Evidence of a key role for photosynthetic oxygen release in oil storage in developing soybean seeds

Based on the topographical analysis of photosynthesis and oil storage, we propose in a companion paper that photosynthetic oxygen release plays a key role in the local energy state, storage metabolism and flux toward lipid biosynthesis in developing soybean seeds. To test this hypothesis, we combined topographical analysis of ATP gradients across tissues, microsensor quantifications of internal O2 levels, assays of energy balance, metabolite profiles and isotope-labelling studies. Seeds show a marked degree of oxygen starvation in vivo (minimum O2 levels 0.1 kPa, approximately 1.3 microm), affecting ATP gradients, overall energy state, metabolite pools and storage activity. Despite the low light availability, photosynthesis supplies significant amounts of oxygen to the hypoxic seed tissue. This is followed by an increase in local ATP levels, most prominently within the lipid-synthesizing (inner) regions of the embryo. Concomitantly, partitioning of 14C-sucrose to lipids is increased, suggesting higher rates of lipid biosynthesis. It is concluded that both respiratory and biosynthetic fluxes are dynamically adjusted to photosynthetic oxygen supply.     

rgf1, a mutation reducing grain filling in maize through effects on basal endosperm and pedicel development

The maize cob presents an excellent opportunity to screen visually for mutations affecting assimilate partitioning in the developing kernel. We have identified a defective kernel mutant termed rgf1, reduced grain filling, with a final grain weight 30% of the wild type. In contrast with most defective endosperm mutants, rgf1 shows gene dosage-dependent expression in the endosperm. rgf1 kernels possess a small endosperm incompletely filling the papery pericarp, but embryo development is unaffected and the seeds are viable. The mutation conditions defective pedicel development and greatly reduces expression of endosperm transfer layer-specific markers. rgf1 exhibits striking morphological similarities to the mn1 mutant, but maps to a locus approximately 4 cM away from mn1 on chromosome 2 of maize. Despite reduced starch accumulation in the mutant, no obvious lesion in starch biosynthesis has been detected. Free sugar levels are unaltered in rgf1 endosperm. Rates of sugar uptake, measured over short (8 h) periods in cultured kernels, are increased in rgf1 compared to the wild type. rgf1 and wild-type kernels, excised at 5 DAP and cultured in vitro also develop differently in response to variations in sugar regime: glucose concentrations above 1% arrest placentochalazal development of rgf1 kernels, but have no effect on cultured wild-type kernels. These findings suggest that either uptake or perception of sugar(s) in endosperm cells at 5-10 DAP determines the rgf1 kernel phenotype.     

Distribution of Abscisic Acid in Maize Kernel during Grain Filling

The distribution of abscisic acid (ABA) within maize (Zea mays L.) kernels was studied in kernels from nontreated plants, from plants in which assimilate supply had been altered by source/sink manipulations, and in kernels cultured in vitro on ABA-free media. Prior to growth of the embryo, both the pedicel/placento-chalazal complex and the endosperm contained high concentration of ABA; however, the quantity of ABA in these tissues declined as the concentration in the embryo increased during the early stages of embryo growth. Peaks in the levels of ABA appeared to occur prior to and not concurrent with physiological events during grain filling. During most of the grain filling period, ABA concentration in the embryo was higher than that found in other kernel components. Altering assimilate supply by partial defoliation at two stages of development resulted in variable and transient effects on the relative distribution and concentration of ABA in kernel components. The concentration and distribution of ABA among components of kernels grown in vitro was similar to that observed for field-grown kernels. On the basis of these findings, in situ synthesis of ABA by kernel components is implicated and the putative role of ABA in the regulation of kernel development is discussed.     

Sugar Uptake and Metabolism in the Developing Endosperm of Tassel-seed Tunicate (Ts-5 Tu) Maize

Factors regulating assimilate transport into developing maize (Zea mays L.) kernels have been difficult to determine because of the structural complexity of basal kernel tissues and the damage that results from tissue dissection. The sensitivity of maize kernels to experimental manipulation is such that substantial maternal tissue is required to support kernel growth in vitro. Consequently, sugar transport experiments with isolated seed tissues or detached kernels have not unequivocally demonstrated how sugar transport occurs. In the present study, Tassel-seed Tunicate (Ts-5 Tu) maize kernels were investigated as a model system for introducing test solutions into the pedicel apoplast with minimal wounding. Transpiration in leafy glumes drew ¹⁴C-sugar solutions up the 8- to 10-millimeter-long pedicel stalks into the basal endosperm transfer cell region. ¹⁴C from fructose was incorporated into starch for 8 days. Sugar uptake into endosperm and embryo tissue showed specificity and inhibitor sensitivity. In particular, p-chloromercuribenzene sulfonate partially inhibited fructose uptake into the endosperm but had no effect on the metabolic conversion of that fructose that entered the endosperm. These results are consistent with active, carrier-mediated sugar transport, but a definitive determination would require more detailed tissue analysis. We propose that further refinement of the incubation solution may allow long-term kernel growth without cob tissue and thus provide a more precise determination of which maternal factors influence seed development.     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

Robustness of central carbohydrate metabolism in developing maize kernels

The central carbohydrate metabolism provides the precursors for the syntheses of various storage products in seeds. While the underlying biochemical map is well established, little is known about the organization and flexibility of carbohydrate metabolic fluxes in the face of changing biosynthetic demands or other perturbations. This question was addressed in developing kernels of maize (Zea mays L.), a model system for the study of starch and sugar metabolism. ¹³C-labeling experiments were carried out with inbred lines, heterotic hybrids, and starch-deficient mutants that were selected to cover a wide range of performances and kernel phenotypes. In total, 46 labeling experiments were carried out using either [U-¹³C₆]glucose or [U-¹³C₁₂]sucrose and up to three stages of kernel development. Carbohydrate flux distributions were estimated based on glucose isotopologue abundances, which were determined in hydrolysates of starch by using quantitative ¹³C-NMR and GC-MS. Similar labeling patterns in all samples indicated robustness of carbohydrate fluxes in maize endosperm, and fluxes were rather stable in response to glucose or sucrose feeding and during development. A lack of ADP-glucose pyrophosphorylase in the bt2 and sh2 mutants triggered significantly increased hexose cycling. In contrast, other mutations with similar kernel phenotypes had no effect. Thus, the distribution of carbohydrate fluxes is stable and not determined by sink strength in maize kernels.     

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II.

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.     

Energy state and its control on seed development: starch accumulation is associated with high ATP and steep oxygen gradients within barley grains

The role of oxygen and energy state in development and storage activity of cereal grains is an important issue, but has remained largely uninvestigated due to the lack of appropriate analytical methods. Metabolic profiling, bioluminescence-based in situ imaging of ATP, and oxygen-sensitive microsensors were combined here to investigate barley seed development. For the first time temporal and spatial maps of O2 and ATP distribution in cereal grains were determined and related to the differentiation pattern. Steep O2 gradients were demonstrated and strongly hypoxic regions were detected within the caryopsis (<0.1% of atmospheric saturation). Growing lateral and peripheral regions of endosperm remained well-supplied with O2 due to pericarp photosynthesis. ATP distribution in the developing grain was coupled to endosperm differentiation. High ATP concentrations were associated with the local onset of starch storage within endosperm, while low ATP overlapped with the hypoxic regions. Temporally, the building of steep gradients in ATP coincided with overall elevating metabolite levels, specific changes in the metabolite profiles (glycolysis and citrate cycle), and channelling of metabolic fluxes towards storage (increase of starch accumulation rate). These findings implicate an inhomogenous spatial arrangement of metabolic activity within the caryopsis. It is suggested that the local onset of starch storage is coupled with the accumulation of ATP and elevated metabolic activity. Thus, the ATP level reflects the metabolic state of storage tissue. On the basis of these findings, a hypothetical model for the regulation of starch storage in barley seeds is proposed.     

Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes

Legume seeds are heterotrophic and dependent on mitochondrial respiration. Due to the limited diffusional gas exchange, embryos grow in an environment of low oxygen. O(2) levels within embryo tissues were measured using microsensors and are lowest in early stages and during night, up to 0.4% of atmospheric O(2) concentration (1.1 micro M). Embryo respiration was more strongly inhibited by low O(2) during earlier than later stages. ATP content and adenylate energy charge were lowest in young embryos, whereas ethanol emission and alcohol dehydrogenase activity were high, indicating restricted ATP synthesis and fermentative metabolism. In vitro and in vivo experiments further revealed that embryo metabolism is O(2) limited. During maturation, ATP levels increased and fermentative metabolism disappeared. This indicates that embryos become adapted to the low O(2) and can adjust its energy state on a higher level. Embryos become green and photosynthetically active during differentiation. Photosynthetic O(2) production elevated the internal level up to approximately 50% of atmospheric O(2) concentration (135 micro M). Upon light conditions, embryos partitioned approximately 3-fold more [(14)C]sucrose into starch. The light-dependent increase of starch synthesis was developmentally regulated. However, steady-state levels of nucleotides, free amino acids, sugars, and glycolytic intermediates did not change upon light or dark conditions. Maturing embryos responded to low O(2) supply by adjusting metabolic fluxes rather than the steady-state levels of metabolites. We conclude that embryogenic photosynthesis increases biosynthetic fluxes probably by providing O(2) and energy that is readily used for biosynthesis and respiration.     

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.     

Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen

This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise.     

Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape

The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds. Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds. (a). By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds. At ambient external oxygen (21% [v/v]) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds. A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds. (b). The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen. (c). To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques. The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations. In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels. The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% [v/v]) external oxygen concentrations. (d). When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v). (e). Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites. (f). Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase. (g). The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state. (h). The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis. The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed.     

Enzymes of sucrose and hexose metabolism in developing kernels of two inbreds of maize

Tissue distribution and activity of enzymes involved in sucrose and hexose metabolism were examined in kernels of two inbreds of maize (Zea mays L.) at progressive stages of development. Levels of sugars and starch were also quantitated throughout development. Enzyme activities studied were: ATP-linked fructokinase, UTP-linked fructokinase, ATP-linked glucokinase, sucrose synthase, UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, PPi-linked phosphofructokinase, ATP-linked phosphofructokinase, NAD-dependent sorbitol dehydrogenase, NADP-dependent 6-P-gluconate dehydrogenase, NADP-dependent Glc-6-P dehydrogenase, aldolase, phosphoglucoisomerase, and phosphoglucomutase. Distribution of invertase activity was examined histochemically. Hexokinase and ATP-linked phosphofructokinase activities were the lowest among these enzymes and it is likely that these enzymes may regulate the utilization of sucrose in developing maize kernels. Most of the hexokinase activity was found in the endosperm, but the embryo had high activity on a dry weight basis. The endosperm, which stores primarily starch, contained high PPi-linked phosphofructokinase and low ATP-linked phosphofructokinase activities, whereas the embryo, which stores primarily lipids, had much higher ATP-linked phosphofructokinase activity than did the endosperm. It is suggested that PPi required by UDP-Glc pyrophosphorylase and PPi-linked phosphofructokinase in the endosperm may be supplied by starch synthesis. Sorbitol dehydrogenase activity was largely restricted to the endosperm, whereas 6-P-gluconate and Glc-6-P dehydrogenase activities were highest in the base and pericarp. A possible metabolic pathway by which sucrose is converted into starch is proposed.