Which Factors Control Starch Granule Initiation?

Down-regulation by small interfering RNA or absence of hypoxia-inducible factor (HIF-1α) has been shown to lead to increased sensitivity to glycolytic inhibitors in hypoxic tumor cells. In surveying a number of tumor types for differences in intrinsic levels of HIF under hypoxia, we find that the reduction of the upstream pathways of HIF, AKT, and mammalian target of rapamycin (mTOR) correlates with increased toxic effects of 2-deoxy-d-glucose (2-DG) in lung cancer cell lines when treated under hypoxia. Because HIF-1α translation is regulated by mTOR, we examined the effects of blocking mTOR under hypoxia with an analogue of rapamycin (CCI-779) in those cell lines that showed increased mTOR and AKT activity and found that HIF-1α down-regulation coincided with increased 2-DG killing. CCI-779, however, was ineffective in increasing 2-DG toxicity in cell lines that did not express HIF. These results support the hypothesis that although mTOR inhibition leads to the blockage of numerous downstream targets, CCI-779 increases the toxicity of 2-DG in hypoxic cells through down-regulation of HIF-1α. Overall, our findings show that CCI-779 hypersensitizes hypoxic tumor cells to 2-DG and suggests that the intrinsic expression of AKT, mTOR, and HIF in lung cancer, as well as other tumor types, may be important in dictating the decision on how best to use 2-DG alone or in combination with CCI-799 to kill hypoxic tumor cells clinically. [Mol Cancer Ther 2008;7(6):1506–13]

Storage and remobilization of sugar molecules play important roles for the growth and survival of living organisms. Besides a few exceptions, animals store carbohydrates in the form of soluble glycogen while green plants and algae bank glucans as insoluble starch. Starch forms the basis of human nutrition and constitutes a renewable raw material for industry, thus highlighting its importance for humankind.
Starch is a nonstructural polymer composed of linked glucose units forming inert granules within the chloroplast. In leaves, transitory starch is synthesized during the day from photosynthetic assimilates and subsequently degraded to provide energy for growth at night (reviewed by Pfister and Zeeman, 2016). The enzymes controlling starch synthesis and degradation have been extensively studied, and recent attention has also been given to factors controlling starch granule initiation. Notably, plants lacking PROTEIN TARGETING TO STARCH2 (PTST2) or its interaction partners MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) and MAR BINDING FILAMENT-LIKE PRO-TEIN1 (MFP1) have reduced numbers of starch granules per chloroplast (Seung et al., 2017(Seung et al., , 2018Vandromme et al., 2019). In this issue of The Plant Cell, Abt et al. (2020) characterize the function of STARCH SYNTHASE5 (SS5) in starch metabolism and show that this enzymatically inactive protein plays an important role within the nexus of factors regulating the number of starch granules formed in chloroplasts.
The plant model organism Arabidopsis (Arabidopsis thaliana) contains six starch synthases. Five of them elongate glucan chains to make amylopectin and amylose, the two polysaccharide types found in starch. Abt and colleagues (2020) observed that the sixth starch synthase, SS5, was present throughout the plant kingdom but lacked a conserved glycosyltransferase1 (GT1) domain (see figure). Structural modeling indicated that SS5 has a surface carbohydrate binding site conserved with SS4. Consistently, recombinant SS5 was able to bind maize (Zea mays) starch but not to actively transfer ADP-glucose onto it. Furthermore, SS5 was not able to produce glucans when expressed in yeast (Saccharomyces cerevisiae), confirming that SS5 was not an enzymatically active glucosyltransferase.
PlantslackingSS5didnotdisplayastrong visible phenotype (see figure) and overall starch levels remained similar to those of the wild type, except at the end of the night, when ss5 2/2 had slightly more starch. Scanning electron micrographs of purified starch granules and light micrographs of embedded tissue sections showed that ss5-deficient plants had fewer but larger starch granules (see figure). Together, these data suggested that SS5 was not primarily involved in starch synthesis or degradation but rather in starch granule initiation.
Immunoprecipitation followed by mass spectrometry of tagged SS5 revealed an interaction with SS4 and MRC. Coimmunoprecipitation experiments validated the interaction between SS5 and MRC and showed that the coiled-coil domain of SS5 was required for this interaction. In vivo localization experiments of fluorescent transcriptional fusions revealed that SS5 Role of SS5 in Starch Granule Initiation.
(A) SS5 is a conserved starch synthase encompassing a chloroplast transit peptide (cTP), a coiledcoil domain required for its physical interaction with MRC, and a GT5 domain. SS5 lacks a conserved GT1 domain (present in SS1 to SS4) and does not function as an active canonical starch synthase. (B) SS5 fused to GFP localizes to small puncta of yet unknown function inside the chloroplast. (C) ss5 2/2 mutant plants do not display a strong developmental phenotype, but the shape and number of starch granules per chloroplast differ from those of the wild type (WT), indicating a function for SS5 in starch granule initiation. (Adapted from Abt et al. [2020], Figures 1, 4, and 6.) [OPEN] Articles can be viewed without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.20.00502 was localized in the chloroplast, consistent with the presence of an N-terminal chloroplast transit peptide (see figure). Interestingly, the authors found that SS5 and MRC colocalized in small puncta, suggesting the existence of a possible structural "platform" regulating starch granule homeostasis.
To understand the functional relationship between SS5 and known granule initiation factors, the authors created several double mutants with ss5 2/2 and quantified the resulting number and size of starch granules per chloroplast. Interestingly, the additional loss of SS5 did not enhance the phenotype of mrc 2/2 , indicating that SS5 and MRC function in the same pathway. A similar epistatic relationship was observed between SS5 and MFP1. In contrast, the loss of SS4 or PTST2 in addition to SS5 enhanced the phenotype of the single ss5 2/2 mutant, suggesting that SS5 may act independently from SS4 and PTST2 in starch granule initiation.
In summary, Abt and colleagues (2020) coined SS5 as a new player in the network of factors determining starch granule initiation. Future investigations will tackle the complex relationships between all these factors and also likely shed light on the exact nature of SS5-MRC-containing puncta, whose function remains enigmatic.

Which Factors Control Starch Granule Initiation?
This information is current as of November 3, 2020