DEFINITION AND STRUCTURE OF STARCH
STARCH
Carbohydrates play a central role in all living organisms; primary metabolism is dependent on carbon and energy conversion, irrespective of autotrophic or heterotrophic nutrition, and is thus centralized around carbohydrates. Therefore, it is not surprising that polysaccharides are the most abundant polymers in the biosphere. Starch is quantitatively the most dominant storage carbohydrate on Earth and is synthesized mostly in plants and some cyanobacteria [1]. Starch is accumulated as water-insoluble particles, i.e., the starch granules, whereas most other species produce water-soluble glycogen as a storage carbohydrate. Both polymers are similar in biological function and chemical composition, consisting of glucose units that are linked by α-1,4 and α-1,6 glycosidic bonds. The physical parameters of starch strongly differ from those of glycogen and are responsible for the high value of starch in various applications. Starch is a versatile biomaterial of special interest due to its abundance, cheapness, non-toxic properties, and biodegradability for various food and non-food industries. Thus, it is used in numerous dairy and bakery goods, soups, and sauces, as well as coatings and meat products. In addition, there is an increasing demand from non-food industries for starch as a renewable material [2]. Starch non-food applications include pharmaceuticals, textiles, alcohol-based fuels, adhesives, dyes, and furniture. Therefore, it is obvious that starch property demands are very different for the various applications.
The physico-chemical properties of native starches are, in many cases, not optimal for further processing and efforts are needed to adapt them for particular industrial use. Native starch granules are water-insoluble, largely inert, and, to a great extent, resistant to enzymatic hydrolysis. They also tend to be rather retrograde with respect to changes in pH, temperature, and shear forces [3], [4]. Thus, starches often have to be modified for better physical and functional characteristics. In principle, this modification can be distinguished into alteration of the starches before isolation from plants, in planta modification during starch metabolism, and modifications following purification from plant material. In general, modification during the isolation process is also possible. The first mentioned situation can be achieved by breeding and molecular biology, whereas in the other cases the modifications include physical, chemical, and enzymatic/biotechnology methods, or even combinations of these [5]. The grade of requisite modifications also depends on the starting source of starch. In general, higher plants form two types of starch, assimilatory (or transitory) and reserve (or storage) starch. Assimilatory starch is synthesized in autotrophic tissues and directly linked to photosynthesis and, thus, the synthesis and degradation follow the diurnal rhythm.
Reserve starch accumulation and degradation take place usually only once, in specific tissues and organs, such as seeds or roots and is indirectly linked to photosynthesis [6]. The two types differ in quantity per plant, shape, size, metabolism, and biochemistry properties. Some examples of starches with different sizes (Table 1) and morphology (Fig. 1) are given.
Table 1. The starch granule from different botanical sources.
| Species | Source part | Granular shape | Allomorph | Relative crystallinity (%)* * | Starch size range (μm) | Reference |
|---|---|---|---|---|---|---|
| Arabidopsis (Arabidopsis thaliana) | Leaves | Discoid | B | NAa | 1–2 | [7], [8] |
| Potato (Solanum tuberosum) | Tubers | Oval, spherical | B | 29.8 | 10–75 | [9] |
| Cassava (Manihot esculenta) | Tubers | Round, irregular | A or C | 35.8 | 5–40 | [9], [10] |
| Maize (Zea mays) | Grain | Round, polygonal | A | 31 | 5–20 | [9] |
| Wheat (Triticum spp.) | Grain | Lenticular-shaped,small round | A | 33.5 | 1.5–20 | [11], [12] |
| Rice (Oryza sativa) | Grain | Round, angular, and polygonal | A | 37.1 | 3–10 | [9] |
| Barley (Hordeum vulgare) | Grain | Large disc-shaped granules, small spherical granules | A | 35.2 | 1–24 | [12], [13] |
| Apple (Malus spp.) | Fruit | Spherical ordome-shaped | C | NAa | 2–12 | [14] |
| Banana (Musa spp.) | Fruit | Elongated ovals with ridges | A, B or C | 2–12.2 | 14–108 | [15], [16] |
| Sago (Metroxylon sagu) | Stem | Oval with a temple bell-like shape | C | 30.5 | 8–240 | [17], [18] |
- a
NA-not available ; * * Relative crystallinity (RC) of starch granule-defined as RC= (Ac/(Ac+Aa))* 100, where Ac is the crystalline area; and Aa is the amorphous area on the X-ray diffractograms [19].
Fig. 1. Starch granules. Starch morphology, distribution in cells and tissues, as well as some starch granule examples from various crops are given. Starch granules from Arabidopsis thaliana leaves (a) wild type (Col-0), (b) mutant lacking α-glucan, water dikinase (GWD), and (c) double mutant lacking both Disproportionating enzyme 2 (DPE2) and GWD, visualized using scanning electron microscopy (SEM). Starch granule number inside mesophyll cells from Arabidopsis thaliana leaves (d) wild type (Col-0), (e) mutant lacking Starch synthase 4 (SS4), and (f) mutant lacking Protein targeting to starch 2 (PTST2), visualized using fluorescence confocal microscopic (red – chlorophyll auto fluorescence, green – Safranin O staining of the starch granules (see also [25]). Starch granule morphology from various crops, for instance, (g) potato (Solanum tuberosum), (h) fonio (Digitaria exilis), and (i) sweet potato (Ipomoea batatas), visualized using SEM. (a), (b), (c) figure bars = 2 µm. (d), (e), (f) figure bars = 5 µm. (g), (h), (i) figure bars = 10 µm.
(a), (b), (c) figure adapted from [26].The quantity of reserve starch per plant mostly exceeds that of assimilatory starch by several orders of magnitude and, therefore, is mainly used for agricultural and industrial applications. As mentioned before, the physical parameters can differ largely, and a balance of the cost for necessary modifications against the higher cost of production/isolation of native starches is thus meaningful. In this context, it is also interesting to consider original, old, rare, and rediscovered crops, as well as crops used in other regions of the world e.g., Africa, which have the potential for starches with unique characteristics, not only in size and morphology but, also in surface properties, inner starch structure, and other physico-chemical parameters. Unfortunately, the starch parameters of many of these crops have not been well analyzed.
The inner structure of starch
From an evolutionary perspective, some features of starch seem to be highly conserved, such as the inner structure of starch [20]. However, even here there is variability that should be considered when starches are used in applications. Starch is formed of two glucan polymers: amylose and amylopectin. The latter has a much higher molecular weight than amylose. On average, the size distribution peaks at about 108 Da, whereas the size distribution of amylose is around two orders of magnitude lower (106 Da.). Amylopectin is quantitatively by far the dominant polysaccharide type, and it primarily determines the characteristics of the starch granule. However, the ratio of amylopectin and amylose can also vary largely. Thus, the proportion of amylose can be from nearly undetectable up to 70% [21]. On average, amylopectin is considerably more branched than amylose, which represents a complex mixture of mostly strictly linear (α-1,4 linked) and some poorly branched polyglucans. In amylopectin, the branching points are clustered and allow the formation of allomorphs. The vicinal glucan chains thereby form double helices. The double helices are formed by neighboring α-1,4-linked glucan chains with a degree of polymerization (DP) from 10 to 20. The non-reducing ends of the chains are orientated towards the granule surface, and several of these helices generate higher-ordered allomorphs. Two main allomorphs - A-type and B-type – have been reported (see Fig. 2). Both allomorphs differ in their resulting thermostability [8], [22], and there are indications in some cases for an altered chain length distribution [23]. Another type of allomorph - C-type - is a mixture of both the A- and B-type [8], [24]. The A-type is more thermostable than the B-type, the latter containing more water molecules within the structure, and thus probably represents a biological function, as the A-type is mainly observed in storage starches that are only synthesized and degraded once in contrast to the B-type which reveal a daily turnover (see Table 1 and references there). Unfortunately, systemic studies of the allomorphs from various plants are missing. Thus, it is largely unknown whether the same allomorph type from various plant species shows variability, which can then be used for starch processing during applications.
Morphology and size of starch granules
The inner structure of starch seems to be highly evolutionarily conserved, yet there is no clue as to how this results in totally different starch granule morphologies and sizes. Thus, flat and discoid but also total spherical starch granules can be found in planta, which also differ totally in size (Fig. 1). Reports exist of tiny starch granules, smaller than 1 µm, up to granules of more than 100 µm (Table 1). Furthermore, also compound granules (formed by the assembly of several smaller starch granules) [49], [50] and granules with extraordinary shape have been described [26], [51]. The size, morphology, and number of starch granules can differ largely within one species regarding organs and tissues. An example of this is potato leaf [52] and tuber starch [9].
As the resulting surface is directly linked to the size and morphology of native starch granules, both parameters are important for starch usage, yet they are only alterable in planta. However, recent evidence reveals that these parameters are not strictly genetically determined, because the morphology can differ with the plant´s metabolic and developmental changes [26], [53], [54], making alterations to these parameters challenging. The possible manipulation of the starch granule morphology and size are very promising for both increasing the starch yield and modifying the starch properties [55]. Thus, further knowledge of the mechanism for size and morphology control is urgently needed.
Existing modifications of native starch granules
For isolated starch granules, in addition to the carbohydrate constituents, amylose and amylopectin, lipids and proteins bound to starch are frequently reported [56], [57]. Both are non-covalently bound to starch and represent parts of the synthesizing and degradation machinery, as well as potential isolation contaminants. With regard to proteins, many of them involved in starch turnover are detectable bound to starch [58]. Here, proteins incorporated into starch and proteins bound on the surface of starch granules can be distinguished. Proteins belonging to the first group are obviously involved in starch metabolism, such as e.g., the granule bound starch synthase (GBSS; see above), whereas for proteins attached to the starch granules, contamination during isolation of the granules from the plants cannot be totally excluded. Because starch granules - more precisely, the polysaccharides amylose and amylopectin - possess mostly three free hydroxyl-groups (at C2, C3, and C6) at glucosyl moieties, in principle, they can be targets of covalent modifications. Thus far, only modifications at C3 and C6 of glucosyl units have been identified and both are phosphate monoesters [59], [60], [61]. The amount of phosphate differs strongly between various starches (Table 2) and is transient, reflecting a metabolic situation [61], [62]. The enzymes involved in starch phosphorylation and dephosphorylation were identified and thus allowed a further detailed analysis and application (see below). Two plastidial dikinases, the α-glucan, water dikinase [GWD; EC 2.7.9.4], and phosphoglucan, water dikinase [PWD; EC 2.7.9.5] [51], [60], [61], as well as two dual specificity phosphatases, starch excess four [SEX4; EC 3.1.3.48] [63], [64], and like starch excess four 2 [LSF2; EC 3.1.3.48] [65] were identified and characterized. However, to date neither the exact action of these enzymes at the starch granule surface nor the exact phosphorylation position within an α-glucan chain, or even the distribution of the phosphorylation at the starch granule surface, is known. It seems that the main glucan molecules of starch, the amylopectins, are exclusively phosphorylated [59], [66], [67].
Natural occurring starch phosphate amounts in planta.
Species Source part C6 phosphate ( P/mg starch) C3 phosphate (P/mg starch) Reference Potato
(Solanum tuberosum)Tubers 7.8–33.1 nmol 6–9.1 nmol [66], [67], [69] Leaves 4.5 nmol NAa Cassava
(Manihot esculenta)Tubers 58 ng / 2.5 nmol 0.4 nmol [66], [70] Leaves 20 ng NAa Rice
(Oryza sativa)Grain 0.2–1 nmol 0.25 nmol [69], [71] Barley
(Hordeum vulgare)Grain 0.35–0.8 nmol NAa [72], [73] - The phosphorylation is the only natural covalent modification of starch that also strongly influences the physico-chemical properties and, thus, the hydrophilicity, the surface charge, the chemical vulnerability, crystallinity, and, thereby, characteristics such as thermal stability, pasting and swelling power, it is of high interest to further elucidate the molecular mechanism. Moreover, it is still unclear whether or not the phosphorylation events for various starches from different species are similar (see also [68]), because the phosphorylation of starch reflects metabolic states of the plant. It was also shown that the phosphate amounts of starches from identical tissues/organs (e.g., potato tuber), but different sizes alter strongly in the total phosphate content [58]. Thus, more data will be helpful for establishing a clear definition of the starting starch material for further use/modification in various sectors.
Conclusion and perspectives
Although starch seems simple from its chemistry, it is a highly complex carbohydrate, and various aspects such its metabolism, inner organization, or connection of structure and physical properties are largely obscure. In addition to the mentioned in planta changes of starches, modifications outside of the plant are a sizeable field allowing alteration of starches via physical, chemical, and enzymatic methods. The objective is to overcome disadvantageous physico-chemical properties of native starches, such as syneresis, retrogradation, low solubility in organic solvents, and breakdown. In some cases, one type of modification does not satisfy the requirements of industry and/or research, so combining different techniques is unavoidable.
Further research should focus on detailed understanding of starch metabolism in plants, possible alterations of specific starch characteristics in planta, development of new methods and innovative technologies both in planta and outside the plant system for starch analytics, to create finally an arsenal of possible and combinable modifications.
An interesting and promising path is the transfer of starch generation per se from plants to other species, especially to fast-growing microorganisms that are, cheap, easy to handle, and reveal low starch extraction efforts, as was initially shown for yeast [94]. In addition, cell-free starch synthesis from carbon dioxide was reported opening a wide field of further research and application [95].
All these options together will allow for a decrease in the cost of starch applications and the development of completely new starch-based products for the future.
References
U. Cenci, F. Nitschke, M. Steup, B.A. Minassian, C. Colleoni, S.G. Ball
Transition from glycogen to starch metabolism in ArchaeplastidaTrends Plant Sci., 19 (1) (2014)- Starch: its metabolism, evolution, and biotechnological modification in plantsAnnu. Rev. Plant Biol., 61 (2010)
- Starch granules in arabidopsis thaliana mesophyll and guard cells show similar morphology but differences in size and numberInt. J. Mol. Sci., 22 (11) (2021)
- Regulation of starch metabolism: the age of enlightenment?Curr. Opin. Plant Biol., 13 (3) (2010), pp. 321-329
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