Irradiated Pollen-Induced Parthenogenesis for Doubled Haploid Production in Sunflowers (Helianthus spp.).

Doubled haploid (DH) technology is a tool used to develop large numbers of inbred lines and increase the rate of genetic gain by shortening the breeding cycles. However, previous attempts to produce DH sunflower plants (Helianthus spp.) have resulted in limited success. In this research, we applied gamma-induced parthenogenesis to assist the production of DH sunflowers. The objectives of the study included (1) identifying optimal gamma ray doses for inducing DH sunflowers using two cytoplasmic male sterility (CMS) lines as female plants and two male pollinators with recognizable morphological markers, (2) selecting new male pollinators from wild sunflower varieties, and (3) testing the efficacy of the selected male pollinators using emasculated non-male sterile sunflower lines as female plants. In these experiments, pollen grains were irradiated with gamma ray doses ranging from 50 to 200 Gy. The optimal gamma ray dose for pollen grain irradiation and DH plant production was identified to be 100 Gy. In addition, a cultivated (G11/1440) and a wild-type (ANN1811) sunflower line can be used as common male pollinators for their distinctive morphological markers and wide capacity for DH induction by gamma-irradiated pollen grains.

Weed-induced changes in the maize root transcriptome reveal transcription factors and physiological processes impacted early in crop-weed interactions.

A new paradigm suggests weeds primarily reduce crop yield by altering crop developmental and physiological processes long before the weeds reduce resources through competition. Multiple studies have implicated stress response pathways are activated when crops such as maize are grown in close proximity with weeds during the first 4-8 weeks of growth-the point at which weeds have their greatest impact on subsequent crop yields. To date, these studies have mostly focused on the response of above-ground plant parts and have not examined the early signal transduction processes associated with maize root response to weeds. To investigate the impact of signals from a below-ground competitor on the maize root transcriptome when most vulnerable to weed pressure, a system was designed to expose maize to only below-ground signals. Gene set enrichment analyses identified over-represented ontologies associated with oxidative stress signalling throughout the time of weed exposure, with additional ontologies associated with nitrogen use and transport and abscisic acid (ABA) signalling, and defence responses being enriched at later time points. Enrichment of promoter motifs indicated over-representation of sequences known to bind FAR-RED IMPAIRED RESPONSE 1 (FAR1), several AP2/ERF transcription factors and others. Likewise, co-expression networks were identified using Weighted-Gene Correlation Network Analysis (WGCNA) and Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) algorithms. WGCNA highlighted the potential roles of several transcription factors including a MYB 3r-4, TB1, WRKY65, CONSTANS-like5, ABF3, HOMEOBOX 12, among others. These studies also highlighted the role of several specific proteins involved in ABA signalling as being important for the initiation of the early response of maize to weeds. SC-ION highlighted potential roles for NAC28, LOB37, NAC58 and GATA2 transcription factors, among many others.

Homozygosity mapping identified loci and candidate genes responsible for freezing tolerance in Camelina sativa.

Homozygosity mapping is an effective tool for detecting genomic regions responsible for a given trait when the phenotype is controlled by a limited number of dominant or co-dominant loci. Freezing tolerance is a major attribute in agricultural crops such as camelina. Previous studies indicated that freezing tolerance differences between a tolerant (Joelle) and susceptible (CO46) variety of camelina were controlled by a small number of dominant or co-dominant genes. We performed whole genome homozygosity mapping to identify markers and candidate genes responsible for freezing tolerance difference between these two genotypes. A total of 28 F3 RILs were sequenced to ∼30× coverage, and parental lines were sequenced to >30-40× coverage with Pacific Biosciences high fidelity technology and 60× coverage using Illumina whole genome sequencing. Overall, about 126k homozygous single nucleotide polymorphism markers were identified that differentiate both parents. Moreover, 617 markers were also homozygous in F3 families fixed for freezing tolerance/susceptibility. All these markers mapped to two contigs forming a contiguous stretch of chromosome 11. The homozygosity mapping detected 9 homozygous blocks among the selected markers and 22 candidate genes with strong similarity to regions in or near the homozygous blocks. Two such genes were differentially expressed during cold acclimation in camelina. The largest block contained a cold-regulated plant thionin and a putative rotamase cyclophilin 2 gene previously associated with freezing resistance in arabidopsis (Arabidopsis thaliana). The second largest block contains several cysteine-rich RLK genes and a cold-regulated receptor serine/threonine kinase gene. We hypothesize that one or more of these genes may be primarily responsible for freezing tolerance differences in camelina varieties.

Overwintering Camelina and Canola/Rapeseed Show Promise for Improving Integrated Weed Management Approaches in the Upper Midwestern U.S.

Winter oilseed cash cover crops are gaining popularity in integrated weed management programs for suppressing weeds. A study was conducted at two field sites (Fargo, North Dakota, and Morris, Minnesota) to determine the freezing tolerance and weed-suppressing traits of winter canola/rapeseed (Brassica napus L.) and winter camelina [Camelina sativa (L.) Crantz] in the Upper Midwestern USA. The top 10 freezing tolerant accessions from a phenotyped population of winter canola/rapeseed were bulked and planted at both locations along with winter camelina (cv. Joelle) as a check. To phenotype our entire winter B. napus population (621 accessions) for freezing tolerance, seeds were also bulked and planted at both locations. All B. napus and camelina were no-till seeded at Fargo and Morris at two planting dates, late August (PD1) and mid-September (PD2) 2019. Data for winter survival of oilseed crops (plants m-2) and their corresponding weed suppression (plants m-2 and dry matter m-2) were collected on two sampling dates (SD) in May and June 2020. Crop and SD were significant (p < 0.05) for crop plant density at both locations, and PD in Fargo and crop x PD interaction in Morris were significant for weed dry matter. At Morris and Fargo, PD1 produced greater winter B. napus survival (28% and 5%, respectively) and PD2 produced higher camelina survival (79% and 72%, respectively). Based on coefficient of determination (r2), ~50% of weed density was explained by camelina density, whereas ≤20% was explained by B. napus density at both locations. Camelina from PD2 suppressed weed dry matter by >90% of fallow at both locations, whereas weed dry matter in B. napus was not significantly different from fallow at either PD. Genotyping of overwintering canola/rapeseed under field conditions identified nine accessions that survived at both locations, which also had excellent freezing tolerance under controlled conditions. These accessions are good candidates for improving freezing tolerance in commercial canola cultivars.

Weed-induced crop yield loss: a new paradigm and new challenges.

Direct competition for resources is generally considered the primary mechanism for weed-induced yield loss. A re-evaluation of physiological evidence suggests weeds initially impact crop growth and development through resource-independent interference. We suggest weed perception by crops induce a shift in crop development, before resources become limited, which ultimately reduce crop yield, even if weeds are subsequently removed. We present the mechanisms by which crops perceive and respond to weeds and discuss the technologies used to identify these mechanisms. These data lead to a fundamental paradigm shift in our understanding of how weeds reduce crop yield and suggest new research directions and opportunities to manipulate or engineer crops and cropping systems to reduce weed-induced yield losses.

Genetic loci associated with freezing tolerance in a European rapeseed (Brassica napus L.) diversity panel identified by genome-wide association mapping.

Winter biotypes of rapeseed (Brassica napus L.) require a vernalization treatment to enter the reproductive phase and generally produce greater yields than spring rapeseed. To find genetic loci associated with freezing tolerance in rapeseed, we first performed genotyping-by-sequencing (GBS) on a diversity panel consisting of 222 rapeseed accessions originating primarily from Europe, which identified 69,554 high-quality single-nucleotide polymorphisms (SNPs). Model-based cluster analysis suggested that there were eight subgroups. The diversity panel was then phenotyped for freezing survival (visual damage and Fv/Fo and Fv/Fm) after 2 months of cold acclimation (5°C) and a freezing treatment (-15°C for 4 h). The genotypic and phenotypic data for each accession in the rapeseed diversity panel was then used to conduct a genome-wide association study (GWAS). GWAS results showed that 14 significant markers were mapped to seven chromosomes for the phenotypes scored. Twenty-four candidate genes located within the mapped loci were identified as previously associated with lipid, photosynthesis, flowering, ubiquitination, and cytochrome P450 in rapeseed or other plant species.

Genome-Wide Association Studies and Transcriptome Changes during Acclimation and Deacclimation in Divergent Brassica napus Varieties.

Information concerning genes and signals regulating cold acclimation processes in plants is abundant; however, less is known about genes and signals regulating the deacclimation process. A population of primarily winter B. napus varieties was used to conduct a genome-wide association study and to compare the transcriptomes from two winter B. napus varieties showing time-dependent differences in response to cold acclimation and deacclimation treatments. These studies helped to identify loci, candidate genes, and signaling processes impacting deacclimation in B. napus. GWAS identified polymorphisms at five different loci associated with freezing tolerance following deacclimation. Local linkage decay rates near these polymorphisms identified 38 possible candidate genes. Several of these genes have been reported as differentially regulated by cold stress in arabidopsis (Arabidopsis thaliana), including a calcium-binding EF-hand family protein (encoded by BnaCnng10250D) that was also differentially expressed during deacclimation in this study. Thousands of other genes differentially expressed during the acclimation and deacclimation treatments implicated processes involving oxidative stress, photosynthesis, light-regulated diurnal responses, and growth regulation. Generally, responses observed during acclimation were reversed within one week of deacclimation. The primary differences between the two winter B. napus varieties with differential deacclimation responses involved protection from oxidative stress and the ability to maintain photosynthesis.

Genes associated with chloroplasts and hormone-signaling, and transcription factors other than CBFs are associated with differential survival after low temperature treatments of Camelina sativa biotypes.

Winter annual biotypes of Camelina sativa regularly survive after winter conditions experienced in northern regions of the U.S., whereas summer annual biotypes do not. To determine potential molecular mechanisms associated with these biotype differences in survival after low temperature treatments, we examined genetic and transcript variations in both a winter- (Joelle) and a summer- (CO46) biotype. It was determined that as few as one or two dominant genes may control differential survival after low temperature treatments. Of the 1797 genes that were differentially expressed in response to cold in both the winter and summer biotypes many COR genes were identified, indicating that the CBF regulon is functional in both. However, only 153 and 76 genes from Joelle and CO46, respectively, were either differentially expressed or not expressed at all in one biotype versus the other following cold acclimation. We hypothesize that these 229 genes play a significant role in, or are primarily responsive to, differences in survival after freezing between these two biotypes. Promoter analysis provided few clues as to the regulation or these genes; however, genes that were down-regulated specifically in the winter biotype Joelle were enriched with the sequence TGGCCCTCGCTCAC, which is over-represented among genes associated with chloroplasts in Arabidopsis. Additionally, several genes involved in auxin signaling were down-regulated specifically in Joelle. A transcription factor with strong similarity to MYB47, known to be up-regulated by salt, drought, and jasmonic acid, but not cold in Arabidopsis, was essentially off in the freezing sensitive biotype CO46, but was cold-induced in the winter biotype Joelle. Several other transcription factors genes including three with similarity to WRKY70, that may be involved in SA/JA-dependent responses, a HOMEOBOX 6 gene involved in ABA signaling, and two others (NUCLEAR FACTOR Y and CONSTANS-like 2) known to be implicated in photoperiodic flowering were also differentially expressed between the two biotypes.

Varying Weed Densities Alter the Corn Transcriptome, Highlighting a Core Set of Weed-Induced Genes and Processes with Potential for Manipulating Weed Tolerance.

CORE IDEAS: Corn increases the number of differentially expressed genes and the intensity of differential gene expression in response to increasing weed density. Genes associated with kinase signaling and transport functions are upregulated by weeds. Genes associated with protein production are downregulated by weeds. A sugar transporter (PMT5) and NUCLEOREDOXIN 1 are upregulated by weeds under diverse conditions. The phenological responses of corn (Zea mays L.) to competition with increasing densities of winter canola (Brassica napus L.) as the weedy competitor were investigated. Changes in the corn transcriptome resulting from varying weed densities were used to identify genes and processes responsive to competition under controlled conditions where light, nutrients, and water were not limited. Increasing densities of weeds resulted in decreased corn growth and development and increased the number and expression intensity of competition-responsive genes. The physiological processes identified in corn that were consistently induced by competition with weeds included protein synthesis and various transport functions. Likewise, numerous genes involved in these processes, as well as several genes implicated in phytochrome signaling and defense responses, were noted as differentially expressed. The results obtained in this study, conducted under controlled (greenhouse) conditions, were compared with a previously published study where the response of corn to competition with other species was evaluated under field conditions. Approximately one-third of the genes were differentially expressed in response to competition under both field and controlled conditions. These competition-responsive genes represent a resource for investigating the signaling processes by which corn recognizes and responds to competition. These results also highlight specific physiological processes that might be targets for mitigating the response of crops to weeds or other competitive plants under field conditions.

Cloning and characterization of the homoeologous genes for the Rec8-like meiotic cohesin in polyploid wheat.

BACKGROUND: Meiosis is a specialized cell division critical for gamete production in the sexual reproduction of eukaryotes. It ensures genome integrity and generates genetic variability as well. The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiotic cell division. The Rec8-like genes and cohesins have been cloned and characterized in diploid models, but not in polyploids. The present study aimed to clone the homoeologous genes (homoeoalleles) for Rec8-like cohesin in polyploid wheat, an important food crop for humans, and to characterize their structure and function under a polyploid condition. RESULTS: We cloned two Rec8-like homoeoalleles from tetraploid wheat (TtRec8-A1 and TtRec8-B1) and one from hexaploid wheat (TaRec8-D1), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat (TaRec8-A1 and TaRec8-B1) and the one in Aegilops tauschii (AetRec8-D1) by referencing the DNA sequences of the Rec8 homoeoalleles cloned in this study. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B (TtRec8-B1 and TaRec8-B1) and those from the subgenomes A and D (TtRec8-A1, TaRec8-A1, and TaRec8-D1). TtRec8-A1 expressed dominantly over TtRec8-B1, but comparably to TaRec8-D1, in polyploid wheat. In addition, we developed the antibody against wheat Rec8 and used the antibody to detect Rec8 cohesin in the Western blotting and subcellular localization analyses. CONCLUSIONS: The Rec8 homoeoalleles from the subgenomes A and D are transcriptionally more active than the one from the subgenome B in polyploid wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in polyploid wheat, and imply the distinction of the wheat subgenome B from the subgenomes A and D in the origin and evolution.

Expression of FLOWERING LOCUS C and a frameshift mutation of this gene on chromosome 20 differentiate a summer and winter annual biotype of Camelina sativa.

The nature of the vegetative to reproductive transition in the shoot apical meristem of Camelina sativa summer annual cultivar CO46 and winter annual cultivar Joelle was confirmed by treating seedlings with or without 8 weeks of vernalization. True to their life cycle classification, Joelle required a vernalization treatment to induce bolting and flowering, whereas CO46 did not. In this study, whole genome sequence, RNAseq, and resequencing of PCR-amplified transcripts for a key floral repressor were used to better understand factors involved in the flowering habit of summer and winter biotypes at the molecular level. Analysis of transcriptome data indicated that abundance for one of the three genes encoding the floral repressor FLOWERING LOCUS C (FLC; Csa20 g015400) was 16-fold greater in Joelle compared to CO46 prior to vernalization. Abundance of this transcript decreased only slightly in CO46 postvernalization, compared to a substantial decrease in Joelle. The results observed in the winter annual biotype Joelle are consistent with repression of FLC by vernalization. Further characterization of FLC at both the genome and transcriptome levels identified a one base deletion in the 5th exon coding for a keratin-binding domain in chromosome 20 of CO46 and Joelle. The one base deletion detected in chromosome 20 FLC is predicted to result in a frameshift that would produce a nonfunctional protein. Analysis of whole genome sequence indicated that the one base deletion in chromosome 20 FLC occurred at a greater ratio in the summer biotype CO46 (2:1) compared to the winter biotype Joelle (1:4); similar trends were also observed for RNAseq and cDNA transcripts mapping to chromosome 20 FLC of CO46 and Joelle.

Comparison of phytohormone levels and transcript profiles during seasonal dormancy transitions in underground adventitious buds of leafy spurge.

Leafy spurge (Euphorbia esula L.) is an herbaceous perennial weed that maintains its perennial growth habit through generation of underground adventitious buds (UABs) on the crown and lateral roots. These UABs undergo seasonal phases of dormancy under natural conditions, namely para-, endo-, and ecodormancy in summer, fall, and winter, respectively. These dormancy phases can also be induced in growth chambers by manipulating photoperiod and temperature. In this study, UABs induced into the three phases of dormancy under controlled conditions were used to compare changes in phytohormone and transcriptome profiles. Results indicated that relatively high levels of ABA, the ABA metabolite PA, and IAA were found in paradormant buds. When UABs transitioned from para- to endodormancy, ABA and PA levels decreased, whereas IAA levels were maintained. Additionally, transcript profiles associated with regulation of soluble sugars and ethylene activities were also increased during para- to endodormancy transition, which may play some role in maintaining endodormancy status. When crown buds transitioned from endo- to ecodormancy, the ABA metabolites PA and DPA decreased significantly along with the down-regulation of ABA biosynthesis genes, ABA2 and NCED3. IAA levels were also significantly lower in ecodormant buds than that of endodormant buds. We hypothesize that extended cold treatment may trigger physiological stress in endodormant buds, and that these stress-associated signals induced the endo- to ecodormancy transition and growth competence. The up-regulation of NAD/NADH phosphorylation and dephosphorylation pathway, and MAF3-like and GRFs genes, may be considered as markers of growth competency.

Foliar Glyphosate Treatment Alters Transcript and Hormone Profiles in Crown Buds of Leafy Spurge and Induces Dwarfed and Bushy Phenotypes throughout its Perennial Lifecycle.

Leafy spurge ( L.) is an invasive weed of North America and its perennial nature attributed to underground adventitious buds (UABs) that undergo seasonal cycles of para-, endo-, and ecodormancy. Recommended rates of glyphosate (∼1 kg ha) destroy aboveground shoots but plants still regenerate vegetatively; therefore, it is considered glyphosate-tolerant. However, foliar application of glyphosate at higher rates (2.2-6.7 kg ha) causes sublethal effects that induce UABs to produce stunted, bushy phenotypes. We investigated the effects of glyphosate treatment (±2.24 kg ha) on vegetative growth, phytohormone, and transcript profiles in UABs under controlled environments during one simulated seasonal cycle. Because shoots derived from UABs of foliar glyphosate-treated plants produced stunted, bushy phenotypes, we could not directly determine if these UABs transitioned through seasonally induced endo- and ecodormancy. However, transcript abundance for leafy spurge dormancy marker genes and principal component analyses suggested that UABs of foliar glyphosate-treated plants transitioned through endo- and ecodormancy. Glyphosate treatment increased shikimate abundance in UABs 7 d after treatment; however, the abundance of shikimate gradually decreased as UABs transitioned through endo- and ecodormancy. The dissipation of shikimate over time suggests that glyphosate's target site was no longer affected, but these changes did not reverse the altered phenotypes observed from UABs of foliar glyphosate-treated leafy spurge. Transcript profiles further indicated that foliar glyphosate treatment significantly affected phytohormone biosynthesis and signaling, particularly auxin transport; gibberellic acid, abscisic acid and jasmonic acid biosynthesis; ethylene responses; and detoxification and cell cycle processes in UABs. These results correlated well with the available phytohormone profiles and altered phenotypes.

Phytohormone balance and stress-related cellular responses are involved in the transition from bud to shoot growth in leafy spurge.

BACKGROUND: Leafy spurge (Euphorbia esula L.) is an herbaceous weed that maintains a perennial growth pattern through seasonal production of abundant underground adventitious buds (UABs) on the crown and lateral roots. During the normal growing season, differentiation of bud to shoot growth is inhibited by physiological factors external to the affected structure; a phenomenon referred to as paradormancy. Initiation of shoot growth from paradormant UABs can be accomplished through removal of the aerial shoots (hereafter referred to as paradormancy release). RESULTS: In this study, phytohormone abundance and the transcriptomes of paradormant UABs vs. shoot-induced growth at 6, 24, and 72 h after paradormancy release were compared based on hormone profiling and RNA-seq analyses. Results indicated that auxin, abscisic acid (ABA), and flavonoid signaling were involved in maintaining paradormancy in UABs of leafy spurge. However, auxin, ABA, and flavonoid levels/signals decreased by 6 h after paradormancy release, in conjunction with increase in gibberellic acid (GA), cytokinin, jasmonic acid (JA), ethylene, and brassinosteroid (BR) levels/signals. Twenty four h after paradormancy release, auxin and ABA levels/signals increased, in conjunction with increase in GA levels/signals. Major cellular changes were also identified in UABs at 24 h, since both principal component and Venn diagram analysis of transcriptomes clearly set the 24 h shoot-induced growth apart from other time groups. In addition, increase in auxin and ABA levels/signals and the down-regulation of 40 over-represented AraCyc pathways indicated that stress-derived cellular responses may be involved in the activation of stress-induced re-orientation required for initiation of shoot growth. Seventy two h after paradormancy release, auxin, cytokinin, and GA levels/signals were increased, whereas ABA, JA, and ethylene levels/signals were decreased. CONCLUSION: Combined results were consistent with different phytohormone signals acting in concert to direct cellular changes involved in bud differentiation and shoot growth. In addition, shifts in balance of these phytohormones at different time points and stress-related cellular responses after paradormancy release appear to be critical factors driving transition of bud to shoot growth.

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze).

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions.

The resemblance and disparity of gene expression in dormant and non-dormant seeds and crown buds of leafy spurge (Euphorbia esula).

BACKGROUND: Leafy spurge (Euphorbia esula L.) is a herbaceous perennial weed and dormancy in both buds and seeds is an important survival mechanism. Bud dormancy in leafy spurge exhibits three well-defined phases of para-, endo- and ecodormancy; however, seed dormancy for leafy spurge is classified as physiological dormancy that requires after-ripening and alternating temperature for maximal germination. Overlaps in transcriptome profiles between different phases of bud and seed dormancy have not been determined. Thus, we compared various phases of dormancy between seeds and buds to identify common genes and molecular processes, which should provide new insights about common regulators of dormancy. RESULTS: Cluster analysis of expression profiles for 201 selected genes indicated bud and seed samples clustered separately. Direct comparisons between buds and seeds are additionally complicated since seeds incubated at a constant temperature of 20°C for 21 days (21d C) could be considered paradormant (Para) because seeds may be inhibited by endosperm-generated signals, or ecodormant (Eco) because seeds germinate after being subjected to alternating temperature of 20:30°C. Since direct comparisons in gene expression between buds and seeds were problematic, we instead examined commonalities in differentially-expressed genes associated with different phases of dormancy. Comparison between buds and seeds ('Para to Endo buds' and '21d C to 1d C seeds'), using endodormant buds (Endo) and dormant seeds (1d C) as common baselines, identified transcripts associated with cell cycle (HisH4), stress response/transcription factors (ICE2, ERFB4/ABR1), ABA and auxin response (ABA1, ARF1, IAA7, TFL1), carbohydrate/protein degradation (GAPDH_1), and transport (ABCB2). Comparison of transcript abundance for the 'Eco to Endo buds' and '21d C to 1d C seeds' identified transcripts associated with ABA response (ATEM6), auxin response (ARF1), and cell cycle (HisH4). These results indicate that the physiological state of 21d C seeds is more analogous to paradormant buds than that of ecodormant buds. CONCLUSION: Combined results indicate that common molecular mechanisms associated with dormancy transitions of buds and seeds involve processes associated with ABA and auxin signaling and transport, cell cycle, and AP2/ERF transcription factors or their up-stream regulators.

Increase in ACC oxidase levels and activities during paradormancy release of leafy spurge (Euphorbia esula) buds.

The plant hormone ethylene is known to affect various developmental processes including dormancy and growth. Yet, little information is available about the role of ethylene during paradormancy release in underground adventitious buds of leafy spurge. In this study, we examined changes in ethylene evolution and the ethylene biosynthetic enzyme ACC oxidase following paradormancy release (growth induction). Our results did not show an obvious increase in ethylene during bud growth. However, when buds were incubated with 1 mM ACC, ethylene levels were higher in growing than non-growing buds, suggesting that the levels of ACC oxidase increased in growing buds. Real-time qPCR indicated that the transcript of a Euphorbia esula ACC oxidase (Ee-ACO) increased up to threefold following growth induction. In addition, a 2.5- to 4-fold increase in ACO activity was observed 4 days after decapitation, and the Ee-ACO accounted for 40 % of the total ACO activity. Immunoblot analyses identified a 36-kD Ee-ACO protein that increased in expression during bud growth. This protein was highly expressed in leaves, moderately expressed in crown buds, stems and meristems, and weakly expressed in roots and flowers. Immunolocalization of Ee-ACO on growing bud sections revealed strong labeling of the nucleus and cytoplasm in cells at the shoot apical meristem and leaf primordia. An exception to this pattern occurred in cells undergoing mitosis, where labeling of Ee-ACO was negligible. Taken together, our results indicated an increase in the levels of Ee-ACO during paradormancy release of leafy spurge that was not correlated with an increase in ethylene synthesis.

Induction of endodormancy in crown buds of leafy spurge (Euphorbia esula L.) implicates a role for ethylene and cross-talk between photoperiod and temperature.

Leafy spurge is a model for studying well-defined phases of dormancy in underground adventitious buds (UABs) of herbaceous perennial weeds, which is a primary factor facilitating their escape from conventional control measures. A 12-week ramp down in both temperature (27 â†’ 10 °C) and photoperiod (16 â†’ 8 h light) is required to induce a transition from para- to endo-dormancy in UABs of leafy spurge. To evaluate the effects of photoperiod and temperature on molecular networks of UABs during this transition, we compared global transcriptome data-sets obtained from leafy spurge exposed to a ramp down in both temperature and photoperiod (RDtp) versus a ramp down in temperature (RDt) alone. Analysis of data-sets indicated that transcript abundance for genes associated with circadian clock, photoperiodism, flowering, and hormone responses (CCA1, COP1, HY5, MAF3, MAX2) preferentially increased in endodormant UABs. Gene-set enrichment analyses also highlighted metabolic pathways involved in endodormancy induction that were associated with ethylene, auxin, flavonoids, and carbohydrate metabolism; whereas, sub-network enrichment analyses identified hubs (CCA1, CO, FRI, miR172A, EINs, DREBs) of molecular networks associated with carbohydrate metabolism, circadian clock, flowering, and stress and hormone responses. These results helped refine existing models for the transition to endodormancy in UABs of leafy spurge, which strengthened the roles of circadian clock associated genes, DREBs, COP1-HY5, carbohydrate metabolism, and involvement of hormones (ABA, ethylene, and strigolactones). We further examined the effects of ethylene by application of 1-aminocyclopropane-1-carboxylate (ACC) to paradormant plants without a ramp down treatment. New vegetative growth from UABs of ACC-treated plants resulted in a dwarfed phenotype that mimicked the growth response in RDtp-induced endodormant UABs. The results of this study provide new insights into dormancy regulation suggesting a short-photoperiod treatment provides an additive cross-talk effect with temperature signals that may impact ethylene's effect on AP2/ERF family members.

The transcriptomes of dormant leafy spurge seeds under alternating temperature are differentially affected by a germination-enhancing pretreatment.

Seed dormancy is an important stage in the life cycle of many non-domesticated plants, often characterized by the temporary failure to germinate under conditions that normally favor the process. Pre-treating dormant imbibed seeds at a constant temperate accelerated germination of leafy spurge seeds under alternating temperatures. However, dormant seeds will also germinate without a pre-treatment, albeit at a much slower rate, which gives rise to longer periods of imbibition before germination. Transcriptome analyses on seeds exposed to prolonged imbibition highlighted pathways associated with phenylpropanoid biosynthesis and interacting networks of genes involved in plant defense. In addition to the many pathways associated with phenylpropanoid biosynthesis enriched with down-regulated genes upon germination, there were also numerous pathways enriched with up-regulated genes associated with energy metabolism, such as glycolysis. Transcriptome data further suggest that metabolism and signaling by the plant hormones ethylene, gibberellin, and abscisic acid are involved in the developmental transition from dormancy to germination. More specifically, sub-network enrichment analysis identified ABI3 as a central hub of a sub-network at germination including several down-regulated genes such as DELLA (i.e., RGL2), which represses gibberellin signaling processes required for germination. The 595-fold increase in the expression of ACC oxidase (ACO4) at germination also suggests an important role for ethylene biosynthesis in germinating leafy surge seeds. Furthermore, the 10-578-fold difference in expression of many genes such as HY5 and Histone H3 between two populations at germination, which were treated with and without a constant temperature germination-enhancing pretreatment, revealed disparate impacts on various biosynthetic, growth, signaling, and response processes. Overall, our results indicate a constant temperature pretreatment (20°C for 21d) is not required for germination of leafy spurge seeds at an alternating temperature. However, the presence or absence of the pretreatment does affect the rate of germination and the germination transcriptional programs.