Relationship between hexokinase and cytokinin in the regulation of leaf senescence and seed germination

Arabidopsis hexokinase (AtHXK1), an enzyme that catalyses hexose phosphorylation, accelerates leaf senescence, whereas the plant hormone cytokinin inhibits senescence. Previous work in our laboratory has shown that isopentenyl transferase (IPT), a key gene in the biosynthesis of cytokinin, expressed under promoters of the senescence-associated genes SAG12 or SAG13 (P(SAG12)::IPT and P(SAG13)::IPT, respectively), inhibits leaf senescence in tomato plants. To study the relationship between hexokinase and cytokinin in the regulation of leaf senescence, we created and analysed double-transgenic tomato plants expressing both AtHXK1 and either P(SAG12)::IPT or P(SAG13)::IPT. We found that expression of IPT in the double-transgenic plants could not prevent the accelerated senescence induced by over-expression of AtHXK1. Since cytokinin inhibits senescence via an apoplastic invertase that produces extracellular hexoses, whereas AtHXK1 is an intracellular mitochondria-associated hexokinase, our results suggest that intracellular sugar sensing via AtHXK1 is dominant over extracellular sugar sensing with regard to leaf senescence. Interestingly, the heterologous SAG12 and SAG13 promoters are also expressed in germinating tomato seed, around the radicle penetration zone, suggesting that seed germination involves a senescence process that is probably necessary for radicle emergence. Indeed, seed expressing P(SAG12)::IPT and P(SAG13)::IPT exhibited delayed radicle emergence, possibly due to delayed endosperm senescence.     

Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence.

Sugars are key regulatory molecules that affect diverse processes in higher plants. Hexokinase is the first enzyme in hexose metabolism and may be a sugar sensor that mediates sugar regulation. We present evidence that hexokinase is involved in sensing endogenous levels of sugars in photosynthetic tissues and that it participates in the regulation of senescence, photosynthesis, and growth in seedlings as well as in mature plants. Transgenic tomato plants overexpressing the Arabidopsis hexokinase-encoding gene AtHXK1 were produced. Independent transgenic plants carrying single copies of AtHXK1 were characterized by growth inhibition, the degree of which was found to correlate directly to the expression and activity of AtHXK1. Reciprocal grafting experiments suggested that the inhibitory effect occurred when AtHXK1 was expressed in photosynthetic tissues. Accordingly, plants with increased AtHXK1 activity had reduced chlorophyll content in their leaves, reduced photosynthesis rates, and reduced photochemical quantum efficiency of photosystem II reaction centers compared with plants without increased AtHXK1 activity. In addition, the transgenic plants underwent rapid senescence, suggesting that hexokinase is also involved in senescence regulation. Fruit weight, starch content in young fruits, and total soluble solids in mature fruits were also reduced in the transgenic plants. The results indicate that endogenous hexokinase activity is not rate limiting for growth; rather, they support the role of hexokinase as a regulatory enzyme in photosynthetic tissues, in which it regulates photosynthesis, growth, and senescence.     

Growth and Phosphate Transport in Barley and Tomato Plants During the Development of, and Recovery from, Phosphate-stress

Barley and tomato plants were cultured in nutrient solutionsincluding 0.15 mol m^–3 H²PO^–₄. The phosphate supplywas discontinued and the subsequent effects on growth, internalphosphorus concentrations, phosphate absorption and translocationwere measured at frequent intervals. Growth rates were at firstunchanged and the internal phosphorus concentration decreased.During this phase the rate of phosphate transport by the rootssometimes increased significantly. Growth slowed more in shootsthan in roots during a second phase of stress development andvisual symptoms of deficiency appeared in tomato but not inbarley. During this phase, enhancement of phosphate uptake capacityreached a maximum in both species. The subsequent decline inuptake capacity was associated with visible symptoms of deficiencydeveloping in barley and intensifying in tomato. When stressedplants were returned to a solution containing 0.15 mol m^–3H₂PO^–₄ rapid absorption continued for several days afterthe internal phosphorus concentration had returned to the levelof the controls. Phosphate toxicity may have been the causeof leaf lesions and necrosis during the ‘recovery’phase. Stomatal conductance in tomato was decreased at an early stageof stress development. Foliar-applied phosphate was absorbedmore rapidly by P-stressed barley leaves than by their controlsand much larger amounts were translocated from the leaves tothe roots.     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.     

Induction of leaf senescence by low nitrogen nutrition in sunflower (Helianthus annuus) plants

Different parameters which vary during the leaf development in sunflower plants grown with nitrate (2 or 20 mM) for a 42‐day period have been determined. The plants grown with 20 mM nitrate (N+) showed greater leaf area and specific leaf mass than the plants grown with 2 mM nitrate (N?). The total chlorophyll content decreased with leaf senescence, like the photosynthetic rate. This decline of photosynthetic activity was greater in plants grown with low nitrogen level (N?), showing more pronounced senescence symptoms than with high nitrogen (N+). In both treatments, soluble sugars increased with aging, while starch content decreased. A significant increase of hexose to sucrose ratio was observed at the beginning of senescence, and this raise was higher in N? plants than in N+ plants. These results show that sugar senescence regulation is dependent on nitrogen, supporting the hypothesis that leaf senescence is regulated by the C/N balance. In N+ and N? plants, ammonium and free amino acid concentrations were high in young leaves and decreased progressively in the senescent leaves. In both treatments, asparagine, and in a lower extent glutamine, increased after senescence start. The drop in the (Glu+Asp)/(Gln+Asn) ratio associated with the leaf development level suggests a greater nitrogen mobilization. Besides, the decline in this ratio occurred earlier and more rapidly in N? plants than in N+ plants, suggesting that the N? remobilization rate correlates with leaf senescence severity. In both N+ and N? plants, an important oxidative stress was generated in vivo during sunflower leaf senescence, as revealed by lipid peroxidation and hydrogen peroxide accumulation. In senescent leaves, the increase in hydrogen peroxide levels occurred in parallel with a decline in the activity of antioxidant enzymes. In N+ plants, the activities of catalase and ascorbate peroxidase (APX) increased to reach their highest values at 28 days, and later decreased during senescence, whereas in N? plants these activities started to decrease earlier, APX after 16 days and catalase after 22 days, suggesting that senescence is accelerated in N‐leaves. It is probable that systemic signals, such as a deficit in amino acids or other metabolites associated with the nitrogen metabolism produced in plants grown with low nitrogen, lead to an early senescence and a higher oxidation state of the cells of these plant leaves.     

Arbuscular mycorrhiza reduces susceptibility of tomato to Alternaria solani

Mycorrhiza frequently leads to the control of root pathogens, but appears to have the opposite effect on leaf pathogens. In this study, we studied mycorrhizal effects on the development of early blight in tomato (Solanum lycopersicum) caused by the necrotrophic fungus Alternaria solani. Alternaria-induced necrosis and chlorosis of all leaves were studied in mycorrhizal and non-mycorrhizal plants over time course and at different soil P levels. Mycorrhizal tomato plants had significantly less A. solani symptoms than non-mycorrhizal plants, but neither plant growth nor phosphate uptake was enhanced by mycorrhizas. An increased P supply had no effect on disease severity in non-mycorrhizal plants, but led to a higher disease severity in mycorrhizal plants. This was parallel to a P-supply-induced reduction in mycorrhiza formation. The protective effect of mycorrhizas towards development of A. solani has some parallels to induced systemic resistance, mediated by rhizobacteria: both biocontrol agents are root-associated organisms and both are effective against necrotrophic pathogens. The possible mechanisms involved are discussed.     

The significance of the magnesium to manganese ratio in plant tissues for growth and alleviation of manganese toxicity in tomato (Lycopersicon esculentum) and wheat (Triticum aestivum) plants

Results are reported for tomato (Lycopersicon esculentum L. var. Ailsa craig) and wheat (Triticum aestivum L. cv. Mara) which demonstrate that increasing concentrations of Mg in the plant raises plant tolerance to Mn toxicity.Water culture experiments with tomato show that under conditions of high Mn supply (200 µM, Mn), not only does increasing Mg application (0.75 mM to 15 mM) depress Mn uptake, but the higher Mg concentrations in the shoot counteract the onset of Mn toxicity when the concentrations of Mn in the shoot are also high. The ratio of Mg: Mn in the tissues is a better indicator of the appearance of toxicity symptoms than Mn concentration alone. Toxicity symptoms were observed when the Mg:Mn ratio in the shoot tissue was from 1.13 to a value between 3.53 and 6.54. The corresponding Mg: Mn ratio in the older leaves was from 0.82 to between 2.27 and 3.51.For wheat grown in soil, analyses of leaves revealed that growth could be expressed by the following relationship: Y=A+B exp(-kX), where Y=growth, X=Mg:Mn ratio, A, B and k=constants. Growth was significantly reduced when the Mg:Mn ratio fell below 20:1. From a measurement of this ratio it is therefore possible to predict the appearance of Mn toxicity and its influence on growth.     

Influence of potassium and manganese on growth and uptake of magnesium by soybeans (Glycine max (L.) Merr. cv. Bragg)

Summary Soybean (Glycine max (L) Merr. cv. Bragg) seedlings were grown in nutrient solutions to evaluate the response to manganese nutrition as affected by potassium supply. In solutions containing 275 M manganese, increasing the solution concentration of potassium from 1 mM to 10 mM alleviated symptoms of manganese toxicity, decreased manganese concentrations in the leaves and increased dry matter yields of the plants. The reduction in manganese toxicity was brought about by a reduced rate of root absorption of manganese at high potassium supply levels.Increasing the supply of either potassium or manganese decreased the leaf concentration of magnesium although there were no apparent symptoms of magnesium deficiency in any treatment. The reduced concentration of magnesium in the leaves was due to effects of potassium and manganese on the rate of root absorption of magnesium.Under manganese deficiency conditions, growth was reduced and manganese concentrations in plant parts were very low; there was no effect of potassium supply when manganese was absent from the nutrient solution.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

Uptake and distribution of phosphorus in tomato plants

Summary The uptake and distribution of phosphorus was examined in tomato plants, cv. Kirdford Cross, grown in peat to which phosphate was added (P₂) or omitted (P₁). The plants received a liquid feed containing either a high (N₂) or low (N₁) concentration of ammonium nitrate. Initially, all plants were grown in peat containing an intermediate level of phosphate.There was a rapid net export of P from the leaves of plants transferred to the P₁ medium resulting in deficiency symptoms before the fruit on the first truss had ripened. Most of the P absorbed by 11-week-old plants in the N₁P₂ and N₂P₂ treatments was located in the developing fruit, in the laminae of the mature leaves and in the lower parts of the stem. In the P₁ treatments, the lowest fruit truss was the dominant sink for the limited supply of P, but there was also a significant concentration of P in the shoot apex and in the laminae. Increasing the supply of N to plants in the P₂ treatment promoted the transport of P to the shoot and to the fruit trusses and also increased the total P uptake. However, plants in the N₂ treatment required a significantly higher level of tissue P to prevent the symptoms of P deficiency occurring in the laminae. Generally, symptoms occurred in laminae of mature leaves containing less than 0.13 per cent P. Increases in concentration of tissue P in response to raising the level of applied P were greatest in the petioles of the mature leaves, and it is suggested that these petioles are the most suitable tissues for the assessment of the P status of tomato plants.     

Quantitative Aspects of Leaf Acid Phosphatase Activity and the Phosphorus Status of Tomato Plants

The relation between tomato leaf acid phosphatase activity andleaf tissue P content has been examined, and a study made ofthe effects of leaf development, variation in nitrogen supply,and variation in the growing medium on this relationship. Tomatoplants were grown in sand and given various concentrations ofphosphate. Plants were also grown for an initial period in peatcontaining an adequate level of phosphate, then transferredto peat to which was added 0 or 2.3 kg superphosphate m^–3and supplied with either 50 of 300 µg N ml^–1. Expressed on a unit tissue f. wt basis, acid phosphatase activityin the control plants in sand (given 41 µg P ml^minus;1)was highest in extracts from the expanding leaves and decreasedwith leaf maturity. However, when given a reduced supply ofphosphate, the enzyme activity in the more mature leaves wasequal to, or greater than, that in the expanding leaves. Thephosphatase activity increased first in the young, fully-expandedleaves and in the mature leaves (with 4.1 µg P ml^–1),but did not increase in the expanding leaves until the supplywas restricted to 2.1 µg P ml^–1. On closer examination,the increase in enzyme activity appeared to be associated withthe P level in the leaf tissues, the activity increasing whenthe level fell below about 0.25 per cent (g P per 100 g drywt tissue). The same relation was found with the plants grownin peat, and was independent of the concentration of nitrogensupplied to the plants. The fully expanded leaves showed the best enzyme response whenthe phosphate supply was restricted and the activity reflectedclosely the local levels of tissue P. The assay of the enzymein unpurified leaf extracts is simple and rapid, and could beused in a test to detect P-deficiency in tomato plants. Lycopersicon esculentum L, tomato, acid phosphatase activity, phosphorus status     

Response of greenhouse tomato to salt stress and K+ supplement

Abstract

The effect of NaCl salinity and potassium supplement on growth, tissue ion concentration, photosynthesis, yield and fruit quality characteristics of tomato plants was studied. Tomato plants, hyb. Belladonna, were grown in 8.5 l pots, filled with 1:3 sand:perlite mixture and irrigated with a half-strength Hoagland solution through a closed hydroponic system. Six irrigation treatments were applied, including combinations of 3 salinity (0, 35 and 70 mM NaCl) and two potassium levels (K1: 200 ppm and K2: 400 ppm) in the nutrient solution. Salinity reduced photosynthesis resulting in reduced plant height and dry weight. Yield was reduced by 25% and 69% at 35 and 70 mM, respectively, as compared to control plants (0 mM NaCl). Both total soluble solids and titratable acidity of the fruit increased with increasing salinity and K levels. The application of high potassium level (K2) reduced the concentration of Na and increased that of K in the leaves and roots of the plants, as compared to K1 treatment. Toxicity symptoms were mostly observed in the leaves of 70K1 plants, while no visual symptoms of toxicity were observed in 70K2 treatment. Despite the positive effects of potassium supplement in reducing Na concentration and the absence of toxicity symptoms in the leaves, plant growth was not improved, while leaf photosynthesis was reduced. Furthermore, no positive effects in the percentage of marketable fruit, mean fruit weight and yield were observed in the plants receiving extra K.     

Increased hexose transport in the roots of tomato plants submitted to prolonged hypoxia

We investigated the effects of prolonged hypoxia on the sugar uptake in tomato (Solanum lycopersicum L. var. MP-1) roots. Hydroponic cultures of whole tomato plants were submitted to hypoxic treatment for 1 week, and the roots were analyzed for sugar concentrations, hexose uptake and hexose transporter expression level. Contrary to what has been observed after anoxic shock or short-term hypoxic treatment, we show that sugar concentrations increase and hexose uptake is up-regulated in the roots after 1 week of hypoxic treatment. Increased hexose transport is concomitant with the induction of the hexose transporter gene LeHT2. These responses may be due either to a direct effect of low O₂ supply, or to a secondary effect associated with the increase in sugar concentrations, which, typically, develops in most hypoxic plants.     

Occurrence,identification and transmission of the phytoplasma associated with tomato big bud disease and identification of its vector and weed host in Pakistan

Abstract

Tomato (Solanum lycopersicum L.) plants showing stunting, big bud, leaves yellowing or reddening and witches’-broom symptoms were observed since 2009 in Pakistan. A weed Parthenium hysterophorus grown in and around tomato fields also exhibited witches’-broom like symptoms. Fluorescence light microscopy of hand-cut stem stalk sections treated with Dienes’ stain showed blue areas in the phloem region of both tomato and P. hysterophorus symptomatic plants that indicated the association of phytoplasma with the complex. Amplification of 1.2?kb 16S rDNA fragment in nested PCR confirmed that the symptomatic tomato and P. hysterophorus plants are infected by a phytoplasma. Partial sequencing of 16S rRNA (GenBank accession: LT671581 and LT671583) and virtual restriction fragment length polymorphism confirmed that the phytoplasma associated with both plant species had the greatest homology to 16SrII-D subgroup. Disease was successfully transmitted by grafting and leafhopper Orosius albicinctus in tomato plants. This is the first report of natural occurrence of 16SrII-D phytoplasma in tomatoes and a weed P. hysterophorus in Pakistan.     

Retardation of bean leaf senescence by benzyladenine and its influence on phosphate metabolism

The levels of glucose, sugar phosphates, and adenosine phosphates were determined in primary leaves of intact bean plants during normal senescence and compared to leaves in which senescence was delayed by application of benzyladenine (BA). In both cases there was a rise with time in the levels of glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate, and a decline in 2-phosphoglyceric acid, inorganic phosphate, and the adenosine phosphates (AMP, ADP, ATP). The levels of fructose 1,6-diphosphate remained fairly constant. Although the levels of hexose phosphates, adenosine phosphates, and inorganic phosphate were lower in the BA-treated leaves, the incorporation of ³²P into these compounds by 3- and 6-week-old plants was higher than in the controls. These results suggest that the retardation of leaf senescence by BA in intact bean plants is associated with increased utilization of metabolites, indicating a more rapid turnover of the adenosine phosphates. It is concluded that this effect is brought about by a regulatory coordination of metabolic processes in relation to energy production and utilization.     

Uptake of the herbicide [14C] 2,4-D iso-octyl in the vapour phase by tomato and lettuce plants and some effects on growth and phytotoxicity

Tomato and lettuce plants were exposed to vapour of the herbicide [¹⁴C-phenyl] 2,4-D iso-octyl at concentrations in the range 0–450 pg litre^-1 for periods of 6, 24 and 72 h in separate experiments using a flowing-air system. The rate of uptake, as indicated by plant content of radiolabel, was both linear with respect to the vapour concentration and independent of the duration of exposure. The uptake rate of tomato was greater than that of lettuce at any given vapour concentration. Uptake by the apical leaves of lettuce was higher than by the older leaves expressed on an area basis; tomato showed no effect of leaf position. Visible symptoms of phytotoxicity were shown by both species 40 days after exposure to the highest doses. Advantages of using the low-volatile ester (iso-octyl) of 2,4-D to reduce vapour drift damage are discussed in terms of plant uptake.     

Transgenic tomato overexpressing ath-miR399d has enhanced phosphorus accumulation through increased acid phosphatase and proton secretion as well as phosphate transporters

By generating and examining transgenic tomato overexpressing ath-miR399d grown in hydroponic conditions, in quartz sand, or in a polytunnel greenhouse vegetable soil culture, this study aimed to investigate the effects of miR399d from Arabidopsis on phosphorus (P) accumulation, P concentrations in transgenic tomato overexpressing ath-miR399d shoots, phosphate transporter expression, and proton secretion and acid phosphatase (APase) activity in roots. In the transgenic tomato, leaf P concentration increased significantly in an agricultural soil, and roots had higher uptake of P, as evidenced by leaf P concentrations and relative expression of the genes LePT1, LePT2, LePT4, and LePT5 in normal-P solution. Enhanced APase activity in transgenic roots and the outside medium led to superior hydrolysis of organic P, and increased proton extrusion by roots led to superior dissolution of AlPO₄. Thus, besides phosphate transporters, higher APase activity and strengthened acidification in the vicinity of the roots may be important mechanisms for transgenic tomato to scavenge or acquire P in soil. These results provide new understanding of miR399-overexpressing plants that accumulate excess P in shoots.     

Reduced inositol content and altered morphology in transgenic potato plants inhibited for 1D- myo -inositol 3-phosphate synthase

Myo -inositol is a precursor of many plant metabolites, including polyols, cell wall components and phosphoinositides. The first committed step in the de novo myo -inositol synthetic pathway is catalysed by the enzyme 1D- myo -inositol 3-phosphate synthase (MIPS; EC 5.5.1.4 ), which converts D-glucose 6-phosphate to 1D- myo -inositol 3-phosphate. Suppression of MIPS activity by an antisense RNA approach in transgenic potato ( Solanum tuberosum L.) plants to below 20% of the wild-type level in leaves resulted in strongly reduced levels of inositol, galactinol and raffinose (approximately 7%, 5% and 12%, respectively, of wild-type values). In contrast, increases were observed for concentrations of hexose phosphates (up to 1.7-fold), sucrose (twofold) and starch (two- to fourfold). Transgenic plants exhibited reduced apical dominance, altered leaf morphology, precocious leaf senescence and a decrease in overall tuber yield. These observations indicate a crucial role for myo -inositol in plant physiology and development.     

P-Nuclear Magnetic Resonance Determination of Phosphate Compartmentation in Leaves of Reproductive Soybeans (Glycine max L.) as Affected by Phosphate Nutrition

Most leaf phosphorus is remobilized to the seed during reproductive development in soybean. We determined, using ³¹P-NMR, the effect phosphorus remobilization has on vacuolar inorganic phosphate pool size in soybean (Glycine max [L.] Merr.) leaves with respect to phosphorus nutrition and plant development. Phosphate compartmentation between cytoplasmic and vacuolar pools was observed and followed in intact tissue grown hydroponically, at the R2, R4, and R6 growth stages. As phosphorus in the nutrient solution decreased from 0.45 to 0.05 millimolar, the vacuolar phosphate peak became less prominent relative to cytoplasmic phosphate and hexose monophosphate peaks. At a nutrient phosphate concentration of 0.05 millimolar, the vacuolar phosphate peak was not detectable. At higher levels of nutrient phosphate, as plants progressed from the R2 to the R6 growth stage, the vacuolar phosphate peak was the first to disappear, suggesting that storage phosphate was remobilized to a greater extent than metabolic phosphate. Under suboptimal phosphate nutrition (≤ 0.20 millimolar), the hexose monophosphate and cytoplasmic phosphate peaks declined earlier in reproductive development than when phosphate was present in optimal amounts. Under low phosphate concentrations (0.05 millimolar) cytoplasmic phosphate was greatly reduced. Carbon metabolism was coincidently disrupted under low phosphate nutrition as shown by the appearance of large, prominent starch grains in the leaves. Cytoplasmic phosphate, and leaf carbon metabolism dependent on it, are buffered by vacuolar phosphate until late stages of reproductive growth.