BACKGROUND
tomato  is one of the most popular vegetable species grown world-wide because of its edible fruit and agronomic importance.
nevertheless, its production is frequently threatened by environmental stresses such as drought and salinity.
high salt concentration reduces tomato germination, leaf number and area, slows down shoot and root growth and increases root/shoot ratio, induces leaf senescence, and ultimately impairs crop production  <cit> .
salinity imposes both an osmotic stress due to water shortage, followed by an ionic stress resulting from disproportionate nutrients accumulation and an augmentation of toxic ion concentrations, such as sodium  <cit> .
modifications of plant physiological processes following salinization are partly attributed to alterations in phytohormone metabolism.
this may be true especially for cytokinins .
indeed, under long-term salt stress the level of bioactive cks in tomato plants was reduced down to 50% both in roots and leaves  <cit> .
the ability to control cell division and differentiation, root growth, leaf senescence, apical dominance, branching, flower and seed development as well as germination and nutrients uptake into sink organs defines cks as essential regulatory substances in plants  <cit> .
chemically, cks are derivatives of adenine substituted at the n <dig> position either with isoprenoid or an aromatic side chain.
both isoprenoid and aromatic cks are present in plants in bioactive forms, as free bases and corresponding nucleosides and nucleotides, and in non-active or storage forms as conjugates with glucose  for instance.
the typical representatives of isoprenoid cks are derivatives of trans-zeatin , cis-zeatin , dihydrozeatin  and n6-adenine , occuring throughout the plant kingdom  <cit> .
the first step of ck biosynthesis is performed by isopentenyltransferases .
plant ipts belong to multigenic families, chiefly described in arabidopsis   <cit>  and in maize, for instance  <cit> .
recently, ipt genes have also been identified in tomato , and their expression patterns specifically investigated during fruit set and development  <cit> .
ipts catalyze the transfer of the isoprenoid moiety from precursors dimethylallyl diphosphate  or -4-hydroxy-3-methyl-but-2-enyl diphosphate  to adenine nucleotide forms   <cit> .
modulation of ck contents through modification of ipt expression strongly impacts plants development.
indeed, while overexpression of ipt in plants resulted in faster shoot formation, shorter internodes, loss of apical dominance, delay of leaf senescence, higher photosynthetic rates and accumulation of tz and its riboside  <cit> , arabidopsis ipt deficient plants showed strong inhibition of shoot growth, elongation of primary and lateral roots, and reduction of tz and ip contents  <cit> .
recent progress in genetic engineering of cks enabled to control plant ck contents, affected plant traits, increased yield production and improved plant adaptation to enviromental stresses such as salinity  <cit> .
indeed, responsiveness of arabidopsis ipt genes to salt stress has been demonstrated by transcriptome analyses, where upregulation of atipt <dig>  atipt <dig> and atipt <dig> and downregulation of atipt <dig>  atipt <dig>  atipt <dig> and atipt <dig> were reported  <cit> .
regulation of ipt expression through inducible promoter in tomato roots improved tolerance to salt stress and led to higher yield compared to the wild-type plants  <cit> .
similarly, plant salinity tolerance was enhanced in transgenic cotton expressing ipt under control of a cysteine proteinase  promoter, delaying the salt-induced senescence of leaves  <cit> .
positive correlation between ipt expression and salt resistance has been demonstrated in transgenic tobacco by introducing agrobacterium tumefaciens ipt under control of the stress-inducible promoter rd29a as well  <cit> .
in addition to ck biosynthesis, ck signaling steps are also critical for plant response to salinity.
cks are perceived at the plasma membrane by specific receptors, and the signal is transduced via type-b arabidopsis response regulators  controlling transcription of type-a arrs, which act as negative feedback regulators of ck signaling  <cit> .
arr transcription factors respond in different ways to salt stress and were reported to regulate sodium accumulation in arabidopsis  <cit> .
moreover, a transcriptome analysis of arabidopsis ck deficient ipt <dig> , <dig>  mutants exposed to salinity revealed the importance of ck regulation on stress-responsive signaling pathways, even under normal conditions of growth  <cit> .
although cks assume crucial functions in tomato salt tolerance, data concerning the underlying molecular cues involved in this process remain unclear.
to better understand salt impact on ck metabolism in tomato, a thorough characterization of genes coding for ipts and their response to salinity is required.
the present study reports the functional characterization of two tomato ipt encoding genes, slipt <dig> and slipt <dig>  both in vitro and in planta.
spatio-temporal expression profiles during plant development, enzymatic activity of both proteins and their involvement in ck biosynthesis were determined.
likewise, slipt <dig> and slipt <dig> participation in response to salt stress was investigated in arabidopsis and tomato.
RESULTS
identification of two putative non-redundant tomato isopentenyltransferases
to identify ipt encoding genes in tomato, arabidopsis ipt1- <dig> coding sequences were used to screen the tomato genome database  <cit>  .
among the selected expressed sequence tags , two were apparently encoding full length proteins and could be amplified by pcr.
for the first gene , a  <dig> bp coding sequence was cloned, including a  <dig> bp open reading frame  defining a  <dig> amino acid  polypeptide of  <dig>  kda .
this gene was recently annotated as slipt <dig>  <cit> .
for the second gene , a  <dig> bp coding sequence was amplified, exhibiting a  <dig> bp orf predicted to encode a  <dig> a protein of  <dig>  kda.
this gene was classified as slipt <dig>  <cit> .figure  <dig>
identification of slipt <dig> and slipt <dig>   full-length protein sequence alignment of slipt <dig>  slipt <dig> and arabidopsis atipt <dig> and atipt <dig>  identical residues are colored in black and conserved residues in dark gray.
the isopentenyltransferase  domain is labeled.
phylogenetic relationships between slipt <dig> , slipt <dig>  and related proteins of cucumis sativus csipt <dig> , ipomoea nil inipt , malus domestica mdipt , fragaria vesca fvipt <dig> , arabidopsis thaliana atipt <dig> , atipt <dig> , atipt <dig> , atipt <dig> , atipt <dig>  and atipt <dig> , glycine max gmipt <dig> , zea mays zmipt <dig> , zmipt <dig>  and zmipt <dig> , solanum lycopersicum slipt <dig> , slipt <dig> , slipt <dig>  and slipt <dig> , nicotiana tabacum ntipt <dig> .
the phylogenetic tree was constructed according to the neighbor-joining method, using mega <dig>  <cit> .
the percentage of reliability of each branch point of the rooted tree, as assessed by the analysis of  <dig> trees , is shown on the branch stem.
slipt <dig> is located on tomato chromosome  <dig>  while slipt <dig> is situated on chromosome  <dig>  in addition, slipt <dig> includes a  <dig> bp intron localized in the 5′ utr region.
slipt <dig> and slipt <dig> display 52% identity and 72% similarity.
alignment of slipt <dig> and slipt <dig> proteins with arabidopsis ipt <dig> and ipt <dig> indicates that the ipt catalytic domain represents almost the entirety of the proteins, with exception of the n- and c-termini .
a phylogenetic analysis of slipt <dig> and slipt <dig> and nineteen additional orthologs belonging to different species was constructed by means of the neighbour-joining method using full-length amino acid sequences .
it indicated that slipt <dig> mainly clusters with soybean gmipt <dig> and arabidopsis atipt <dig>  while slipt <dig> clusters with arabidopsis atipt <dig> and japanese morning glory inipt.
except from arabidopsis and maize, full characterization of ipt enzymes in other plant species is missing.
tomato slipt <dig> and slipt <dig> show isopentenyltransferase activity in vitro
to examine the ability of slipt <dig> and slipt <dig> to catalyze ck biosynthesis, a functional analysis of both proteins was carried out.
as slipt <dig> and slipt <dig> are highly insoluble when produced in bacteria, both proteins were produced in vitro.
the enzymatic activity of slipt <dig> and slipt <dig> was determined using in vitro assays based on conversion of radiolabeled adenylated substrates  in the presence of dmapp to isopentenylated products.
both enzymes were able to convert tritium-labeled amp/adp/atp to the corresponding iprmp/iprdp/iprtp in time-dependent manner .
the prevailing metabolites formed by both slipt <dig> and slipt <dig> were iprmp and iprdp, with almost 10-fold higher catalytic activity shown by slipt <dig> compared to slipt <dig>  these results clearly demonstrate that slipt <dig> and slipt <dig> display ck biosynthetic activity in in vitro conditions.figure  <dig>
in vitro
determination of dmapp:amp, adp and atp isopentenyltransferase activity of slipt <dig>  and slipt <dig>  proteins.
adp: adenosine diphosphate, atp: adenosine triphosphate, ipmp: isopentenyladenosine-5′monophosphate; ipdp: isopentenyladenosine-5′-diphosphate, iptp: isopentenyladenosine-5′-triphosphate.
note that vertical scales are not the same for slipt <dig> and slipt <dig>
subcellular localization of slipt <dig> and slipt4
an analysis of the deduced slipt <dig> and slipt <dig> sequences revealed the presence of chloroplast transit peptides  <cit>  .
to investigate protein subcellular localization, slipt <dig> and slipt <dig> cdna were fused at their c-terminal end to green fluorescent protein  coding sequence, placed under cauliflower mosaic virus 35s promoter and used to agro-infiltrate tomato leaves.
as observed in figure  <dig> , despite a strong auto-fluorescence, slipt <dig> showed localization predominantly in chloroplasts and in the cytosolic area surrounding the chloroplast.
indeed, gfp fluorescence colocalized with chlorophyll fluorescence in this cellular compartment.
in contrast, free gfp was restricted to the cytoplasm ), while no fluorescence at  <dig> nm  was detected in untransformed tomato leaves ).
despite several attempts, we could not establish subcellular localization for slipt <dig>  and no gfp signal of the recombinant protein could be detected in transformed tomato cells.
these data show that at least slipt <dig> is localized in the chloroplast and in the cytosolic area surrounding chloroplasts.figure  <dig>
subcellular localization of slipt4-gfp by tomato leaves agroinfiltration.
slipt4-gfp fluorescence and chlorophyll/gfp fluorescence, respectively.
control gfp fluorescence and chlorophyll/gfp fluorescence, respectively.
control untransformed tomato leaves and chlorophyll autofluorescence, respectively.
bar =  <dig> µm.
characterization of slipt <dig> and slipt <dig> in arabidopsis ipt <dig> mutants
in parallel, tomato slipt <dig> and slipt <dig> were functionally characterized in planta via complementation of arabidopsis ipt <dig> knock-out  mutants.
five days after sowing , the arabidopsis wild-type  plants, ipt <dig> ko mutants and arabidopsis ipt <dig> ko complemented plants showed a similar phenotype on half-strength ms medium .
we thus suggested to determine the function of slipt <dig> and slipt <dig> in arabidopsis complemented ipt <dig> ko mutants grown on salt medium and to follow their response to salinity.
based on the highest phenotype discrimination,  <dig> mm nacl medium was used for further testing of seed germination and plant survival percentages, primary root elongation, seedlings cks content and ck-related genes expression profiles.
on the control medium, germination and survival frequencies were not significantly different in between the variants reaching 100%.
on the saline medium, seed germination percentage was reduced to 67% for both lines of arabidopsis slipt <dig> complemented plants , significantly less than that of ipt <dig> ko and wt plants .
however, in arabidopsis slipt <dig> complemented plants , seed germination reached 89% and 90%, respectively, while wt seeds displayed 78% and ipt <dig> ko 93% of germination rate, respectively.
the same trend was detected for seedlings’ survival percentage on  <dig> mm nacl  .
similarly, primary root elongation was estimated by screening  <dig> das arabidopsis slipt <dig> and slipt <dig> complemented plants .
on control medium, the primary root length among the 9at and 10at lines significantly differed compared to wt while on the salt-containing medium, no significant changes between the slipt <dig> complemented and wt plants were observed.
arabidopsis plants complemented with slipt <dig> showed significantly longer primary root  than wt  on control medium, while antagonistic effects were detected on salt-containing medium .
these results demonstrate that both tomato ipt enzymes are functional in heterologous system.
however, in reaction to salt stress the slipt <dig> complemented plants displayed the same characteristics as wt in germination and survival assays, while both of the slipt <dig> complemented lines differed in their response to salinity.figure  <dig>
salinity response of slipt <dig> and slipt <dig> in arabidopsis.
germination percentage  and survival percentage  of slipt <dig>  and slipt <dig>  arabidopsis ipt <dig> complemented plants.
**significantly lower germination and survival percentages compared to ipt <dig> ko.
***significantly lower survival percentages compared to wt and ipt <dig> ko.
primary root length of arabidopsis slipt <dig> and slipt <dig> complemented plants.
*significantly longer primary root compared to wt.
**significantly shorter primary root compared to wt.
***significantly shorter primary root compared to ipt <dig> ko plants .
to assess whether salt treatment affected ck metabolism in arabidopsis slipt <dig> and slipt <dig> complemented plants, ck contents and gene expression profiles of ck biosynthetic genes were investigated on control and salt medium .
generally, in both types of media, the total content of cks of arabidopsis slipt <dig> and slipt <dig> complemented plants exceeded that of ipt <dig> ko plants.
however, in comparison to wt plants, arabidopsis ipt <dig> complemented plants showed lower ck concentrations in transgenic lines with exception of the 9at line on salt medium .
proportionally, ck-n-glucosides accumulated strongly in all tested variants , with ip7g as the predominant metabolite regardless of growth medium .
salt treatment substantially increased the levels of ck free bases and ribosides, while tz-type cks were mainly enhanced among the ck groups .figure  <dig>
cks accumulation in arabidopsis
ipt3
complemented plants.
total endogenous ck levels of slipt <dig>  and slipt <dig>  ipt <dig> ko complemented plants divided according to the chemical structure  and contents of bioactive cks .
relative expression of ck biosynthetic genes in arabidopsis ipt <dig> ko mutants complemented plants cultivated on the control medium  and on the salt-containing  medium .
actin and ef <dig> were used as internal controls for normalization of atipt transcript levels.
data represent means and sd of two replicates.
*statistically significant difference from ipt <dig> ko .
**statistically significant difference from ipt <dig> ko after Šidák correction for testing of multiple lines .
the expression profiles of ck biosynthetic genes were investigated in arabidopsis seedlings.
no expression in vegetative phase of development was detected for atipt <dig>  atipt <dig>  and atipt <dig>  as had been previously observed  <cit> .
on the control medium, complemented plants showed differential expression of the remaining ipt genes compared to wt or ipt <dig> ko plants.
atipt <dig> and atipt <dig> were repressed in arabidopsis slipt <dig> complemented plants, while their expression increased in arabidopsis slipt <dig> complemented plants, in comparison with wt or ipt <dig> ko plants .
salinity strongly downregulated atipt <dig> and atipt <dig> expression, while it increased expression of atipt <dig> in wt, ipt <dig> ko and slipt <dig> complemented plants .
in slipt <dig> complemented plants, genes were differentially regulated depending on the transgenic line considered .
these results demonstrate that salinity differently affects ipt genes expression and ck status in arabidopsis, and that slipt <dig> and slipt <dig> show distinct regulatory mechanisms in heterologous system.
therefore we propose that arabidopsis plants respond to salt stress by the elevation of ck levels, with predominant accumulation of n-glucosides suggesting overabundance of cks that is followed by downregulation of some ipt encoding genes to maintain ck homeostasis.
slipt <dig> and slipt <dig> are differently expressed in tomato organs and participate in cytokinin homeostasis
the expression patterns of slipt <dig> and slipt <dig> in various tomato vegetative and reproductive organs from plants cultivated in the greenhouse under normal growth conditions, including three stages of fruit development and ripening, were investigated by qrt-pcr .
in general, slipt <dig> transcripts were much more abundant than slipt <dig> ones in all the tested organs.
both genes were expressed in young and old leaves, roots and stems of tomato plants.
in vegetative tissues, slipt <dig> transcripts were weakly detected in young leaves but highly accumulated in stems, roots and old leaves.
in contrast, slipt <dig> was preferentially expressed in young leaves .
in reproductive tissues, slipt <dig> transcripts were abundant in buds and flowers, then rapidly decreased during tomato fruit maturation with higher levels of transcripts in the breaker stage.
slipt <dig> was preferentially expressed in buds, then continuously declined until the breaker stage and reached its peak expression at the red stage of tomato fruit .
taken together, these data indicate that slipt <dig> and slipt <dig> display distinct expression profiles during tomato development.figure  <dig>
qrt-pcr analysis of
slipt3
and
slipt4
expression.
spatio-temporal expression profile during tomato plant development in vegetative organs  and reproductive organs .
regulation of slipt <dig> and slipt <dig> expression by ck treatment: effects of tz  treatment on slipt <dig> and slipt <dig> expression  and the expression levels of ck response regulators slarr <dig>  slarr <dig> and slarr12
.
actin and gapdh were used as internal controls for normalization of slipt <dig> and slipt <dig> transcript levels.
data represent means and sd of three replicates.
*statistically significant difference from time  <dig> h .
**statistically significant difference from time  <dig> h after Šidák correction for testing of multiple points .
yl, young leaves; ol, old leaves; ro, roots; st, stem; bud, bud; flo, flower; gre, green stage; bre, breaker stage; red, red stage of tomato development.
in order to clarify a putative feedback regulation by cks on slipt <dig> and slipt <dig> expression, wt tomato roots from hydroponic plants were treated with tz.
ck treatment gradually repressed slipt <dig>  and slipt <dig>  expression, the inhibitory effect being most noticeable  <dig> h after treatment onset.
however, slipt <dig> transcripts then started to accumulate again, which was not the case for slipt <dig> .
likewise, the exogenous application of tz strongly induced expression of the ck negative-feedback arr type-a slarr <dig>  in contrast to its more discrete effect on arr type-b slarr <dig> and slarr <dig> .
altogether, these data suggest a regulation of slipt <dig> and slipt <dig> transcripts accumulation through plant endogenous ck status, in addition to the feedback regulation via the ck signaling pathway.
overexpression of slipt <dig> strongly impacts tomato phenotype and cytokinin status
to understand the impact of slipt <dig> and slipt <dig> overexpression on ck metabolism and verify the enzymes activity in planta, we attempted to generate transgenic lines constitutively expressing slipt <dig> or slipt <dig>  several 35s::slipt <dig> transgenic lines were able to develop and survive repotting into soil, while no 35s::slipt <dig> tomato plants could be regenerated from transgenic calli in vitro.
for further characterization, three 35s::slipt <dig> transgenic lines  were selected, with transgene expression in leaves increasing up to 14-fold  compared to wt .figure  <dig>
relative expression of ck metabolic and response genes in tomato young leaves of
35s::slipt3
and wt plants.
transcript abundance of ck biosynthetic genes , genes involved in ck signaling  and degradation  pathways.
actin and gapdh were used as internal controls for normalization of candidate genes expression.
data represent means and sd of two replicates.
*statistically significant difference from wt .
**statistically significant difference from wt after Šidák correction for testing of multiple lines .
the phenotype of 35s::slipt <dig> tomatoes substantially differed from that of the wt plants.
slipt <dig> overexpression induced plant dwarfism, release from apical dominance with a branchy phenotype, thick and ligneous stems, thicker leaves with punctual shape modification and shorter internodes ).
a significant yellowing of leaves, as well as accumulation of anthocyanin, was also observed.
additionally, despite inflorescence formation, no flowers were visible in most of slipt <dig> transgenic lines ) compared to wt plants ).
only a single transgenic line  showing the weakest phenotype  was able to develop normal fruits containing seeds.
generally, during several months of cultivation, fruit formation was either significantly reduced or completely aborted.
when fruits did develop, ripening was incomplete in transgenic plants.
taken together, these observations indicated that constitutive overexpression of slipt <dig> strongly affects tomato plant development, morphogenesis and reproduction.figure  <dig>
phenotype of t0-generation
35s::slipt3
tomato transformants.
transformants showed dwarfed aerial part with a branching phenotype , modified leaf shape  and flower inflorescence  compared to wt plants  .
endogenous ck levels in tomatoes divided according to the chemical structure  and contents of bioactive cks .
wt: wild type; l <dig>  l <dig> and l9: independently regenerated 35s::slipt <dig> transformants.
data represent means and sd of two replicates.
*statistically significant difference datasets from wt .
**statistically significant difference from wt after Šidák correction for testing of multiple lines .
the strong effects of slipt <dig> overexpression on tomato phenotype were simultaneously reflected in ck metabolism.
the total ck contents extracted from young and fully expanded tomato leaves increased up to 12-fold in 35s::slipt <dig> lines  compared to wt plants.
proportionally, ck-n-glucosides represented the main group of cks, especially in 35s::slipt <dig> lines, with ip7-glucoside  being the predominant metabolite .
in addition, the highest concentration of tz was observed in wt plants .
interestingly, the ck storage forms  only slightly increased  in all transformants .
these results show that slipt <dig> overexpression leads to a dramatic increase in cks, particularly to the enhanced formation of ip7g throughout n7-glucosylation pathway, which seems to be the major metabolic pathway for bioactive cks inactivation.
indeed, no significant differences in ck oxidase/dehydrogenase  activity were found between the control and transgenic tomatoes .
slipt <dig> overexpression modifies cytokinin-related genes expression and plant tolerance to salinity
to elucidate the role of ck biosynthesis , signaling  and catabolism  related genes in the response to slipt <dig> overexpression in tomatoes, the expression patterns of corresponding genes were determined by qrt-pcr in young leaves of transgenic tomatoes and wt plants.
in 35s::slipt <dig> lines, the transcript abundance of slipt <dig>  was higher than in wt, whereas lower expression of slipt <dig> was observed in all transgenic lines .
likewise, upregulation of slarr <dig>  functioning in feedback regulatory loops of cks , and downregulation of ck deactivating genes slckx <dig> and slckx <dig>  were observed in all transformants, relative to wt plants.
thus, ck-related genes expression analysis revealed upregulation of some genes involved in ck biosynthesis  and signaling , but downregulation of genes involved in ck degradation pathways .
we hypothesized that the upregulation of some ck metabolic genes and enhancement of cks content could be elementary prerequisites of tomato salt stress tolerance.
as only l <dig> could successfully reproduce, t <dig> homozygous transformants from this transgenic line were further analyzed for their tolerance to salinity.
for this purpose, l <dig> and wt lines were cultivated in vitro for  <dig> weeks on the  <dig> mm nacl medium.
compared to wt plants, 35s::slipt <dig> l <dig> plants exhibited a larger shoot with extending leaves, whereas wt plants showed more compact shoot and smaller leaves as a result of limited growth in presence of salt .
subsequently, the effects of salt stress induced either by  <dig> mm and  <dig> mm nacl concentration on photosynthesis and nutrients balance were evaluated in wt and l <dig> by measurements of photosynthetic pigments and macronutrients contents.figure  <dig>
effect of salt stress on wt and
35s::slipt3
tomato plants cultivated on control and salt-containing growth media.
phenotype of wt and 35s::slipt <dig>  transgenic plants grown  <dig> weeks on the  <dig> mm nacl medium.
chlorophyll content , carotenoid content , cation concentration  and k+/na+ ratio  in  <dig> weeks-old tomato developing leaves of wt and trangenic plants.
sum vaz, sum violaxanthin, antheraxanthin and zeaxanthin.
data represent means and sd of two replicates for photosynthetic pigments while three replicates represent data from cation concentration.
1statistically significant difference from the same treatment of wt .
2statistically significant difference from ms medium within the same line , 2statistically significant difference from ms medium within the same line after Šidák correction for testing of multiple nacl concentrations .
in wt plants, chlorophyll a  and chlorophyll b  significantly decreased with the addition of nacl into the growth media.
in l <dig> plants, the content of both types of chlorophyll was significantly lower on control medium relative to wt, but remained nearly constant on nacl containing media, and a significant decrease in chl a content was only apparent on  <dig> mm nacl .
likewise, the levels of carotenoids , especially lutein and β-carotene displaying photoprotective roles in photosynthesis, exhibited almost identical contents in 35s::slipt <dig> l <dig> plants regardless of growth media.
in contrast, wt plants showed a significant decrease of β-carotene on both types of nacl media, and of lutein on  <dig> mm nacl.
increasing contents of car involved in the xanthophyll cycle, violaxanthin , antheraxanthin  and zeaxanthin  were as well observed in presence of salt in both wt and transgenic tomatoes, significant for wt at  <dig> mm nacl and 35s::slipt <dig> grown on both concentrations of salt .
shoot sodium , potassium , magnesium  and calcium  concentrations were assessed in plants exposed to  <dig> ,  <dig> mm and  <dig> mm nacl .
as observed in figure 9d, na+ and k+ concentrations were similar in wt and l <dig> under control conditions of growth.
salinity induced na+ accumulation and substantially reduced k+ contents in both types of tomato plants.
however, na+ accumulated to a lesser extent in l <dig>  than in wt  on the  <dig> and  <dig> mm nacl medium, respectively.
similarly, k+ concentration was significantly higher in l <dig>  than in wt  in presence of  <dig> and  <dig> mm nacl, respectively, and these results are also visible by the k+/na+ ratio, which remained significantly higher in l <dig> than in wt in salt stress conditions .
finally, no difference of ca2+contents between wt and transgenic plants was noticed, while the mg2+ concentration was significantly higher in transgenic tomatoes on control and  <dig> mm nacl conditions, compared to wt .
overall, plant morphological changes in presence of salt, roughly constant levels of photosynthetic pigments, especially chl a, chl b, lutein and β-carotene in 35s::slipt <dig> tomatoes regardless on the growth medium and higher k+ contents in presence of nacl indicate that slipt <dig> overexpression could raise plant tolerance to salt stress.
both slipt <dig> and slipt <dig> are involved in salt stress response
to further understand the participation of slipt <dig> and slipt <dig> in salt stress response, their expression pattern was examined by qrt-pcr in roots, young leaves and old leaves of tomato wt plants exposed to salinity  for  <dig> h. in roots , which are the first organ perceiving salinity, slipt <dig> and slipt <dig> shared very similar expression profiles.
a rapid decrease in transcript levels of both genes within  <dig> h after salt exposure was followed by a very small peak at  <dig> h for both genes, before a new decline.
in young leaves, slipt <dig> and slipt <dig> showed partially divergent transcript accumulation profiles .
indeed, slipt <dig> transcripts gradually and regularly increased with incubation time.
in contrast, slipt <dig> was repressed  <dig> -fold as rapidly as  <dig> h after stress initiation.
this repression seemed transient, however, as a slight increase in slipt <dig> transcripts was visible  <dig> h after stress onset .
similar expression profiles were observed in old leaves .
these results indicate that slipt <dig> and slipt <dig> directly participate in salt stress response in tomato via a differential modulation of their expression, depending on the organ and duration of the stress.figure  <dig>
salinity response of
slipt3
and
slipt4
in tomato wt plants.
analysis of slipt <dig> and slipt <dig> expression levels in tomato wt roots , young leaves  and old leaves  in response to salt stress .
actin and gapdh were used as internal controls for normalization of slipt <dig> and slipt <dig> transcript levels.
data represent means and sd of two replicates.
*statistically significant difference from time  <dig> h .
**statistically significant difference from time  <dig> h after Šidák correction for testing of multiple time points .
discussion
ck homeostasis during plant development and in response to changing environmental conditions is, among other mechanisms, properly maintained by the activity of ck biosynthetic enzymes, ipts.
it was previously reported that root-localized ipt expression essentially improved salt stress tolerance in tomato  <cit> .
however, the genes involved and the mechanisms of cks regulation under salt stress conditions have not yet been elucidated.
therefore, based on functional characterization of slipt <dig> and slipt <dig> we provide insights into both genes involvement in the response to salt stress in tomato plants.
tomato slipt <dig> and slipt <dig> are non-redundant functional isopentenyltransferases
in the present study, we have demonstrated that tomato slipt <dig> and slipt <dig> encode two functional isopentenyltransferases .
multiple alignment of proteins sequences and phylogenetic analysis revealed that slipt <dig> and slipt <dig> are closely related to arabidopsis ortholog atipt <dig> and atipt <dig> , as previously reported  <cit> .
interestingly, we observed identical chloroplast localization of gfp-tagged slipt <dig> protein in tomato leaf cells , as described in arabidopsis where atipt <dig> was localized to plastids in roots and leaf cells  <cit> .
these data indicate that slipt <dig> and atipt <dig> partly share similar function and protein subcellular localization.
we therefore speculate that slipt <dig>  and probably also slipt <dig>  due to the presence of a chloroplastic transit peptide as well, may have other comparable regulatory mechanisms for protein chloroplast targeting such as farnesylation for instance, as was demonstrated for atipt <dig>  <cit> .
distinct spatio-temporal expression profiles of slipt <dig> and slipt <dig> have been observed in tomato vegetative organs and during fruit onset, development and ripening, indicating that these two genes have non-redundant functions .
in general, slipt <dig> expression was lower relative to that of slipt <dig> and showed its highest levels in young leaves and red fruits whereas slipt <dig> substantially predominated in the remaining tested organs.
our data are in agreement with a previous report describing very low transcript abundance of slipt <dig> in all tomato tested organs with flowers exhibiting the highest slipt <dig> expression, and a greater accumulation of slipt <dig> transcripts, especially in the very early stages of flower and fruit development  <cit> .
interestingly, although slipt <dig> is less expressed than slipt <dig> in tomato, slipt <dig> showed ten times greater activity in vitro than slipt <dig>  which may explain why no transgenic plants could be regenerated from calli.
indeed, slipt <dig> is probably at the origin of a greater hormonal imbalance than slipt <dig>  which may have impaired proper shoot and root systems regeneration.
differences between slipt <dig> and slipt <dig> in organ expression specificity point out their distinct physiological roles in tomato developmental processes.
however, it seems rather difficult to assign a specific role to each ipt during tomato plant development, due to their overlapping functions.
indeed, although atipt <dig> is one of the three most expressed ipt genes in arabidopsis during the vegetative phase, ipt <dig> knock-out mutant exhibited no distinguishable phenotype from wt plants, indicating functional redundancy between the arabidopsis ipt proteins  <cit> .
therefore, none of the visible phenotypes or characteristics in slipt <dig> and slipt <dig> complemented arabidopsis ipt <dig> knock-out mutants were apparent in comparison to wt plants under control conditions .
ectopic expression of slipt <dig> alters tomato phenotype and ck metabolism
overexpression of slipt <dig> under 35s camv promoter and consequent ck over-accumulation had extensive negative effects on tomato growth under control conditions , resulting in dwarf phenotype and delayed senescence, substantially limiting formation of tomato generative organs.
only one transgenic line  showing the weakest phenotypic differences from wt was able to produce seeds .
a similar effect of ck overabundance in flowering delay was reported in stably transformed tobacco plants with 35s::mdipt3a  <cit> .
likewise, phenotype alterations such as a strong delay in growth, shorter rosettes, serrated leaves and decreased apical dominance were observed in arabidopsis plants overexpressing maize zmipt <dig>  <cit> .
in contrast, no changes in flowering time were evident in arabidopsis and tobacco transgenic plants expressing ipt under hsp <dig>  <cit> , underlining the crucial role of cks accumulation and homeostasis during flowering.
we found out that constitutive expression of slipt <dig> increased ck production in tomatoes by up to 12-fold relative to wt plants, primarily ip7g .
similarly, high concentrations of ip7g in arabidopsis complemented plants cultivated on both control and salt media suggest that the formation of ck-n7-glucosides may be a sufficient pathway for downregulation of bioactive cks because no significant enhancement of ckx activity was detected in 35s::slipt <dig> tomatoes when compared to wt .
in accordance with our results, ipt overexpression resulted in increased levels of bioactive ip, tz and their ribosides in tomato  <cit>  and tobacco  <cit> .
however, detailed data regarding all ck derivatives were not reported in these previous studies.
based on the observation that iprmp/iprdp/iprtp are the first products in ck biosynthesis  <cit> , that ip and ipr are the best substrates for ckx activity  <cit>  and are in massive amounts metabolized to ck-n-glucoside forms , we conclude that ip-type cks have a prominent role in fast regulation and balancing of the ck metabolism.
slipt <dig> and slipt <dig> participate in salinity response in tomato and arabidopsis
expression analyses of slipt <dig> and slipt <dig> in tomato vegetative organs confirmed the participation of both genes in response to salt stress.
after stress onset, salinity evoked repression of slipt <dig> and slipt <dig> in roots, and differently affected slipt <dig> and slipt <dig> in young and old leaves.
after  <dig> to  <dig> h of exposure to salinity, increasing levels of both transcripts were detected in young leaves, suggesting preferential ck biosynthesis in photosynthetic organs and thus underlining cks stress-prevention and anti-senescence properties .
similarly, an augmentation of ipt expression was apparent both in maize leaves and roots following longer exposure to salinity whereas shorter salt treatment  induced a repression of ipt encoding genes  in maize leaves  <cit> .
likewise, two weeks-old arabidopsis plants showed downregulation of atipt <dig> and atipt <dig> as early as  <dig> h after salt treatment, whereas the expression of atipt <dig> and atipt <dig> was elevated.
conversely, with longer salt stress, the majority of atipt transcripts were repressed  <cit> .
together, these data indicate a dynamic regulation of ck biosynthesis/metabolism over the stress period.
moreover, multiple putative stress-responsive cis-regulatory elements were identified in the promoter regions of bripts  promoters, pointing to the response of these genes to stress stimuli and a possible response to unfavourable conditions via changing ck status  <cit> .
similarly in this study, an in silico analysis of slipt <dig> and slipt <dig> promoters  <cit>   revealed the presence of putative stress response elements and many arr binding cis-elements, indicating at least for slipt <dig> the existence of salt stress tolerance mechanisms similar to those in bripts.
our findings in tomato plants illustrated that the regulation of slipt <dig> and slipt <dig> in response to salt stress is organ specific, depends on the duration of stress exposure and potentially also on the presence of cis-regulatory elements in the promoters of ck-related genes.
in addition, diverse roles of both tomato slipts in salt stress responses have been characterized in arabidopsis ipt <dig> ko complemented plants under longer period of salt treatment .
transgenic plants ectopically expressing slipt <dig> under 35s promoter showed superior above-ground growth after several weeks on salt medium, relative to wt plants .
beneficial effects of cks overproduction in response to salt stress were reported in tomato  <cit> .
cks elevation enhanced shoot growth and prevented senescence under salinity conditions due to the specific induction of ipt in roots by a heat-shock promoter .
likewise, higher ck concentration was associated with salt tolerance in tomatoes overexpressing a cysteine-2/histidine-2-type zinc finger transcription factor  <cit> .
similarly, expression of agrobacterium tumefaciens ipt under the stress inducible promoter rd29a resulted in higher salinity tolerance in transgenic tobacco  <cit> .
using specific promoters for ipt overexpression also successfully improved tolerance to drought in rice  <cit> , peanut  <cit> , cassava  <cit> , tobacco  <cit>  and to the cold stress in tobacco  <cit> .
on the other hand, tobacco plants with reduced ck contents showed smaller osmotic potential and higher drought resistance than wt plants  <cit> .
likewise, arabidopsis knock-out mutants for two ck histidine kinase receptors  showed tolerance to drought and salinity  <cit> .
aspects of plant phenotype, such as root architecture modifications and growth inhibition in transgenic plants, influenced by imbalance between cks accumulation and distribution can partly explain tolerance to various environmental stresses.
thus, root-specific ckx overexpression enhanced root system development and consequently increased drought tolerance, nutrient uptake and leaf nutrient enrichment in arabidopsis  <cit> .
consequently, it is still difficult to estimate the precise nature of ck effects due to the complexity of phytohormones metabolism and other cross-talk networks.
salinity tolerance of 35s::slipt <dig> plants is correlated, among others, with improved nutrients balance and retention of the photosynthesis capacity, in addition to the senescence-protective effects of the photosynthetic pigments .
indeed, salinity strongly impacts tomato plants ionic status, as na+ concentration considerably increases in leaves, inducing a premature senescence, while k+ content differently declines depending on the leaf age  <cit> .
transgenic tomato plants hsp70::ipt showing improved growth and final yield under long-term salinity treatment consequently accumulate less na+ in leaves and roots than wt, but higher k+ contents, underlining the importance of the limitation of toxic ions accumulation for plant development  <cit> .
in addition, salinity strongly affects plant photosynthesis, and salt-induced reduction in photosynthetic pigments such as chl and car have been reported in several studies  <cit> .
variations in chl and car contents under different salt concentrations and time exposure were observed in three varieties of tomato cultivars, suggesting that sensitivity to salt stress depends on plant genotype  <cit> .
our results indicate that the contents of chl a and ch b as well as lutein and β-carotene are maintained in 35s::slipt <dig> plants at the same levels regardless of the growth media in contrast to wt, in which salt treatments decreased both chl and car contents .
similarly, transgenic tomatoes overexpressing ipt within the root zone maintained their photosynthesis ability under long-term salinity treatement  <cit> , while transgenic tobacco expressing stress-inducible promoter rd29a-ipt showed only slight decrease in chl contents in comparison to wt plants  <cit> .
significant accumulation of pigments involved in the xanthophyll cycle in 35s::slipt <dig> plants exposed to salt may reflect higher intensity in photoprotective function of car in transgenic plants than in wt plants.
in summary, our results indicate that constitutive expression of slipt <dig> induces several changes in plant growth, nutrients accumulation, hormonal metabolism and photosynthesis maintenance, leading to enhanced tolerance to salt stress.
mechanism of slipt <dig> and slipt <dig> regulation under salt and ck treatments
the comprehensive mechanism of ck regulatory feedback loop is supported by our findings that exogenous application of tz inhibited the expression of slipt <dig> and slipt <dig> within  <dig> h, enhanced the expression of negative-feedback regulator type-a slarr <dig> and slightly moderated the expression levels of ck primary response genes type-b slarr <dig> and slarr <dig> .
comparable data reporting downregulation of the ipt genes after ck treatment have been reported for chinese cabbage  <cit>  and maize  <cit> .
the connection between ck homeostasis maintenance via type-b response regulators and salinity response was reported in arabidopsis where arr <dig> and arr <dig> were shown to control sodium accumulation in shoots by regulating expression of genes encoding high-affinity k+ transporter 1; <dig> responsible for na+ exclusion from the root xylem  <cit> .
these data suggest that ck homeostasis may be effectively regulated, not only by cks molecules, but also environmental stimuli.
taken together, we have shown the mechanism of slipt <dig> and slipt <dig> response to early salt stress and their feedback regulation by exogenous ck treatment in tomato plants.
based on the data obtained in this study, we propose a model illustrating the complexity of ck networks in tomato plants in response to salinity .
in this model, we only included relations supported by our experimental data.
we outline an organ specific down-regulation of slipt <dig> and slipt <dig> transcripts immediately after stress onset, followed by transcripts increase with the duration of the stress treatment.
the complexity of ck networks is demonstrated by the repression of slipt <dig> and slipt <dig> after exogenous application of tz, while an overabundance of cks  in 35s::slipt <dig> may ensure a fitter phenotype under salt stress conditions.
similarly, the response to salinity by slipt <dig> and slipt <dig> was paralleled in arabidopsis ipt <dig> ko complemented plants where salinity resulted in elevation of cks  with successive downregulation of atipts to maintain ck homeostasis .
CONCLUSIONS
in summary, we have characterized slipt <dig> and slipt <dig> response to early salt stress in tomato, and identified slipt <dig> as a key player in the ck metabolism of tomato plants under salinity conditions.
our results contribute to the understanding of ck regulation at the molecular level and provide a potentially useful tool to obtain and improve high-quality stress-tolerant crops in agriculture.

 tomato is one of the most popular vegetable species grown world-wide because of its edible fruit and agronomic importance. nevertheless, its production is frequently threatened by environmental stresses such as drought and salinity. high salt concentration reduces tomato germination, leaf number and area, slows down shoot and root growth and increases root/shoot ratio, induces leaf senescence, and ultimately impairs crop production. salinity imposes both an osmotic stress due to water shortage, followed by an ionic stress resulting from disproportionate nutrients accumulation and an augmentation of toxic ion concentrations, such as sodium. modifications of plant physiological processes following salinization are partly attributed to alterations in phytohormone metabolism. this may be true especially for cytokinins. indeed, under long-term salt stress the level of bioactive cks in tomato plants was reduced down to 50% both in roots and leaves. the ability to control cell division and differentiation, root growth, leaf senescence, apical dominance, branching, flower and seed development as well as germination and nutrients uptake into sink organs defines cks as essential regulatory substances in plants. chemically, cks are derivatives of adenine substituted at the n position either with isoprenoid or an aromatic side chain. both isoprenoid and aromatic cks are present in plants in bioactive forms, as free bases and corresponding nucleosides and nucleotides, and in non-active or storage forms as conjugates with glucose for instance. the typical representatives of isoprenoid cks are derivatives of trans-zeatin, cis-zeatin, dihydrozeatin and n6-adenine, occuring throughout the plant kingdom. the first step of ck biosynthesis is performed by isopentenyltransferases. plant ipts belong to multigenic families, chiefly described in arabidopsis and in maize, for instance. recently, ipt genes have also been identified in tomato, and their expression patterns specifically investigated during fruit set and development. ipts catalyze the transfer of the isoprenoid moiety from precursors dimethylallyl diphosphate or -4-hydroxy-3-methyl-but-2-enyl diphosphate to adenine nucleotide forms. modulation of ck contents through modification of ipt expression strongly impacts plants development. indeed, while overexpression of ipt in plants resulted in faster shoot formation, shorter internodes, loss of apical dominance, delay of leaf senescence, higher photosynthetic rates and accumulation of tz and its riboside, arabidopsis ipt deficient plants showed strong inhibition of shoot growth, elongation of primary and lateral roots, and reduction of tz and ip contents. recent progress in genetic engineering of cks enabled to control plant ck contents, affected plant traits, increased yield production and improved plant adaptation to enviromental stresses such as salinity. indeed, responsiveness of arabidopsis ipt genes to salt stress has been demonstrated by transcriptome analyses, where upregulation of atipt atipt and atipt and downregulation of atipt atipt atipt and atipt were reported. regulation of ipt expression through inducible promoter in tomato roots improved tolerance to salt stress and led to higher yield compared to the wild-type plants. similarly, plant salinity tolerance was enhanced in transgenic cotton expressing ipt under control of a cysteine proteinase promoter, delaying the salt-induced senescence of leaves. positive correlation between ipt expression and salt resistance has been demonstrated in transgenic tobacco by introducing agrobacterium tumefaciens ipt under control of the stress-inducible promoter rd29a as well. in addition to ck biosynthesis, ck signaling steps are also critical for plant response to salinity. cks are perceived at the plasma membrane by specific receptors, and the signal is transduced via type-b arabidopsis response regulators controlling transcription of type-a arrs, which act as negative feedback regulators of ck signaling. arr transcription factors respond in different ways to salt stress and were reported to regulate sodium accumulation in arabidopsis. moreover, a transcriptome analysis of arabidopsis ck deficient ipt, mutants exposed to salinity revealed the importance of ck regulation on stress-responsive signaling pathways, even under normal conditions of growth. although cks assume crucial functions in tomato salt tolerance, data concerning the underlying molecular cues involved in this process remain unclear. to better understand salt impact on ck metabolism in tomato, a thorough characterization of genes coding for ipts and their response to salinity is required. the present study reports the functional characterization of two tomato ipt encoding genes, slipt and slipt both in vitro and in planta. spatio-temporal expression profiles during plant development, enzymatic activity of both proteins and their involvement in ck biosynthesis were determined. likewise, slipt and slipt participation in response to salt stress was investigated in arabidopsis and tomato.  identification of two putative non-redundant tomato isopentenyltransferases to identify ipt encoding genes in tomato, arabidopsis ipt1- coding sequences were used to screen the tomato genome database. among the selected expressed sequence tags, two were apparently encoding full length proteins and could be amplified by pcr. for the first gene, a bp coding sequence was cloned, including a bp open reading frame defining a amino acid polypeptide of kda. this gene was recently annotated as slipt. for the second gene, a bp coding sequence was amplified, exhibiting a bp orf predicted to encode a a protein of kda. this gene was classified as slipt.figure identification of slipt and slipt full-length protein sequence alignment of slipt slipt and arabidopsis atipt and atipt identical residues are colored in black and conserved residues in dark gray. the isopentenyltransferase domain is labeled. phylogenetic relationships between slipt, slipt and related proteins of cucumis sativus csipt, ipomoea nil inipt, malus domestica mdipt, fragaria vesca fvipt, arabidopsis thaliana atipt, atipt, atipt, atipt, atipt and atipt, glycine max gmipt, zea mays zmipt, zmipt and zmipt, solanum lycopersicum slipt, slipt, slipt and slipt, nicotiana tabacum ntipt. the phylogenetic tree was constructed according to the neighbor-joining method, using mega. the percentage of reliability of each branch point of the rooted tree, as assessed by the analysis of trees, is shown on the branch stem. slipt is located on tomato chromosome while slipt is situated on chromosome in addition, slipt includes a bp intron localized in the 5′ utr region. slipt and slipt display 52% identity and 72% similarity. alignment of slipt and slipt proteins with arabidopsis ipt and ipt indicates that the ipt catalytic domain represents almost the entirety of the proteins, with exception of the n- and c-termini. a phylogenetic analysis of slipt and slipt and nineteen additional orthologs belonging to different species was constructed by means of the neighbour-joining method using full-length amino acid sequences. it indicated that slipt mainly clusters with soybean gmipt and arabidopsis atipt while slipt clusters with arabidopsis atipt and japanese morning glory inipt. except from arabidopsis and maize, full characterization of ipt enzymes in other plant species is missing. tomato slipt and slipt show isopentenyltransferase activity in vitro to examine the ability of slipt and slipt to catalyze ck biosynthesis, a functional analysis of both proteins was carried out. as slipt and slipt are highly insoluble when produced in bacteria, both proteins were produced in vitro. the enzymatic activity of slipt and slipt was determined using in vitro assays based on conversion of radiolabeled adenylated substrates in the presence of dmapp to isopentenylated products. both enzymes were able to convert tritium-labeled amp/adp/atp to the corresponding iprmp/iprdp/iprtp in time-dependent manner. the prevailing metabolites formed by both slipt and slipt were iprmp and iprdp, with almost 10-fold higher catalytic activity shown by slipt compared to slipt these results clearly demonstrate that slipt and slipt display ck biosynthetic activity in in vitro conditions.figure in vitro determination of dmapp:amp, adp and atp isopentenyltransferase activity of slipt and slipt proteins. adp: adenosine diphosphate, atp: adenosine triphosphate, ipmp: isopentenyladenosine-5′monophosphate; ipdp: isopentenyladenosine-5′-diphosphate, iptp: isopentenyladenosine-5′-triphosphate. note that vertical scales are not the same for slipt and slipt subcellular localization of slipt and slipt4 an analysis of the deduced slipt and slipt sequences revealed the presence of chloroplast transit peptides. to investigate protein subcellular localization, slipt and slipt cdna were fused at their c-terminal end to green fluorescent protein coding sequence, placed under cauliflower mosaic virus 35s promoter and used to agro-infiltrate tomato leaves. as observed in figure , despite a strong auto-fluorescence, slipt showed localization predominantly in chloroplasts and in the cytosolic area surrounding the chloroplast. indeed, gfp fluorescence colocalized with chlorophyll fluorescence in this cellular compartment. in contrast, free gfp was restricted to the cytoplasm ), while no fluorescence at nm was detected in untransformed tomato leaves ). despite several attempts, we could not establish subcellular localization for slipt and no gfp signal of the recombinant protein could be detected in transformed tomato cells. these data show that at least slipt is localized in the chloroplast and in the cytosolic area surrounding chloroplasts.figure subcellular localization of slipt4-gfp by tomato leaves agroinfiltration. slipt4-gfp fluorescence and chlorophyll/gfp fluorescence, respectively. control gfp fluorescence and chlorophyll/gfp fluorescence, respectively. control untransformed tomato leaves and chlorophyll autofluorescence, respectively. bar = µm. characterization of slipt and slipt in arabidopsis ipt mutants in parallel, tomato slipt and slipt were functionally characterized in planta via complementation of arabidopsis ipt knock-out mutants. five days after sowing, the arabidopsis wild-type plants, ipt ko mutants and arabidopsis ipt ko complemented plants showed a similar phenotype on half-strength ms medium. we thus suggested to determine the function of slipt and slipt in arabidopsis complemented ipt ko mutants grown on salt medium and to follow their response to salinity. based on the highest phenotype discrimination, mm nacl medium was used for further testing of seed germination and plant survival percentages, primary root elongation, seedlings cks content and ck-related genes expression profiles. on the control medium, germination and survival frequencies were not significantly different in between the variants reaching 100%. on the saline medium, seed germination percentage was reduced to 67% for both lines of arabidopsis slipt complemented plants, significantly less than that of ipt ko and wt plants. however, in arabidopsis slipt complemented plants, seed germination reached 89% and 90%, respectively, while wt seeds displayed 78% and ipt ko 93% of germination rate, respectively. the same trend was detected for seedlings’ survival percentage on mm nacl. similarly, primary root elongation was estimated by screening das arabidopsis slipt and slipt complemented plants. on control medium, the primary root length among the 9at and 10at lines significantly differed compared to wt while on the salt-containing medium, no significant changes between the slipt complemented and wt plants were observed. arabidopsis plants complemented with slipt showed significantly longer primary root than wt on control medium, while antagonistic effects were detected on salt-containing medium. these results demonstrate that both tomato ipt enzymes are functional in heterologous system. however, in reaction to salt stress the slipt complemented plants displayed the same characteristics as wt in germination and survival assays, while both of the slipt complemented lines differed in their response to salinity.figure salinity response of slipt and slipt in arabidopsis. germination percentage and survival percentage of slipt and slipt arabidopsis ipt complemented plants. **significantly lower germination and survival percentages compared to ipt ko. ***significantly lower survival percentages compared to wt and ipt ko. primary root length of arabidopsis slipt and slipt complemented plants. *significantly longer primary root compared to wt. **significantly shorter primary root compared to wt. ***significantly shorter primary root compared to ipt ko plants. to assess whether salt treatment affected ck metabolism in arabidopsis slipt and slipt complemented plants, ck contents and gene expression profiles of ck biosynthetic genes were investigated on control and salt medium. generally, in both types of media, the total content of cks of arabidopsis slipt and slipt complemented plants exceeded that of ipt ko plants. however, in comparison to wt plants, arabidopsis ipt complemented plants showed lower ck concentrations in transgenic lines with exception of the 9at line on salt medium. proportionally, ck-n-glucosides accumulated strongly in all tested variants, with ip7g as the predominant metabolite regardless of growth medium. salt treatment substantially increased the levels of ck free bases and ribosides, while tz-type cks were mainly enhanced among the ck groups.figure cks accumulation in arabidopsis ipt3 complemented plants. total endogenous ck levels of slipt and slipt ipt ko complemented plants divided according to the chemical structure and contents of bioactive cks. relative expression of ck biosynthetic genes in arabidopsis ipt ko mutants complemented plants cultivated on the control medium and on the salt-containing medium. actin and ef were used as internal controls for normalization of atipt transcript levels. data represent means and sd of two replicates. *statistically significant difference from ipt ko. **statistically significant difference from ipt ko after Šidák correction for testing of multiple lines. the expression profiles of ck biosynthetic genes were investigated in arabidopsis seedlings. no expression in vegetative phase of development was detected for atipt atipt and atipt as had been previously observed. on the control medium, complemented plants showed differential expression of the remaining ipt genes compared to wt or ipt ko plants. atipt and atipt were repressed in arabidopsis slipt complemented plants, while their expression increased in arabidopsis slipt complemented plants, in comparison with wt or ipt ko plants. salinity strongly downregulated atipt and atipt expression, while it increased expression of atipt in wt, ipt ko and slipt complemented plants. in slipt complemented plants, genes were differentially regulated depending on the transgenic line considered. these results demonstrate that salinity differently affects ipt genes expression and ck status in arabidopsis, and that slipt and slipt show distinct regulatory mechanisms in heterologous system. therefore we propose that arabidopsis plants respond to salt stress by the elevation of ck levels, with predominant accumulation of n-glucosides suggesting overabundance of cks that is followed by downregulation of some ipt encoding genes to maintain ck homeostasis. slipt and slipt are differently expressed in tomato organs and participate in cytokinin homeostasis the expression patterns of slipt and slipt in various tomato vegetative and reproductive organs from plants cultivated in the greenhouse under normal growth conditions, including three stages of fruit development and ripening, were investigated by qrt-pcr. in general, slipt transcripts were much more abundant than slipt ones in all the tested organs. both genes were expressed in young and old leaves, roots and stems of tomato plants. in vegetative tissues, slipt transcripts were weakly detected in young leaves but highly accumulated in stems, roots and old leaves. in contrast, slipt was preferentially expressed in young leaves. in reproductive tissues, slipt transcripts were abundant in buds and flowers, then rapidly decreased during tomato fruit maturation with higher levels of transcripts in the breaker stage. slipt was preferentially expressed in buds, then continuously declined until the breaker stage and reached its peak expression at the red stage of tomato fruit. taken together, these data indicate that slipt and slipt display distinct expression profiles during tomato development.figure qrt-pcr analysis of slipt3 and slipt4 expression. spatio-temporal expression profile during tomato plant development in vegetative organs and reproductive organs. regulation of slipt and slipt expression by ck treatment: effects of tz treatment on slipt and slipt expression and the expression levels of ck response regulators slarr slarr and slarr12. actin and gapdh were used as internal controls for normalization of slipt and slipt transcript levels. data represent means and sd of three replicates. *statistically significant difference from time h. **statistically significant difference from time h after Šidák correction for testing of multiple points. yl, young leaves; ol, old leaves; ro, roots; st, stem; bud, bud; flo, flower; gre, green stage; bre, breaker stage; red, red stage of tomato development. in order to clarify a putative feedback regulation by cks on slipt and slipt expression, wt tomato roots from hydroponic plants were treated with tz. ck treatment gradually repressed slipt and slipt expression, the inhibitory effect being most noticeable h after treatment onset. however, slipt transcripts then started to accumulate again, which was not the case for slipt. likewise, the exogenous application of tz strongly induced expression of the ck negative-feedback arr type-a slarr in contrast to its more discrete effect on arr type-b slarr and slarr. altogether, these data suggest a regulation of slipt and slipt transcripts accumulation through plant endogenous ck status, in addition to the feedback regulation via the ck signaling pathway. overexpression of slipt strongly impacts tomato phenotype and cytokinin status to understand the impact of slipt and slipt overexpression on ck metabolism and verify the enzymes activity in planta, we attempted to generate transgenic lines constitutively expressing slipt or slipt several 35s::slipt transgenic lines were able to develop and survive repotting into soil, while no 35s::slipt tomato plants could be regenerated from transgenic calli in vitro. for further characterization, three 35s::slipt transgenic lines were selected, with transgene expression in leaves increasing up to 14-fold compared to wt.figure relative expression of ck metabolic and response genes in tomato young leaves of 35s::slipt3 and wt plants. transcript abundance of ck biosynthetic genes, genes involved in ck signaling and degradation pathways. actin and gapdh were used as internal controls for normalization of candidate genes expression. data represent means and sd of two replicates. *statistically significant difference from wt. **statistically significant difference from wt after Šidák correction for testing of multiple lines. the phenotype of 35s::slipt tomatoes substantially differed from that of the wt plants. slipt overexpression induced plant dwarfism, release from apical dominance with a branchy phenotype, thick and ligneous stems, thicker leaves with punctual shape modification and shorter internodes ). a significant yellowing of leaves, as well as accumulation of anthocyanin, was also observed. additionally, despite inflorescence formation, no flowers were visible in most of slipt transgenic lines ) compared to wt plants ). only a single transgenic line showing the weakest phenotype was able to develop normal fruits containing seeds. generally, during several months of cultivation, fruit formation was either significantly reduced or completely aborted. when fruits did develop, ripening was incomplete in transgenic plants. taken together, these observations indicated that constitutive overexpression of slipt strongly affects tomato plant development, morphogenesis and reproduction.figure phenotype of t0-generation 35s::slipt3 tomato transformants. transformants showed dwarfed aerial part with a branching phenotype, modified leaf shape and flower inflorescence compared to wt plants. endogenous ck levels in tomatoes divided according to the chemical structure and contents of bioactive cks. wt: wild type; l l and l9: independently regenerated 35s::slipt transformants. data represent means and sd of two replicates. *statistically significant difference datasets from wt. **statistically significant difference from wt after Šidák correction for testing of multiple lines. the strong effects of slipt overexpression on tomato phenotype were simultaneously reflected in ck metabolism. the total ck contents extracted from young and fully expanded tomato leaves increased up to 12-fold in 35s::slipt lines compared to wt plants. proportionally, ck-n-glucosides represented the main group of cks, especially in 35s::slipt lines, with ip7-glucoside being the predominant metabolite. in addition, the highest concentration of tz was observed in wt plants. interestingly, the ck storage forms only slightly increased in all transformants. these results show that slipt overexpression leads to a dramatic increase in cks, particularly to the enhanced formation of ip7g throughout n7-glucosylation pathway, which seems to be the major metabolic pathway for bioactive cks inactivation. indeed, no significant differences in ck oxidase/dehydrogenase activity were found between the control and transgenic tomatoes. slipt overexpression modifies cytokinin-related genes expression and plant tolerance to salinity to elucidate the role of ck biosynthesis, signaling and catabolism related genes in the response to slipt overexpression in tomatoes, the expression patterns of corresponding genes were determined by qrt-pcr in young leaves of transgenic tomatoes and wt plants. in 35s::slipt lines, the transcript abundance of slipt was higher than in wt, whereas lower expression of slipt was observed in all transgenic lines. likewise, upregulation of slarr functioning in feedback regulatory loops of cks, and downregulation of ck deactivating genes slckx and slckx were observed in all transformants, relative to wt plants. thus, ck-related genes expression analysis revealed upregulation of some genes involved in ck biosynthesis and signaling, but downregulation of genes involved in ck degradation pathways. we hypothesized that the upregulation of some ck metabolic genes and enhancement of cks content could be elementary prerequisites of tomato salt stress tolerance. as only l could successfully reproduce, t homozygous transformants from this transgenic line were further analyzed for their tolerance to salinity. for this purpose, l and wt lines were cultivated in vitro for weeks on the mm nacl medium. compared to wt plants, 35s::slipt l plants exhibited a larger shoot with extending leaves, whereas wt plants showed more compact shoot and smaller leaves as a result of limited growth in presence of salt. subsequently, the effects of salt stress induced either by mm and mm nacl concentration on photosynthesis and nutrients balance were evaluated in wt and l by measurements of photosynthetic pigments and macronutrients contents.figure effect of salt stress on wt and 35s::slipt3 tomato plants cultivated on control and salt-containing growth media. phenotype of wt and 35s::slipt transgenic plants grown weeks on the mm nacl medium. chlorophyll content, carotenoid content, cation concentration and k+/na+ ratio in weeks-old tomato developing leaves of wt and trangenic plants. sum vaz, sum violaxanthin, antheraxanthin and zeaxanthin. data represent means and sd of two replicates for photosynthetic pigments while three replicates represent data from cation concentration. 1statistically significant difference from the same treatment of wt. 2statistically significant difference from ms medium within the same line, 2statistically significant difference from ms medium within the same line after Šidák correction for testing of multiple nacl concentrations. in wt plants, chlorophyll a and chlorophyll b significantly decreased with the addition of nacl into the growth media. in l plants, the content of both types of chlorophyll was significantly lower on control medium relative to wt, but remained nearly constant on nacl containing media, and a significant decrease in chl a content was only apparent on mm nacl. likewise, the levels of carotenoids, especially lutein and β-carotene displaying photoprotective roles in photosynthesis, exhibited almost identical contents in 35s::slipt l plants regardless of growth media. in contrast, wt plants showed a significant decrease of β-carotene on both types of nacl media, and of lutein on mm nacl. increasing contents of car involved in the xanthophyll cycle, violaxanthin, antheraxanthin and zeaxanthin were as well observed in presence of salt in both wt and transgenic tomatoes, significant for wt at mm nacl and 35s::slipt grown on both concentrations of salt. shoot sodium, potassium, magnesium and calcium concentrations were assessed in plants exposed to, mm and mm nacl. as observed in figure 9d, na+ and k+ concentrations were similar in wt and l under control conditions of growth. salinity induced na+ accumulation and substantially reduced k+ contents in both types of tomato plants. however, na+ accumulated to a lesser extent in l than in wt on the and mm nacl medium, respectively. similarly, k+ concentration was significantly higher in l than in wt in presence of and mm nacl, respectively, and these results are also visible by the k+/na+ ratio, which remained significantly higher in l than in wt in salt stress conditions. finally, no difference of ca2+contents between wt and transgenic plants was noticed, while the mg2+ concentration was significantly higher in transgenic tomatoes on control and mm nacl conditions, compared to wt. overall, plant morphological changes in presence of salt, roughly constant levels of photosynthetic pigments, especially chl a, chl b, lutein and β-carotene in 35s::slipt tomatoes regardless on the growth medium and higher k+ contents in presence of nacl indicate that slipt overexpression could raise plant tolerance to salt stress. both slipt and slipt are involved in salt stress response to further understand the participation of slipt and slipt in salt stress response, their expression pattern was examined by qrt-pcr in roots, young leaves and old leaves of tomato wt plants exposed to salinity for h. in roots, which are the first organ perceiving salinity, slipt and slipt shared very similar expression profiles. a rapid decrease in transcript levels of both genes within h after salt exposure was followed by a very small peak at h for both genes, before a new decline. in young leaves, slipt and slipt showed partially divergent transcript accumulation profiles. indeed, slipt transcripts gradually and regularly increased with incubation time. in contrast, slipt was repressed -fold as rapidly as h after stress initiation. this repression seemed transient, however, as a slight increase in slipt transcripts was visible h after stress onset. similar expression profiles were observed in old leaves. these results indicate that slipt and slipt directly participate in salt stress response in tomato via a differential modulation of their expression, depending on the organ and duration of the stress.figure salinity response of slipt3 and slipt4 in tomato wt plants. analysis of slipt and slipt expression levels in tomato wt roots, young leaves and old leaves in response to salt stress. actin and gapdh were used as internal controls for normalization of slipt and slipt transcript levels. data represent means and sd of two replicates. *statistically significant difference from time h. **statistically significant difference from time h after Šidák correction for testing of multiple time points. discussion ck homeostasis during plant development and in response to changing environmental conditions is, among other mechanisms, properly maintained by the activity of ck biosynthetic enzymes, ipts. it was previously reported that root-localized ipt expression essentially improved salt stress tolerance in tomato. however, the genes involved and the mechanisms of cks regulation under salt stress conditions have not yet been elucidated. therefore, based on functional characterization of slipt and slipt we provide insights into both genes involvement in the response to salt stress in tomato plants. tomato slipt and slipt are non-redundant functional isopentenyltransferases in the present study, we have demonstrated that tomato slipt and slipt encode two functional isopentenyltransferases. multiple alignment of proteins sequences and phylogenetic analysis revealed that slipt and slipt are closely related to arabidopsis ortholog atipt and atipt, as previously reported. interestingly, we observed identical chloroplast localization of gfp-tagged slipt protein in tomato leaf cells, as described in arabidopsis where atipt was localized to plastids in roots and leaf cells. these data indicate that slipt and atipt partly share similar function and protein subcellular localization. we therefore speculate that slipt and probably also slipt due to the presence of a chloroplastic transit peptide as well, may have other comparable regulatory mechanisms for protein chloroplast targeting such as farnesylation for instance, as was demonstrated for atipt. distinct spatio-temporal expression profiles of slipt and slipt have been observed in tomato vegetative organs and during fruit onset, development and ripening, indicating that these two genes have non-redundant functions. in general, slipt expression was lower relative to that of slipt and showed its highest levels in young leaves and red fruits whereas slipt substantially predominated in the remaining tested organs. our data are in agreement with a previous report describing very low transcript abundance of slipt in all tomato tested organs with flowers exhibiting the highest slipt expression, and a greater accumulation of slipt transcripts, especially in the very early stages of flower and fruit development. interestingly, although slipt is less expressed than slipt in tomato, slipt showed ten times greater activity in vitro than slipt which may explain why no transgenic plants could be regenerated from calli. indeed, slipt is probably at the origin of a greater hormonal imbalance than slipt which may have impaired proper shoot and root systems regeneration. differences between slipt and slipt in organ expression specificity point out their distinct physiological roles in tomato developmental processes. however, it seems rather difficult to assign a specific role to each ipt during tomato plant development, due to their overlapping functions. indeed, although atipt is one of the three most expressed ipt genes in arabidopsis during the vegetative phase, ipt knock-out mutant exhibited no distinguishable phenotype from wt plants, indicating functional redundancy between the arabidopsis ipt proteins. therefore, none of the visible phenotypes or characteristics in slipt and slipt complemented arabidopsis ipt knock-out mutants were apparent in comparison to wt plants under control conditions. ectopic expression of slipt alters tomato phenotype and ck metabolism overexpression of slipt under 35s camv promoter and consequent ck over-accumulation had extensive negative effects on tomato growth under control conditions, resulting in dwarf phenotype and delayed senescence, substantially limiting formation of tomato generative organs. only one transgenic line showing the weakest phenotypic differences from wt was able to produce seeds. a similar effect of ck overabundance in flowering delay was reported in stably transformed tobacco plants with 35s::mdipt3a. likewise, phenotype alterations such as a strong delay in growth, shorter rosettes, serrated leaves and decreased apical dominance were observed in arabidopsis plants overexpressing maize zmipt. in contrast, no changes in flowering time were evident in arabidopsis and tobacco transgenic plants expressing ipt under hsp, underlining the crucial role of cks accumulation and homeostasis during flowering. we found out that constitutive expression of slipt increased ck production in tomatoes by up to 12-fold relative to wt plants, primarily ip7g. similarly, high concentrations of ip7g in arabidopsis complemented plants cultivated on both control and salt media suggest that the formation of ck-n7-glucosides may be a sufficient pathway for downregulation of bioactive cks because no significant enhancement of ckx activity was detected in 35s::slipt tomatoes when compared to wt. in accordance with our results, ipt overexpression resulted in increased levels of bioactive ip, tz and their ribosides in tomato and tobacco. however, detailed data regarding all ck derivatives were not reported in these previous studies. based on the observation that iprmp/iprdp/iprtp are the first products in ck biosynthesis, that ip and ipr are the best substrates for ckx activity and are in massive amounts metabolized to ck-n-glucoside forms, we conclude that ip-type cks have a prominent role in fast regulation and balancing of the ck metabolism. slipt and slipt participate in salinity response in tomato and arabidopsis expression analyses of slipt and slipt in tomato vegetative organs confirmed the participation of both genes in response to salt stress. after stress onset, salinity evoked repression of slipt and slipt in roots, and differently affected slipt and slipt in young and old leaves. after to h of exposure to salinity, increasing levels of both transcripts were detected in young leaves, suggesting preferential ck biosynthesis in photosynthetic organs and thus underlining cks stress-prevention and anti-senescence properties. similarly, an augmentation of ipt expression was apparent both in maize leaves and roots following longer exposure to salinity whereas shorter salt treatment induced a repression of ipt encoding genes in maize leaves. likewise, two weeks-old arabidopsis plants showed downregulation of atipt and atipt as early as h after salt treatment, whereas the expression of atipt and atipt was elevated. conversely, with longer salt stress, the majority of atipt transcripts were repressed. together, these data indicate a dynamic regulation of ck biosynthesis/metabolism over the stress period. moreover, multiple putative stress-responsive cis-regulatory elements were identified in the promoter regions of bripts promoters, pointing to the response of these genes to stress stimuli and a possible response to unfavourable conditions via changing ck status. similarly in this study, an in silico analysis of slipt and slipt promoters revealed the presence of putative stress response elements and many arr binding cis-elements, indicating at least for slipt the existence of salt stress tolerance mechanisms similar to those in bripts. our findings in tomato plants illustrated that the regulation of slipt and slipt in response to salt stress is organ specific, depends on the duration of stress exposure and potentially also on the presence of cis-regulatory elements in the promoters of ck-related genes. in addition, diverse roles of both tomato slipts in salt stress responses have been characterized in arabidopsis ipt ko complemented plants under longer period of salt treatment. transgenic plants ectopically expressing slipt under 35s promoter showed superior above-ground growth after several weeks on salt medium, relative to wt plants. beneficial effects of cks overproduction in response to salt stress were reported in tomato. cks elevation enhanced shoot growth and prevented senescence under salinity conditions due to the specific induction of ipt in roots by a heat-shock promoter. likewise, higher ck concentration was associated with salt tolerance in tomatoes overexpressing a cysteine-2/histidine-2-type zinc finger transcription factor. similarly, expression of agrobacterium tumefaciens ipt under the stress inducible promoter rd29a resulted in higher salinity tolerance in transgenic tobacco. using specific promoters for ipt overexpression also successfully improved tolerance to drought in rice, peanut, cassava, tobacco and to the cold stress in tobacco. on the other hand, tobacco plants with reduced ck contents showed smaller osmotic potential and higher drought resistance than wt plants. likewise, arabidopsis knock-out mutants for two ck histidine kinase receptors showed tolerance to drought and salinity. aspects of plant phenotype, such as root architecture modifications and growth inhibition in transgenic plants, influenced by imbalance between cks accumulation and distribution can partly explain tolerance to various environmental stresses. thus, root-specific ckx overexpression enhanced root system development and consequently increased drought tolerance, nutrient uptake and leaf nutrient enrichment in arabidopsis. consequently, it is still difficult to estimate the precise nature of ck effects due to the complexity of phytohormones metabolism and other cross-talk networks. salinity tolerance of 35s::slipt plants is correlated, among others, with improved nutrients balance and retention of the photosynthesis capacity, in addition to the senescence-protective effects of the photosynthetic pigments. indeed, salinity strongly impacts tomato plants ionic status, as na+ concentration considerably increases in leaves, inducing a premature senescence, while k+ content differently declines depending on the leaf age. transgenic tomato plants hsp70::ipt showing improved growth and final yield under long-term salinity treatment consequently accumulate less na+ in leaves and roots than wt, but higher k+ contents, underlining the importance of the limitation of toxic ions accumulation for plant development. in addition, salinity strongly affects plant photosynthesis, and salt-induced reduction in photosynthetic pigments such as chl and car have been reported in several studies. variations in chl and car contents under different salt concentrations and time exposure were observed in three varieties of tomato cultivars, suggesting that sensitivity to salt stress depends on plant genotype. our results indicate that the contents of chl a and ch b as well as lutein and β-carotene are maintained in 35s::slipt plants at the same levels regardless of the growth media in contrast to wt, in which salt treatments decreased both chl and car contents. similarly, transgenic tomatoes overexpressing ipt within the root zone maintained their photosynthesis ability under long-term salinity treatement, while transgenic tobacco expressing stress-inducible promoter rd29a-ipt showed only slight decrease in chl contents in comparison to wt plants. significant accumulation of pigments involved in the xanthophyll cycle in 35s::slipt plants exposed to salt may reflect higher intensity in photoprotective function of car in transgenic plants than in wt plants. in summary, our results indicate that constitutive expression of slipt induces several changes in plant growth, nutrients accumulation, hormonal metabolism and photosynthesis maintenance, leading to enhanced tolerance to salt stress. mechanism of slipt and slipt regulation under salt and ck treatments the comprehensive mechanism of ck regulatory feedback loop is supported by our findings that exogenous application of tz inhibited the expression of slipt and slipt within h, enhanced the expression of negative-feedback regulator type-a slarr and slightly moderated the expression levels of ck primary response genes type-b slarr and slarr. comparable data reporting downregulation of the ipt genes after ck treatment have been reported for chinese cabbage and maize. the connection between ck homeostasis maintenance via type-b response regulators and salinity response was reported in arabidopsis where arr and arr were shown to control sodium accumulation in shoots by regulating expression of genes encoding high-affinity k+ transporter 1; responsible for na+ exclusion from the root xylem. these data suggest that ck homeostasis may be effectively regulated, not only by cks molecules, but also environmental stimuli. taken together, we have shown the mechanism of slipt and slipt response to early salt stress and their feedback regulation by exogenous ck treatment in tomato plants. based on the data obtained in this study, we propose a model illustrating the complexity of ck networks in tomato plants in response to salinity. in this model, we only included relations supported by our experimental data. we outline an organ specific down-regulation of slipt and slipt transcripts immediately after stress onset, followed by transcripts increase with the duration of the stress treatment. the complexity of ck networks is demonstrated by the repression of slipt and slipt after exogenous application of tz, while an overabundance of cks in 35s::slipt may ensure a fitter phenotype under salt stress conditions. similarly, the response to salinity by slipt and slipt was paralleled in arabidopsis ipt ko complemented plants where salinity resulted in elevation of cks with successive downregulation of atipts to maintain ck homeostasis.  in summary, we have characterized slipt and slipt response to early salt stress in tomato, and identified slipt as a key player in the ck metabolism of tomato plants under salinity conditions. our results contribute to the understanding of ck regulation at the molecular level and provide a potentially useful tool to obtain and improve high-quality stress-tolerant crops in agriculture. 
in response to salt stress, slipt and slipt were strongly repressed in tomato roots, and differently affected in young and old leaves. it has been previously reported that enhancing ck contents improved salt stress tolerance in tomato. the substantial participation of slipt in ck metabolism during salt stress has been determined in 35s::slipt tomato transformants, where enhancement of cks accumulation significantly improved plant tolerance to salinity, underlining the importance of this phytohormone in stress response. involvement of slipt and slipt in salt stress response was also observed in arabidopsis ipt knock-out complemented plants, through maintenance of ck homeostasis. however, the underlying mechanisms of ck metabolism and signaling under salt stress conditions remain to be deciphered. both proteins displayed isopentenyltransferase activity in vitro, while their encoding genes exhibited different spatio-temporal expression patterns during tomato plant development. slipt and slipt are functional ipts encoded by differently expressed genes, distinctively taking part in the salinity response. in addition, 35s::slipt tomato plants displayed improved tolerance to salinity consecutive to photosynthetic pigments and k+/na+ ratio retention.
 cytokinins are involved in response to various environmental cues, including salinity. it has been previously reported that enhancing ck contents improved salt stress tolerance in tomato. however, the underlying mechanisms of ck metabolism and signaling under salt stress conditions remain to be deciphered.  two tomato isopentenyltransferases, slipt and slipt were characterized in tomato and arabidopsis. both proteins displayed isopentenyltransferase activity in vitro, while their encoding genes exhibited different spatio-temporal expression patterns during tomato plant development. slipt and slipt were affected by the endogenous ck status, tightly connected with cks feedback regulation, as revealed by hormonal treatements. in response to salt stress, slipt and slipt were strongly repressed in tomato roots, and differently affected in young and old leaves. slipt overexpression in tomato resulted in high accumulation of different ck metabolites, following modifications of ck biosynthesis-, signaling- and degradation-gene expression. in addition, 35s::slipt tomato plants displayed improved tolerance to salinity consecutive to photosynthetic pigments and k+/na+ ratio retention. involvement of slipt and slipt in salt stress response was also observed in arabidopsis ipt knock-out complemented plants, through maintenance of ck homeostasis.  slipt and slipt are functional ipts encoded by differently expressed genes, distinctively taking part in the salinity response. the substantial participation of slipt in ck metabolism during salt stress has been determined in 35s::slipt tomato transformants, where enhancement of cks accumulation significantly improved plant tolerance to salinity, underlining the importance of this phytohormone in stress response. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cytokininisopentenyltransferasesalt stressslipt3slipt4tomatoissue-copyright-statement© the author 2015 