BACKGROUND
phosphorus is a vital plant macronutrient, functioning as a component in essential biomolecules such as phospholipids and atp.
inorganic phosphate  plays central roles in virtually all of the major metabolic processes in plants, particularly photosynthesis  <cit> .
to further increase crop yields will require improving photosynthesis  <cit> .
thus, the efficient use of phosphorus during photosynthesis is a potentially important determinant of crop growth and yield.
plants have evolved a series of strategies to cope with inadequate phosphate conditions while maintaining a proper balance of internal phosphorus levels  <cit> .
these adjustments include  reducing phosphorus consumption by the plant  <cit> ,  increasing plant internal phosphorus recycling  <cit> , and  improving phosphorus use in metabolic pathways  <cit> .
physiological and molecular adaptations that improve the phosphorus use efficiency include accelerated leaf senescence combined with the redirection of resources to growing tissues, as well as changes to metabolic pathways, such as primary carbon metabolism and phospholipid metabolism  <cit> .
the release of phosphorus from membrane phospholipids by lipid remodeling is an important mechanism used by plants to adapt to low-phosphate conditions .
sulfoquinovosyl diglyceride  is a non-phosphorus lipid associated with several protein complexes in photosynthetic membranes, such as chloroplasts cf0-cf <dig> of atpase, light harvesting complex ii-apoproteins, and d1/d <dig> heterodimers  <cit> .
the glycerophosphodiester-mediated lipid metabolic pathway may be involved in phosphorus release from phospholipids under low-phosphate stress.
sulfolipids and galactolipids, rather than phospholipids, are the major lipids of the thylakoid membrane in plants subjected to phosphate-deficiency stress.
under these conditions, plants can replace the phospholipids in photosynthetic membranes with specific non-phosphorus lipids  <cit> .
these changes prolong and enhance the productive use of phosphorus during photosynthesis.
starch accumulation in the shoots is another common reaction of all plants to long-term phosphate deficiency  <cit> .
one of the effects associated with starch accumulation is the release of phosphorus from chloroplasts to the cytoplasm for phosphorus recycling  <cit> .
the accumulation of starch in phosphate-deficient leaves may help maintain the phosphorus balance between the cytoplasm and chloroplasts  <cit> .
increasing phosphorus recycling and phosphorus release from the vacuole may increase the phosphorus use efficiency.
the vacuole is an important organelle involved in maintaining cytoplasmic phosphorus homeostasis  <cit> .
excess cellular phosphorus in the cytoplasm is stored in the vacuole and is used to buffer the phosphorus demands of the cytoplasm  <cit> .
the influx of phosphorus into the vacuole moderates phosphorus fluctuations by controlling the external intake of phosphorus and influencing cell metabolism.
under phosphorus deficiency, the v-atpase gene may improve the proton transport to maintain an electrochemical gradient across the tonoplast by increasing its expression level, thereby providing the required energy to facilitate phosphorus transport  <cit> .
previously, we obtained the low-phosphate-tolerant mutant qi319- <dig> from qi <dig> using cellular engineering, therefore, they have a common genetic background.
a comparison of the low-phosphate responses in these two maize genotypes indicated that low-phosphate tolerance is greater in the qi319- <dig> genotype than in qi <dig>  the light energy conversion efficiency and pi contents are higher in qi319- <dig> than in qi <dig> under low-phosphate conditions  <cit> .
we previously performed a systematic proteome analysis of qi <dig> maize leaves in response to phosphate starvation, finding that the phosphate starvation response is a complicated process involving several metabolic reactions, such as photosynthesis, carbohydrate metabolism, energy metabolism, secondary metabolism, signal transduction, and protein synthesis.
after being subjected to a long period of phosphorus stress, the internal phosphorus use efficiency in qi <dig> maize may increase through altered photorespiration and lipid composition, along with increased starch synthesis  <cit> .
to elucidate the molecular mechanisms of the different tolerance levels to low-p conditions between qi319- <dig> and qi <dig>  a comparative proteome analysis should be performed.
in this study, we performed comparative proteome analyses of leaves from mutant qi319- <dig> and wild-type qi <dig> plants treated with 1000 μm  and 5 μm  kh2po <dig> over a long time period.
the objectives of this study were  to determine the reasons behind the differences in leaf pi levels between the two genotypes;  to investigate the mechanism behind the high photosynthetic efficiency levels in qi319-96; and  to provide information for further research into the functions of genes involved in phosphate-stress responses.
RESULTS
differential growth and physiological responses to phosphate deprivation between qi <dig> and qi319-96
after treatment with 5 μmol kh2po <dig> for 25 days, qi <dig> and qi319- <dig> maize plants grew to the six- to seven-leaf stage but exhibited apparent phosphorus deficiency symptoms, such as reduced overall phosphorus contents, marked changes in biomass , heliotrope-colored stems, and restricted growth .table  <dig> influence of different phosphate treatments on biomass, inorganic phosphorus concentration and phosphorus contents of qi <dig> and qi319-96
three seedlings per bottle were cultured in phosphorus saturation solution  to the three-leaf stage, followed by low phosphate stress solution  for an additional 25 days to the six–seven-leaf stage.
values represent the means of nine seedlings ± sd.
values with different letters within a row are significantly different  by multiple comparison analysis
fig.
<dig> qi <dig> and qi319- <dig> maize plants grown in two growth conditions + p and -p. a: qi <dig> and qi319- <dig> grew under -p conditions .
b: qi <dig> and qi319- <dig> grew under + p conditions
the phosphorus contents, and root and shoot biomasses, were not significantly different between qi319- <dig> and qi <dig> under the sufficient phosphate  treatment.
however, the root biomasses were significantly higher in qi319- <dig> than in qi <dig> after phosphorus stress treatments.
the phosphorus content in roots was significantly higher in qi319- <dig> than in qi <dig>  while the overall phosphorus content of the shoots did not significantly differ between genotypes .
however, the pi levels of qi319- <dig> and qi <dig> decreased by  <dig>  % and  <dig>  %, respectively, after the phosphorus deficiency treatment, but the pi content in shoots was still  <dig>  % higher in qi319- <dig> than in qi <dig> .
low-phosphate stress limits plant photosynthesis .
under phosphate-deficiency conditions, the net photosynthesis rate  decreased by  <dig>  % in qi <dig> and  <dig>  % in qi319- <dig>  under low-phosphate stress, the pn of qi319- <dig> was  <dig>  % higher than that of qi <dig>  phosphate deficiency also reduced the stomatal limitation value .
the ls values of the two genotypes, when subjected to the sufficient phosphorus treatment, were not significantly different, but the intercellular co <dig> concentration  increased in both genotypes after low-phosphate stress .
the chlorophyll content in leaves was  <dig>  % higher in qi319- <dig> than in qi <dig> , indicating that photo-absorption by the chloroplasts was better in qi319- <dig> under low-phosphate conditions.table  <dig> influence of different phosphate treatments on photosynthesis in qi <dig> and qi319-96
three seedlings per bottle were cultured in phosphorus saturation solution  to the three-leaf stage, followed by low phosphate stress solution  for an additional 25 days to the six–seven-leaf stage.
values represent the means of nine seedlings ± sd.
values with different letters within a row are significantly different  by multiple comparison analysis
three seedlings per bottle were cultured in phosphorus saturation solution  to the three-leaf stage, followed by low phosphate stress solution  for an additional 25 days to the six–seven-leaf stage.
values represent the means of nine seedlings ± sd.
values with different letters within a row are significantly different  by multiple comparison analysis
the sucrose contents in the leaves significantly declined under low-phosphate stress, but the starch contents were still higher than the contents detected under phosphate-sufficient conditions .
the sucrose contents in the leaves were higher in qi319- <dig> than in qi <dig>  more of the photosynthetic products were used for sucrose biosynthesis in qi319- <dig> than in qi <dig>  these data indicated that the distribution of the photosynthetic carbon metabolism between sucrose and starch was altered in both genotypes under low-phosphate conditions.
thus, the low-phosphate-tolerant mutant qi319- <dig> had a higher photosynthetic co <dig> fixation rate and plant biomass compared with wild-type qi <dig>  although the phosphorus level in shoots did not differ between genotypes, qi319- <dig> had significantly higher levels of pi than qi <dig>
differential analysis of leaf protein profiles
we performed comparative proteomic studies of qi <dig> and qi319- <dig> maize leaves subjected to two different phosphate levels using immobilized ph gradient  strips , with three biological replicates.
the number of protein spots detected in the gels and the proteins that differentially accumulated in the two genotypes are summarized in table  <dig>  approximately  <dig> spots were detected under the phosphate saturation treatment.
of these,  <dig>  spots differentially accumulated between qi <dig> and qi319- <dig>  of the  <dig> spots, nine spots  accumulated to a greater extent in qi <dig> than in qi319- <dig>  whereas  <dig> spots  accumulated to a greater extent in qi319- <dig> .
after the phosphate-deficiency treatment,  <dig> spots were detected,  <dig>  of which differentially accumulated between qi <dig> and qi319- <dig>  of these,  <dig> spots  accumulated to a greater extent in qi319- <dig> than in qi <dig>  whereas  <dig> spots  accumulated to a greater extent in qi <dig> .table  <dig> number of differentially accumulated proteins between the two genotypes and in response to phosphate stress on 2-de gels
fig.
<dig> comparison 2-de protein gel maps taken from qi <dig> and qi319- <dig> maize leaves that had been subjected to two different phosphorus concentrations.
the proteins were extracted by tca/acetone from the middle of the fourth leaf and the  <dig>  mg protein samples were separated in ief using 17 cm ph 5– <dig> ipg strips.
then they were put on a 12 % polyacrylamide gel for the second dimensional separation and stained with cbb.
the gel image analysis was carried out using pdquest software .
the spots marked with numbers  indicated proteins that were differentially accumulated in leaves of qi319- <dig> and qi- <dig> under + p conditions identified by maldi-tof ms; the spots marked with numbers  indicate proteins that were differentially accumulated in leaves of qi319- <dig> and qi- <dig> under -p conditions identified by maldi-tof ms. a: the image of the + p treatment qi319- <dig> protein; b: the image of the + p treatment qi <dig> protein; c: the image of the –p treatment qi319- <dig> protein; d: the image of the –p treatment qi <dig> protein
identification and classification of phosphate-stress-responsive proteins
we identified the proteins that were differentially expressed after the two phosphate treatments using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry  to gain a better understanding of the mechanisms involved in phosphorus stress and the differences in phosphorus tolerance between qi319- <dig> and qi <dig> maize plants.
in total,  <dig> proteins were identified using the ncbi databases .
the detailed peptide sequences are shown in additional file  <dig>  we classified these proteins based on the tair  and kegg  databases.
the proteins were classified into protein fate, protein synthesis, cell rescue/defense/virulence, metabolism, energy and transcription/signal transduction mechanisms .
to confirm the results produced by peptide mass finger printing , eight randomly selected spots from among these proteins were subjected to maldi-tof-tof ms analysis.
the results for all eight were consistent with the pmf results , which confirmed their reliability.table  <dig> differentially accumulated proteins with similar functionsin qi319- <dig> and qi <dig> leaves under both + p and − p conditions
matched protein namea
a: name of protein identified by maldi-tof ms
b: database accession numbers from ncbinr
c: assigned spot number, as indicated in fig.
2
d: “increase” indicates significance at p <  <dig>  and an increase in amount of at least  <dig> -fold on the qi319- <dig> gel under + p or –p conditions; “decrease”indicates significance at p <  <dig>  and a decrease in amount of at least  <dig> -fold on the qi319- <dig> gel under + p or –p conditions compared withqi319
e: specificity indicates the ratio of accumulation of a particular protein in leavesbetween qi319-96 + p and qi319- <dig> –p
f: specificity indicates the ratio of accumulation of a particular protein in leavesbetween qi319 + p and qi <dig> – p
differentially accumulated proteins and their effects on photosynthesis in the low-phosphate-tolerant mutant and wild-type maize
the levels of several proteins that participate in photosynthesis were significantly different between the two genotypes under the two phosphate treatments.
under low phosphate conditions, the levels of rubisco , nadp-malic enzyme , n <dig> and n36), pyruvate orthophosphate dikinase ), delta-aminolevulinic acid dehydratase , sucrose-phosphatase , cytoplasm- phosphoglucomutase , fructose-bisphosphate  aldolase ), nadp-glyceraldehyde-3-phosphate dehydrogenase , nadph dihydroethidium , plastoquinone-dehydrogenase , and chlorophyll a/b binding protein  increased significantly compared with qi <dig>  to verify these differences, we performed several physiological and biochemical experiments.
the rubisco, pgm, fbp aldolase, nadp-me and ppdk activities were also higher in qi319- <dig> than in qi <dig> under low phosphate stress , which was consistent with the two-dimensional gel electrophoresis  results.
fbp aldolase catalyzes the reaction between fructose- <dig> -diphosphate and sedoheptulose- <dig> -diphosphate during ribulose- <dig> -bisphosphate  regeneration.
nadp-malic enzyme and ppdk play important roles in co <dig> fixation in bundle sheath cells.
the chlorophyll content in leaves from qi319- <dig> was higher than from qi <dig> by 50 % , which may be related to the significant increase in delta-aminolevulinic acid dehydratase expression.
furthermore, the photosynthetic rate was  <dig>  % higher in qi319- <dig> than in qi <dig> .
the proteome and physiological data showed that qi319- <dig> has a higher photosynthetic rate due to its higher chlorophyll content, and the higher expression levels and activities of calvin cycle and “co <dig> pump” enzymes during phosphate stress.fig.
<dig> comparison of  <dig> important protein between qi <dig> and qi319- <dig> under pi-deficient condition.
n <dig>  uridine-5’-diphospho-sulfoquinovose  synthase; n <dig>  fructose-bisphosphate  aldolase; n <dig>  nadp-malic enzyme; n <dig>  pyruvate orthophosphate dikinase; n <dig>  delta-aminolevulinic acid dehydratase; n <dig>  v-atpase
three seedlings per bottle were cultured in phosphorus saturation solution  to the three-leaf stage, followed by low phosphate stress solution  for an additional 25 days to the six–seven-leaf stage.
values represent the means of nine seedlings ± sd.
values with different letters within a row are significantly different  by multiple comparison analysis
differentially accumulated proteins are involved in energy metabolism between the low-phosphate-tolerant mutant and wild-type maize
the levels of several proteins that participate in energy metabolism were significantly different between the two genotypes under the two phosphorus treatments.
these proteins are involved in the tricarboxylic acid  cycle, the pentose phosphate pathway and glycolysis .
under the phosphorus deficiency treatment, nadp-non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase  accumulated, which may have allowed emp to proceed smoothly under very low phosphate conditions  <cit> .
increases in the levels of aconitase  and the pyruvate dehydrogenase complex  in qi319- <dig> may accelerate atp synthesis through the tca cycle in this line compared with in qi <dig>  the physiological data showed that the amount of atp in maize leaves was  <dig>  % higher in qi319- <dig> than in qi <dig> under low phosphate stress .table  <dig> influence of different phosphorus concentrations on atp, sqdg, pg content and v-atpase activity
three seedlings per bottle were cultured in phosphorus saturation solution  to the three-leaf stage, followed by low phosphate stress solution  for an additional 25 days to the six–seven-leaf stage.
values represent the means of nine seedlings ± sd.
values with different letters within a row are significantly different  by multiple comparison analysis
differentially accumulated proteins associated with increased phosphorus utilization between the low-phosphate-tolerant mutant and wild-type maize
under low phosphate stress, the level of uridine-5’-diphospho-sulfoquinovose  synthase  increased significantly in qi319- <dig> leaves compared with in qi <dig> leaves.
udp-sq synthase may increase the production of udp-sq, leading to an increase in available sq, which is then used to produce sqdg.
the increase in udp-sq synthase in qi319- <dig> suggests that qi319- <dig> may produce more sqdg than qi <dig> under low phosphate stress.
the accumulation of sqdg in the photo-membrane may displace phosphatidylglycerols , which would accelerate the transformation of organic phosphorus and the utilization of internal phosphorus.
the sqdg contents were  <dig>  % higher in qi319- <dig> than in qi <dig>  which is consistent with the udp-sq expression patterns for qi319- <dig> and qi <dig> .
v-atpase levels were not significantly different between qi319- <dig> and qi <dig> under the high phosphate treatment.
however, under the low phosphate treatment, the increase in v-atpase  levels was greater in qi319- <dig> than in qi <dig>  suggesting that qi319- <dig> might release phosphorus from the vacuole to increase the metabolic reaction rate in the cell, which would mitigate the symptoms caused by low phosphorus stress.
the v-atpase activity in the leaves was  <dig>  % higher in qi319- <dig> than in qi <dig>  which is consistent with the 2-de results .
other differentially accumulated proteins between the low-phosphate-tolerant mutant and wild-type maize
the abundance of some proteins, including molecular chaperones , resistance-related proteins , and proteins involved in protein synthesis , signal transduction , and secondary metabolism , were significantly different between qi319- <dig> and qi <dig> under different phosphate levels.
several proteins involved in protein folding and assembly, protein synthesis and mediating signal transduction accumulated differently between the two genotypes under different phosphate levels.
discussion
in this study, we applied comparative proteomics to gain insights into the phosphate-stress tolerance of qi319- <dig> compared with qi <dig>  compared with qi <dig>  the majority of the phosphate-stress-responsive proteins in qi319- <dig> are involved in photosynthesis and internal phosphorus mobilization.
altered membrane lipid compositions, and increased v-atpase activities and abundances in qi319- <dig>  increased the phosphorus use efficiency under low phosphate stress
to counteract the detrimental effects of phosphate stress, higher plants have evolved a series of mechanisms for maintaining internal phosphorus levels.
an important adaptive strategy used by plants subjected to low phosphate conditions is to increase the internal phosphorus utilization efficiency by remodeling the lipid membrane  <cit> .
one-third of the total organophosphate contents in plants is present as phospholipids.
when plants are subjected to low phosphate stress, membrane phospholipids are replaced by non-phosphorus glycerolipids, which promotes the mobilization of organophosphates  <cit> .
sqdg and pg are thought to be involved in maintaining phosphorus levels in the thylakoid membrane, and their contents are known to be regulated by the phosphorus level  <cit> .
under the low phosphate treatment, the expression of udp-sq synthase increased in qi319- <dig>  to supply sq polar groups for sqdg biosynthesis.
indeed, the physiological data showed that the sqdg content was higher in qi319- <dig> than in qi <dig> under phosphate deprivation, indicating that sqdg accumulates in the photo-membrane, which could then be used to supplement pg levels.
these changes may accelerate organophosphate conversion and increase available phosphorus recycling during periods of low phosphate stress.
there is a great interest in the ability of qi <dig> to increase internal phosphorus utilization through lipid remodeling  <cit> .
compared with wild-type qi <dig>  qi319- <dig> exhibited an increase in udp-sq expression in response to low phosphate stress, suggesting that udp-sq synthase plays a crucial role in maintaining an internal pi balance in this maize mutant.
phosphorus in the vacuole acts as a cushion under fluctuating external and internal phosphorus levels.
phosphorus may move from the vacuole to the cytoplasm and chloroplasts, thereby participating in metabolic reactions during phosphate stress  <cit> .
therefore, the vacuole plays a key role in maintaining cytoplasmic phosphorus homeostasis  <cit> .
phosphorus movement across the membrane depends on an electrochemical h+-gradient across the membrane, which is maintained by v-atpase  <cit> .
v-atpase is more highly accumulated in qi319- <dig> than in qi <dig> during phosphate stress.
the mutant may utilize the additional v-atpase to intensify vacuolar-membrane proton transport, forming an electrochemical gradient across the membrane, and thus, increasing the energy supply for phosphorus transport across the membrane.
this would significantly improve the tolerance of qi319- <dig> to low phosphate stress.
qi319- <dig> plants exhibited much better lipid composition remodeling and higher v-atpase expression and activity levels than qi <dig> plants, which could facilitate phosphorus utilization during periods of phosphate stress.
furthermore, these changes could be responsible for the increased pi levels in qi319- <dig> leaves compared with in qi <dig> leaves.
increased expression of enzymes involved in the calvin cycle and co <dig> fixation may enhance photosynthesis in qi319- <dig> compared with in qi319
phosphate deprivation has an adverse effect on plant photosynthesis  <cit> .
the decline in photosynthetic efficiency in maize leaves under low phosphate stress is related to the reduced levels of proteins involved in co <dig> enrichment, the calvin cycle and the electron transport system.
the reduced abundance of these proteins correlates well with the rates of photochemical reactions, co <dig> assimilation, the calvin cycle and rubp regeneration  <cit> .
several enzymes that play key roles in photosynthesis, such as rubisco and 3-phosphoglyceric phosphokinase, are involved in plant responses to low phosphate conditions  <cit> .
rubisco plays an important role in carbon fixation during the calvin cycle  <cit> .
reduced rubisco activity is one of the non-stomatal factors that leads to a substantial decline in photosynthesis  <cit> .
in the current study, rubisco was more highly expressed in qi319- <dig> than in qi <dig>  and rubisco carboxylase activity was significantly higher in qi319- <dig> than in qi <dig>  which may be related to the increased rate of photosynthesis in qi319- <dig>
a reduction in rubp regeneration has a considerable impact on photosynthesis.
the rate of photosynthesis may decrease if rubp regeneration declines  <cit> .
reductions in rubp regeneration may be due to reduced atp levels when plants are subjected to low phosphate conditions.
reduced atp levels inhibit the reduction of 3-phosphoglycerate to triose phosphate through 3-phosphoglyceric phosphokinase, and they also inhibit the phosphorylation of ribulose-5-phosphate to rubp through phosphoribulokinase  <cit> .
the increased accumulation of atp in the leaves of qi319- <dig> compared with qi <dig> under low phosphate stress may be beneficial for rubp regeneration.
in addition, the reduced rubp regeneration may be due to the reduced initial activities of several enzymes, such as nadp-gapdh and fbp aldolase.
rubp regeneration is also affected by the increased amount of photosynthetic carbon that is diverted to starch formation and away from rubp regeneration   <cit> .
the higher level and activity of fbp aldolase in the chloroplasts of qi319- <dig> versus qi <dig> under phosphorus deprivation conditions may have led to a greater regeneration of rubp in qi319- <dig> compared with in qi <dig>
maize is a c <dig> plant with “co <dig> pumps” that have a “floral hoop structure”.
the “co <dig> pump” mechanism in c <dig> plants increases the level of co <dig> around rubisco, which may increase the carboxylation reaction rate and reduce the alternative fixation of o <dig>  c <dig> photosynthesis is more efficient than c <dig> photosynthesis under low atmospheric co <dig> conditions, which suggests that the “co <dig> pump” is an important mechanism that leads to an increase in photosynthesis  <cit> .
the accumulation and activity levels of nadp-me and ppdk were higher in qi319- <dig> than in qi <dig> under phosphate deprivation conditions, which favored a greater capture rate and level of co <dig> in vascular bundle sheath cells.
higher co <dig> levels not only increase rubisco carboxylase activity, but they also suppress the oxidation of rubisco and reduce photorespiration.
the higher co <dig> level in qi319- <dig> helped the plants increase carbon assimilation and counteract phosphate deprivation.
in conclusion, qi319- <dig> had a higher rate of photosynthesis than qi <dig> under low phosphate conditions, which could be attributed to its higher chlorophyll content and increased levels/activities of photosynthesis-related enzymes, such as rubisco, ppdk and nadp-me.
CONCLUSIONS
qi319- <dig> had a significantly higher level of pi than qi <dig> under low-phosphate stress, which may be related to qi319-96’s remodeled membrane lipids and the increase in v-atpase activity, which releases phosphorus from plant organophosphorus to the cytoplasm.
a physiological analysis showed that the carbon assimilation rate of qi319- <dig> was significantly higher than that of qi <dig> under low-phosphate stress, which may be owing to the increased accumulation of several photosynthesis-related in the mutant qi319- <dig> compared with wild-type qi <dig>  our results clearly indicated that the differences in increasing the internal p-use efficiency are the main reasons for the higher tolerance to low-p conditions in the mutant compared with the wild-type.
at the same time, this study suggests that the inbred lines of maize with low-p tolerant traits could be obtained effectively through cellular engineering.

 phosphorus is a vital plant macronutrient, functioning as a component in essential biomolecules such as phospholipids and atp. inorganic phosphate plays central roles in virtually all of the major metabolic processes in plants, particularly photosynthesis. to further increase crop yields will require improving photosynthesis. thus, the efficient use of phosphorus during photosynthesis is a potentially important determinant of crop growth and yield. plants have evolved a series of strategies to cope with inadequate phosphate conditions while maintaining a proper balance of internal phosphorus levels. these adjustments include reducing phosphorus consumption by the plant, increasing plant internal phosphorus recycling, and improving phosphorus use in metabolic pathways. physiological and molecular adaptations that improve the phosphorus use efficiency include accelerated leaf senescence combined with the redirection of resources to growing tissues, as well as changes to metabolic pathways, such as primary carbon metabolism and phospholipid metabolism. the release of phosphorus from membrane phospholipids by lipid remodeling is an important mechanism used by plants to adapt to low-phosphate conditions. sulfoquinovosyl diglyceride is a non-phosphorus lipid associated with several protein complexes in photosynthetic membranes, such as chloroplasts cf0-cf of atpase, light harvesting complex ii-apoproteins, and d1/d heterodimers. the glycerophosphodiester-mediated lipid metabolic pathway may be involved in phosphorus release from phospholipids under low-phosphate stress. sulfolipids and galactolipids, rather than phospholipids, are the major lipids of the thylakoid membrane in plants subjected to phosphate-deficiency stress. under these conditions, plants can replace the phospholipids in photosynthetic membranes with specific non-phosphorus lipids. these changes prolong and enhance the productive use of phosphorus during photosynthesis. starch accumulation in the shoots is another common reaction of all plants to long-term phosphate deficiency. one of the effects associated with starch accumulation is the release of phosphorus from chloroplasts to the cytoplasm for phosphorus recycling. the accumulation of starch in phosphate-deficient leaves may help maintain the phosphorus balance between the cytoplasm and chloroplasts. increasing phosphorus recycling and phosphorus release from the vacuole may increase the phosphorus use efficiency. the vacuole is an important organelle involved in maintaining cytoplasmic phosphorus homeostasis. excess cellular phosphorus in the cytoplasm is stored in the vacuole and is used to buffer the phosphorus demands of the cytoplasm. the influx of phosphorus into the vacuole moderates phosphorus fluctuations by controlling the external intake of phosphorus and influencing cell metabolism. under phosphorus deficiency, the v-atpase gene may improve the proton transport to maintain an electrochemical gradient across the tonoplast by increasing its expression level, thereby providing the required energy to facilitate phosphorus transport. previously, we obtained the low-phosphate-tolerant mutant qi319- from qi using cellular engineering, therefore, they have a common genetic background. a comparison of the low-phosphate responses in these two maize genotypes indicated that low-phosphate tolerance is greater in the qi319- genotype than in qi the light energy conversion efficiency and pi contents are higher in qi319- than in qi under low-phosphate conditions. we previously performed a systematic proteome analysis of qi maize leaves in response to phosphate starvation, finding that the phosphate starvation response is a complicated process involving several metabolic reactions, such as photosynthesis, carbohydrate metabolism, energy metabolism, secondary metabolism, signal transduction, and protein synthesis. after being subjected to a long period of phosphorus stress, the internal phosphorus use efficiency in qi maize may increase through altered photorespiration and lipid composition, along with increased starch synthesis. to elucidate the molecular mechanisms of the different tolerance levels to low-p conditions between qi319- and qi a comparative proteome analysis should be performed. in this study, we performed comparative proteome analyses of leaves from mutant qi319- and wild-type qi plants treated with 1000 μm and 5 μm kh2po over a long time period. the objectives of this study were to determine the reasons behind the differences in leaf pi levels between the two genotypes; to investigate the mechanism behind the high photosynthetic efficiency levels in qi319-96; and to provide information for further research into the functions of genes involved in phosphate-stress responses.  differential growth and physiological responses to phosphate deprivation between qi and qi319-96 after treatment with 5 μmol kh2po for 25 days, qi and qi319- maize plants grew to the six- to seven-leaf stage but exhibited apparent phosphorus deficiency symptoms, such as reduced overall phosphorus contents, marked changes in biomass, heliotrope-colored stems, and restricted growth.table influence of different phosphate treatments on biomass, inorganic phosphorus concentration and phosphorus contents of qi and qi319-96 three seedlings per bottle were cultured in phosphorus saturation solution to the three-leaf stage, followed by low phosphate stress solution for an additional 25 days to the six–seven-leaf stage. values represent the means of nine seedlings ± sd. values with different letters within a row are significantly different by multiple comparison analysis fig. qi and qi319- maize plants grown in two growth conditions + p and -p. a: qi and qi319- grew under -p conditions. b: qi and qi319- grew under + p conditions the phosphorus contents, and root and shoot biomasses, were not significantly different between qi319- and qi under the sufficient phosphate treatment. however, the root biomasses were significantly higher in qi319- than in qi after phosphorus stress treatments. the phosphorus content in roots was significantly higher in qi319- than in qi while the overall phosphorus content of the shoots did not significantly differ between genotypes. however, the pi levels of qi319- and qi decreased by  % and  %, respectively, after the phosphorus deficiency treatment, but the pi content in shoots was still  % higher in qi319- than in qi. low-phosphate stress limits plant photosynthesis. under phosphate-deficiency conditions, the net photosynthesis rate decreased by  % in qi and  % in qi319- under low-phosphate stress, the pn of qi319- was  % higher than that of qi phosphate deficiency also reduced the stomatal limitation value. the ls values of the two genotypes, when subjected to the sufficient phosphorus treatment, were not significantly different, but the intercellular co concentration increased in both genotypes after low-phosphate stress. the chlorophyll content in leaves was  % higher in qi319- than in qi, indicating that photo-absorption by the chloroplasts was better in qi319- under low-phosphate conditions.table influence of different phosphate treatments on photosynthesis in qi and qi319-96 three seedlings per bottle were cultured in phosphorus saturation solution to the three-leaf stage, followed by low phosphate stress solution for an additional 25 days to the six–seven-leaf stage. values represent the means of nine seedlings ± sd. values with different letters within a row are significantly different by multiple comparison analysis three seedlings per bottle were cultured in phosphorus saturation solution to the three-leaf stage, followed by low phosphate stress solution for an additional 25 days to the six–seven-leaf stage. values represent the means of nine seedlings ± sd. values with different letters within a row are significantly different by multiple comparison analysis the sucrose contents in the leaves significantly declined under low-phosphate stress, but the starch contents were still higher than the contents detected under phosphate-sufficient conditions. the sucrose contents in the leaves were higher in qi319- than in qi more of the photosynthetic products were used for sucrose biosynthesis in qi319- than in qi these data indicated that the distribution of the photosynthetic carbon metabolism between sucrose and starch was altered in both genotypes under low-phosphate conditions. thus, the low-phosphate-tolerant mutant qi319- had a higher photosynthetic co fixation rate and plant biomass compared with wild-type qi although the phosphorus level in shoots did not differ between genotypes, qi319- had significantly higher levels of pi than qi differential analysis of leaf protein profiles we performed comparative proteomic studies of qi and qi319- maize leaves subjected to two different phosphate levels using immobilized ph gradient strips, with three biological replicates. the number of protein spots detected in the gels and the proteins that differentially accumulated in the two genotypes are summarized in table  approximately spots were detected under the phosphate saturation treatment. of these, spots differentially accumulated between qi and qi319- of the spots, nine spots accumulated to a greater extent in qi than in qi319- whereas spots accumulated to a greater extent in qi319-. after the phosphate-deficiency treatment, spots were detected, of which differentially accumulated between qi and qi319- of these, spots accumulated to a greater extent in qi319- than in qi whereas spots accumulated to a greater extent in qi.table number of differentially accumulated proteins between the two genotypes and in response to phosphate stress on 2-de gels fig. comparison 2-de protein gel maps taken from qi and qi319- maize leaves that had been subjected to two different phosphorus concentrations. the proteins were extracted by tca/acetone from the middle of the fourth leaf and the  mg protein samples were separated in ief using 17 cm ph 5– ipg strips. then they were put on a 12 % polyacrylamide gel for the second dimensional separation and stained with cbb. the gel image analysis was carried out using pdquest software. the spots marked with numbers indicated proteins that were differentially accumulated in leaves of qi319- and qi- under + p conditions identified by maldi-tof ms; the spots marked with numbers indicate proteins that were differentially accumulated in leaves of qi319- and qi- under -p conditions identified by maldi-tof ms. a: the image of the + p treatment qi319- protein; b: the image of the + p treatment qi protein; c: the image of the –p treatment qi319- protein; d: the image of the –p treatment qi protein identification and classification of phosphate-stress-responsive proteins we identified the proteins that were differentially expressed after the two phosphate treatments using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry to gain a better understanding of the mechanisms involved in phosphorus stress and the differences in phosphorus tolerance between qi319- and qi maize plants. in total, proteins were identified using the ncbi databases. the detailed peptide sequences are shown in additional file we classified these proteins based on the tair and kegg databases. the proteins were classified into protein fate, protein synthesis, cell rescue/defense/virulence, metabolism, energy and transcription/signal transduction mechanisms. to confirm the results produced by peptide mass finger printing, eight randomly selected spots from among these proteins were subjected to maldi-tof-tof ms analysis. the results for all eight were consistent with the pmf results, which confirmed their reliability.table differentially accumulated proteins with similar functionsin qi319- and qi leaves under both + p and − p conditions matched protein namea a: name of protein identified by maldi-tof ms b: database accession numbers from ncbinr c: assigned spot number, as indicated in fig. 2 d: “increase” indicates significance at p <  and an increase in amount of at least -fold on the qi319- gel under + p or –p conditions; “decrease”indicates significance at p <  and a decrease in amount of at least -fold on the qi319- gel under + p or –p conditions compared withqi319 e: specificity indicates the ratio of accumulation of a particular protein in leavesbetween qi319-96 + p and qi319- –p f: specificity indicates the ratio of accumulation of a particular protein in leavesbetween qi319 + p and qi – p differentially accumulated proteins and their effects on photosynthesis in the low-phosphate-tolerant mutant and wild-type maize the levels of several proteins that participate in photosynthesis were significantly different between the two genotypes under the two phosphate treatments. under low phosphate conditions, the levels of rubisco, nadp-malic enzyme, n and n36), pyruvate orthophosphate dikinase ), delta-aminolevulinic acid dehydratase, sucrose-phosphatase, cytoplasm- phosphoglucomutase, fructose-bisphosphate aldolase ), nadp-glyceraldehyde-3-phosphate dehydrogenase, nadph dihydroethidium, plastoquinone-dehydrogenase, and chlorophyll a/b binding protein increased significantly compared with qi to verify these differences, we performed several physiological and biochemical experiments. the rubisco, pgm, fbp aldolase, nadp-me and ppdk activities were also higher in qi319- than in qi under low phosphate stress, which was consistent with the two-dimensional gel electrophoresis results. fbp aldolase catalyzes the reaction between fructose- -diphosphate and sedoheptulose- -diphosphate during ribulose- -bisphosphate regeneration. nadp-malic enzyme and ppdk play important roles in co fixation in bundle sheath cells. the chlorophyll content in leaves from qi319- was higher than from qi by 50 %, which may be related to the significant increase in delta-aminolevulinic acid dehydratase expression. furthermore, the photosynthetic rate was  % higher in qi319- than in qi. the proteome and physiological data showed that qi319- has a higher photosynthetic rate due to its higher chlorophyll content, and the higher expression levels and activities of calvin cycle and “co pump” enzymes during phosphate stress.fig. comparison of important protein between qi and qi319- under pi-deficient condition. n uridine-5’-diphospho-sulfoquinovose synthase; n fructose-bisphosphate aldolase; n nadp-malic enzyme; n pyruvate orthophosphate dikinase; n delta-aminolevulinic acid dehydratase; n v-atpase three seedlings per bottle were cultured in phosphorus saturation solution to the three-leaf stage, followed by low phosphate stress solution for an additional 25 days to the six–seven-leaf stage. values represent the means of nine seedlings ± sd. values with different letters within a row are significantly different by multiple comparison analysis differentially accumulated proteins are involved in energy metabolism between the low-phosphate-tolerant mutant and wild-type maize the levels of several proteins that participate in energy metabolism were significantly different between the two genotypes under the two phosphorus treatments. these proteins are involved in the tricarboxylic acid cycle, the pentose phosphate pathway and glycolysis. under the phosphorus deficiency treatment, nadp-non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase accumulated, which may have allowed emp to proceed smoothly under very low phosphate conditions. increases in the levels of aconitase and the pyruvate dehydrogenase complex in qi319- may accelerate atp synthesis through the tca cycle in this line compared with in qi the physiological data showed that the amount of atp in maize leaves was  % higher in qi319- than in qi under low phosphate stress.table influence of different phosphorus concentrations on atp, sqdg, pg content and v-atpase activity three seedlings per bottle were cultured in phosphorus saturation solution to the three-leaf stage, followed by low phosphate stress solution for an additional 25 days to the six–seven-leaf stage. values represent the means of nine seedlings ± sd. values with different letters within a row are significantly different by multiple comparison analysis differentially accumulated proteins associated with increased phosphorus utilization between the low-phosphate-tolerant mutant and wild-type maize under low phosphate stress, the level of uridine-5’-diphospho-sulfoquinovose synthase increased significantly in qi319- leaves compared with in qi leaves. udp-sq synthase may increase the production of udp-sq, leading to an increase in available sq, which is then used to produce sqdg. the increase in udp-sq synthase in qi319- suggests that qi319- may produce more sqdg than qi under low phosphate stress. the accumulation of sqdg in the photo-membrane may displace phosphatidylglycerols, which would accelerate the transformation of organic phosphorus and the utilization of internal phosphorus. the sqdg contents were  % higher in qi319- than in qi which is consistent with the udp-sq expression patterns for qi319- and qi. v-atpase levels were not significantly different between qi319- and qi under the high phosphate treatment. however, under the low phosphate treatment, the increase in v-atpase levels was greater in qi319- than in qi suggesting that qi319- might release phosphorus from the vacuole to increase the metabolic reaction rate in the cell, which would mitigate the symptoms caused by low phosphorus stress. the v-atpase activity in the leaves was  % higher in qi319- than in qi which is consistent with the 2-de results. other differentially accumulated proteins between the low-phosphate-tolerant mutant and wild-type maize the abundance of some proteins, including molecular chaperones, resistance-related proteins, and proteins involved in protein synthesis, signal transduction, and secondary metabolism, were significantly different between qi319- and qi under different phosphate levels. several proteins involved in protein folding and assembly, protein synthesis and mediating signal transduction accumulated differently between the two genotypes under different phosphate levels. discussion in this study, we applied comparative proteomics to gain insights into the phosphate-stress tolerance of qi319- compared with qi compared with qi the majority of the phosphate-stress-responsive proteins in qi319- are involved in photosynthesis and internal phosphorus mobilization. altered membrane lipid compositions, and increased v-atpase activities and abundances in qi319- increased the phosphorus use efficiency under low phosphate stress to counteract the detrimental effects of phosphate stress, higher plants have evolved a series of mechanisms for maintaining internal phosphorus levels. an important adaptive strategy used by plants subjected to low phosphate conditions is to increase the internal phosphorus utilization efficiency by remodeling the lipid membrane. one-third of the total organophosphate contents in plants is present as phospholipids. when plants are subjected to low phosphate stress, membrane phospholipids are replaced by non-phosphorus glycerolipids, which promotes the mobilization of organophosphates. sqdg and pg are thought to be involved in maintaining phosphorus levels in the thylakoid membrane, and their contents are known to be regulated by the phosphorus level. under the low phosphate treatment, the expression of udp-sq synthase increased in qi319- to supply sq polar groups for sqdg biosynthesis. indeed, the physiological data showed that the sqdg content was higher in qi319- than in qi under phosphate deprivation, indicating that sqdg accumulates in the photo-membrane, which could then be used to supplement pg levels. these changes may accelerate organophosphate conversion and increase available phosphorus recycling during periods of low phosphate stress. there is a great interest in the ability of qi to increase internal phosphorus utilization through lipid remodeling. compared with wild-type qi qi319- exhibited an increase in udp-sq expression in response to low phosphate stress, suggesting that udp-sq synthase plays a crucial role in maintaining an internal pi balance in this maize mutant. phosphorus in the vacuole acts as a cushion under fluctuating external and internal phosphorus levels. phosphorus may move from the vacuole to the cytoplasm and chloroplasts, thereby participating in metabolic reactions during phosphate stress. therefore, the vacuole plays a key role in maintaining cytoplasmic phosphorus homeostasis. phosphorus movement across the membrane depends on an electrochemical h+-gradient across the membrane, which is maintained by v-atpase. v-atpase is more highly accumulated in qi319- than in qi during phosphate stress. the mutant may utilize the additional v-atpase to intensify vacuolar-membrane proton transport, forming an electrochemical gradient across the membrane, and thus, increasing the energy supply for phosphorus transport across the membrane. this would significantly improve the tolerance of qi319- to low phosphate stress. qi319- plants exhibited much better lipid composition remodeling and higher v-atpase expression and activity levels than qi plants, which could facilitate phosphorus utilization during periods of phosphate stress. furthermore, these changes could be responsible for the increased pi levels in qi319- leaves compared with in qi leaves. increased expression of enzymes involved in the calvin cycle and co fixation may enhance photosynthesis in qi319- compared with in qi319 phosphate deprivation has an adverse effect on plant photosynthesis. the decline in photosynthetic efficiency in maize leaves under low phosphate stress is related to the reduced levels of proteins involved in co enrichment, the calvin cycle and the electron transport system. the reduced abundance of these proteins correlates well with the rates of photochemical reactions, co assimilation, the calvin cycle and rubp regeneration. several enzymes that play key roles in photosynthesis, such as rubisco and 3-phosphoglyceric phosphokinase, are involved in plant responses to low phosphate conditions. rubisco plays an important role in carbon fixation during the calvin cycle. reduced rubisco activity is one of the non-stomatal factors that leads to a substantial decline in photosynthesis. in the current study, rubisco was more highly expressed in qi319- than in qi and rubisco carboxylase activity was significantly higher in qi319- than in qi which may be related to the increased rate of photosynthesis in qi319- a reduction in rubp regeneration has a considerable impact on photosynthesis. the rate of photosynthesis may decrease if rubp regeneration declines. reductions in rubp regeneration may be due to reduced atp levels when plants are subjected to low phosphate conditions. reduced atp levels inhibit the reduction of 3-phosphoglycerate to triose phosphate through 3-phosphoglyceric phosphokinase, and they also inhibit the phosphorylation of ribulose-5-phosphate to rubp through phosphoribulokinase. the increased accumulation of atp in the leaves of qi319- compared with qi under low phosphate stress may be beneficial for rubp regeneration. in addition, the reduced rubp regeneration may be due to the reduced initial activities of several enzymes, such as nadp-gapdh and fbp aldolase. rubp regeneration is also affected by the increased amount of photosynthetic carbon that is diverted to starch formation and away from rubp regeneration. the higher level and activity of fbp aldolase in the chloroplasts of qi319- versus qi under phosphorus deprivation conditions may have led to a greater regeneration of rubp in qi319- compared with in qi maize is a c plant with “co pumps” that have a “floral hoop structure”. the “co pump” mechanism in c plants increases the level of co around rubisco, which may increase the carboxylation reaction rate and reduce the alternative fixation of o c photosynthesis is more efficient than c photosynthesis under low atmospheric co conditions, which suggests that the “co pump” is an important mechanism that leads to an increase in photosynthesis. the accumulation and activity levels of nadp-me and ppdk were higher in qi319- than in qi under phosphate deprivation conditions, which favored a greater capture rate and level of co in vascular bundle sheath cells. higher co levels not only increase rubisco carboxylase activity, but they also suppress the oxidation of rubisco and reduce photorespiration. the higher co level in qi319- helped the plants increase carbon assimilation and counteract phosphate deprivation. in conclusion, qi319- had a higher rate of photosynthesis than qi under low phosphate conditions, which could be attributed to its higher chlorophyll content and increased levels/activities of photosynthesis-related enzymes, such as rubisco, ppdk and nadp-me.  qi319- had a significantly higher level of pi than qi under low-phosphate stress, which may be related to qi319-96’s remodeled membrane lipids and the increase in v-atpase activity, which releases phosphorus from plant organophosphorus to the cytoplasm. a physiological analysis showed that the carbon assimilation rate of qi319- was significantly higher than that of qi under low-phosphate stress, which may be owing to the increased accumulation of several photosynthesis-related in the mutant qi319- compared with wild-type qi our results clearly indicated that the differences in increasing the internal p-use efficiency are the main reasons for the higher tolerance to low-p conditions in the mutant compared with the wild-type. at the same time, this study suggests that the inbred lines of maize with low-p tolerant traits could be obtained effectively through cellular engineering. 
chlorophyll biosynthesis was improved and the levels, and activities, of several calvin cycle and “co pump” enzymes were greater in qi319- than in qi which led to a higher rate of photosynthesis under low-phosphate stress in this line compared with in qi proteome and physiological analyses revealed that qi319- could better remodel the lipid composition of membranes and had higher v-atpase activity levels than qi under low-phosphate starvation, which enhanced the recycling of intracellular pi, as reflected by its increased pi levels. to elucidate the molecular mechanisms underlying the low-phosphate tolerance of this mutant, we performed comparative proteome analyses of the leaves of qi319- and qi under inorganic phosphate -sufficient and pi-deficient conditions. our results suggest that the increased tolerance of the maize mutant qi319- to low-phosphate levels is owing to its ability to increase pi availability. the changes in starch and sucrose contents under these conditions also differed between genotypes. the low-phosphate-tolerant maize mutant qi319- was obtained from qi through cellular engineering. additionally, inbred lines of maize with low-p-tolerant traits could be obtained effectively through cellular engineering. although the overall phosphorus contents of shoots were not significantly different between qi and qi319- the pi level of qi319- was  % higher than that of qi under low phosphorus conditions, qi319- had increased chlorophyll levels and enhanced photosynthesis.
 the low-phosphate-tolerant maize mutant qi319- was obtained from qi through cellular engineering. to elucidate the molecular mechanisms underlying the low-phosphate tolerance of this mutant, we performed comparative proteome analyses of the leaves of qi319- and qi under inorganic phosphate -sufficient and pi-deficient conditions.  low-phosphorus levels limit plant growth and metabolism. although the overall phosphorus contents of shoots were not significantly different between qi and qi319- the pi level of qi319- was  % higher than that of qi under low phosphorus conditions, qi319- had increased chlorophyll levels and enhanced photosynthesis. the changes in starch and sucrose contents under these conditions also differed between genotypes. the proteomic changes included and differentially expressed proteins involved in numerous metabolic processes. proteome and physiological analyses revealed that qi319- could better remodel the lipid composition of membranes and had higher v-atpase activity levels than qi under low-phosphate starvation, which enhanced the recycling of intracellular pi, as reflected by its increased pi levels. chlorophyll biosynthesis was improved and the levels, and activities, of several calvin cycle and “co pump” enzymes were greater in qi319- than in qi which led to a higher rate of photosynthesis under low-phosphate stress in this line compared with in qi  our results suggest that the increased tolerance of the maize mutant qi319- to low-phosphate levels is owing to its ability to increase pi availability. additionally, inbred lines of maize with low-p-tolerant traits could be obtained effectively through cellular engineering. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords maizeinorganic phosphoruslow-phosphate-tolerantproteomeleafhttp://dx.doi.org/ /501100001809national natural science foundation of china31172028zhang kewei shandong province agricultural breeding projectprograms involved in research on transgenic plants in china2014zx0800504bzhang kewei issue-copyright-statement© the author 2016 