Kristy Nguyen, Diane M. BecklesDepartment of Plant Sciences University of California, One Shields Avenue, Davis CA 95616

Tomato (Solanum lycopersicum L.) is one of the most popular vegetables globally with healthful attributes, but like many horticultural crops, it requires high-nitrogen fertilizer to maintain yields. In order to combat the environmental degradation contributed by fertilizer, this study aims to investigate the efficiency ofa transgenic tomato line for growth on low nitrogen (N). The tomato line chosen for this study, 4080, ectopically expresses the gene TF1 cloned fromArabidopsis (AtTF1). Previous studies showed that the TF1 transcription factor enhances N assimilation. This transgenic line showed increased fruit yield of30% compared to the control, and preliminary results indicate increased tissue N and an elaborate root system. In this study, biomass measurements and theexpression of Fd-GOGAT, a key N-assimilation gene and target of TF1, will be evaluated at varying N concentrations (5%,100%) in 4080. In this preliminaryexperiment, the anticipated outcome is that when N is reduced, biomass will be maintained in 4080 because of its increased efficiency of N-uptake,assimilation, and metabolism and that this will be brought on by higher expression of the Fd-GOGAT gene which should be targeted by both the native TF1(SlTF1) and transgenic AtTF1. In contrast, the control tomato line which only contains SlTF1 will do poorly. The findings may be useful in determining if futureresearch should be invested in generating high N-use efficiency, non-transgenic to efficiently absorb N by modification of this gene.

RESULTSEXPERIMENTAL PLAN/METHODS

Agriculture will be negatively impacted byclimate change, requiring proactive andinnovative ideas for adaptation. Globally, 100million tons of fertilizer is used to promote cropproductivity at a cost of $100 billion per year.About two-thirds of this N-fertilizer is wastedand contributes to environmental degradation.1

Agronomic measures are needed to reduce N-fertilizer use while maintaining crop production.Breeding tomatoes that can grow efficiently atlow N would be a good strategy to enablesustainable agriculture in expectation of globalwarming.

My proposed project is to test the efficiency ofa transgenic tomato line for growth on low N.The transgenic line, 4080, ectopically expressesa transcription factor TF1 cloned fromArabidopsis. This transcription factor causesphysiological and metabolic effects on growthby binding to N and carbon assimilation genes.2

Ferredoxin-dependent glutamate synthase (FD-GOGAT) is a key enzyme in N assimilation andis a known target of TF1.3

HYPOTHESIS

ACKNOWLEDGEMENTSResearch supported by Provost Undergraduate Fellowship # F12-42. Maysaya Thitisaksakul, Mandy Wang, John Curato, and WanlingAng are recognized.

ROLE OF TRANSCRIPTION FACTORS

Figure 1: Transcription factors bind to the promoterregion of multiple genes and thereby regulate theirexpression. TF1, the gene studied, is involved in Nand carbon assimilation and binds several genes inthose pathways. In this example, TF1 is shownbinding to the Fd-GOGAT gene, one of its knowntargets.

CONCLUSIONS

At low N levels, I hypothesize that the 4080 linewill maintain growth better than the controltomato line. Both the 4080 and control line willhave enhanced SlTF1 expression, which willtarget the Fd-GOGAT gene, stimulating itsexpression and leading to better N-assimilation.However, since 4080 has additional TF1 (AtTF1)introduced by genetic engineering, this will leadto even higher expression of the Fd-GOGATgene in 4080 compared to the control and thusthe transgenic 4080 line will grow better at lowN when compared to the control. This will bereflected in higher biomass, plant height, freshand dry weight in 4080.

The Effect of Varying Nitrogen on TF1 Gene Expression and the Productivity of Transgenic Tomato Lines

• Our preliminary data showed no difference inheight or fresh weight between lines ortreatments, however dry weights have not yetbeen calculated and this could indicatedifferences.

• Early flowering in 4080 is a sign of accelerateddevelopment due to AtTF1 expression.

• All plants showed signs of stress. The build upof N (even 5%) may have exceeded the upperthreshold for plants, due to small pots used.

• The experiment should be repeated using largerpots, half strength N, and more N treatments.

Figure 5: At 7 weeks, the only visual difference isflowering in the 4080. The control did not indicatethis advanced development (data not shown).

Figure 2: The study group involved five replicates of each line (control and 4080) and each treatment (5% and 100%) of N.

Physiological Measurements: A total of 40plants (five replicates of each line/ each Ntreatment of 5% and 100%) were planted in 4”pots with fritted clay soil. Plants were fertilizedwith equal volume of 5% N or 100% N in solutionevery four days. The growth of tissue aboveground was directly observed and measuredperiodically. Fresh weight and dry weight will alsobe measured. All plants were grown in arandomized block design. Data is statisticallysignificant if P<0.05.

Control 4080

Figure 4: Plants were measured every week. Growth was identical between treatment and genotype n=5 (P>0.05).

1. Chardon, F. et al. (2010) J. of Experimental Botany. (9): 2293.2. Yanagisawa, S. et al. (2004). Proc Natl Acad Sci USA. 101: 78333. Kumar R et al. (2009) Mol Biol Rep. 36 (8): 2209.

5% N-Control

5% N-4080

RESULTS

I. Fresh weight of above (shoot) and below(root) ground tissue in 4080 and controlplants at normal and low N

III. Tomato plant growth at 6 weeks

5 replicates 5 replicates5 replicates 5 replicates

II. Average plant height in 4080 and control plants at normal and low N

Figure 3: At 7 weeks, there was no significantdifference in fresh weight between treatments orgenotypes (P<0.05).

The following experiments will be conducted on afresh set of plants:

Biochemical Measurements: N content will beused to assess the nitrogen use efficiency (NUE)of the plants, defined as the amount of N pergram of fresh weight.

Gene Expression: RT- PCR will be used to testthe relative levels of Fd-GOGAT gene expressionin control and 4080. Optimization of PCR cyclesand amount of RNA is necessary for RT-PCR toprevent saturation. Using regression analysis, themean relative expression levels of Fd-GOGAT atvarying concentrations of N.

ONGOING/FUTURE WORK

0

5

10

15

20

25

30

.05

N-A

.05

N-B

.05

N-A

.05

N-B

.05

N-A

.05

N-B

.05

N-A

.05

N-B

.05

N-A

.05

N-B

.05

N-A

.05

N-B

.05

N-A

.05

N-B

.05

N-A

.05

N-B

.05

N-A

.05

N-B

.05

N-A

.05

N-B

Day 4 Day 10 Day 15 Day 19 Day 23 Day 27 Day 30 Day 34 Day 38 Day 42

He

igh

t (c

m)

Day

Average Plant Height

Control

4080

0

5

10

15

20

25

30

35

40

Above Below Above Below

Control 4080

Fre

sh W

eig

ht

(gra

m)

Line

Fresh Weight of Plant Tissue

5%

100%

INTRODUCTION

REFERENCES