Induction And Regulation Of Fungal Defense Related Compounds In Sorghum Bicolor

Open Access
Liu, Bin
Graduate Program:
Plant Biology
Master of Science
Document Type:
Master Thesis
Date of Defense:
February 05, 2016
Committee Members:
  • Surinder Chopra, Thesis Advisor/Co-Advisor
  • Lavanya Reddivari, Thesis Advisor/Co-Advisor
  • Majid R Foolad, Thesis Advisor/Co-Advisor
  • Sorghum bicolor
  • 3-deoxyanthocyanidin
  • HPLC
  • phytohormones
  • salicylic acid
  • jasmonic acid
  • yellow seed1
  • ft-nir
Sorghum (Sorghum bicolor L. Moench) is an important member of the Poaceae family. It is a highly resilient crop that is drought and cold tolerant, and capable of surviving in the areas that would otherwise not support other cereal crops like maize. One of the major challenges in sorghum production is anthracnose leaf blight caused by Colletotrichum sublineolum. Understanding the mechanisms by which sorghum combat anthracnose is key to breeding for resistant cultivars. Plants combat disease infection through several ways: Passive defense mechanisms are thickening of cell wall, growth of waxy layers, epidermal outgrowth etc. Active defense mechanisms leading to hypersensitive response, which appears as localized cell death, synthesis of phytoalexins and antibiotic compounds. Sorghum is unique in the Poaceae family in its ability to synthesize 3-deoxyanthocyanidins (3-DAs), a sub-class of flavonoids. These compounds have been shown to act as phytoalexins that accumulate as brick red pigments at the primary site of attempted penetration by the fungus. Because of their anti-fungal properties, 3-DA phytoalexins can limit fungal progression. Sorghum phytoalexins include luteolinidin, apigeninidin and their methoxylated derivatives. We are interested in investigating detailed biosynthetic pathways of 3-DAs in sorghum as well as signal transduction events that lead to the resistance responses imparted via the 3-DAs. Following are my thesis objectives: 1. Interaction of sorghum Y1 transcription factor with maize flavonoid structural genes during pathogen defense response. Based on the structural similarities of 3-DAs and flavan-4-ols (see Figure 1 in Chapter 1), it has been hypothesized that 3-DAs are also synthesized via the flavonoid pathway branch which leads to the production of phlobaphenes in sorghum. An R2R3 MYB transcription factor encoded by yellow seed1 (y1) has been shown to be required for the biosynthesis of flavan-4-ols and phlobaphenes in the pericarp and leaf tissues. Using a transposon insertion mutant in y1, our lab has shown that the y1 gene is also required for the accumulation of 3-DAs in Colletotrichum sublineolum challenged sorghum leaves. Further, transformation of sorghum y1 gene into maize showed induced disease resistance response to Colletotrichum graminicola, establishing that the y1 gene can induce maize structural genes to accumulate 3-DAs in maize. In addition, that study indicated the y1 promoter contains cis- regulatory elements that are possibly involved in regulation of y1 during fungal challenges. Despite the above-mentioned results, we do not exactly know the flavonoid branch and the structural genes that are required for the biosynthesis of 3-DAs. It is also possible that 3-DAs either originate from a separate branch from naringenin, or these are simply derived from flavan-4-ols as a precursor. To identify the flavonoid branch leading to 3-DAs, we first transferred sorghum y1 to maize and then used maize mutants of flavonoid structural genes because of the non-availability of such mutants in sorghum. Our results showed that y1 transgene can regulate the expression of all the maize flavonoid genes tested. We also found that this induced expression was correlated with the observed disease resistance phenotype. This study indicates that the anthocyanidin synthase encoded by a2 in maize also have novel influence on flavan-4-ols and 3-DAs accumulation. Furthermore, through the accumulation of total phenolics, we have found that the y1-driven flavonoid pathway as a whole promoted the total phenolic accumulation. 2. Expression of phytohormones salicylic acid and jasmonic acid genes during the 3-DA biosynthesis in sorghum and maize. Currently, the upstream regulation of y1 remains relatively unclear. We attempted to understand signal transduction events upstream of y1 by measuring phytohormones that may have a role in early signaling events. Two phytohormones particularly important in plant defense are jasmonic acid (JA) and salicylic acid (SA). Previous studies focusing on the effect of exogenous phytohormones on 3-DA pathway have yielded conflicting results. In this work, sorghum mesocotyls and transgenic y1 maize lines were inoculated to study the internal interactions between the transcription of key genes in the SA and JA phytohormonal pathways and the accumulation of 3-DAs. Our results indicate a dual mode of 3-DAs regulation: JA is involved in disease triggered 3-DAs accumulation, and SA is involved in wounding triggered 3-DAs accumulation. 3. Cell wall component profiling for biomass improvement in sorghum using Fourier-Transform Near-infrared Spectroscopy. Cell wall composition plays a major role in the plant immune system, both as a source of primary, and secondary defenses. It is also the basis for lignocellulose biomass. This objective of my thesis was to explore biomass diversity cell wall composition in sorghum. My goal is to correlate this information with anthracnose resistance in sorghum. Currently, we have a collection of over 800 sorghum lines, representing a wide variety of grain, forage, bioenergy, and sweet sorghum in diversity. To fully exploit these lines and to understand the genetic variation contributing to sorghum lignocellulosic biomass accumulation, we developed and tested a high throughput method. In collaboration with Dr. Seong Kim’s Laboratory, Fourier-transform Near Infrared (FT-NIR) Spectroscopy was used to characterize the cell wall composition in sorghum. Cell wall content assayed by FT-NIR showed high fidelity compared to that produced by wet chemistry laboratory while reducing both analysis time and sample size needed by order of magnitudes. Our results show FT-NIR is a useful method to fully exploit the large number of germplasm lines available in our collection. In conclusion, by understanding the regulatory role of y1 transcription factor in phytoalexins biosynthesis, and the role of phytohormones synthesized during 3-DAs accumulation, we will gain a clearer picture of the active defense responses in sorghum. By quantifying cell wall component in a vast library of available germplasm, we can gain a better understanding of passive defense mechanisms. Breeding for increased defense against plant pathogens should be a holistic approach that combines both passive and active defense mechanisms.