AN EVALUTION OF THE INDUCTION AND METABOLIC ENGINEERING OF FLAVONOID DEFENSE COMPOUNDS IN SORGHUM AND MAIZE
Abstract:
Sorghum is closely related to maize but has a better ability to withstand biotic and abiotic stress. To gain insights into the possible reasons underlying such difference, a comparative characterization of genes and traits has been conducted. We have investigated the flavonoid pathway in these two species. This research indicated that sorghum and maize differ substantially in their ability to synthesize flavonoid compounds. For example, sorghum plants respond to anthracnose and leaf blight fungi by synthesis of flavonoid antifungal compounds (phytoalexins). However, maize plants do not produce simillar compounds in response to fungal attack. Therefore, we used sorghum as a model system to understand the biosynthetic routes of the flavonoid phytoalexins and attempted to engineer the production of these compounds in maize. Sorghum phytoalexins belong to the 3-deoxyanthocyanidin class which includes luteolinidin, apigeninidin and their derivatives. These compounds are produced in response to fungal attack and appear as red-brown pigments at the primary infection sites. Their antifungal activity against many fungi has been demonstrated; however, the lack of well defined phytoalexin deficient mutants has hampered the understanding of the mechanisms underlying their biosynthesis. These compounds have chemical structures similar to that of flavan-4-ols, the precursor of phlobaphene pigments that appear in sorghum floral tissues and mature leaves, suggesting that these two flavonoid classes may be synthesized via a common or an overlapping pathway. The biosynthesis of flavan-4-ols is under the control of an R2R3 myb transcription factor encoded by yellow seed1 (y1). To gain a better understanding of the possible role of y1 in controlling sorghum phytoalexins, we used a transposon-based approach and developed near-isogenic sorghum lines that differ in the functionality of y1. Molecular and phenotypic analyses of one of these lines (y1-ww) revealed the presence of an internal deletion in y1 and such deletion rendered y1 non-functional. The gene expression analysis of the fungal inoculated isogenic lines indicated that the expression of the phytoalexin biosynthetic genes is y1 dependent. Comparison of the phytoalexin biosynthetic ability of these lines demonstrated that y1 mutant is phytoalexin deficient. The y1 activity was also positively correlated with the resistance against anthracnose disease. These results provide direct genetic evidence that y1 is necessary for the biosynthesis of sorghum 3-deoxyanthocyanidins and resistance against anthracnose disease. Like sorghum, maize accumulates flavan-4-ols in the floral tissues but not in leaves. The biosynthesis of these compounds in maize is under the control of pericarap color1 (p1). p1 and y1 are orthologues with a high degree of similarity in their coding sequences but very low similarity in the regulatory regions . These two genes have almost the same pattern of expression except that y1 is significantly expressed in leaf and is induced by fungal infection. Therefore, we developed transgenic maize lines expressing either an Y1::GUS fusion or the intact y1 gene driven by its own promoter. These plants were used to test the hypothesis that the expression of y1 in leaves and its induction by the fungus is a property of the y1 promoter. Analysis of the Y1::GUS plants revealed that y1 promoter has constitutive low expression in maize floral and vegetative tissues and is responsive to fungal infection. Interestingly, y1 induced phlobaphene pigmentation in maize floral tissues and these phenotypes were stably inherited through generations. In addition, y1 successfully drives the flavonoid pathway in maize leaves towards the biosynthesis of flavan-4-ols, and induces the biosynthesis of indigenous as well as novel flavonoid and phenylpropanoid compounds in maize leaves. The accumulation of these defense-related compounds in transgenic maize leaves resulted in an enhanced resistance against corn southern leaf blight. These results suggest that possible evolutionary modifications in y1 and p1 promoters might be responsible for the differential flavonoid profiles of sorghum and maize in response to fungal attack. The biosynthesis of sorghum phytoalexins occurs rapidly after fungal attack and involves activation of y1 and its target genes. The signaling cascades upstream of the biosynthesis of these compounds are unknown. The dissection of the y1 promoter revealed the presence of cis recognition sequences that have been shown to be targets of signaling compounds such as JA, ABA, and WRKY proteins. Simultaneous phytohormonal analysis during 3-deoxyanthocyanidins biosynthesis indicated that sorghum phytoalexins are associated with early induction of JA and IAA, and either no change or reduction of ABA respectively. A hypothetical model for signal transduction during sorghum phytoalexin biosynthesis is proposed