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For a long time, plastid transformation has been a routine technology only in tobacco due to lack of effective selection and regeneration protocols, and, for some species, due to inefficient recombination using heterologous flanking regions in transformation vectors. Nevertheless, the availability of this technology to economically important crops offers new possibilities in plant breeding to manage pathogen resistance or improve nutritional value. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum), achieved by the optimization of the tissue culture procedures and using transformation vectors carrying homologous potato flanking sequences. This protocol allowed to obtain up to one shoot per shot, an efficiency comparable to that usually accomplished in tobacco. Further, the method described in this chapter has been successfully used to regenerate potato transplastomic plants expressing recombinant GFP protein in chloroplasts and amyloplasts or long double-stranded RNAs for insect pest control.

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Cell suspension cultures represent a widely used experimental tool suitable to perform a variety of structural and physiological studies in a more simplified system compared to the organism in toto. In this chapter we describe the methods routinely used in our laboratory to establish and maintain Arabidopsis photosynthetic and heterotrophic cell suspension cultures, containing either chloroplasts or amyloplasts, respectively. The use of these in vitro systems may allow to obtain insights into the unique features of chloroplasts versus non-green plastids, as well as their integration in the structural and metabolic compartmentalization of the plant cell.

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Starch synthesis is an elaborate process employing several isoforms of starch synthases (SSs), starch branching enzymes (SBEs) and debranching enzymes (DBEs). In cereals, some starch biosynthetic enzymes can form heteromeric complexes whose assembly is controlled by protein phosphorylation. Previous studies suggested that SSIIa forms a trimeric complex with SBEIIb, SSI, in which SBEIIb is phosphorylated. This study investigates the post-translational modification of SSIIa, and its interactions with SSI and SBEIIb in maize amyloplast stroma. SSIIa, immunopurified and shown to be free from other soluble starch synthases, was shown to be readily phosphorylated, affecting Vmax but with minor effects on substrate Kd and Km values, resulting in a 12-fold increase in activity compared with the dephosphorylated enzyme. This ATP-dependent stimulation of activity was associated with interaction with SBEIIb, suggesting that the availability of glucan branching limits SSIIa and is enhanced by physical interaction of the two enzymes. Immunoblotting of maize amyloplast extracts following non-denaturing polyacrylamide gel electrophoresis identified multiple bands of SSIIa, the electrophoretic mobilities of which were markedly altered by conditions that affected protein phosphorylation, including protein kinase inhibitors. Separation of heteromeric enzyme complexes by GPC, following alteration of protein phosphorylation states, indicated that such complexes are stable and may partition into larger and smaller complexes. The results suggest a dual role for protein phosphorylation in promoting association and dissociation of SSIIa-containing heteromeric enzyme complexes in the maize amyloplast stroma, providing new insights into the regulation of starch biosynthesis in plants.

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Starch is a water-insoluble polymer of glucose synthesized as discrete granules inside the stroma of plastids in plant cells. Starch reserves provide a source of carbohydrate for immediate growth and development, and act as long term carbon stores in endosperms and seed tissues for growth of the next generation, making starch of huge agricultural importance. The starch granule has a highly complex hierarchical structure arising from the combined actions of a large array of enzymes as well as physicochemical self-assembly mechanisms. Understanding the precise nature of granule architecture, and how both biological and abiotic factors determine this structure is of both fundamental and practical importance. This review outlines current knowledge of granule architecture and the starch biosynthesis pathway in relation to the building block-backbone model of starch structure. We highlight the gaps in our knowledge in relation to our understanding of the structure and synthesis of starch, and argue that the building block-backbone model takes accurate account of both structural and biochemical data.

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Plant organs control their growth orientation in response to gravity. Within gravity-sensing cells, the input (gravity sensing) and signal conversion (gravity signalling) progress sequentially. The cells contain a number of high-density, starch-accumulating amyloplasts, which sense gravity when they reposition themselves by sedimentation to the bottom of the cell when the plant organ is re-orientated. This triggers the next step of gravity signalling, when the physical signal generated by the sedimentation of the amyloplasts is converted into a biochemical signal, which redirects auxin transport towards the lower flank of the plant organ. This review focuses on recent advances in our knowledge of the regulatory mechanisms that underlie amyloplast sedimentation and the system by which this is perceived, and on recent progress in characterising the factors that play significant roles in gravity signalling by which the sedimentation is linked to the regulation of directional auxin transport. Finally, we discuss the contribution of gravity signalling factors to the mechanisms that control the gravitropic set-point angle.

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The legume-rhizobium symbiotic relationship has been widely studied and characterized. However, little information is available about the role of histone lysine methyltransferases in the legume-rhizobium interaction and in the formation of nitrogen-fixing nodules in the common bean. Thus, this study aimed to gain a better understanding of the epigenetic control of nodulation in the common bean. Specifically, we studied the role of PvTRX1h, a histone lysine methyltransferase coding gene, in nodule development and auxin biosynthesis. Through a reverse genetics approach, we generated common bean composite plants to knock-down PvTRX1h expression. Here we found that the down-regulation of PvTRX1h increased the number of nodules per plant, but reduced the number of colony-forming units recovered from nodules. Genes coding for enzymes involved in the synthesis of the indole-3-acetic acid were up-regulated, as was the concentration of this hormone. In addition, PvTRX1h down-regulation altered starch accumulation as determined by the number of amyloplasts per nodule. Metabolic fingerprinting by direct liquid introduction-electrospray ionization-mass spectrometry (DLI-ESI-MS) revealed that the root nodules were globally affected by PvTRX1h down-regulation. Therefore, PvTRX1h likely acts through chromatin histone modifications that alter the auxin signaling network to determine bacterial colonization, nodule number, starch accumulation, hormone levels, and cell proliferation.

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Plastids, a wide family of plant specific organelles, exist in all plant cells in a number of different forms with different functions essential for plant life. Among them, chloroplasts are by far the more extensively studied owing to their central role in photosynthesis. However, other plastid family members, often referred to as nongreen plastids, play also major roles in the physiology of higher plants and could be better suited for studies of specific metabolic processes in heterotrophic plant cells. Unfortunately, serious technical problems are frequently encountered with separating intact, active nongreen plastids from contaminating membranes and mitochondria. Here, we provide detailed protocols suitable for the large scale preparation of intact and highly pure proplastids from cauliflower buds, as well as amyloplasts from sycamore cultured cells, and for the subsequent separation of their surrounding envelope membranes from the stroma and other plastid fractions. Both methods proved to be highly reliable and have been instrumental for in-depth investigations on biochemistry and physiology of nongreen plastids.

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Starch is a water-insoluble polyglucan synthesized inside the plastid stroma within plant cells, serving a crucial role in the carbon budget of the whole plant by acting as a short-term and long-term store of energy. The highly complex, hierarchical structure of the starch granule arises from the actions of a large suite of enzyme activities, in addition to physicochemical self-assembly mechanisms. This review outlines current knowledge of the starch biosynthetic pathway operating in plant cells in relation to the micro- and macro-structures of the starch granule. We highlight the gaps in our knowledge, in particular, the relationship between enzyme function and operation at the molecular level and the formation of the final, macroscopic architecture of the granule.

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Hydrogen peroxide (H2O2) is the key factor in many physiological and metabolic processes in plants. During seed germination, exogenous H2O2 application influences gravitropism and induces curvature of the primary root in grass pea and pea seedlings. However, it remains unclear whether and how this happens in the model plant Arabidopsis thaliana. In the present study, the effect of exogenous H2O2 on the gravitropic response of primary roots during Arabidopsis seed germination was studied using histology and molecular biology approaches. Appropriate H2O2 treatment not only restrained primary root growth, but also disrupted gravitropism and induced root curvature. Histological staining and molecular analysis demonstrated that exogenous H2O2 correlated with lack of starch-dense amyloplasts in root tip columella cells, which ultimately results in the lack of gravisensing. Detection of calcium ion (Ca2+) by a fluorescent probe showed that Ca2+ distribution changed and intracellular Ca2+ concentration increased in H2O2-treated primary root, which was consistent with alterations in auxin distribution and concentration triggered by H2O2 treatment. Furthermore, the normally polar localization of Pin-formed 1 (PIN1) and PIN2 became uniformly distributed on root tip cell membranes after treatment with H2O2. This leads to speculation that the IAA signaling pathway was affected by exogenous H2O2, causing asymmetrical distribution of IAA on both sides of the primary root, which would influence the gravitropic response.

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Using plants as hosts for production of complex, high-value compounds and therapeutic proteins has gained increasing momentum over the past decade. Recent advances in metabolic engineering techniques using synthetic biology have set the stage for production yields to become economically attractive, but more refined design strategies are required to increase product yields without compromising development and growth of the host system. The ability of plant cells to differentiate into various tissues in combination with a high level of cellular compartmentalization represents so far the most unexploited plant-specific resource. Plant cells contain organelles called plastids that retain their own genome, harbour unique biosynthetic pathways and differentiate into distinct plastid types upon environmental and developmental cues. Chloroplasts, the plastid type hosting the photosynthetic processes in green tissues, have proven to be suitable for high yield protein and bio-compound production. Unfortunately, chloroplast manipulation often affects photosynthetic efficiency and therefore plant fitness. In this respect, plastids of non-photosynthetic tissues, which have focused metabolisms for synthesis and storage of particular classes of compounds, might prove more suitable for engineering the production and storage of non-native metabolites without affecting plant fitness. This review provides the current state of knowledge on the molecular mechanisms involved in plastid differentiation and focuses on non-photosynthetic plastids as alternative biotechnological platforms for metabolic engineering.

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Starch commands a central role in the carbon budget of the majority of plants on earth, and its biological role changes during development and in response to the environment. Throughout the life of a plant, starch plays a dual role in carbon allocation, acting as both a source, releasing carbon reserves in leaves for growth and development, and as a sink, either as a dedicated starch store in its own right (in seeds and tubers), or as a temporary reserve of carbon contributing to sink strength, in organs such as flowers, fruits, and developing non-starchy seeds. The presence of starch in tissues and organs thus has a profound impact on the physiology of the growing plant as its synthesis and degradation governs the availability of free sugars, which in turn control various growth and developmental processes. This review attempts to summarize the large body of information currently available on starch metabolism and its relationship to wider aspects of carbon metabolism and plant nutrition. It highlights gaps in our knowledge and points to research areas that show promise for bioengineering and manipulation of starch metabolism in order to achieve more desirable phenotypes such as increased yield or plant biomass.

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Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 µm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception.

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Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants.

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Calcium is used by plants as an intracellular messenger in the detection of and response to a plethora of environmental stimuli and contributes to a fine-tuned internal regulation. Interest in the role of different subcellular compartments in Ca(2+) homeostasis and signalling has been growing in recent years. This work has evaluated the potential participation of non-green plastids and chloroplasts in the plant Ca(2+) signalling network using heterotrophic and autotrophic cell suspension cultures from Arabidopsis thaliana plant lines stably expressing the bioluminescent Ca(2+) reporter aequorin targeted to the plastid stroma. Our results indicate that both amyloplasts and chloroplasts are involved in transient Ca(2+) increases in the plastid stroma induced by several environmental stimuli, suggesting that these two functional types of plastids are endowed with similar mechanisms for handling Ca(2+) A comparison of the Ca(2+) trace kinetics recorded in parallel in the plastid stroma, the surface of the outer membrane of the plastid envelope, and the cytosol indicated that plastids play an essential role in switching off different cytosolic Ca(2+) signals. Interestingly, a transient stromal Ca(2+) signal in response to the light-to-dark transition was observed in chloroplasts, but not amyloplasts. Moreover, significant differences in the amplitude of specific plastidial Ca(2+) changes emerged when the photosynthetic metabolism of chloroplasts was reactivated by light. In summary, our work highlights differences between non-green plastids and chloroplasts in terms of Ca(2+) dynamics in response to environmental stimuli.

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Myosins and actin filaments in the actomyosin system act in concert in regulating cell structure and dynamics and are also assumed to contribute to plant gravitropic response. To investigate the role of the actomyosin system in the inflorescence stem gravitropism, we used single and multiple mutants affecting each of the 17 Arabidopsis myosins of class VIII and XI. We show that class XI but not class VIII myosins are required for stem gravitropism. Simultaneous loss of function of myosins XI1, XI2, and XIK leads to impaired gravitropic bending that is correlated with altered growth, stiffness, and insufficient sedimentation of gravity sensing amyloplasts in stem endodermal cells. The gravitropic defect of the corresponding triple mutant xi1 xi2 xik could be rescued by stable expression of the functional XIK:YFP in the mutant background, indicating a role of class XI myosins in this process. Altogether, our results emphasize the critical contributions of myosins XI in stem gravitropism of Arabidopsis.

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Isolated amyloplasts from cauliflower (Brassica oleracea L. var botrytis) buds are able to export orthophosphate unidirectionally into the incubation medium. This orthophosphate transport appears to be protein-mediated, as indicated by the following observations: (i) low temperature and the presence of inhibitors of protein-mediated transport reduced the rate of orthophosphate export, and (ii) the rate of orthophosphate export became saturated with rising internal substrate concentrations. Micromolar concentrations of 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid inhibited the rate of unidirectional orthophosphate export, thus indicating the involvement of the amyloplastic glucose-6-phosphate (Glc6P)translocator in the unidirectional export of orthophosphate. The effect of rising concentrations of orthophosphate upon the activity of ADP glucose pyrophosphorylase in desalted extracts was determined. Orthophosphate given in concentrations similar to those measured in the amyloplastic stroma under conditions of steady-state rates of Glc6P-dependent starch synthesis inhibited the activity of ADP-glucose pyrophosphorylase significantly. However, even under strong limiting substrate conditions the residual activity was sufficient to catalyze the flux of carbon into starch. The maximal rates of orthophosphate transport (in the counter-exchange mode) by isolated spinach (Spinacia oleracea L.) chloroplasts and by isolated cauliflower-bud amyloplasts were also determined. These rates were compared with the maximal rates of undirectional orthophosphate export by these plastids. From these measurements we can conclude that, compared with spinach chloroplasts, isolated amyloplasts of cauliflower exhibit a fivefold greater ratio of unidirectional orthophosphate transport to maximal rate of orthophosphate transport in the counter-exchange mode compared to spinach chloroplasts. The determined rate of maximal unidirectional orthophosphate export is sufficient to catalyze the release of additional inorganic phosphate liberated in the amyloplastic stroma during the process of Glc6P-dependent starch synthesis.