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Sjoberg, S., Carter, A. H., Steber, C. M., & Garland-Campbell, K. A. (2020).
Unravelling Complex Traits in Wheat: Approaches for Analyzing Genotype × Environment Interactions in a Multi-environment Study of Falling Numbers.
Crop Science, 60, 3013–3026.
(Show/Hide Abstract)
Abstract:
Multi-environment trials provide useful information about highly variable, complex plant traits like yield
and quality, but are difficult to analyze due to their frequently unbalanced nature, with genotypes and
locations varying from year to year. Our objectives were to use multiple approaches, including joint
regression and principal components analysis, to characterize patterns in the genotype by environment
interactions across an unbalanced three-year multi-environment wheat variety trial dataset, examining
falling numbers (FN) test results in wheat. The FN test measures the decrease in flour gelling capacity
resulting from starch digestion by the enzyme α-amylase. Low FN/high α-amylase grain is discounted
because it is associated with poor end-use quality. Low FN can be caused by susceptibility either to
preharvest sprouting when it rains before harvest or to late maturity α-amylase induction by
temperature fluctuations during grain maturation. The most effective and visually intuitive approaches
for selecting varieties with high FN across variable environments was a combination of joint regression,
such as Finlay-Wilkinson and Eberhart and Russell, with biplot methods such as the additive main effects
and multiplicative interaction model (AMMI) and the genotype main effects and genotype ×
environment interaction model (GGE). These results identified stable lines for FN resistance and provide
a means to analyze unbalanced, multi-environment data from breeding and variety trials.
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Ghimire, B., Hulbert, S., Steber, C. M., Garland-Campbell, K., & Sanguinet, K. A. (2020).
Characterization of root traits for improvement of spring wheat in the Pacific Northwest.
Agronomy Journal, 112, 228–240.
(Show/Hide Abstract)
Abstract:
Understanding the genetic basis of root traits and overall root physiology provides
essential information on a largely untapped resource for crop improvement as roots are
instrumental for the uptake of water and nutrients. However, breeding for improved root traits is
challenging due to laborious and time-consuming root phenotyping in soil. Our studies sought to
uncover spatiotemporal root growth dynamics of mature plant root systems in five spring wheat
(Triticum aestivum L.) cultivars - Louise, Alpowa, Hollis, Drysdale, and Dharwar Dry - and a
facultative spring landrace, AUS28451 using the in situ minirhizotron technique. The two-year
greenhouse study revealed that the root system grows rapidly after early node elongation to gain
maximum size during anthesis after which root growth slows and transitions to senescence. We
were able to detect quantifiable differences among wheat cultivars in root traits in both 5-day old
seedlings and root systems at anthesis. Furthermore, the positive correlation of the observed root
traits with grain yield and the consistency in root traits observed using minirhizotrons and
through extraction of young and mature root systems has reinforced the experimental results. A
negative correlation was found between root number, area, and length and root diameter. We
found that the spring wheat cultivars - AUS28451, Dharwar Dry, and Alpowa - had increased
root number, area, and length, but also increased time to heading. The results from this study can
be further leveraged to screen breeding lines for root traits of interest as well as assess the
heritability of root traits for dryland farming in the inland Pacific Northwest.
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Shrestha, S. L., Garland-Campbell, K. A., Steber, C. M., & Hulbert, S. H. (2020).
Carbon isotope discrimination association with yield and test weight in Pacific Northwest-adapted spring and winter wheat.
Agrosystems, Geosciences & Environment, 3(1), e20052, (15 pages).
(Show/Hide Abstract)
Abstract:
Due to considerable influence of environment on yield, breeding for drought tolerance could benefit from focusing on selection of more heritable physiological traits, such as carbon isotope discrimination (as measured by delta, Δ) for
indirectly assessing water use efficiency (WUE) of wheat (Triticum aestivum L.).
Spring and winter wheat cultivars were assayed for ∆, and these values were
used to determine the relationships with performance in over 13 environments
in the U.S. Pacific Northwest. The correlation coefficients of Δ values between the
wheat cultivars grown in different environments ranged from 0.11 to 0.73 for both
spring and winter wheat. There was significant genotypic variation for Δ in soft
spring and hard winter wheat but not in hard spring and soft winter wheat. The
Δ values were poor indicators of yield for this set of wheat cultivars in most environments, although low values (better WUE) were sometimes correlated with
yield. A population of 165 hard spring wheat recombinant inbred lines derived
from a cross between two hard spring wheat varieties that differed in Δ was also
screened in low-rainfall dryland and irrigated environments. High yields in this
recombinant inbred population were weakly correlated with high Δ values or
low WUE in most environments.
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Hauvermale, A. L., & Steber, C. M. (2020).
GA signaling is essential for the embryo-to-seedling transition during Arabidopsis seed germination, a ghost story.
Plant Signaling & Behavior, 15(1), e1705028 (9 pages).
(Show/Hide Abstract)
Abstract:
The plant hormone gibberellin (GA) stimulates developmental transitions including seed germination,
flowering, and the transition from juvenile to adult growth stage. This study provided evidence that GA
and the GA receptor GID1 (GA-INSENSITIVE DWARF1) are also needed for the embryo-to-seedling transition in
Arabidopsis. The ga1-3 GA biosynthesis mutant fails to germinate unless GA is applied, whereas the gid1abc
triple mutant fails to germinate because it cannot perceive endogenous or applied GA. Overexpression of
the GID1a, GID1b, and GID1c GA receptors rescued the germination of a small percentage of ga1-3 seeds
without GA application, and this rescue was improved by dormancy-breaking treatments, after-ripening and
cold stratification. While GID1 overexpression stimulated ga1-3 seed germination, this germination was
aberrant suggesting incomplete rescue of the germination process. Cotyledons emerged before the radicle,
and the resulting “ghost” seedlings failed to develop a primary root, lost green coloration, and eventually
died. The development of ga1-3 seedlings overexpressing GID1 was rescued by pre-germinative but not
post-germinative GA application. Since the gid1abc mutant also exhibited a ghost phenotype after germination
was rescued by cutting the seed coat, we concluded that both GA and GID1 are needed for the embryo-to-seedling
transition prior to emergence from the seed coat.
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Martinez, S. A., Shorinola, O., Conselman, S., See, D., Skinner, D. Z., Uauy, C., & Steber, C. M. (2020).
Exome sequencing of bulked segregants identified a novel TaMKK3‐A allele linked to the wheat ERA8 ABA‐hypersensitive germination phenotype.
Theoretical and Applied Genetics, 133, 719–736.
(Show/Hide Abstract)
Abstract:
Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest.
The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft
white wheat 'Zak'. ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant
analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb
region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a
missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat.
The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is
likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS-
tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population
likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent
complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a
known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation
in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results
from the MKK3-A-G1093A mutation.
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Sjoberg, S. M., Carter, A. H., Steber, C. M., & Garland-Campbell, K. A. (2020)
Application of the factor analytic model to assess wheat falling number performance and stability in multi‐environment trials.
Crop Science, doi:10.1002/csc2.20293.
(Show/Hide Abstract)
Abstract:
A factor analytic model was used to characterize data generated with the Hagberg‐Perten Falling Number (FN) method, a measure of wheat quality influenced by genotype‐by‐environment interactions. The FN method detects starch degradation due to the presence of the enzyme alpha‐amylase in wheat grain such that a low FN indicates high alpha‐amylase activity and high risk of poor end‐product quality. Because farmers receive severe discounts for low FN, FN data has been collected over multiple years for the Washington State University multilocation variety trials to help farmers and breeders identify lower risk varieties. Analysis of this data to objectively rank varieties is challenging because the dataset is unbalanced and because FN is subject to complex genotype‐by‐environment interactions. Low FN can result from environmental differences at multiple stages in grain development because there are two major causes of alpha‐amylase accumulation in grain, late‐maturity alpha‐amylase (LMA) and preharvest sprouting (PHS). A five‐factor analytic model extracted explicit measures of overall performance and of stability in variable environments from historical falling number data from the multilocation trial, providing a basis for breeding and planting decisions. Whereas a linear model explained 70.3% of the variation, the five‐factor analytic model accounted for 92.5% of variation in the data. Examination of factor loadings enabled us to separate environments and genotype response to either PHS or LMA, specifically. This is the first application of a factor analytic model to evaluate the end‐use quality trait, FN, providing a method to rank varieties for grower decisions and for breeder selections.
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Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2018, January).
Hunting for genes: falling numbers
project seeks to reduce risk by breeding for genetic resistance.
Wheat Life, 61(1), 50–51.
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Martinez, S. A., Godoy, J., Huang, M., Zhang, Z., Carter, A. H., Garland Campbell, K. A., & Steber, C. M. (2018).
Genome-Wide Association Mapping
for Tolerance to Preharvest Sprouting
and Low Falling Numbers in Wheat.
Frontiers in Plant Science, 9, 1–16, https://doi.org/10.3389/fpls.2018.00141.
(Show/Hide Abstract)
Abstract:
Preharvest sprouting (PHS), the germination of grain on the mother plant under cool and
wet conditions, is a recurring problem for wheat farmers worldwide. α-amylase enzyme
produced during PHS degrades starch resulting in baked good with poor end-use
quality. The Hagberg-Perten Falling Number (FN) test is used to measure this problem
in the wheat industry, and determines how much a farmer’s wheat is discounted for
PHS damage. PHS tolerance is associated with higher grain dormancy. Thus, breeding
programs use germination-based assays such as the spike-wetting test to measure
PHS susceptibility. Association mapping identified loci associated with PHS tolerance
in U.S. Pacific Northwest germplasm based both on FN and on spike-wetting test data.
The study was performed using a panel of 469 white winter wheat cultivars and elite
breeding lines grown in six Washington state environments, and genotyped for 15,229
polymorphic markers using the 90k SNP Illumina iSelect array. Marker-trait associations
were identified using the FarmCPU R package. Principal component analysis was directly
and a kinship matrix was indirectly used to account for population structure. Nine loci
were associated with FN and 34 loci associated with PHS based on sprouting scores.
None of the QFN.wsu loci were detected in multiple environments, whereas six of the 34
QPHS.wsu loci were detected in two of the five environments. There was no overlap
between the QTN detected based on FN and PHS, and there was little correlation
between the two traits. However, both traits appear to be PHS-related since 19 of the 34
QPHS.wsu loci and four of the nine QFN.wsu loci co-localized with previously published
dormancy and PHS QTL. Identification of these loci will lead to a better understanding
of the genetic architecture of PHS and will help with the future development of genomic
selection models.
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Martinez, S. A., Thompson, A. L., Wen, N., Murphy, L., Sanguinet, K. A., Steber, C. M., & Garland Campbell, K. A. (2018).
Registration of the Louise/Alpowa Wheat Recombinant Inbred Line Mapping Population.
Journal of Plant Registrations, doi:10.3198/jpr2017.08.0053crmp.
(Show/Hide Abstract)
Abstract:
A mapping population was developed from the cross of soft
white spring wheat (Triticum aestivum L.) cultivars 'Louise'
and 'Alpowa' for use in investigating the genetic architecture
of drought tolerance in the US Pacific Northwest. The Louise/Alpowa (Reg. No. MP-8, NSL 520824 MAP) recombinant inbred
line mapping population was developed through single
seed descent from the F2 generation to the F5 generation.
The population consists of 141 F5:6 recombinant inbred lines,
of which 132 were used to construct the genetic linkage
map. The 32 linkages groups included 882 single nucleotide
polymorphism markers and one simple sequence repeat
marker spanning 18 of 21 chromosomes. The Louise/Alpowa
population was characterized for variation in agronomic
traits, phenology, and end-use quality traits. This population
will be used for identification and introgression of multiple
loci providing resistance to environmental stress such as
drought, stripe rust, and high temperatures.
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Aramrak, A., Lawrence, N. C., Demacon, V. L., Carter, A. H., Kidwell, K. K., Burke, I. C., & Steber, C. M. (2018).
Isolation of Mutations Conferring Increased Glyphosate Resistance in Spring Wheat.
Crop Science, 58, 84–97.
(Show/Hide Abstract)
Abstract:
A mutation breeding approach was used to
explore the feasibility of isolating glyphosate-
resistant (GR) wheat (Triticum aestivum L.) lines.
Although transgenic GR wheat cultivars were
developed, they were never introduced due to
lack of consumer acceptance and concern over
management of volunteer wheat in rotation.
Large-scale screening experiments recovered
ethyl methanesulfonate mutants able to resist
360 to 480 g acid equivalent (ae) ha-1 glyphosate in four spring wheat cultivars, 'Hollis', 'Louise', 'Macon', and 'Tara2002', indicating that it is
possible to recover resistance in a wide range
of genetic backgrounds (glyphosate is typically
applied at 840 g ae ha-1 in transgenic crops).
Glyphosate rates of 420 to 530 g ae ha-1 were sufficient to kill the susceptible wild-type parents.
Seven GR mutants were characterized: GRH9-5, GRH9-8, GRL1, GRL33, GRL65, GRM14, and
GRT20. Glyphosate resistance was examined at
the whole-plant level in dose-response experiments. Three mutant lines – GRL33, GRH9-5,
and GRT20 – exhibited resistance based on
a significant increase in the dose required to
retard growth compared with the corresponding susceptible wild type. According to F2 segregation analysis, GRL1, GRL65, and GRT20
segregated as a single dominant gene, whereas
GRL33, GRH9-5, and GRH9-8 appeared to be
either a single semidominant or polygenic trait.
Although GRL1 was associated with an amino
acid substitution (L239F) in TaEPSPS-7D1, no
nucleotide changes were observed in the coding regions of wheat 5-enolpyruvylshikimate-
3-phosphate synthase (EPSPS) gene in GRL33
and GRH9-8. Results suggest that glyphosate
resistance can result from multiple genetic
mechanisms in wheat.
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Ge, W., & Steber, C. M. (2018).
Positive and negative regulation of seed germination by the
Arabidopsis GA hormone receptors, GID1a, b, and c.
Plant Direct, 2(9), 1–11.
(Show/Hide Abstract)
Abstract:
Epistasis analysis of gid1 single and double mutants revealed that GID1c is a key
positive regulator of seed germination, whereas the GID1b receptor can negatively
regulate germination in dormant seeds and in the dark. The GID1 GA receptors were
expected to positively regulate germination because the plant hormone gibberellin
(GA) is required for seed germination in Arabidopsis thaliana. The three GA hormone
receptors, GID1a, GID1b, and GID1c, positively regulate GA responses via GA/GID1‐
stimulated destruction of DELLA (Asp‐Glu‐Leu‐Leu‐Ala) repressors of GA responses.
The fact that the gid1abc triple mutant but not gid1 double mutants fail to germi-
nate indicates that all three GA receptors can positively regulate non‐dormant seed
germination in the light. It was known that the gid1abc triple mutant fails to lose
dormancy through the dormancy breaking treatments of cold stratification (moist
chilling of seeds) and dry after‐ripening (a period of dry storage). Previous work sug-
gested that there may be some specialization of GID1 gene function during germina-
tion because GID1b mRNA expression was more highly induced by after‐ripening,
whereas GID1a and GID1c mRNA levels were more highly induced by cold stratifica-
tion. In light‐germinated dormant seeds, the gid1b mutation can partly rescue the
germination efficiency of gid1a but not of gid1c seeds. Thus, GID1b can function as
an upstream negative regulator GID1c, a positive regulator of dormant seed germina-
tion. Further experiments showed that GID1b can negatively regulate dark germina-
tion. Wild‐type Arabidopsis seeds do not germinate well in the dark. The gid1b and
gid1ab double mutants germinated much more efficiently than wild type, gid1c, or
gid1ac mutants in the dark. The observation that the gid1ab double mutant also
shows increased dark germination suggests that GID1b, and to some extent GID1a,
can act as upstream negative regulators of GID1c. Since the gid1abc triple mutant
failed to germinate in the dark, it appears that GID1c is a key downstream positive
regulator of dark germination. This genetic analysis indicates that the three GID1
receptors have partially specialized functions in GA signaling.
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Delwiche, S. R., Higginbotham, R. W., & Steber, C. M.(2018).
Falling number of soft white wheat by near-infrared spectroscopy: A challenge revisited.
Cereal Chemistry, 95(3), 469–477.
(Show/Hide Abstract)
Abstract:
Background and objectives: Wheat Hagberg falling number (FN) is a long-
standing quality test that, by means of measuring the viscosity of a heated water-meal or water-flour mixture, characterizes the activity of endogenous α-amylase,
the enzyme primarily responsible for starch hydrolysis. The accuracy, time
requirement, and cost of this test have come under heightened scrutiny, particularly in seasons when weather conditions have been favorable to preharvest
sprouting or late maturity amylase. Near-infrared (NIR) spectroscopy, an analytical approach routinely used in the grain industry to measure contents of protein
and moisture, was reexamined as a possible alternative to the FN procedure.
Findings: Partial least squares (PLS) regression quantitative models developed on
a genetically diverse set of Washington grown white wheat demonstrated low
accuracy, with standard errors of performance ranging from 40 to 77 s. Alternatively, linear discriminant analysis and PLS discriminant analysis (PLSDA) qualitative models, developed and tested using a FN cutoff (pass/fail) value, also
demonstrated low accuracy, with the best model correctly identifying 67% and
71% of the samples, respectively, above and below a threshold value established
as the median value of FN in a calibration set of several hundred samples.
Conclusions: Replacement of the FN test with one based on NIR spectroscopy
on either whole grain or ground meal for making decisions on segregating wheat
lots according to α-amylase activity is not recommended.
Significance and novelty: Because NIR spectroscopy is not sufficiently accurate
to quantitatively model FN or differentiate low from high FN grain, viscometry procedures for starch integrity, such as FN, will continue their use in grain commerce.
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Steber, C. M. (2022, November).
Rising to the challenge of falling numbers.
Wheat Life, 65(10), 44–45.
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Hu, Y., Sjoberg, S. M., Chen, C., Hauvermale, A. L., Morris, C. F., Delwiche, S. R., Cannon, A. E., Steber, C. M., & Zhang, Z. (2022).
As the number falls, alternatives to the Hagberg–Perten falling number method: A review.
Comprehensive Reviews in Food Science and Food Safety
, 1–13.
(Show/Hide Abstract)
Abstract:
This review examines the application, limitations, and potential alternatives to
the Hagberg–Perten falling number (FN) method used in the global wheat industry
for detecting the risk of poor end-product quality mainly due to starch degradation by the enzyme α-amylase.
By viscometry, the FN test indirectly detects
the presence of α-amylase, the primary enzyme that digests starch. Elevated α-amylase
results in low FN and damages wheat product quality resulting in cakes
that fall, and sticky bread and noodles. Low FN can occur from preharvest sprouting (PHS) and late
maturity α-amylase (LMA). Moist or rainy conditions before
harvest cause PHS on the mother plant. Continuously cool or fluctuating temperatures during the
grain filling stage cause LMA. Due to the expression of addi-
tional hydrolytic enzymes, PHS has a stronger negative impact than LMA. Wheat
grain with low FN/high α-amylase results in serious losses for farmers, traders,
millers, and bakers worldwide. Although blending of low FN grain with sound
wheat may be used as a means of moving affected grain through the marketplace,
care must be taken to avoid grain lots from falling below contract-specified FN.
A large amount of sound wheat can be ruined if mixed with a small amount of
sprouted wheat. The FN method is widely employed to detect α-amylase after
harvest. However, it has several limitations, including sampling variability, high
cost, labor intensiveness, the destructive nature of the test, and an inability to
differentiate between LMA and PHS. Faster, cheaper, and more accurate alternatives could
improve breeding for resistance to PHS and LMA and could preserve the value of wheat grain by avoiding inadvertent mixing of high- and low-
FN grain by enabling testing at more stages of the value stream including at
harvest, delivery, transport, storage, and milling. Alternatives to the FN method
explored here include the Rapid Visco Analyzer, enzyme assays, immunoassays,
near-infrared spectroscopy, and hyperspectral imaging.
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Liu, C., Tuttle, K., Garland Campbell, K., Pumphrey, M., & Steber, C. (2021).
Investigating conditions that induce late maturity alpha-amylase (LMA) using Northwestern US spring wheat (Triticum aestivum L.).
Seed Science Research, 1–9. https://doi.org/10.1017/S0960258521000052
(Show/Hide Abstract)
Abstract:
The wheat industry rejects grain with unacceptably high α-amylase enzyme levels due to the risk of poor end product quality. There are two main causes of elevated grain α-amylase: (1) preharvest sprouting in response to rain before harvest and (2) late maturity α-amylase (LMA) induction in response to a cool temperature shock during late grain development. LMA induction was detected in a panel of 24 Northwestern US spring wheat lines. Thus, this problem previously described in Australian and U.K. varieties also exists in U.S. varieties. Because LMA induction results were highly variable using published methods, a characterization of LMA-inducing conditions was conducted in an LMA-susceptible soft white spring wheat line, WA8124. Problems with elevated α-amylase in untreated controls were reduced by raising the temperature, 25°C day/18°C night versus 20°C day/10°C night. LMA induction was not improved by colder temperatures (15°C day/4°C night) versus moderately cold temperatures (18°C day/7.5°C night or 10°C day/10°C night). While previous studies observed LMA induction by heat stress, it failed to induce LMA in WA8124. Thus, not all LMA-susceptible cultivars respond to heat. The timing of LMA susceptibility varied between two cultivars and within a single cultivar grown at slightly different temperatures. Thus, variability in LMA induction likely results from variability in the timing of the grain developmental stage during which cold shock induces LMA. Thus, it was concluded that the visual inspection of grain is needed to correctly identify LMA-sensitive spikes at the soft dough stage of grain development (Zadok's stage 85).
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Liu C., Parveen R. S., Revolinski S. R., Garland Campbell K. A., Pumphrey M. O., & Steber C. M. (2021).
The genetics of late maturity alpha-amylase (LMA) in North American spring wheat (Triticum aestivum L.).
Seed Science Research, 1–10. https://doi.org/10.1017/S0960258521000064
(Show/Hide Abstract)
Abstract:
Genetic susceptibility to late maturity alpha-amylase (LMA) in wheat (Triticum aestivum L.)
results in increased alpha-amylase activity in mature grain when cool conditions occur during
late grain maturation. Farmers are forced to sell wheat grain with elevated alpha-amylase at a
discount because it has an increased risk of poor end-product quality. This problem can result
from either LMA or preharvest sprouting, grain germination on the mother plant when rain
occurs before harvest. Whereas preharvest sprouting is a well-understood problem, little is
known about the risk LMA poses to North American wheat crops. To examine this, LMA susceptibility was characterized in a panel of 251 North American hard spring wheat lines, representing ten geographical areas. It appears that there is substantial LMA susceptibility in North
American wheat since only 27% of the lines showed reproducible LMA resistance following
cold-induction experiments. A preliminary genome-wide association study detected six significant marker-trait associations. LMA in North American wheat may result from genetic
mechanisms similar to those previously observed in Australian and International Maize
and Wheat Improvement Center (CIMMYT) germplasm since two of the detected QTLs,
QLMA.wsu.7B and QLMA.wsu.6B, co-localized with previously reported loci. The Reduced
height (Rht) loci also influenced LMA. Elevated alpha-amylase levels were significantly associated with the presence of both wild-type and tall height, rht-B1a and rht-D1a, loci in both
cold-treated and untreated samples.
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Sexton, T. M., Steber, C. M., & Cousins, A. B. (2021).
Leaf temperature impacts canopy water use efficiency independent of
changes in leaf level water use efficiency.
Journal of Plant Physiology, 25, 1–10.
(Show/Hide Abstract)
Abstract:
Canopy water use efficiency (above-ground biomass over lifetime water loss, WUEcanopy) can influence yield in
wheat and other crops. Breeding for WUEcanopy is difficult because it is influenced by many component traits. For
example, intrinsic water use efficiency (WUEi), the ratio of net carbon assimilation (Anet) over stomatal
conductance, contributes to WUEcanopy and can be estimated from carbon isotope discrimination (Δ). However, Δ
is not sensitive to differences in the water vapor pressure deficit between the air and leaf (VPDleaf). Alternatively,
measurements of instantaneous leaf water use efficiency (WUEleaf) are defined as Anet over transpiration and can
be determined with gas exchange, but the dynamic nature of field conditions are not represented. Specifically,
fluctuations in canopy temperature lead to changes in VPDleaf that impact transpiration but not Anet. This alters
WUEleaf and in turn affects WUEcanopy. To test this relationship, WUEcanopy was measured in conjunction with
WUEi, WUEcanopy, and canopy temperature under well-watered and water-limited conditions in two drought-
tolerant wheat cultivars that differ in canopy architecture. In this experiment, boundary layer conductance
was low and significant changes in leaf temperature occurred between cultivars and treatments that correlated
with WUEcanopy likely because of the effect of canopy temperature on VPDleaf driving T. However, deviations
between WUEi, WUEleaf, and WUEcanopy were present because measurements made at the leaf level do not account for variations in leaf temperature. This uncoupled the relationship of measured WUE and WUEi from
WUEcanopy and emphasizes the importance of canopy temperature on carbon uptake and transpired water loss.
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Horgan, A. M., Garland-Campbell, K. A., Carter, A. H., & Steber, C. M. (2021).
Seedling elongation responses to gibberellin seed treatments in wheat.
Agrosystems, Geosciences & Environment, 4, 1–13.
(Show/Hide Abstract)
Abstract:
The wheat (Triticum aestivum L.) Reduced height (Rht) alleles are widely used to prevent lodging through semi-dwarfism. Seedling elongation and coleoptile length can
be significantly decreased by some of these alleles, leading to reduced soil emergence
after deep sowing in certain semi-arid environments. Application of the elongation-promoting hormone gibberellin A3 (GA3) as a seed treatment has been used as an
alternative to improve seedling emergence. Seedling responses to GA3 seed treatment
were investigated under controlled conditions in a collection of varieties differing for
Rht dwarfing alleles and the ability to emerge from deep planting. Data between
treated and untreated seed were collected on overall coleoptile length and emergence
from deep planting in simulated pot studies. Gibberellin-sensitive varieties, carrying either no dwarfing gene or the Rht8 dwarfing gene, responded to the GA3 seed
treatment with increased coleoptile and subcrown internode elongation. Comparison of near-isogenic lines carrying no dwarfing allele or the GA-insensitive Rht-B1b
and/or Rht-D1b semi-dwarfing alleles showed that GA insensitivity was associated
with reduced seedling response to GA3 seed treatment. However, there was variation
in seedling elongation and GA3 response in a collection of GA-insensitive varieties.
Thus, it cannot be assumed that all Rht-B1b and Rht-D1b varieties will fail to respond
to GA3 seed treatment. Interestingly, some better emerging GA-insensitive varieties
had longer coleoptiles after treatment, suggesting that selection for better emergence
in semi-arid regions of the U.S. Pacific Northwest may have led to responses in GA3
application independent of the dwarfing gene used.
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Sjoberg, S., Carter, A. H., Steber, C. M., & Garland-Campbell, K. A. (2020).
Unravelling Complex Traits in Wheat: Approaches for Analyzing Genotype × Environment Interactions in a Multi-environment Study of Falling Numbers.
Crop Science, 60, 3013–3026.
(Show/Hide Abstract)
Abstract:
Multi-environment trials provide useful information about highly variable, complex plant traits like yield
and quality, but are difficult to analyze due to their frequently unbalanced nature, with genotypes and
locations varying from year to year. Our objectives were to use multiple approaches, including joint
regression and principal components analysis, to characterize patterns in the genotype by environment
interactions across an unbalanced three-year multi-environment wheat variety trial dataset, examining
falling numbers (FN) test results in wheat. The FN test measures the decrease in flour gelling capacity
resulting from starch digestion by the enzyme α-amylase. Low FN/high α-amylase grain is discounted
because it is associated with poor end-use quality. Low FN can be caused by susceptibility either to
preharvest sprouting when it rains before harvest or to late maturity α-amylase induction by
temperature fluctuations during grain maturation. The most effective and visually intuitive approaches
for selecting varieties with high FN across variable environments was a combination of joint regression,
such as Finlay-Wilkinson and Eberhart and Russell, with biplot methods such as the additive main effects
and multiplicative interaction model (AMMI) and the genotype main effects and genotype ×
environment interaction model (GGE). These results identified stable lines for FN resistance and provide
a means to analyze unbalanced, multi-environment data from breeding and variety trials.
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Shrestha, S. L., Garland-Campbell, K. A., Steber, C. M., & Hulbert, S. H. (2020).
Carbon isotope discrimination association with yield and test weight in Pacific Northwest-adapted spring and winter wheat.
Agrosystems, Geosciences & Environment, 3(1), e20052, (15 pages).
(Show/Hide Abstract)
Abstract:
Due to considerable influence of environment on yield, breeding for drought tolerance could benefit from focusing on selection of more heritable physiological traits, such as carbon isotope discrimination (as measured by delta, Δ) for
indirectly assessing water use efficiency (WUE) of wheat (Triticum aestivum L.).
Spring and winter wheat cultivars were assayed for ∆, and these values were
used to determine the relationships with performance in over 13 environments
in the U.S. Pacific Northwest. The correlation coefficients of Δ values between the
wheat cultivars grown in different environments ranged from 0.11 to 0.73 for both
spring and winter wheat. There was significant genotypic variation for Δ in soft
spring and hard winter wheat but not in hard spring and soft winter wheat. The
Δ values were poor indicators of yield for this set of wheat cultivars in most environments, although low values (better WUE) were sometimes correlated with
yield. A population of 165 hard spring wheat recombinant inbred lines derived
from a cross between two hard spring wheat varieties that differed in Δ was also
screened in low-rainfall dryland and irrigated environments. High yields in this
recombinant inbred population were weakly correlated with high Δ values or
low WUE in most environments.
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Martinez, S. A., Shorinola, O., Conselman, S., See, D., Skinner, D. Z., Uauy, C., & Steber, C. M. (2020).
Exome sequencing of bulked segregants identified a novel TaMKK3‐A allele linked to the wheat ERA8 ABA‐hypersensitive germination phenotype.
Theoretical and Applied Genetics, 133, 719–736.
(Show/Hide Abstract)
Abstract:
Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest.
The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft
white wheat 'Zak'. ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant
analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb
region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a
missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat.
The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is
likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS-
tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population
likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent
complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a
known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation
in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results
from the MKK3-A-G1093A mutation.
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Sjoberg, S. M., Carter, A. H., Steber, C. M., & Garland-Campbell, K. A. (2020)
Application of the factor analytic model to assess wheat falling number performance and stability in multi‐environment trials.
Crop Science, doi:10.1002/csc2.20293.
(Show/Hide Abstract)
Abstract:
A factor analytic model was used to characterize data generated with the Hagberg‐Perten Falling Number (FN) method, a measure of wheat quality influenced by genotype‐by‐environment interactions. The FN method detects starch degradation due to the presence of the enzyme alpha‐amylase in wheat grain such that a low FN indicates high alpha‐amylase activity and high risk of poor end‐product quality. Because farmers receive severe discounts for low FN, FN data has been collected over multiple years for the Washington State University multilocation variety trials to help farmers and breeders identify lower risk varieties. Analysis of this data to objectively rank varieties is challenging because the dataset is unbalanced and because FN is subject to complex genotype‐by‐environment interactions. Low FN can result from environmental differences at multiple stages in grain development because there are two major causes of alpha‐amylase accumulation in grain, late‐maturity alpha‐amylase (LMA) and preharvest sprouting (PHS). A five‐factor analytic model extracted explicit measures of overall performance and of stability in variable environments from historical falling number data from the multilocation trial, providing a basis for breeding and planting decisions. Whereas a linear model explained 70.3% of the variation, the five‐factor analytic model accounted for 92.5% of variation in the data. Examination of factor loadings enabled us to separate environments and genotype response to either PHS or LMA, specifically. This is the first application of a factor analytic model to evaluate the end‐use quality trait, FN, providing a method to rank varieties for grower decisions and for breeder selections.
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Steber, C. M., & Garland Campbell, K. A. (2019, April).
Rising optimism fuels falling numbers research.
Wheat Life, 62(4), 40–52.
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Liu, C. (2019).
Late maturity alpha-amylase in north American spring wheat (Triticum aestivum L.).
(Master's thesis). Washington State University.
(Show/Hide Abstract)
Abstract:
Late maturity alpha-amylase (LMA) is considered a genetic defect in wheat (Triticum aestivum
L.), resulting in the induction of alpha-amylase enzyme in response to a low or high temperature
shock during late grain filling. The wheat industry uses the Hagberg-Perten Falling Number
(FN) method to detect loss of gelling capacity due to the presence of alpha-amylase in meal or
flour. Low FN is associated with higher risk of poor end-product quality, such as cakes that fall
and sticky bread or noodles. To improve selection for LMA resistance, LMA testing methods
were optimized and then used to characterize the LMA susceptibility in North American wheat.
Preliminary LMA testing results were highly variable in cold-treated and in untreated controls.
Warmer (25°C day/ 18°C night) and drier (˜55-65% relative humidity) conditions reduced alpha-
amylase levels in untreated controls. Colder LMA-induction experiments did not result in
stronger or more consistent LMA-induction in spring wheat variety, WA8124. The most
significant cause of LMA phenotypic variability appeared to be variability in the developmental
window during which a 7-day low temperature shock triggered LMA. This window varied with
environmental conditions prior to grain development and by genotype, such that WA8124
induced LMA at 20-24 days past anthesis (dpa) whereas Australian ‘Kennedy’ induced at 25-29
dpa. Only 21% of the 256 North American breeding lines characterized showed LMA
resistance, suggesting that improved selection for LMA resistance is needed. In this panel, the
tall rht-B1a rht-D1a genotype was associated with higher LMA in both cold-treated and
untreated experiments, suggesting the presence of a constitutive LMA phenotype that did not
require cold treatment. However, some rht-B1a rht-D1a lines required cold induction whereas
some semi-dwarf lines had constitutive LMA, suggesting that the constitutive LMA phenotype is
genetically complex. A preliminary genome-wide association study identified six significant
marker-trait associations on chromosomes 2B, 3A, 3B, 5A, 7B, and 7D. The QLMA.wsu.7B
locus detected in this study co-localized a QTL detected in four previous studies of Australian
and CIMMYT germplasm. Future work will determine if these molecular markers are effective
in selecting LMA resistance in U.S. wheat.
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Delwiche, S. R., Higginbotham, R. W., & Steber, C. M.(2018).
Falling number of soft white wheat by near-infrared spectroscopy: A challenge revisited.
Cereal Chemistry, 95(3), 469–477.
(Show/Hide Abstract)
Abstract:
Background and objectives: Wheat Hagberg falling number (FN) is a long-
standing quality test that, by means of measuring the viscosity of a heated water-meal or water-flour mixture, characterizes the activity of endogenous α-amylase,
the enzyme primarily responsible for starch hydrolysis. The accuracy, time
requirement, and cost of this test have come under heightened scrutiny, particularly in seasons when weather conditions have been favorable to preharvest
sprouting or late maturity amylase. Near-infrared (NIR) spectroscopy, an analytical approach routinely used in the grain industry to measure contents of protein
and moisture, was reexamined as a possible alternative to the FN procedure.
Findings: Partial least squares (PLS) regression quantitative models developed on
a genetically diverse set of Washington grown white wheat demonstrated low
accuracy, with standard errors of performance ranging from 40 to 77 s. Alternatively, linear discriminant analysis and PLS discriminant analysis (PLSDA) qualitative models, developed and tested using a FN cutoff (pass/fail) value, also
demonstrated low accuracy, with the best model correctly identifying 67% and
71% of the samples, respectively, above and below a threshold value established
as the median value of FN in a calibration set of several hundred samples.
Conclusions: Replacement of the FN test with one based on NIR spectroscopy
on either whole grain or ground meal for making decisions on segregating wheat
lots according to α-amylase activity is not recommended.
Significance and novelty: Because NIR spectroscopy is not sufficiently accurate
to quantitatively model FN or differentiate low from high FN grain, viscometry procedures for starch integrity, such as FN, will continue their use in grain commerce.
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Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2018, January).
Hunting for genes: falling numbers
project seeks to reduce risk by breeding for genetic resistance.
Wheat Life, 61(1), 50–51.
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Martinez, S. A., Godoy, J., Huang, M., Zhang, Z., Carter, A. H., Garland Campbell, K. A., & Steber, C. M. (2018).
Genome-Wide Association Mapping
for Tolerance to Preharvest Sprouting
and Low Falling Numbers in Wheat.
Frontiers in Plant Science, 9, 1–16, https://doi.org/10.3389/fpls.2018.00141.
(Show/Hide Abstract)
Abstract:
Preharvest sprouting (PHS), the germination of grain on the mother plant under cool and
wet conditions, is a recurring problem for wheat farmers worldwide. α-amylase enzyme
produced during PHS degrades starch resulting in baked good with poor end-use
quality. The Hagberg-Perten Falling Number (FN) test is used to measure this problem
in the wheat industry, and determines how much a farmer’s wheat is discounted for
PHS damage. PHS tolerance is associated with higher grain dormancy. Thus, breeding
programs use germination-based assays such as the spike-wetting test to measure
PHS susceptibility. Association mapping identified loci associated with PHS tolerance
in U.S. Pacific Northwest germplasm based both on FN and on spike-wetting test data.
The study was performed using a panel of 469 white winter wheat cultivars and elite
breeding lines grown in six Washington state environments, and genotyped for 15,229
polymorphic markers using the 90k SNP Illumina iSelect array. Marker-trait associations
were identified using the FarmCPU R package. Principal component analysis was directly
and a kinship matrix was indirectly used to account for population structure. Nine loci
were associated with FN and 34 loci associated with PHS based on sprouting scores.
None of the QFN.wsu loci were detected in multiple environments, whereas six of the 34
QPHS.wsu loci were detected in two of the five environments. There was no overlap
between the QTN detected based on FN and PHS, and there was little correlation
between the two traits. However, both traits appear to be PHS-related since 19 of the 34
QPHS.wsu loci and four of the nine QFN.wsu loci co-localized with previously published
dormancy and PHS QTL. Identification of these loci will lead to a better understanding
of the genetic architecture of PHS and will help with the future development of genomic
selection models.
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Martinez, S. A., Thompson, A. L., Wen, N., Murphy, L., Sanguinet, K. A., Steber, C. M., & Garland Campbell, K. A. (2018).
Registration of the Louise/Alpowa Wheat Recombinant Inbred Line Mapping Population.
Journal of Plant Registrations, doi:10.3198/jpr2017.08.0053crmp.
(Show/Hide Abstract)
Abstract:
A mapping population was developed from the cross of soft
white spring wheat (Triticum aestivum L.) cultivars 'Louise'
and 'Alpowa' for use in investigating the genetic architecture
of drought tolerance in the US Pacific Northwest. The Louise/Alpowa (Reg. No. MP-8, NSL 520824 MAP) recombinant inbred
line mapping population was developed through single
seed descent from the F2 generation to the F5 generation.
The population consists of 141 F5:6 recombinant inbred lines,
of which 132 were used to construct the genetic linkage
map. The 32 linkages groups included 882 single nucleotide
polymorphism markers and one simple sequence repeat
marker spanning 18 of 21 chromosomes. The Louise/Alpowa
population was characterized for variation in agronomic
traits, phenology, and end-use quality traits. This population
will be used for identification and introgression of multiple
loci providing resistance to environmental stress such as
drought, stripe rust, and high temperatures.
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Steber, C. M., & Campbell, K. G. (2017, November).
All hands on deck: USDA-ARS, WSU scientists tackle falling numbers.
Wheat Life, 60(10), 40–41.
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Steber, C. M. (2017, March-April).
Avoiding problems in wheat with low Falling Numbers.
Crops & Soils Magazine, (50)2, 22–25.
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Martinez, S. A., Tuttle K. M., Takebayashi, Y., Seo, M., Campbell, K. G., & Steber, C. M. (2016).
The wheat ABA hypersensitive ERA8 mutant is associated with increased preharvest sprouting tolerance and altered hormone accumulation.
Euphytica, 212(2), 229–245.
(Show/Hide Abstract)
Abstract:
Wheat preharvest sprouting (PHS) is the germination of mature grain on the mother plant when rain occurs before harvest. Higher abscisic acid (ABA) hormone levels and sensitivity are associated with higher seed dormancy and PHS tolerance. Consistent with this, the ABA hypersensitive ENHANCED RESPONSE TO ABA8 (ERA8) mutant resulted in increased dormancy and PHS tolerance in soft white spring wheat 'Zak'. ERA8 seeds were initially less responsive to germination-rescue by the hormone gibberellin (GA). ERA8 gained GA and lost ABA sensitivity more slowly than wild-type during dormancy loss through after-ripening and cold imbibition. This study examined if increased ABA sensitivity in ERA8 likely resulted from increased ABA signaling or increased ABA hormone levels. Zak ERA8 had higher initial grain dormancy although endogenous embryo ABA levels were similar in Zak ERA8 and wild-type, suggesting that increased dormancy was due to increased ABA signaling rather than increased ABA accumulation. ABA levels declined with Zak ERA8 after-ripening, suggesting that ABA turnover is not defective. Elevated ERA8 dormancy was also associated with increased embryonic jasmonic acid-Ile and aleurone indole-3-acetic acid (IAA) levels. The possible implication that other plant hormones may influence wheat seed dormancy and germination are discussed.
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Steber, C. M., Carter, A. H., & Pumphrey, M. O. (2016, December).
Get out your hard hats, the numbers are falling.
Wheat Life, 59(11), 47–49.
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Steber, C. M. (2016).
Managing the risk of low falling numbers in wheat.
Washington State University Extension, FS242E, 1–6.
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Tuttle, K. M., Martinez, S. A., Schramm, E. C., Takebayashi, Y., Seo, M., & Steber, C. M. (2015).
Grain dormancy loss is associated with changes in ABA and GA sensitivity and hormone accumulation in bread wheat, Triticum aestivum (L.).
Seed Science Research, 25(2), 179–193.
(Show/Hide Abstract)
Abstract: Knowledge about the hormonal control of grain dormancy and dormancy loss is essential in wheat, because low grain dormancy at maturity is associated with the problem of pre-harvest sprouting (PHS) when cool and rainy conditions occur before harvest. Low GA (gibberellin A) hormone sensitivity and high ABA (abscisic acid) sensitivity were associated with higher wheat grain dormancy and PHS tolerance. Grains of two PHS-tolerant cultivars were very dormant at maturity, and insensitive to GA stimulation of germination. More PHS-susceptible cultivars were less sensitive to ABA inhibition of germination, and were either more GA sensitive or germinated efficiently without GA at maturity. As grain dormancy was lost through dry afterripening or cold imbibition, grains first gained GA sensitivity and then lost ABA sensitivity. These changes in GA and ABA sensitivity can serve as landmarks defining stages of dormancy loss that cannot be discerned without hormone treatment. These dormancy stages can be used to compare different cultivars, seed lots and studies. Previous work showed that wheat afterripening is associated with decreasing ABA levels in imbibing seeds. Wheat grain dormancy loss through cold imbibition also led to decreased endogenous ABA levels, suggesting that reduced ABA signalling is a general mechanism triggering dormancy loss.
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Martinez, S. A., Schramm, E. C., Harris, T. J., Kidwell, K. K., Garland-Campbell, K., & Steber, C. M. (2014).
Registration of Zak ERA8 Soft White Spring Wheat Germplasm with Enhanced Response to ABA and Increased Seed Dormancy.
Journal of Plant Registrations, 8(2), 217–220.
(Show/Hide Abstract)
Zak ERA8 (ENHANCED RESPONSE to ABA8) (Reg. No. GP-966, PI 669443) is a unique line derived from soft white spring wheat (Triticum aestivum L.) cultivar Zak that has increased seed dormancy but after-ripens within 10 to 16 wk. The goal in developing this germplasm was to use increased seed dormancy to improve tolerance to preharvest sprouting, a problem that can cause severe economic losses. This germplasm was developed by USDA–ARS, Pullman, WA, in collaboration with Washington State University. Zak ERA8 was tested under experimental number 60.1.27.10. The ERA8 mutation was generated by chemical mutagenesis followed by selection for the inability to germinate on abscisic acid (ABA) concentrations too low to inhibit wild-type Zak seed germination. The semidominant Zak ERA8 line has been backcrossed twice to wild-type Zak. Following the first backcross, Zak ERA8 showed similar morphological and grain quality traits to the original Zak cultivar.
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Schramm, E. C., Nelson, S. K., Kidwell, K. K., & Steber, C. M. (2013).
Increased ABA sensitivity results in higher seed dormancy in soft white spring wheat cultivar ‘Zak’.
Theoretical and Applied Genetics, 126(3), 791–803.
(Show/Hide Abstract)
Abstract: As a strategy to increase the seed dormancy of soft
white wheat, mutants with increased sensitivity to the plant hormone abscisic
acid (ABA) were identified in mutagenized grain of soft white spring wheat
“Zak”. Lack of seed dormancy is correlated with increased susceptibility to
preharvest sprouting in wheat, especially those cultivars with white kernels.
ABA induces seed dormancy during embryo maturation and inhibits the germination
of mature grain. Three mutant lines called Zak ERA8, Zak ERA19A, and Zak ERA19B
(Zak ENHANCED RESPONSE to ABA) were recovered based on failure to germinate on 5
μM ABA. All three mutants resulted in increased ABA sensitivity over a wide
range of concentrations such that a phenotype can be detected at very low ABA
concentrations. Wheat loses sensitivity to ABA inhibition of germination with
extended periods of dry after-ripening. All three mutants recovered required
more time to after-ripen sufficiently to germinate in the absence of ABA and to
lose sensitivity to 5 μM ABA. However, an increase in ABA sensitivity could
be detected after as long as 3 years of after-ripening using high ABA
concentrations. The Zak ERA8 line showed the strongest phenotype and segregated
as a single semi-dominant mutation. This mutation resulted in no obvious
decrease in yield and is a good candidate gene for breeding preharvest sprouting
tolerance.
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Schramm, E. C., Nelson, S. K., & Steber, C. M. (2012).
Wheat ABA-insensitive mutants result in reduced grain dormancy.
Euphytica, 188(1), 35–49.
(Show/Hide Abstract)
Abstract:
This paper describes the isolation of wheat mutants in the hard red
spring Scarlet resulting in reduced sensitivity to the plant hormone abscisic
acid (ABA) during seed germination. ABA induces seed dormancy during embryo
maturation and inhibits the germination of mature seeds. Wheat sensitivity to
ABA gradually decreases with dry after-ripening. Scarlet grain normally fails to
germinate when fully dormant, shows ABA sensitive germination when partially
after-ripened, and becomes ABA insensitive when after-ripened for 8–12 months.
Scarlet ABA-insensitive (ScABI) mutants were isolated based on the ability to
germinate on 5 μM ABA after only 3 weeks of after-ripening, a condition under
which Scarlet would fail to germinate. Six independent seed-specific mutants
were recovered. ScABI1, ScABI2, ScABI3 and ScABI4 are able to germinate more
efficiently than Scarlet at up to 25 μM ABA. The two strongest ABA
insensitive lines, ScABI3 and ScABI4, both proved to be partly dominant
suggesting that they result from gain-of-function mutations. The ScABI1, ScABI2,
ScABI3, ScABI4, and ScABI5 mutants after-ripen more rapidly than Scarlet. Thus,
ABA insensitivity is associated with decreased grain dormancy in Scarlet wheat.
This suggests that ABA sensitivity is an important factor controlling grain
dormancy in wheat, a trait that impacts seedling emergence and pre-harvest
sprouting resistance.
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Schramm, E. C., Abellera, J. C., Strader, L. C., Campbell, K. G., & Steber, C. M. (2010).
Isolation of ABA-responsive mutants in allohexaploid bread wheat ( Triticum
aestivum L.): Drawing connections to grain dormancy, preharvest sprouting,
and drought tolerance.
Plant Science, 179(6), 620–629.
(Show/Hide Abstract)
Abstract:
This paper describes the isolation of Wheat ABA-responsive mutants (Warm)
in the Chinese Spring background of allohexaploid Triticum aestivum L.
Because the hormone abscisic acid (ABA) is required for the
induction of seed dormancy, stomatal closure, and drought, cold and salt
tolerance, ABA-hypersensitive
wheat mutants were expected to show increased seed dormancy and decreased leaf
transpiration in drying soils. Lack of wheat grain dormancy is associated with a
propensity for preharvest sprouting (PHS),
the germination of seed on the mother plant when moist conditions persist before
harvest. PHS tolerance correlates with higher seed dormancy resulting from red
grain color, higher ABA accumulation and
sensitivity. Wheat grain loses dormancy and sensitivity to ABA inhibition of
seed germination with afterripening. Warm lines maintained higher ABA
sensitivity when partially after-ripened. The Warm1 and
Warm4 mutants showed the strongest and most reproducible increase in ABA
sensitivity, accompanied
by a requirement for more prolonged after-ripening to break dormancy. Warm2,
Warm3, Warm5, and
Warm6 showed normal germination without ABA but increased sensitivity to
inhibition of germination
by applied ABA. The Warm4 mutant showed decreased whole plant
transpiration in drying soil, consistent
with the role of ABA in inhibiting vegetative leaf transpiration.
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Schramm, E. C. (2010).
Isolation of
ABA-response mutants in allohexaploid bread wheat (Triticum aestivum L.):
Drawing connections to grain dormancy, preharvest sprouting, and drought
tolerance
(Doctoral dissertation). Washington State University.
(Show/Hide Abstract)
Abstract:
This dissertation describes the characterization of seed dormancy and drought
tolerance
phenotypes of abscisic acid (ABA) response mutants in allohexaploid wheat
(Triticum aestivum
L.). The plant hormone ABA is required for the induction of seed dormancy,
stomatal closure in
drying soils, and drought tolerance. Preharvest sprouting (PHS), the
germination of seed on the
mother plant, occurs when cool moist conditions persist before harvest. PHS
resistance is
correlated with seed dormancy resulting from red grain color, higher ABA
accumulation or
sensitivity. ABA only inhibits the germination of imbibing dormant wheat grain,
not of afterripened grain. ABA hypersensitive lines were isolated based on the
phenotype of maintaining
higher ABA sensitivity when partially after-ripened. ABA hypersensitive
phenotypes were
stronger and persisted longer in the soft white spring 'Zak' compared to the
hard red springs
Chinese Spring and 'Scarlet'. ZakERA (Zak
Enhanced Response to ABA) 0 and 19A mutants
showed increased dormancy associated with a reproducible increase in ABA
sensitivity in
germination. These mutants required more time to after-ripen than Zak wild type
(WT), and still
showed ABA sensitivity after 3 years after-ripening. Warm1 (Wheat
ABA-responsive mutant)
and Warm4 showed the strongest ABA-hypersensitive germination phenotype
among Chinese
Spring mutants, and were accompanied by decreased germination in the absence of
ABA.
Although these mutants required more time to after-ripen than WT, they lost ABA
sensitivity
within 3-12 months. ScERA (Scarlet
Enhanced Response to ABA) 10A, 68A, 10C, and 4A
showed the strongest ABA-hypersensitive germination phenotype. ScERA 4A is
unique in
having enhanced ABA sensitivity without any apparent increase in seed dormancy.
ABA
insensitive mutants in Scarlet, ScABI (Scarlet
ABA-Insensitive) 2A, 2C, 1B, and 1C, were
insensitive to ABA inhibition of germination with little or no after-ripening.
ABA mutants are
also expected to show altered water relations. All of the ScERA or ScABI
mutants tested so far
appear to have only seed germination phenotypes. Consistent with the role of
ABA in downregulating transpiration in drying soils, Warm3, Warm4, and
ZakERA 19A are associated with
decreased leaf transpiration. In Warm4 and ZakERA 19A this decrease in
transpiration is
associated with higher canopy temperatures under field conditions.
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Schramm, E. C., Abellera, J. C., Strader, L. C., Campbell, K. G., & Steber,
C. M. (2008).
Can ABA signaling be used to develop drought tolerant wheat?
In R. Appels, R. Eastwood, E. Lagudah, P. Langridge, M. Mackay, L. MacIntyre
& P. Sharp (Eds.)
11th International Wheat Genetics Symposium 2008 Proceedings – Volume
1 (pp. 116–118).
Sydney, Australia: Sydney University Press.
(Show/Hide Abstract)
Abstract:
Wheat yield and quality can be compromised by drought
stress and preharvest sprouting (PHS) on the mother
plant. It is believed that problems with PHS are due to
lack of grain dormancy partly resulting from lack of
ABA sensitivity. The long term goal is to use wheat
mutants with increased sensitivity to ABA to increase
grain dormancy and drought tolerance. ABA is required
to set up grain dormancy and embryo desiccation
tolerance during embryo maturation, and stimulate
storage of nutrients. ABA also inhibits germination of
mature seeds and stimulates stomatal closure in response
to drought stress. A screen for wheat mutants with
altered response to ABA in seed germination has been
used as a first step to isolate wheat plants with increased
ABA sensitivity. Mutants have been characterized for
changes in ABA dose-response. In addition, ABA
hypersensitive grain germination appears to correspond
to reduced vegetative transpiration under drought stress
and decreased carbon isotope discrimination.
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Abellera, J. C. (2006).
ABA Responsive when Afterripened (ARA) mutants increase seed dormancy, ABA
sensitivity, and improve water relations: towards wheat varieties with improved
preharvest sprouting and drought tolerance.
(Master's thesis). Washington State University.
(Show/Hide Abstract)
Abstract:
This thesis explores the hypothesis that mutants isolated based on ABA (abscisic
acid) hypersensitive seed germination can be used to obtain drought tolerance
and resistance to preharvest sprouting through increased seed dormancy in allohexaploid wheat.
The plant hormone ABA is required for establishment of dormancy during seed development,
stimulates stomatal closure in response to drought stress and induces expression
of genes that promote osmotic adjustment. Arabidopsis mutants isolated for ABA hypersensitive
seed germination have increased seed dormancy and drought tolerance resulting from
increased sensitivity of stomates to ABA. Based on this result, wheat mutants showing
increased sensitivity to ABA in germination of afterripened grain have been isolated and
tested for improved vegetative drought tolerance. A total of 25 mutants were isolated from
fastneutron mutagenized Chinese Spring, whereas four and 27 mutants were
isolated from EMS mutagenized cv. Zak and cv. Scarlet, respectively. Of the 25
Chinese Spring mutants, 4 demonstrated a clear and reproducible hypersensitive response to ABA in
dose-response germination assays, whereas 2 lines showed embryo dormancy. The remaining
mutants showed inconsistent phenotypes over seven retest experiments suggesting
variable expressivity. Genetic analysis of Chinese Spring mutants showed that two are
the result of a single dominant mutation, whereas three others may be single semi-dominant
mutations. Mutants with a vegetative ABA-hypersensitive phenotype should close their
stomates earlier in response to drought stress and have slower transpiration. Drought tolerance
was evaluated by comparing the mutant rate of soil moisture loss during drought stress,
stomatal conductance, and carbon isotope discrimination (Δ13C) to
wild-type. Of eight Chinese Spring mutants tested, four showed a slower transpiration rate under drought stress,
and one of the four showed reduced stomatal conductance compared to wild-type. The four
mutants with ABA-hypersensitive seed germination were the same four mutants that showed lower
rate of soil moisture loss under drought stress. Five of 19 mutants appeared to have
reduced Δ13C relative to wild type, suggesting that they may have improved transpiration
efficiency.
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Nelson, S. (2005).
Isolating drought tolerant wheat using germination screens for increased ABA hormone sensitivity
(Honors thesis). Washington State University.
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Strader, L. C., Zale, J., & Steber, C. M. (2004).
Sivb 2003 congress symposium proceeding: Mutation- and transposon-based
approaches for the identification of genes for pre-harvest sprouting in
wheat.
In Vitro Cellular & Developmental Biology - Plant, 40(3), 256–259.
(Show/Hide Abstract)
Abstract:
This article reviews techniques for gene identification and cloning in
allohexaploid bread wheat (Triticum aestivum L.).
Gene identification and cloning in wheat are complicated by the large size and
high redundancy of the genome. Both
classical mutagenesis and transposon tagging are important tools for the study
of grain dormancy and plant hormone
signaling in wheat. While classical mutagenesis can be used to identify wheat
mutants with altered hormone sensitivity, it
can be difficult to clone the corresponding genes. We review the techniques
available for gene identification in wheat, and
propose that transposon-based activation tagging will be an important tool for
wheat genetics.
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Hauvermale, A. L., Cárdenas, J. J., Bednarek, S. Y., & Steber, C. M. (2022).
GA signaling expands: The plant UBX domain-containing protein 1 is a binding partner for the GA receptor.
Plant Physiology, 190(4), 2651–2670.
(Show/Hide Abstract)
Abstract:
The plant Ubiquitin Regulatory X (UBX) domain-containing protein 1 (PUX1) functions as a negative regulator of gibberel-
lin (GA) signaling. GAs are plant hormones that stimulate seed germination, the transition to flowering, and cell elongation
and division. Loss of Arabidopsis (Arabidopsis thaliana) PUX1 resulted in a ''GA-overdose'' phenotype including early flowering, increased stem and root elongation, and partial resistance to the GA-biosynthesis inhibitor paclobutrazol during seed
germination and root elongation. Furthermore, GA application failed to stimulate further stem elongation or flowering onset suggesting that elongation and flowering response to GA had reached its maximum. GA hormone partially repressed
PUX1 protein accumulation, and PUX1 showed a GA-independent interaction with the GA receptor GA-INSENSITIVE
DWARF-1 (GID1). This suggests that PUX1 is GA regulated and/or regulates elements of the GA signaling pathway.
Consistent with PUX1 function as a negative regulator of GA signaling, the pux1 mutant caused increased GID1 expression
and decreased accumulation of the DELLA REPRESSOR OF GA1-3, RGA. PUX1 is a negative regulator of the hexameric
AAA + ATPase CDC48, a protein that functions in diverse cellular processes including unfolding proteins in preparation
for proteasomal degradation, cell division, and expansion. PUX1 binding to GID1 required the UBX domain, a binding motif
necessary for CDC48 interaction. Moreover, PUX1 overexpression in cell culture not only stimulated the disassembly of
CDC48 hexamer but also resulted in co-fractionation of GID1, PUX1, and CDC48 subunits in velocity sedimentation assays.
Based on our results, we propose that PUX1 and CDC48 are additional factors that need to be incorporated into our understanding of GA signaling.
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Hauvermale, A. L., & Steber, C. M. (2020).
GA signaling is essential for the embryo-to-seedling transition during Arabidopsis seed germination, a ghost story.
Plant Signaling & Behavior, 15(1), e1705028 (9 pages).
(Show/Hide Abstract)
Abstract:
The plant hormone gibberellin (GA) stimulates developmental transitions including seed germination,
flowering, and the transition from juvenile to adult growth stage. This study provided evidence that GA
and the GA receptor GID1 (GA-INSENSITIVE DWARF1) are also needed for the embryo-to-seedling transition in
Arabidopsis. The ga1-3 GA biosynthesis mutant fails to germinate unless GA is applied, whereas the gid1abc
triple mutant fails to germinate because it cannot perceive endogenous or applied GA. Overexpression of
the GID1a, GID1b, and GID1c GA receptors rescued the germination of a small percentage of ga1-3 seeds
without GA application, and this rescue was improved by dormancy-breaking treatments, after-ripening and
cold stratification. While GID1 overexpression stimulated ga1-3 seed germination, this germination was
aberrant suggesting incomplete rescue of the germination process. Cotyledons emerged before the radicle,
and the resulting “ghost” seedlings failed to develop a primary root, lost green coloration, and eventually
died. The development of ga1-3 seedlings overexpressing GID1 was rescued by pre-germinative but not
post-germinative GA application. Since the gid1abc mutant also exhibited a ghost phenotype after germination
was rescued by cutting the seed coat, we concluded that both GA and GID1 are needed for the embryo-to-seedling
transition prior to emergence from the seed coat.
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Ge, W., & Steber, C. M. (2018).
Positive and negative regulation of seed germination by the
Arabidopsis GA hormone receptors, GID1a, b, and c.
Plant Direct, 2(9), 1–11.
(Show/Hide Abstract)
Abstract:
Epistasis analysis of gid1 single and double mutants revealed that GID1c is a key
positive regulator of seed germination, whereas the GID1b receptor can negatively
regulate germination in dormant seeds and in the dark. The GID1 GA receptors were
expected to positively regulate germination because the plant hormone gibberellin
(GA) is required for seed germination in Arabidopsis thaliana. The three GA hormone
receptors, GID1a, GID1b, and GID1c, positively regulate GA responses via GA/GID1‐
stimulated destruction of DELLA (Asp‐Glu‐Leu‐Leu‐Ala) repressors of GA responses.
The fact that the gid1abc triple mutant but not gid1 double mutants fail to germi-
nate indicates that all three GA receptors can positively regulate non‐dormant seed
germination in the light. It was known that the gid1abc triple mutant fails to lose
dormancy through the dormancy breaking treatments of cold stratification (moist
chilling of seeds) and dry after‐ripening (a period of dry storage). Previous work sug-
gested that there may be some specialization of GID1 gene function during germina-
tion because GID1b mRNA expression was more highly induced by after‐ripening,
whereas GID1a and GID1c mRNA levels were more highly induced by cold stratifica-
tion. In light‐germinated dormant seeds, the gid1b mutation can partly rescue the
germination efficiency of gid1a but not of gid1c seeds. Thus, GID1b can function as
an upstream negative regulator GID1c, a positive regulator of dormant seed germina-
tion. Further experiments showed that GID1b can negatively regulate dark germina-
tion. Wild‐type Arabidopsis seeds do not germinate well in the dark. The gid1b and
gid1ab double mutants germinated much more efficiently than wild type, gid1c, or
gid1ac mutants in the dark. The observation that the gid1ab double mutant also
shows increased dark germination suggests that GID1b, and to some extent GID1a,
can act as upstream negative regulators of GID1c. Since the gid1abc triple mutant
failed to germinate in the dark, it appears that GID1c is a key downstream positive
regulator of dark germination. This genetic analysis indicates that the three GID1
receptors have partially specialized functions in GA signaling.
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Nelson, S. K., Ariizumi, T., & Steber, C. M. (2017).
Biology in the Dry Seed:
Transcriptome Changes Associated
with Dry Seed Dormancy and
Dormancy Loss in the Arabidopsis
GA-Insensitive sleepy1-2 Mutant.
Frontiers in Plant Science, 8, 1–21, https://doi.org/10.3389/fpls.2017.02158.
(Show/Hide Abstract)
Abstract:
Plant embryos can survive years in a desiccated, quiescent state within seeds. In
many species, seeds are dormant and unable to germinate at maturity. They acquire
the capacity to germinate through a period of dry storage called after-ripening (AR),
a biological process that occurs at 5–15% moisture when most metabolic processes
cease. Because stored transcripts are among the first proteins translated upon water
uptake, they likely impact germination potential. Transcriptome changes associated with
the increased seed dormancy of the GA-insensitive sly1-2 mutant, and with dormancy
loss through long sly1-2 after-ripening (19 months) were characterized in dry seeds.
The SLY1 gene was needed for proper down-regulation of translation-associated genes
in mature dry seeds, and for AR up-regulation of these genes in germinating seeds.
Thus, sly1-2 seed dormancy may result partly from failure to properly regulate protein
translation, and partly from observed differences in transcription factor mRNA levels.
Two positive regulators of seed dormancy, DELLA GAI (GA-INSENSITIVE) and the
histone deacetylase HDA6/SIL1 (MODIFIERS OF SILENCING1) were strongly AR-down-
regulated. These transcriptional changes appeared to be functionally relevant since loss
of GAI function and application of a histone deacetylase inhibitor led to decreased
sly1-2 seed dormancy. Thus, after-ripening may increase germination potential over
time by reducing dormancy-promoting stored transcript levels. Differences in transcript
accumulation with after-ripening correlated to differences in transcript stability, such
that stable mRNAs appeared AR-up-regulated, and unstable transcripts AR-down-regulated. Thus, relative transcript levels may change with dry after-ripening partly as
a consequence of differences in mRNA turnover.
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Nelson, S. K, & Steber, C. M. (2017).
Transcriptional mechanisms associated with seed dormancy and dormancy loss in the gibberellin-insensitive sly1-2 mutant of Arabidopsis thaliana.
PLoS ONE 12(6), 1–32, https://doi.org/10.1371/journal.pone.0179143.
(Show/Hide Abstract)
Abstract:
While widespread transcriptome changes were previously observed with seed dormancy loss,
this study specifically characterized transcriptional changes associated with the increased
seed dormancy and dormancy loss of the gibberellin (GA) hormone-insensitive sleepy1-2
(sly1-2) mutant. The SLY1 gene encodes the F-box subunit of an SCF E3 ubiquitin ligase
needed for GA-triggered proteolysis of DELLA repressors of seed germination. DELLA over-
accumulation in sly1-2 seeds leads to increased dormancy that can be rescued without
DELLA protein destruction either by overexpression of the GA receptor, GA-INSENSITIVE
DWARF1b (GID1b-OE) (74% germination) or by extended dry after-ripening (11 months, 51%
germination). After-ripening of sly1 resulted in different transcriptional changes in early versus
late Phase II of germination that were consistent with the processes known to occur. Approxi-
mately half of the transcriptome changes with after-ripening appear to depend on SLY1-trig-
gered DELLA proteolysis. Given that many of these SLY1/GA-dependent changes are genes
involved in protein translation, it appears that GA signaling increases germination capacity in
part by activating translation. While sly1-2 after-ripening was associated with transcript-level
changes in 4594 genes over two imbibition timepoints, rescue of sly1-2 germination by GID1b-
OE was associated with changes in only 23 genes. Thus, a big change in sly1-2 germination
phenotype can occur with relatively little change in the global pattern of gene expression during
the process of germination. Most GID1b-OE-responsive transcripts showed similar changes
with after-ripening in early Phase II of imbibition, but opposite changes with after-ripening by
late Phase II. This suggests that GID1b-OE stimulates germination early in imbibition, but may
later trigger negative feedback regulation.
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Nelson, S. K, & Steber, C. M. (2016).
Gibberellin hormone signal perception: down-regulating DELLA repressors of plant growth and development.
In P. Hedden & S. G. Thomas (Eds), Annual Plant Reviews, Volume 49, The Gibberellins (pp. 153–187). Chichester, UK: Wiley Blackwell.
(Show/Hide Abstract)
Abstract:
The gibberellin (GA) hormone signal is perceived by a receptor with
homology to hormone-sensitive lipases, GID1 (GA-INSENSITIVE DWARF1). This
leads to GA-stimulated responses, including stem elongation, seed germination
and the transition to flowering. GA-binding enables GID1 to interact with and
block the function of the DELLA repressors of GA responses. DELLA repression
can be blocked both by proteolytic and non-proteolytic mechanisms triggered by
the formation of a GID1-GA-DELLA complex. DELLA is down-regulated by the
SLEEPY1/GID2 F-box proteins via the ubiquitin-proteasome pathway, and can
be regulated by other post-translational modifications. This chapter reviews the
structural requirements for GA-binding by GID1 and for GID1-GA-DELLA protein complex formation, and reviews the current understanding of the mechanisms
regulating DELLA repressors.
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Hauvermale, A. L., Tuttle, K. M., Takebayashi, Y., Seo, M., and Steber, C. M. (2015).
Loss of Arabidopsis thaliana seed dormancy is associated with increased accumulation of the GID1 GA hormone receptors.
Plant and Cell Physiology, 56(9), 1773–1785.
(Show/Hide Abstract)
Abstract:
Dormancy prevents seeds from germinating under favorable conditions until they have experienced dormancy-breaking conditions, such as after-ripening through a period of dry storage or cold imbibition. Abscisic acid (ABA) hormone signaling establishes and maintains seed dormancy, whereas gibberellin (GA) signaling stimulates germination. ABA levels decrease and GA levels increase with after-ripening and cold stratification. However, increasing GA sensitivity may also be critical to dormancy loss since increasing seed GA levels are detectable only with long periods of after-ripening and imbibition. After-ripening and cold stratification act additively to enhance GA hormone sensitivity in ga1-3 seeds that cannot synthesize GA. Since the overexpression of the GA receptor GID1 (GA-INSENSITIVE DWARF1) enhanced this dormancy loss, and because gid1a gid1b gid1c triple mutants show decreased germination, the effects of dormancy–breaking treatments on GID1 mRNA and protein accumulation were examined. Partial after-ripening resulted in increased GID1b, but not GID1a or GID1c mRNA levels. Cold imbibition stimulated the accumulation of all three GID1 transcripts, but resulted in no increase in GA sensitivity during ga1-3 seed germination unless seeds were also partially after-ripened. This is likely because after-ripening was needed to enhance GID1 protein accumulation, independently of transcript abundance. The rise in GID1b transcript with after-ripening was not associated with decreased ABA levels, suggesting there is ABA-independent GID1b regulation by after-ripening. GA and the DELLA RGL2 repressor of GA responses differentially regulated the three GID1 transcripts. Moreover, DELLA RGL2 appeared to switch between positive and negative regulation of GID1 expression in response to dormancy breaking treatments. The rise in GID1b transcript with after-ripening was not associated with decreased ABA levels, suggesting that after-ripening regulates GID1 independently of ABA.
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Hauvermale, A. L., Ariizumi, T., & Steber, C. M. (2014). The roles of the GA receptors GID1a, GID1b, and GID1c in sly1-independent GA signaling. Plant Signaling & Behavior 9:e28030.
(Show/Hide Abstract)
Abstract: Gibberellin (GA) hormone signaling occurs through proteolytic and non-proteolytic mechanisms. GA binding to the GA receptor GID1 (GA-INSENSITIVE DWARF1) enables GID1 to bind negative regulators of GA responses called DELLA proteins. In proteolytic GA signaling, the SLEEPY1 (SLY1) F-box protein targets DELLA proteins in the GID1-GA-DELLA complex for destruction through the ubiquitin-proteasome pathway. Non-proteolytic GA signaling occurs in sly1 mutants when GA cannot target DELLA proteins for destruction, and requires GA and GID1 gene function. Based on comparison of gid1 multiple mutants to sly1 gid1 mutants, GID1a is the primary GA receptor stimulating stem elongation in proteolytic and non-proteolytic signaling, and stimulating fertility in proteolytic GA signaling. GID1b plays the primary role in fertility, and a secondary role in elongation during non-proteolytic GA signaling. The stronger role of GID1b in non-proteolytic GA signaling
may result from the fact that GID1b has higher affinity for DELLA protein than GID1a and GID1c.
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Ariizumi, T., Hauvermale, A. L., Nelson, S. K., Hanada, A., Yamaguchi, S., & Steber, C. M. (2013).
Lifting DELLA repression of Arabidopsis seed germination by non-proteolytic GA signaling.
Plant Physiology, 162(4), 2125–2139.
(Show/Hide Abstract)
Abstract: DELLA repression of Arabidopsis thaliana seed germination can be lifted either through DELLA proteolysis by the ubiquitin-proteasome pathway or through proteolysis-independent GA hormone signaling. GA-binding to the GID1 (GIBBERELLIN-INSENSITIVE DWARF1) GA receptors stimulates GID1-GA-DELLA complex formation which in turn triggers DELLA protein ubiquitination and proteolysis via the SCFSLY1 E3 ubiqutin ligase and 26S proteasome. Although DELLA cannot be destroyed in the sly1-2 F-box mutant, long dry after-ripening and GID1 overexpression can relieve the strong sly1-2 seed dormancy phenotype. It appears that sly1-2 seed dormancy results from ABA signaling downstream of DELLA since dormant sly1-2 seeds accumulate high levels of ABA hormone and loss of ABA sensitivity rescues sly1-2 seed germination. DELLA positively regulates the expression of XERICO, an inducer of ABA biosynthesis. GID1b overexpression rescues
sly1-2 germination through proteolysis-independent DELLA down-regulation associated with increased expression of GA-inducible genes and decreased ABA accumulation, apparently as a result of decreased XERICO mRNA levels. Higher levels of GID1 overexpression are associated with more efficient sly1 germination and increased GID1-GA-DELLA complex formation, suggesting that GID1 downregulates DELLA through protein binding. After-ripening results in increased GA accumulation and GID1a-dependent GA signaling, suggesting that after-ripening triggers GA-stimulated GID1-GA-DELLA protein complex formation which in turn blocks DELLA transcriptional activation of the XERICO inhibitor of seed germination.
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Ariizumi, T., Lawrence, P. K., & Steber, C. M. (2011).
The role of two F-box proteins, SLEEPY1 and SNEEZY, in arabidopsis gibberellin
signaling.
Plant Physiology, 155(2), 765–775.
(Show/Hide Abstract)
Abstract:
The SLEEPY1 (SLY1) F-box gene is a positive
regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana).
Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced
fertility, delayed flowering, and increased seed
dormancy. These sly1 phenotypes are partially rescued by overexpression
of the SLY1 homolog SNEEZY (SNE)/SLY2,
suggesting that SNE can functionally replace SLY1. GA responses
are repressed by DELLA family proteins. GA relieves
DELLA repression when the SCFSLY1
(for Skp1, Cullin, F-box) E3 ubiquitin ligase ubiquitinates DELLA protein,
thereby targeting it for proteolysis. Coimmunoprecipitation experiments using
constitutively expressed 35S:hemagglutinin (HA)-SLY1
and 35S:HA-SNE translational fusions in the sly1-10 background suggest
that SNE can function similarly to SLY1 in GA
signaling. Like HA-SLY1, HA-SNE interacted with the CULLIN1 subunit of the SCF
complex, and this interaction required the
F-box domain. Like HA-SLY1, HA-SNE coimmunoprecipitated with the DELLA REPRESSOR
OF GA1-3 (RGA), and this
interaction required the SLY1 or SNE carboxyl-terminal domain. Whereas
HA-SLY1 overexpression resulted in a decrease in
both DELLA RGA and RGA-LIKE2 (RGL2) protein levels, HA-SNE caused a
decrease in DELLA RGA but not in RGL2 levels.
This suggests that one reason HA-SLY1 is able to effect a stronger rescue
of sly1-10 phenotypes than HA-SNE is because SLY1
regulates a broader spectrum of DELLA proteins. The FLAG-SLY1 fusion protein was
found to coimmunoprecipitate with the
GA receptor HA-GA-INSENSITIVE DWARF1b (GID1b), supporting the model that SLY1
regulates DELLA through interaction
with the DELLA-GA-GID1 complex.
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Ariizumi, T., & Steber, C. M. (2011).
Mutations in the F-box gene SNEEZY result in decreased Arabidopsis GA signaling.
Plant Signaling & Behavior, 6(6), 831–833.
(Show/Hide Abstract)
Abstract:
We previously reported that the
SLEEPY1 (SLY1) homolog, F-box
gene SNEEZY/SLEEPY2 (SNE/SLY2),
can partly replace SLY1 in gibberellin
(GA) hormone signaling through interaction with DELLAs RGA and GAI. To
determine whether SNE normally functions in GA signaling, we
characterized
the phenotypes of two T-DNA alleles,
sne-t2 and sne-t3. These mutations result
in no apparent vegetative phenotypes,
but do result in increased ABA sensitivity in seed germination. Double mutants
sly1-t2 sne-t2 and sly1-t2 sne-t3 result in a
significant decrease in plant fertility and
final plant height compared to sly1-t2.
The fact that sne mutations have an additive effect with sly1
suggests that SNE
normally functions as a redundant positive regulator of GA signaling.
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Ariizumi, T., Murase, K., Sun, T.-p., & Steber, C. M. (2008).
Proteolysis-independent downregulation of DELLA repression in Arabidopsis
by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1.
The Plant Cell, 20(9), 2447–2459.
(Show/Hide Abstract)
Abstract:
This article presents evidence that DELLA repression of gibberellin (GA)
signaling is relieved both by proteolysis-dependent and -independent pathways in
Arabidopsis thaliana. DELLA proteins are negative regulators of GA
responses, including seed germination, stem elongation, and fertility. GA
stimulates GA responses by causing DELLA repressor degradation via the
ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three
functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1
(GID1a, b, and c), and the SLEEPY1 (SLY1) F-box
subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more
DELLA proteins but display less severe dwarf and germination phenotypes than the
GA biosynthesis mutant ga1-3 or the gid1abc triple mutant.
Interestingly,
GID1 overexpression rescued the sly1 dwarf and infertility
phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF
ga1-3. GID1 rescue of sly1 mutants was dependent on the level of
GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA
shows increasing interaction with GID1 with increasing GA levels, it appears
that GA-bound GID1 can block DELLA repressor activity by direct protein–protein
interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA
signaling may function without DELLA degradation.
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Ariizumi, T., & Steber, C. M. (2007).
Seed germination of GA-insensitive sleepy1 mutants does not require RGL2
protein disappearance in Arabidopsis.
The Plant Cell, 19(3), 791–804.
(Show/Hide Abstract)
Abstract:
We explore the roles of gibberellin (GA) signaling genes SLEEPY1
(SLY1) and RGA-LIKE2 (RGL2) in regulation of seed
germination in Arabidopsis thaliana, a plant in which the hormone GA is
required for seed germination. Seed germination failure
in the GA biosynthesis mutant ga1-3 is rescued by GA and by mutations in
the DELLA gene RGL2, suggesting that RGL2
represses seed germination. RGL2 protein disappears before wild-type seed
germination, consistent with the model that GA
stimulates germination by causing the SCFSLY1
E3 ubiquitin ligase complex to trigger ubiquitination and destruction of RGL2.
Unlike ga1-3, the GA-insensitive sly1 mutants show variable seed
dormancy. Seed lots with high seed dormancy after-ripened
slowly, with stronger alleles requiring more time. We expected that if RGL2
negatively controls seed germination, sly1 mutant
seeds that germinate well should accumulate lower RGL2 levels than those failing
to germinate. Surprisingly, RGL2
accumulated at high levels even in after-ripened sly1 mutant seeds with
100% germination, suggesting that RGL2 disappearance is not a prerequisite for
seed germination in the sly1 background. Without GA, several GA-induced
genes show increased
accumulation in sly1 seeds compared with ga1-3. It is possible
that the RGL2 repressor of seed germination is inactivated by
after-ripening of sly1 mutant seeds.
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Dill, A., Thomas, S. G., Hu, J., Steber, C. M., & Sun, T.-p. (2004).
The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation.
The Plant Cell, 16(6), 1392–1405.
(Show/Hide Abstract)
Abstract:
The nuclear DELLA proteins are highly conserved repressors of hormone
gibberellin (GA) signaling in plants. In Arabidopsis
thaliana, GA derepresses its signaling pathway by inducing proteolysis of
the DELLA protein REPRESSOR OF ga1-3 (RGA).
SLEEPY1 (SLY1) encodes an F-box–containing protein, and the
loss-of-function sly1 mutant has a GA-insensitive dwarf
phenotype and accumulates a high level of RGA. These findings suggested that SLY1
recruits RGA to the SCFSLY1
E3 ligase
complex for ubiquitination and subsequent degradation by the 26S proteasome. In
this report, we provide new insight into
the molecular mechanism of how SLY1 interacts with the DELLA proteins for
controlling GA response. By yeast two-hybrid
and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with
RGA and GA INSENSITIVE (GAI, a closely
related DELLA protein) via their C-terminal GRAS domain. The rga and
gai null mutations additively suppressed the
recessive sly1 mutant phenotype, further supporting the model that
SCFSLY1
targets both RGA and GAI for degradation. The
N-terminal DELLA domain of RGA previously was shown to be essential for
GA-induced degradation. However, we found
that this DELLA domain is not required for protein–protein interaction with SLY1
in yeast (Saccharomyces cerevisiae),
suggesting that its role is in a GA-triggered conformational change of the DELLA
proteins. We also identified a novel gain-of-function sly1-d mutation that
increased GA signaling by reducing the levels of the DELLA protein in plants.
This effect of
sly1-d appears to be caused by an enhanced interaction between sly1-d and
the DELLA proteins.
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Strader, L. C., Ritchie, S., Soule, J. D., McGinnis, K. M., & Steber, C. M. (2004).
Recessive-interfering mutations in the gibberellin signaling gene SLEEPY1 are rescued by overexpression of its homologue, SNEEZY.
Proc. Natl. Acad. Sci. U.S.A., 101(34), 12771–12776.
(Show/Hide Abstract)
Abstract:
This article reports the genetic interaction of two F-box genes,
SLEEPY1 (SLY1) and SNEEZY (SNE), in Arabidopsis
thaliana gibberellin (GA) signaling. The SLY1 gene encodes an F-box
subunit of
a Skp1–cullin–F-box (SCF) E3 ubiquitin ligase complex that
positively regulates GA signaling. The sly1-2 and sly1-10 mutants
have
recessive, GA-insensitive phenotypes including delayed germination, dwarfism,
reduced fertility, and overaccumulation of the
DELLA proteins RGA (Repressor of ga1–3), GAI (GA-Insensitive), and
RGL2 (RGA-Like 2). The DELLA domain proteins are putative transcription factors
that negatively regulate GA signaling. The requirement for
SLY1 in GA-stimulated disappearance of DELLA
proteins suggests that GA targets DELLA proteins for destruction
via SCFSLY1-mediated ubiquitylation. Overexpression of SLY1 in
sly1-2 and sly1-10 plants rescues the recessive GA-insensitive
phenotype of these mutants. Surprisingly, antisense expression of
SLY1 also suppresses these mutants. This result caused us to
hypothesize that the SLY1 homologue SNE can functionally replace
SLY1 in the absence of the recessive interfering sly1-2 or
sly1-10
genes. This hypothesis was supported because overexpression of
SNE in sly1-10 rescues the dwarf phenotype. In addition to
rescuing
the sly1-10 dwarf phenotype, SNE overexpression also restored
normal RGA protein levels, suggesting that the SNE F-box protein
can replace SLY1 in the GA-induced proteolysis of RGA. If the
C-terminal truncation in the sly1-2 and sly1-10 alleles interferes
with SNE rescue, we reasoned that overexpression of sly1-2 might
interfere with wild-type SLY1 function. Indeed, overexpression of
sly1-2 in wild-type Ler (Landsberg erecta) yields dwarf plants.
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Strader, L. C. (2004).
Hormonal control of seed dormancy and germination: drawing connections
between Arabidopsis thaliana L. and Triticum aestivum L.
(Doctoral dissertation). Washington State University.
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McGinnis, K. M., Thomas, S. G., Soule, J. D., Strader, L. C., Zale, J. M., Sun, T.-p., & Steber, C. M. (2003).
The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase.
The Plant Cell, 15(5), 1120–1130.
(Show/Hide Abstract)
Abstract:
The Arabidopsis SLY1 (SLEEPY1) gene positively regulates
gibberellin (GA) signaling. Positional cloning of SLY1 revealed
that it encodes a putative F-box protein. This result suggests that SLY1 is the
F-box subunit of an SCF E3 ubiquitin ligase
that regulates GA responses. The DELLA domain protein RGA (repressor of
ga1-3) is a repressor of GA response that appears to undergo
GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because
sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This
result suggests SCFSLY1-targeted degradation of RGA through the 26S proteasome pathway. Further support
for this model is provided by the observation that an rga
null allele partially suppresses the sly1-10 mutant phenotype. The
predicted SLY1 amino acid sequence is highly conserved
among plants, indicating a key role in GA response.
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Steber, C. M., & McCourt, P. (2001).
A role for Brassinosteroids in germination in Arabidopsis.
Plant Physiology, 125(2), 763–769.
(Show/Hide Abstract)
Abstract:
This paper presents evidence that plant brassinosteroid (BR) hormones play a
role in promoting germination. It has long
been recognized that seed dormancy and germination are regulated by the plant
hormones abscisic acid (ABA) and
gibberellin (GA). These two hormones act antagonistically with each other. ABA
induces seed dormancy in maturing
embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes
germination. Severe mutations in GA
biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement
for GA application to germinate. Whereas previous
work has shown that BRs play a critical role in controlling cell elongation,
cell division, and skotomorphogenesis, no
germination phenotypes have been reported in BR mutants. We show that BR rescues
the germination phenotype of severe
GA biosynthetic mutants and of the GA-insensitive mutant
sleepy1. This result shows that BR stimulates germination and
raises the possibility that BR is needed for normal germination. If true, we
would expect to detect a germination phenotype
in BR mutants. We found that BR mutants exhibit a germination phenotype in the
presence of ABA. Germination of both the
BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1
is more strongly inhibited by ABA than is germination
of wild type. Thus, the BR signal is needed to overcome inhibition of
germination by ABA. Taken together, these results
point to a role for BRs in stimulating germination.
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Steber, C. M., Cooney, S. E., & McCourt, P. (1998).
Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1
in Arabidopsis thaliana.
Genetics, 149(2), 509–521.
(Show/Hide Abstract)
Abstract:
Seed dormancy and germination in higher plants are partially controlled by the
plant hormones abscisic
acid (ABA) and gibberellic acid (GA). ABA establishes dormancy during embryo
maturation, whereas GA
breaks dormancy and induces germination. Previous attempts to identify GA
response genes were confounded because GA mutants are not expected to germinate
and, unlike GA auxotrophs, should fail to
be rescued by exogenous GA. Here, we describe a screen for suppressors of the
ABA-insensitive mutant
ABI1-1 that enriches for GA auxotrophs and GA-insensitive mutants. The
vast majority (76%) of the
suppressors of ABI1-1 strongly resemble GA auxotrophs in that they are
severely dwarfed and have dark
green foliage and flowers with underdeveloped petals and stamen. Three isolates
were alleles of the GA
auxotroph ga1. The remaining severe dwarves were not rescued by GA and
belong to a single complementation group that we designate
sly1 (Sleepy 1). The alleles of sly1 identified are
the first recessive GAinsensitive mutations to reflect the full spectrum of
GA-associated phenotypes, including the failure to
germinate in the absence of the ABI1-1 lesion. Thus, we postulate that
SLY1 is a key factor in GA reception.