Publications

2014
Nasr, I, Messing J, Ciclitira PJ.  2014.  Novel and Experimental Therapies on the Horizon. Celiac Disease, Clinical Gastroenterology. :193-208.
Wu, Y, Messing J.  2014.  Proteome balancing of the maize seed for higher nutritional value. Front Plant Sci. 5:240. AbstractWebsite
Most flowering plant seeds are composed of the embryo and endosperm, which are surrounded by maternal tissue, in particular the seed coat. Whereas the embryo is the dormant progeny, the endosperm is a terminal organ for storage of sugars and amino acids in proteins and carbohydrates, respectively. Produced in maternal leaves during photosynthesis, sugars, and amino acids are transported to developing seeds after flowering, and during germination they nourish early seedlings growth. Maize endosperm usually contains around 10% protein and 70% starch, and their composition ratio is rather stable, because it is strictly regulated through a pre-set genetic program that is woven by networks of many interacting or counteracting genes and pathways. Endosperm protein, however, is of low nutritional value due mainly to the high expression of the alpha-zein gene family, which encodes lysine-free proteins. Reduced levels of these proteins in the opaque 2 (o2) mutant and alpha-zein RNAi (RNA interference) transgenic seed is compensated by an increase of non-zein proteins, leading to the rebalancing of the nitrogen sink and producing more or less constant levels of total proteins in the seed. The same rebalancing of zeins and non-zeins has been observed for maize seeds bred for 30% protein. In contrast to the nitrogen sink, storage of sulfur is controlled through the accumulation of specialized sulfur-rich proteins in maize endosperm. Silencing the synthesis of alpha-zeins through RNAi fails to raise sulfur-rich proteins. Although overexpression of the methionine-rich delta-zein can increase the methionine level in seeds, it occurs at least in part at the expense of the cysteine-rich beta- and gamma-zeins, demonstrating a balance between cysteine and methionine in sulfur storage. Therefore, we propose that the throttle for the flow of sulfur is placed before the synthesis of sulfur amino acids when sulfur is taken up and reduced during photosynthesis.
Wang, W, Wu Y, Messing J.  2014.  RNA-Seq transcriptome analysis of Spirodela dormancy without reproduction. BMC Genomics. 15:60. AbstractWebsite
BACKGROUND: Higher plants exhibit a remarkable phenotypic plasticity to adapt to adverse environmental changes. The Greater Duckweed Spirodela, as an aquatic plant, presents exceptional tolerance to cold winters through its dormant structure of turions in place of seeds. Abundant starch in turions permits them to sink and escape the freezing surface of waters. Due to their clonal propagation, they are the fastest growing biomass on earth, providing yet an untapped source for industrial applications. RESULTS: We used next generation sequencing technology to examine the transcriptome of turion development triggered by exogenous ABA. A total of 208 genes showed more than a 4-fold increase compared with 154 down-regulated genes in developing turions. The analysis of up-regulated differential expressed genes in response to dormancy exposed an enriched interplay among various pathways: signal transduction, seed dehydration, carbohydrate and secondary metabolism, and senescence. On the other side, the genes responsible for rapid growth and biomass accumulation through DNA assembly, protein synthesis and carbon fixation are repressed. Noticeably, three members of late embryogenesis abundant protein family are exclusively expressed during turion formation. High expression level of key genes in starch synthesis are APS1, APL3 and GBSSI, which could artificially be reduced for re-directing carbon flow from photosynthesis to create a higher energy biomass. CONCLUSIONS: The identification and functional annotation of differentially expressed genes open a major step towards understanding the molecular network underlying vegetative frond dormancy. Moreover, genes have been identified that could be engineered in duckweeds for practical applications easing agricultural production of food crops.
Wang, W, Haberer G, Gundlach H, Glasser C, Nussbaumer T, Luo MC, Lomsadze A, Borodovsky M, Kerstetter RA, Shanklin J et al..  2014.  The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nat Commun. 5:3311. AbstractWebsite
The subfamily of the Lemnoideae belongs to a different order than other monocotyledonous species that have been sequenced and comprises aquatic plants that grow rapidly on the water surface. Here we select Spirodela polyrhiza for whole-genome sequencing. We show that Spirodela has a genome with no signs of recent retrotranspositions but signatures of two ancient whole-genome duplications, possibly 95 million years ago (mya), older than those in Arabidopsis and rice. Its genome has only 19,623 predicted protein-coding genes, which is 28% less than the dicotyledonous Arabidopsis thaliana and 50% less than monocotyledonous rice. We propose that at least in part, the neotenous reduction of these aquatic plants is based on readjusted copy numbers of promoters and repressors of the juvenile-to-adult transition. The Spirodela genome, along with its unique biology and physiology, will stimulate new insights into environmental adaptation, ecology, evolution and plant development, and will be instrumental for future bioenergy applications.
2013
Wang, C, Shi X, Liu L, Li H, Ammiraju JS, Kudrna DA, Xiong W, Wang H, Dai Z, Zheng Y et al..  2013.  Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives. Genetics. 195:723-37. AbstractWebsite
Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.
Goettel, W, Messing J.  2013.  Epiallele biogenesis in maize. Gene. 516:8-23. AbstractWebsite
We have correlated cytosine methylation of two epialleles, P1-rr and P1-pr, with variation in gene expression and therefore phenotype. The p1 gene in maize encodes a transcription factor that controls phlobaphene pigment accumulation in floral tissues. While cytosine methylation was assayed in various regions spanning 17 kb, the only difference in DNA methylation pattern between the expressed P1-rr allele and the silenced P1-pr allele was detected in a region that consists of a complex arrangement of transposons and adjacent repeats. This region, which comprises the distal enhancer element of P1-rr, is hypermethylated in P1-pr compared to P1-rr. Based on other precedents, we hypothesize that DNA methylation spreads from the transposable elements into the flanking P1-rr enhancer, thereby transcriptionally silencing the gene. Interestingly, P1-pr is reactivated in mutants of the dominant epigenetic modifier Ufo1. DNA methylation in the distal enhancer sequence is significantly reduced, which inversely correlates with increased transcript levels and pigmentation in P1-pr Ufo1 plants. If in general DNA methylation spreads from transposons into adjacent sequences containing regulatory elements for neighboring genes, the corresponding genes could be silenced by chance. Given the large amount of transposable elements in the maize genome, epialleles may be far more frequent than previously estimated.
Zhang, W, Sangtong V, Peterson J, Scott MP, Messing J.  2013.  Divergent properties of prolamins in wheat and maize. Planta. 237:1465-73. AbstractWebsite
Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. Wheat, barley, and oats belong to a different subfamily of the grasses than rice and in addition to maize, millets, sugarcane, and sorghum. All their seeds, however, are largely devoid of free amino acids because they are stored during dormancy in specialized storage proteins. Prolamins, the major class of storage proteins in cereals with preponderance of proline and glutamine, are synthesized at the endoplasmic reticulum during seed development and deposited into subcellular structures of the immature endosperm, the protein bodies. Prolamins have diverged during the evolution of the grass family in their structure and their properties. Here, we used the expression of wheat glutenin-Dx5 in maize to examine its interaction with maize prolamins during endosperm development. Ectopic expression of Dx5 alters protein body morphology in a way that resembles non-vitreous kernel phenotypes, although Dx5 alone does not cause an opaque phenotype. However, if we lower the amount of gamma-zeins in Dx5 maize through RNAi, a non-vitreous phenotype emerges and the deformation on the surface of protein bodies is enhanced, indicating that Dx5 requires gamma-zeins for its proper subcellular organization in maize.
Goettel, W, Messing J.  2013.  Paramutagenicity of a p1 epiallele in maize. Theor Appl Genet. 126:159-77. AbstractWebsite
Complex silencing mechanisms in plants and other kingdoms target transposons, repeat sequences, invasive viral nucleic acids and transgenes, but also endogenous genes and genes involved in paramutation. Paramutation occurs in a heterozygote when a transcriptionally active allele heritably adopts the epigenetic state of a transcriptionally and/or post-transcriptionally repressed allele. P1-rr and its silenced epiallele P1-pr, which encode a Myb-like transcription factor mediating pigmentation in floral organs of Zea mays, differ in their cytosine methylation pattern and chromatin structure at a complex enhancer site. Here, we tested whether P1-pr is able to heritably silence its transcriptionally active P1-rr allele in a heterozygote and whether DNA methylation is associated with the establishment and maintenance of P1-rr silencing. We found that P1-pr participates in paramutation as the repressing allele and P1-rr as the sensitive allele. Silencing of P1-rr is highly variable compared to the inducing P1-pr resulting in a wide range of gene expression. Whereas cytosine methylation at P1-rr is negatively correlated with transcription and pigment levels after segregation of P1-pr, methylation lags behind the establishment of the repressed p1 gene expression. We propose a model in which P1-pr paramutation is triggered by changing epigenetic states of transposons immediately adjacent to a P1-rr enhancer sequence. Considering the vast amount of transposable elements in the maize genome close to regulatory elements of genes, numerous loci could undergo paramutation-induced allele silencing, which could also have a significant impact on breeding agronomically important traits.
Calvino, M., Messing J.  2013.  Discovery of MicroRNA169 gene copies in genomes of flowering plants through positional information. Genome Biol Evol. 5:402-17. AbstractWebsite
Expansion and contraction of microRNA (miRNA) families can be studied in sequenced plant genomes through sequence alignments. Here, we focused on miR169 in sorghum because of its implications in drought tolerance and stem-sugar content. We were able to discover many miR169 copies that have escaped standard genome annotation methods. A new miR169 cluster was found on sorghum chromosome 1. This cluster is composed of the previously annotated sbi-MIR169o together with two newly found MIR169 copies, named sbi-MIR169t and sbi-MIR169u. We also found that a miR169 cluster on sorghum chr7 consisting of sbi-MIR169l, sbi-MIR169m, and sbi-MIR169n is contained within a chromosomal inversion of at least 500 kb that occurred in sorghum relative to Brachypodium, rice, foxtail millet, and maize. Surprisingly, synteny of chromosomal segments containing MIR169 copies with linked bHLH and CONSTANS-LIKE genes extended from Brachypodium to dictotyledonous species such as grapevine, soybean, and cassava, indicating a strong conservation of linkages of certain flowering and/or plant height genes and microRNAs, which may explain linkage drag of drought and flowering traits and would have consequences for breeding new varieties. Furthermore, alignment of rice and sorghum orthologous regions revealed the presence of two additional miR169 gene copies (miR169r and miR169s) on sorghum chr7 that formed an antisense miRNA gene pair. Both copies are expressed and target different set of genes. Synteny-based analysis of microRNAs among different plant species should lead to the discovery of new microRNAs in general and contribute to our understanding of their evolution.
Messing, J, Holding D.  2013.  Evolution, Structure, and Function of Prolamin Storage Proteins.. Seed Genomics. :139-158.
Wu, Y, Yuan L, Guo X, Holding DR, Messing J.  2013.  Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum. Nat Commun. 4:2217. AbstractWebsite
Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.
2012
Wu, Y, Messing J.  2012.  Rapid divergence of prolamin gene promoters of maize after gene amplification and dispersal. Genetics. 192:507-19. AbstractWebsite
Seeds have evolved to accommodate complicated processes like senescence, dormancy, and germination. Central to these is the storage of carbohydrates and proteins derived from sugars and amino acids synthesized during photosynthesis. In the grasses, the bulk of amino acids is stored in the prolamin superfamily that specifically accumulates in seed endosperm during senescence. Their promoters contain a conserved cis-element, called prolamin-box (P-box), recognized by the trans-activator P-box binding factor (PBF). Because of the lack of null mutants in all grass species, its physiological role in storage-protein gene expression has been elusive. In contrast, a null mutant of another endosperm-specific trans-activator Opaque2 (O2) has been shown to be required for the transcriptional activation of subsets of this superfamily by binding to the O2 box. Here, we used RNAi to knockdown Pbf expression and found that only 27-kDa gamma- and 22-kDa alpha-zein gene expression were affected, whereas the level of other zeins remained unchanged. Still, transgenic seeds had an opaque seed phenotype. Combination of PbfRNAi and o2 resulted in further reduction of alpha-zein expression. We also tested the interaction of promoters and constitutively expressed PBF and O2. Whereas transgenic promoters could be activated, endogenous promoters appeared to be not accessible to transcriptional activation, presumably due to differential chromatin states. Although analysis of the methylation of binding sites of PBF and O2 correlated with the expression of endogenous 22-kDa alpha-zein promoters, a different mechanism seems to apply to the 27-kDa gamma-zein promoter, which does not undergo methylation changes.
Xu, JH, Bennetzen JL, Messing J.  2012.  Dynamic gene copy number variation in collinear regions of grass genomes. Mol Biol Evol. 29:861-71. AbstractWebsite
A salient feature of genomes of higher organisms is the birth and death of gene copies. An example is the alpha prolamin genes, which encode seed storage proteins in grasses (Poaceae) and represent a medium-size gene family. To better understand the mechanism, extent, and pace of gene amplification, we compared prolamin gene copies in the genomes of two different tribes in the Panicoideae, the Paniceae and the Andropogoneae. We identified alpha prolamin (setarin) gene copies in the diploid foxtail millet (Paniceae) genome (490 Mb) and compared them with orthologous regions in diploid sorghum (730 Mb) and ancient allotetraploid maize (2,300 Mb) (Andropogoneae). Because sequenced genomes of other subfamilies of Poaceae like rice (389 Mb) (Ehrhartoideae) and Brachypodium (272 Mb) (Pooideae) do not have alpha prolamin genes, their collinear regions can serve as "empty" reference sites. A pattern emerged, where genes were copied and inserted into other chromosomal locations followed by additional tandem duplications (clusters). We observed both recent (species-specific) insertion events and older ones that are shared by these tribes. Many older copies were deleted by unequal crossing over of flanking sequences or damaged by truncations. However, some remain intact with active and inactive alleles. These results indicate that genomes reflect only a snapshot of the gene content of a species and are far less static than conventional genetics has suggested. Nucleotide substitution rates for active alpha prolamins genes were twice as high as for low copy number beta, gamma, and delta prolamin genes, suggesting that gene amplification accelerates the pace of divergence.
Goettel, W, Messing J.  2012.  Paramutagenicity of a p1 epiallele in maize. Theoretical and Applied Genetics. (Epub Sep 18) AbstractWebsite
Complex silencing mechanisms in plants and other kingdoms target transposons, repeat sequences, invasive viral nucleic acids and transgenes, but also endogenous genes and genes involved in paramutation. Paramutation occurs in a heterozygote when a transcriptionally active allele heritably adopts the epigenetic state of a transcriptionally and/or post-transcriptionally repressed allele. P1-rr and its silenced epiallele P1-pr, which encode a Myb-like transcription factor mediating pigmentation in floral organs of Zea mays, differ in their cytosine methylation pattern and chromatin structure at a complex enhancer site. Here, we tested whether P1-pr is able to heritably silence its transcriptionally active P1-rr allele in a heterozygote and whether DNA methylation is associated with the establishment and maintenance of P1-rr silencing. We found that P1-pr participates in paramutation as the repressing allele and P1-rr as the sensitive allele. Silencing of P1-rr is highly variable compared to the inducing P1-pr resulting in a wide range of gene expression. Whereas cytosine methylation at P1-rr is negatively correlated with transcription and pigment levels after segregation of P1-pr, methylation lags behind the establishment of the repressed p1 gene expression. We propose a model in which P1-pr paramutation is triggered by changing epigenetic states of transposons immediately adjacent to a P1-rr enhancer sequence. Considering the vast amount of transposable elements in the maize genome close to regulatory elements of genes, numerous loci could undergo paramutation-induced allele silencing, which could also have a significant impact on breeding agronomically important traits.
Liao, JC, Messing J.  2012.  Energy biotechnology. Current opinion in biotechnology. 23(3):287-9.Website
Calviño, M, Messing J.  2012.  Sweet sorghum as a model system for bioenergy crops.. Current opinion in biotechnology. 23(3):323-9. AbstractWebsite
Bioenergy is the reduction of carbon via photosynthesis. Currently, this energy is harvested as liquid fuel through fermentation. A major concern, however, is input cost, in particular use of excess water and nitrogen, derived from an energy-negative process, the Haber-Bosch method. Furthermore, the shortage of arable land creates competition between uses for food and fuel, resulting in increased living expenses. This review seeks to summarize recent knowledge in genetics, genomics, and gene expression of a rising model species for bioenergy applications, sorghum. Its diploid genome has been sequenced, it has favorable low-input cost traits, and genetic crosses between different cultivars can be used to study allelic variations of genes involved in stem sugar metabolism and incremental biomass.
Wu, Y, Wang W, Messing J.  2012.  Balancing of sulfur storage in maize seed. BMC plant biology. 12:77. AbstractWebsite
A balanced composition of amino acids in seed flour is critical because of the demand on essential amino acids for nutrition. However, seed proteins in cereals like maize, the crop with the highest yield, are low in lysine, tryptophan, and methionine. Although supplementation with legumes like soybean can compensate lysine deficiency, both crops are also relatively low in methionine. Therefore, understanding the mechanism of methionine accumulation in the seed could be a basis for breeding cultivars with superior nutritional quality.
Wang, W, Wu Y, Messing J.  2012.  The Mitochondrial Genome of an Aquatic Plant, Spirodela polyrhiza.. PloS one. 7(10):e46747. AbstractWebsite
Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant.
Wang, W, Messing J.  2012.  Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed). BMC Plant Biol. 12:5. AbstractWebsite
BACKGROUND: Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season. RESULTS: To begin a characterization of the transition from the growth to the dormant phase we used abscisic acid (ABA), a plant hormone, to induce controlled turion formation in Spirodela polyrhiza and investigated their differentiation from fronds, representing their growth phase, into turions with respect to morphological, ultra-structural characteristics, and starch content. Turions were rich in anthocyanin pigmentation and had a density that submerged them to the bottom of liquid medium. Transmission electron microscopy (TEM) of turions showed in comparison to fronds shrunken vacuoles, smaller intercellular space, and abundant starch granules surrounded by thylakoid membranes. Turions accumulated more than 60% starch in dry mass after two weeks of ABA treatment. To further understand the mechanism of the developmental switch from fronds to turions, we cloned and sequenced the genes of three large-subunit ADP-glucose pyrophosphorylases (APLs). All three putative protein and exon sequences were conserved, but the corresponding genomic sequences were extremely variable mainly due to the invasion of miniature inverted-repeat transposable elements (MITEs) into introns. A molecular three-dimensional model of the SpAPLs was consistent with their regulatory mechanism in the interaction with the substrate (ATP) and allosteric activator (3-PGA) to permit conformational changes of its structure. Gene expression analysis revealed that each gene was associated with distinct temporal expression during turion formation. APL2 and APL3 were highly expressed in earlier stages of turion development, while APL1 expression was reduced throughout turion development. CONCLUSIONS: These results suggest that the differential expression of APLs could be used to enhance energy flow from photosynthesis to storage of carbon in aquatic plants, making duckweeds a useful alternative biofuel feedstock.
Wu, Y, Messing J.  2012.  RNA Interference Can Rebalance the Nitrogen Sink of Maize Seeds without Losing Hard Endosperm. PLoS One. 7:e32850. AbstractWebsite
BACKGROUND: One of the goals of plant breeding is to create crops to provide better nutrition for humans and livestock. Insufficient intake of protein is one of the most severe factors affecting the growth and development of children in developing countries. More than a century ago, in 1896, Hopkins initiated the well-known Illinois long-term selection for maize seed protein concentration, yielding four protein strains. By continuously accumulating QTLs, Illinois High Protein (IHP) reached a protein level 2.5-fold higher than normal maize, with the most increased fraction being the zein protein, which was shown to contain no lysine soon after the long-term selection program initiated. Therefore, IHP is of little value for feeding humans and monogastric animals. Although high-lysine lines of non-vitreous mutants were based on reduced zeins, the kernel soft texture precluded their practical use. Kernel hardness in opaque 2 (o2) could be restored in quality protein maize (QPM) with quantitative trait loci called o2 modifiers (Mo2s), but those did not increase total protein levels. METHODS: The most predominant zeins are the 22- and 19-kDa alpha-zeins. To achieve a combination of desired traits, we used RNA interference (RNAi) against both alpha-zeins in IHP and evaluated the silencing effect by SDS-PAGE. Total protein, amino acid composition and kernel texture were analyzed. CONCLUSIONS: The alpha-zeins were dramatically reduced, but the high total seed protein level remained unchanged by complementary increase of non-zein proteins. Moreover, the residual zein levels still allowed for a vitreous hard seed. Such dramatic rebalancing of the nitrogen sink could have a major impact in world food supply.
2011
Wu, Y, Messing J.  2011.  Novel Genetic Selection System for Quantitative Trait Loci of Quality Protein Maize. Genetics. 188:1019-1022. AbstractWebsite
Quality Protein Maize combines a high-lysine trait with kernel hardness, for which a new simpler genetic selection was designed.
Miclaus, M, Xu JH, Messing J.  2011.  Differential gene expression and epiregulation of alpha zein gene copies in maize haplotypes. PLoS Genet. 7:e1002131. AbstractWebsite
Multigenic traits are very common in plants and cause diversity. Nutritional quality is such a trait, and one of its factors is the composition and relative expression of storage protein genes. In maize, they represent a medium-size gene family distributed over several chromosomes and unlinked locations. Two inbreds, B73 and BSSS53, both from the Iowa Stiff Stock Synthetic collection, have been selected to analyze allelic and non-allelic variability in these regions that span between 80-500 kb of chromosomal DNA. Genes were copied to unlinked sites before and after allotetraploidization of maize, but before transposition enlarged intergenic regions in a haplotype-specific manner. Once genes are copied, expression of donor genes is reduced relative to new copies. Epigenetic regulation seems to contribute to silencing older copies, because some of them can be reactivated when endosperm is maintained as cultured cells, indicating that copy number variation might contribute to a reserve of gene copies. Bisulfite sequencing of the promoter region also shows different methylation patterns among gene clusters as well as differences between tissues, suggesting a possible position effect on regulatory mechanisms as a result of inserting copies at unlinked locations. The observations offer a potential paradigm for how different gene families evolve and the impact this has on their expression and regulation of their members.
Miclaus, M, Wu Y, Xu JH, Dooner HK, Messing J.  2011.  The maize high-lysine mutant opaque7 is defective in an acyl-CoA synthetase-like protein. Genetics. 189:1271-80. AbstractWebsite
Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from approximately 1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.
Wu, Y, Messing J.  2011.  Novel genetic selection system for quantitative trait loci of quality protein maize. Genetics. 188:1019-22. AbstractWebsite
Quality protein maize combines a high-lysine trait with kernel hardness, for which a new, simpler genetic selection was designed.