Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed)

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>Spirodela polyrhiza </it>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 (<it>APLs</it>). 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. <it>APL</it>2 and <it>APL</it>3 were highly expressed in earlier stages of turion development, while <it>APL</it>1 expression was reduced throughout turion development.</p> <p>Conclusions</p> <p>These results suggest that the differential expression of <it>APL</it>s could be used to enhance energy flow from photosynthesis to storage of carbon in aquatic plants, making duckweeds a useful alternative biofuel feedstock.</p

Divergence of gene regulation through chromosomal rearrangements

<p>Abstract</p> <p>Background</p> <p>The molecular mechanisms that modify genome structures to give birth and death to alleles are still not well understood. To investigate the causative chromosomal rearrangements, we took advantage of the allelic diversity of the duplicated <it>p1 </it>and <it>p2 </it>genes in maize. Both genes encode a transcription factor involved in maysin synthesis, which confers resistance to corn earworm. However, <it>p1 </it>also controls accumulation of reddish pigments in floral tissues and has therefore acquired a new function after gene duplication. <it>p1 </it>alleles vary in their tissue-specific expression, which is indicated in their allele designation: the first suffix refers to red or white pericarp pigmentation and the second to red or white glume pigmentation.</p> <p>Results</p> <p>Comparing chromosomal regions comprising <it>p1-ww[4Co63]</it>, <it>P1-rw1077 </it>and <it>P1-rr4B2 </it>alleles with that of the reference genome, <it>P1-wr[B73]</it>, enabled us to reconstruct additive events of transposition, chromosome breaks and repairs, and recombination that resulted in phenotypic variation and chimeric regulatory signals. The <it>p1-ww[4Co63] </it>null allele is probably derived from <it>P1-wr[B73] </it>by unequal crossover between large flanking sequences. A transposon insertion in a <it>P1-wr</it>-like allele and NHEJ (non-homologous end-joining) could have resulted in the formation of the <it>P1-rw1077 </it>allele. A second NHEJ event, followed by unequal crossover, probably led to the duplication of an enhancer region, creating the <it>P1-rr4B2 </it>allele. Moreover, a rather dynamic picture emerged in the use of polyadenylation signals by different <it>p1 </it>alleles. Interestingly, <it>p1 </it>alleles can be placed on both sides of a large retrotransposon cluster through recombination, while functional <it>p2 </it>alleles have only been found proximal to the cluster.</p> <p>Conclusions</p> <p>Allelic diversity of the <it>p </it>locus exemplifies how gene duplications promote phenotypic variability through composite regulatory signals. Transposition events increase the level of genomic complexity based not only on insertions but also on excisions that cause DNA double-strand breaks and trigger illegitimate recombination.</p

Characterization of the small RNA component of the transcriptome from grain and sweet sorghum stems

Background: Sorghum belongs to the tribe of the Andropogoneae that includes potential biofuel crops like switchgrass, Miscanthus and successful biofuel crops like corn and sugarcane. However, from a genomics point of view sorghum has compared to these other species a simpler genome because it lacks the additional rounds of whole genome duplication events. Therefore, it has become possible to generate a high-quality genome sequence. Furthermore, cultivars exists that rival sugarcane in levels of stem sugar so that a genetic approach can be used to investigate which genes are differentially expressed to achieve high levels of stem sugar. Results: Here, we characterized the small RNA component of the transcriptome from grain and sweet sorghum stems, and from F2 plants derived from their cross that segregated for sugar content and flowering time. We found that variation in miR172 and miR395 expression correlated with flowering time whereas variation in miR169 expression correlated with sugar content in stems. Interestingly, genotypic differences in the ratio of miR395 to miR395* were identified, with miR395* species expressed as abundantly as miR395 in sweet sorghum but not in grain sorghum. Finally, we provided experimental evidence for previously annotated miRNAs detecting the expression of 25 miRNA families from the 27 known and discovered 9 new miRNAs candidates in the sorghum genome. Conclusions: Sequencing the small RNA component of sorghum stem tissue provides us with experimental evidence for previously predicted microRNAs in the sorghum genome and microRNAs with a potential role in stem sugar accumulation and flowering time

γ-Zeins are essential for endosperm modification in quality protein maize

Essential amino acids like lysine and tryptophan are deficient in corn meal because of the abundance of zein storage proteins that lack these amino acids. A naturalmutant, opaque 2 (o2) causes reduction of zeins,anincreaseofnonzeinproteins,andas a consequence, adoubling of lysine levels.However, o2’s soft inferior kernels precluded its commercial use. Breeders subsequently overcame kernel softness, selectingseveral quantitative loci (QTLs), called o2modifiers,without losing the high-lysine trait. These maize lines are known as “quality protein maize” (QPM). One of the QTLs is linked to the 27-kDa γ-zein locus on chromosome 7S. Moreover, QPM lines have 2- to 3-fold higher levels of the 27-kDa γ-zein, but the physiological significance of this increase is not known. Because the 27- and 16-kDa γ-zein genes are highly conserved in DNA sequence, we introduced a dominant RNAi transgene into a QPM line (CM105Mo2) to eliminate expression of them both. Elimination of γ-zeins disrupts endosperm modification by o2 modifiers, indicating their hypostatic action to γ-zeins. Abnormalities in protein body structure and their interaction with starch granules in the F1 with Mo2/+; o2/o2; γRNAi/+ genotype suggests that γ-zeins are essential for restoring protein body density and starch grain interaction in QPM. To eliminate pleiotropic effects caused by o2, the 22-kDa α-zein, γ-zein, and β-zein RNAis were stacked, resulting in protein bodies forming as honeycomb-like structures. We are unique in presenting clear demonstration that γ-zeins play a mechanistic role in QPM, providing a previously unexplored rationale for molecular breeding

Novel Genetic Selection System for Quantitative Trait Loci of Quality Protein Maize

Quality protein maize combines a high-lysine trait with kernel hardness, for which a new, simpler genetic selection was designed

Divergent properties of prolamins in wheat and maize

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 γ-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 γ-zeins for its proper subcellular organization in maize

Balancing of sulfur storage in maize seed

Abstract Background 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. Results In maize (Zea mays), the 22- and 19-kDa α-zeins are the most prominent storage proteins, nearly devoid of lysine and methionine. Although silencing synthesis of these proteins through RNA interference (RNAi) raises lysine levels in the seed, it fails to do so for methionine. Computational analysis of annotated gene models suggests that about 57% of all proteins exhibit a lysine content of more than 4%, whereas the percentage of proteins with methionine above 4% is only around 8%. To compensate for this low representation, maize seeds produce specialized storage proteins, the 15-kDa β-, 18-kDa and 10-kDa δ-zeins, rich in methionine. However, they are expressed at variant levels in different inbred lines. A654, an inbred with null δ-zein alleles, methionine levels are significantly lower than when the two intact δ-zein alleles are introgressed. Further silencing of β-zein results in dramatic reduction in methionine levels, indicating that β- and δ-zeins are the main sink of methionine in maize seed. Overexpression of the 10-kDa δ-zein can increase the methionine level, but protein analysis by SDS-PAGE shows that the increased methionine levels occur at least in part at the expense of cysteines present in β- and γ-zeins. The reverse is true when β- and γ-zein expression is silenced through RNAi, then 10-kDa δ-zein accumulates to higher levels. Conclusions Because methionine receives the sulfur moiety from cysteine, it appears that when seed protein synthesis of cysteine-rich proteins is blocked, the synthesis of methionine-rich seed proteins is induced, probably at the translational level. The same is true, when methionine-rich proteins are overexpressed, synthesis of cysteine-rich proteins is reduced, probably also at the translational level. Although we only hypothesize a translational control of protein synthesis at this time, there are well known paradigms of how amino acid concentration can play a role in differential gene expression. The latter we think is largely controlled by the flux of reduced sulfur during plant growth.</p

On the Tetraploid Origin of the Maize Genome

Data from cytological and genetic mapping studies suggest that maize arose as a tetraploid. Two previous studies investigating the most likely mode of maize origin arrived at different conclusions. Gaut and Doebley [7] proposed a segmental allotetraploid origin of the maize genome and estimated that the two maize progenitors diverged at 20.5 million years ago (mya). In a similar study, using larger data set, Brendel and colleagues (quoted in [8]) suggested a single genome duplication at 16 mya. One of the key components of such analyses is to examine sequence divergence among strictly orthologous genes. In order to identify such genes, Lai and colleagues [10] sequenced five duplicated chromosomal regions from the maize genome and the orthologous counterparts from the sorghum genome. They also identified the orthologous regions in rice. Using positional information of genetic components, they identified 11 orthologous genes across the two duplicated regions of maize, and the sorghum and rice regions. Swigonova et al. [12] analyzed the 11 orthologues, and showed that all five maize chromosomal regions duplicated at the same time, supporting a tetraploid origin of maize, and that the two maize progenitors diverged from each other at about the same time as each of them diverged from sorghum, about 11.9 mya