CYTOKININ RESPONSE FACTOR 2 is involved in modulating the salt stress response.

Cytokinin has strong connections to development and a growing role in the abiotic stress response. Here we show that CYTOKININ RESPONSE FACTOR 2 (CRF2) is additionally involved in the salt (NaCl) stress response. CRF2 promoter-GUS expression indicates CRF2 involvement in the response to salt stress as well as the previously known cytokinin response. Interestingly, CRF2 mutant seedlings are quite similar to the wild type (WT) under non-stressed conditions yet have many distinct changes in response to salt stress. Cytokinin levels measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) that increased in the WT after salt stress are decreased in crf2, potentially from CRF2 regulation of cytokinin biosynthesis genes. Ion content measured by inductively coupled plasma optical emission spectrometry (ICP-OES) was increased in the WT for Na, K, Mn, Ca and Mg after salt stress, whereas the corresponding Ca and Mg increases are lacking in crf2. Many genes examined by RNA-seq analysis were altered transcriptionally by salt stress in both the WT and crf2, yet interestingly approximately one-third of salt-modified crf2 transcripts (2655) showed unique regulation. Different transcript profiles for salt stress in crf2 compared with the WT background was further supported through an examination of co-expressed genes by weighted gene correlation network analysis (WGCMA) and principal component analysis (PCA). Additionally, Gene Ontology (GO) enrichment terms found from salt-treated transcripts revealed most photosynthesis-related terms as only being affected in crf2, leading to an examination of chlorophyll levels and the efficiency of photosystem II (via the ratio of variable fluorescence to maximum fluorescence, Fv /Fm ) as well as physiology after salt treatment. Salt stress-treated crf2 plants had both reduced chlorophyll levels and lower Fv /Fm values compared with the WT, suggesting that CRF2 plays a role in the modulation of salt stress responses linked to photosynthesis.

Detection of subgenome bias using an anchored syntenic approach in Eleusine coracana (finger millet).

BACKGROUND: Finger millet (Eleusine coracana 2n = 4x = 36) is a hardy, nutraceutical, climate change tolerant, orphan crop that is consumed throughout eastern Africa and India. Its genome has been sequenced multiple times, but A and B subgenomes could not be separated because no published genome for E. indica existed. The classification of A and B subgenomes is important for understanding the evolution of this crop and provide a means to improve current and future breeding programs. RESULTS: We produced subgenome calls for 704 syntenic blocks and inferred A or B subgenomic identity for 59,377 genes 81% of the annotated genes. Phylogenetic analysis of a super matrix containing 455 genes shows high support for A and B divergence within the Eleusine genus. Synonymous substitution rates between A and B genes support A and B calls. The repetitive content on highly supported B contigs is higher than that on similar A contigs. Analysis of syntenic singletons showed evidence of biased fractionation showed a pattern of A genome dominance, with 61% A, 37% B and 1% unassigned, and was further supported by the pattern of loss observed among cyto-nuclear interacting genes. CONCLUSION: The evidence of individual gene calls within each syntenic block, provides a powerful tool for inference for subgenome classification. Our results show the utility of a draft genome in resolving A and B subgenomes calls, primarily it allows for the proper polarization of A and B syntenic blocks. There have been multiple calls for the use of phylogenetic inference in subgenome classification, our use of synteny is a practical application in a system that has only one parental genome available.

A chromosome-scale assembly for 'd'Anjou' pear.

Cultivated pear consists of several Pyrus species with Pyrus communis (European pear) representing a large fraction of worldwide production. As a relatively recently domesticated crop and perennial tree, pear can benefit from genome-assisted breeding. Additionally, comparative genomics within Rosaceae promises greater understanding of evolution within this economically important family. Here, we generate a fully phased chromosome-scale genome assembly of P. communis 'd'Anjou.' Using PacBio HiFi and Dovetail Omni-C reads, the genome is resolved into the expected 17 chromosomes, with each haplotype totaling nearly 540 Megabases and a contig N50 of nearly 14 Mb. Both haplotypes are highly syntenic to each other and to the Malus domestica 'Honeycrisp' apple genome. Nearly 45,000 genes were annotated in each haplotype, over 90% of which have direct RNA-seq expression evidence. We detect signatures of the known whole-genome duplication shared between apple and pear, and we estimate 57% of d'Anjou genes are retained in duplicate derived from this event. This genome highlights the value of generating phased diploid assemblies for recovering the full allelic complement in highly heterozygous crop species.

Vascular expression and C-terminal sequence divergence of cytokinin response factors in flowering plants.

Cytokinin response factors (CRFs) are important transcription factors that form a side branch of the cytokinin signaling pathway and have been linked to cytokinin-regulated processes during development. CRF proteins are defined as belonging to a specific transcription factor family by the presence of an AP2/ERF DNA-binding domain and can be distinguished within this family by a group-specific CRF domain involved in protein-protein interactions. Here we further delimit CRFs into five distinct clades (I-V) represented across all major angiosperm lineages. Protein sequences within each clade contain a clade-specific C-terminal region distinct from other CRFs, suggesting ancient evolutionary divergence and specialization within this gene family. Conserved patterns of transcriptional regulation support these clade divisions. Despite these important differences, CRFs appear to show preferential localization or targeting to vascular tissue in quantitative real-time PCR and reporter line analyses of Arabidopsis thaliana and Solanum lycopersicum (tomato). Phloem tissue expression within the vasculature often appears the strongest in CRF reporter lines, and an analysis of CRF promoter sequences revealed conservation and significant enrichment of phloem targeting cis-elements, suggesting a potential role for CRFs in this tissue. An examination of CRF loss-of-function mutants from cytokinin-regulated clades revealed alterations in higher order vein patterning. This supports both the general link of CRFs to vascular tissue and clade-specific differences between CRFs, since alterations in vascular patterning appear to be clade specific. Together these findings indicate that CRFs are potential regulators of developmental processes associated with vascular tissues.

The CRF domain defines cytokinin response factor proteins in plants.

BACKGROUND: Cytokinin Response Factors (CRFs) are a small subset of AP2/ERF transcription factor genes shown in Arabidopsis to regulate leaf development as part of the cytokinin signal transduction pathway. This study examines the phylogenetic distribution of CRF genes in other plant species, and attempts to identify the extent of sequence conservation and potential gene function among all CRF genes. RESULTS: We identified CRF genes in representatives of all major land plant lineages, including numerous flowering plant taxa in addition to the model systems in which ERF genes have been catalogued. Comparative analysis across this broader sampling has identified strongly conserved amino acid motifs other than the AP2/ERF domain for all CRF proteins as well as signature sequences unique to specific clades of CRF genes. One of these motifs, here designated as the CRF domain, is conserved in and unique to CRF proteins distinguishing them from related genes. We show that this novel domain of approximately 65 amino acids is found in CRF proteins from all groups of land plants and only in CRF genes. Phylogenetic analyses suggest that the evolution of CRF genes has included numerous duplication events. In this phylogenetic context we examine protein evolution including the gain and loss of accessory domains, correlate these molecular evolutionary events with experimental data on cytokinin regulation and speculate on the function and evolution of the CRF domain within AP2/ERF transcription factor proteins. We also tested a prediction drawn from the phylogenetic analyses that four CRF domain containing genes from Tomato, previously unexamined for cytokinin response, are transcriptionally inducible by cytokinin, supporting the link between CRF genes, CRF-specific domains and cytokinin regulation. CONCLUSION: CRF genes can be identified in all lineages of land plants, as a distinct subset of AP2/ERF proteins containing a specific and unique CRF domain. The CRF domain can be used to identify previously unclassified predicted genes or genes identified only as members of the AP2/ERF protein family. CRF domain presence and phylogenetic relatedness to known Arabidopsis CRF genes predicts gene function to some extent.

The Mitochondrial Genome of Eleusine indica and Characterization of Gene Content within Poaceae.

Plant mitochondrial (mt) genome assembly provides baseline data on size, structure, and gene content, but resolving the sequence of these large and complex organelle genomes remains challenging due to fragmentation, frequent recombination, and transfers of DNA from neighboring plastids. The mt genome for Eleusine indica (Poaceae: goosegrass) is comprehensibly analyzed here, providing key reference data for an economically significant invasive species that is also the maternal parent of the allotetraploid crop Finger millet (Eleusine coracana). The assembled E. indica genome contains 33 protein coding genes, 6 rRNA subunits, 24 tRNA, 8 large repetitive regions 15 kb of transposable elements across a total of 520,691 bp. Evidence of RNA editing and loss of rpl2, rpl5, rps14, rps11, sdh4, and sdh3 genes is evaluated in the context of an updated survey of mt genomic gene content across the grasses through an analysis of publicly available data. Hypothesized patterns of Poaceae mt gene loss are examined in a phylogenetic context to clarify timing, showing that rpl2 was transferred to the nucleus from the mitochondrion prior to the origin of the PACMAD clade.

Lineage specific conservation of cis-regulatory elements in Cytokinin Response Factors.

Expression patterns of genes are controlled by short regions of DNA in promoter regions known as cis-regulatory elements. How expression patterns change due to alterations in cis-regulatory elements in the context of gene duplication are not well studied in plants. Over 300 promoter sequences from a small, well-conserved family of plant transcription factors known as Cytokinin Response Factors (CRFs) were examined for conserved motifs across several known clades present in Angiosperms. General CRF and lineage specific motifs were identified. Once identified, significantly enriched motifs were then compared to known transcription factor binding sites to elucidate potential functional roles. Additionally, presence of similar motifs shows that levels of conservation exist between different CRFs across land plants, likely occurring through processes of neo- or sub-functionalization. Furthermore, significant patterns of motif conservation are seen within and between CRF clades suggesting cis-regulatory regions have been conserved throughout CRF evolution.

Identification and functional characterization of the Marshallia (Asteraceae) Clade III Cytokinin Response Factor (CRF).

Cytokinin Response Factor (CRF) genes are a subgroup of AP2/ERF domain-containing transcription factors that are defined by the CRF domain, from which five clades of CRF genes have been identified. Clade III CRFs are strongly induced by cytokinin, as well as other abiotic stress factors, such as oxidative stress. While this appears well studied for the Clade III CRFs in Arabidopsis and tomato, there have been almost no studies done outside of these model systems. This study expands upon that and represents the first CRF research in the Sunflower family, Asteraceae. Fifty Asterid Clade III CRF protein sequences were examined, and novel Clade III CRF C-terminus motifs were identified. Clade III CRF genes of Marshallia mohrii and M. caespitosa were assembled from genome-skimming and transcriptomic data. Expression experiments were conducted on M. caespitosa to test responsiveness to both cytokinin and oxidative stress. Low levels of basal expression for the McCRF1 were found to be strongly induced in both treatment groups. These are the first experiments to show regulation of a nuclear gene in a Marshallia species, and these results suggest there is broad conservation in the sequence, form, and regulation of Clade III CRF genes and proteins.

Transcriptome Analysis Reveals Unique Relationships Among Eleusine Species and Heritage of Eleusine coracana.

Relationships in the genus Eleusine were obtained through transcriptome analysis. Eleusine coracana (E. coracana ssp. coracana), also known as finger millet, is an allotetraploid minor crop primarily grown in East Africa and India. Domesticated E. coracana evolved from wild E. africana (E. coracana ssp. africana) with the maternal genome donor largely supported to be E. indica; however, the paternal genome donor remains elusive. We developed transcriptomes for six Eleusine species from fully developed seedlings using Illumina technology and three de novo assemblers (Trinity, Velvet, and SOAPdenovo2) with the redundancy-reducing EvidentialGene pipeline. Mapping E. coracana reads to the chloroplast genes of all Eleusine species detected fewer variants between E. coracana and E. indica compared to all other species. Phylogenetic analysis further supports E. indica as the maternal parent of E. coracana and E. africana, in addition to a close relationship between E. indica and E. tristachya, and between E. floccifolia and E. multiflora, and E. intermedia as a separate group. A close relationship between E. floccifolia and E. multiflora was unexpected considering they are reported to have distinct nuclear genomes, BB and CC, respectively. Further, it was expected that E. intermedia and E. floccifolia would have a closer relationship considering they have similar nuclear genomes, AB and BB, respectively. A rethinking of the labeling of ancestral genomes of E. floccifolia, E. multiflora, and E. intermedia is maybe needed based on this data.

Development of a goosegrass (Eleusine indica) draft genome and application to weed science research.

BACKGROUND: Genomes are vital to the study of genomics, population genetics, and evolution of species. To date, only one genome (Echinochloa crus-galli) for C4 annual weedy grass species has been sequenced. Research was conducted to develop a draft genome of goosegrass (Eleusine indica; 2n = 2x = 18), one of the most common and troublesome weeds in the world. RESULTS: A draft assembly of an approximately 492 Mb whole-genome sequence of goosegrass was obtained by de novo assembly of paired-end and mate-paired reads generated by Illumina sequencing of total genomic DNA. The genome was assembled into 24,072 scaffolds with N50 = 233,459 bp. More than 99% of transcriptome sequences were mapped to the goosegrass draft genome, and 95% of the commonly conserved plant genes were present. The assembled genome contains 25,467 unique protein-coding genes. Genes associated with herbicide resistance were obtained and variant calling allowed the detection of 754,409 single nucleotide polymorphisms. In addition, we also report 115,417 simple sequence repeats which can be deployed in population genetics and phylogenetic analysis. CONCLUSION: This is the first report of genome sequence of goosegrass. Our assembly was able to identify all major herbicide-resistance related genes and develop a useful tool for other genomic and evolutionary analysis. © 2019 Society of Chemical Industry.

Cytokinin Response Factor 5 has transcriptional activity governed by its C-terminal domain.

Cytokinin Response Factors (CRFs) are AP2/ERF transcription factors involved in cytokinin signal transduction. CRF proteins consist of a N-terminal dimerization domain (CRF domain), an AP2 DNA-binding domain, and a clade-specific C-terminal region of unknown function. Using a series of sequential deletions in yeast-2-hybrid assays, we provide evidence that the C-terminal region of Arabidopsis CRF5 can confer transactivation activity. Although comparative analyses identified evolutionarily conserved protein sequence within the C-terminal region, deletion experiments suggest that this transactivation domain has a partially redundant modular structure required for activation of target gene transcription.

Complete plastid genome sequence of goosegrass (Eleusine indica) and comparison with other Poaceae.

Eleusine indica, also known as goosegrass, is a serious weed in at least 42 countries. In this paper we report the complete plastid genome sequence of goosegrass obtained by de novo assembly of paired-end and mate-paired reads generated by Illumina sequencing of total genomic DNA. The goosegrass plastome is a circular molecule of 135,151bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 20,919 bases. The large (LSC) and the small (SSC) single-copy regions span 80,667 bases and 12,646 bases, respectively. The plastome of goosegrass has 38.19% GC content and includes 108 unique genes, of which 76 are protein-coding, 28 are transfer RNA, and 4 are ribosomal RNA. The goosegrass plastome sequence was compared to eight other species of Poaceae. Although generally conserved with respect to Poaceae, this genomic resource will be useful for evolutionary studies within this weed species and the genus Eleusine.

Transcriptome Assembly and Comparison of an Allotetraploid Weed Species, Annual Bluegrass, with its Two Diploid Progenitor Species, Schrad and Kunth.

Annual bluegrass ( L.) is one of the most widespread weed species in this world. As a young allotetraploid, has occupied diverse environments from Antarctic area to subtropical regions. To unveil the evolutionary mystery behind 's wide distribution, extensive adaptability and phenotypic plasticity needs collaboration from multiple research scopes from ecology and plant physiology to population genetics and molecular biology. However, the lack of omic data and reference has greatly hampered the study. This is the first comprehensive transcriptome study on species. Total RNA was extracted from and its two proposed diploid parents, Schrad and Kunth, and sequenced in Illumina Hiseq2000. Optimized, nonredundant transcriptome references were generated for each species using four de novo assemblers (Trinity, Velvet, SOAPdenovo, and CLC Genomics Workbench) and a redundancy-reducing pipeline (CD-HIT-EST and EvidentialGene tr2aacds). Using the constructed transcriptomes together with sequencing reads, we found high similarity in nucleotide sequences and homeologous polymorphisms between and the two proposed parents. Comparison of chloroplast and mitochondrion genes further confirmed as the maternal parent. Less nucleotide percentage differences were observed between and homeologs than between and homeologs, indicating a higher nucleotide substitution rates in homeologs than in homeologs. Gene ontology (GO) enrichment analysis suggested the more compatible cytoplasmic environment and cellular apparatus for homeologs as the major cause for this phenomenon.

The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

MicroRNAs (miRNAs) and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs) are two distinct subfamilies of small RNAs (sRNAs) that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs) are processed from longer RNA precursors by DICER-LIKE proteins (DCLs). Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs) and 108 novel lineage-specific miRNAs (ls-miRNAs). Along with miRNAs, 2,033 miRNA variants (isomiRNAs) were identified as well. Those isomiRNAs with variation at the 3'-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs) were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers of a second round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.

Digitization workflows for flat sheets and packets of plants, algae, and fungi.

Effective workflows are essential components in the digitization of biodiversity specimen collections. To date, no comprehensive, community-vetted workflows have been published for digitizing flat sheets and packets of plants, algae, and fungi, even though latest estimates suggest that only 33% of herbarium specimens have been digitally transcribed, 54% of herbaria use a specimen database, and 24% are imaging specimens. In 2012, iDigBio, the U.S. National Science Foundation's (NSF) coordinating center and national resource for the digitization of public, nonfederal U.S. collections, launched several working groups to address this deficiency. Here, we report the development of 14 workflow modules with 7-36 tasks each. These workflows represent the combined work of approximately 35 curators, directors, and collections managers representing more than 30 herbaria, including 15 NSF-supported plant-related Thematic Collections Networks and collaboratives. The workflows are provided for download as Portable Document Format (PDF) and Microsoft Word files. Customization of these workflows for specific institutional implementation is encouraged.

Analysis of the Citrullus colocynthis transcriptome during water deficit stress.

Citrullus colocynthis is a very drought tolerant species, closely related to watermelon (C. lanatus var. lanatus), an economically important cucurbit crop. Drought is a threat to plant growth and development, and the discovery of drought inducible genes with various functions is of great importance. We used high throughput mRNA Illumina sequencing technology and bioinformatic strategies to analyze the C. colocynthis leaf transcriptome under drought treatment. Leaf samples at four different time points (0, 24, 36, or 48 hours of withholding water) were used for RNA extraction and Illumina sequencing. qRT-PCR of several drought responsive genes was performed to confirm the accuracy of RNA sequencing. Leaf transcriptome analysis provided the first glimpse of the drought responsive transcriptome of this unique cucurbit species. A total of 5038 full-length cDNAs were detected, with 2545 genes showing significant changes during drought stress. Principle component analysis indicated that drought was the major contributing factor regulating transcriptome changes. Up regulation of many transcription factors, stress signaling factors, detoxification genes, and genes involved in phytohormone signaling and citrulline metabolism occurred under the water deficit conditions. The C. colocynthis transcriptome data highlight the activation of a large set of drought related genes in this species, thus providing a valuable resource for future functional analysis of candidate genes in defense of drought stress.

Spliceosomal intron size expansion in domesticated grapevine (Vitis vinifera).

BACKGROUND: Spliceosomal introns are important components of eukaryotic genes as their structure, sizes and contents reflect the architecture of gene and genomes. Intron size, determined by both neutral evolution, repetitive elements activities and potential functional constraints, varies significantly in eukaryotes, suggesting unique dynamics and evolution in different lineages of eukaryotic organisms. However, the evolution of intron size, is rarely studied. To investigate intron size dynamics in flowering plants, in particular domesticated grapevines, a survey of intron size and content in wine grape (Vitis vinifera Pinot Noir) genes was conducted by assembling and mapping the transcriptome of V. vinifera genes from ESTs to characterize and analyze spliceosomal introns. RESULTS: Uncommonly large size of spliceosomal intron was observed in V. vinifera genome, otherwise inconsistent with overall genome size dynamics when comparing Arabidopsis, Populus and Vitis. In domesticated grapevine, intron size is generally not related to gene function. The composition of enlarged introns in grapevines indicated extensive transposable element (TE) activity within intronic regions. TEs comprise about 80% of the expanded intron space and in particular, recent LTR retrotransposon insertions are enriched in these intronic regions, suggesting an intron size expansion in the lineage leading to domesticated grapevine, instead of size contractions in Arabidopsis and Populus. Comparative analysis of selected intronic regions in V. vinifera cultivars and wild grapevine species revealed that accelerated TE activity was associated with grapevine domestication, and in some cases with the development of specific cultivars. CONCLUSIONS: In this study, we showed intron size expansion driven by TE activities in domesticated grapevines, likely a result of long-term vegetative propagation and intensive human care, which simultaneously promote TE proliferation and repress TE removal mechanisms such as recombination. The intron size expansion observed in domesticated grapevines provided an example of rapid plant genome evolution in response to artificial selection and propagation, and may shed light on the important genomic changes during domestication. In addition, the transcriptome approach used to gather intron size data significantly improved annotations of the V. vinifera genome.

Expression, in vivo localization and phylogenetic analysis of a pyridoxine 5'-phosphate oxidase in Arabidopsis thaliana.

Pyridoxal phosphate (PLP), a vitamin B(6) vitamer, is an essential cofactor for numerous enzymes. Pyridoxine/pyridoxamine phosphate oxidase (PPOX) catalyzes the synthesis of pyridoxal phosphate from pyridoxine phosphate (PNP) and/or pyridoxamine phosphate (PMP). The At5g49970 locus in Arabidopsis thaliana encodes an AtPPOX, a PNP/PMP oxidase. The expression of the AtPPOX gene varied in different tissues of Arabidopsis examined, being up-regulated by light, heat shock, ABA, and ethylene treatments, and down-regulated by exposure to drought and NaCl. Monoclonal antibodies raised against two different domains of AtPPOX recognized different sizes of AtPPOX, suggesting that AtPPOX proteins are produced as splice variants of the AtPPOX gene in Arabidopsis. Phylogenetic analysis of AtPPOX across all domains of life demonstrated that plant AtPPOX homologs have an additional Yjef_N domain preceding the Pyridox_Oxidase domain at the C-terminal end of the protein, while AtPPOX homologs from bacteria, fungi and animals have only Pyridox_Oxidase domain. The presence of the Yjef_N domain in plant AtPPOX homologs suggests that acquisition of this domain, and its fusion with the pyridox_oxidase domain began with the endosymbiotic acquisition of the chloroplast. Bioinformatic analysis suggested that AtPPOX is localized in chloroplast, but the monoclonal antibody could not be used for subcellular localization of this protein. A GFP-AtPPOX fusion construct introduced into the Arabidopsis protoplast confirmed localization of AtPPOX into the chloroplast. An RNAi mutant of AtPPOX showed sensitivity to high light suggesting a role for PPOX in resistance to photooxidative damage, and alteration in root growth in the presence of sucrose suggests a role for PPOX in root development.

Grand challenges in organismal biology: The need to develop both theory and resources.

This contribution is fifth in a series of articles in Integrative and Comparative Biology that was initiated by Schwenk et al. (2009) and followed by Satterlie et al. (2009), Denny and Helmuth (2009), and Denver et al. (2009). Here, our intent is to be provocative and to stimulate further discourse. Like other contributors we have our own biases, and as it should be clear to the readers, we approach this task as evolutionary biologists, specifically systematists.

Bi-parental cytoplasmic DNA inheritance in Wisteria (Fabaceae): evidence from a natural experiment.

Cytoplasmic inheritance was investigated in interspecific hybrids of Wisteria sinensis and W. floribunda. Species-specific nuclear, mitochondrial and plastid DNA markers were identified from wild-collected plants of each species in its native range. These markers provide evidence for the bi-parental transmission of plastids in hybrid swarms of these two species in the southeastern USA. These population level molecular data corroborate previous cytological evidence of this phenomenon in Wisteria.