Dormancy Release and Apical Dominance in Postharvest Potato: the Role of Auxin and Programmed Cell Death in Sprouting Induction

Non-Technical Summary:<br/>
Potato (Solanum tuberosum L.) is an important crop in the United States and approximately 38,000 tons were produced in Massachusetts in 2006, with a value of $7.5M (National Agricultural Statistics Service, USDA). About 92% of the Massachusetts crop is consumed fresh or marketed as processed products, while 8% is re-used as seed tubers. Tuber sprouting during storage, caused by dormancy release of tuber buds, leads to undesirable loss of weight, turgidity and texture alterations. Undesirable sprouting during storage is a serious problem for the fresh market, prior to industrial processing, and in storage of seed-tubers (Coleman, 2000). Ultimately, breakthroughs in table potato and seed-tuber marketing will depend on controlling sprouting with breeding, transgenic strategies, or environmentally friendly agents that pose low health risks. Therefore it is necessary to further define the molecular mechanisms that control dormancy release and apical dominance of the potato tuber. This research has the potential to improve food quality, safety and security, which are relevant to USDA priorities.
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Approach:<br/>
The Eshel group will grow the minitubers, conduct the CK, GA and BE treatments, carry out tissue sampling and labeling and extraction of total RNA and total soluble proteins. After tissue and cDNA lyophilization, samples will be sent to the Normanly and Campbell labs for IAA analysis and deep sequencing, respectively. Data will be analyzed together and then validated by qRT-PCR in Eshel lab. The Eshel lab will do the IAA transport inhibition and VPE inhibition treatments and send samples to the Normanly lab for further IAA analysis. We propose to examine IAA synthesis rates and pathway utilization in tuber meristems undergoing dormancy release as a result of CK, GA or BE treatments. The Eshel lab will carry out labeling using methods that account for the effect of wounding on Trp-dependent IAA synthesis (Sztein et al., 2002). All experiments will be conducted on Nicola cv. minitubers and key findings will be verified on commercial tubers of Nicola and Desiree cvs. Nicola has a short dormancy period with strong apical dominance when it sprouts while Desiree manifests long dormancy and weaker apical dominance. Similar size and age minitubers, grown in controlled environment conditions, will be used immediately after harvest to minimize the variability of physiological age. In some of the experiments we will used detached apical buds, as described before (Hartmann et al., 2011; Rentzsch et al., 2011). The apical and lateral bud (position 5 buds from the apical bud, down the tuber) will be sampled from each tuber to eliminate potential variability between bud positions. The sampled meristems will be frozen immediately for further analysis of transcriptome, enzyme activity and IAA levels (Sorce et al., 2009). Meristems will be sampled from 120 tubers, corresponding to each time point after CK, GA or BE treatments; 40 tubers will be pooled for each replicate (3 replicates of 40 tubers). Tissue will be flash frozen immediately in liquid nitrogen, powdered and shipped on dry ice to the Normanly lab. We have already tested the feasibility of shipping lyophilized samples from Israel to the US and are confident that sample integrity is not affected. In the Normanly lab, the incorporation of label from 15N-ANA and 13C1115N2-Trp into IAA will be followed to determine a) rates of IAA synthesis after various treatments and b) any changes in the utilization of Trp-dependent or Trp-independent IAA synthesis pathways. The Normanly lab has many years experience in the extraction and analysis of IAA precursors and metabolites (Normanly et al., 1993; Ilic et al., 1996; Normanly et al., 1997; Tam and Normanly, 1998; Tam et al., 2000; Ljung et al., 2005a; Barkawi et al., 2008). In preliminary work the Normanly lab measured free-IAA in sections of apical and lateral meristems of potato tubers according to a novel high throughput method that was developed in Normanly lab (Barkawi et al., 2008).
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Progress:<br/>
2011/10 TO 2012/09<br/>
OUTPUTS: This project is a collaboration with a colleague from the Department of Postharvest Science at the Volcani Institute in Israel. We applied for and received a travel grant from the United States-Israel Binational Science Foundation for a graduate student on the project to travel from Israel to UMass Amherst in April of 2012 to carry out analysis of potato tuber samples that had been subjected to various treatments that impact dormancy release. No results have been disseminated at this point.
<br/>PARTICIPANTS: Bukola Fatunmbi was hired as a Research Fellow at UMass Amherst to carry out method refinements. Mr. Fatunmbi has a B.S. in chemistry and has received training in plant metabolite profiling. Yossi Buskila is a graduate student from the Department of Postharvest Science, Volcani Institute, Israel, who visited the Normanly lab at UMass Amherst to receive training in robotic extraction and analysis of indole-3-acetic acid from potato tuber tissues.
<br/>TARGET AUDIENCES: Nothing significant to report during this reporting period.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: We have spent this reporting period refining our analytical methods in order to meet project objectives. We are adapting a robotic method for extraction of small molecule metabolites from small amounts of plant tissue, specifically the growth regulator indole-3-acetic acid (IAA) for potato tuber tissue. Our initial findings were that the variation between replicate samples was higher than 10 percent and we are devising sampling and extraction methods that will bring the variation between replicates to less than 10 percent.