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Seed priming is a simple biotechnological tool which is potentially able to promote seed germination and invigoration as well as seedlings establishment and stress tolerance. In this study, the eff...

The utilization of the nutrient potential of alfalfa ( Medicago sativa L.) cannot be maximized because of its rapidly degradable protein content in the rumen, leading to waste and various digestive disorders. This might be alleviated if protein-binding proanthocyanidins are present in aerial parts of alfalfa forage in adequate amounts. The Lc (bHLH) and C1 (MYB) genes of maize are transcription factors known to be collectively involved in the regulation of anthocyanin biosynthetic pathways. The objective of this study was to investigate the effect of Lc and C1 gene transformations on the proanthocyanidin content, nutrient composition, and degradation characteristics of proteins and carbohydrates by comparing the transgenic alfalfa with its parental nontransgenic (NT) alfalfa and commercial AC-Grazeland cultivar. The DNA extracted from transgenic plants was tested for the presence of respective transgenes by amplification with specific primers of respective transgenes using PCR. Both Lc-single and LcC1-double transgenic alfalfa accumulated both monomeric and polymeric proanthocyanidins with total proanthocyanidins ranging from ca. 460 to 770 μg/g of DM. The C1-transgenic alfalfa did not accumulate proanthocyanidins similar to NT alfalfa. The C1 gene increased the NPN content significantly only in C1-single and Lc1C1-double transgenic alfalfa. The LcC1 combination seemed to have a synergic effect on reducing sugar in alfalfa. In contrast, the Lc gene appears to have a negative effect on starch content. The C1 gene tended to lower the PB3 content irrespective of the presence of the Lc gene. Although the cotransformation of Lc and C1 increased the total N/CHO ratio compared to Lc single gene transformation, the total N/CHO ratio of transgenic alfalfa was not significantly different from NT. In conclusion, Lc-bHLH single and LcC1 double gene transformation resulted in the accumulation of proanthocyanidins and affected the chemical profiles in alfalfa, which altered ruminal degradation patterns and impacted the nutrient availability of alfalfa in ruminant livestock systems.

Growth of plants in soil inoculated with plant growth promoting bacteria (PGPB) producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase or expression of the corresponding acdS gene in transgenic lines reduces the decline in shoot length, shoot weight and photosynthetic capacity triggered by salt stress in Camelina sativa. Reducing the levels of ethylene attenuated the salt stress response as inferred from decreases in the expression of genes involved in development, senescence, chlorosis and leaf abscission that are highly induced by salt to levels that may otherwise have a negative effect on plant growth and productivity. Growing plants in soil treated with Pseudomonas migulae 8R6 negatively affected ethylene signaling, auxin and JA biosynthesis and signalling, but had a positive effect on the regulation of genes involved in GA signaling. In plants expressing acdS, the expression of the genes involved in auxin signalling was positively affected, while the expression of genes involved in cytokinin degradation and ethylene biosynthesis were negatively affected. Moreover, fine-tuning of ABA signaling appears to result from the application of ACC deaminase in response to salt treatment. Moderate expression of acdS under the control of the root specific rolD promoter or growing plants in soil treated with P. migulae 8R6 were more effective in reducing the expression of the genes involved in ethylene production and/or signaling than expression of acdS under the more active Cauliflower Mosaic Virus 35S promoter.

In the present study, the effects of elevated zinc concentrations on germination, physiological and biochemical parameters were investigated in basil (Ocimum basilicum L.). Results indicate that zinc excess (1–5 mM ZnSO4) did not affect germination process, but it drastically reduced vigor index and radicle elongation, and induced oxidative stress. Exposure of basil plants to 400 and 800 µM Zn decreased aerial parts and roots dry biomass, root length and leaf number. Under these conditions, the reduction of plant growth was associated with the formation of branched and abnormally shaped brown roots. Translocation factor 1 was observed for 100 µM Zn suggested the possible use of basil as a phytostabiliser. Excess of Zn supply (> 100 µM) decreased chlorophyll content, total phenol and total flavonoid contents. Additionally, an increased TBARS levels reflecting an oxidative burst was observed in Zn-treated plants. These findings suggest that excess Zn adversely affects plant growth, photosynthetic pigments, phenolic and flavonoid contents, and enhances oxidative stress in basil plants.

Background Abiotic stress, including heat, is one of the major factors that affect alfalfa growth and forage yield. The small RNA, microRNA156 (miR156), regulates multiple traits in alfalfa during abiotic stress. The aim of this study was to explore the role of miR156 in regulating heat response in alfalfa at the protein level. Results In this study, we compared an empty vector control and miR156 overexpressing (miR156OE) alfalfa plants after exposing them to heat stress (40 °C) for 24 h. We measured physiological parameters of control and miR156OE plants under heat stress, and collected leaf samples for protein analysis. A higher proline and antioxidant contents were detected in miR156OE plants than in controls under heat stress. Protein samples were analyzed by label-free quantification proteomics. Across all samples, a total of 1878 protein groups were detected. Under heat stress, 45 protein groups in the empty vector plants were significantly altered (P 2FC| > 2). Conversely, 105 protein groups were significantly altered when miR156OE alfalfa was subjected to heat stress, of which 91 were unique to miR156OE plants. The identified protein groups unique to miR156OE plants were related to diverse functions including metabolism, photosynthesis, stress-response and plant defenses. Furthermore, we identified transcription factors in miR156OE plants, which belonged to squamosa promoter binding-like protein, MYB, ethylene responsive factors, AP2 domain, ABA response element binding factor and bZIP families of transcription factors. Conclusions These results suggest a positive role for miR156 in heat stress response in alfalfa. They reveal a miR156-regulated network of mechanisms at the protein level to modulate heat responses in alfalfa.

Background Previously, transgenic trichome-bearing (hairy leaf) Brassica napus lines expressing either the Arabidopsis thaliana GL3 gene (line AtGL3+) [1] or the AtGL3 gene in combination with an RNAi construct to down-regulate TTG1 (line K-5-8) [2] were developed. The leaves of these lines exhibited altered insect feeding (flea beetle) and oviposition (diamondback moth) behaviour compared to the non-transgenic semi-glabrous leaves of B. napus cv. Westar. Interestingly, the cotyledons of these lines remained glabrous, but also showed reduced feeding by flea beetles. Here we examine the composition and global transcriptome of the glabrous cotyledons from these transgenic lines to ascertain the mechanism(s) underlying this unexpected phenomenon. Results Approximately, 7500 genes were up-regulated in cotyledons of each hairy line, compared with

Our results show SPL13 plays a crucial role in regulating vegetative and reproductive development in Medicago sativa L. (alfalfa), and that MYB112 is targeted and downregulated by SPL13 in alfalfa. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing microRNA156 (miR156) show a bushy phenotype, reduced internodal length, delayed flowering time, and enhanced biomass yield. In alfalfa, transcripts of seven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, including SPL13, are targeted for cleavage by miR156. Thus, association of each target SPL gene to a trait or set of traits is essential for developing molecular markers for alfalfa breeding. In this study, we investigated SPL13 function using SPL13 overexpression and silenced alfalfa plants. Severe growth retardation, distorted branches and up-curled leaves were observed in miR156-impervious 35S::SPL13m over-expression plants. In contrast, more lateral branches and delayed flowering time were observed in SPL13 silenced plants. SPL13 transcripts were predominantly present in the plant meristems, indicating that SPL13 is involved in regulating shoot branch development. Accordingly, the shoot branching-related CAROTENOID CLEAVAGE DIOXYGENASE 8 gene was found to be significantly downregulated in SPL13 RNAi silencing plants. A R2R3-MYB gene MYB112 was also identified as being directly silenced by SPL13 based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays, suggesting that MYB112 may be involved in regulating alfalfa vegetative growth.

Volatile terpenoids produced in tea plants (Camellia sinensis) are airborne signals interacting against other ecosystem members, but also pleasant odorants of tea products. Transcription regulation (including transcript processing) is pivotal for plant volatile terpenoid production. In this study, a terpene synthase gene CsLIS/NES was recovered from tea plants (C. sinensis cv. ‘Long-Men Xiang’). CsLIS/NES transcription regulation resulted in two splicing forms: CsLIS/NES-1 and CsLIS/NES-2 lacking a 305 bp-fragment at N-terminus, both producing (E)-nerolidol and linalool in vitro. Transgenic tobacco studies and a gene-specific antisense oligo-deoxynucleotide (AsODN) suppression applied in tea leaves indicated that CsLIS/NES-1, localized in chloroplasts, acted as linalool synthase while CsLIS/NES-2 localized in cytosol, functioned as a potential nerolidol synthase, but not linalool synthase. Expression patterns of the two transcript isoforms in tea were distinctly different and responded differentially to the application of stress signal molecule methyl jasmonate (MeJA). Leaf expression of CsLIS/NES-1, but not CsLIS/NES-2, was significantly induced by MeJA. Our data indicated that distinct transcript splicing regulation patterns, together with subcellular compartmentation of CsLIS/NE-1 and CsLIS/NE-2 implemented the linalool biosynthesis regulation in tea plants in responding to endogenous and exogenous regulatory factors.

MicroRNA156 (miR156) regulates a network of downstream genes to affect plant growth and development. We previously generated alfalfa (Medicago sativa) plants that overexpress homologous miR156 (MsmiR156OE), and identified three of its SPL target genes. These plants exhibited increased vegetative yield, delayed flowering and longer roots. In this study, we aimed to elucidate the effect of miR156 on the root system, including effect on nodulation and nitrogen fixation. We found that MsmiR156 overexpression increases root regeneration capacity in alfalfa, but with little effect on root biomass at the early stages of root development. MsmiR156 also promotes nitrogen fixation activity by upregulating expression of nitrogenase-related genes FixK, NifA and RpoH in roots inoculated with Sinorrhizobium meliloti. Furthermore, we conducted transcriptomics analysis of MsmiR156OE alfalfa roots and identified differentially expressed genes belonging to 132 different functional categories, including plant cell wall organization, peptidyl-hypusine synthesis, and response to water stress. Expression analysis also revealed miR156 effects on genes involved in nodulation, root development and phytohormone biosynthesis. The present findings suggest that miR156 regulates root development and nitrogen fixation activity. Taken together, these findings highlight the important role that miR156 may play as a tool in the biotechnological improvement of alfalfa, and potentially other crops.

With the growing limitations on arable land, alfalfa (a widely cultivated, low-input forage) is now being selected to extend cultivation into saline lands for low-cost biofeedstock purposes. Here, minerals and transcriptome profiles were compared between two new salinity-tolerant North American alfalfa breeding populations and a more salinity-sensitive western Canadian alfalfa population grown under hydroponic saline conditions. All three populations accumulated two-fold higher sodium in roots than shoots as a function of increased electrical conductivity. At least 50% of differentially expressed genes (p < 0.05) were down-regulated in the salt-sensitive population growing under high salinity, while expression remained unchanged in the saline-tolerant populations. In particular, most reduction in transcript levels in the salt-sensitive population was observed in genes specifying cell wall structural components, lipids, secondary metabolism, auxin and ethylene hormones, development, transport, signalling, heat shock, proteolysis, pathogenesis-response, abiotic stress, RNA processing, and protein metabolism. Transcript diversity for transcription factors, protein modification, and protein degradation genes was also more strongly affected in salt-tolerant CW064027 than in salt-tolerant Bridgeview and salt-sensitive Rangelander, while both saline-tolerant populations showed more substantial up-regulation in redox-related genes and B-ZIP transcripts. The report highlights the first use of bulked genotypes as replicated samples to compare the transcriptomes of obligate out-cross breeding populations in alfalfa.

Biotic stresses negatively affect canola growth and production. Flea beetle damage and Sclerotinia sclerotiorum (S. sclerotiorum) infection are two of the worst biotic stresses for canola. Auxin Repressed Proteins (ARPs) responsive to several abiotic stresses have been reported. However, information about ARPs induced by Flea beetle damage and S. sclerotiorum infection, their roles in biotic stress tolerance are still lacking in canola. ESTs for an Auxin Repressed Protein 1 (BnARP1) were highly represented (expressed) in a Brassica napus subtractive library developed after leaf damage by the crucifer flea beetle (Phyllotreta cruciferae). Expression of this gene was under different developmental control in B. napus, and it was co-induced in B. napus by flea beetle feeding, S. sclerotiorum infection, drought and cold. A total of 25 BnARP genes were represented in different B. napus stress and development EST libraries and indicated larger, diversified families than known earlier. Dwarf phenotypes, primary root growth inhibition, lateral root enhancement, reduced sensitivity to 2, 4-D, and reduced PIN1 and LOX expression in transgenic Arabidopsis expression lines suggest that BnARP1 is an auxin repressor that prevents auxin transport and supports an interaction between the auxin and jasmonate signalling pathways. And the increased survival after S. sclerotiorum infection in transgenic over-expression Arabidopsis suggests that BnARP1 could play a role in S. sclerotiorum tolerance through connecting auxin and jasmonate signalling pathways.

Despite numerous studies on ABI4, its role in plant secondary metabolism has not been fully investigated. Here, we used metabolite profiling together with transcriptome analysis to demonstrate that ABI4 transcript levels regulate a host of secondary metabolite pathways and growth modalities in ABI4 over-expression (ABI4_OE) lines of Arabidopsis thaliana. This strategy provided a unique and comprehensive overview of the regulation of metabolic shifts in response to ABI4 transcription. We show that enhancement of ABI4 transcript levels changed seed proanthocyanidin (PA), flavonoid, and carotenoid levels in ABI4_OE seeds and 30-day-old shoots, as well as the expression of genes encoding enzymes involved in the production of these and other secondary metabolites in ABI4_OE shoots. In seeds, PA accumulated in very large uneven patches, which was dramatically different from the even distribution of PA in wild-type seeds. Shoots of ABI4_OE lines also exhibited altered expression of a range of genes involved in several aspects of plant development, including hormone and cell-wall synthesis. Alteration of such disparate secondary metabolite pathways, along with hormone and developmental pathways, suggests that ABI4 is a master regulator integrating these compounds with plant development.

Plant growth and insect resistance characteristics were determined for two Brassica napus Linnaeus (Brassicaceae) lines, AtGL3+ and K-5-8, developed for enhanced trichome densities relative to their parental cultivar Westar. In the field, both transgenic lines had glabrous cotyledons that curled upwards at emergence but flattened with time, and young leaves with elevated trichome density. Flea beetle (Phyllotreta cruciferae (Goeze) and Phyllotreta striolata (Fabricius); Coleoptera: Chrysomelidae) feeding was reduced on true leaves of both lines by 30–50% compared with insecticide-free Westar. Flea beetle feeding levels on cotyledons of the two hairy-leaved lines were lower than on unprotected Westar and similar to those seen on insecticide-treated Westar. Antixenosis and antibiosis resistance was observed when diamondback moths (Plutella xylostella (Linnaeus); Lepidoptera: Plutellidae) interacted with the hairy AtGL3+ and K-5-8 lines in the laboratory. Although the numbers of eggs laid by female diamondback moths on the transformed lines were similar to or higher than on Westar, in feeding bioassays larvae moved off AtGL3+ plants and larval feeding injury decreased on the transformed lines compared with Westar leaves. No agronomic or seed yield penalties were found for plants of K-5-8. These data highlight the utility of manipulating trichome regulatory genes to increase plant resistance against brassicaceous insect pests.

We compared the secondary metabolite composition in seeds of Camelina sativa and its wild relatives to identify potential germplasm with reduced levels of antinutritional compounds. Twenty Camelina accessions, from five different species, were analyzed by liquid chromatography mass spectrometry and subjected to principal component analysis, which revealed that Camelina spp. separated into distinct chemotaxonomic groups. Three major glucosinolates (GSs) were identified in our study, namely, 9-methylsulfinylnonyl GS (GS9), 10-methylsulfinyldecyl GS (GS10), and 11-methylsulfinylundecyl GS (GS11). While there were differences in total GS levels, species-specific patterns for GS9 and GS11 were noted. Sinapine content ranged between 1.4 and 5.6 mg/g FW, with the lowest levels observed in C. laxa and C. sativa. Lignin levels were also lowest in C. sativa, with most accessions containing less than 6 mg/g FW. Our results show that wild Camelina spp. have distinct metabolomes, and based on their levels of major antinutritionals, some could be incorporated into breeding programs with C. sativa.

Drought is one of the major abiotic stresses that negatively impact alfalfa growth and productivity. The role of microRNA156 (miR156) in drought has been demonstrated in plants. To date, there are no published studies investigating the role of miR156 in regulating global gene expression in alfalfa under drought. In our study, alfalfa genotypes overexpressing miR156 (miR156OE) exhibited reduced water loss, and enhanced root growth under drought. Our RNA-seq data showed that in response to drought, a total of 415 genes were upregulated and 169 genes were downregulated specifically in miR156OE genotypes. Genotypic comparison revealed that 285 genes were upregulated and 253 genes were downregulated in miR156OE genotypes relative to corresponding WT under drought. Gene Ontology enrichment analysis revealed that the number of differentially expressed genes belonging to biological process, molecular function and cell component functional groups was decreased in miR156OE genotypes under drought. Furthermore, RNA-Seq data showed downregulation of a gene encoding WD40 repeat in a miR156-specific manner. 5′ RACE experiments verified cleavage of WD40-2 transcript under drought. Moreover, alfalfa plants overexpressing WD40-2 showed drought sensitive, whereas those with silenced WD40-2 exhibited drought tolerant phenotypes. These findings suggest that miR156 improves drought tolerance in alfalfa by targeting WD40-2.

MicroRNA156 (miR156) is a regulator of flowering time and biomass production through regulation of members of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family. To expand our investigations on the utility of miR156 in alfalfa, we generated transgenic alfalfa expressing a heterologous miR156 from Lotus japonicus (LjmiR156a). 5′ RACE and qRT-PCR analysis confirmed that the same three SPL genes (MsSPL6, MsSPL12, and MsSPL13) targeted by MsmiR156d are also targets of LjmiR156a in alfalfa. Ectopic expression of LjmiR156a down-regulated these SPL genes in transgenic alfalfa, coupled with reduced internode length, a more extreme delay in flowering time than occurred with MsmiR156d, enhanced shoot branching, and elevated biomass production. While root length was maintained, nodulation was reduced in some transgenic genotypes. Furthermore, heterologous expression of LjmiR156a enhanced the contents of starch, soluble sugars, and phenolics in all transgenic genotypes in contrast to the impact from MsmiR156d enhancement, even though the effects on lignin, cellulose, pectin, structural sugars, flavonoids, and carotenoids were variable among the new alfalfa genotypes. The variations among the traits/genotypes reflect the change in expression of alfalfa SPL genes targeted by LjmiR156a and show that LjmiR156a could be employed as a tool to improve quality and yield of alfalfa biomass.

The discovery of small RNAs in plants has opened a new field in the study of gene regulation, and its application in plant genetic engineering and crop improvement. Of small RNAs, plant microRNAs have been used to increase crop productivity and enhance other traits, such as crop quality and stress tolerance. MicroRNA156 (miR156) and its target SPL genes are highly conserved in the plant kingdom, and together they form an extensive gene regulatory network that controls various aspects of plant growth and development. These include strong impacts on crop yield and quality, flowering time, root development and nodulation, reproduction capacity, secondary metabolism, and plant stress. Here, we review the most recent understanding of miR156 function in plants, and highlight the major impact we are now seeing from its application to crop germplasm improvement and its prospects for future use in plant molecular breeding.

Complex T-DNA repeat structures are a frequent outcome of Agrobacterium-mediated plant transformation and often lead to co-suppression of gene expression, even gene silencing. Thus, they are undesired in transgenic plants for commercial or research purposes and frequently need to be efficiently identified in a large transgenic plant population. Application of conventional Southern blot analysis is limited because it is laborious and time consuming. In this study, a new advancement that enables for high throughput determination of T-DNA repeat copy numbers in large scale screening of transgenic plant populations was developed by improving on the standard addition quantitative PCR method with specific reference plasmids. The plasmids contained the Arabidopsis single copy gene encoding high mobility group A and either a T-DNA direct repeat or T-DNA BAR gene. The improvement measured complex T-DNA repeat numbers quickly and accurately. Discrepancies in qPCR detected T-DNA copy number versus Southern detected T-DNA insertion number were largely a result of complex T-DNA repeats. Sequencing results revealed the existence in the repeat junctions of similar T-DNA insertion patterns commonly found in transgenic plants, including precise RB structure and LB deletion. Moreover, the end-joining nucleotides from both RB and LB in many repeat junctions were exactly at sites immediately adjacent to a homologous segment in the two borders, suggesting possible involvement of homologous recombination in the repeat formation. Our study demonstrates that this new advancement in high throughput quantification of T-DNA tandem repeats is useful for a large scale transgenic plant population analysis and in elucidating the mechanism of T-DNA tandem repeat formation.

MicroRNAs (miRNAs) are small endogenous RNA molecules that regulate gene expression at the posttranscriptional level. Recently, studies have discovered that miRNAs are induced by various stresses, suggesting that miRNAs may be an efficient target to improve crop resilience. Studies that are more recent have demonstrated that the response of miRNAs to stresses stimuli depends on type and level of stress, tissue, and genotype. Stress conditions induce several miRNAs, which alter downstream signaling cascades by regulating target genes and lead to adaptive responses to stress. During the past decade, research was focused on identification of plant miRNAs in response to environmental stressors, dissecting their expression patterns and studying their role in plant stress responses and tolerance. This has been accomplished principally by using next-generation sequencing (NGS) technologies, which have proven to be a powerful tool for this purpose. Altering miRNAs expression resulted in changes in plant growth and development under abiotic stress including cold, drought, salinity, nutrient, high temperature, and heavy metal stress as well as biotic stress such as fungus, bacteria, and virus infection. These findings implicate potential targets for genetic manipulation to improve stress tolerance in plants. This review is aimed to provide updates on recent miRNA research in improving plant resistance to biotic and abiotic stress. Moreover, we discuss availability of computational tools and genomics platforms for identification of stress-related miRNAs in plants.

The response of Camelina sativa to salt stress was examined. Salt reduced shoot, but not root length. Root and shoot weight were affected by salt, as was photosynthetic capacity. Salt did not alter micro-element concentration in shoots, but increased macro-element (Ca and Mg) levels. Gene expression patterns in shoots indicated that salt stress may have led to shuttling of Na+ from the cytoplasm to the tonoplast and to an increase in K+ and Ca+2 import into the cytoplasm. In roots, gene expression patterns indicated that Na+ was exported from the cytoplasm by the SOS pathway and that K+ was imported in response to salt. Genes involved in chelation and storage were up-regulated in shoots, while metal detoxification appeared to involve various export mechanisms in roots. In shoots, genes involved in secondary metabolism leading to lignin, anthocyanin and wax production were up-regulated. Partial genome partitioning was observed in roots and shoots based on the expression of homeologous genes from the three C. sativa sub-genomes. Sub-genome I and II were involved in the response to salinity stress to about the same degree, while about 10% more differentially-expressed genes were associated with sub-genome III.

A total of 88 new Arabidopsis lines with trichome variation were recovered by screening 49,200 single-seed descent T3 lines from the SK activation-tagged population and from a new 20,000-line T-DNA insertion population (called pAG). Trichome variant lines were classified into 12 distinct phenotype categories. Single or multiple T-DNA insertion sites were identified for 89% of these mutant lines. Alleles of the well-known trichome genes TRY, GL2 and TTG1 were recovered with atypical phenotype variation not reported previously. Moreover, atypical gene expression profiles were documented for two additional mutants specifying TRY and GL2 disruptions. In remaining mutants, ten lines were disrupted in genes coding for proteins not implicated in trichome development, five were disrupted in hypothetical proteins and 11 were disrupted in proteins with unknown function. The collection represents new opportunities for the plant biology community to define trichome development more precisely and to refine the function of individual trichome genes.

The effects of microRNA156 overexpression on general plant architecture, branching, flowering time and nodulation were investigated in the model legume, Lotus japonicus. We cloned an miR156 homolog, LjmiR156a, from L. japonicus, and investigated its SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes and its biological function at enhancing vegetative biomass yield, extending flowering time, and its impact on nodulation. Thirteen potential targets for LjmiR156 were identified in vitro and their expression profiles were determined in aerial and underground parts of mature plants, including genes coding for eight SPLs, one WD-40, one RNA-directed DNA polymerase, two transport proteins, and one histidine-phosphotransfer protein. Two SPL and one WD-40 cleavage targets for LjmiR156—TC70253, AU089191, and TC57859—were identified. Transgenic plants with ectopic expression of LjmiR156a showed enhanced branching, dramatically delayed flowering, underdeveloped roots, and reduced nodulation. We also examined the transcript levels of key genes involved in nodule organogenesis and infection thread formation to determine the role of miR156 in regulating symbiosis. Overexpression of LjmiR156a led to repression of several nodulation genes during the early stages of root development such as three ENOD genes, SymPK, POLLUX, CYCLOPS, Cerberus, and Nsp1, and the stimulation of NFR1. Our results show that miR156 regulates vegetative biomass yield, flowering time and nodulation by silencing downstream target SPLs and other genes, suggesting that the miR156 regulatory network could be modified in forage legumes (such as alfalfa and trefoils) and in leafy vegetables (like lettuce and spinach) to positively impact economically valuable crop species. Electronic supplementary material The online version of this article (doi:10.1007/s00438-014-0931-4) contains supplementary material, which is available to authorized users.

Zinc finger proteins comprise a large family and function in various developmental processes. CCCH type zinc finger protein is one kind of zinc finger protein, which function is little known. MsZFN gene encoding a CCCH type zinc finger protein was first discovered by its elevated transcript level in a salt-induced alfalfa SSH cDNA library. The previous experiment had showed that MsZFN protein was localized to the nucleus and little is known about the function of MsZFN protein and its homologous proteins in other plants including model plant, Arabidopsis thaliana . In the current study, we found that MsZFN transcript levels increased in alfalfa under continuous dark conditions and that expression was strongest in leaves and weakest in unopened flowers under light/dark conditions. Expression of MsZFN in transgenic Arabidopsis plants resulted in late flowering phenotypes under long day conditions. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that MsZFN protein can interact with itself. Transcript analyses of floral regulatory genes in MsZFN + transgenic Arabidopsis showed enhanced expression of the flowering repressor FLOWERING LOCUS C and decreased expression of three key flowering time genes, FLOWERING LOCUS T, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS and GIGANTEA . These results suggest that MsZFN primarily controls flowering time by repressing flowering genes expression under long day conditions.

Nasturtium officinale R. Br. seedlings were treated with a range of NaCl concentrations (0, 50, 100 and 150 mM) for 21 days after seedling emergence. Physiological analysis based on growth and mineral nutrition, showed a substantial decrease in leaf dry matter with 150 mM NaCl treatment. The growth decrease was correlated with nutritional imbalance and a reduction in potassium accumulation and transport to the leaves. At the same time, we noted an increase in leaf sodium and chloride accumulation and transport. Salt tolerance of N. officinale under 100 mM NaCl was associated with osmotic adjustment via Na+ and Cl− and the maintenance of high K+/Na+ selectivity. Salt decreased carotenoid content more than chlorophylls and also disturbed membrane integrity by increasing malondialdehyde content and electrolyte leakage. At 150 mM NaCl, an increase in antioxidant enzyme-specific activities for superoxide dismutase, catalase and guaiacol peroxidase occurred in concert with a decrease in ascorbic acid, polyphenol, tannin and flavonoid content. These results indicate that N. officinale can maintain growth and natural antioxidant defense compounds such as, vitamin C, carotenoids, and polyphenols, when cultivated in 100 mM NaCl, but not at higher salt levels.

In this study, shade-induced conversion from a young pale/yellow leaf phenotype to a green leaf phenotype was studied using metabolic and transcriptomic profiling and the albino cultivar ‘Yu-Jin-Xiang’ (‘YJX’) of Camellia sinensis for a better understanding of mechanisms underlying the phenotype shift and the altered catechin and theanine production. Shaded leaf greening resulted from an increase in leaf chlorophyll and carotenoid abundance and chloroplast development. A total of 1,196 differentially expressed genes (DEGs) were identified between the ‘YJX’ pale and shaded green leaves, and these DEGs affected ‘chloroplast organization’ and ‘response to high light’ besides many other biological processes and pathways. Metabolic flux redirection and transcriptomic reprogramming were found in flavonoid and carotenoid pathways of the ‘YJX’ pale leaves and shaded green leaves to different extents compared to the green cultivar ‘Shu-Cha-Zao’. Enhanced production of the antioxidant quercetin rather than catechin biosynthesis was correlated positively with the enhanced transcription of FLAVONOL SYNTHASE and FLAVANONE/FLAVONOL HYDROXYLASES leading to quercetin accumulation and negatively correlated to suppressed LEUCOANTHOCYANIDIN REDUCTASE, ANTHOCYANIDIN REDUCTASE and SYNTHASE leading to catechin biosynthesis. The altered levels of quercetin and catechins in ‘YJX’ will impact on its tea flavor and health benefits.

Canola (Brassica napus L.) productivity is severely affected by various biotic and abiotic stresses such as insects, pathogens, drought, and cold. Characterization of genes with specific effects on...

Tea volatiles’ generation and retention over manufacturing processes are crucial for tea quality. In this study, floral volatile adsorption and retention in green tea scented with Jasminum sambac flowers were examined over the scenting process. Out of 34 enhanced volatiles in the scented tea, β-ionone, β-linalool, indole, and methyl anthranilate were the most potent odorants with 5.1–45.2-fold higher odor activity values than the corresponding controls in the nonscented tea. Scenting efficiencies for the floral volatiles retained in the scented tea (the percentage of volatile abundance over its corresponding amount in jasmine flowers) ranged from 0.22% for α-farnesene to 75.5% for β-myrcene. Moreover, due to additional rounds of heat treatment for scented green tea manufacturing, some volatiles such as carotenoid-derived geraniol and β-ionone and lipid-derived (Z)-jasmone were heat-enhanced and others such as nonanal were heat-desorbed in the scented green tea. Our study revealed that dynamic volatile absorption and desorption collectively determined tea volatile retention and tea aroma. Our findings may have a great potential for practical improvement of tea aroma.

Camelina sativa (camelina) is an oilseed crop touted for use on marginal lands; however, it is no more tolerant of soil salinity than traditional crops, such as canola. Plant growth-promoting bacteria (PGPB) that produce 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase) facilitate plant growth in the presence of abiotic stresses by reducing stress ethylene. Rhizospheric and endophytic PGPB and the corresponding acdS- mutants of the latter were examined for their ability to enhance tolerance to salt in camelina. Stimulation of growth and tolerance to salt was correlated with ACC deaminase production. Inoculation of soil with wild-type PGPB led to increased shoot length in the absence of salt, and increased seed production by approximately 30-50 percent under moderately saline conditions. The effect of ACC deaminase was further examined in transgenic camelina expressing a bacterial gene encoding ACC deaminase (acdS) under the regulation of the CaMV 35S promoter or the root-specific rolD promoter. Lines expressing acdS, in particular those using the rolD promoter, showed less decline in root length and weight, increased seed production, better seed quality and higher levels of seed oil production under salt stress. This study clearly demonstrates the potential benefit of using either PGPB that produce ACC deaminase or transgenic plants expressing the acdS gene under the control of a root-specific promoter to facilitate plant growth, seed production and seed quality on land that is not normally suitable for the majority of crops due to high salt content.

In this study, we report on the relationship between individual antioxidant carotenoids, phenolic fractions, and flavonoids and attenuating NaCl toxicity in two contrasting varieties of lettuce (Lactuca sativa L., var. Verte and Romaine), as a means of developing more precise targets for lettuce breeding. In seedling leaves, carotenoid levels increased under NaCl stress in Verte, but remained unchanged in Romaine. Chlorogenic acid (CGA), a soluble polyphenol abundantly present in leaves, was the only phenolic acid that accumulated significantly in both varieties and was predominant under salinity. Remarkably, CGA was more prominent in Verte than Romaine at moderate salinity levels (100 mM). Foliar flavonoid levels were also differentially increased in both seedling types grown under 100 mM NaCl, but dropped significantly in both varieties under 200 mM NaCl. Germination, which was overall more salt-resistant in Romaine, was significantly increased under salt stress by application of low dosage (10–6 to10–5 M) of CGA to Verte, but not to Romaine. These results suggest that CGA is involved in modulating NaCl-induced oxidative-stress responses during seed germination in Verte.

An Arabidopsis thaliana mutant, cbd (carotenoid biosynthesis deficient), was recovered from a mutant population based on its yellow cotyledons, yellow-first true leaves, and stunted growth. Seven-day-old seedlings and mature seeds of this mutant had lower chlorophyll and total carotenoids than the wild type (WT). Genetic and molecular characterization revealed that cbd was a recessive mutant caused by a T-DNA insertion in the gene cpSRP54 encoding the 54 kDa subunit of the chloroplast signal recognition particle. Transcript levels of most of the main carotenoid biosynthetic genes in cbd were unchanged relative to WT, but expression increased in carotenoid and abscisic acid catabolic genes. The chloroplasts of cbd also had developmental defects that contributed to decreased carotenoid and chlorophyll contents. Transcription of AtGLK1 (Golden 2-like 1), AtGLK2, and GUN4 appeared to be disrupted in the cbd mutant suggesting that the plastid-to-nucleus retrograde signal may be affected, regulating the changes in chloroplast functional and developmental states and carotenoid content flux. Transformation of A. thaliana and Brassica napus with a gDNA encoding the Arabidopsis cpSRP54 showed the utility of this gene in enhancing levels of seed carotenoids without affecting growth or seed yield.

Extracts of quercetin glycosides from three-week old leaves of Camelina sativa were tested for their effects on crucifer flea beetle feeding. The extract fractions moderately deterred flea beetle feeding in mixtures containing rutin, but not when the glycosides were presented as individual compounds. Leaves and cotyledons of C. sativa contain large amounts of quercetin glycosides. In contrast, Brassica napus leaves and cotyledons contain only traces of quercetin glycosides, while leaves accumulate large amounts of kaempferol glycosides. The results suggest that quercetin glycosides form part of the arsenal of C. sativa chemical defenses against flea beetles, and that B. napus is devoid of this form of defense.

Foam stability related to pasture bloat from alfalfa pastures might be reduced by introducing a gene that stimulates the accumulation of mono-/polymeric-anthocyanidin. The objective of this study was to determine the foam formation (at 0 min) and stability (at 150 min) from aqueous leaf extracts of three transgenic Lc-alfalfa progeny (BeavLc1, RambLc3 and RangLc4), parental non-transgenic (NT) alfalfa and AC Grazeland (bloat-reduced cultivar) harvested at 07:00 or 18:00 h. Initial and final foam volumes at 07:00 h were lower for AC Grazeland compared with all other treatments and lower for RangLc4 compared with the other two Lc-progeny at 0 min and NT-alfalfa at 150 min. At 18:00 h, initial foam volume was larger for NT-alfalfa and final foam volume was larger for RambLc3 compared with AC Grazeland, BeavLc1 and RangLc4. Spectroscopic vibration associated with carbohydrates increased initial foam volume and stability. More amide I relative to amide II, fewer α-helices relative to β-sheets and leaf extract ethanol-film and protein content increased initial foam volume but did not stabilize it. Spectroscopic vibration associated with all carbohydrates other than structural carbohydrates was more important than vibration from protein structures for foam formation and stabilization. In conclusion, Lc-alfalfa accumulated anthocyanidin and had reduced foaming properties compared with parental NT alfalfa but AC Grazeland and RangLc4 had the lowest foaming properties.

The molecular basis of canola ( Brassica napus L.) susceptibility to the crucifer flea beetle (FB, Phyllotreta cruciferae Goeze) was investigated by comparing transcript representation in FB-damaged and undamaged cotyledons. The B. napus cotyledon transcriptome increased and diversified substantially after FB feeding damage. Twenty-two genes encoding proteins with unknown function, six encoding proteins involved in signaling, and a gene encoding a B-box zinc finger transcription factor were moderately or strongly changed in representation with FB feeding damage. Zinc finger and calcium-dependent genes formed the largest portion of transcription factors and signaling factors with changes in representation. Six genes with unknown function, one transcription factor, and one signaling gene specific to the FB-damaged library were co-represented in a FB-damaged leaf library. Out of 188 transcription factor and signaling gene families screened for “early” expression changes, 16 showed changes in expression within 8 h. Four of these early factors were zinc finger genes with representation only in the FB-damaged cotyledon. These genes are now available to test their potential at initiating or specifying cotyledon responses to crucifer FB feeding.

Plant carotenoid derived β-ionone has been shown to have diverse biological effects on some insect herbivores and herbivore parasitoids. In this study, Arabidopsis transgenic plants over-expressing a carotenoid cleavage dioxygenasel gene (AtCCD1) were generated to test whether β-ionone emissions could be enhanced and used to control feeding by the crucifer flea beetle (Phyllotreta cruciferae Goeze). The transgenic plants exhibited a morphological phenotype indistinguishable from the wild type (WT) control over their complete life cycle. Gas chromatography and mass spectrometry analyses of headspace volatiles collected from 6-wk-old intact flowering plants revealed substantially enhanced β-ionone emission from transgenic plants compared with WT, but no β-ionone enhancement occurred at a young vegetative stage (4-wk-old seedlings). Bioassays in an enclosed environment showed that ATCCD1 over-expression resulted in ≈50% less leaf area damage by flea beetles on transgenic plants compared with WT plants. The mean number of damaged transgenic leaves per plant also was significantly lower in transgenic plants (P < 0.05). Our results indicate that AtCCD1 over-expression and induced β-ionone emission might find application in the control of pests for Brassica crops grown in greenhouse operations. Potentially, β-ionone also could be used on crops grown in open-air ecosystems if this allomone is released in sufficient quantities to discourage herbivore foragers.

Brassinosteroids (BRs) play a crucial role in plant growth and development and DIMINUTO 1 (DIM1), a protein involved in BR biosynthesis, was previously identified as a cell elongation factor in Arabidopsis thaliana. Through promoter expression analysis, we showed that DIM1 was expressed in most of the tissue types in seedlings and sectioning of the inflorescence stem revealed that DIM1 predominantly localizes to the xylem vessels and in the interfascicular cambium. To investigate the role of DIM1 in cell wall formation, we generated loss-of-function and gain-of-function mutants. Disruption of the gene function caused a dwarf phenotype with up to 38 and 23% reductions in total lignin and cellulose, respectively. Metabolite analysis revealed a significant reduction in the levels of fructose, glucose and sucrose in the loss-of-function mutant compared to the wild type control. The loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type, but no changes were detected in the gain-of-function mutant. Phloroglucinol and toluidine blue staining showed a size reduction of the vascular apparatus with smaller and disintegrated xylem vessels in the inflorescence stem of the loss-of-function mutant. Taken together, these data indicate a role for DIM1 in secondary cell wall formation. Moreover, this study demonstrated the potential role of BR hormones in modulating cell wall structure and composition.

The objective of this study was to determine more indepth physiological and antioxidant responses in two Medicago ciliaris lines (a salt-tolerant line TNC 1.8 and a salt-sensitive line TNC 11.9) with contrasting responses to 100 mM NaCl. Under salt stress, both lines showed a decrease in total biomass and in the growth rate for roots, but TNC 1.8 was less affected by salt than TNC 11.9 in that it maintained leaf growth even in the presence of added salt. In both lines, salt stress mainly affected micronutrient status (Fe, Mn, Cu and Zn) rather than K nutrition, but the tolerant line TNC 1.8 accumulated more Na in leaves and less in roots compared with TNC 11.9. Salt stress decreased total soluble sugars (TSS) in all organs of the sensitive line TNC 11.9, whereas TSS was only reduced in roots of the tolerant line. The salt-induced drop in growth was linked to an increase in lipid peroxidation in roots of both lines and in leaves of the sensitive line. The salt-tolerant line TNC 1.8 was more efficient at managing salt-induced oxidative damage in leaves and to a lesser extent in roots than the salt-sensitive line TNC 11.9, by preserving higher phenolic compound and superoxide dismutase levels in both organs.

The root system is central for plant adaptation to soil heterogeneity and is organized primarily by root branching. To search for compounds that regulate root branching, a forward chemical genetics screen was employed, and 4-methylumbelliferone (4-MU), a coumarin derivative, was found to be a potent regulator of lateral root formation. Exogenous application of 4-MU to Arabidopsis thaliana seeds affected germination and led to reduced primary root growth, the formation of bulbous root hairs, and irregular detached root caps accompanied by reorganization of the actin cytoskeleton in root tips before seedling establishment. Abundant lateral roots formed after exposure to 125 μM 4-MU for 22 days. Molecular, biochemical, and phytochemical approaches were used to determine the effect of 4-MU on root growth and root branching. Arabidopsis seedlings grown in the presence of 4-MU accumulated this compound only in roots, where it was partially transformed by UDP-glycosyltransferases (UGTs) into 4-methylumbelliferyl-β-D-glucoside (4-MU-Glc). The presence of 4-MU-Glc in seedling roots was consistent with the upregulation of several genes that encode UGTs in the roots. This shows that UGTs play an integral role in the detoxification of 4-MU in plants. The increased expression of two auxin efflux facilitator genes (PIN2 and PIN3) in response to 4-MU and the lack of response of the auxin receptor TIR1 and the key auxin biosynthetic gene YUCCA1 suggest that auxin redistribution, rather than auxin biosynthesis, may directly or indirectly mediate 4-MU-induced root branching.

A full-length cDNA of 1,728 nt, called MsLEA3-1, was cloned from alfalfa by rapid amplification of cDNA ends from an expressed sequence tag homologous to soybean pGmPM10 (accession No. AAA91965.1). MsLEA3-1, encodes a deduced protein of 436 amino acids, a calculated molecular weight of 47.0 kDa, a theoretical isoelectric point of 5.18, and closest homology with late embryogenesis abundant proteins in soybean. Sequence homology suggested a signal peptide in the N terminus, and subcellular localization with GFP revealed that MsLEA3-1 was localized preferentially to the nucleolus. The transcript titre of MsLEA3-1 was strongly enriched in leaves compared with roots and stems of mature alfalfa plants. Gene expression of MsLEA3-1 was strongly induced when seedlings were treated with NaCl and ABA. Expression of the MsLEA3-1 transgenic was detected in transgenic tobacco. Malondialdehyde content and, electrical conductivity content were reduced and electrical conductivity and proline content were increased in transgenic tobacco compared with non-transgenic tobacco under salt stress. The results showed that accumulation of the MsLEA3-1 protein in the vegetative tissues of transgenic plants enhanced their tolerance to salt stress. These results demonstrate a role for the MsLEA3-1 protein in stress protection and suggest the potential of the MsLEA3-1 gene for genetic engineering of salt tolerance. Electronic supplementary material The online version of this article (doi:10.1007/s11033-011-1048-z) contains supplementary material, which is available to authorized users.

Genotypic variability was assessed within six Medicago ciliaris genotypes growing symbiotically with Sinorhizobium medicae in order to identify physiological criteria (growth, ion content, and plant health) associated with salt tolerance. Response to salt stress depended on the line and the level of salt. Two lines with lower dry biomass under non-saline conditions (TNC 1.8 from a semi-arid area and TNC 10.8 from a sub-humid area), were more tolerant to NaCl, whereas the most productive lines (TNC 11.5 and TNC 11.9 from a humid bioclime) were more sensitive in terms of growth and nitrogen fixation. Susceptibility of symbiotic nitrogen fixation to saline stress was not associated with a higher accumulation of Na+ in nodules, since the most tolerant lines TNC 1.8 and TNC 10.8 accumulated the highest Na+ amount in nodules. Leaf area and net photosynthate assimilation rate were conserved in line TNC 1.8 and to a lesser extent in line TNC 10.8 potentially owing to a greater ability to protect aerial organs and nodules from Na+ damage and to insure a better supply of leaves with nitrogen. Our results suggest that nodule growth and number and nodule Na+ content should not be used as selection tools for tolerance or susceptibility, since two of the tested lines maintained consistent growth in spite of reduced nodule and high Na+ content. Instead, the most reliable physiological indicators for tolerance appear to be consistent growth (i.e., no growth changes) and reduced leaf Na+ accumulation with increasing concentrations of NaCl.

The aim of this study was to investigate the effect of NaCl salinity on symbiotic nitrogen fixation (SNF) in a Medicago ciliaris – Sinorhizobium medicae symbiosis system, with special emphasis on two genetically pure lines of M. ciliaris developed from germplasm recovered from saline and non-saline lands. Saline treatment (100 mM NaCl) was applied to these two contrasting lines growing in pots in a greenhouse after the appearance of nodules. At the end of the vegetative period, both lines under salt stress registered a decrease in nitrogenase activity, whole plant and organ biomass production and chlorophyll concentration, but the tolerant line TNC 1.8 was much less affected by salt than the sensitive line TNC 11.9. Salinity also provoked a decrease in sucrose phosphate synthase activity (SPS), mainly in TNC 11.9. The decline in leaf SPS in TNC 11.9 was accompanied by salinity-induced membrane oxidative damage in source leaves of the sensitive line, probably induced by a disturbance in soluble sugar/starch partitioning. We report these physiological and biochemical parameters to promote these useful lines as useful germplasm with potential to reveal detailed transcriptomic and metabolomic information and new genetic tools which could assist breeders in improving salinity tolerance and biomass for pasture legumes.

The Arabidopsis transparent testa (tt) mutant tt19-4 shows reduced seed coat colour, but stains darkly with DMACA and accumulates anthocyanins in aerial tissues. Positional cloning showed that tt19-4 was allelic to tt19-1 and has a G-to-T mutation in a conserved 3-domain in the TT19-4 gene. Soluble and unextractable seed proanthocyanidins and hydrolysis of unextractable proanthocyanidin differ between wild-type Col-4 and both mutants. However, seed quercetins, unextractable proanthocyanidin hydrolysis, and seedling anthocyanin content, and flavonoid gene expres- sion differ between tt19-1 and tt19-4. Transformation of tt19-1 with a TT19-4 cDNA results in vegetative anthocya- nins, whereas TT19-4 cDNA cannot complement the proanthocyanidin and pale seed coat phenotype of tt19-1. Both recombinant TT19 and TT19-4 enzymes are func- tional GSTs and are localized in the cytosol, but TT19 did not function with wide range of flavonoids and natural products to produce conjugation products. We suggest that the dark seed coat of Arabidopsis is related to soluble proanthocyanidin content and that quercetin holds the key to the function of TT19. In addition, TT19 appears to have a5 GSH-binding domain influencing both anthocyanin and proanthocyanidin accumulation and a 3 domain affecting proanthocyanidin accumulation by a single amino acid substitution.