Many factors can influence starch synthesis and grain filling in wheat. Some are intrinsic, such as development of reproductive structures, mechanisms of assimilate utilization, and phytohormones within the seed. Others relate to environmental factors, such as nitrogen (N) availability and high temperature stress. Regardless of the particular component, these controls are often evaluated for their influence on seed dry matter accumulation, as a whole, and not starch biosynthesis, per se. Because starch is the primary constituent of the seed, it is fair to presuppose that circumstances which influence the overall growth of single grain are also involved, either directly or indirectly, in the regulation of starch biosynthesis.
Regulation of starch accumulation in the kernel can fall within the constraints of mechanisms not directly related to starch metabolism. For instance, Cottrell and Dale (128) studied the variation in size and development of spikelets within the ear of barley and found a strong correlation between the width of the spikelet at the double ridge stage (pre-pollination) and the final weight of the grain. For wheat, a study with multiple cultivars sampled at several stages of development showed that grain weight and volume are highly correlated (129). The volume, in turn, may be controlled by nonfilial factors. Millet (130) measured the volume of the floret cavity of mature grain from multiple lines and found that differences, both within the spike and among the genotypes, closely reflected variation in grain size and shape. Additional work (131) using aneuploids to provide a dosage effect of group 5 chromosome showed that a reduced dosage resulted in smaller floral organs, floret cavities, and lighter grains, while increased dosage had the opposite effects. Because the weight of the kernel appears to be affected by allometric relations to the volume of the floret cavity, regulation of starch biosynthesis in the seed must be indirectly coordinated by developmental mechanisms operative in the sporophyte, even before pollination. One way this could occur is through linked metabolism of the pericarp, because a close correspondence exists between the rate of dry matter accumulation and glycosidase activities in developing wheat seed (44). Hence, cell wall loosening enzymes may have an indirect role of influencing the amount of starch which can accumulate in the seed. Although the causal relationship between floret cavity volume or wall-bound enzyme activities and grain weight provide only a partial explanation for differences in seed growth, it demonstrates how factors other than direct elements of starch biosynthesis may participate in controlling seed growth and composition.
Availability of carbohydrate assimilate is another factor which governs grain starch synthesis. The precise influence of C supply has been examined in many fashions. One way has entailed manipulation of assimilate availability through either shading or partial defoliation of the plant to restrict C supply. Another has been by partial degraining of the spike to enhance assimilate availability to remaining florets. The reader is referred to publication (132) for a summary of earlier source-sink experiments of this type. In a more recent study, however, shading during the grain filling portion of seed development decreased the weight of kernels at all floret positions, primarily by a reduction in the number of large type-A starch granules (133). Low levels of irradiance has also been shown to reduce the rate, but not duration, of dry matter accumulation in the grain (14, 134, 135). The reduced rate has been associated with a reduction in B-type starch granule number.
Light, per se, does not have a direct effect on grain growth, but instead exerts its influence through provision of C precursors via photosynthesis. Starch synthesis in vivo is not dependent upon oxygen production by photosynthesis in the green layer of the pericarp (136) and light does not have a photomorphogenic effect upon ear development (137). This is exemplified by the fact that variation of light intensity has no effect on the synthesis of starch in ears when they are grown detached from the plant and supplied with sucrose (138). But the provision of C, up to some minimum level, is important for seed fill. That is why the response to sucrose is greater in ears cultured in the shade than those cultured in higher light (137).
The detached ear culture technique has been used in many additional studies to explore the effect of assimilate availability on starch synthesis. Use of the technique is justified since seed development is functionally the same for grain of intact plants and those grown on detached ears (139). Overall, the studies provide a number of insights into the relationship between C assimilate availability and starch synthesis and/or grain growth. For instance, the kernel has a capacity to uptake sucrose in a concentration dependent manner (140). Despite this fact, the rate of starch synthesis follows the typical pattern of being low initially, increasing to a maximum in mid-development, and subsequently declining towards maturation. Although low levels of C supply can limit starch deposition, it was realized early that assimilate supply may not "normally" be the factor limiting starch accumulation in the seed (141). Others have also found that the supply of sugar precursors does not limit starch synthesis or grain size of wheat (142). Instead, the decline in starch synthesis in the latter portion of grain development seems to be due to a decrease in the enzymatic capacity of the seed (43, 143) and perhaps similar limitations restrict greater rates of starch deposition in early growth. The overall result is that the duration of seed growth and starch deposition is set by the length of enzymatic activity occurring within endosperm cells (140).
Provision of sugars through in vitro culture of wheat spikes shows that starch synthesis in the grain shows limited response to increasing C supply and does not appear to be restrained by substrate availability. In contrast, grain with the outer pericarp and vascular bundle of the furrow removed respond linearly to sucrose concentration when it is directly available (144). The question of whether kernels grown on intact plants may be restricted in starch synthesis because of factors limiting the transport capacity of the ear needed to be answered.
Sucrose is transported to developing endosperm without inversion, so hydrolysis of the sugar is not a likely limitation to starch metabolism (145, 146). It is also clear that provision of sucrose to the seed does not appear to be limited by vascular connection of the kernels to the plant. Lingle and Chevalier (147) studied the dynamics of grain filling and development of vascular tissue in wheat and barley and found that a marked decline in the rate of dry matter accumulation occurred prior to the collapse of the phloem and chalaza. Also, the number of sieve tubes was not related to the rate of grain filling (147) and grain growth is not even affected by severing half of the vascular bundles in the peduncle (148). These facts, along with many reports that have found superfluous sugar present in endosperm that are slowing starch synthesis as maturation approaches, support the notion that under normal conditions starch synthesis in developing grain is not restricted by a lack of carbohydrate precursor.
The amount of protein in the grain follows a dose response curve, up to a plateau, with the amount of N available to the developing seed (149-152). The genotype of the plant and position of the seed within the wheat head can also influence the level of stored protein-N (9, 20, 153, 154). Generally, grain from the third floret of a spikelet tends to have a lower concentration and content of N than seed of the lower florets. Kernels of the distal floret are also typically smaller.
It is widely believed that the deposition of protein and starch in the seed occurs by independent mechanisms. Provided as evidence, protein accumulation shows more flexibility in response to N supply than starch deposition shows to provision of sugar (155). Also, under normal levels of N fertility there is a difference in the profile of the rate and duration of N versus dry weight accumulation in wheat seed (153). The independence of starch/protein accumulation becomes even more pronounced when N rates reach high levels or when comparing high- and low-protein cultivars (156). Examination of the latter indicates that variation in carbohydrate metabolism is not responsible for differences in protein content (157).
Furthermore, manipulation of post anthesis N availability increases N import by the grain more than storage of carbohydrate, so that grain N concentration increases over time (151).
Despite the presumed independence of the two storage processes, N availability still has the potential to indirectly regulate the amount of starch synthesized in the seed. Deprivation of N can significantly reduce the number of endosperm cells which form in the seed and, therefore, restrict the number of sites where starch can be made (158, 159). Small additions of N can overcome this problem (160). Also, Solfield et al. (135) reported that there is an association between accumulation of dry matter and N in seed which have been influenced by varying temperatures and illumination. In other experiments where grain growth was manipulated by N supply and illumination, Barneix et al. (152) suggested there is a dependence of N transport on C transport and that the final grain N concentration is determined by the C/N ratio exported from vegetative tissues. This effect of precursor C/N ratio on seed growth and composition has been clearly shown for maize kernels grown in vitro, but the authors believe that the apparent influence of C/N ratio over growth was only coincidental and that, in fact, starch and storage protein synthesis have a mutually exclusive dependency upon N supply (161).
Although most evidence suggests that N only limits starch synthesis by restricting the synthesis of metabolic proteins, it cannot be ignored that N supply can impact the expression of genes related to both starch and N storage metabolism. Doehlert et al. (162) cultured maize kernels in vitro with different levels of available N and found that some genes of carbohydrate metabolism and storage protein synthesis were increased 2-7 fold in expression, while others were unaffected. Coordinated control of starch and storage protein gene expression has also been shown in several other types of plants (163-165).
Probably no phenomenon of wheat grain carbohydrate metabolism has been studied more than the effect of high temperature stress on starch biosynthesis. The extensiveness of studies ensures that many permutations of high temperature treatment on developing wheat grain have been performed. As a result, we know that as temperature increases, the rate of dry matter accumulation in the endosperm increases and the duration of grain filling decreases (166, 167). At temperatures above approximately 30°C, the rate of fill also begins to decline (168). The degree at which heat stress restricts starch accumulation is dependent on the severity of the treatment, the stage of development at which high temperatures occur, and the genotype of the plant (135, 169-171). At the gross level, high temperatures do not affect endosperm cell number but cause a reduction in the number and/or size of starch granules within the cells (172-175).
There is an extensive amount of information on the direct effect of high temperature on enzymes involved in starch production. Rijven found that pretreatment of grain slices with high temperature reduced the conversion of sucrose to starch (176). The sensitivity of starch synthesis was reflected by the in vitro response of soluble starch synthase activity to temperature. Rijven suggested that in vivo effects of heat may be caused by inactivation of this enzyme. Since Rijven's work, others have further examined the heat sensitivity of soluble starch synthase and other enzymes involved in starch metabolism. One example is that of Keeling et al. (177), where they confirmed the sensitivity of starch synthase and reported that activity of AGP, UDP-glucose pyrophosphorylase, sucrose synthase, PGM, phosphoglucoisomerase, GBSS, and hexokinase were not affected by elevated temperatures. They believed that soluble starch synthase is a major sight of regulating starch synthesis in wheat grain. A similar comparison of enzyme activities and high temperature (178), as well as the influence of temperature upon 14C-starch synthesis and soluble starch synthase activity (179), led to similar conclusions.
The thermal characteristics of soluble starch synthase from wheat endosperm have been studied in detail by Jenner et al. (180). The Km values of amylopectin and ADP-glucose increased with elevated temperature, indicating that the affinity of the enzyme for its substrates was reduced at high temperature. Soluble starch synthase has also been implicated in the control of starch synthesis in heat stressed barley (181).
Although a strong case can be made for soluble starch synthase limiting starch synthesis in heat stressed wheat, there are several reasons to question the exact importance of the enzyme. Several cultivars of wheat have been well characterized for their degree of response to high temperature stress and Jenner and Sharma (182) have measured the kinetic properties of soluble starch synthase from grain of these contrasting seed genotypes, as well as seed of barley, rice, and bean. They found that the kinetic properties of soluble starch synthase from all the species are sensitive to temperature, but the rank order of sensitivity did not coincide with the known degrees of heat tolerance exhibited by these crops. Nor did the comparison of the two cultivars of wheat show a direct link between temperature response of grain filling and Vmax of the enzyme. In addition, there was no distinction in wheat or rice soluble starch synthase Km values for amylopectin.
Wardlaw et al. (43) extended this work and looked at the in vitro rate of starch synthesis in grain having the outer pericarp and embryo removed and the kernel split along the crease region. Differences between cultivars in the accumulation of 14C-sucrose and starch synthesis followed the same contrasts as growth rate of kernels on the plant. However, differences in their response to high temperature were not evident in the 14C-sucrose uptake studies. This calls into question whether any of the enzymes of starch metabolism, per se, in the endosperm actually serve as a control point of starch synthesis under elevated temperatures.
Two other points are worth mentioning. First, the temperature reactivity of soluble starch synthase measured in vitro can be altered by exogenous materials such as PEG, sucrose, sodium citrate, or type of glucan primer (62, 183). The sensitivity of the enzyme to catalytic conditions calls into question how relevant in vitro activity measurements are to catalysis that occurs in the undefined environment of endosperm amyloplasts. Secondly, many studies examining the response of wheat grain and starch synthesis to high temperature stress have employed whole plants treated in growth cabinets. This means that root temperatures are also elevated. Kuroyanagi and Paulsen (184) demonstrated more than 10 years ago that heat stress of wheat roots, alone or in combination with stress on the shoot, during grain filling severely reduces grain growth. It is possible that the effect of high temperature on grain filling in wheat is actually a reflection of stress to the roots and not directly pertaining to the metabolism of starch in the endosperm. In fact, maize, also known through growth cabinet studies for its sensitivity to heat stress, shows little response to high temperature during grain filling when roots are not stressed (185).
Although for many years phytohormones have been known to occur in cereal grain, their role in controlling growth has remained unclear. The pattern of their levels during seed development in wheat has been reported by a number of researchers (186-189). In general, cytokinins display a transient increase early in grain development, when endosperm cell number is being determined. Gibberellic acid (GA) reaches a peak during mid-grain development and rapid starch synthesis, before subsequently declining. Auxin reaches a peak slightly beyond that of GA and then declines towards maturation.
Many believe that the spike of cytokinin concentration in early seed growth has a casual role in controlling the number of endosperm cells that form in the kernel. Accordingly, cytokinins could have an indirect bearing on the amount of starch deposited in the endosperm. While this may be true, Mengel et al. (188) regulated growth and starch accumulation of wheat grain by providing low and high levels of illumination and found that the profde and amount of cytokinin in seed of both treatments was alike. The same was not true for GA content. In this case, higher levels occurred in grain of plants receiving high light. In the opposite way, Mengel and Judel (14) showed that reduced light intensity decreased the amount of starch produced in grain, as well as the GA content and activity of several enzymes of starch synthesis. Reports generally agree that GA may partake in regulating dry matter accumulation in seed, but this may not always be the case. Radely (8) showed that degraining treatments, which produced "giant" grains in remaining florets, had no marked effect on the pattern of change in GA content.
Some have also speculated that auxin content may have a positive impact on the intensity of grain filling (13) and ABA a detrimental effect on grain growth. Regarding the latter, ABA concentration goes up as grain growth is reduced under conditions of limited sucrose or reduced illumination (188, 189). Explicit information regarding the role of auxin in controlling grain growth, and its possible influence over starch synthesis, remains unreported.
In short, very few studies have simultaneously examined the pattern of phytohormone accumulation and starch synthesizing capability of developing grain. This is the case despite abundant circumstantial evidence which infers that regulation of grain growth may be a function of the interaction between these two factors.
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