nitrogen applications & soybean yields

Illinois Soybean Association Summary Report: Can applying nitrogen increase yields and improve compositional quality?

Background: As Illinois growers continue working toward higher soybean yields, many are questioning the role fertility may play in breaking current yield barriers. To help growers make informed fertility decisions, ISA recently hosted a summit focused on the use of nitrogen in soybean production. Participants in the day-long discussion included key soybean researchers from universities and industry and industry partners, as well as ISA directors and local agronomists. The heart of the question, “Can Illinois farmers expect to see a yield response to supplemental nitrogen?” still remains unanswered. Complicating the answer is an apparent disconnect between the nitrogen (N) needed to support higher soybean yields and the inconsistent response to additional nitrogen in field trials. Researchers cited several examples showing that the amount of nitrogen removed by a 60 bu/a yield clearly exceeds the amount that can be fixed by the nodules on the soybean plant and that soybeans do not leave residual nitrogen for the next corn crop. Yet, field trials results are often inconclusive regarding whether to apply N on soybeans. As a result of this roundtable discussion, attendees offered perspectives on best management practices for growers, as well as suggestions for areas where additional research is needed.

Key Takeaways for Growers:

Always start by looking at the overall system for managing soybeans and be sure that no factors other than N are limiting.

Supplemental nitrogen typically is not needed for yields in the 60- to 70-bushel range.

High-yielding beans (greater than 70 bushels) may need supplemental nitrogen because the nitrogen needed by the plant will exceed the amount available from fixation.

Early N applications may inhibit nodulation, so current research is looking at 60 to 70 pounds of urea or UAN (urea ammonium nitrate) per acre with a urease inhibitor at the R3 stage when N demand begins to increase while N fixation begins to wane.

More research and local, on-farm trials are needed before best management practices or recommendations on applying N are finalized.

Due to environmental concerns regarding the use of nitrogen on corn, care should be used before making added nitrogen a standard practice on soybeans.

Key highlights by presentation: Howard Brown, Growmark: Status of soybean fertility

Fertilizing soybeans is not common

Deficiencies are rare and farmers should use a soil test for fertilizer application rates

Soybeans fix up to 50% of their own N with the rest coming from the soil

Iron and manganese deficiencies occur in areas with high pH

Potassium deficiencies occur in some areas

Fertilizer is usually not considered a limiting factor in soybean production

A limiting factor(s) can have the greatest impact on yield, even with the best conditions for all other growing factors

On-farm trials are important as we move forward with addressing the N questions Harold Reetz, Reetz Agronomics: Soybean’s Nitrogen Requirements

100-bushel soybean crop requires 548 lbs. of N (5 to 5.5 lbs. per bushel)

58% of the N requirements on average come from N fixation

Soybeans can fix enough N for a 60-bushel soybean crop

Any additional N comes from the soil

N uptake takes off by mid July as N fixation begins to slow

Too much soil N (nitrate or NO3) will depress N fixation Larry Purcell, University of Arkansas: Understanding soybean growth and nitrogen fixation

and what it tells us about N uptake and partitioning

Soybeans generally take up 3.6 to 5.4 lbs. N per acre per day

If a foliar fertilizer is 20% N and the application rate is 2 quarts per acre, this provides only ~0.8 lbs. N

Due to the low amount of N delivered, foliar N fertilization cannot contribute meaningful amounts of N to the crop

N fixation is more sensitive to drought than photosynthesis, so you will see a greater response to N fertilizer during a drought due to greater crop growth rates

N fixation (at least in Arkansas conditions) continues through R6 and R7 if the plants are not stressed and well-watered

Individual nodules last 4 to 5 weeks, total nodulation lasts 8 to 12 weeks

N fixation begins about 3 weeks after emergence

Large amounts of N are required for soybean seed production, but under low soil N conditions, about 90% of the N in seed was derived from N2 fixation

N2 fixation is capable of providing essentially all the N required for today’s high seed yields, and fixation continued at high rates almost until maturity

Seed N accumulation rates are considerably greater than rates of N2 fixation

N is remobilized from leaves and stems to meet seed demand

N does not appear to be a limitation to yield under most conditions today

However, under drought conditions, high rates of N fertilization can increase yield Fred Below, University of Illinois: Soybean Response to Fertilizer N

62-bushel crop requires 272 lbs. of N and 80% of it goes with the seed

N fixation peaks at R5 and then declines (in Illinois observations)

70 bushels is the upper yield limit that N fixation can support

Soybeans fix N into uriedes, and soybeans can take up uriedes from the soil; uriedes do not depress nodule formation or N fixation

Deep placement of N below the nodule zone has some promise as a fertility approach

Only 50 to 60% of soybean N is obtained from N2 fixation, and the proportion decreases with increasing yield levels

Low N supply requires remobilization from leaf N, which decreases photosynthesis and yield potential

N is obtained from soluble N (nitrate) in the soil, soil organic matter mineralization, N2 fixation and supplemental fertilizer N sources

An antagonism exists between NO3- and N2 fixation in nodules with fertilizer application

Observations show that urea-N is perceived differently by the plant than other forms of N and ammonia

Seth Naeve, University of Minnesota: Nitrogen on Soybean: Protein and Oil

Compositional quality response to N is more unpredictable than yield

There is a greater opportunity for N response during grain fill (R5)

Seed protein response to N was more common in in vitro (60% increase) and hydroponic experiments (20% increase) and less common in field experiments (1% or less increase)

Natural variation in oil and protein concentrations in the seed can mask the impact of N

Newer varieties appear to respond better to N than older varieties Lila Vodkins, University of Minnesota: Soybean Genetics

Apply N and look at gene response to see what the plant is doing in the roots, leaves and seed

Gene expression profiles show when protein is deposited in the seed

Expression analysis can look at every gene in the amino acid and protein pathways

Downward trends in seed protein are gene regulatory activities

Identified soybean line with increased protein levels of about 50%

Areas needing additional research Overall:

Do we need to optimize N fixation before considering N supplementation?

Who is researching N fixation today? What are the best options for field measurement?

Can farmers inoculate with more efficient rhizobia than what we’re using today?

Do we need more site-specific rhizobia?

What is the level of nitrate in the soil that can depress N fixation?

Is N fixation being suppressed in the soil environment in other ways?

Should we approach the N concerns as a “system” solution on how to increase yield and N is one component?

Lack of Response to N:

What other yield-limiting factors exist?

How does this work in areas that are not high-yield environments?

Are N fixation and/or soil reserves currently meeting the crop’s N needs?

How does N mineralization from organic matter affect outcomes? Role of environment on response to N:

How does drought affect response to N?

Are yield responses different in marginal soils?

How do high-yield environments respond to N?

What considerations are needed for certain lower-yield environments? Poor nodule establishment Extremely low soil N at planting Plant water stress Soil pH problems Low soil temperature Absence of native Bradyrhizobium

Nitrogen fertilizer: Source, form and timing:

Do organic forms of N provide a more steady supply of N that soybeans respond to during pod fill than inorganic sources applied supplementally?

What is it about organic forms of N that make it more effective?

How much N is being mineralized in soybean fields in season?

When are preplant or at-plant N applications on soybeans effective and under what conditions?

Is applying N at planting on double crop soybeans beneficial?

What are the other yield-limiting factors?

What are the high-yield scenarios where N is limiting?

What is the right high-yield system where N is required?

What is the relationship between C and N in the soil and nutrient release?

How can yield responses to N applications be more consistent?

Keep in mind that N on corn and water quality is a major concern. Research activities that ISA could consider: Research first needs to focus on understanding more about applying N on beans, then on developing recommendations on when to apply and how much. All N recommendations for soybeans need to be based on science.

Develop a field test for measuring N fixation in the field.

Determine why N responses are inconsistent in the field.

Look at soil health relationship to nodulation and N fixation. A good soil health environment is conducive to better nodulation and N fixation.

Assess nitrogen budgets including soluble N forms in the soil and mineralization N under the soybean crop during the season to see if soil N is plentiful in different environments.

Looking at different rates, forms and timing (preplant or at plant) of N and how it affects stand establishment, vigor, N fixation and yield.

Investigate whether supplement N is more beneficial to double-cropped soybeans.

What’s the “scoop” on manure and impact on nodulation and fixation?

Harold ReetzReetz AgronomicsMonticello, Illinois

February 14, 2014

Nitrogen Distribution in Soybeans (Above Ground)

Iowa State University Extension

Generalized N Budget for Soybeans

Summary of Soybean N ResearchReviewed by University of Nebraska Staff

• 637 data sets (site‐year‐treatment)‐‐‐1986‐2006• 0.013 Mg soybean seed yield per kg increase in N accumulation• 50‐60% of N demand met by biological N2 fixation• N fixed in most cases not sufficient to replace N exported in harvested seed• Partial N balance (fixed N in above‐ground biomass‐N in seeds) was negative in 80% of the data sets; mean N mining of ‐40kg ha‐1 (about 36 lb/A)

• Below ground N contribution of 24% brought N balance to ‐4kg ha‐1 (3.6 lb/A)• N fixation tended to be reduced when N fertilizer was applied• Deep‐placed (below nodulation zone) and late season (during grain fill) application may increase yield response to N fertilization in high‐yield environments.

High Yield Soybean Nutrient Uptake (101 bu/A)

Source: Spectrum Analytic Lab

Table 20 shows nutrient uptake requirements for soybeans under maximum economic yield conditions at various stages of growth, based on research by - Dr. Ray Flannery, who raised 101 bu/A soybeans in 1986. The research was performed on a Freehold sandy loam soil, testing very high in P and K, with a pH of 6.0 to 6.5. Total nutrient applications were 150 lbs/A N, 200 lbs/A P2O5, and 300 lbs/A K2O applied preplant, and two fertigations. Figure 13 shows nutrient storage in plant parts at the green stage of development.

Total Nutrient Accumulation for 101 bu/A Soybeans

Nutrient Distribution in 101 bu/A Soybeans

Nitrogen Distribution in Soybeans (Above Ground)

N Requirement Relative to Yield Level

Yield Above Ground N Uptake@ 4.73 lb N/bu

Seed N Removed@ 3.3 lb N/bu

40 bu/A 189 lb/A 132 lb/A

88 bu/A 415 lb/A 290 lb/A

N in seed was 6.34%

• 3 primary sources of N in Soybeans• N2 fixation Bradyrhizobium• Nitrate and ammonium in the soil• Fertilizer N

• 50‐60% of N comes from N2 fixation; remainder from the soil • Maximum N2 fixation is about 300 lb N/A.• When fertilizer N is applied, N2 fixation can be reduced

• @ 45 lb N/A, maximum fixation is about 190 lb N/A• @ 90 lb N/A, maximum fixation is about 125 lb N/A

Summary of Soybean N ResearchReviewed by University of Nebraska (Salvagiotti, et al)

• About half of 67 studies reported positive response to N fertilizer.• Average yield response was 8 bu/A• 10 bu/A response when < 45 lb N/A was applied at growth stage R3 (beginning pod)• 9 of 12 studies (75%) with yields >67 bu/A had positive response to N

• In high yield environments, fixed N and soil N may not meet the N demands of soybean plants.

• High yield environments have a higher probability of response to N fertilizer.


• High yielding environments• Certain Lower yield environments

• Poor nodule establishment• Extremely low soil N at planting• Plant water stress• Soil pH problems• Low soil temperature• Absence of native Bradyrhizobium

Conditions favoring soybean response to N fertilizer


Do high yielding soybeans need N fertilizer?• Maybe• Yield response may only be marginally profitable• Depends on risk from N price vs soybean price• Local trials are an important guide

Dr. T. Scott Murrell. 2012. Do High Yielding Soybeans Need to Be Fertilized with Nitrogen? Plant Nutrition Today, 2012 #4 IPNI PNT Spring2012.pdf. International Plant Nutrition Institute.www.ipni.net .

Dr. Harold F. Reetz, Jr.Reetz Agronomics107 S. State Street

Monticello, Illinois 61856

Phone: 217‐762‐2074

e‐mail: [email protected]

SOYBEAN NUTRIENT REQUIREMENTS

• Fertilizing soybeans not common

• Deficiencies rare and use soil test for fert. appl.

• Fix up to 50% of own N

• Iron/Manganese deficiencies with high pH

• Potassium deficiencies in some areas

• “Usually not a limiting factor”

Essential Nutrients

• Maximum demand during seed fill

• Demand for N high at seed formation (protein)

• Late season remobilization from older tissues

NITROGEN and GROWTH

• High N levels in soil limit nodulation

• Incr. dependence on soil N

• Use inoculum where soybeans not freq. crop

• N at planting only decr. Nodulation/dependence

• No consistent yield results in research

• Beneficial specific years/specific locations

• N during season may help in low organic soils

FOLIAR FERTILIZATION• Hope for delayed senescence/incr. yield

• Partially fix deficiencies during growing season

• Not during hot part of day

• On‐farm results show inconsistent results

• Low probability of increasing yields

• No diff. between products, rates, or freq.

• May be beneficial for sandy soils or high‐yielding fields that are irrigated.

IL AGRON. HANDBOOK

• Split 90 lbs N• Foliar fung. (2)• Micronutrients• Two locations• Response limited

SOURCES:

Soybean Nutrient RequirementsPalle Pedersen, Iowa State University8/7/2007http://extension.agron.iastate.edu/soybean/production_soilfert.html

Illinois Agronomy Handbook, 24th EditionDr. Emerson Nafziger, Univ. of IllinoisChapter 3: Soybean, page 36.http://extension.cropsci.illinois.edu/handbook/pdfs/chapter03.pdf

Understanding soybean growth and nitrogen fixation and what it tells us about N uptake and partitioning

Larry Purcell

N-on-Beans Roundtable Bloomington, IL 14 February 2014

I. Review importance of N and N2 fixation to soybean production

II. Evaluation of N2 fixation, seed N, vegetative N under well-watered conditions

III. Yield response to N fertilizer under drought conditions

• N2 fixation reaches maximum during early- to mid-podfill and then rapidly declines (Harper and Hageman, 1974; Zapata et al., 1987).

• N2 fixation estimated to provide 80-110 kg N ha-1 (71 to 98 lbs N ac-1, Hardy et al., 1968).

• N2 fixation under well-watered conditions continued to occur until senescence (Denison and Sinclair, 1985; Nelson et al., 1984).

Previous research has:• relied heavily upon acetylene reduction assays,

• been conducted in soils with relatively high organic matter and high concentrations of mineral N,

• and/or been conducted in controlled environments.

Sinclair and deWit (1975) Science 189:565

Implications of Self Destruct Hypothesis

• Increasing the amount of vegetative N at the beginning of seed fill will provide a greater pool of N that can be remobilized to seed.

• Later-maturing isolines would expectantly have larger amounts of N to remobilize and increased yields.

• Field experiment at Fayetteville, AR. Fully irrigated conditions.

• Rye sown fall before experiment; removed in spring at heading.

• 5 genotypes: Near isolines for MG IV, V, and VI, Lee-nonnod, and R01-416F

• RCB, 4 reps

• Plots: seven 19-cm rows x 9.1 m

• When MG IV isoline was at beginning R5, 1 m2

harvested from each plot. Subsample separated into leaves, stems, pods, seeds.

• Total N determined on all plant fractions.

• Repeated ~ every 10 days.

Genotype MG Pedigree

Lee Nonnod (D68-0099) VI Lee(6) x T201

D49-2491 VI S100 x CNS (same F2 as Lee)

D66-5566 IV D49-2491(4) x Hawkeye

D61-1513 V D49-2491(5) x Hawkeye

R01-416F V Jackson x KS4895

Mastrodominico and Purcell (2012) Crop Sci. 52:1281


2.7 lbs N ac-1

y = 0.4x ‐ 12Adj. R² = 0.61

0

5

10

15

20

25

30

35

55 65 75 85 95 105 115

N2fix

ation (g

N m

‐2)

DAE

Isoline (MG IV)Isoline (MG V)Isoline (MG VI)R01 416F (MG V)


3.6 lb N m-2 d-1

Side Comments about Foliar Nitrogen

N accumulation rates of 3.6 to 5.4 lbs N ac-1 day-1 are typical

18 lbs N ac-1 d-1 at Kip Cullers

If a foliar fertilizer is 20% N, and you put out 2 quarts ac-1, this provides only ~0.8 lbs N

Foliar N fertilization cannot contribute meaningful amounts of N to the crop

‐ 36 (MG IV)y = 0.59x ‐ 44 (MG V)

‐ 48 (MG VI)

Adj. R² = 0.840

5

10

15

20

25

30

35

60 80 100 120 140

N con

tent (g

N m

‐2)

DAE

Isoline (MG IV)Isoline (MG V)Isoline (MG VI)R01‐416F (MG V)

Seed


5.3 lb N ac-1 d-1

+ 22 (MG IV)y = ‐0.17x + 24 (MG V)

+ 26 (MG VI)Adj. R² = 0.67

0

2

4

6

8

10

12

14

16

60 80 100 120

N co

nten

t (g m

‐2)

DAE


Leaves


1.5 lb N ac-1 d-1

+ 15 (MG IV)y = ‐0.14x + 18 (MG V)

+ 20 (MG VI)Adj. R² = 0.68

0

2

4

6

8

10

12

14

16

60 80 100 120 140

N co

nten

t (g m

‐2)

DAE


Stems


1.2 lb N ac-1 d-1

10

12

14

16

18

20

22

24

MG IV MG V MG VI R01‐416F

N co

nten

t at b

eginning

seed fill (g N m

‐2)

20082009


98 to 196 lb N ac-1

80

85

90

95

100

105

110

115

120

MG IV MG V MG VI R01‐416F

Days a

fter emergence

End of seedfillEnd of N2 fixation


Genotype

Yield NHI Stem N Seed N SenescedLeaf N

bu ac-1 ---- lb N ac-1 ---- %

MG IV Iso 52 b 0.89 a 22 c 190 b 1.5 c

MG V Iso 56 b 0.85 b 33 b 194 b 1.7 b

MG V Iso 50 b 0.78 c 52 a 180 b 2.1 a

R01-416F 69 a 0.86 b 37 b 226 a 1.9 a

Lee NonNod

12 0.59 15 17 ---


00

Increasing resource availability

Cro

p re

spon

se

y = β0 + α(1 - e-β1 * x)

Nutrients

• Large amounts of N are required for soybean seed production

• N2 fixation is capable of providing essentially all the N required for high seed yields

• Seed N accumulation rates are considerably greater than rates of N2 fixation

• N2 fixation continues until the end of seedfill

• No indication of N shortage under these conditions

Sinclair et al. (1987) Agron J 79:986-991

Seed

Yie

ld (k

g ha

-1)

2000

2250

2500

2750

30000 N ha-1

336 kg N ha-1

b

aa

ab

Drought Well-watered

Purcell and King (1996) J Plant Nutr 19:969-993

29.8

44.7

+18%

Egli and Zhen-wen. 1991. Crop Sci. 31:439

0

5

10

15

20

25

30

BM R2 N content Yield Seed No.

Chan

ge with

N fertilizatio

n (%

)Irrigated

Nonirrigated

Ray et al. (2006) Crop Sci. 46:52

Final Thoughts

Under low soil N conditions, ~90% of the N in seed was derived from N2 fixation.

N2 fixation continued at high rates almost until maturity.

Current N2 fixation was inadequate for the seed N accumulation rate.

N was remobilized from both leaves and stems.

N does not appear to be a limitation to yield under these conditions.

Under drought conditions high rates of N fertilization increase yield.

Soybean Response to Fertilizer N

ISA N on Beans Workshop, Bloomington, IL February 14, 2014

Fred Below, Jason Haegele, and Ross Bender

Crop Physiology LaboratoryDepartment of Crop Sciences

University of Illinois at Urbana-Champaign

Soybean Gets Some N from fixation by Nodules

Planting V3 V7 R2 R4 R5 R6 R8

Growth Stage

Days After Planting0 20 40 60 80 100 120

N Up

take

(lb

N ac

-1)

0

40

80

120

160

200

240

280

Perc

ent o

f Tot

al (%

)

0

20

40

60

80

100GrainFlowers, PodsStem, PetiolesLeaves

N Uptake & Partitioning for 62 Bushel Soybean

Average of 2 varieties at DeKalb, IL 2012

Nitrogen Needs and Removal by 62 Bushel Soybean Crop

Amount Required

*Amount from

Nodules

Removed with Grain

NetRemoval from Soil

lb acre-1

271 135 194 59

Average of two varieties at DeKalb, IL 2012

* Assuming 50% of total N accumulation supplied by N fixation from nodules

Soybean Needs More N than it Can Fix

Adapted from Salvagiotti et al. 2008. Field Crops Res. 108:1-13.

Yield (bu acre-1)10 30 50 70 90 110N

upt

ake

or fi

xatio

n (lb

acr

e-1)

0

100

200

300

400

500 Total N uptakeBiological N fixation

Soybean Nitrogen Basics•Only 50-60% of soybean N is obtained from N2 fixation and the proportion decrease s with increasing yield levels

•Low N supply requires remobilization from leaf N (decreasing photosynthesis and yield potential)

Soybean Nitrogen Basics•N obtained from: 1) soil/organic matter mineralization, 2) N2 fixation, and 3) fertilizer N sources

•An antagonism exists between NO3-

and N2 fixation in nodules with fertilizer application

Can supplemental fertilizer N sources be applied that don’t inhibit N2 fixation?

Soybean Gets Some N from fixation by Nodules

Nitrogen from Nodules is Transported From the Roots to the Shoot as Ureides

Allantoic acid Allantoin acid

Ureides

Does soybean also convert urea-N to ureides?

Fig. 1. Effect of N source and concentration on nodule mass of soybean grown in nutrient solutions.

Urea-N Does Not Inhibit Nodule Development

Plant Physiol. 17:169-172 (1977)

Fig. 2. Depletion of NO3- and urea from nutrient solutions by soybean plants.

Concentration of N sources was determined on days 19, 21, 25, and 28 ofgrowth.

Urea-N Uptake by Plants is Slower than Nitrate-N

Plant Physiol. 17:169-172 (1977)

Deep Placement of Coated Urea Enhances Dry Weight Production, N Accumulation, and Grain Yield

Stage TreatmentDry

Weight N

Accumulation Seed Yield------------------------------------------- g m-2 ---------------

-------------------Mg ha-1 bu ac-1

R3 Control 316a 8.3a -- --

Sidedress(100 kg N ha-1 at R1) 394 10.8 -- --

Deep Placed(100 kg N ha-1 at planting)

368a 9.7a -- --

R7 Control 740a 23.5a 3.17a 54.1aSidedress(100 kg N ha-1 at R1) 756 24.4 3.05a 52.1aDeep Placed(100 kg N ha-1 at planting) 957b 31.4b 3.60b 61.5b

Adapted from Soil Sci. Plant Nutr. 37:223-231 (1991)

Soybean Response to N Study 2013

• Unfertilized Control

• Urea and ESN to provide 75 lbs N

• Banded & Broadcast Before Planting

• Broadcast at V3

• Sidedress at R1 and R3

• Four locations in Illinois

Champaign

Harrisburg

Four Locations in IllinoisDeKalb• 5.3% OM• 14.4 ppm NO3-N• 65.6 bushels/acre

Rushville• 1.8% OM• 11.0 ppm NO3-N• 76.8 bushels/acre

Champaign• 4.4% OM• 9.2 ppm NO3-N• 60.0 bushels/acre

Harrisburg• 3.4% OM• 10.2ppm NO3-N• 65.0 bushels/acre

Rushville

DeKalb

Response to N on SoybeanChampaign, IL 2013 (75 lbs of N applied)

No significant differences.

Application method Urea ESN–––––– bushels acre–1 ––––––

Unfertilized check 59.5Pre-plant banded 60.1 61.4Pre-plant broadcast 60.4 60.3V3 broadcast 59.8 58.8R1 sidedress 58.1 61.0R3 sidedress 59.8 61.6

Response to N on SoybeanHarrisburg, IL 2013 (75 lbs of N applied)

No significant differences.


Unfertilized check 65.7Pre-plant banded 66.6 64.1Pre-plant broadcast 65.1 68.1V3 broadcast 65.3 64.5R1 sidedress 62.4 65.1R3 sidedress 65.0 62.4

Response to N on SoybeanRushville, IL 2013 (75 lbs of N applied)

* Significantly different from UTC at P < 0.10.


Unfertilized check 77.5Pre-plant banded 74.0* 76.2Pre-plant broadcast 75.1 78.8V3 broadcast 73.4* 73.8*R1 sidedress 78.6 79.0R3 sidedress 78.6 79.7

Response to N on SoybeanDeKalb, IL 2013 (75 lbs of N applied)

* Significantly different from UTC at P < 0.05.


Unfertilized check 60.0Pre-plant banded 64.0 69.5*Pre-plant broadcast 74.7* 69.5*V3 broadcast 64.1 66.8*R1 sidedress 64.3 62.9R3 sidedress 65.9* 65.8*

QUIZ Time

2003: What cost $1 billion dollars over the prior 2 decades of work to complete in 2003?

2013: What cost $4000 and took less than 2 weeks to do the same thing in 2013?

ANSWER: The First Human Genome Sequence

2003: It cost $1 billion dollars over the prior 2 decades of work to complete the first human genome sequence 2 billion assembled base pairs of DNA

2014: It is projected to cost only $1000 in 2014 and take only days to complete a human genome sequence .

Now thousands of people are having their DNA sequenced for diagnosing genetic and non genetic diseases including cancer at major medical centers although not yet in routine clinical use. The new term in use is “personalized medicine”.

QUIZ Time

2010: What cost >$10 million and several years prior to complete?

2013: What costs $4000 and take less than 2 weeks to do the same thing in 2014?

ANSWER:

The First Soybean Genome Sequence

2010: What cost >$10 million and several years to complete?

2013: What costs $4000 and take less than 2 weeks to do the same thing in 2014?

The Public/Private Effort Drove a Revolution in Sequencing Technology

Now Referred to as “Next Generation” SequencingThe $1000 human genome sequence is predicted for 2014

Illumina GAII or HiSeq Instruments at Keck Center, UI

DNA mRNA Proteins Traits

Whole genome sequencing of DNA

Transcriptomics: Sequencing of all the mRNA (messenger RNAs) of different tissues

The proteins can be predicted from the DNA or RNA sequences

The goals is to understand traits with these new sequencing approaches that were not feasible to do just 5 years ago

Current project goals in our lab: -Find all the mRNAs and small RNAs expressed in a seed - Dissect how they control important traits as protein and oil composition in the developing seed

Approaches:

•There are potentially 78,000 genes (gene models) that code for proteins in soybean as shown by the recent sequencing of the soybean genome

•We determine the expression of all 78,000 genes in different developmental times of seed development by sequencing the messenger RNA populations yielding up to 100 million raw sequence reads from each sample.

•The sequence reads are aligned to the 78,000 gene models to find which ones match and determine their relative expression in the different tissues or conditions.

Next Generation Illumina Deep Sequencing Technology Used to Sequence mRNAs or Small RNA Populations

Yields 200 Million Sequence Reads Per Sample

Illumina GAII or HiSeq Instruments at Keck Center, UI

Williams cultivar is used as the standard cultivar source for seed and seedling tissues

Seed Coat Cotyledon

Shoot tips

Unifoliates

StemGerminated Cotyledons Roots

Immature SeedYoung Seedling

5-6mgReference

Day 0Fertilization/Flowering

4 DAFGlobular Stage

8-10 DAFHeart Stage

12-14 DAF 17-19 DAF 22-24 DAF 5-6mgReference

Day 0Fertilization/Flowering

4 DAFGlobular Stage

8-10 DAFHeart Stage

12-14 DAF 17-19 DAF 22-24 DAF

Global Expression Analyses of Soybean Seed Development Including Very Early Stages Post-Fertilization Using

Illumina Next Generation Sequencing

• Total RNA from the younger whole seeds was extracted and subjected to high throughput sequencing (RNA seq)

•12 to 77 million sequence reads of the mRNAs from each of 7 stages using Illumina “Next Generation” Sequencing, Keck Center, University of Illinois

- 4 DAF (days after flowering)-12-14 DAF-22-24 DAF-5-6 mg fresh weight per seed -100-200 mg fresh weight per seed -400-500 mg fresh weight per seed-mature dry seed

• Seed coats and cotyledons are dissected from immature soybean seed at various stages of fresh weight during seed development including the mid-maturation stages when soybean accumulates proteins and oils.

• The mRNAs and small RNA populations were sequenced.

Selection of Genes with Highest Expression During Different Stages (Each line represents expression of a different gene)

12-14 Days After Flowering

Young Cotyledons, 5-6 mg

Mid to Late Maturation Cotyledons

Mature Seed Cotyledons

Beta‐conglycinin gene models (black) peak in RPKM at 100‐200mg cotyledonGlycinin gene models (pink) peak in RPKM at 400‐500mg cotyledon

Glycinin and Beta‐Conglycinin Storage Protein Gene Models Have High Expression in Mid‐to Late Maturation Seeds

Omega‐6‐fatty acid desaturases (black) tend to have higher RPKMs throughout development than omega‐3‐fatty acid desaturases (pink)

Expression of Omega Fatty Acid Desaturase Gene Models Throughout Seed Development

aspartate kinase (2.7.2.4)

aspartate-semialdehyde dehydrogenase (1.2.1.11)

homoserine dehydrogenase (1.1.1.3)

threonine synthase (4.2.3.1)

cystathionine beta-lyase (4.4.1.8)

homocysteine S-methyltransferase (2.1.1.10) (2 purple stars)

5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (2.1.1.14)

LL-diaminopimelate aminotransferase (2.6.1.83)

diaminopimelate epimerase (5.1.1.7)

diaminopimelate decarboxylase (4.1.1.20)

Enzymes in the methione, threonine, lysine superpathway with gene models identified in SoyBase

dihydrodipicolinate synthase (4.2.1.52)

dihydrodipicolinate reductase (1.3.1.26)homoserine kinase (2.7.1.39)

Pathway to Methionine

dark blue: aspartate kinaselight blue: aspartate kinase/homoserinedehydrogenasered: aspartate-semialdehyde dehydrogenasegreen: homoserine kinaseorange: Cystathionine beta-lyasepurple: Homocysteine S-methyltransferasepink: 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase

Bio Rep 1

Bio Rep 2

A display of mRNA levels from RNA Seq data for genes encoding enzymes in the pathway to methionine

1. Aspartate kinase

2. Aspartate semi-aldehyde dehydrogenase

Bio Rep 1

3. Homoserinedehydrogenase

5. Cystathioninebeta-lyase

RNA Seq Data for Genes Encoding Enzymes in the Pathway to Methionine

Bio Rep 2

6a. Homocysteine S-methyltransferase

4. Homoserinekinase

6b. 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase

Bio Rep 1 Bio Rep 2

1. Aspartate kinase

2. Aspartate semi-aldehyde dehydrogenase

Bio Rep 1

3. dihydrodipicolinate synthase

5. LL-diaminopimelateaminotransferase

Bio Rep 2

6. diaminopimelate epimerase

4. dihydrodipicolinate reductase

7. diaminopimelate decarboxylase

Bio Rep 1 Bio Rep 2

RNA Seq Data for Genes Encoding Enzymes in the Pathway to Lysine

Aspartate kinase

Aspartate semi-aldehyde dehydrogenase

Bio Rep 1

Homoserine dehydrogenase

Homoserine kinase

Enzymes in the Pathway to Threonine

Bio Rep 2

Threonine synthase

Graphs depict our RNA Seq data from developing seed for genes encoding enzymes

in the pathway to threonine

Using transcriptome sequencing of developing seed to examine expression of enzymes in the pathway to nitrogen accumulation

Could be done with different applications of nitrogen or growth conditions to genes that are changed in gene expression

Could use genetic lines that differ in nitrogen response

Glutamate-ammonia ligase

Glutamate synthase (ferredoxin)

Glutamate synthase (NADH)

Ammonia Assimilation Pathway

Source: Soybase

Soak in water 24hrs

Glutamate-Ammonia Ligase

23.9 22.3 21.8 11.5 0.7 0.1 0.0

0.5 10.1 25.6 89.6 249.0587.4 400.0

14.0

320.0

Glutamate-Ammonia Ligase

Seed Development Seedling Cotyledons

Soak in water 24hrs

Glutamate Synthase

5.6 5.1 7.1 4.6 4.7 7.5 2.5

2.0 14.2 22.0 27.4 70.892.3 119.3

27.0

19.0

Glutamate Synthase


Urea Cycle

Argininosuccinatesynthase

Argininosuccinate lyase

ArginaseCarbamoyl-phosphate synthase

Ornithine carbamoyltransferase

Asparagine Biosynthesis

Source: Soybase, superpathway of asparagine biosynthesisBoth enzymes give an identical list of gene models.

Asparagine synthase (glutamine-hydrolysing)

Aspartate-Ammonia Ligase

Aspartate-Ammonia Ligase/ Asparagine Synthase


Combined data

ii ki iI

Recently, we have shown it to be a tissue-specific example of gene silencing or naturally occurring RNA interference by CHS siRNAs that are produced in a tissue specific manner, Tuteja et al, Plant Cell 21, 3063-3077, 2009.

Therefore all commercially-grown yellow soybeans grown on millions of acres are the result of a naturally generated, non-transgenic siRNAmediated gene silencing locus.

This example shows how small RNAs can control traits. Does a similar process regulate other gene networks to influence protein composition and quantity?

Small RNAs lead to yellow seed coats produced by the naturally occurring alleles of the I (Inhibitor) locus in soybean

Yellow Hilum Saddle Self color

RNA Seq Reveals Quantitative Differences in Expression Levels of the CHS7 and CHS8 Genes of Yellow and Pigmented Genotypes

CHS7 and CHS8 are 6-8 fold more highly expressed in the pigmented genotypes

Gene Model Length Gene i i i i i i

Glyma08g11620 1167 CHS1 7 14 0.5 0.9Glyma05g28610 1167 CHS2 4 7 12 10Glyma08g11630 1167 CHS3 8 18 1 1Glyma08g11520 1167 CHS5 11 17 0.6 0.6Glyma08g11610 1167 CHS9 6 12 0.6 0.9Glyma01g22880 1167 CHS6 0.3 0.5 1 0.9Glyma01g43880 1170 CHS7 64 85 517 400Glyma11g01350 1170 CHS8 23 30 198 167

Phenotype & Genotype at I LocusYellow Pigmented

Reads per Kilobase per Million (RPKM)

Richland (I)Seed Coat

yellow

Williams (ii)Seed Coat

yellow

Williams (ii)Cotyledon

yellow

Williams (i)Seed Coat

black

CHS Gene CHS siRNA Counts per 3 Million Sequence Reads

CHS1 17277 30094 33 86

CHS2 16891 28547 38 76

CHS3 18595 31679 63 102

CHS4 18368 31841 43 112

CHS5 18368 31825 43 112

CHS6 14066 24006 83 187

CHS7 30712 41937 61 110

CHS8 30382 42045 77 137

CHS9 18886 32131 13 50Adapted from Vodkin Lab – Tuteja et al., Plant Cell 2009

Comparison of siRNA Counts from Seed Coat and Cotyledon Libraries that Map to the Coding Regions of the Nine Member

CHS Gene Family

I

Tissue Specific Inhibition of the Flavonoid Pathway by Endogenous Chalcone Synthase siRNAs in Yellow Seed Coats

Seed Size Difference in a Transgenic Line also with High Protein

Gene Model High protein Control AnnotationGlyma03g32030.1 39933 38418 Glycinin seed proteinGlyma10g04280.1 32953 28357 Glycinin seed proteinGlyma19g00200.1 1859 1909 Unnamed proteinGlyma02g42600.1 1835 199 Unknown proteinGlyma09g06130.1 1822 56 Unknown proteinGlyma02g45050.1 1655 634 Unknown proteinGlyma09g06100.1 1274 38 Unknown proteinGlyma02g45070.1 1258 204 Unknown proteinGlyma07g37250.1 1125 0.5 Gly M4 subunitGlyma05g01850.1 1030 36 Unknown proteinGlyma11g26470.1 952 156 Putative proteinGlyma10g02210.1 776 1 Unknown proteinGlyma09g12260.1 716 166 Proline rich proteinGlyma13g35320.1 680 0 Unknown protein

Comparison of some soybean genes with higher normalized expression (sequence reads per million) in high protein transgenic line versus control line

using “Next Generation” sequencing.

Gene models shaded in green show enhanced expression in the high protein sublines

5608 Transcription Factors Displayed by Their Expression Levels at Different Stages of Soybean Development

Transcription factors (TFs) are “master regulators” controlling the expression of many other genes to either increase or decrease their levels of gene expression.

The networks of genes whose expression is regulated by TFs control plant traits including protein and oil amounts and likely can influence the amino acid composition of the seed.

Expression of a TF generally would precede the change in expression of the genes in the networks they control, ie storage proteins, oils, amino acid biosynthetic enzymes.

Therefore, the TFs expressed early in seed development are likely to have the greatest influence on the ultimate composition of the seed.

The key is finding the TFs critical in this process especially as their expression levels are often low compared to other genes as those coding for enzymes and certainly compared to the major seed storage proteins.

We have completed high throughput sequencing the entire complement of TFs in normal development of soybean seeds.

Examination of lines that vary in protein and oil content and amino acid composition with high throughput RNA sequencing is one approach to the goal of improving seed composition. Particular emphasis should be placed on transcription factors in these lines.

Why Transcription Factors Are Important in Seed Composition

Nitrogen on Soybean: Protein and Oil

Nitrogen on Soybean: Protein and Oil

Seth NaeveUniversity of MinnesotaSeth NaeveUniversity of Minnesota

Assumption and background• N fertilizer will be most effective for

soybean yield (and presumably seed [N]) when applied during seed filling– Deibert et al. (1979)– Afza et al. (1987)– Shibles (1998)

• A review of the literature shows the range of expected responses– Yield + / Protein +– Yield + / Protein 0– Yield 0 / Protein +– Yield 0 / Protein 0

Rotundo and Westgate (2009)

Relative response of soybean seed protein content (mg per seed) and concentration (%) to nitrogen supply. Symbols and bars represent mean response and 95% confidence interval, respectively. The number of studies included in the meta-analysis are indicated in the figure.


• In vitro studies increased [protein] by ~ 60%• Hydroponic studies resulted in a ~20%

increase in [protein]• The average [protein] increase in field

studies was only ~1%• However, the response appeared to be rate

dependent. – Applications of <100kg ha-1 resulted no response– >100kg ha-1 (planting-V6) = ~2%– >100kg ha-1 (R1-R4) = ~3%


• Rotundo and Westgate also examined environmental variability in seed quality through the Uniform Variety Trials.– They found a 18% variation in seed protein and

23% variation in seed oil– This ‘natural’ variation dwarfed any treatment

effects noted in their meta-analysis

Rotundo and Westgate (2009)• Oil

Genetic Gain X Management• Eric Wilson, et al. (2014)• 59 MG II’s and 57 MG III’s released from

1928-2007)• Minnesota, Wisconsin, Illinois, and Indiana• Lines were either planted with or without

added urea (total of 560 kg N ha-1)– Split application (pre-plant and V5)– Split forms (ESN and Urea + Agrotain)

Wilson et al – observations• Oil concentration increased with release

dates, but no N affect• N fixation at MG II locations was smaller

(23% of total N compared with ~60% in MG III locations).

• Longer leaf retention in fertilized plots

Casteel et al. (2013)• Paper presented at the ASA meetings• Casteel partitioned plants from the

genetic gain study at V4, R2, R4, and R6

• Found increased leaf biomass and N concentration across year of release at all stages sampled

• Found increased allocation of whole-plant N to leaves at R6 over release date– MG II – from 25 to 36% (from 1928 to 2011)– MG III – from 21 to 32% (from 1923 to 2011)

Thank YouThank You

Seth [email protected] [email protected]

nitrogen applications & soybean yields

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