Regulatory Aspects Of Starch And Carotenoid Accumulation During Fruit Development Of Novel Inter-Specific Inbred Squash Lines

Cucurbita species rank collectively among the10leading vegetable crops worldwide. China has become the first in the ranking of squash producing countries with6,506,966tons since2009. The productivity of squashes can’t be properly evaluated only by comparing yield unless considering the quality attributes of the fruits. Fruit quality is a major concern of current squash breeders. Moreover, the consumer’s demand for fruit quality is increasing and there is a critical need to provide a consistently good product. A key property of a desirable vegetable product is a palatable texture. Furthermore, it is considered crucial while planning for healthy products to retain high nutrient content, and to ensure high bioavailability of the nutrients.In squash, eating acceptability is most often related to flesh color, texture and sweetness traits. People differ in their preference for different degree of texture in squash. Chinese squash (C. moschata) is a fairly moist squash, with dry matter between10to15%. The market for Chinese squash has been stable, but adversely affected by low dry matter and therefore led to poor eating quality in many of the modern squash hybrids. Indian squash (C. maxima), popular in many central and northern Asian countries, have high dry matter about20to33%, contributing to their excellent eating quality. Since dry matter content varies according to the balance of starch and sugar content in squash fruits and starch represents the predominant substance in squash fruits, it would seem reasonable that starch content could also influence squash texture. Hence, the differences in starch content and physicochemical properties of their fruit should be taken into consideration while explaining the variations in squash fruit texture.Besides carbohydrates, another main nutritional component of squash is the carotenoids. These crops play an important role in local diets and could provide a viable solution to nutritional deficiency in developing countries. Squashes are available in a wide range of flesh fruit colors. A flesh color is one of most important attribute for consumer acceptability. The total carotenoid content has a positive association with the intensity of fruit-flesh color. It is suggested that, intense yellow or orange flesh is an important positive component of eating quality in squash. So because of these important functional and nutritional characteristics, it is important to understand the mechanisms of starch and carotenoid biosynthesis regulation to help developing breeding strategies for increased squash fruit nutritional value.Crossing among cultivated Cucurbita species has also excited interest in breeding for high fruit quality. This study used inter-specific inbred lines which were jointly developed at Extension Station of Agricultural Technology, Agriculture Bureau of Huzhou City, Huzhou. These inbred lines were obtained from interspecific crosses between C. maxima and C. moschata using hybridization, back-crossing and self-pollination approaches. This inter-specific inbred squash lines namely "Maxchata" appear to be potentially valuable to produce a high fruit quality. The overall objective of this research work was to further understand the starch and carotenoid accumulation differences in fruits of inter-specific inbred line and their parents.In first experiment, squash fruits from two different species and their inter-specific inbred line ("Maxchata1") were analyzed for their starch granule morphology, dry matter, amylose and total starch contents. Moreover, cooked squashes were observed for the sensory analysis, cooking properties, and texture characteristics of raw and cooked squashes. The main results are as follows (1) The starch granules of all squashes had less smooth granule surfaces and appeared as a mixture of spherical, polyhedral and dome shaped granules with sizes ranging from1to17μm when observed by scanning electron microscopy.(2) Granule size of C. maxima was noticeable larger than C. moschata and "Maxchata1".(3) C. maxima and "Maxchatal" were significantly higher in dry matter and starch content than C. moschata.(4) Dry matter, amylose and total starch contents were positively correlated with extent of mealiness.(5) For cooking properties, squashes with higher mealiness scores (C. maxima and "Maxchatal") had longer cooking times and higher firmness force than squash with lower mealiness scores (C. moschata).(6) C maxima and "Maxchatal" also showed higher total solid loss and water uptake values during cooking than C. moschata.(7) Texture of raw and cooked squashes was evaluated by using a Texture Analyzer. Texture parameters such as fracturability, hardness, adhesiveness, springiness, cohesiveness, gumminess, chewiness were higher for C maxima and "Maxchatal" for both raw and cooked squashes and were found to be related to mealiness scores, dry matter and starch contents. An additional experiment was conducted on isolation and characterization of starch and carotenoid related genes with the aim to understand the regulation of starch and carotenoid accumulation at gene expression levels during squash fruit development. The full-length cDNAs encoding PSY1, PDS, ZDS, CHYB, LCYB, ZEP, GBSSI and partial cDNAs encoding LCYE, CCD, AGPaseL, SSII, SBEII and ISA1genes were isolated from C. moschata. The sequence analysis showed that they shared high sequence identity and conserved motifs with other orthologous genes.Second experiment was conducted to evaluate the correlation of starch accumulation with transcriptional regulation of starch related genes during squash fruit development among two different species as well as their inter-specific inbred lines. Dry matter and starch composition during squash fruit development were observed in C. maxima, C. moschata and their inter-specific inbred lines, namely "Maxchata1" and "Maxchata2". Five genes associated with starch biosynthesis were analyzed for their expression patterns to elucidate the molecular mechanism of starch synthesis in squash fruit by quantitative RT-PCR. The two squash species and their inter-specific inbred lines exhibited different quantitative starch composition and regulatory mechanisms. The main results are as follows (1) C. moschata had the lowest total starch content as expected from comparatively low transcript levels of AGPaseL gene compared to the other squashes.(2) Starch content in C. maxima,"Maxchatal" and "Maxchata2" were not significantly different in mature fruits.(3) The higher expression of the AGPaseL genes resulted in more starch accumulation in these squashes.(4) AGPaseL gene exhibited high expression levels at early fruit development that led to rapid increase in starch accumulation at early stage of fruit development in "Maxchatal".(5) Likewise, GBSSI gene exhibited high expression level in C. maxima and inter-specific inbred lines that was correlated with high accumulation of amylose content.(6) In contrast, SSII, SBEII and ISAI genes, responsible for amylopectin synthesis, were expressed late during fruit development and showed similar transcript levels among squashes.The final experiment was to investigate the role of transcriptional regulation of carotenogenic genes responsible for different carotenoid composition in squash fruits. Carotenoid levels and composition during squash fruit development were compared among those squashes. Eight genes associated with carotenoid biosynthesis were analyzed by quantitative RT-PCR. The two squash species and their inter-specific inbred lines exhibited different qualitative and quantitative carotenoid profiles and regulatory mechanisms.(1) C. moschata had the lowest total carotenoid content and mainly accumulated a-carotene and β-carotene, as predicted from its fruit with pale-orange flesh.(2) Low carotenoid content in this species was due to the comparatively low expression of all genes investigated, especially PSY1gene compared to the other squashes.(3) The predominant carotenoids in C. maxima were violaxanthin and lutein which produce a corresponding yellow flesh color in mature fruit.(4) The relationship between the expression of the CHYB and ZEP genes may result in almost equal concentrations of violaxanthin and lutein in C. maxima at fruit ripening.(5) In contrast, their inter-specific inbred lines principally accumulated lutein and β-carotene, leading to deep orange flesh color.(6) PSY1gene exhibited high expression level at the early stage of fruit development that might have triggered high accumulation of carotenoid in "Maxchatal" and "Maxchata2".(7) Likewise, higher transcription level of CHYB gene observed in the inter-specific inbred lines might be correlated with high lutein in these inter-specific inbred lines.(8) However, this study could not explain the observed P-carotene accumulation on the basis of gene expression solely.These findings allow us to develop an understanding of the regulation of starch and carotenoids biosynthesis during squash fruit development of novel inter-specific inbred lines. Moreover, this information may provide a source for further study regarding improvement in eating quality of squash fruit.