Starch is the major constituent of matured wheat grain. The details of subtle localized differences in the evolution of the structure of starch are important for an understanding of starch biosynthesis. However, the distinct stages involved in the formation and transformation of the molecular structure of starch during starch biosynthesis are still not fully understood. In this study, starches extracted from wheat grains harvested at 3, 7, 14, 28, and 49 days after anthesis (DAA) were used as a means of examining the molecular structure of starch from developing wheat grain. Gel-permeation chromatography and high-performance anion-exchange chromatography were employed for the analysis of the structure of both whole starch and its isolated amylopectin (AP) component. Scanning electron microscopy of 3 DAA wheat grain cross-sections revealed the absence of endosperm but the presence of spherical transitory-type small starch granules in the pericarp. Endosperm was present at 7 DAA and contained lenticular-shaped developing large granules. From 14 DAA onward, spherical-shaped small granules coexisted with large granules in the endosperm. The structure of transitory pericarp starch (PS) was compared with that of matured endosperm storage starch (ES). The composition of PS and ES differed: PS granules contained 14 % apparent amylose (AAM), whereas ES granules contained 33 % AAM. The AAM fractions of PS showed characteristics similar to those of intermediate-type materials with short branches, whereas ES contained both linear and branched amylose (AM). Differences in the amylopectin component of PS and ES were also apparent, especially in their internal structures. PS amylopectin had longer chains and fewer A-chains, resulting in a structure less branched than that of ES amylopectin. Starches isolated from 7 DAA to 49 DAA were studied with respect to endosperm development. The AAM of both large and small granules increased with increasing maturity. The AAM fraction of starch granules at early maturity (7 DAA and 14 DAA) consisted of intermediate-type materials in addition to linear AM, whereas starch at later maturity stages (28 and 49 DAA) contained linear and branched AM. During granule development, the fine structure of AP varied with the maturity level as well as with the size of the granule. During the post-physiological maturity stage, when the net accumulation of sugars ceases, the grain dries out; however, structural changes occurred in AP at this stage, possibly due to the action of starch branching and debranching enzymes. In both large and small granules, the external AP structure was more organized at post-physiological maturity (49 DAA) than at pre-physiological maturity (7 DAA to 28 DAA). Compared to their characteristics at post-physiological maturity, at the pre-physiological maturity stage, isolated clusters of AP were larger with more branches and building blocks. In addition to the time-dependent discrepancies in the AP structure of developing starch, differences were also evident between large and small granules with regard to glucan trimming and the type of new chains produced. The clusters isolated from small starch granules were more tightly branched than those isolated from large granules

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Angiosperm seed development requires co-ordinated development of the embryo and a second zygotic tissue, the endosperm. In Arabidopsis thaliana, the endosperm is ephemeral and is largely consumed by the embryo during seed development. In addition to a role in embryo nutrition, it is also likely that the endosperm may play a more direct role in signalling to the embryo to regulate development. Despite their importance for embryo development, these processes are very poorly understood. The ZHOUPI (ZOU) gene provides an important tool to address these problems. Firstly, ZOU likely regulates endosperm breakdown. Whereas wild-type seed have a single layer of endosperm at maturity, zou seed has a large persistent endosperm and a correspondingly small embryo. The small zou embryo does not fill the seed so that the seed shrivels as it desiccates during maturation. Secondly, zou embryos have defects in their cuticle, so that the endosperm adheres to the embryo throughout seed development. After seed germination, zou cotyledons develop holes in their epidermis as they expand, probably due to the defects in the cuticle. ZHOUPI (ZOU) encodes a bHLH transcription factor and is expressed in the embryo surrounding region (ESR) of endosperm but not in the embryo itself. The role of ZOU in cuticle development is partly mediated by the ABNORMAL LEAF SHAPE1 (ALE1) gene. Thus, ale1 mutants also show defects in embryonic cuticle development and ALE1 is specifically expressed in ESR in a ZOU-dependent fashion. It was unclear whether the effects of ZOU upon embryo development are an indirect consequence of the persistent endosperm mechanically impeding embryo expansion, or rather reflect a more direct role of the ESR in signalling to the embryo. The main aims of this thesis were 1) to provide evidence that ZOU regulates endosperm cell death and 2) to test whether ZOU function in controlling endosperm cell death could be separated from that in embryonic epidermal cuticle development. To achieve this goal, 1) TUNEL assays were performed in the seeds to confirm the zou endosperm cell death phenotype, 2) ALE1 expression in the ESR in zou mutants was rescued using the ZOU-independent AtSUC5 promoter to investigate whether one or both of zou phenotypes were complemented, 3) Candidate ZOU target genes were validated and characterized to determine their functions in endosperm cell death and/or embryonic epidermal cuticle development. The TUNEL assays revealed that zou mutants display less DNA fragmentation in the ESR than that of the wild-type, but that zou did not have defects in cell death outside the seeds suggesting ZOU specifically regulated endosperm cell death. The AtSUC5::ALE1 transgene partially rescued zou defects in epidermal cuticle but not in endosperm cell death. This shows that the defects in the zou cuticle are not caused by the defective endosperm, rather zou has distinct, separable functions. Lastly, I characterised several novel ZOU targets and showed that RGP3 may be a direct ZOU target as it is expressed in ESR in ZOU dependent fashion, whereas RGP4 is likely indirect as it is expressed in the testa and up-regulated in zou mutants. In conclusion, ZOU has independent roles in endosperm cell death and embryonic epidermal cuticle development. Because ALE1, which largely mediates the role in cuticle development, is less widely conserved than is ZOU, the role in promoting endosperm cell death may be the ancestral function of ZOU.

BACKGROUND:The opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary.RESULTS:In this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch.CONCLUSIONS:The results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 4 uppsatser.

No description available.

In der vorliegenden Arbeit wurden Prozesse untersucht, welche die Akkumulation von Kohlenstoff und Stickstoff im Endosperm von Gerste im frühen Zeitraum nach der Anthese beeinflussen. Es wurde die C- und N-Verlagerung in Pflanzen der Sorte ´Bomi` sowie den low-starch-Mutanten Risoe 16 und Risoe 13 zu 8, 14 und 20 Tagen nach Anthese untersucht. Im Fokus standen die Vorgänge im Endosperm. Für die Termine 14 und 20 Tage nach Anthese wurde daher die Zusammensetzung des Proteins nach Fraktionierung in Albumine/Globuline, Hordeine und Gluteline analysiert. Mittels 13C- und 15N in vivo Doppel-Pulsmarkierung ganzer Pflanzen erfolgte die Untersuchung des Beitrages der aktuellen C- und N-Assimilation zur Endospermentwicklung. Im Untersuchungszeitraum konnte keine C-Remobilisierung festgestellt werden. Dahingegen wurde bei ´Bomi` und Risoe 16 eine Genotyp-abhängige Mobilisierung von Stickstoff in den vegetativen Organen nachgewiesen. Die Analyse von Frisch- und Trockenmassen der Endosperme offenbarte ein um 17 % niedrigeres Vermögen zur Akkumulation von Trockenmasse bei Risoe 16 und Risoe 13 gegenüber bei gleicher Sink-Kapazität. Es zeigte sich weiterhin, dass die C- und N-Einlagerung in das Endosperm nicht komplett unabhängig erfolgten. Die Analyse der Proteinfraktionen des Endosperms offenbarte, dass deren Bildungsdynamik und Zusammensetzung von Veränderungen im Stärkemetabolismus beeinflusst wird. Die Albumin/Globulin-Menge pro Endosperm betrug bei Risoe 16 bzw. Risoe 13 zu 14 dpa das 3,5- und 1,9-fache des Wertes von ´Bomi`. Sie stieg beim Wildtyp bis 20 dpa an, während sie bei den Mutanten zumindest tendenziell sank. Weiterhin fiel die Hordeinmenge im Mittel beider Mutanten gegenüber ´Bomi` zu 14 dpa 57 % und 20 dpa 38 % niedriger aus. Für die Glutelinmenge zeigte sich erst zu 20 dpa ein Unterschied, wobei der Wert von Risoe 16 und Risoe 13 37 % bzw. 29 % höher als jener des Wildtyps lag. / This thesis investigates processes that influence the accumulation of carbon and nitrogen in the early stage of endosperm development. The C- and N allocation was examined at 8, 14 and 20 days post anthesis (dpa) in the barley cultivar ´Bomi` and the low-starch mutants Risoe 16 as well as Risoe 13. The endosperm was in the focus of interest. Therefore the composition of its protein was determined at 14 and 20 dpa by fractionation in albumins/globulins, hordeins and glutelins. Furthermore the contribution of current C-and N assimilation to endosperm development was investigated by employing 13C- and 15N in vivo double puls-labelling of whole plants. During the investigation period no C remobilisation occurred. In contrast a genotype dependent mobilization of nitrogen in the vegetative organs was proved for ´Bomi` and Risoe 16. The analysis of endosperm fresh and dry weight revealed a 17 % lower ability to accumulate dry matter for Risoe 16 and Risoe 13 as compared to ´Bomi` although sink capacity was comparable. Furthermore there werevstrong indications that C- and N incorporation in the endosperm were not completely independent from each other. The analysis of the endosperm protein revealed that its composition as well as the formation dynamics and the amount of the single fractions were influenced by the changes in starch metabolism. The amount of albumin/globulin per endosperm of Risoe 16 and Risoe 13 at 14 dpa was 3.5 and 1.9-fold higher as compared with ´Bomi`. In the following 6 days it rose for the wildtype, while it tended to decrease for the mutants. Furthermore the amount of hordeins (mean value of both) in the endosperm of the mutants was 57 % and 38 % lower than that of ´Bomi` at 14 dpa and 20 dpa, respectively. The amounts of glutelins were not distinct between genotypes at 14 dpa but at 20 dpa with 37 % and 29 % higher values compared with ´Bomi` for Risoe 16 and Risoe 13, respectively.

Il mais è uno dei cereali maggiormente diffusi perché utilizzato in ambito alimentare umano e animale, per la produzione di materiale biodegradabile e di bioetanolo. I processi fisiologici che coordinano la crescita della cariosside vengono regolati principalmente dall’auxina che agisce a livello trascrizionale e post-traduzionale. Attraverso analisi comparative tra il mutante de18 (defective endosperm 18), deficitario nella produzione di acido indolo-3-acetico (IAA), e del suo corrispettivo wild-type, è stato possibile individuare i geni coinvolti nella determinazione delle dimensioni della cariosside. In particolare, sono state effettuate analisi morfologiche e di quantificazione dell’amido su cariossidi de18 e wild type negli stadi precoci di sviluppo. E’ stato inoltre allestito un esperimento di RNA sequencing sull’endosperma dei due genotipi a 8 e 12 DAP (Days After Pollination). L’analisi dei geni differenzialmente espressi attraverso la classificazione GO (Gene Ontology) ha permesso di studiare l’effetto della carenza di auxina sull’espressione genica. Nel mutante si riscontra l’attivazione tardiva della sintesi dell’amido e l’incremento delle proteine di riserva. Inoltre, la carenza di auxina determina una riduzione dell’attività mitotica ed endoreduplicativa, confermata dalla repressione dell’attività di geni legati al ciclo cellulare. / Maize is one of the world’s leading cereal grains due to its diverse functionality as a food source for both humans and animals, as well as a source of raw materials and biofuel. The physiological processes responsible for the growth of the kernel are regulated mainly by auxin acting at the transcriptional and post-translational level. Through comparative analysis between mutant de18 (defective endosperm 18), defective in indol-3-acetic acid (IAA) production, and its wild-type B37, it has been possible to identify the genes involved in the determination of the final seed size. Morphological analysis and quantification of starch were done on seed mutant and wild-type in the early stages of their development. Finally, an RNA sequencing analysis was carried out on mutant and wild-type endosperm at 8 and 12 DAP (Days After Pollination) and differentially expressed genes were classified by Gene Ontology. Down-regulation of genes related to sugar metabolism suggested a delayed activation of starch biosynthesis. This finding was confirmed by the determination of starch content that was lower in the mutant endosperms respect to the normal in the early stages of gran filling (12 and 16 DAP). The reduced auxin level affected the mitotic and endoreduplication activities as suggested by the repression of genes involved in the cell cycle.

It is becoming increasingly important to improve the yield of seed crops to feed an expanding population and, more recently, to cope with additional strains on food-oriented agriculture posed by biofuel production and global climate change. One strategy to increase yield is to increase seed size. However in nature, due to the limited resources of the mother plant, an enlarged seed size is often associated with a concomitant reduction in seed number. Using the model plant Arabidopsis thaliana, a biotechnological approach to increase seed size through the modification of the triploid endosperm or the ovule integuments was shown to be a viable strategy to improve seed yield. Targeted over-expression of the ANT transcription factor specifically within the endosperm significantly increased seed size without negative effects on fertility. Overcoming compromised fertility in arf2 mutants established that ARF2 and the integuments are important factors in determining seed size and yield. To ensure agronomic relevance, both the gross yield and the harvest index (HI, ratio of seed yield to biological yield) were used to assess the impact of traits, such as increased integument size, introduced into Arabidopsis. To uncover novel regulators of seed size and further develop the current understanding of seed development, second-site mutations were induced in the auxin response factor 2 (arf2) mutant, which produces large seeds due to extra cell division in the ovule integuments. The ARABIDOPSIS CRINKLY 4 (ACR4) gene was identified as a suppressor of the arf2 seed phenotype. In arf2-8 acr4 double mutants, an additive effect on cell proliferation was observed, indicating that ACR4 affects the seed coat through a developmental pathway independent of ARF2. Natural variation present in Arabidopsis was used to study seed yield and its components. Considerable variation in seed size, yield and HI was revealed. Significantly, high seed weight was not associated with high yield or high HI. In contrast, high seed number and reduced plant stature were revealed as important components of high yield and yield efficiency. Additionally, ERECTA was identified as a potential ‘Green Revolution’ gene. Seed size in Arabidopsis was shown to be an extremely plastic trait in response to alterations in the post-flowering source-sink ratio, increasing by 35.0 – 66.2% in the ecotypes tested. Furthermore, a difference in seed weight between restricted (high source-sink ratio) and unrestricted (low source-sink ratio) pollinations was first observed remarkably early in seed development. However, reducing seed number by restricting pollination did not substantially alter integument or endosperm development in order to facilitate increased nutrient uptake.

The protein quality of maize can be improved by replacing normal Opaque2 alleles with non-functional recessive alleles opaque2 (o2). The allele o2 produces a severe phenotype with soft endosperm enhancing its protein quality but decreasing its agronomical value. Plant breeders have restored a desirable ratio of hard to soft endosperm in o2 germplasm known as Quality Protein Maize (QPM). Neither the mechanism nor the genetic components by which the modification of the endosperm in QPM lines occurs are well understood. To increase the understanding of the genetics of endosperm modification, a population of 146 recombinant inbred lines derived from a cross between the o2 inbred line B73o2 and the QPM inbred line CML161 was evaluated in two Texas locations from 2004 to 2006. Four traits related to endosperm texture were measured and showed significant effect of the inbred lines, high heritability estimates and high genetic correlations. Relative content of the essential amino acids lysine, tryptophan and methionine were measured and showed significant effects of the lines and considerable high genetic correlations and heritabilities. Negative correlation was observed between endosperm texture traits and amino acid content. Quantitative trait loci (QTL) were mapped for traits related to the modification of endosperm texture and the content of lysine, tryptophan and methionine. QTLs clusters for endosperm texture traits were detected on chromosomes 3, 5, 6 and 8 explaining 62-68% of the variation. QTLs clusters for amino acid contents were located on chromosomes 7 and 8 that explained up to 39% of the observed variation. The product of the O2 gene is a transcription factor that affects the expression of a number of endosperm genes. A group of 29 endosperm genes associated with the O2 activity were evaluated in developing endosperm of the recombinant inbred lines. Genomic regions controlling gene transcript abundance in developing endosperm were identified by expression QTL mapping. Evidence is presented of QTL hot spots that segregate in association with endosperm texture modification or amino acid contents and are associated with the regulation of the expression of a group of endosperm genes.