Functional genomics of plant compensatory responses

Siddappaji, Madhura

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https://hdl.handle.net/2142/45576 Copy

Description

Title

Functional genomics of plant compensatory responses

Author(s)

Siddappaji, Madhura

Issue Date

2013-08-22T16:48:21Z

Director of Research (if dissertation) or Advisor (if thesis)

Paige, Ken N.

Doctoral Committee Chair(s)

Paige, Ken N.

Committee Member(s)

• Bohn, Martin O.
• Clough, Steven J.
• Suarez, Andrew V.

Department of Study

School of Integrative Biology

Discipline

Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Plant compensatory responses
• Arabidopsis
• Quantitative Trait Locus (QTL) mapping
• Columbia
• Landsberg erecta
• Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
• invertase
• glucose-6-phosphate-1-dehydrogenase
• Glucose-6-phosphate dehydrogenase (G6PDH1)
• fitness
• Ipomopsis aggregata
• mammalian herbivory
• ungulate herbivory
• endoreduplication
• transcriptional reprogramming
• oxidative pentose phosphate pathway (OPPP)
• invertase isoenzymes

Abstract

Numerous studies have shown that some plant genotypes can compensate for tissues lost with little or no decrement in fitness relative to those that are undamaged (see Stowe et al. 2000 for a review); such plants are termed as tolerant. This trait motivated many empirical studies demonstrating that herbivore damage can, under certain circumstances, increase, rather than decrease, plant reproductive success (a specialized case termed overcompensation, i.e., increased flower, fruit, and seed production following herbivory). Specifically, studies by Paige and Whitham (1987) showed that when mule deer and elk removed 95% or more of the aboveground biomass of the monocarpic biennial scarlet gilia, Ipomopsis aggregata, the lifetime seed production, seed germination, and seedling survival averaged 3.0 times that of uneaten controls (Paige 1992, 1994, 1999). Despite evidence that genetic variation for tolerance exists, the underlying genetic basis of compensation is not known. My research focuses on understanding the molecular basis of plant compensatory responses using suite of quantitative and molecular techniques. As a first step, QTL mapping was combined with results from a microarray analysis to identify potential candidate genes viz., G6PDH1 and invertase. These genes were then characterized using recombinant DNA techniques. A set of recombinant inbred lines developed from a cross between Columbia X Landsberg erecta, of the annual plant Arabidopsis thaliana were screened initially to identify QTLs. A total of three QTL located on chromosomes 1, 4 and 5 explaining 48.2% of the variation in fitness compensation were found. The microarray experiment revealed 109 genes that were differentially expressed between clipped and unclipped plants of the overcompensating ecotype Columbia. Combining the results from the QTL and microarray data, two genes were uncovered that appeared to play a significant role in the phenomenon of overcompensation, a cytosolic glucose-6-phosphate-1-dehydrogenase (G6PDH1) and an invertase. G6PDH1 T-DNA knockout studies of the overcompensatory accession Columbia-4 accession showed patterns of equal/under-compensation verifying its role in the compensatory response. G6PDH1 is a key enzyme in the oxidative pentose-phosphate pathway that plays a central role in plant metabolism, converting glucose to ribose-5-phosphate. The role of G6PDH1 in plant compensation was further verified by complementing G6PDH1 to reinstate its function in a G6PDH1 knockout and to localize where it is expressed by creating chimeric promoter-reporter (GUS) fusion constructs. Results from one of four complementation lines showed a partial rescue effect of G6PDH1, showing patterns more similar to the overcompensating Columbia line than either Landsberg erecta or the knockout line. Furthermore, results of our promoter-reporter fusion studies (G6PDH1 promoter: β-glucuronidase (GUS)) and subsequent histochemical staining revealed that G6PDH1 is expressed in virtually all tissues rather than localized to any specific tissue. These results are consistent with patterns of regrowth observed following clipping in Arabidopsis, reconstituting the entire plant with greater biomass and higher fitness. The importance of invertase isoenzymes in the compensatory response of the two Arabidopsis thaliana ecotypes Columbia and Landsberg erecta (overcompensating and undercompensating genotypes, respectively) were also determined. Invertases represent one class of enzymes that shunt glucose to activate the oxidative pentose phosphate pathway, therefore expression of invertase isoenzymes over developmental time and fitness analysis of T-DNA knockout mutants was examined. Results showed differences in plasticity in the expression of invertases following the removal of apical dominance. In Columbia, an overcompensating genotype, nine of twelve invertase isoenzymes were significantly up-regulated one to five days after the removal of apical dominance. In, Landsberg erecta, an undercompensating genotype, only two neutral invertases showed a decline in expression at 15 days post-clipping. These results were consistent with patterns observed for G6PDH1, showing up-regulation at five days post-clipping in Columbia. This is possibly due, in part, to an increase in glucose fed from invertase isoenzymes into the OPP pathway, facilitating the rapid regrowth and greater biomass accumulation observed in the overcompensating genotype Columbia. Furthermore, there was a general trend toward higher expression at 50% flowering for both clipped and unclipped plants (with no significant differences in expression between treatments or between genotypes) in six of twelve Columbia isoenzymes and three of twelve for Landsberg erecta. These results suggested Columbia and to a lesser degree Landsberg erecta may up-regulate gene expression over earlier time periods in order to facilitate flower and fruit development. These results were also consistent with the patterns observed for G6PDH1, showing greater up-regulation at 50% flowering post-clipping in Columbia (i.e., twice the number of invertases up-regulating to supply the added glucose for increased flower and fruit production in the overcompensating genotype, Columbia versus the undercompensating genotype, Landsberg erecta). The T-DNA knockout experiments on the two invertase genes, a vacuolar invertase and a neutral invertase, and their isoforms confirmed their importance in plant growth and fitness in Arabidopsis thaliana following the removal of apical dominance. Of particular note, there did not appear to be any functional redundancy of other invertases or any of the sucrose synthases. Thus, all invertase isoenzymes seem to be necessary for normal growth, development and reproduction and, most importantly here, for growth and fitness compensation following apical damage. Based on these results, it is proposed that plants with the capability of overcompensating reprogram their transcriptional activity in three important ways: a) through defensive mechanisms, b) through increased expression of genes involved in energy metabolism and c) through an increase in DNA content (via endoreduplication) that feed metabolites to pathways involved in defense through transcriptional programming (Scholes and Paige, 2011). Collectively, these results, indicate the direct or indirect importance of G6PDH1and invertase isoenzymes in regulating the compensatory response following the removal of apical dominance.

Graduation Semester

2013-08

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Copyright 2013 Madhura Siddappaji

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