Three Diverse Granule Preparation Methods for Proteomic Analysis of Mature Rice (Oryza sativa L.) Starch Grain.

Starch is the primary form of reserve carbohydrate storage in plants. Rice (Oryza sativa L.) is a monocot whose reserve starch is organized into compounded structures within the amyloplast, rather than a simple starch grain (SG). The mechanism governing the assembly of the compound SG from polyhedral granules in apposition, however, remains unknown. To further characterize the proteome associated with these compounded structures, three distinct methods of starch granule preparation (dispersion, microsieve, and flotation) were performed. Phase separation of peptides (aqueous trypsin-shaving and isopropanol solubilization of residual peptides) isolated starch granule-associated proteins (SGAPs) from the distal proteome of the amyloplast and the proximal 'amylome' (the amyloplastic proteome), respectively. The term 'distal proteome' refers to SGAPs loosely tethered to the amyloplast, ones that can be rapidly proteolyzed, while proximal SGAPs are those found closer to the remnant amyloplast membrane fragments, perhaps embedded therein-ones that need isopropanol solvent to be removed from the mature organelle surface. These two rice starch-associated peptide samples were analyzed using nano-liquid chromatography-tandem mass spectrometry (Nano-HPLC-MS/MS). Known and novel proteins, as well as septum-like structure (SLS) proteins, in the mature rice SG were found. Data mining and gene ontology software were used to categorize these putative plastoskeletal components as a variety of structural elements, including actins, tubulins, tubulin-like proteins, and cementitious elements such as reticulata related-like (RER) proteins, tegument proteins, and lectins. Delineating the plastoskeletal proteome begins by understanding how each starch granule isolation procedure affects observed cytoplasmic and plastid proteins. The three methods described herein show how the technique used to isolate SGs differentially impacts the subsequent proteomic analysis and results obtained. It can thus be concluded that future investigations must make judicious decisions regarding the methodology used in extracting proteomic information from the compound starch granules being assessed, since different methods are shown to yield contrasting results herein. Data are available via ProteomeXchange with identifier PXD032314.

Comparative Analysis of In Vitro Responses and Regeneration between Diverse Bioenergy Sorghum Genotypes.

Sorghum has been considered a recalcitrant plant in vitro and suffers from a lack of regeneration protocols that function broadly and efficiently across a range of genotypes. This study was initiated to identify differential genotype-in vitro protocol responses across a range of bioenergy sorghum parental lines and the common grain sorghum genotype Tx430 in order to characterize response profiles for use in future genetic studies. Two different in vitro protocols, LG and WU, were used for comparisons. Distinct genotype-protocol responses were observed, and the WU protocol performed significantly better for plantlet regeneration. Most bioenergy genotypes performed as well, if not better than Tx430, with Rio and PI329311 as the top regenerating lines. Genotypes displayed protocol-dependent, differential phenolic exudation responses, as indicated by medium browning. During the callus induction phase, genotypes prone to medium browning exhibited a response on WU medium which was either equal or greater than on LG medium. Genotype- and protocol-dependent albino plantlet regeneration was also noted, with three of the bioenergy genotypes showing albino plantlet regeneration. Grassl, Rio and Pink Kafir were susceptible to albino plantlet regeneration, with the response strongly associated with the WU protocol. These bioenergy parental genotypes, and their differential responses under two in vitro protocols, provide tools to further explore and assess the role of genetic loci, candidate genes, and allelic variants in the regulation of in vitro responsiveness in sorghum.

Integrated Genome-Scale Analysis Identifies Novel Genes and Networks Underlying Senescence in Maize.

Premature senescence in annual crops reduces yield, while delayed senescence, termed stay-green, imposes positive and negative impacts on yield and nutrition quality. Despite its importance, scant information is available on the genetic architecture of senescence in maize (Zea mays) and other cereals. We combined a systematic characterization of natural diversity for senescence in maize and coexpression networks derived from transcriptome analysis of normally senescing and stay-green lines. Sixty-four candidate genes were identified by genome-wide association study (GWAS), and 14 of these genes are supported by additional evidence for involvement in senescence-related processes including proteolysis, sugar transport and signaling, and sink activity. Eight of the GWAS candidates, independently supported by a coexpression network underlying stay-green, include a trehalose-6-phosphate synthase, a NAC transcription factor, and two xylan biosynthetic enzymes. Source-sink communication and the activity of cell walls as a secondary sink emerge as key determinants of stay-green. Mutant analysis supports the role of a candidate encoding Cys protease in stay-green in Arabidopsis (Arabidopsis thaliana), and analysis of natural alleles suggests a similar role in maize. This study provides a foundation for enhanced understanding and manipulation of senescence for increasing carbon yield, nutritional quality, and stress tolerance of maize and other cereals.

A new reference genome for Sorghum bicolor reveals high levels of sequence similarity between sweet and grain genotypes: implications for the genetics of sugar metabolism.

BACKGROUND: The process of crop domestication often consists of two stages: initial domestication, where the wild species is first cultivated by humans, followed by diversification, when the domesticated species are subsequently adapted to more environments and specialized uses. Selective pressure to increase sugar accumulation in certain varieties of the cereal crop Sorghum bicolor is an excellent example of the latter; this has resulted in pronounced phenotypic divergence between sweet and grain-type sorghums, but the genetic mechanisms underlying these differences remain poorly understood. RESULTS: Here we present a new reference genome based on an archetypal sweet sorghum line and compare it to the current grain sorghum reference, revealing a high rate of nonsynonymous and potential loss of function mutations, but few changes in gene content or overall genome structure. We also use comparative transcriptomics to highlight changes in gene expression correlated with high stalk sugar content and show that changes in the activity and possibly localization of transporters, along with the timing of sugar metabolism play a critical role in the sweet phenotype. CONCLUSIONS: The high level of genomic similarity between sweet and grain sorghum reflects their historical relatedness, rather than their current phenotypic differences, but we find key changes in signaling molecules and transcriptional regulators that represent new candidates for understanding and improving sugar metabolism in this important crop.

Global gene expression profiling of two switchgrass cultivars following inoculation with Burkholderia phytofirmans strain PsJN.

Improvement and year-to-year stabilization of biomass yields are primary objectives for the development of a low-input switchgrass feedstock production system using microbial endophytes. An earlier investigation of the effect of Burkholderia phytofirmans strain PsJN on switchgrass germplasm demonstrated differential responses between genotypes. PsJN inoculation of cv. Alamo (lowland ecotype) increased the plant root system, shoot length, and biomass yields, whereas it had no beneficial effect on cv. Cave-in-Rock (upland ecotype). To understand the gene networks governing plant growth promotion responses triggered by PsJN, the gene expression profiles were analysed in these two hosts, following seedling inoculation. The Affymetrix platform switchgrass expressed sequence tag (EST) microarray chip representing 122 972 probe sets, developed by the DOE BioEnergy Science Center, was employed to assess transcript abundance at 0.5, 2, 4, and 8 DAI (days after PsJN inoculation). Approximately 20 000 switchgrass probe sets showed significant responses in either cultivar. Switchgrass identifiers were used to map 19 421 genes in MapMan software. There were apparent differences in gene expression profiling between responsive and non-responsive cultivars after PsJN inoculation. Overall, there were 14 984 and 9691 genes affected by PsJN inoculation in Alamo and Cave-in-Rock, respectively. Of these, 394 are annotated as pathogenesis-related genes. In the responsive cv. Alamo, 68 pathogenesis-related genes were affected, compared with only 10 in the non-responsive cv. Cave-in-Rock. At the very early stage at 0.5 DAI, both cultivars exhibited similar recognition and defence responses, such as genes in signalling and proteolysis, after which the defence reaction in the responsive cv. Alamo became weaker while it was sustained in non-responsive cv. Cave-in-Rock.

Characterization of a potato activation-tagged mutant, nikku, and its partial revertant.

MAIN CONCLUSION: A potato mutant with a strong stress-response phenotype, and a partial mutant revertant, were characterized. Gene expression patterns and DNA cytosine methylation varied between these and wild-type, indicating a role for DNA cytosine methylation changes in the gene expression and visible phenotypes. Morphological and molecular studies were conducted to compare potato cv. Bintje, a Bintje activation-tagged mutant (nikku), and nikku revertant phenotype plants. Morphological studies revealed that nikku plants exhibited an extremely dwarf phenotype, had small hyponastic leaves, were rootless, and infrequently produced small tubers compared to wild-type Bintje. The overall phenotype was suggestive of a constitutive stress response, which was further supported by the greater expression level of several stress-responsive genes in nikku. Unlike the nikku mutant, the revertant exhibited near normal shoot elongation, larger leaves and consistent rooting. The reversion appeared partial, and was not the result of a loss of 35S enhancer copies from the original nikku mutant. Southern blot analyses indicated the presence of a single T-DNA insertion on chromosome 12 in the mutant. Gene expression studies comparing Bintje, nikku and revertant phenotype plants indicated transcriptional activation/repression of several genes flanking both sides of the insertion in the mutant, suggesting that activation tagging had pleiotropic effects in nikku. In contrast, gene expression levels for many, but not all, of the same genes in the revertant were similar to Bintje, indicating some reversion at the gene expression level as well. DNA methylation studies indicated differences in cytosine methylation status of the 35S enhancers between the nikku mutant and its revertant. In addition, global DNA cytosine methylation varied between Bintje, the nikku mutant and the revertant, suggesting involvement in gene expression changes, as well as mutant phenotype.

Characterization and RNA-seq analysis of underperformer, an activation-tagged potato mutant.

The potato cv. Bintje and a Bintje activation-tagged mutant, underperformer (up) were compared. Mutant up plants grown in vitro were dwarf, with abundant axillary shoot growth, greater tuber yield, altered tuber traits and early senescence compared to wild type. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant remained, but the up plants exhibited a lower tuber yield and fewer axillary shoots compared to wild type. Southern blot analyses indicated a single T-DNA insertion in the mutant, located on chromosome 10. Initial PCR-based gene expression studies indicated transcriptional activation/repression of several genes in the mutant flanking the insertion. The gene immediately flanking the right border of the T-DNA insertion, which encoded an uncharacterized Broad complex, Tramtrac, Bric-a-brac; also known as Pox virus and Zinc finger (BTB/POZ) domain-containing protein (StBTB/POZ1) containing an Armadillo repeat region, was up-regulated in the mutant. Global gene expression comparisons between Bintje and up using RNA-seq on leaves from 60 day-old plants revealed a dataset of over 1,600 differentially expressed genes. Gene expression analyses suggested a variety of biological processes and pathways were modified in the mutant, including carbohydrate and lipid metabolism, cell division and cell cycle activity, biotic and abiotic stress responses, and proteolysis.

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection.

KEY MESSAGE : We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics. Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12-16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1-2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.

Growth promotion and colonization of switchgrass (Panicum virgatum) cv. Alamo by bacterial endophyte Burkholderia phytofirmans strain PsJN.

BACKGROUND: Switchgrass is one of the most promising bioenergy crop candidates for the US. It gives relatively high biomass yield and can grow on marginal lands. However, its yields vary from year to year and from location to location. Thus it is imperative to develop a low input and sustainable switchgrass feedstock production system. One of the most feasible ways to increase biomass yields is to harness benefits of microbial endophytes. RESULTS: We demonstrate that one of the most studied plant growth promoting bacterial endophytes, Burkholderia phytofirmans strain PsJN, is able to colonize and significantly promote growth of switchgrass cv. Alamo under in vitro, growth chamber, and greenhouse conditions. In several in vitro experiments, the average fresh weight of PsJN-inoculated plants was approximately 50% higher than non-inoculated plants. When one-month-old seedlings were grown in a growth chamber for 30 days, the PsJN-inoculated Alamo plants had significantly higher shoot and root biomass compared to controls. Biomass yield (dry weight) averaged from five experiments was 54.1% higher in the inoculated treatment compared to non-inoculated control. Similar results were obtained in greenhouse experiments with transplants grown in 4-gallon pots for two months. The inoculated plants exhibited more early tillers and persistent growth vigor with 48.6% higher biomass than controls. We also found that PsJN could significantly promote growth of switchgrass cv. Alamo under sub-optimal conditions. However, PsJN-mediated growth promotion in switchgrass is genotype specific. CONCLUSIONS: Our results show B. phytofirmans strain PsJN significantly promotes growth of switchgrass cv. Alamo under different conditions, especially in the early growth stages leading to enhanced production of tillers. This phenomenon may benefit switchgrass establishment in the first year. Moreover, PsJN significantly stimulated growth of switchgrass cv. Alamo under sub-optimal conditions, indicating that the use of the beneficial bacterial endophytes may boost switchgrass growth on marginal lands and significantly contribute to the development of a low input and sustainable feedstock production system.

Potato cytosine methylation and gene expression changes induced by a beneficial bacterial endophyte, Burkholderia phytofirmans strain PsJN.

Burkholderia phytofirmans strain PsJN is a highly effective plant-beneficial endophyte. We have used a combination of capillary electrophoresis and methylation-sensitive amplification length polymorphism (CE-MSAP) analysis to investigate the potato genomic DNA cytosine methylation changes that occur in response to PsJN bacterization. Six weeks after PsJN inoculation, over 6800 loci were identified and assessed in two in vitro grown potato varieties, the strongly-responsive Red Pontiac and the poorly-responsive Superior. Compared to non-bacterized control, bacterized Red Pontiac exhibited little change in the overall cytosine methylation, although methylation polymorphisms did occur. In contrast, poorly-responsive Superior exhibited significantly higher levels of overall cytosine methylation and a decrease in the number of non-methylated sites in the bacterized plants compared to controls. Superior had significantly higher DNA methylation and DNA hyper-methylation than Red Pontiac, suggesting that enhanced DNA loci methylation is involved in the suppression of PsJN-induced plant growth stimulation. Several DNA fragments, corresponding to different open reading frames exhibiting methylation polymorphisms in Red Pontiac or Superior were sequenced. Gene expression analysis of a subset of those genes was carried out using real time PCR. We identified several genes whose transcript levels were either enhanced or decreased in response to PsJN in a variety-specific way, as well as genes that were specifically enhanced in both varieties in response to the endophyte.

Initiation of somatic embryogenesis from immature zygotic embryos of oocarpa pine (Pinus oocarpa Schiede ex Schlectendal).

The focus of the current project was to establish somatic embryogenesis protocols for the tropical pine species Pinus oocarpa using immature zygotic embryos (ZEs) as explants. Somatic embryogenesis is best supported by mimicking natural seed-embryo developmental conditions, through a tissue culture medium formulation based on the mineral content of the seed nutritive tissue [megagametophyte (MG)]. A novel culture medium (P. oocarpa medium, PO) was tested in combination with different plant growth regulator (PGR) concentrations and compared with standard Pinus taeda media for the initiation of somatic embryogenesis from immature ZEs of P. oocarpa. Immature MGs containing immature ZEs of two mother trees were used with 12 and 8% extrusion rates for mother tree genotypes 3 and 5, respectively. In both mother trees the percentage capture was 2%. Multiplication of two captured cell lines (T5C2S01 and T5C1S12) was improved by lowering the concentrations of PGRs to 2.5 µM each 2,4-dichlorophenoxyacetic acid and abscisic acid (ABA) plus 1.0 µM each 6-benzylaminopurine and kinetin. Mature somatic embryos formed on 40 µM ABA, 6% (w/v) maltose, 12% (w/v) PEG 8000 and 0.6% (w/v) Phytagel. While PO medium appeared suboptimal for somatic embryo induction, it did exhibit potential for enhanced culture proliferation and subsequent improved maturation with cell line T5C2S01, where microscopic analysis revealed better embryo morphology on PO medium than on 1250 medium. However, this enhancement was not observed with cell line T5C1S12. Germination was preceded by partial desiccation for a period of 2-3 weeks before transferring the embryos to germination medium. Germination was observed after 7 days under low light, and apical primordia slowly expanded after transfer to ex vitro conditions. To our knowledge, this is the first report on the production of somatic seedlings in P. oocarpa.

The use of beneficial microbial endophytes for plant biomass and stress tolerance improvement.

Endophytes are microorganisms that live within host plants for at least part of their life and do not cause apparent symptoms of diseases. In general, beneficial endophytes promote host plant growth, increase plant nutrient uptake, inhibit plant pathogen growth, reduce disease severity, and enhance tolerance to environmental stresses. As sustainable and renewable agricultural production (including current biofuel and bioenergy crops) increases in prominence, endophytic microorganisms will play important roles and offer environmentally-friendly methods to increase productivity while reducing chemical inputs. This review discusses various aspects of beneficial fungal and bacterial endophyte interactions with plants, including the physiological and molecular mechanisms by which they benefit plant performance. We also discuss the potential for genetic modification of endophytes with useful genes, which could be used to impart additional traits following inoculation with the genetically engineered endophytes. Finally, we review US-issued patents over the past decade which relate to the use of fungal and bacterial endophytes for plant growth and stress tolerance improvement.

Identification and characterization of a matrix metalloproteinase (Pta1-MMP) expressed during Loblolly pine (Pinus taeda) seed development, germination completion, and early seedling establishment.

Extracellular matrix (ECM) modifications occur during plant growth, development, and in response to environmental stimuli. Key modulators of ECM modification in vertebrates, the extracellular matrix metalloproteinases (MMPs), have also been described in a few plants. Here, we report the identification of Loblolly pine (Pinus taeda) Pta1-MMP and its characterization during seed development and germination. Pta1-MMP protein has the structural characteristics of other plant MMPs, the recombinant protein exhibits Zn(2+)-dependent protease activity, and is inhibited by EDTA and the active site-binding hydroxamate inhibitor GM6001. The Pta1-MMP gene is expressed in both embryo and megagametophyte, with transcript levels increasing in both during the period from proembryo to early cotyledonary stage, then declining during late embryogenesis and maturation drying. Protein extracts exhibited similar developmental-stage MMP-like activity. Seed germination was stimulated by GA(3) and inhibited by ABA, and the timing of germination completion was mirrored by the presence of MMP-like protease activity in both water- and GA(3)-imbibed embryos. Pta1-MMP gene transcript levels increased in association with radicle protrusion for both GA(3)- and water-treated embryos, in agreement with MMP-like activity. In contrast, by 11 days after imbibition, Pta1-MMP gene transcripts in ABA-treated embryos were at levels similar to the other treatments, although MMP-like activity was not observed. The application of GM6001 during Loblolly pine seed germination inhibited radicle protrusion. Our results suggest that MMP activity may be involved in ECM modification, facilitating the cell division and expansion required during seed development, germination completion, and subsequent seedling establishment.

Genes driving potato tuber initiation and growth: identification based on transcriptional changes using the POCI array.

The increasing amount of available expressed gene sequence data makes whole-transcriptome analysis of certain crop species possible. Potato currently has the second largest number of publicly available expressed sequence tag (EST) sequences among the Solanaceae. Most of these ESTs, plus other proprietary sequences, were combined and used to generate a unigene assembly. The set of 246,182 sequences produced 46,345 unigenes, which were used to design a 44K 60-mer oligo array (Potato Oligo Chip Initiative: POCI). In this study, we attempt to identify genes controlling and driving the process of tuber initiation and growth by implementing large-scale transcriptional changes using the newly developed POCI array. Major gene expression profiles could be identified exhibiting differential expression at key developmental stages. These profiles were associated with functional roles in cell division and growth. A subset of genes involved in the regulation of the cell cycle, based on their Gene Ontology classification, exhibit a clear transient upregulation at tuber onset indicating increased cell division during these stages. The POCI array allows the study of potato gene expression on a much broader level than previously possible and will greatly enhance analysis of transcriptional control mechanisms in a wide range of potato research areas. POCI sequence and annotation data are publicly available through the POCI database ( http://pgrc.ipk-gatersleben.de/poci ).

Plant extracellular matrix metalloproteinases.

The plant extracellular matrix (ECM) includes a variety of proteins with critical roles in the regulation of plant growth, development, and responses to pests and pathogens. Several studies have shown that various ECM proteins undergo proteolytic modification. In mammals, the extracellular matrix metalloproteinases (MMPs) are known modifiers of the ECM, implicated in tissue architecture changes and the release of biologically active and/or signalling molecules. Although plant MMPs have been identified, little is known about their activity and function. Plant MMPs show structural similarity to mammalian MMPs, including the presence of an auto-regulatory cysteine switch domain and a zinc-binding catalytic domain. Plant MMPs are differentially expressed in cells and tissues during plant growth and development, as well as in response to several biotic and abiotic stresses. The few gene expression and mutant analyses to date indicate their involvement in plant growth, morphogenesis, senescence and adaptation and response to stress. In order to gain a further understanding of their function, an analysis and characterisation of MMP proteins, their activity and their substrates during plant growth and development are still required. This review describes plant MMP work to date, as well as the variety of genomic and proteomic methodologies available to characterise plant MMP activity, function and potential substrates.

Potato expressed sequence tag generation and analysis using standard and unique cDNA libraries.

To help develop an understanding of the genes that govern the developmental characteristics of the potato (Solanum tuberosum), as well as the genes associated with responses to specified pathogens and storage conditions, The Canadian Potato Genome Project (CPGP) carried out 5' end sequencing of regular, normalized and full-length cDNA libraries of the Shepody potato cultivar, generating over 66,600 expressed sequence tags (ESTs). Libraries sequenced represented tuber developmental stages, pathogen-challenged tubers, as well as leaf, floral developmental stages, suspension cultured cells and roots. All libraries analysed to date have contributed unique sequences, with the normalized libraries high on the list. In addition, a low molecular weight library has enhanced the 3' ends of our sequence assemblies. Using the combined assembly dataset, unique tuber developmental, cold storage and pathogen-challenged sequences have been identified. A comparison of the ESTs specific to the pathogen-challenged tuber and foliar libraries revealed minimal overlap between these libraries. Mixed assemblies using over 189,000 potato EST sequences from CPGP and The Institute for Genomics Research (TIGR) has revealed common sequences, as well as CPGP- and TIGR-unique sequences.