expression in leaves, bracts, and young fruits but low expression in roots, false stems, and approximately mature fruits. the expression level of maga20ox was relatively high in all tested plant tissues, presenting the highest expression in young fruits and the lowest expression in leaves. maga20ox was prominently expressed in leaves, false stems, and approximately mature fruits and lowly expressed in roots. maga20ox was also expressed in all tissues, and showed extremely high levels in young fruits and extremely low levels in roots. these results reveal obvious tissue specificity. maga20ox and maga20ox expression levels were relatively similar, showing evident tissue specificity, particularly high expression in young fruits and low expression in roots, false stems, and approximately mature fruits. maga20ox expression was higher than those of maga20ox and but lower than those of abundant genes, such as maga20ox maga20ox and so on. tissue specificity among these genes was evident. in general, young fruits contained abundant genes, except maga20ox and maga20ox and other maga20ox genes all demonstrated maximum expression levels in young fruits. fourteen maga2ox genes could generally be divided into two categories. genes included in the first category were strongly expressed in most tissues and expressed differently in most tissues. this group included maga2ox maga2ox maga2ox maga2ox maga2ox maga2ox maga2ox and maga2ox genes in the second category were weakly expressed and slightly high expression in individual tissues. this group included maga2ox maga2ox maga2ox maga2ox and maga2ox maga2ox demonstrated the highest expression level in roots, followed maga2ox14; other maga2ox genes were weakly expressed. in false stems, maga2ox was the most abundant gene, although maga2ox maga2ox maga2ox and maga2ox were also strongly expressed. other maga2ox genes were weakly expressed. maga2ox showed the highest expression in leaves, followed by maga2ox maga2ox maga2ox and maga2ox8; by contrast, maga2ox maga2ox maga2ox and maga2ox were lowly expressed in this tissue. in bracts, the top three most abundantly expressed genes included maga2ox maga2ox and maga2ox numbers of high-expression genes in young fruits exceeded those in other tissues. maga2ox was the most high- expression gene, followed by maga2ox maga2ox maga2ox maga2ox maga2ox maga2ox and maga2ox in approximately mature fruits, highly expressed maga2ox genes were few. maga2ox was the most strongly expressed gene, followed by maga2ox14; all other genes were weakly expressed. the analysis described above reveals that genes expressed strongly in most tissues, namely, maga2ox maga2ox and maga2ox and so on, may be the key genes regulating ga content. higher expression levels of genes were found in false stems, bracts, leaves, especially in young fruits, and such expression was of considerable significance for regulating ga contents in these tissues. maga2ox gene family expression patterns in different tissues in 8818- may explain changes in the morphological characteristics of 8818- to a certain extent, such as dwarf false stems and shorter fruits, in comparison with those of differential expression analysis of ga metabolism genes in the false stem of williams banana and 8818-1 the differential expression of macps maks mako and makao in false stems of williams banana and its mutant 8818- was analyzed. makao was the most highly expressed gene among the four early ga biosynthesis genes in the false stems of and 8818- compared with makao was more highly expressed in 8818- by contrast, 8818- showed lower expression levels of macps than and the difference observed was significant. maks and mako did’t show significantly different expression levels between and 8818- fig. expression analysis of ga metabolism genes in false stems of williams banana and its mutant 8818- a early ga biosynthesis genes. b maga3ox. c maga20ox. d maga2ox. total rnas were isolated from to 8818- false stems when the plant had grown to eight leaves and qrt-pcr was conducted. relative expression value was calculated using ‘actin’ by 2-ΔΔct method. expression level of maks maga20ox maga3ox maga2ox in were respectively defined as in a, b, c and d. the ct value of each gene was the average of three technical replicates with the standard error indicated. significant difference of relative expression value in the same target gene estimated by duncan’s test was reported on the graphics. means labeled by the same letter are not significantly different maga3ox and maga3ox of the four ga3ox-like genes showed higher expression levels than maga3ox and maga3ox in false stems. the expression levels of the four genes in and 8818- showed differences that were not significant. after exogenous ga application on 8818- expression levels of four genes were all more or less higher than those in 8818- treated with water, had no significant difference between them. ga3ox gene expression may not be the main cause of total gas and active ga contents differences in the false stems of and 8818- analysis of the expression levels of ten maga20ox-like genes in the false stems of williams banana and its mutant 8818- revealed that maga20ox was the most prominently expressed ga20ox family member, being expressed 18-fold higher in than maga20ox in and 3-fold higher than maga20ox in. maga20ox expression in 8818- was significantly lower than that in and only 65 % of the expression value in moreover, the expression levels of maga20ox maga20ox and maga20ox were higher than those of other maga20ox genes. the maga20ox and maga20ox expression levels in was -fold and 3-fold higher than those in 8818- respectively. the expression level of maga20ox in was higher than that in 8818- but no significant difference between them. other maga20ox genes, such as maga20ox maga20ox maga20ox maga20ox maga20ox and maga20ox showed similar expression levels in and 8818- with no significant difference. after exogenous ga application on 8818- the results revealed the expression levels of maga20ox maga20ox and maga20ox increased significantly than those in 8818- treated with water. although expression levels of other maga20ox genes were more or less increased after exogenous ga application, they had no significant difference. these results suggested that maga20ox maga20ox and maga20ox in maga20ox gene family were the main genes induced by exogenous active ga in false stems of banana. overall, expression levels of maga20ox maga20ox and maga20ox in 8818- were significantly lower than those in meanwhile, these three genes were dramatically induced in 8818- false stems. therefore we speculate that maga20ox maga20ox and maga20ox may play important regulating roles in ga synthesis of banana false stems, and their expression differences may cause differences of total gas and active ga contents between and 8818- as shown in fig. 5d, maga2ox maga2ox maga2ox and maga2ox exhibited higher expression levels among maga20ox-like gene family members. the expression levels of maga2ox maga2ox and maga2ox in 8818- were prominently lower than those in respectively only 15 %, 25 %, and 63 % of the expression levels in 8818; maga2ox expression level was not significantly different between and 8818- maga2ox maga2ox maga2ox and maga2ox demonstrated lower expression levels than the above four genes in false stems with significant difference between and 8818- maga2ox maga2ox maga2ox maga2ox maga2ox and maga2ox also showed lower expression levels than the above four genes in false stems, but no significant differences between and 8818- were observed. in 8818- false stems, most genes of maga2ox gene family were inhibited by exogenous ga treatment. expression of maga2ox maga2ox maga2ox maga2ox maga2ox maga2ox maga2ox and maga2ox were inhibited and had significant difference in 8818- false stems between ga and water treatment. maga2ox and maga2ox were significantly inhibited by ga treatment, but they showed no significant difference between and 8818- so they may be not the key regulating genes. maga2ox maga2ox and maga2ox were significantly inhibited by ga treatment and their expression levels had significant differences between and 8818- but they showed lower expression levels, perhaps not the main regulating genes. maga2ox maga2ox and maga2ox not only had higher expression levels, but also their expression levels in 8818- were prominently lower than those in probably play important regulating roles in ga synthesis in banana false stems. discussion phylogenetic analysis of ga metabolism enzyme genes in banana early ga biosynthesis genes in banana a genome, genes corresponding to enzymes involved in the early stages of ga biosynthesis pathway have several homologous sequences, which is also reported in other plants. although multiple cps-like, ks-like, kao-like, and ko-like genes are present, only one of these genes is a ga metabolism enzyme in most plants. in arabidopsis, the cps, ks, and ko enzymes involved in ga biosynthesis are encoded by single genes. the rice genome contains two genes that encode a functional cps, but only oscps has been proven to participate in ga biosynthesis by mutant studies; oscps is involved in the biosynthesis of diterpene phytoalexins. prisic and xu compared oscps1ent, oscps2ent, and oscpssyn sequences with an1/zmcps1ent from maize and found that oscps1ent was more similar to an1/zmcps1ent than to either one of its paralogs. three cps-like genes were screened in banana a genome; macps and macps possess particularly high homology, whereas compared with macps and macps macps shares higher identity with oscps macps probably involves in ga synthesis, whereas macps and macps may involve in the biosynthesis of phytoalexins in banana. however, this hypothesis needs further investigation. although ks and ko each possess two homologous genes with low identity, both enzymes probably perform different functions, similar to multiple ks-like and ko-like sequences existing in the rice genome. however, ks and ko in ga biosynthesis are also encoded by single genes. late ga biosynthesis and inactivation genes we studied the evolutionary properties of ga oxidase gene family in rice, arabidopsis, and banana to elucidate the expansion patterns of banana ga oxidase genes after species split. according to their phylogeny, most of ga oxidase genes were mainly divided into four subgroups, thus reflecting functional classifications corresponding to ga20ox, ga3ox, ga2ox, and c ga2ox. these results are consistent with a study on rice, arabidopsis, and soybean by han and zhu. previous results suggest that and ga oxidase genes are discovered in arabidopsis, rice, grapevine, and soybean, respectively. in comparison, banana a genome contains up to ga oxidase genes, thereby suggesting that ga oxidases in banana are more divergent than those in arabidopsis and rice. given the common conservative protein domain observed, these four subgroups perform similar functions. few sequence changes occur in ga3ox in arabidopsis, rice, and banana, thus implying that ga3ox may be under greater pressure and more conserved in comparison with other ga oxidases. however, more copies of ga20ox and ga2ox exist, and the sequences of these genes are more divergent. ga2ox gene family is divided into two subgroups, namely, subgroups iii and c ga2ox, and several ga oxidase genes are not included in the four subgroups. these genes, including osga20ox osga20ox osga20ox osga20ox maga20ox and maga20ox evolved from ga20ox. these results suggest that ga20ox and ga2ox likely undergo a more dynamic evolutionary route, resulting in greater functional redundancy, especially in banana. more copies of ga20ox and ga2ox probably cause relaxed selective pressure or fewer constraints during evolution. on the other hand, ga20ox and ga3ox are separated by a relatively small distance. together with the positive regulation of ga20ox and ga3ox in ga biosynthesis, the results indicate that genes essential for survival are preferentially produced. although the ga2ox gene family possesses more divergent sequences, subgroups iii and c ga2ox may share similar functions. overexpression of c ga2ox in wild-type is found to cause semi-dwarfism by inactivating c ga precursors. these findings imply that ga2ox genes perform important functions in a variety of approaches. differential expression of ga metabolism genes in dwarf and normal banana cultivars the tissue-specific expression patterns of ga metabolism genes suggest that each member of them may perform different physiological functions. precise regulation of ga content appears to be critical in plant normal development. early ga biosynthesis genes are often single-copy genes in most plants. although several homologous genes exist, only one gene is proven by mutant studies to participate in the ga metabolic pathway. as these genes are expressed in the early ga synthesis phase, extreme dwarf plants easily occur with mutation. however, if only the expression level of one gene is changed, dwarfism will probably not occur, because control of the ga biosynthesis pathway is mainly exerted by ga oxidases through feedback loop mechanisms and localization of their expression to limited tissues. early ga biosynthesis genes are broadly expressed in all tested tissues of williams banana 8818- macps is the first gene in ga synthesis pathway and the expression level of macps is lower in 8818- in comparison with while, makao shows higher expression in 8818- therefore, the expression difference of macps may not be enough to decrease gas and active ga contents in 8818- in ga3ox gene family, maga3ox and maga3ox present higher expression levels and evident tissue specificity. thus, the two genes likely perform important functions in different tissues during certain development stages in banana. the expression levels of the four genes in and 8818- show statistically insignificant differences. therefore, changes in the expression levels of these genes may not lead to ga content reduction in false stems of 8881- the gene families of ga20ox and ga2ox include more family members than ga3ox gene family. previous research suggests that functional diversification of surviving duplicated genes is an important feature of long-term evolution of polyploids. this finding implies that the ga20ox and ga2ox gene families probably perform diverse and important functions in banana. most of the evidences point to the dynamic regulation of ga biosynthesis through regulation of 2-odd genes in contrast to terpene cyclase and cytochrome p genes in earlier stages of the pathway. alteration of the expression levels of ga oxidases has been proven to be successful in controlling plant stature in most plants and often affects several traits. loss of function of osga20ox can generate dwarf phenotypes. overexpression of ptaga2ox in poplar produce a short and stout phenotype, whereas a dwarf plum hybrid shows enhanced expression of pslga2ox. except for maga20ox and other maga20ox genes show high expression levels in several tissues, thus revealing evident tissue specificity. maga20ox genes present the highest expression levels in young fruits, possibly because the young fruit is in a critical period of growth and development and needs more active ga to promote fruit elongation. by contrast, high-expression genes in roots are few, indicating that the root in detected period is not the main area of ga synthesis. maga20ox and maga20ox were the most highly expressed genes among the detected tissues, meanwhile, they show significantly different expression levels between and 8818- false stems. these observations suggest that the two ga20ox genes not only perform important functions in the development of whole plant organs but also may be the core genes regulating ga content in banana false stems. it is noteworthy that maga20ox and maga20ox are excluded from the four subgroups, along with osga20ox osga20ox and osga20ox and don’t cluster together with other ga20ox. recent research has also demonstrated that osga20ox and osga20ox are expressed in callus, root, leaf, and flower in rice. these results imply that this type of genes have important functions in the plant development. moreover, some researchers have postulated that multiple functionalization of ga20ox can lead to sequence divergence. on the basis of the distances in the phylogenetic tree, maga2ox genes are divided into subgroups iii and c ga2ox. according to the expression levels in detected tissues, maga2ox genes are divided into high-expression and low-expression genes. subgroups iii and c ga2ox both include high-expression and low-expression genes with similar numbers, maybe the two subgroups both play important roles in banana growth and development. in rice, improvements in plant architecture, such as semi-dwarfism, increased root systems, and higher tiller numbers, can be induced by overexpression of c ga2ox or modified c ga2ox. c ga2ox provides an alternative mechanism for controlling endogenous ga levels. tissue specificity analysis reveals that more high-expression ga oxidase genes are present in young fruits and false stems. in young fruits in particular, the genes are of considerable significance for regulating activity of active ga content. multiple genes regulation may greatly aid in maintaining ga contents at the appropriate levels and satisfying the needs of cells and organs at different developmental stages. ga concentrations can be regulated precisely at this stage and transported to tissues requiring further elongation. bioactive hormone concentrations are regulated at the level of hormone synthesis through controlled inactivation. down-regulation of certain ga20ox and ga3ox genes and up-regulation of several ga2ox genes can control bioactive ga content. maga2ox maga2ox and maga2ox of the maga2ox gene family present significantly different expressions with high-expression levels in and 8818- false stems, and are markedly inhibited by exogenous ga maga20ox maga20ox and maga20ox also present significantly different expressions with high- expression levels in and 8818- false stems, and are markedly induced by exogenous ga the low expression of maga20ox genes in 8818- and the high expression of maga2ox genes in 8818- can both lead to reducing active ga content. to a certain extent, the different expression between and 8818- and the expression patterns of maga20ox and maga2ox genes in different tissues of 8818- can explain the changes in morphological characteristics of 8818- such as dwarf false stems and shorter fruits, in comparison with these genes may be the key genes regulating ga content in williams banana false stems.  in this research, we screened all banana ga metabolism genes in a genome, as well as analyzed their homologous evolutionary relationship and identified the tissue specificity of these genes. these analyses may help elucidate metabolism genes in other monocotyledonous herbs. we found ga metabolism genes and gene features and phylogenetic analysis showed their conservation and divergence in williams banana. moreover, our study found that the most high-expression ga oxidase genes existed in young fruits. this finding demonstrates that young fruits are the most active areas of ga metabolism, which contributes to fruit length regulation. we investigated the key genes regulating ga content in banana false stem by expression difference analysis not only between and its dwarf mutant 8818- but also in 8818- treated with water and ga ga oxidase genes, including maga2ox maga2ox maga2ox maga20ox maga20ox and maga20ox may perform key regulating functions. differences in the expression levels of these genes in terms of ga content in 8818- false stems are lower than those in thus explaining the semi-dwarf phenotype of the 8818- mutant. based on tissue specificity and expression level differences, identifying important ga metabolism enzyme genes can help us obtain new banana cultivars with stocky features but comparable fruit lengths through genetic engineering. the present results will also benefit research on other closely related species with significant agricultural importance. 
phylogenetic tree and conserved protein domain analyses showed sequence conservation and divergence. ga metabolism genes exhibited tissue-specific expression patterns. expression patterns between and 8818- revealed that maga20ox maga20ox and maga20ox of the maga20ox gene family and maga2ox maga2ox and maga2ox of the maga2ox gene family exhibited significant differential expression and high-expression levels in false stems. gibberellinsbananaga oxidase genesearly ga biosynthesis genesexpression patternstissue specificityhainan provincial natural science foundation314109chen jingjing fundamental research funds for south subtropical crops research institute1630062013005chen jingjing issue-copyright-statement© the author 2016 the present results revealed that maga20ox maga20ox maga20ox maga2ox maga2ox and maga2ox were the main genes regulating ga content difference between and 8818- all of these genes may perform important functions in the developmental processes of banana, but each gene may perform different functions in different tissues or during different developmental stages. early ga biosynthesis genes were constitutively expressed but presented differential regulation in different tissues in williams banana. overall, phylogenetic evolution, tissue specificity and differential expression analyses of ga metabolism genes can provide a better understanding of ga-regulated development in banana. ga oxidase family genes were mainly transcribed in young fruits, thus suggesting that young fruits were the most active tissue involved in ga metabolism, followed by leaves, bracts, and finally approximately mature fruits. genome-wide screening revealed candidate ga metabolism genes were systematically identified for the first time; these genes included macps, maks, mako, makao, maga20ox, maga3ox, and maga2ox genes. we obtained a dwarf mutant ‘8818-1’ through ems mutagenesis of williams banana. these genes are likely to be responsible for the regulation of gas content in 8818- false stems. our research have shown that gibberellins content in 8818- false stems was significantly lower than that in its parent and the dwarf type of 8818- could be restored by application of exogenous ga although ga exerts important impacts on the 8818- dwarf type, our understanding of the regulation of ga metabolism during banana dwarf mutant development remains limited.
 dwarfism is one of the most valuable traits in banana breeding because semi-dwarf cultivars show good resistance to damage by wind and rain. moreover, these cultivars present advantages of convenient cultivation, management, and so on. we obtained a dwarf mutant ‘8818-1’ through ems mutagenesis of williams banana. our research have shown that gibberellins content in 8818- false stems was significantly lower than that in its parent and the dwarf type of 8818- could be restored by application of exogenous ga although ga exerts important impacts on the 8818- dwarf type, our understanding of the regulation of ga metabolism during banana dwarf mutant development remains limited.  genome-wide screening revealed candidate ga metabolism genes were systematically identified for the first time; these genes included macps, maks, mako, makao, maga20ox, maga3ox, and maga2ox genes. phylogenetic tree and conserved protein domain analyses showed sequence conservation and divergence. ga metabolism genes exhibited tissue-specific expression patterns. early ga biosynthesis genes were constitutively expressed but presented differential regulation in different tissues in williams banana. ga oxidase family genes were mainly transcribed in young fruits, thus suggesting that young fruits were the most active tissue involved in ga metabolism, followed by leaves, bracts, and finally approximately mature fruits. expression patterns between and 8818- revealed that maga20ox maga20ox and maga20ox of the maga20ox gene family and maga2ox maga2ox and maga2ox of the maga2ox gene family exhibited significant differential expression and high-expression levels in false stems. these genes are likely to be responsible for the regulation of gas content in 8818- false stems.  overall, phylogenetic evolution, tissue specificity and differential expression analyses of ga metabolism genes can provide a better understanding of ga-regulated development in banana. the present results revealed that maga20ox maga20ox maga20ox maga2ox maga2ox and maga2ox were the main genes regulating ga content difference between and 8818- all of these genes may perform important functions in the developmental processes of banana, but each gene may perform different functions in different tissues or during different developmental stages. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords gibberellinsbananaga oxidase genesearly ga biosynthesis genesexpression patternstissue specificityhainan provincial natural science foundation314109chen jingjing fundamental research funds for south subtropical crops research institute1630062013005chen jingjing issue-copyright-statement© the author 2016 