Progress 03/01/11 to 02/28/16
Outputs
Target Audience:The target audience for this project for 2015 comprises the international readership of publications resulting from the work. We previously described the first definitive identification of the major determining factor in the Quality Protein Maize phenotype. This can be used as an 'easy to assay' molecular marker for those engaged in QPM breeding worldwide. We also described how novel maize variants created with deletion mutagenesis can be characterized at the molecular genetic level using modern DNA and RNA sequencing technologies. In the 2015 reporting period we have made good progress in combining a Bulked Segregant Analysis mapping procedure based on RNA-seq reads for mapping of causative mutations with exon-seq, a method for identifying mutations within the mapping interval. We are preparing a paper for Nucleic Acids Research, summarizing our new functional genomics methodology. This is of interest to the maize genetics community. We published a paper describing a novel RNA-seq adaptation for analyzing such mutants. We also published a paper describing the utilization of a new proteomics method for comparing the whole proteome across multiple such mutants. This work reaches a global audience since its present new information on how maize endosperm develops, its influence on grain protein quality and provides new resources in maize functional genomics. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has involved the training of two graduate students fundedby a combination of support from the UNL Department of Agronomy and Horticulture, the UNL Center for Plant Science Innovation andby the Agriculture and Food Research Initiative competitive grant no. 2013-02278 of the USDA National Institute of Food and Agriculture. Two post-docs, funded as described above, have devoted part of their time to this Hatch Project. All have worked together with the PI as a close-knit team, and derived considerable benefit from sharing each other's experience. Kyla Morton and Lingling Yuan gained very substantial experience in molecular genetics, bioinformatics and in analyzing and interpreting proteomics datasets derived from a separate facility. Aixia Li, a post-doc who started in August 2014, worked effectively in this project. She has expertise in molecular and classical genetics and working on this project will advance her skills in genomics and bioinformatics. She works closely with the other post-doctoral associate, Shangang Jia, who started working on this project in Dec 2014. He has considerable skills in plant bioinformatics and extensive experience in RNA-seq data sets and genome assembly. He has been the driving force in facilitating our BSR-seq/exon-seq platform. Working on this project is giving him experience in cereal genomics and classical maize genetics as well as hands on training in field and lab work. He is developing highly valuable skills and I expect him to have excellent opportunities in his future career, whether in maize or in a broader crop genomics field. I also trained a freshman undergraduate in the Agronomy and Horticulture department as well as a high school senior who is a gifted budding plant scientist. The high school student (now a UNL freshman) has assisted in the field pollination season and screening the mutant populations in the lab. He is very inquisitive about the science of the project and now plans a career in plant science. The freshman, now Junior, works in the lab performing screens and biochemical profiling of the lines as a UNL UCARE scholar. He completed a summer internship at the Danforth center based in interests gained in my lab and as a student and then a TA in my undergraduate Plant Science course. How have the results been disseminated to communities of interest?The work has been disseminated through the papers entered in the products section What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project enhanced our basic knowledge of genes and gene products controlling the essential process of maize grain hardening, how this relates to the lack of certain essential amino acids in the grain, and how we can optimize maize for both hardness and protein quality. The endosperm is the major nutrient storage organ in the maize grain. Endosperm hardening during kernel maturation is essential for most of maize's agronomic and industrial uses but usually confers sub-optimal amino acid composition (protein quality) on the grain. This is because the dominantly abundant zein seed storage proteins, which drive vitreous (hard) endosperm formation, are devoid of lysine and tryptophan, two amino acids essential in the diets of humans and mono-gastric livestock. The maize opaque-2 mutant, shuts down zein synthesis thereby improving protein quality, but produces unacceptably soft mature kernels. However, modified opaque-2 lines (Quality Protein Maize; QPM), which have restored kernel hardness, maintain the high-protein quality trait. Unfortunately, the genetic basis of this modification process is not well understood, which has contributed to the underutilization of QPM. The real world impact of the work described below is best described in terms of basic knowledge and the creation of new and useful maize germplasm. First, by using the latest methods in targeted DNA sequencing and RNA sequencing to investigate the mutational repertoire in mutagenized lines, we are generating technical knowledge and mutational resources that may be of use to others in the maize community. Second, because of the plasticity of the maize genome for change, viable new lines with loss of genes or groups of genes can exhibit beneficial changes in the proteome (the sum of the total proteins in a cell or tissue). This is especially true in the case of the prolamin storage proteins. With our method, we are creating new prolamin variants that often display increased protein quality with respect to wild type maize and even, the high-lysine QPM parent. Objective 1. The PhD student who worked on this objective tried crossing the events we had received for the PPDK-2 overexpression construct to the opaque-2 mutant to determine if PPDK alone is able to contribute to o2 endosperm modification. The transgenic events of this construct yielded inconclusive results. Second, she used ATP and PPi measurement kits to establish if there is a general ATP deficiency in the developing endosperms of all opaque mutants and if so, can we detect alterations in the PPi pool that might suggest a switch to PPi usage for high energy phosphate. The ATP detection was very unreliable using this biochemical method because of the lability of ATP in the biological samples. We were not able to detect differences in the PPi pool. The third, and most fruitfulobjective was to complete the interpretation of the proteomics comparison data we had generated for the isogenic opaque mutant series and publish a paper describing these results. We demonstrated that shotgun proteomics is a very useful tool for detecting qualitative and quantitative differences in specific proteins derived from whole proteome when comparing multiple isogenic lines. Furthermore, we discovered enrichment of novel proteins are specifically enriched in lysine that contribute to the improved protein quality of several of the mutants we profiled. This work was published in 2015 in Journal of Experimental Botany Objective 2. As previously reported, this aim was largely completed with the publication of another Plant Physiology paper in 2013 which described new findings on the non-redundant functions of different zein species in protein body formation in the maize endosperm. The total alpha-zein RNAi line described in the paper was partly intended as a dominant opaque test line that we could cross with transgenic lines over-expressing candidate o2 modifier genes. We completed this test for both the PFP alpha and the 27-kD gamma zein overexpression lines. Both of these failed to even partially reverse the dominant opaque phenotype caused by complete alpha zein suppression. This suggests that residual alpha zein, especially the 19-kD alpha zein, is necessary for the o2 modifiers to work and possibly that the o2 phenotype results from more than just low levels of alpha zeins (for example, low PPDK activity may also be partly responsible for opacity in the o2 mutant. Objective 3. As part of a USDA NIFA funded project, with which this Hatch Project overlaps, we focused on the characterization of several opaque QPM reversion lines and this work will continue after the conclusion of the hatch project. We focused on seven mutant lines (12, 44, 112, 121, 134, 198 and 223). From our first exon capture DNA sequencing experiment and concurrent RNA-seq analysis, we generated data for lines 112, 121 and 198. This data has been useful to identify a strong candidate deletion in lines 112 but not for 121 and 198. Illumina Hi-seq genomic DNA sequencing was used for 121 and we are searching for candidate deletions. For line 112, we have a strong candidate gene deletion of a lactoglutathione lyase (LGL; glyoxalase). The role of LGL is to metabolize methylglyoxal, a cytotoxic product elicited by multiple stresses. We have described the stress response in opaque-2 mutant endosperm and it's amelioration in modified o2 (QPM) endosperm and LGL is a transcriptional target for O2. The loss of modification in line 112 may suggest LGL is important in this amelioration. We made an antibody LGL and shown that LGL is reduced in o2 endosperm and is undetectable in 112 endosperm. We have obtained a Uniform Mu transposon insertional allele which we are currently propagating. With one or both of these mutants, we should be able to confirm whether LGL is essential for normal endosperm development or if it functions as an o2 modifier gene. In 2016, we utilized Bulked Segregant Exon Sequencing (BSEx-seq) of WT and Mutant pool collected from F3 (B73 x QPM) population to perform preliminary gene mapping for mutants 44,112 and 121. The mapping results were partially informative. Candidate genes regions of mutant 44 were located to chr5, chr7 and chr10. Candidate genes of 121 were mapped to chr2 and chr7. The causal mutation in mutant 112 maps to a region of chromosome 8 where we had already identified a large deletion containing a lactoglutathione lyase (LGL) gene candidate. In summer 2016 we outcrossed several Uniform Mu transposon mutant alleles of this gene since and selfed ears from these plants were often poor because the genetic background struggled under our field conditions. We are further propagating the crossed materials for complementation analysis in the greenhouse. For our sgRNA/CAS9 knock-out construct for LGL, we recently received the first transgenic events back and sequencing has revealed target site mutations. In general, we are continuing to interpret the mapping results and identify candidate genes in these QPM deletion mutants and beyond the life of this hatch project and the related USDA NIFA foundational project which expires in December 2017. At the end of the project, we used BSR-seq of QPM RILs previously generated to refine previously identified QTLs. In addition to confirming the QTLs, we identified a very strong candidate gene for the second major QPM QTL. This is a version of the Waxy1, granule bound starch synthase gene, which encodes an enzyme with a five amino acid deletion specific to QPM and QPM RILs with this QTL. We are performing functional analysis to find out how this affects starch composition and its role in endosperm modifcation and will publish a paper on this soon.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Morton, K., Jia, S., Zhang, C., Holding, D.R. (2015). Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins. Journal of Experimental Botany. doi: 10.1093/jxb/erv532
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Y. Dou, X. Guo, L. Yuan, D.R. Holding, C. Zhang. Differential Expression Analysis in RNA-Seq by a Naive Bayes Classifier
with Local Normalization. BioMed Research International (2015) Article ID 789516, doi:10.1155/2015/789516.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Jia S., Li A., Morton K., Avoles-Kianian P., Kianian S.F., Zhang C., Holding D. (2016) A Population of Deletion Mutants and an Integrated Mapping and Exome-seq Pipeline for Gene Discovery in Maize. G3: Genes|Genomes|Genetics. DOI: 10.1534/g3.116.030528.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Yuan, L., Dou, Y., Kianian, S., Zhang, C. and Holding, D.R. (2014) Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification. Plant Physiol. 164: 119-130.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Holding, D.R. (2014). Recent advances in the study of prolamin storage protein organization and function. Frontiers in Plant Science. Special issue: Advances in Seed Biology, 5:276. doi: 10.3389/fpls.2014.00276. Combined review and original research paper.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Shangang Jia, Aixia Li, Chi Zhang and David Holding
Deletion mutagenesis and identification of causative mutations in maize
Methods in Molecular Biology
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience for this project for 2015 comprises the international readership of publications resulting from the work. We previously described the first definitive identification of the major determining factor in the Quality Protein Maize phenotype. This can be used as an 'easy to assay' molecular marker for those engaged in QPM breeding worldwide. We also described how novel maize variants created with deletion mutagenesis can be characterized at the molecular genetic level using modern DNA and RNA sequencing technologies. In the 2015 reporting period we have made good progress in combining a Bulked Segregant Analysis mapping procedure based on RNA-seq reads for mapping of causative mutations with exon-seq, a method for identifying mutations within the mapping interval. We are preparing a paper for Nucleic Acids Research, summarizing our new functional genomics methodology. This is of interest to the maize genetics community. We published a paper describing a novel RNA-seq adaptation for analyzing such mutants. We also published a paper describing the utilization of a new proteomics method for comparing the whole proteome across multiple such mutants. This work reaches a global audience since its present new information on how maize endosperm develops, its influence on grain protein quality and provides new resources in maize functional genomics. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In 2015, a graduate student was working on one part of this project for the first half of the year. Two post-docs have devoted part of their time to this Hatch Project. All have worked together with the PI as a close-knit team, and derived considerable benefit from sharing each other's experience. Kyla Morton gained very substantial experience in analyzing and interpreting proteomics datasetsderived from a separate facility. She also was able to immensely improve as an all-roundscientist and took the lead role in the paper describing this work.Aixia Li, a post-doc who started in August 2014, is working very effectively in this project. She has expertise in molecular and classical genetics and working on this project will advance her skills in genomics and bioinformatics. She works closely with the other post-doctoral associate, Shangang Jia, who started working on this project in Dec 2014. He has considerable skills in plant bioinformatics and extensive experience in RNA-seq data sets and genome assembly. He has been the driving force in facilitating our BSR-seq/exon-seq platform. Working on this project is giving him experience in cereal genomics and classical maize genetics as well as hands on training in field and lab work. He is developing highly valuable skills and I expect him to have excellent opportunities in his future career, whether in maize or in a broader crop genomics field. I also employed a freshman undergraduate in the Agronomy and Horticulture department as well as a high school senior who is a gifted budding plant scientist. The high school student has assisted in the field pollination season and screening the mutant populations in the lab. He is very inquisitive about the science of the project and now plans a career in plant science. The freshman, now sophomore, works in the lab performing screens and biochemical profiling of the lines and plans to continue next year as a UNL UCARE scholar, which involves a more formalized lab research project. He is applying for summer internships based in interests gained in my lab in my undergraduate Plant Science course. How have the results been disseminated to communities of interest?The work hasbeen disseminated through the papers entered in the products section What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. The PhD student who has been working on this objective graduated in the summer of 2015 and completed her lab work in the spring of 2015. Before then, she completed the following tasks. She tried crossing the events we had received for the PPDK-2 overexpression construct to the opaque-2 mutant to determine if PPDK alone is able to contribute to o2 endosperm modification. The results are so far inconclusive since we are still waiting for enough transgenic events of this construct. Second, she used ATP and PPi measurement kits to establish if there is a general ATP deficiency in the developing endosperms of all opaque mutants and if so, can we detect alterations in the PPi pool that might suggest a switch to PPi usage for high energy phosphate. The ATP detection was very unreliable using this biochemical method because of the lability of ATP in the biological samples. We were not able to detect differences in the PPi pool. The third objective was to complete the interpretation of the proteomics comparison data we had generated for the isogenic opaque mutant series and publish a paper describing these results. This was a very fruitful objective in 2015. We demonstrated that shotgun proteomics is a very useful tool for detecting qualitative and quantitative differences in specific proteins derived from whole proteome when comparing multiple isogenic lines. Furthermore, we discovered enrichment of novel proteins are specifically enriched in lysine that contribute to the improved protein quality of several of the mutants we profiled. This work is currently in press with Journal of Experimental Botany Objective 2. As previously reported, this aim was largely completed with the publication of another Plant Physiology paper in 2013 which described new findings on the non-redundant functions of different zein species in protein body formation in the maize endosperm. The total alpha-zein RNAi line described in the paper was partly intended as a dominant opaque test line that we could cross with transgenic lines over-expressing candidate o2 modifier genes. We completed this test for both the PFP alpha and the 27-kD gamma zein overexpression lines. Both of these failed to even partially reverse the dominant opaque phenotype caused by complete alpha zein suppression. This suggests that residual alpha zein, especially the 19-kD alpha zein, is necessary for the o2 modifiers to work and possibly that the o2 phenotype results from more than just low levels of alpha zeins (for example, low PPDK activity may also be partly responsible for opacity in the o2 mutant. Objective 3. As previously reported, these results were included in the 2012 Plant Physiology paper. However, although we gained significant insight into correlative metabolic changes associated with unmodified and modified o2 using this expression profiling approach, it is difficult to infer causal relationships. As explained in previous reports, the focus of this aim shifted towards a deletion mutagenesis approach which has shown good potential for identifying opaque-2 modifier genes as well as genes more generally required for endosperm maturation. As part of a USDA NIFA funded project, with which this Hatch Project overlaps, we are focusing on the characterization of several opaque QPM reversion lines and this work will continue after the conclusion of the hatch project. The current focus is on seven mutant lines (12, 44, 112, 121, 134, 198 and 223). From our first exon capture DNA sequencing experiment and concurrent RNA-seq analysis, we generated data for lines 112, 121 and 198. This data has been useful to identify a strong candidate deletion in lines 112 but not for 121 and 198. Illumina Hi-seq genomic DNA sequencing was used for 121 and we are searching for candidate deletions. For line 112, we have a strong candidate gene deletion of a lactoglutathione lyase (LGL; glyoxalase). The role of LGL is to metabolize methylglyoxal, a cytotoxic product elicited by multiple stresses. We have described the stress response in opaque-2 mutant endosperm and it's amelioration in modified o2 (QPM) endosperm and LGL is a transcriptional target for O2. The loss of modification in line 112 may suggest LGL is important in this amelioration. We made an antibody LGL and shown that LGL is reduced in o2 endosperm and is undetectable in 112 endosperm. We are currently transforming a sgRNA/CAS9 construct to knock out this protein. We have obtained a Uniform Mu transposon insertional allele which we are currently propagating. With one or both of these mutants, we should be able to confirm whether LGL is essential for normal endosperm development or if it functions as an o2 modifier gene. In 2015 we used the University of Minnesota Genomics Center exon capture service in combination with the new chemistry Zeanome exon capture kit made by Roche to generate a second round of exon capture results. This included the B73 deletion mutants (below) and QPM deletion mutants 12, 44 and 134. Line 12 has a strong candidate deletion of a protein disulfide isomerase (PDI) gene. Previous research has shown that PDI proteins are essential in the oxidative folding, processing and assembly of storage proteins. Since line 12 does not show a marked reduction in zein abundance, further characterization of this mutant should provide insight into storage protein deposition and organization in the developing protein body. Consistent with the unchanged zein profile, we determined that 12 does not have an increase in kernel lysine content. We are preparing to analyze protein bodies in this mutant using immunogold electron microscopy. We have ordered a Uniform Mu transposon insertion line in this gene to perform complementation crosses with line 12. Although we have not yet identified a candidate deletion in line 44, we are pursuing it since this line shows partial o2 modification and lysine content even higher than the value seen in QPM. In addition to its potential to identify a new o2 modifier gene, line 44 with its partially modified endosperm could have application as a feed where increased protein quality and digestibility are desirable.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Kyla J. Morton, Shangang Jia, Chi Zhang and David R. Holding,
Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins 2015 Journal of Experimental Botany. In press
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Y. Dou, X. Guo, L. Yuan, D.R. Holding, C. Zhang. Differential Expression Analysis in RNA-Seq by a Naive Bayes Classifier with Local Normalization. BioMed Research International (2015) Article ID 789516, doi:10.1155/2015/789516.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The target audience for this project for 2014 comprises the international readership of publications resulting from the work. A paper published in Plant Physiology in 2014 described the first definitive identification of the major determining factor in the Quality Protein Maize phenotype. This can be used as an ‘easy to assay’ molecular marker for those engaged in QPM breeding worldwide. The paper also described how novel maize variants created with deletion mutagenesis can be characterized at the molecular genetic level using modern DNA and RNA sequencing technologies. This is of interest to the maize genetics community. I also published a combined review and original data article in Frontiers in Plant Science in 2014. This reviewed the current insight into the function and organization of prolamin storage proteins. Both papers reach a global audience since they present new information on how prolamins are stored and their effect on protein quality in maize. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? In this reporting period, theprojects outlined have led to the training of two graduate students,one of whom graduated in August 2014.This studentwas first author on a Plant Physiology paper published this year. A post-doctoral associate has recently continued working on this project and she is advancing her skils in molecular and classical genetics, genomics and bioinformatics. How have the results been disseminated to communities of interest? The results have been disseminated through a paper in Plant Physiology and a paper in Frontiers in Plant Science in 2014 as well as through an invited oral presentation at the 2014 CSSA meeting in Long Beach California. What do you plan to do during the next reporting period to accomplish the goals? Objective 1. The graduate student who has been working on this objective is due to graduate in the summer of 2015. Before that time, we aim to complete the following tasks. First, she will cross the events we have received for the PPDK-2 overexpression construct to the opaque-2 mutant to determine if PPDK alone is able to contribute to o2 endosperm modification. Second, she will use ATP and PPi measurement kits to establish if there is a general ATP deficiency in the developing endosperms of all opaque mutants and if so, can we detect alterations in the PPi pool that might suggest a switch to PPi usage for high energy phosphate. Third, she will complete the interpretation of the proteomics comparison data we have generated for the isogenic opaque mutant series and publish a paper describing these results. Objective 2. This objective has run its course. Objective 3. We have seven full or partial opaque reversion deletion mutants in our QPM population that we are in the process of molecular characterization. We will prioritize and test candidate genes from exon-seq and RNA seq data sets that are already generated. We will generated new exon-seq and RNA-seq datasets where they have not already been generated. This will utilize the new Roche Nimblegen Zeanome exon capture array which contains gene sequences from B73 as well as several other common maize inbred lines. This will help our analysis of our QPM line which is not in the B73 background. Exon capture and RNA-seq will be carried out that the University of Minnesota Genomics Facility who offer a full exon capture service including the HiSeq sequencing.
Impacts
What was accomplished under these goals?
This project is enhancing our basic knowledge of genes and gene products controlling the essential process of maize grain hardening, how this relates to the lack of certain essential amino acids in the grain, and how we can optimize maize for both hardness and protein quality. The endosperm is the major nutrient storage organ in the maize grain. Endosperm hardening during kernel maturation is essential for most of maize’s agronomic and industrial uses but usually confers sub-optimal amino acid composition (protein quality) on the grain. This is because the dominantly abundant zein seed storage proteins, which drive vitreous (hard) endosperm formation, are devoid of lysine and tryptophan, two amino acids essential in the diets of humans and mono-gastric livestock. The maize opaque-2 mutant, shuts down zein synthesis thereby improving protein quality, but produces unacceptably soft mature kernels. However, modified opaque-2 lines (Quality Protein Maize; QPM), which have restored kernel hardness, maintain the high-protein quality trait. Unfortunately, the genetic basis of this modification process is not well understood, which has contributed to the underutilization of QPM. The real world impact of the work described below is best described in terms of basic knowledge and the creation of new and useful maize germplasm. First, by using the latest methods in targeted DNA sequencing and RNA sequencing to investigate the mutational repertoire in mutagenized lines, we are generating technical knowledge and mutational resources that may be of use to others in the maize community. Second, because of the plasticity of the maize genome for change, viable new lines with loss of genes or groups of genes can exhibit beneficial changes in the proteome (the sum of the total proteins in a cell or tissue). This is especially true in the case of the prolamin storage proteins. With our method, we are creating new prolamin variants that often display increased protein quality with respect to wild type maize and even, the high-lysine QPM parent. Objective 1. As previously reported, this aim was completed with the publication of a Plant Physiology paper in 2012. In this paper, we also described some data implicating the possible role in QPM endosperm of another pyrophosphate dependent, central glycolytic enzyme called pyruvate Pi dikinase (PPDK). We presented data suggesting that PPDK-1, Opaque-2 (O2) regulated, isoform is down-regulated in endosperms of both opaque-2 and modified opaque-2 (QPM). However, the PPDK-2 isoform is not regulated by O2, and its expression is increased in QPM compared with o2. Consequently we planned to make a transgenic PPDK-2 over-expression line to see it could be used to effect endosperm modification when crossed to opaque-2 mutants. Since we were unable to amplify a full length open reading frame for this gene, we ultimately made the construct with direct synthesis of the gene and finally, we have just received the first transgenic events. We are currently growing this in the greenhouse and plan to cross it into the opaque-2 mutant to address the above question. Related to Objective 1, we previously proposed that opaque2 and other opaque mutants likely have an endosperm ATP deficiency resulting from induction of chaperones (ATPases) and that this energy shortfall may contribute to the opaque phenotype. We tested this hypothesis using an ATP colorimetric assay to test developing endosperm tissue of wild-type, o2 and QPM. The results showed o2 endosperm has significantly lower levels of available ATP than wild-type and QPM. Furthermore, we wondered whether PPi could serve as an alternative source of high energy phosphate as might be inferred from differential expression and activity of PFP and PPDK that we have previously reported. However, we were not able to detect significant differences in PPi levels between these genotypes. Also related to Objective 1, in 2013 I reported plans for complete proteomics analysis for a series of isogenic opaque mutants using the shot-gun proteomics core facility at UC-Davis. The graduate student is currently analyzing the data we received for this experiment. We found all opaque mutants have elevated accumulation of a number of ATP-requiring heat shock chaperones which may contribute to the energy crisis by exhausting an already low supply of ATP. We hope to gain insight into additional processes that contribute to the opaque phenotype and endosperm maturation in general. Objective 2. As previously reported, this aim was largely completed with the publication of another Plant Physiology paper in 2013 which described new findings on the non-redundant functions of different zein species in protein body formation in the maize endosperm. The total alpha-zein RNAi line described in the paper was partly intended as a dominant opaque test line that we could cross with transgenic lines over-expressing candidate o2 modifier genes. We completed this test for both the PFP alpha and the 27-kD gamma zein overexpression lines. Both of these failed to even partially reverse the dominant opaque phenotype caused by complete alpha zein suppression. This suggests that residual alpha zein, especially the 19-kD alpha zein, is necessary for the o2 modifiers to work and possibly that the o2 phenotype results from more than just low levels of alpha zeins (for example, low PPDK activity may also be partly responsible for opacity in the o2 mutant. Objective 3. As previously reported, these results were included in the 2012 Plant Physiology paper. However, although we gained significant insight into correlative metabolic changes associated with unmodified and modified o2 using this expression profiling approach, it is difficult to infer causal relationships. This is partly because of the large number of differentially expressed we detected and the difficulty and expense associated with making transgenic events for these genes. As explained in previous reports, the focus of this aim has shifted towards a deletion mutagenesis approach which has shown good potential for identifying opaque-2 modifier genes as well as genes more generally required for endosperm maturation. In a population of 300 M3 families, we identified several opaque revertants including one line in which the 27-kD gamma-zein gene, whose product is known to be essential for o2 modification, is deleted. We have shown using a hemizygous version of the mutant, that gamma zein acts in a dosage dependent, haploinsufficient manner in endosperm modification. Of the mutants we are focusing on, one accumulates very low levels of 19-kD alpha zein and has increased lysine and may lead to insight in o2-independent alpha zein regulation. We have mapped this mutation to a region on chromosome 3. Exon-equencing and RNA-seq did not suggest any deleted genes in this interval so we are currently conducting HiSeq full genome sequencing to scan this mapping interval with greater resolution. Six other QPM deletion mutants have been selected based on their being partial opaque revertants. These mutants offer the potential to uncover the location of other modifier genes, whose existence was suggested by QTL mapping studies. All these six lines have increased lysine compared with QPM and we are in the process of prioritizing these lines for new exon capture DNA sequencing and RNA-seq studies. Therefore, these lines have the potential to identify other genes responsible for the QPM phenotype and thus facilitate its future breeding. Furthermore, these partially vitreous lines with increased lysine may have use as corn varieties that have increased digestibility, while offering acceptable physical kernel properties. An example of one such application is for dairy cattle which can have higher milk production when fed more digestible corn.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Yuan, L., Dou, Y., Kianian, S., Zhang, C. and Holding, D.R. (2014) Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification. Plant Physiol. 164: 119-130.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Holding, D.R. (2014). Recent advances in the study of prolamin storage protein organization and function. Frontiers in Plant Science. Special issue: Advances in Seed Biology, 5:276. doi: 10.3389/fpls.2014.00276.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Holding, D. Nov 2014 invitation, A novel functional genomics platform for dissecting maize kernel maturation and protein quality Crop Science Society of America, Annual Meeting, Long Beach California
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The target audience for this project for 2013 comprises the international readership of publications resulting from the work. A book chapter published in Seed Genomics focusing on the evolution, structure and function of prolamin storage proteins has a global audience as evidenced by several requests for PDFs that have been received from international institutions whose access to the on-line article is restricted. Other 2013 manuscripts listed in products reach a global audience since they present new information on how prolamins are stored and their effect on protein quality and grain digestibility in maize and sorghum. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The projects outlined have led to the training of a post-doctoral scholar whom was involved in all aspects of the project with first authorship on two papers and co-authorship on two papers. The post-doc completed a three year term, funded by the PI's start up funds. The project has also led to the training of two PhD graduate students one of which has co-authorship on three papers and a first author paper in revision. The other graduate student is a second author on a Plant Phsiology paper. How have the results been disseminated to communities of interest? The results have been disseminated in 2013 through publications in Plant Physiology, Nature Communications and the Book Chapter, previously reported, has now been published in 2013. What do you plan to do during the next reporting period to accomplish the goals? In the first project area, we plan to complete our analysis of central carbon metabolites including ATP in developing endosperm of an isogenic series of opaque mutants. We will also supplement this with shot-gun proteomics analysis using the UC-Davis core facility. This will allow us to obtain further insight into the processes controlling endosperm maturation in maize and provide valuable incremental knowledge from previous transcriptional profiling experiments performed on these mutants. For the transgenic zein lines, we will utilize gamma-zein overexpression constructs to try to complement our gamma zein null deletion line. Furthermore, using synthetic gamma zein constructs which were engineered to contain lysine and tryptophan amino acids, we will test their ability to replace the native gamma zein with nutritionally enhanced versions. For the QPM deletion lines, we have created F2 mapping populations for several mutants including the 19-kD alpha-zein deficient mutant. We will conduct exon and RNA-sequencing on 5-10 more mutants and use the obtained map positions to guide selection of candidate gene deletions for further testing. Specific functional testing and in depth phenotypic analyses of mutants will be decided on a case by case basis.
Impacts
What was accomplished under these goals?
In the 2012 report, we described progress in understanding changes in the regulation and activity of glycolytic enzymes in quality protein maize (QPM) seeds in relation to our proposal that maintenance of energy (ATP) supply in the endosperm is a critical aspect of endosperm modification. This work was published in Plant Physiology in 2012. In 2013, the work to test the link between endosperm stress and ATP deficiency in different opaque endosperm mutants, mentioned in the 2012 report, has continued. However, this represents a lower overall proportion of the lab focus since the post-doc whom completed the bulk of that work as moved on. We have generated a collection of developing kernels from an isogenic series of known opaque endosperm mutants. We are in the process of trying to measure ATP and PPi levels in these mutants to try to establish if altered energy currency balance accompanies the common global Hsp response in these mutants. These opaque mutants are also being prepared for shot-gun proteomics to be carried out at the UC-Davis proteomics core facility. The transgenic approach to address the relative roles of PFP and PPDK is also ongoing. We crossed a PFPα overexpression line to o2 to determine if it is able to modify the opaque phenotype. F2 plants are currently being grown to see if the transgenic PFP subunit is able to reduce the expected 25% opaque kernels. Making the overexpression construct for PPDK2 was reported in 2012. We finally have submitted this construct for maize transformation after considerable difficulty in amplifying the full length open reading frame, which we eventually overcame by synthesizing the gene. We will then cross this event to o2 and survey the F2 segregation ratios in the same way as the PFP overexpression construct. The progress studying the role of different zein family members in protein body formation is largely complete and has been published in Plant Physiology in 2013. Zein protein body formation is crucial for proper kernel maturation and has a close relationship with kernel protein quality and thus a full understanding of this process is required. Using various zein RNAi and overexpression transgenic lines, we defined the redundant and non-redundant roles of different zein family members in protein body initiation and expansion. A related project involved the molecular characterization ofthe High Digestibly High Lysine sorghum mutant. In collaboration with Joachim Messing's lab, we determined that the phenotypes results from a single amino acid substitution in a prolamin storage protein that causes a dominant negative phenotype of reticulated, highly digestible protein bodies. The work was published in Nature Communications in 2013. The overall emphasis in this project has shifted towards deletion mutagenesis approach which has shown good potential for identifying opaque-2 modifier genes as well as genes more generally required for endosperm maturation. In a population of 300 M3 families, we identified several opaque revertants including one line in which the 27-kD gamma-zein gene, whose product is known to be essential for o2 modification, is deleted. We have shown using a hemizygous version of the mutant, that gamma zein acts in a dosage dependent, haploinsufficient manner in endosperm modification. Using a sequencing approach which combines genomic sequencing of exome captured DNA and RNA-seq we implemented a novel system for functional genomics of specific kernel traits such as endosperm texture and protein quality but also general seed and whole plant traits. For dissemination of these results, the proof of concept experiment focusing on the gamma-zein deletion mutant is currently in revision at Plant Physiology. We have secured funding from USDA AFRI to characterize addition opaque revertants from the QPM deletion population. One of these mutants accumulates very low levels of 19-kD alpha zein. We are particularly interested in the molecular characterization of this mutant since it has lysine levels elevated above that of QPM, and also has the potential to identify an Opaque-2 unrelated transcription factor, which acts in specifically regulating the 19-kD alpha zein families. We have made an F2 mapping population for this and other opaque mutants in the collection.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Guo, X, Yuan, L, Chen, H, Sato, S.J., Clemente, T.E., and Holding, D.R. (2013). Non-redundant function of zeins and their correct stoichiometric ratio drive protein body formation in maize endosperm. Plant Physiol. 162, 13591369
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wu, Y., Yuan, L., Guo, X., Holding, D.R., and Messing, J. (2013). Mutation Causing a Single Amino Acid Substitution Creates High Food Value Trait in Sorghum. Nature Communications, Volume 4, Published 8-16-2013
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: We have made progress in understanding changes in the regulation and activity of glycolytic enzymes in quality protein maize (QPM) seeds in relation to our proposal that maintenance of energy (ATP) supply in the endosperm is a critical aspect of endosperm modification. We identified a generalized Heat Shock Protein (Hsp) stress response in o2 and its attenuation in QPM which is predicted to reverse or prevent a state of seed energy crisis. We have shown that 20% of Hsp genes in maize are increased in o2 and returned to normal expression in QPM endosperm. This work, published this year in Plant Physiology, focuses mainly on the involvement of PFP and PPDK as alternative glycolytic enzymes in maize endosperm. We showed QPM specific expression and activity induction of the regulatory PFP alpha subunit as well as activation of an O2 non-regulated PPDK2 isoform. We are currently testing the link between endosperm stress and ATP deficiency in different opaque endosperm mutants. To address the relative roles of PFP and PPDK we have taken a transgenic approach. We now have a PFPα overexpression line which we are in the process of crossing to o2 to determine if it is able to modify the opaque phenotype. We are currently making a similar overexpression construct for PPDK to ask the same question. Transgenic events expressing seed specific PPDK and PFPalpha RNAi constructs are being made with the aim of determining if they abolish vitreous endosperm formation in wild type and QPM. While events for the PFPalpha RNAi are being recovered, the PPDK RNAi appears to be lethal suggesting some leakiness of this promoter in vegetative tissue. We have made progress studying the role of different zein family members in protein body formation. Zein protein body formation is crucial for proper kernel maturation and has a close relationship with kernel protein quality and thus a full understanding of this process is required. Using various zein RNAi and overexpression transgenic lines, we have made good progress in defining the redundant and non-redundant roles of different zein family members in protein body formation and this work has recently been submitted for publication in Plant Physiology. We have also determined that over-expression of the 27-kD gamma-zein alone, although an essential o2 modifier, is not able to modify a dominant alpha-zein RNAi phenotype and we are currently seeing if this line can modify opacity in the o2 mutant. Another productive approach to identifying genes behind the QPM QTL regions has been gamma-radiation mutagenesis of a QPM line to generate opaque revertants. In a population of 300 M3 families, we have characterized several opaque revertants including one line in which the major chromosome 7 QTL including 27-kD gamma-zein is deleted. Using a sequencing approach which combines Exome capture and RNA-seq, we are implementing a novel system for functional genomics of specific kernel traits such as endosperm texture and protein quality but also general seed and whole plant traits. For dissemination, the proof of concept experiment focusing on the gamma-zein deletion mutant is currently being prepared for publication. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: A new functional genomics approach for identifying genes involved in kernel maturation QPM and normal maize has been added to this project. This utilizes gamma-radiation mutagenesis of a QPM line to generate opaque revertants. In a population of 300 M3 families, we have characterized several opaque revertants including one line in which the major chromosome 7 QTL including 27-kD gamma-zein is deleted. Using a sequencing approach which combines Exome capture and RNA-seq, we are implementing this new system for functional genomics of specific kernel traits such as endosperm texture and protein quality but also general seed and whole plant traits.
Impacts
We have and continue to elucidate the significance of alternative, pyrophosphate utilizing enzymes, PFP and PPDK for central carbon metabolism in maize endosperm both in normal development and during stressed invoked by opaque endosperm mutations We have deciphered non-redundant functions of zein sub-family members in high-level accumulation of storage proteins in the endosperm. This work has important biotechnological implications for expression of engineered proteins at significant levels. We have pioneered a novel functional genomics tool for studying various traits in maize which we are developing as a publicly available resource.
Publications
- Holding, D.R., and Messing, J. (2013) Evolution, structure and function of prolamin storage proteins. Invited book chapter in: Seed Genomics, Wiley-Blackwell Publishing, Editor, Becraft, P. in press
- Guo, X., Ronhovde, K., Yuan, L., Yao, B., Soundararajan, M.P., Elthon, T.E., Zhang, C., and Holding. D.R. (2012) Pyrophosphate dependent fructose-6-phosphate 1-phosphotransferase induction and attenuation of Hsp gene expression during endosperm modification in Quality Protein Maize. Plant Physiology 158: 917-929
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Progress 03/01/11 to 12/31/11
Outputs
OUTPUTS: We have made progress in understanding changes in the regulation and activity of glycolytic enzymes in quality protein maize (QPM) seeds in relation to our proposal that maintenance of energy (ATP) supply in the endosperm is a critical aspect of endosperm modification. We identified a generalized Heat Shock Protein (Hsp) stress response in o2 and its attenuation in QPM which is predicted to reverse or prevent a state of seed energy crisis. We have shown that 20% of Hsp genes in maize are increased in o2 and returned to normal expression in QPM endosperm. PFP is a major ATP non-requiring glycolytic enzyme which functions as a backup in stressful cellular conditions that lead to ATP deficiency such as phosphate deficiency or hypoxia in roots. Since the interior of maize seeds (the endosperm) becomes hypoxic during seed expansion, glycolysis alone is increasingly important for supplying ATP in the central seed. A global stress response is thus predicted to have severe consequences in ATP availability which could be buffered by alternative enzymes such as PFP. We have shown in addition to large increases in PFP RNA and protein, the active PFP enzyme complex is specifically formed in QPM endosperm and not in wild type or o2. Furthermore, forward PFP enzyme activity is specifically increased in QPM endosperm. Another reversible PPi dependent glycolytic enzyme, encoded by the O2 regulated PPDK1 gene, is reduced in abundance o2 and QPM. However, the O2 independent PPDK2 gene is increased in expression in QPM and could function as an additional glycolytic bypass to maintain ATP production. Reverse PPDK activity is reduced in QPM thus supporting an increased forward glycolytic flux. Our preliminary metabolic profiling of central carbon glycolytic intermediates showed accumulation or deficiency at key steps which supports the observations of enzyme abundance and activity. Detailed metabolic profiling through Metabolon is in progress with multiple seed developmental stages and QPM genotypes. Our transgenic plant pipeline is allowing us to assess the function of various genes in relation to QPM as well as normal endosperm maturation. We have multiple independent lines for our dominant total alpha zein RNAi line which totally suppress alpha-zein expression and result in opaque endosperm. We have also received T1 seed for lines which over express 27-kD gamma-zein and PFP-alpha and we have crossed these to the alpha-zein null to determine if they are able to modify its opaque phenotype. We have also generated transgenic seeds with synthetic lysine and tryptophan-containing gamma-zein proteins and we are determining if they are able to further enrich seed protein quality when combined with the alpha-zein RNAi line at the same time as modifying its opaque phenotype. Our dissemination of these results is primarily through publication and a paper describing the role of PFP in QPM has been submitted to Plant Physiology and several papers with relevance to this work have been published in the last year. Large gene expression datasets are being placed on a local database (http://sysbio.unl.edu). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results of our investigation of the induction of PFP in QPM endosperm has two major impacts: First, we have developed a model in which an endosperm ATP deficiency in the high-lysine opaque2 mutant contributes to the undesirable seed characteristics which outweigh its improved nutritional quality. Further, in QPM endosperm, a switch to the use of pyrophosphate as an energy source through the PFP enzyme, enables the energy deficit to be ameliorated. By fully characterizing the role of PFP and other PPi dependent glycolytic enzymes such as PPDK, through thorough profiling of gene expression, enzyme activities and metabolite flux, and through transgenic manipulation, we are beginning to understand the basis of endosperm modification in QPM. Though our understanding is not complete, we have already produced transgenic plants with the potential to recreate vitreous endosperm in a dominant high lysine, low alpha-zein background, through physical (protein body) and biochemical (ATP) restoration and if successful we will seek industry collaboration to develop useful products for release to U.S. agriculture. Secondly, our study of PFP and PPi dependent glycolysis has relevance for other aspects of crop response to stress. We have determined that the PFP alpha subunit is co-induced with Hsp proteins in maize roots in response to heat shock, cold shock and endoplasmic reticulum stress. This not only supports our model that maintenance of ATP is essential for cell survival during stress but raises the possibility that we can enhance stress tolerance in crop plants by manipulating PFP in vegetative root tissues. Our dominant alpha-zein null line is predicted to have seed protein quality as good as or better than o2 but without the disadvantage of being recessive or pleiotropic. It is therefore an ideal test case for overexpression of candidate QPM genes. If such crosses result in lines with agronomic potential, we will swiftly seek industrial collaboration to introgress them into elite maize lines while aggressively protecting our intellectual property with the assistance of NUtech Ventures. Homozygous alpha-zein RNAi lines will be crossed to maize diversity lines and NAM parents to mine additional modifier loci which we expect to have swift impact in identifying and breeding genome regions containing novel modifier loci.
Publications
- Wu, R., Holding, D.R., and Messing, J. (2010) Gamma zeins are essential for endosperm modification in Quality Protein Maize. Proceedings of the National Academy of Sciences, USA. 107: 12,810-12,815
- Guo, X., Ronhovde, K., Yuan, L., Yao, B., Soundararajan, M.P., Elthon, T.E., Zhang, C., and Holding. D.R. (2012) Pyrophosphate dependent fructose-6-phosphate 1-phosphotransferase induction and attenuation of Hsp gene expression during endosperm modification in Quality Protein Maize. Pending with Plant Physiology (resubmitted with encouragement).
- Holding, D.R., and Messing, J. (2012) Evolution, structure and function of prolamin storage proteins. Invited book chapter in: Seed Genomics, Wiley-Blackwell Publishing, Editor, Becraft, P.
- Holding, D.R., Hunter, B.G., Klingler, J.P., Wu, S., Guo, X., Gibbon, B.C., Wu, R., Schulze, J., Jung, R., and Larkins, B.A. (2011). Characterization of opaque2 modifier QTLs and candidate genes in recombinant inbred lines derived from the K0326Y Quality Protein Maize inbred. Theor. Appl. Gen. 122, 783-794.
- Reyes, F.C., Chung, T., Holding, D., Jung, R., Vierstra, R., and Otegui, M.S. (2011) Delivery of Prolamins to the Protein Storage Vacuole in Maize Aleurone Cells, The Plant Cell 23, 769-784.
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