SummaryIt is widely known that some of the starch synthases and starch-branching enzymes are trapped inside the starch granule matrix during the course of starch deposition in amyloplasts. The objective of this study was to use maize SSI to further our understanding of the protein domains involved in starch granule entrapment and identify the chain-length speci®cities of the enzyme. Using af®nity gel electrophoresis, we measured the dissociation constants of maize SSI and its truncated forms using various glucans. The enzyme has a high degree of speci®city in terms of its substrate±enzyme dissociation constant, but has a greatly elevated af®nity for increasing chain lengths of a-1, 4 glucans. Deletion of the N-terminal arm of SSI did not affect the K d value. Further small deletions of either N-or C-terminal domains resulted in a complete loss of any measurable af®nity for its substrate, suggesting that the starch-af®nity domain of SSI is not discrete from the catalytic domain. Greater af®nity was displayed for the amylopectin fraction of starch as compared to amylose, whereas glycogen revealed the lowest af®nity. However, when the outer chain lengths (OCL) of glycogen were extended using the phosphorylase enzyme, we found an increase in af®nity for SSI between an average OCL of 7 and 14, and then an apparently exponential increase to an average OCL of 21. On the other hand, the catalytic ability of SSI was reduced several-fold using these glucans with extended chain lengths as substrates, and most of the label from [ 14 C]ADPG was incorporated into shorter chains of dp < 10. We conclude that the rate of catalysis of SSI enzyme decreases with the OCL of its glucan substrate, and it has a very high af®nity for the longer glucan chains of dp »20, rendering the enzyme catalytically incapable at longer chain lengths. Based on the observations in this study, we propose that during amylopectin synthesis shorter A and B 1 chains are extended by SSI up to a critical chain length that soon becomes unsuitable for catalysis by SSI and hence cannot be elongated further by this enzyme. Instead, SSI is likely to become entrapped as a relatively inactive protein within the starch granule. Further glucan extension for continuation of amylopectin synthesis must require a handover to other SS enzymes which can extend the glucan chains further or for branching by branching enzymes. If this is correct, this proposal provides a biochemical basis to explain how the speci®cities of various SS enzymes determine and set the limitations on the length of A, B, C chains in the starch granule.

This study reports the ultrastructural changes in maize endosperm that result from exposure to high temperature during cell division. Kernels were grown in vitro at 25 ºC continuously (control) and at 5 d after pollination (DAP) subsamples were transferred to continuous 35 ºC for either 4 or 6 d. The 4 d treatment reduced kernel mass by 40% and increased kernel abortion three-fold. The 6-d high-temperature treatment resulted in a 77% reduction in kernel mass and a 12-fold increase in kernel abortion. Evaluation of the kernels at 11 DAP using scanning and transmission electron microscopy revealed that the reduced kernel mass and/or abortion was associated with the disruption of cell division and amyloplast biogenesis in the periphery of the endosperm. This was further confirmed by the presence of an irregular-shaped nucleus, altered size of the nucleolus, highly dense nucleoplasm, and a decrease in the number of proplastids and amyloplasts. Thus, the endosperm cavity was not filled, the total number of endosperm cells was reduced by 35 and 70%, and the number of starch granules was decreased by 45 and 80% after exposure to 4 and 6 d of high-temperature treatments, respectively. This also resulted in a 35-70% reduction in total starch accumulation. KI/I 2 staining and light microscopy revealed that starch accumulation in the peripheral endosperm cells was reduced more severely than in the central zones. However, the scanning electron micrographs of cells from the central endosperm showed that the number and the size of apparently viable amyloplasts were reduced and isolated granules were smaller and/or showed enhanced pitting. These ultrastructural data support the hypothesis that high temperature during endosperm cell division reduces kernel sink potential and subsequently mature kernel mass, mainly by disrupting cell division and amyloplast biogenesis in the peripheral and central endosperm.Key-words: Zea mays L.; amyloplast number; cell division; corn; high temperature; kernel morphology; kernel sink potential; kernel ultrastructure. INTRODUCTIONIn many of the world's maize-producing areas, high temperature is a common abiotic stress and is a major cause of decreased grain yield (Dale 1983). The thermal optimum for grain development in maize (Zea mays L.) has been shown to be between 27 and 32 ºC (Keeling & Greaves 1990; Teixiera & Jones, unpublished results). However, an average temperature of 32 ºC or more during reproductive development is common across many parts of the USA corn belt (Thompson 1968). High temperature, especially during the endosperm cell division is highly detrimental to the subsequent starch deposition and the grain yield (Jones, Gengenbach & Cardwell 1981;Jones, Ouattar & Crookston 1984;Jones, Roessler & Ouattar 1985). Elevated temperature (35 ºC) even for 3-4 d during this stage can reduce kernel mass at maturity and may even result in kernel abortion (Jones et al. 1984;Cheikh & Jones 1994). In cereal grains, kernel sink capacity is an important physiological determinant of the ...

High temperature during endosperm cell division reduces grain yield of maize (Zea mays L.). The objective of the study was to determine if there were differences in tolerance of two inbred lines (B73 and Mo17) to exposure to brief high temperature treatments (HTTs). Beginning 5 d after pollination (DAP), kernels were exposed to a continuous 35°C temperature for either 4 or 6 d. The effects of HTTs on kernel development, ultrastructure, and sink capacity were evaluated under both in vitro and field conditions. In B73, the 4 and 6 d HTT reduced final kernel dry weights >40 to 60% under in vitro and 79 to 95% under field conditions, compared with the controls. The HTT‐induced reduction in kernel mass was due mainly to reduction in starch granule number, since by 16 DAP the endosperm cell number had recovered and was not significantly different from the controls. In contrast, in Mo17 both the number of endosperm cells and starch granules were reduced by >45 to 80% by the 4 and 6 d HTT imposed under the two growing conditions. Hence, these data and kernel ultrastructure evidence confirm that kernel development is more tolerant to high temperature in B73 than in Mo17. The difference appears to be due mainly to the ability of B73 to maintain a higher kernel sink capacity after exposure to HTT during endosperm cell division. Exploiting the differential response of these genotypes appears to be a viable approach to further elucidate the physiological basis for heat tolerance during early kernel development.

No abstract

Heat stress during the early formative stages of maize (Zea mays L.) kernel development is detrimental to subsequent growth and grain yield. During this period, even a brief exposure to temperature above the optimum (25°C) can result in significant yield losses. The mechanisms involved in this response are not well understood, but appear to be associated with a thermally induced shift in hormone balance (cytokinin/ABA), which results in a reduction in kernel sink capacity (i.e. number of endosperm cells and starch granules formed) and the disruption of sugar metabolism and starch biosynthesis. However, the effect of heat stress on the morphology and ultra-structure of kernel component tissues (pedicel, pericarp, endosperm, and embryo) has not been studied. Therefore, the objective of our current study was to characterize heat stress induced changes in the pericarp (maternal) and the embryo (embryonic) tissues using SEM.