Journal of Experimental Botany - current issue

New insights on the effects of heat stress on crops

Halford, N. G. — Fri, 30 Oct 2009 06:38:02 PDT

Symbolism of plants: examples from European-Mediterranean culture presented with biology and history of art: DECEMBER: Cross-roses and rose crosses

Kandeler, R., Ullrich, W. R. — Fri, 30 Oct 2009 06:38:02 PDT

Symbolism of plants: examples from European-Mediterranean culture presented with biology and history of art: EPILOGUE

Kandeler, R., Ullrich, W. R. — Fri, 30 Oct 2009 06:38:02 PDT

Protein targets of tyrosine nitration in sunflower (Helianthus annuus L.) hypocotyls

Fri, 30 Oct 2009 06:38:02 PDT

Tyrosine nitration is recognized as an important post-translational protein modification in animal cells that can be used as an indicator of a nitrosative process. However, in plant systems, there is scant information on proteins that undergo this process. In sunflower hypocotyls, the content of tyrosine nitration (NO₂-Tyr) and the identification of nitrated proteins were studied by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and proteomic approaches, respectively. In addition, the cell localization of nitrotyrosine proteins and peroxynitrite were analysed by confocal laser-scanning microscopy (CLSM) using antibodies against 3-nitrotyrosine and 3'-(p-aminophenyl) fluorescein (APF) as the fluorescent probe, in that order. The concentration of Tyr and NO₂-Tyr in hypocotyls was 0.56 µmol mg^–1 protein and 0.19 pmol mg^–1 protein, respectively. By proteomic analysis, a total of 21 nitrotyrosine-immunopositive proteins were identified. These targets include proteins involved in photosynthesis, and in antioxidant, ATP, carbohydrate, and nitrogen metabolism. Among the proteins identified, S-adenosyl homocysteine hydrolase (SAHH) was selected as a model to evaluate the effect of nitration on SAHH activity using SIN-1 (a peroxynitrite donor) as the nitrating agent. When the hypocotyl extracts were exposed to 0.5 mM, 1 mM, and 5 mM SIN-1, the SAHH activity was inhibited by some 49%, 89%, and 94%, respectively. In silico analysis of the barley SAHH sequence, characterized Tyr448 as the most likely potential target for nitration. In summary, the present data are the first in plants concerning the content of nitrotyrosine and the identification of candidates of protein nitration. Taken together, the results suggest that Tyr nitration occurs in plant tissues under physiological conditions that could constitute an important process of protein regulation in such a way that, when it is overproduced in adverse circumstances, it can be used as a marker of nitrosative stress.

The alternative respiratory pathway allows sink to cope with changes in carbon availability in the sink-limited plant Erythronium americanum

Gandin, A., Lapointe, L., Dizengremel, P. — Fri, 30 Oct 2009 06:38:02 PDT

Mechanisms that allow plants to cope with a recurrent surplus of carbon in conditions of imbalance between source and sink activity has not received much attention. The response of sink growth and metabolism to the modulation of source activity was investigated using elevated CO₂ and elevated O₃ growth conditions in Erythronium americanum. Sink activity was monitored via slice and mitochondrial respiratory rates, sucrose hydrolysis activity, carbohydrates, and biomass accumulation throughout the growth season, while source activity was monitored via gas exchanges, rubisco and phosphoenolpyruvate carboxylase activities, carbohydrates, and respiratory rates. Elevated CO₂ increased the net photosynthetic rate by increasing substrate availability for rubisco. Elevated O₃ decreased the net photosynthetic rate mainly through a reduction in rubisco activity. Despite this modulation of the source activity, neither plant growth nor starch accumulation were affected by the treatments. Sucrose synthase activity was higher in the sink under elevated CO₂ and lower under elevated O₃, thereby modulating the pool of glycolytic intermediates. The alternative respiratory pathway was similarly modulated in the sink, as seen with both the activity and capacity of the pathway, as well as with the alternative oxidase abundance. In this sink-limited species, the alternative respiratory pathway appears to balance carbon availability with sink capacity, thereby avoiding early feedback-inhibition of photosynthesis in conditions of excess carbon availability.

Polyamine oxidase activity contributes to sustain maize leaf elongation under saline stress

Rodriguez, A. A., Maiale, S. J., Menendez, A. B., Ruiz, O. A. — Fri, 30 Oct 2009 06:38:02 PDT

The possible involvement of apoplastic reactive oxygen species produced by the oxidation of free polyamines in the leaf growth of salinized maize has been studied here. Salt treatment increased the apoplastic spermine and spermidine levels, mainly in the leaf blade elongation zone. The total activity of polyamine oxidase was up to 20-fold higher than that of the copper-containing amine oxidase. Measurements of H₂O₂, ·O₂^–, and HO· production in the presence or absence of the polyamine oxidase inhibitors 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane suggest that, in salinized plants, the oxidation of free apoplastic polyamines by polyamine oxidase by would be the main source of reactive oxygen species in the elongation zone of maize leaf blades. This effect is probably due to increased substrate availability. Incubation with 200 µM spermine doubled segment elongation, whereas the addition of 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane to 200 µM spermine attenuated and reversed the last effect, respectively. Similarly, the addition of MnCl₂ (an ·O₂^– dismutating agent) or the HO· scavenger sodium benzoate along with spermine, annulled the elongating effect of the polyamine on the salinized segments. As a whole, the results obtained here demonstrated that, under salinity, polyamine oxidase activity provides a significant production of reactive oxygen species in the apoplast which contributes to 25–30% of the maize leaf blade elongation.

Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain

Waters, B. M., Uauy, C., Dubcovsky, J., Grusak, M. A. — Fri, 30 Oct 2009 06:38:02 PDT

The NAM-B1 gene is a NAC transcription factor that affects grain nutrient concentrations in wheat (Triticum aestivum). An RNAi line with reduced expression of NAM genes has lower grain protein, iron (Fe), and zinc (Zn) concentrations. To determine whether decreased remobilization, lower plant uptake, or decreased partitioning to grain are responsible for this phenotype, mineral dynamics were quantified in wheat tissues throughout grain development. Control and RNAi wheat were grown in potting mix and hydroponics. Mineral (Ca, Cu, Fe, K, Mg, Mn, P, S, and Zn) and nitrogen (N) contents of organs were determined at regular intervals to quantify the net remobilization from vegetative tissues and the accumulation of nutrients in grain. Total nutrient accumulation was similar between lines, but grain Fe, Zn, and N were at lower concentrations in the NAM knockdown line. In potting mix, net remobilization of N, Fe, and Zn from vegetative tissues was impaired in the RNAi line. In hydroponics with ample nutrients, net remobilization was not observed, but grain Fe and Zn contents and concentrations remained lower in the RNAi line. When Fe or Zn was withheld post-anthesis, both lines demonstrated remobilization. These results suggest that a major effect of the NAM genes is an increased efflux of nutrients from the vegetative tissues and a higher partitioning of nutrients to grain.

Axillary bud outgrowth potential is determined by parent apical bud activity

Thomas, R. G., Hay, M. J. M. — Fri, 30 Oct 2009 06:38:03 PDT

Axillary buds within a plant shoot system are known to differ in their ability to respond to treatments favouring their development. This ability is referred to as their outgrowth potential. Using two species of prostrate nodally-rooting herbs, dicotyledonous Trifolium repens and monocotyledonous Tradescantia fluminensis, grown throughout in a strictly vegetative state, this study tested two hypotheses. Hypothesis 1: that each axillary bud exhibits an outgrowth potential that is directly related to the growth rate of its parent apical bud, and Hypothesis 2: that the growth rate attained by an axillary bud depends upon both its outgrowth potential and the local supply of stimulatory root-derived signal (NRS) available to it. Activation levels (growth rates) of apical buds were varied by differential exposure to nodal roots and the outgrowth responses of axillary buds recently emerged from them were then measured under standardized conditions of NRS supply. Hypothesis 1 was shown to be correct for both species. Hypothesis 2, tested only in T. repens, was supported by results showing that an axillary bud's outgrowth potential and the NRS supply to it each independently influenced its growth rate, there being no significant interaction between the two. These results emphasize the significant role the physiological state/activity of apical buds has on the outgrowth potential of axillary buds formed within them. The fact that similar relationships were observed on axillary buds on stems of differing developmental maturity and branching hierarchy, and in two taxonomically diverse species, suggests they might be widespread among morphologically similar species.

Effects of {beta}-1,3-glucan from Septoria tritici on structural defence responses in wheat

Fri, 30 Oct 2009 06:38:03 PDT

The accumulation of the pathogenesis-related (PR) proteins β-1,3-glucanase and chitinase and structural defence responses were studied in leaves of wheat either resistant or susceptible to the hemibiotrophic pathogen Septoria tritici. Resistance was associated with an early accumulation of β-1,3-glucanase and chitinase transcripts followed by a subsequent reduction in level. Resistance was also associated with high activity of β-1,3-glucanase, especially in the apoplastic fluid, in accordance with the biotrophic/endophytic lifestyle of the pathogen in the apoplastic spaces, thus showing the highly localized accumulation of defence proteins in the vicinity of the pathogen. Isoform analysis of β-1,3-glucanase from the apoplastic fluid revealed that resistance was associated with the accumulation of an endo-β-1,3-glucanase, previously implicated in defence against pathogens, and a protein with identity to ADPG pyrophosphatase (92%) and germin-like proteins (93%), which may be involved in cell wall reinforcement. In accordance with this, glycoproteins like extensin were released into the apoplast and callose accumulated to a greater extent in cell walls, whereas lignin and polyphenolics were not found to correlate with defence. Treatment of a susceptible wheat cultivar with purified β-1,3-glucan fragments from cell walls of S. tritici gave complete protection against disease and this was accompanied by increased gene expression of β-1,3-glucanase and the deposition of callose. Collectively, these data indicate that resistance is dependent on a fast, initial recognition of the pathogen, probably due to β-1,3-glucan in the fungal cell walls, and this results in the accumulation of β-1,3-glucanase and structural defence responses, which may directly inhibit the pathogen and protect the host against fungal enzymes and toxins.

The 'trade-off' between synthesis of primary and secondary compounds in young tomato leaves is altered by nitrate nutrition: experimental evidence and model consistency

Bot, J. L., Benard, C., Robin, C., Bourgaud, F., Adamowicz, S. — Fri, 30 Oct 2009 06:38:03 PDT

Plants allocate internal resources to fulfil essential, yet possibly conflicting, demands such as defence or growth, as hypothesized by the ‘growth–differentiation balance theory’ (GDB). This trade-off was examined in young tomato plants grown for 25 d using the nutrient film technique with seven nitrate concentrations ([NO₃]). The modification of primary (growth-related: organic acids, carbohydrates) and secondary (defence-related: phenolics) metabolite concentrations in leaves was assessed. Then a simple model was devised to simulate the trade-off between growth and secondary metabolism in response to N nutrition. N affected growth and metabolite concentrations in the leaves. Dry biomass, leaf area, and concentrations of nitrate and organic acid (malic, citric) increased with rising [NO₃], up to a threshold, above which they remained constant. Starch, sucrose, and organic N concentrations were invariant with [NO₃]. Glucose, fructose, and phenolic (chlorogenic acid, rutin, and kaempferol-rutinoside) concentrations were highest at lowest [NO₃]. They declined progressively with rising [NO₃] until a threshold, above which they remained constant. Model predictions are in phase with experimental phenolic concentration data although the simulated metabolic rates differ from the GDBH proposals depicted in the literature. From the model output it is shown that a careful definition of the C reserve compounds is required.

Biochemical and proteomic analysis of 'Dixiland' peach fruit (Prunus persica) upon heat treatment

Fri, 30 Oct 2009 06:38:03 PDT

Shipping of peaches to distant markets and storage require low temperature; however, cold storage affects fruit quality causing physiological disorders collectively termed ‘chilling injury’ (CI). In order to ameliorate CI, different strategies have been applied before cold storage; among them heat treatment (HT) has been widely used. In this work, the effect of HT on peach fruit quality as well as on carbon metabolism was evaluated. When fruit were exposed to 39 °C for 3 d, ripening was delayed, with softening inhibition and slowing down of ethylene production. Several differences were observed between fruit ripening at ambient temperature versus fruit that had been heat treated. However, the major effects of HT on carbon metabolism and organoleptic characteristics were reversible, since normal fruit ripening was restored after transferring heated peaches to ambient temperature. Positive quality features such as an increment in the fructose content, largely responsible for the sweetness, and reddish coloration were observed. Nevertheless, high amounts of acetaldehyde and low organic acid content were also detected. The differential proteome of heated fruit was characterized, revealing that heat-induced CI tolerance may be acquired by the activation of different molecular mechanisms. Induction of related stress proteins in the heat-exposed fruits such as heat shock proteins, cysteine proteases, and dehydrin, and repression of a polyphenol oxidase provide molecular evidence of candidate proteins that may prevent some of the CI symptoms. This study contributes to a deeper understanding of the cellular events in peach under HT in view of a possible technological use aimed to improve organoleptic and shelf-life features.

Differences in C metabolism of ash species and provenances as a consequence of root oxygen deprivation by waterlogging

Jaeger, C., Gessler, A., Biller, S., Rennenberg, H., Kreuzwieser, J. — Fri, 30 Oct 2009 06:38:03 PDT

The waterlogging tolerance and the physiological responses to this stress were tested in seedlings of Fraxinus angustifolia, an ash tree inhabiting riparian forests, and two provenances of the closely related Fraxinus excelsior, one derived from a riparian forest (FER) and one from a mountainous region (FEM). Besides visible damage, physiological parameters reflecting adaptations of plants to waterlogging such as net CO₂ assimilation, alcoholic fermentation, and the concentrations of metabolites related to flooding responses were studied. Consistent with the higher flooding tolerance of F. angustifolia and FER compared with FEM, net assimilation remained unaffected in F. angustifolia, was slightly reduced in FER, but was strongly affected in FEM. Altered carbohydrate concentrations in the roots of the seedlings suggest differences in the ability to supply alcoholic fermentation with substrate during prolonged periods of soil anoxia. Another difference between the seedlings was connected to the -aminobutyric acid (GABA) shunt which resulted in alanine accumulation in the flooding-tolerant trees, but strong GABA accumulation in the more sensitive FEM seedlings. This finding indicates differences in GABA conversion into alanine which might result in an accumulation of phytotoxic levels of intermediates. Such provenance-specific differences in Common ash suggest that the selection of appropriate provenances is essential for forest management in flood-prone areas.

Brassinosteroids interact negatively with jasmonates in the formation of anti-herbivory traits in tomato

Fri, 30 Oct 2009 06:38:04 PDT

Given the susceptibility of tomato plants to pests, the aim of the present study was to understand how hormones are involved in the formation of tomato natural defences against insect herbivory. Tomato hormone mutants, previously introgressed into the same genetic background of reference, were screened for alterations in trichome densities and allelochemical content. Ethylene, gibberellin, and auxin mutants indirectly showed alteration in trichome density, through effects on epidermal cell area. However, brassinosteroids (BRs) and jasmonates (JAs) directly affected trichome density and allelochemical content, and in an opposite fashion. The BR-deficient mutant dpy showed enhanced pubescence, zingiberene biosynthesis, and proteinase inhibitor expression; the opposite was observed for the JA-insensitive jai1-1 mutant. The dpyxjai1-1 double mutant showed that jai1-1 is epistatic to dpy, indicating that BR acts upstream of the JA signalling pathway. Herbivory tests with the poliphagous insect Spodoptera frugiperda and the tomato pest Tuta absoluta clearly confirmed the importance of the JA–BR interaction in defence against herbivory. The study underscores the importance of hormonal interactions on relevant agricultural traits and raises a novel biological mechanism in tomato that may differ from the BR and JA interaction already suggested for Arabidopsis.

Variability among species in the apoplastic pH signalling response to drying soils

Sharp, R. G., Davies, W. J. — Fri, 30 Oct 2009 06:38:04 PDT

After the imposition of soil drying treatments, an elevation of xylem sap pH is one of the earliest observable responses in many herbaceous model plant species. It is theorized that alkalization of sap results in a concurrent elevation in abscisic acid (ABA) concentration delivered to transpiring tissues by preventing Henderson–Hasselbalch-regulated partitioning between the apoplast and symplast. However, here it is demonstrated that the sap alkalzation response to soil drying is far from universal in higher plant species. Tests were conducted to determine how universal the pH response to drying soil was in a range of perennial species from a diverse range of plant families. The response was not found in the majority of the 22 species tested. Four species exhibited significant increases in pH, but the majority showed no significant change in xylem sap pH. There was no evolutionary relationship between the species that showed alkalization under drought stress. However, the species that alkalized sap also exhibited good control over internal water status and were the most isohydric species of those tested. None of the species exhibiting anisohydric responses alkalized xylem sap under drought stress. Regardless of alkalization response, plants still retain the ability to respond to changes in xylem sap pH when manipulated by alkaline buffer foliar sprays. This finding indicates that plants have conserved the ability to respond to changes in xylem pH and redistribute ABA, even if they do not currently utilize the mechanism when exposed to drought stress. It was found in Buddleja davidii, Euonymus fortunei, and Hydrangea serrata that the xylem sap pH response to water deficits mirrored the natural pH changes that occur as sap is transported to the leaves, indicating that plants need to be able to have naturally occurring alkalization processes in place for them to be up-regulated under drought stress.

Regulation of oleosin expression in developing peanut (Arachis hypogaea L.) embryos through nucleosome loss and histone modifications

Fri, 30 Oct 2009 06:38:04 PDT

Nucleosome loss and histone modifications are important mechanisms for transcriptional regulation. Concomitant changes in chromatin structures of two peanut (Arachis hypogaea L.) oleosin genes, AhOleo17.8 and AhOleo18.5, were examined in relation to transcriptional activity. Spatial and temporal expression analyses showed that both AhOleo17.8 and AhOleo18.5 promoters can adopt three conformational states, an inactive state (in vegetative tissues), a basal activated state (in early maturation embryos), and a fully activated state (in late maturation embryos). Chromatin immunoprecipitation assays revealed an increase of histone H3 acetylation levels at the proximal promoters and coding regions of AhOleo17.8 and AhOleo18.5 associated with basal transcription in early maturation embryos. Meanwhile, a decrease of histone H3K9 dimethylation levels at coding regions of oleosins was observed in early maturation embryos. However, a dramatic decrease in the histone acetylation signal was observed at the core promoters and the coding regions of the two oleosins in the fully activated condition in late maturation embryos. Although a small decrease of histone H3 levels of oleosins chromatin was detected in early maturation embryos, a significant loss of histone H3 levels occurred in late maturation embryos. These analyses indicate that the histone eviction from the proximal promoters and coding regions is associated with the high expression of oleosin genes during late embryos maturation. Moreover, the basal expression of oleosins in early maturation embryos is accompanied by the increase of histone H3 acetylation and decrease of histone H3K9me2.

Arabidopsis L-type lectin receptor kinases: phylogeny, classification, and expression profiles

Bouwmeester, K., Govers, F. — Fri, 30 Oct 2009 06:38:04 PDT

In plants, lectin receptor kinases are considered to play crucial roles during development and in the adaptive response to various stimuli. Arabidopsis lectin receptor kinases can be divided into three type-classes based on sequence similarity of their extracellular lectin motifs. The current study focuses on the legume-like lectin receptor kinases (LecRKs), which are regarded as ideal candidates for monitoring cell wall integrity and are possibly functional in adaptive responses. An inventory of the Arabidopsis LecRK gene family is presented here. It consists of 45 members including three that were recently identified; two encode N-terminal truncated variants one of which has two in tandem kinase domains. Phylogenetic trees derived from full-length amino acid sequence alignments were highly concordant to phylograms that were purely based on lectin motifs or kinase domains. The phylograms allowed reclassification of the LecRK genes and hence a new proposal for gene nomenclature was suggested. In addition, a comprehensive expression analysis was executed by exploring public repositories. This revealed that several LecRK genes are differentially expressed during plant growth and development. Moreover, multiple LecRKs appear to be induced upon treatment with elicitors and pathogen infection. Variation in gene expression was also analysed in seedlings of diverse Arabidopsis accessions. Taken together, this study provides a genome-wide overview of the LecRK gene family and an up-to-date classification using a novel and systematic gene nomenclature.

Functional diversity in gravitropic reaction among tropical seedlings in relation to ecological and developmental traits

Almeras, T., Derycke, M., Jaouen, G., Beauchene, J., Fournier, M. — Fri, 30 Oct 2009 06:38:04 PDT

Gravitropism is necessary for plants to control the orientation of their axes while they grow in height. In woody plants, stem re-orientations are costly because they are achieved through diameter growth. The functional diversity of gravitropism was studied to check if the mechanisms involved and their efficiency may contribute to the differentiation of height growth strategies between forest tree species at the seedling stage. Seedlings of eight tropical species were grown tilted in a greenhouse, and their up-righting movement and diameter growth were measured over three months. Morphological, anatomical, and biomechanical traits were measured at the end of the survey. Curvature analysis was used to analyse the up-righting response along the stems. Variations in stem curvature depend on diameter growth, size effects, the increase in self-weight, and the efficiency of the gravitropic reaction. A biomechanical model was used to separate these contributions. Results showed that (i) gravitropic movements were based on a common mechanism associated to similar dynamic patterns, (ii) clear differences in efficiency (defined as the change in curvature achieved during an elementary diameter increment for a given stem diameter) existed between species, (iii) the equilibrium angle of the stem and the anatomical characters associated with the efficiency of the reaction also differed between species, and (iv) the differences in gravitropic reaction were related to the light requirements: heliophilic species, compared to more shade-tolerant species, had a larger efficiency and an equilibrium angle closer to vertical. This suggests that traits determining the gravitropic reaction are related to the strategy of light interception and may contribute to the differentiation of ecological strategies promoting the maintenance of biodiversity in tropical rainforests.

Transcriptomic profiling of heat-stress response in potato periderm

Fri, 30 Oct 2009 06:38:04 PDT

Potato (Solanum tuberosum L.) periderm is composed of the meristematic phellogen that gives rise to an external layer of suberized phellem cells (the skin) and the internal parenchyma-like phelloderm. The continuous addition of new skin layers and the sloughing of old surface layers during tuber maturation results in smooth, shiny skin. However, smooth-skin varieties frequently develop unsightly russeting in response to high soil temperatures. Microscopic observation of microtubers exposed to high temperatures (37°C) suggested heat-enhanced development and accumulation of suberized skin-cell layers. To identify the genes involved in the periderm response to heat stress, skin and phelloderm samples collected separately from immature tubers exposed to high soil temperatures (33°C) and controls were subjected to transcriptome profiling using a potato cDNA array. As expected, the major functional group that was differentially expressed in both skin and phelloderm consisted of stress-related genes; however, while the major up-regulated phelloderm genes coded for heat-shock proteins, many of the skin's most up-regulated sequences were similar to genes involved in the development of protective/symbiotic membranes during plant–microbe interactions. The primary activities regulated by differentially expressed peridermal transcription factors were response to stress (33%) and cell proliferation and differentiation (28%), possibly reflecting the major processes occurring in the heat-treated periderm and implying the integrated activity of the stress response and tissue development. Accumulating data suggest that the periderm, a defensive tissue, responds to heat stress by enhancing the production and accumulation of periderm/skin layers to create a thick protective cover. Skin russeting may be an indirect outcome; upon continued expansion of the tuber, the inflexible skin cracks while new layers are produced below it, resulting in a rough skin texture.

The amylose extender mutant of maize conditions novel protein-protein interactions between starch biosynthetic enzymes in amyloplasts

Liu, F., Makhmoudova, A., Lee, E. A., Wait, R., Emes, M. J., Tetlow, I. J. — Fri, 30 Oct 2009 06:38:04 PDT

The amylose extender (ae^–) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein–protein interactions in amyloplasts of ae^– mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein–protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae^– amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae^– endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae^–), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis.