Progress 07/01/12 to 07/01/17
Outputs
Target Audience:Our findings are of interest to those interested in strategies to improve crop tolerance to environmental stress through altering factors that alter photosynthesis and abiotic stress responses, specifically with regard to water limitations, issues highly relevant to the irrigation-based agriculture of California. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training to a graduate student during their first year rotation in which they learned techniques involving genotyping mutants. How have the results been disseminated to communities of interest?Our findings were published in a peer reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?With these objectives, we will determine whether genetic interactions between PABP and other factors required for protein expression may also affect plant growth and yield under normal growth conditions and in response to conditions of limited water. This will provide a more complete picture of the requirements for maintaining VDE expression, photoprotection, and crop yield during abiotic stress conditions that increasingly challenge California agriculture.
Impacts
What was accomplished under these goals?
Following transcription and mRNA processing, during which a poly(A) tail is added to most eukaryotic, nuclear-encoded mRNAs, the level of protein expressed is determined by the stability and translational efficiency of an mRNA. The PABP, which binds to the poly(A) tail was first identified as important in determining the stability of an mRNA. Subsequently, PABP, which is bound at the 3'-end of an mRNA, was found to promote translation initiation through its physical and functional interaction with the translation initiation factor (eIF) 4G. PABP is highly conserved throughout eukaryotes as is its physical and functional interaction with eIF4G. In addition to its role in translation, PABP increases mRNA stability by inhibiting DCP1/2-mediated decapping. While yeast expresses just a single and essential PABP, PABP in Arabidopsis is expressed by a highly divergent, multigene family composed of eight members that can be grouped into three classes based on sequence similarity and gene structure. Three members of the family, i.e., PAB2, PAB4, and PAB8, comprise class II PABP genes which are widely expressed, particularly in vegetative tissues. Expression of class I genes (PAB3 and PAB5) is restricted to reproductive tissues whereas class III genes (PAB6 and PAB7) exhibit weak and restricted expression. PAB1, originally considered an "orphan gene" is actually a class I gene expressed weakly in roots, pollen, and during late embryo development. The presence of a large gene family containing divergent members in plants and in the Brassicaceae in particular, raises the possibility of functional specialization as PABP is involved in the processing, nucleocytoplasmic transport, stability, and translation of mRNAs. Given the importance of factors regulating protein expression to photosynthesis and abiotic stress responses, the function of the widely-expressed class II PABP family members were examined to determine how individual class II members contribute to plant growth and development. Of the three class II PABP members, PAB2 and PAB4 contribute most to vegetative growth and vegetative-to-floral transition whereas PAB2, and the recently-evolved third class II member, PAB8, contribute to inflorescence and silique growth. Interestingly, although class I and class III PABP members are expressed specifically in reproductive organs, class II PABP members are also necessary for fertility in that the combinatorial loss of PAB2 and either PAB4 or PAB8 expression resulted in reduced fertility. Although all three class II members are required for protein expression, PAB4 contributes most to the steady-state level of a reporter mRNA and to protein expression. These findings suggest that class II PABP members are partially overlapping in function but also involved in distinct aspects of plant growth and development.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Gallie D.R. 2017. Class II members of the poly(A) binding protein family exhibit distinct functions during Arabidopsis growth and development. Translation 5(1): e1295129. PMID: 28702277
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Our findings are of interest to those interested in strategies to improve crop tolerance to environmental stress through altering factors that alter photosynthesis and abiotic stress responses, specifically with regard to water limitations, issues highly relevant to the irrigation-based agriculture of California. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training to a visiting professor from Brazil during his sabbatical year in my laboratory (concluded June 30, 2016) in which he learned techniques involving measuring ethylene responses. How have the results been disseminated to communities of interest?Our findings were published in a peer reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?With these objectives, we will determine whether alterations in other factors associated with eIFiso4G may also affect plant growth and yield under normal growth conditions and in response to conditions of limited water. This will provide a more complete picture of the requirements for maintaining VDE expression, photoprotection, and crop yield during abiotic stress conditions that increasingly challenge California agriculture.
Impacts
What was accomplished under these goals?
The synthesis of proteins from cellular mRNAs requires several translation initiation factors that assist in recruiting the 40S ribosomal subunit to an mRNA as well as in the recognition of the initiation codon and in the assembly of the 80S ribosome. Eukaryotic initiation factor (eIF) 4F is essential to promote 40S subunit binding to an mRNA and to assist in 40S subunit scanning of the 5′-leader in search of the initiation codon. eIF4F is a multisubunit factor composed of eIF4E, which binds the 5′-cap structure of an mRNA; the RNA helicase, eIF4A, which unwinds the secondary structure in a 5′-leader in an mRNA that would otherwise inhibit 40S subunit scanning during protein synthesis; and the scaffolding protein, eIF4G, which interacts with several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, differences do exist between these two isoform classes as loss of eIFiso4G but not eIF4G results in reduced photosynthesis and growth. Loss of eIFiso4G expression results in an increase in the transcript and protein levels of violaxanthin de-epoxidase, which as part of the xanthophyll cycle, is critical in protecting photosystem (PS) II function and protecting photosynthetic membranes against photodamage. Loss of eIFiso4G but not eIF4G also results in changes to abiotic stress such as water stress. Additionally, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5'-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. Given the importance of eIFiso4G to photosynthesis and abiotic stress responses, we have investigated the roles that the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation. This may have implications for the role of each eIFiso4G isoform in photosynthesis and abiotic stress responses.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Gallie D.R. 2016. Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis. J. Biol. Chem. 291(3):1501-13. PMID: 26578519
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Our findings are of interest to those interested in strategies to improve crop tolerance to environmental stress through altering ethylene hormone signaling by targeting those regulators of the ethylene signaling pathway involved in controlling drought tolerance and photosynthesis, issues highly relevant to the irrigation-based agriculture of California. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training to one junior level scientist who was involved in working on the control of photoprotective mechanisms controlled by ethylene that affect crop yield in response to abiotic stress. The project also provided training to a visiting professor from Brazil during his sabbatical year in my laboratory in which he learned techniques involving measuring ethylene responses. How have the results been disseminated to communities of interest?Our findings were published in three peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?With these objectives, we will determine whether restoring VDE expression maintains better growth and yield in crop species under normal growth conditions and in response to conditions of limited water. This will identify best practices for maintaining VDE expression, photoprotection, and crop yield during ethylene-mediated conditions of stress that increasingly challenge California agriculture. We will also examine whether 1-MCP application or the use of ethylene receptor mutants that confer a state of ethylene insensitivity maintain VDE expression under normal growth conditions and in response to conditions of limited water in order to provide protection against the deleterious effects of ethylene on crop growth.
Impacts
What was accomplished under these goals?
Photosynthesis converts absorbed light energy into chemical energy. The capacity of a plant to use absorbed light energy for photochemistry, however, is limited. Consequently, plants have evolved mechanisms to avoid the over reduction of the photosystems that would otherwise result in the generation of triplet state chlorophyll that can transfer energy to ground-state oxygen to produce highly destructive singlet oxygen and other reactive oxygen species (ROS). Over reduction of the photosystems can also result in the generation of destructive ROS through the Mehler reaction. One mechanism that functions to prevent the over excitation of photosystem (PS) II involves pH-dependent, feedback de-excitation of singlet excited chlorophyll molecules in PSII. This feedback de-excitation, referred to as qE, is one of several processes that include photoinhibition (qI) that contribute to non-photochemical quenching (NPQ). qE requires de-epoxidized xanthophyll pigments, PsbS which is a PSII subunit belonging to the light harvesting complex protein superfamily, and an acidic thylakoid lumen. Stress conditions can inhibit photosynthesis and limit the amount of absorbed light energy that can be used for photochemistry. Such conditions can result in an elevated induction of qE in an attempt to dissipate excess excitation energy to protect against the over reduction of PSII. Under conditions of excess light, however, qI, which includes quenching resulting from damage to PSII reaction centers, can also contribute substantially to NPQ. Abiotic stresses, such as drought and elevated temperatures increase ROS generation and photodamage. The xanthophyll cycle contributes significantly to qE for which violaxanthin de-epoxidase (VDE) is critical and not only protects PSII function but also protects photosynthetic membranes against against photodamage. We have shown that drought-induced increases in ethylene signaling impairs leaf performance and thus crop yield in response to drought. We also demonstrated that ethylene represses photosynthesis in part through repressing expression of VDE resulting in reduced photosynthetic activity and growth. This ethylene-mediated repression of photosynthesis can be corrected by restoring VDE expression which reverses growth inhibition imposed by ethylene without affecting ethylene production. In related work, we developed related technology to target stress responses in corn by blocking binding to ethylene receptors either pharmacologically with the application of 1-methylcyclopropene (1-MCP) which binds to and blocks the binding of ethylene to the receptor binding site or through the generation of ethylene receptor mutants that inhibit transmission of the signal. Our findings demonstrate by targeting ethylene through several difference approaches, we can limit the repression of photosynthesis imposed by ethylene during adverse environmental conditions. We had previously shown how eIFiso4G, a scaffold protein that organizes the assembly of those initiation factors needed to recruit the 40S ribosomal subunit to an mRNA, regulates VDE expression. In the current period, we show that of the two eIF4G isoforms expressed in plants, loss of the eIFiso4G1 isoform did not substantially affect translation from an unstructured mRNA whereas loss of the eIFiso4G2 isoform substantially reduced such translation. We discovered that eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. We demonstrated that loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during translation of specific mRNAs.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Gallie D.R. 2015. Appearance and elaboration of the ethylene receptor family during land plant evolution. Plant Mol. Biol. 87:521-539. PMID: 25682121.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Gallie D.R. 2015. Ethylene receptors in plants - why so much complexity? F1000 Prime Rep. 7:39. PMID: 26171216.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Chen, Z., and Gallie, D.R. 2015. Ethylene regulates energy-dependent non-photochemical quenching in Arabidopsis through repression of the xanthophyll cycle. PLoS One 10:e0144209. PMID: 26630486.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Our findings are of interest to those interested in strategies to improve crop tolerance to environmental stress through altering ethylene hormone signaling by targeting those regulators of the ethylene signaling pathway involved in controlling drought tolerance and photosynthesis, issues highly relevant to the irrigation-based agriculture of California. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided training to two junior level scientists who were involved in working on the control of photoprotective mechanisms controlled by ethylene that affect crop yield in response to abiotic stress. The project also provided training to an undergraduate student in learning the techniques involved in research involving ethylene responses. How have the results been disseminated to communities of interest? Our findings were published in peer reviewed journals and as two patents. What do you plan to do during the next reporting period to accomplish the goals? We will continue the work on controlling ethylene responses that affect crop yield in response to abiotic stress by examining how ethylene affects crop production through photosynthesis that drives growth and ultimately crop yield. We will examine how ethylene affects maize growth and how inhibiting ethylene responses can provide protection against the deleterious effects of ethylene on growth. We will continue our work with plant PDCD4-like proteins in how they regulate the ethylene response and how mutants of plant PDCD4-like proteins may aid in altering ethylene responses during exposure to abiotic stress.
Impacts
What was accomplished under these goals?
The autotrophic nature of plants relies on their ability to transform absorbed light energy into chemical energy through photosynthesis in order to fix atmospheric CO2 into sugars which are used to support growth. Plants use only a fraction of the total light energy they absorb as the capacity of photosynthesis is limited. Levels of light energy exceeding the capacity of photosynthesis can result in the production of reactive oxygen species that can damage the photosynthetic machinery. Given the potential for light-induced damage (ROS), plants have evolved mechanisms to deal with excess absorbed light energy to reduce ROS generation. In addition to quenching as a result of photochemistry (qP), absorbed light energy can be quenched through its dissipation as heat (qE), used in photoinhibition (qI), or re-emitted as light as a consequence of chlorophyll fluorescence. qE and qI are components of non-photochemical quenching (NPQ) which limits the generation of ROS by protecting the photosystems from over reduction. Under most growth conditions, qE predominates, is rapidly reversible, and results in the pH-dependent dissipation of excess excitation energy as heat before it is used to generate ROS. Under high light conditions or during conditions of stress, e.g., drought, ozone exposure, or chilling, qI, which mostly results from photodamage to the reaction centers, can be become significant. Thus, NPQ plays an important role in modulating photosynthetic performance. Induction of qE is accompanied by de-epoxidation of violaxanthin into zeaxanthin (or antheraxanthin) as part of the xanthophyll cycle. Xanthophyll pigments are required for NPQ and can also scavenge ROS directly. Violaxanthin de-epoxidase (VDE) is the enzyme responsible for the de-epoxidation of violaxanthin and antheraxanthin into zeaxanthin. We discovered that this process is regulated by ethylene such that during environmental stress such as drought, this photoprotective mechanism is repressed resulting in increased ROS production. Moreover, we investigated how this process is controlled at the level of protein synthesis by examining the role of eIFiso4G in plant growth. Protein synthesis is facilitated through the action of several translation initiation factors. Recruitment of the 40 S subunit eIF4G which is a scaffolding protein that interacts with several other initiation factors. Plants express an isoform of of eIF4G called eIFiso4G which is unique in that it is substantially smaller than eIF4G and shares only limited similarity to other eIF4G proteins. We discovered that an eIFiso4G loss-of-function mutant exhibits substantially reduced photosynthetic activity that correlates with a smaller cell, leaf, plant size, and biomass accumulation. The reduced photochemistry of eifiso4g1/2 plants was not a result of a change in the level of photorespiration or dark respiration but did correlate with lower chlorophyll levels and elevated levels of the qE and qI components of NPQ, processes that divert absorbed light energy from use in photochemistry. The increase in qE correlated with an increase in the activity of VDE which was a result of its greater expression in the mutant which was reflected at the transcript and protein levels. Our findings indicate that loss of eIFiso4G expression affects the appropriate regulation of VDE expression which is not affected by loss of eIF4G expression and that eIFiso4G expression is important to maintain photosynthetic activity.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Chen Z, Jolley B, Caldwell C, Gallie D.R. 2014. Eukaryotic translation initiation factor eIFiso4G is required to regulate violaxanthin de-epoxidase expression in Arabidopsis. J. Biol. Chem. 289:13926-13936. PMID: 24706761.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Gallie, D.R. 2014. Insights from a paradigm shift: how the poly(A) binding protein brings translating mRNAs full circle. New Journal of Science 2014: 1-16. Article ID 873084
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Gallie, D.R. 2014. The role of the poly(A) binding protein in the assembly of the cap-binding complex during translation initiation in plants. Translation 2: 1-14. e959378
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Our findings are of interest to those interested in strategies to improve crop tolerance to environmental stress through altering ethylene hormone signaling by targeting those regulators of the ethylene signaling pathway involved in controlling salt tolerance, an issue highly relevant to the irrigation-based agriculture of California. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project provided training to two junior level scientists who were involved in working onbreeding approaches to control ethylene responses that affect crop yield in response to abiotic stress.The project also provided training to an undergraduate student in learning the techniques involved in research involvingethylene responses. How have the results been disseminated to communities of interest? The work was published in peer reviewed journals and as an patent application. What do you plan to do during the next reporting period to accomplish the goals? We will continue the work on controlling ethylene responses that affect crop yield in response to abiotic stress by examining how ethylene affects crop production through photosynthesis that drives growth and ultimately crop yield. We will examine howethylene affects maize growth and how 1-MCP can provide protection against the deleterious effects ofethylene on maize growth. We will continue our work with theplant PDCD4-like proteins in how they regulate theethylene response and how mutants ofplant PDCD4-like proteins may aid in alteringethylene responses during exposure to abiotic stress.
Impacts
What was accomplished under these goals?
The programmed cell death 4 (PDCD4) protein is induced upon programmed cell death in several cell types in mice, including lymphoid and neuronal cells. Increasing PDCD4 expression inhibits tumor promoter-induced neoplastic transformation while reducing PDCD4 expression resulted in a transformation-sensitive phenotype and the promotion of tumor invasion. PDCD4 proteins in higher plants are unique in that they contain four tandem MA3 domains. One such four MA3 domain PDCD4 protein (ECIP1) binds Arabidopsis thaliana ethylene receptors, ETR2 and EIN4, as well as EIN2, a downstream component of the ethylene signaling pathway required for the induction of ethylene responses. Loss of ECIP1 expression resulted in increased ethylene sensitivity and tolerance to salt. When the PDCD4 homolog containing four MA3 domains first appeared during plant evolution has not been examined. We examined the evolution of plant PDCD4-like proteins from a two MA3 domain form in prasinophytes to the appearance of a four MA3 domain form in charophytes and land plants and the likely independent appearance of a four MA3 domain form in Chlamydomonadales. We also examined the expansion and divergence of the PDCD4-like gene family during plant evolution and identify how one distinct gene family member appeared following the evolution of basal angiosperms. The plant PDCD4-like proteins also bind the eukaryotic translation initiation factor (eIF) 4G which functions as a scaffold protein that assembles components of the translation initiation complex to an mRNA. Therefore, we examined how eIF4G assembles these components in plants. We discovered that in older algal lineages, PDCD4 contains two MA3 domains similar to the homolog in animals. By the appearance of early land plants, however, PDCD4 is composed of four MA3 domains which likely is the result of a duplication of the two MA3 domain form of the protein. Evidence from fresh water algae, from which land plants evolved, suggests that the duplication event occurred prior to the colonization of land. PDCD4 in more recently evolved chlorophytes also contains four MA3 domains but this may have resulted from an independent duplication event. Expansion and divergence of the PDCD4 gene family occurred during land plant evolution with the appearance of a distinct gene member following the evolution of basal angiosperms. We also discovered that eIF4G differs from the eIF4G isoform, eIFiso4G, in that it contains two distinct interaction domains for the poly(A) binding protein (PABP) and eIF4B but is similar to eIFiso4G in having two eIF4A interaction domains. PABP and eIF4B bind the same N-terminal region of eIF4G as they do to a region C-proximal to the HEAT-1 domain in the middle domain of eIF4G, resulting in competitive binding between eIF4B and PABP to each site. eIF4G also differs from eIFiso4G in that no competitive binding was observed between PABP and eIF4A or between eIF4B and eIF4A to its HEAT-1-containing region. These results demonstrate that despite substantial differences in size, sequence, and domain organization, PABP and eIF4B bind to eIF4G and eIFiso4G competitively.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Cheng, S., Liu, R., and Gallie, D.R. 2013. The unique evolution of the programmed cell death 4 protein in plants. BMC Evolutionary Biology 13(1):199, 1-16.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Cheng, S., and Gallie, D.R. 2013. Eukaryotic initiation factor 4B and the poly(A)-binding protein bind eIF4G competitively. Translation 1:1, 1-13.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Gallie, D.R. 2013. The role of L-ascorbic acid recycling in responding to environmental stress and in promoting plant growth. Journal of Experimental Botany 64: 433-443.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Gallie, D.R. 2013. L-Ascorbic acid: a multifunctional molecule supporting plant growth and development. Scientifica 2013: 124.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Gallie, D.R. 2013. Increasing vitamin c content in plant foods to improve their nutritional value successes and challenges. Nutrients 5: 3424-3446.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Tiruneh BS, Kim BH, Gallie, D.R. Roy B, and von Arnim AG. 2013. The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant. BMC Biology 11(1):123. PMID: 24377433
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Although plants absorb light energy for the purpose of converting it to chemical energy through photosynthesis, the absorption of light energy can also be deleterious when it exceeds the capacity to use it for photochemistry. Plants have evolved multiple mechanisms to avoid over reduction of the photosystems. The xanthophyll cycle functions to thermally dissipate excess absorbed light energy through the pH-dependent, feedback de-excitation of singlet excited chlorophyll molecules in Photosystem II. Violaxanthin de-epoxidase (VDE) is responsible for the de-epoxidation of xanthophyll pigments. While inactive in the dark, VDE is activated by light and requires vitamin C (ascorbic acid) as a co-factor. The goal of our work was to examine whether the endogenous level of VDE was limiting with regard to phototolerance and whether increasing expression of VDE can provide greater phototolerance to improve crop productivity. Ascorbic acid is thought to play a role in pathogenesis by reducing reactive oxygen species produced by the host as part of its defense response. To examine its role in infection by Alternaria brassicicola, an ascomycete fungus that causes black spot disease on cultivated Brassicas, we examined the effect of changes in ascorbic acid on pathogenesis A. brassicicola. Once ascorbic acid has been used, it is oxidized to eventually produce dehydroascorbate which can be reduced to ascorbic acid by dehydroascorbate reductase (DHAR) using glutathione as the reductant. Because ascorbic acid has been reported to promote cell division, we also investigated whether ascorbic acid regulates cell division during embryo development in plants overexpressing DHAR which increases the level of ascorbic acid. PARTICIPANTS: Dr. Daniel R. Gallie, PI Dr. Zhong Chen, Associate Research Biochemist - performed experiments on VDE regulation of photosynthesis and the role of ascorbic acid on infection and embryo development. Jane Glazebrook, Professor in the Department of Plant Biology and Microbial and Plant Genomics Institute, University of Minnesota. Christopher J. Botanga, Gerit Bethke, You Lv, staff in the Department of Plant Biology and Microbial and Plant Genomics Institute, University of Minnesota. Oliver Fiehn, Professor in the University of California at Davis Genome Center. TARGET AUDIENCES: Our findings are of interest to those interested in strategies to improve tolerance to environmental stress and fungal pathogens as well to those interested in strategies to increase seed production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We show that decreasing VDE expression increases photosensitivity whereas overexpression of VDE increased the de-epoxidation state of xanthophyll pigments as well as the initial induction of NPQ and its level achieved under subsaturating light conditions. It did not substantially alter the xanthophyll de-epoxidation state or level of NPQ achieved under saturating light. Under conditions of chilling which reduce VDE activity, however, increasing VDE expression did result in greater phototolerance. Thus increasing VDE expression is a useful strategy in improving tolerance to high light in combination with chilling. We also show that mutants deficient in ascorbic acid were more susceptible to infection by the fungal pathogen, A. brassicicola. Ascorbate levels declined during infection demonstrating that the level of ascorbic acid determines disease susceptibility. We also observed that a high level of DHAR expression induced monozygotic twinning and polycotyly. Twinning induced by DHAR resulted from altered cell polarity and longitudinal instead of transverse cell division of the zygote, generating embryos of equal size. Direct injection of ascorbic acid into ovaries phenocopied the DHAR-induced twinning demonstrating that it was the product of DHAR activity that was responsible for altered embryo development. Monozygotic twinning induced by ascorbic acid was developmentally limited to the first two days after pollination, consistent with its role in altering zygotic division. Similarly, polycotyly was induced when ascorbic acid levels were elevated just prior to cotyledon initiation. Our results demonstrate that the level of DHAR expression, and therefore, the level of ascorbic acid regulates cell polarity during embryo development.
Publications
- Chen, Z., and Gallie, D.R. 2012. Violaxanthin de-epoxidase is rate-limiting for non-photochemical quenching under subsaturating light or during chilling in Arabidopsis. Plant Physiol Biochem. 58C: 66-82.
- Botanga, C.J., Bethke, G., Chen, Z., Gallie, D.R., Fiehn, O., Glazebrook, J. 2012. Metabolite profiling of Arabidopsis inoculated with Alternaria brassicicola reveals that ascorbate reduces disease severity. Mol. Plant Microbe Interact. 25: 1628-1638.
- Chen, Z., and Gallie, D.R. 2012. Induction of monozygotic twinning by ascorbic Acid in tobacco. PLoS One.7: e39147.
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Ethylene serves as an important hormone controlling several aspects of plant growth and development which is perceived following its binding to membrane-localized receptors. Although the effect of ethylene on photosynthesis had been little studied and what had been reported was inconclusive, our work in maize, in which ethylene production had been reduced through the loss of expression of members of the ACS gene family, demonstrated that ethylene does indeed regulate photosynthetic activity. The xanthophyll cycle contributes significantly to qE and involves the light-induced de-epoxidation of violaxanthin (Vx) to antheraxanthin (Ax) and zeaxanthin (Zx) by violaxanthin de-epoxidase (VDE). The generation of Zx is critical for the full induction of NPQ and is involved in singlet oxygen scavenging as well as singlet chlorophyll (1Chl*) quenching. VDE, a 43 kD protein encoded by the nuclear gene, NPQ1, is transported to the thylakoid lumen where its activity is light regulated. While inactive in the dark, VDE is activated by the reduction in pH resulting from proton pumping across the thylakoid membrane that occurs commensurate with the light-driven electron movement through the photosynthetic electron transport chain. As a consequence, VDE associates with the thylakoid membrane where it interacts with its substrate Vx. The activation of VDE in the acidified lumen also involves a conformational change of the protein and its dimerization that enables simultaneous access to the two epoxide rings of Vx for their de-epoxidation. We have investigated how ethylene regulates photosynthesis through its regulation of the xanthophyll cycle. As a consequence of our findings, we have generated ethylene insensitive maize to examine the effects on photosynthesis and on yield. PARTICIPANTS: Dr. Daniel R. Gallie, PI Dr. Zhong Chen, Post-doc - performed experiments on ethylne regulation of photosynthesis Ms. Juifen Chen, Graduate student - performed experiments on ethylene receptors in maize Christian Caldwell, Technician - assisted with experiments on maize ethylene receptors TARGET AUDIENCES: Our findings increase the understanding of how ethylene regulates photosynthesis and thus increase the ability to manage stress responses in maize in such a way that responses to environmental stress can be altered in a tissue-specific manner in order to maintain crop yield. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have discovered that ethylene regulates photosynthesis by repressing VDE expression which results in a defective functioning of the xanthophyll cycle, aberrant induction of NPQ following exposure to light, generation of reactive oxygen species, which in turn damages the photosynthetic machinery, and results in reduced growth and lower yield. We have also observed that the ethylene-mediated repression of photosynthesis can be corrected by inhibiting ethylene perception, e.g., following the application of 1-MCP. Restoring VDE expression reduced photoinhibition to wild-type levels. Restoring VDE expression also reversed the repression of photosynthesis without affecting ethylene production. Our results demonstrate that the functioning of the xanthophyll cycle is impaired by increased ethylene signaling which, as a consequence, increases photosensitivity and contributes to reduced crop performance. These observations suggest that ethylene regulates photosynthesis through mechanisms that protect the photosynthetic machinery from photodamage and increased ethylene production results in increased photosensitivity and a reduction in crop performance. Moreover, maize insensitive to ethylene showed increases in yield.
Publications
- No publications reported this period
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Ethylene serves as an important hormone controlling several aspects of plant growth and development which is perceived following its binding to membrane-localized receptors. One such mutant, i.e., etr1-1, results in a strong ethylene insensitive phenotype. With this work, regulated expression of the Arabidopsis etr1-1 in tomato was achieved using an inducible promoter. In the absence of the inducer, transgenic seedlings remained sensitive to ethylene but in its presence, the level of ethylene sensitivity could be controlled by the amount of inducer used, repressing the epinastic response to ethylene as well as delaying fruit ripening. Restoration of ethylene sensitivity was achieved following the cessation of the induction. We also introduced a Cys to Tyr mutation in the transmembrane domain of the maize ethylene receptors and ZmETR2b that is present in the etr1-1 dominant negative mutant and expressed each protein in Arabidopsis. Mutant Zmers1b and Zmetr2b receptors conferred ethylene insensitivity and functioned equally well in a hemizygous or homozygous state. Expression of the Zmers1b N-terminal transmembrane domain was sufficient to exert dominance over endogenous Arabidopsis ethylene receptors whereas the Zmetr2b N-terminal domain failed to do so. Neither Zmers1b nor Zmetr2b functioned in the absence of subfamily 1 ethylene receptors, i.e., ETR1 and ERS1. PARTICIPANTS: Dr. Daniel R. Gallie, PI Dr. Shijun Cheng, Post-doc - performed experiments on protein synthesis in wheat Ms. Juifen Chen, Graduate student - performed experiments on ethylene receptors in maize Christian Caldwell, Technician - performed experiments on protein synthesis in Arabidopsis TARGET AUDIENCES: This information will increase the ability to manage stress responses in plants in such a way that responses to environmental stress can be altered in a tissue-specific manner or in the degree of the response. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These results demonstrate a linear relationship between the degree of insensitivity and etr1-1 expression and provides the ability to control ethylene responses temporally and in amount through the control of mutant receptor expression. Our results also demonstrate that the mutant maize ethylene receptors are functionally dependent on subfamily 1 ethylene receptors in Arabidopsis, indicating substantial functional conservation between maize and Arabidopsis ethylene receptors despite their sequence divergence.
Publications
- Gallie DR. 2010. Regulated ethylene insensitivity through the inducible expression of the Arabidopsis etr1-1 mutant ethylene receptor in tomato. Plant Physiol. 152: 1928-1939.
- Lellis AD, Allen ML, Aertker AW, Tran JK, Hillis DM, Harbin CR, Caldwell C, Gallie DR, and Browning KS. 2010. Deletion of the eIFiso4G subunit of the Arabidopsis eIFiso4F translation initiation complex impairs health and viability. Plant Mol. Biol. 74: 249-263.
- Chen JF, and Gallie DR. 2010. Analysis of the functional conservation of ethylene receptors between maize and Arabidopsis. Plant Mol. Biol. 74:405-421.
- Cheng S, and Gallie DR. 2010. Competitive and non-competitive binding of eIF4B, eIF4A, and the poly(A)-binding protein to wheat translation initiation factor eIFiso4G. Biochemistry 49:8251-8265.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Ethylene serves as an important hormone controlling several aspects of plant growth and development including fruit ripening and leaf and petal senescence. This includes inducing the formation of air spaces, or aerenchyma, in roots of some species subjected to flooding in an effort to maintain oxygen levels. Because relatively little is known about the role that ethylene plays in cereal root development, we investigated how the ethylene biosynthetic machinery is spatially regulated in maize roots. ACC synthase (ZmACS) expression was observed in the root cap and in cortical cells whereas ACC oxidase (ZmACO) expression was detected in the root cap, protophloem sieve elements, and the companion cells associated with metaphloem sieve elements. Hypoxic treatment induced expression to a higher level in these cell types as well as inducing expression in the root cortex. The transient induction of the ethylene biosynthetic machinery in hypoxic roots correlated with a transient induction of ethylene evolution. Roots from Zmacs6 mutants exhibited significantly reduced ethylene production, and increased growth when largely unimpeded, a phenotype complemented by exogenous ACC, whereas loss of ZmACS2 expression had less of an effect. In contrast, Zmacs6 plants exhibited reduced root growth in soil. PARTICIPANTS: Dr. Daniel R. Gallie, PI Dr. Jane Geisler-Lee, Post-doc - performed experiments on gene expression in maize roots Ms. Juifen Chen, Graduate student - performed experiments on gene expression in maize roots Blair Jolley, Technician - performed experiments on mutant root growth Christian Caldwell, Technician - performed experiments on mutant root growth TARGET AUDIENCES: Provided important knowledge on factors controlling root growth in crop species under normal growth conditions and in response to flooding. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These results demonstrate that reduced root biomass accumulation was observed in soil-grown Zmacs6 plants, indicating that ZmACS6 is responsible for maintaining root growth in soil. Thus, expression of ZmACS6 is important in regulating growth of maize roots in response to physical resistance. Our results also suggest that maize roots respond to conditions of hypoxia by transiently inducing the spatially restricted expression of the ethylene biosynthetic machinery which results in increased ethylene production.
Publications
- Gallie DR, Geisler-Lee J, Chen J, Jolley B. 2009. Tissue-specific expression of the ethylene biosynthetic machinery regulates root growth in maize. Plant Mol Biol. 69: 195-211.
- Geisler-Lee J, Caldwell C, Gallie DR. 2010. Expression of the ethylene biosynthetic machinery in maize roots is regulated in response to hypoxia. J. Exp. Bot. 61: 857-871.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Ascorbic acid (Asc), the most abundant antioxidant in plants, is important in maintaining photosynthetic function. We examined the role of Asc and dehydroascorbate reductase (DHAR), which recycles Asc from its oxidized state. We investigated how the efficiency of Asc recycling affects non-photochemical quenching (NPQ). We examined how reducing DHAR expression reduced the induction of NPQ which correlated with reductions in photosynthetic functioning while the level of reactive oxygen species (ROS) increased. The quickly reversible component of NPQ was lowered but the slowly reversible component of NPQ increased with reduced DHAR expression. Leaves exhibited significant photoinhibition following exposure to high light. We examined how increasing DHAR expression increased photosynthetic functioning, particularly at high light intensities, while the level of ROS was reduced and leaves experienced less photoinhibition following exposure to high light. We also examined the role of ethylene in photosynthesis by measuring photosynthetic functioning in plants with higher or lower ethylene signaling. The results have been disseminated in the form of scientific publications, interviews to the media, and patent applications. PARTICIPANTS: Daniel R. Gallie PI. Oversaw the project. Dr. Shijun Cheng, Postdoctoral scientist. Performed the experiments. Dr. Zhong Chen, Postdoctoral scientist. Performed the experiments. Christian Caldwell. Technician. Provided assistance with the experiments. TARGET AUDIENCES: Provided basic knowledge for the scientific community. Developed technologies that formed the basis for patent applications. PROJECT MODIFICATIONS: None.
Impacts
These observations demonstrate that through its Asc recycling function, DHAR affects the level of reactive oxygen species and photosynthetic functioning and serves to protect the photosynthetic apparatus from damage by photoinhibition. We also found that changes in DHAR expression affect how NPQ is induced and affect the level of photoprotection. Finally, we observed that ethylene plays an important role in regulating photosynthetic functioning, particularly in response to stress conditions.
Publications
- Cheng, S., Sultana, S., Goss, D.J. and Gallie DR. 2008. Translation initiation factor 4B homodimerization, RNA binding, and interaction with poly(A)-binding protein are enhanced by zinc. J. Biol. Chem. 283:36140-36153.
- Khan, M.A., Yumak, H., Gallie, D.R., and Goss, D.J. 2008. Effects of poly(A)-binding protein on the interactions of translation initiation factor eIF4F and eIF4F.4B with internal ribosome entry site (IRES) of tobacco etch virus RNA. Biochim. Biophys. Acta. 1779:622-627.
- Chen, Z., and Gallie, D.R. 2008. Dehydroascorbate reductase affects non-photochemical quenching and photosynthetic performance. J. Biol. Chem. 283:21347-21361.
- Liu, Z., Cheng, S., Gallie, D.R., and Julian, R.R. 2008. Exploring the mechanism of selective noncovalent adduct protein probing mass spectrometry utilizing site-directed mutagenesis to examine ubiquitin. Anal. Chem. 80:3846-3852.
- Khan, M.A., Miyoshi, H., Gallie, D.R., and Goss, D.J. 2008. Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and eIFiso4F. J. Biol. Chem. 283:1340-1349.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We examined the role of ascorbic acid (Asc), which serves as a major antioxidant in photoprotection and photosynthetic function in plants, and dehydroascorbate reductase (DHAR), which catalyzes the regeneration of Asc from its oxidized state and serves as an important regulator of Asc recycling. We have used a molecular biochemical approach to investigate how the efficiency of Asc recycling affects non-photochemical quenching (NPQ). Suppression of DHAR expression resulted in reduced induction of NPQ that correlated with reductions in chlorophyll and xanthophyll pigments, quantum yield of photosystem II (PSII), electron transport rate (ETR), and CO2 assimilation while the level of reactive oxygen species (ROS) increased. The quickly reversible component of NPQ was reduced and the slowly reversible or irreversible component of NPQ increased in plants with reduced DHAR expression and leaves exhibited significant photoinhibition following exposure to high light. In
contrast, increasing DHAR expression increased the xanthophyll pigment and chlorophyll pool size, the rate of CO2 assimilation, and ETR, particularly at high light intensities, while the level of ROS was reduced. Leaves with increased DHAR expression experienced less photoinhibition than did wild-type plants following exposure to high light.
PARTICIPANTS: Daniel R. Gallie PI. Oversaw the project. Dr. Shijun Cheng Postdocatoral scientist. Performed the experiments. Christian Caldwell. Technician. Provided assistance with the experiments.
TARGET AUDIENCES: Provided basic knowledge for the scientific community. Generated inventions for patent applications.
PROJECT MODIFICATIONS: None
Impacts
These observations support the conclusion that through its Asc recycling function, DHAR affects the level of foliar ROS and photosynthetic activity. Moreover, changes in DHAR expression affect the appropriate induction of NPQ and photoprotection.
Publications
- Cheng, S., and Gallie, D.R. 2007. eIF4G, eIFiso4G, and eIF4B bind the poly(A)-binding protein through overlapping sites within the RNA recognition motif domains. J. Biol. Chem. 282:25247-25258.
- Gallie, D.R. 2007. Use of in vitro translation extract depleted in specific initiation factors for the investigation of translational regulation. Methods Enzymol. 429:35-51.
- Gallie, D.R. 2007. Translational Control in Plants and Chloroplasts. In: Translational Control in Biology and Medicine. M.B. Mathews, N. Sonenberg, J.W.B. Hershey, ed. Cold Spring Harbor Press, Pp. 747-774.
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Progress 01/01/06 to 12/31/06
Outputs
Ascorbic acid (Asc) is a major antioxidant in plants that detoxifies reactive oxygen species (ROS) and maintains photosynthetic functioning. Expression of dehydroascorbate reductase (DHAR), responsible for regenerating Asc from an oxidized state, regulates the cellular Asc redox state, which in turn, affects cell responsiveness and tolerance to environmental ROS. Because of its role in Asc recycling, we examined whether DHAR is important for plant growth. Suppression of DHAR expression resulted in a preferential loss of chlorophyll a, a lower steady state of ribulose bisphosphate carboxylase/oxygenase as measured by the amount of the large subunit (RbcL), and a lower rate of CO2 assimilation. As a consequence, a slower rate of leaf expansion and reduced foliar dry weight was observed. In addition, an accelerated rate of loss of chlorophyll, RbcL, light-harvesting complex II (LHCII), and photosynthetic functioning was observed in mature leaves resulting in premature
leaf aging. Reduced growth rate as measured by plant height and leaf number was consistent with the DHAR-mediated reduction of photosynthetic functioning. Increasing DHAR expression maintained higher levels of chlorophyll, RbcL, LHCII, and photosynthetic functioning resulting in delayed leaf aging. The effect of DHAR expression on leaf aging inversely correlated with the level of lipid peroxidation indicating that DHAR functions to protect against ROS-mediated damage.
Impacts
These observations support the conclusion that through its Asc recycling function, DHAR affects the level of foliar ROS and photosynthetic activity during leaf development and as a consequence, influences the rate of plant growth and leaf aging.
Publications
- Chen, Z., and Gallie, D.R. 2006. Dehydroascorbate reductase affects leaf growth, development, and function. Plant Physiol. 142: 775-787.
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Progress 01/01/05 to 12/31/05
Outputs
Plants can limit the damage caused by exposure to oxidizing environmental pollutants such as ozone by avoidance or tolerance. Avoidance involves limiting entry of ozone to the interior of the leaf through stomatal closure. Tolerance includes the detoxification of ozone typically by reactions requiring ascorbic acid (Asc). We examined whether increasing the level of Asc through enhanced Asc recycling using dehydroascorbate reductase (DHAR) would limit the deleterious effects of environmental oxidative stress. DHAR overexpressing plants had a lower oxidative load, lower level of oxidative-related enzyme activities, greater Chl a content, and a higher level of photosynthetic activity following an acute exposure to ozone despite exhibiting a larger open stomatal area. Reducing the size of the Asc pool size through suppression of DHAR expression had the opposite effect. Following a chronic exposure to ozone, plants with a larger Asc pool size had a larger stomatal area and
a higher oxidative load but retained a higher level of photosynthetic activity than control plants whereas DHAR-suppressed plants had a substantially reduced stomatal area but also a substantially lower level of photosynthetic activity.
Impacts
These data indicate that despite a reduced ability to respond to ozone through stomatal closure, enhancing Asc recycling in overexpressing plants provides greater protection against oxidative damage than does reducing total stomatal area.
Publications
- Chen, Z., Ling, J., and Gallie, D.R. 2004. RNase activity requires formation of disulfide bonds and is regulated by the redox state. Plant Mol. Biol. 55: 83-96. Chen, Z., and Gallie, D.R. 2005. Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than does increasing avoidance. Plant Physiol. 138: 1673-1689. Geisler-Lee, J., and Gallie, D.R. 2005. Aleurone cell identity is suppressed following connation in maize kernels. Plant Physiol. 139: 204-212.
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Progress 01/01/04 to 12/31/04
Outputs
We isolated the maize gene families for ACC synthase, ACC oxidase, the ethylene receptor, and EIN2 and EIL, which act downstream of the receptor. We show that ACC oxidase is expressed primarily in the endosperm. ACC synthase is expressed throughout endosperm development. Only two ethylene receptor gene families were identified in maize, in contrast to the five types previously identified in Arabidopsis. Members of both ethylene receptor families were expressed to substantially higher levels in the developing embryo than in the endosperm, as were members of the EIN2 and EIL gene families. Plants with an increased guard cell Asc redox state were generated by increasing DHAR expression. The plants exhibited greater water loss under drought conditions, whereas suppressing DHAR expression conferred increased drought tolerance. Our analyses suggest that DHAR serves regulate Asc recycling in guard cells that is involved in responding to drought stress. Expression from the
cytokinin-synthesizing isopentenyl transferase (IPT) enzyme under the control of the Arabidopsis senescence-inducible promoter SAG (senescence associated gene)12 rescued the lower floret in maize spikelets from abortion, resulting in two functional florets per spikelet. The pistil in each floret was fertile, but the spikelet produced just one kernel composed of a fused endosperm with two viable embryos. The two embryos were genetically distinct, indicating that they had arisen from independent fertilization events.
Impacts
These results suggest that the endosperm and embryo both contribute to the synthesis of ethylene, and they provide a basis for understanding why the developing endosperm is especially sensitive to ethylene-induced cell death while the embryo is protected. Our results also suggest that cytokinin can determine pistil cell fate during maize floret development and can be used to re-engineer maize grain to increase its protein and oil content. Finally, DHAR expression can be used to improve drought tolerance.
Publications
- Young, T.E., and Gallie, D.R. 2004. The ethylene biosynthetic and perception machinery is differentially expressed during maize endosperm and embryo development. Mol. Gen. Genomics 271:267-281.
- Chen, Z., and Gallie, D.R. 2004. The ascorbic acid redox state controls guard cell signaling and stomatal movement. Plant Cell 16:1143-1162.
- Young, T.E., Giesler-Lee, J., and Gallie, D.R. 2004. Senescence induced expression of cytokinin reverses pistil abortion during maize flower development. Plant J. 38:910-922.
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