4. Background
• Early maize reproduction is particularly
sensitive to perturbations in the stream of
assimilate carbon (shade, drought, etc.)
• Water deficits cause water potentials to
decrease which inhibits photosynthesis
• Ovary/Kernel abortion results
• Yield potential is often unrealized
5. Background
• Only a few days of low water potentials at
flowering are sufficient to lead to a high
proportion of aborted kernels
• Sucrose infusion during this period of
development was found to limit kernel
abortion (60-70% seed set vs. ~0 to 5%) (M.
Boyle)
• The water deficit treatment is not inherently
lethal to the developing kernels
6. Background
• Kernel abortion is largely a biochemical problem.
• Acid invertase is the first enzyme to act on sucrose
at the early stage of ovary/embryo development in
maize.
• Acid invertase activities (soluble and insoluble)
were repressed in low water potential treatments
(C. Zinselmeier).
• A lesion at the invertase step was determined to be
a likely candidate that limited carbon flow during
ovary/kernel development.
7. Goal of Research
• Identify metabolic and genetic factors,
which through manipulation could
minimize the negative effect of dehydration
during the critical early stages of
reproduction.
8.
9. Materials and Methods
• DE2 X H99 Hybrid
• Three Treatments (7 Day Time Course)
– Control (High water potential)
– Drought (Low water potential)
– Drought + Infused Sucrose (Low water potential)
• Environmental Control Chamber
• 14 hour photoperiod (30/20 ºC, 40/95% RH)
• Sucrose Infusion (5.3 to 6.8 g sucrose daily)
10.
11. DE2 X H99:
Hybrid developed
at the University of
Delaware by
James Hawk
12.
13. Supplying a Photosynthetic Product (Sucrose)
During Drought
Infuse sucrose
here on day 1
Withhold water
for five days
14.
15. Materials and Methods
• Water Potential Measurements
– Isopiestic Thermocouple Psychrometry
– Leaf disc removal
• Net Photosynthesis
– Li-Cor LI-6400 Photosynthesis Meter
• Water Usage
– Monitored Gravimetrically for the Whole Plant System
• Green Leaf Measurement
– Percentage Healthy Green Leaf (Whole Plant)
16.
17.
18.
19.
20.
21. Materials and Methods
• Ovary Histology
• Invertase Localization (In vivo and In situ)
– Nitroblue Tetrazolium Stain
– Fresh tissue sections (insoluble invertase) In vivo
– Frozen tissue sections (souble invertase) In situ
• Insoluble Invertase
– Cell wall-bound (phloem termini, upper pedicle area of
the ovary)
– Hydrolyze sucrose in the apoplast
• Soluble Invertase
– Nucellus localization
22. Materials and Methods
• Glucose Localization (In situ)
– Cryosections/ Enzyme Gel (pH 7.4 to inhibit acid
invertase)
– Freeze dry sections to preserve (lock sugar location)
– Hydrogen Peroxide (H2O2) detection reagent
– (Glucose oxidase, Horseradish Peroxidase, Gelatin,
Amplex Red)
– Fluorescent Resorufin (epi-fluorescence microscopy)
23. Materials and Methods
• Quantitative PCR (Real time PCR)
• SybrGreen/ABI 7700
• Time course for mRNA abundance
• Gene specific primers used to generate cDNA
• Sucrose processing enzymes
– The invertase gene family (Key regulatory enzymes)
– Sucrose synthase
• Some ovary genes for senescence were tested
– RIP2 (Based on microarray study)
– PLD1 (Based on the literature)
45. Conclusions
• Water deficits inhibit photosynthesis and causes abortion
when starch is depleted in ovary.
• Infusing sucrose returns carbon stream but invertase
activity remains low, preventing full recovery.
• Sucrose infusion partially restored both insoluble and
soluble invertase activity in developing ovaries/kernels
during a period of low water potential.
• The glucose gradient that exists between the pedicel and
nucellus tissue at high water potential is largely lost at low
water potential. Sucrose infusion partially restores this
gradient. Disappearance of the glucose gradient in the
developing ovary plays a role in the kernel abortion
process (sugar signaling and metabolic role).
46. Conclusions cont.
• All of the tested ovary genes for sucrose
processing enzymes were down-regulated at low
water potential.
• Some of the ovary genes for putative senescence
genes were up-regulated at low water potential.
• Some of the ovary genes for sucrose processing
and for the putative senescence response were
sugar responsive.
47. Conclusions
• Partial recovery of gene expression (ex. INCW2,
IVR2) to control levels occurs during sucrose
feeding.
• This helps to explain why invertase activity
responded only partially to the feeding, and
abortion was only partially prevented.
• The late up-regulation of genes like RIP2 may be
the irreversible component of abortion.
48. Summary of events leading to abortion of
maize ovaries when plants are subjected to
low W around the time of pollination.
49. •Call for research to understand why the maternal plant
survives but extensive kernel abortion occurs during
periods of low water potential (PCD vs. necrosis).
•Transgenic approach to modify putative senescence gene
expression (ex. RIP2)
•Attempt to enhance tolerance to drought by decreasing
the sensitivity of the developing kernels to short
interruptions in the carbon steam. Later kernel
development is not as sensitive to periods of water deficits.
The ability to incorporate this “quiescent” ability into early
reproductive development has potential agricultural value.
Future Research
