Endogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. In plants, the RNA-dependent RNA polymerases (RDRs) are essential for smRNA biogenesis. It has been established that RDR2 is involved in the 24 nt siRNA-dependent RNA-directed DNA methylation (RdDM) pathway. Recent studies have suggested that RDR1 is involved in a second RdDM pathway that relies mostly on 21 nt smRNAs and functions to silence a subset of genomic loci that are usually refractory to the normal RdDM pathway in Arabidopsis. Whether and to what extent the homologs of RDR1 may have similar functions in other plants remained unknown.
We characterized a loss-of-function mutant (Osrdr1) of the OsRDR1 gene in rice (Oryza sativa L.) derived from a retrotransposon Tos17 insertion. Microarray analysis identified 1,175 differentially expressed genes (5.2% of all expressed genes in the shoot-tip tissue of rice) between Osrdr1 and WT, of which 896 and 279 genes were up- and down-regulated, respectively, in Osrdr1. smRNA sequencing revealed regional alterations in smRNA clusters across the rice genome. Some of the regions with altered smRNA clusters were associated with changes in DNA methylation. In addition, altered expression of several miRNAs was detected in Osrdr1, and at least some of which were associated with altered expression of predicted miRNA target genes. Despite these changes, no phenotypic difference was identified in Osrdr1 relative to WT under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions.
Our results showed that OsRDR1 plays a role in regulating a substantial number of endogenous genes with diverse functions in rice through smRNA-mediated pathways involving DNA methylation, and which participates in abiotic stress response.
Gene expression; Epigenetics; Small RNA; DNA methylation; RDR1; Oryza sativa L
Pollen of common ragweed (Ambrosia artemisiifolia) is a main cause of allergic diseases in Northern America. The weed has recently become spreading as a neophyte in Europe, while climate change may also affect the growth of the plant and additionally may also influence pollen allergenicity. To gain better insight in the molecular mechanisms in the development of ragweed pollen and its allergenic proteins under global change scenarios, we generated SuperSAGE libraries to identify differentially expressed transcripts.
Ragweed plants were grown in a greenhouse under 380 ppm CO2 and under elevated level of CO2 (700 ppm). In addition, drought experiments under both CO2 concentrations were performed. The pollen viability was not altered under elevated CO2, whereas drought stress decreased its viability. Increased levels of individual flavonoid metabolites were found under elevated CO2 and/or drought. Total RNA was isolated from ragweed pollen, exposed to the four mentioned scenarios and four SuperSAGE libraries were constructed. The library dataset included 236,942 unique sequences, showing overlapping as well as clear differently expressed sequence tags (ESTs). The analysis targeted ESTs known in Ambrosia, as well as in pollen of other plants. Among the identified ESTs, those encoding allergenic ragweed proteins (Amb a) increased under elevated CO2 and drought stress. In addition, ESTs encoding allergenic proteins in other plants were also identified.
The analysis of changes in the transcriptome of ragweed pollen upon CO2 and drought stress using SuperSAGE indicates that under global change scenarios the pollen transcriptome was altered, and impacts the allergenic potential of ragweed pollen.
Ambrosia artemisiifolia; Allergen; Allergy; CO2; Drought; Flavonoids; Pollen; Ragweed; Scanning electron microscopy; Transcriptome
The Medicago truncatula 2HA seed line is highly embryogenic while the parental line Jemalong rarely produces embryos. The 2HA line was developed from one of the rare Jemalong regenerates and this method for obtaining a highly regenerable genotype in M. truncatula is readily reproducible suggesting an epigenetic mechanism. Microarray transcriptomic analysis showed down regulation of an ETHYLENE INSENSITIVE 3-like gene in 2HA callus which provided an approach to investigating epigenetic regulation of genes related to ethylene signalling and the 2HA phenotype. Ethylene is involved in many developmental processes including somatic embryogenesis (SE) and is associated with stress responses.
Microarray transcriptomic analysis showed a significant number of up-regulated transcripts in 2HA tissue culture, including nodule and embryo specific genes and transposon-like genes, while only a few genes were down-regulated, including an EIN3-like gene we called MtEIL1. This reduced expression was associated with ethylene insensitivity of 2HA plants that was further investigated. The weak ethylene insensitivity affected root and nodule development. Sequencing of MtEIL1 found no difference between 2HA and wild-type plants. DNA methylation analysis of MtEIL1 revealed significant difference between 2HA and wild-type plants. Tiling arrays demonstrated an elevated level of miRNA in 2HA plants that hybridised to the antisense strand of the MtEIL1 gene. AFLP-like methylation profiling revealed more differences in DNA methylation between 2HA and wild-type. Segregation analysis demonstrated the recessive nature of the eil1 phenotype and the dominant nature of the SE trait.
We have demonstrated that EIL1 of Medicago truncatula (MtEIL1) is epigenetically silenced in the 2HA seed line. The possible cause is an elevated level of miRNA that targets its 3’UTR and is also associated with DNA methylation of MtEIL1. Down regulation of MtEIL1 makes it possible to form nodules in the presence of ethylene and affects root growth under normal conditions. Segregation analysis showed no association between MtEIL1 expression and SE in culture but the role and mechanism of ethylene signalling in the process of plant regeneration through SE requires further investigation. The work also suggests that epigenetic changes to a particular gene induced in culture can be fixed in regenerated plants.
2HA seed line; Somatic embryogenesis; Ethylene insensitivity; Nodulation; Root growth; Medicago truncatula; Epigenetics; DNA methylation; miRNA
The induction of plant defenses in response to herbivory is well documented. In addition, many plants prime their anti-herbivore defenses following exposure to environmental cues associated with increased risk of subsequent attack, including induced volatile emissions from herbivore-damaged plant tissues. Recently, we showed in both field and laboratory settings that tall goldenrod plants (Solidago altissima) exposed to the putative sex attractant of a specialist gall-inducing fly (Eurosta solidaginis) experienced less herbivory than unexposed plants. Furthermore, we observed stronger induction of the defense phytohormone jasmonic acid in exposed plants compared to controls. These findings document a novel class of plant-insect interactions mediated by the direct perception, by plants, of insect-derived olfactory cues. However, our previous study did not exclude the possibility that the fly emission (or its residue) might also deter insect feeding via direct effects on the herbivores.
Here we show that the E. solidaginis emission does not (directly) deter herbivore feeding on Cucurbita pepo or Symphyotrichum lateriflorum plants—which have no co-evolutionary relationship with E. solidaginis and thus are not expected to exhibit priming responses to the fly emission. We also document stronger induction of herbivore-induced plant volatiles (HIPV) in S. altissima plants given previous exposure to the fly emission relative to unexposed controls. No similar effect was observed in maize plants (Zea mays), which have no co-evolutionary relationship with E. solidaginis.
Together with our previous findings, these results provide compelling evidence that reduced herbivory on S. altissima plants exposed to the emission of male E. solidaginis reflects an evolved plant response to olfactory cues associated with its specialist herbivore and does not involve direct effects of the fly emission on herbivore feeding behavior. We further discuss mechanisms by which the priming of HIPV responses documented here might contribute to enhanced S. altissima defense against galling.
Solidago altissima; Eurosta solidaginis; Priming; Herbivore-induced plant volatiles
In saffron (Crocus sativus), new corms develop at the base of every shoot developed from the maternal corm, a globular underground storage stem. Since the degree of bud sprouts influences the number and size of new corms, and strigolactones (SLs) suppress growth of pre-formed axillary bud, it was considered appropriate to investigate SL involvement in physiology and molecular biology in saffron. We focused on two of the genes within the SL pathway, CCD7 and CCD8, encoding carotenoid cleavage enzymes required for the production of SLs.
The CsCCD7 and CsCCD8 genes are the first ones isolated and characterized from a non-grass monocotyledonous plant. CsCCD7 and CsCCD8 expression showed some overlapping, although they were not identical. CsCCD8 was highly expressed in quiescent axillary buds and decapitation dramatically reduced its expression levels, suggesting its involvement in the suppression of axillary bud outgrowth. Furthermore, in vitro experiments showed also the involvement of auxin, cytokinin and jasmonic acid on the sprouting of axillary buds from corms in which the apical bud was removed. In addition, CsCCD8 expression, but not CsCCD7, was higher in the newly developed vascular tissue of axillary buds compared to the vascular tissue of the apical bud.
We showed that production and transport of auxin in saffron corms could act synergistically with SLs to arrest the outgrowth of the axillary buds, similar to the control of above-ground shoot branching. In addition, jasmonic acid seems to play a prominent role in bud dormancy in saffron. While cytokinins from roots promote bud outgrowth. In addition the expression results of CsCCD8 suggest that SLs could positively regulate procambial activity and the development of new vascular tissues connecting leaves with the mother corm.
Auxin; Buds; Carotenoid cleavage oxygenases; Corm; Saffron; Strigolactones
Plant GSK-3/Shaggy-like kinases are key players in brassinosteroid (BR) signalling which impact on plant development and participate in response to wounding, pathogens and salt stress. Bikinin was previously identified in a chemical genetics screen as an inhibitor targeting these kinases. To dissect the structural elements crucial for inhibition of GSK-3/Shaggy-like kinases by bikinin and to isolate more potent compounds we synthesised a number of related substances and tested their inhibitory activity in vitro and in vivo using Arabidopsis thaliana.
A pyridine ring with an amido succinic acid residue in position 2 and a halogen in position 5 were crucial for inhibitory activity. The compound with an iodine substituent in position 5, denoted iodobikinin, was most active in inhibiting BIN2 activity in vitro and efficiently induced brassinosteroid-like responses in vivo. Its methyl ester, methyliodobikinin, showed improved cell permeability, making it highly potent in vivo although it had lower activity in vitro. HPLC analysis revealed that the methyl residue was rapidly cleaved off in planta liberating active iodobikinin. In addition, we provide evidence that iodobikinin and bikinin are inactivated in planta by conjugation with glutamic acid or malic acid and that the latter process is catalysed by the malate transferase SNG1.
Brassinosteroids participate in regulation of many aspects of plant development and in responses to environmental cues. Thus compounds modulating their action are valuable tools to study such processes and may be an interesting opportunity to modify plant growth and performance in horticulture and agronomy. Here we report the development of bikinin derivatives with increased potency that can activate BR signalling and mimic BR action. Methyliodobikinin was 3.4 times more active in vivo than bikinin. The main reason for the superior activity of methyliodobikinin, the most potent compound, is its enhanced plant tissue permeability. Inactivation of bikinin and its derivatives in planta involves SNG1, which constitutes a novel pathway for modification of xenobiotic compounds.
Brassinosteroid; GSK-3/shaggy-like kinase; Inhibitor; Protein phosphorylation; Signal transduction
Soybean is one of the most important crops, providing large amounts of dietary proteins and edible oil, and is also an excellent model for studying evolution of duplicated genes. However, relative to the model plants Arabidopsis and rice, the present knowledge about soybean transcriptome is quite limited.
In this study, we employed RNA-seq to investigate transcriptomes of 11 soybean tissues, for genome-wide discovery of truly expressed genes, and novel and alternative transcripts, as well as analyses of conservation and divergence of duplicated genes and their functional implications. We detected a total of 54,132 high-confidence expressed genes, and identified 6,718 novel transcriptional regions with a mean length of 372 bp. We also provided strong evidence for alternative splicing (AS) events for ~15.9% of the genes with two or more exons. Among them, 1,834 genes exhibited stage-dependent AS, and 202 genes had tissue-biased exon-skipping events. We further defined the conservation and divergence in expression patterns between duplicated gene pairs from recent whole genome duplications (WGDs); differentially expressed genes, tissue preferentially expressed genes, transcription factors and specific gene family members were identified for shoot apical meristem and flower development.
Our results significantly improved soybean gene annotation, and also provide valuable resources for functional genomics and studies of the evolution of duplicated genes from WGDs in soybean.
Soybean; RNA-seq; Transcriptome; Novel transcriptional regions; Alternative splicing; Meristem; Transcription factors
In flowering plants a number of genes have been identified which control the transition from a vegetative to generative phase of life cycle. In bryophytes representing basal lineage of land plants, there is little data regarding the mechanisms that control this transition. Two species from bryophytes - moss Physcomitrella patens and liverwort Marchantia polymorpha are under advanced molecular and genetic research. The goal of our study was to identify genes connected to female gametophyte development and archegonia production in the dioecious liverwort Pellia endiviifolia species B, which is representative of the most basal lineage of the simple thalloid liverworts.
The utility of the RDA-cDNA technique allowed us to identify three genes specifically expressed in the female individuals of P.endiviifolia: PenB_CYSP coding for cysteine protease, PenB_MT2 and PenB_MT3 coding for Mysterious Transcripts1 and 2 containing ORFs of 143 and 177 amino acid residues in length, respectively. The exon-intron structure of all three genes has been characterized and pre-mRNA processing was investigated. Interestingly, five mRNA isoforms are produced from the PenB_MT2 gene, which result from alternative splicing within the second and third exon. All observed splicing events take place within the 5′UTR and do not interfere with the coding sequence. All three genes are exclusively expressed in the female individuals, regardless of whether they were cultured in vitro or were collected from a natural habitat. Moreover we observed ten-fold increased transcripts level for all three genes in the archegonial tissue in comparison to the vegetative parts of the same female thalli grown in natural habitat suggesting their connection to archegonia development.
We have identified three genes which are specifically expressed in P.endiviifolia sp B female gametophytes. Moreover, their expression is connected to the female sex-organ differentiation and is developmentally regulated. The contribution of the identified genes may be crucial for successful liverwort sexual reproduction.
Liverwort; Pellia; Archegonia development; Sexual reproduction; Dioecious gametophytes; Gene expression
Leguminous plants are able to form a root nodule symbiosis with nitrogen-fixing soil bacteria called rhizobia. This symbiotic association shows a high level of specificity. Beyond the specificity for the legume family, individual legume species/genotypes can only interact with certain restricted group of bacterial species or strains. Specificity in this system is regulated by complex signal exchange between the two symbiotic partners and thus multiple genetic mechanisms could be involved in the recognition process. Knowledge of the molecular mechanisms controlling symbiotic specificity could enable genetic improvement of legume nitrogen fixation, and may also reveal the possible mechanisms that restrict root nodule symbiosis in non-legumes.
We screened a core collection of Medicago truncatula genotypes with several strains of Sinorhizobium meliloti and identified a naturally occurring dominant gene that restricts nodulation by S. meliloti Rm41. We named this gene as Mt-NS1 (for M.truncatulanodulation specificity 1). We have mapped the Mt-NS1 locus within a small genomic region on M. truncatula chromosome 8. The data reported here will facilitate positional cloning of the Mt-NS1 gene.
Evolution of symbiosis specificity involves both rhizobial and host genes. From the bacterial side, specificity determinants include Nod factors, surface polysaccharides, and secreted proteins. However, we know relatively less from the host side. We recently demonstrated that a component of this specificity in soybeans is defined by plant NBS-LRR resistance (R) genes that recognize effector proteins delivered by the type III secretion system (T3SS) of the rhizobial symbionts. However, the lack of a T3SS in many sequenced S. meliloti strains raises the question of how the specificity is regulated in the Medicago-Sinorhizobium system beyond Nod-factor perception. Thus, cloning and characterization of Mt-NS1 will add a new dimension to our knowledge about the genetic control of nodulation specificity in the legume-rhizobial symbiosis.
Legume; Medicago truncatula; Nodulation specificity; Nitrogen fixation
Drought is a major constraint that leads to extensive losses to agricultural yield worldwide. The potential yield is largely determined during inflorescence development. However, to date, most investigations on plant response to drought have focused on vegetative development. This study describes the morphological changes of reproductive development and the comparison of transcriptomes under various drought conditions.
The plants grown were studied under two drought conditions: minimum for successful reproduction (45-50% soil water content, moderate drought, MD) and for survival (30-35%, severe drought, SD). MD plants can produce similar number of siliques on the main stem and similar number of seeds per silique comparing with well-water plants. The situation of SD plants was much worse than MD plants. The transcriptomes of inflorescences were further investigated at molecular level using microarrays. Our results showed more than four thousands genes with differential expression under severe drought and less than two thousand changed under moderate drought condition (with 2-fold change and q-value < 0.01). We found a group of genes with increased expression as the drought became more severe, suggesting putative adaptation to the dehydration. Interestingly, we also identified genes with alteration only under the moderate but not the severe drought condition, indicating the existence of distinct sets of genes responsive to different levels of water availability. Further cis-element analyses of the putative regulatory sequences provided more information about the underlying mechanisms for reproductive responses to drought, suggesting possible novel candidate genes that protect those developing flowers under drought stress.
Different pathways may be activated in response to moderate and severe drought in reproductive tissues, potentially helping plant to maximize its yield and balance the resource consumption between vegetative and reproductive development under dehydration stresses.
Moderate drought; Severe drought; Reproductive development; Transcriptome; Arabidopsis
The American cranberry (Vaccinium macrocarpon Ait.) is one of only three widely-cultivated fruit crops native to North America- the other two are blueberry (Vaccinium spp.) and native grape (Vitis spp.). In terms of taxonomy, cranberries are in the core Ericales, an order for which genome sequence data are currently lacking. In addition, cranberries produce a host of important polyphenolic secondary compounds, some of which are beneficial to human health. Whereas next-generation sequencing technology is allowing the advancement of whole-genome sequencing, one major obstacle to the successful assembly from short-read sequence data of complex diploid (and higher ploidy) organisms is heterozygosity. Cranberry has the advantage of being diploid (2n = 2x = 24) and self-fertile. To minimize the issue of heterozygosity, we sequenced the genome of a fifth-generation inbred genotype (F ≥ 0.97) derived from five generations of selfing originating from the cultivar Ben Lear.
The genome size of V. macrocarpon has been estimated to be about 470 Mb. Genomic sequences were assembled into 229,745 scaffolds representing 420 Mbp (N50 = 4,237 bp) with 20X average coverage. The number of predicted genes was 36,364 and represents 17.7% of the assembled genome. Of the predicted genes, 30,090 were assigned to candidate genes based on homology. Genes supported by transcriptome data totaled 13,170 (36%).
Shotgun sequencing of the cranberry genome, with an average sequencing coverage of 20X, allowed efficient assembly and gene calling. The candidate genes identified represent a useful collection to further study important biochemical pathways and cellular processes and to use for marker development for breeding and the study of horticultural characteristics, such as disease resistance.
Vaccinium macrocarpon; Ericaceae; Transcriptome; COSII; Polyphenolics; Resistance genes; SSRs; SNPs; Inbred
Grain chalkiness is a complex trait adversely affecting appearance and milling quality, and therefore has been one of principal targets for rice improvement. Eliminating chalkiness from rice has been a daunting task due to the complex interaction between genotype and environment and the lack of molecular markers. In addition, the molecular mechanisms underlying grain chalkiness formation are still imperfectly understood.
We identified a notched-belly mutant (DY1102) with high percentage of white-belly, which only occurs in the bottom part proximal to the embryo. Using this mutant, a novel comparison system that can minimize the effect of genetic background and growing environment was developed. An iTRAQ-based comparative display of the proteins between the bottom chalky part and the upper translucent part of grains of DY1102 was performed. A total of 113 proteins responsible for chalkiness formation was identified. Among them, 70 proteins are up-regulated and 43 down-regulated. Approximately half of these differentially expressed proteins involved in central metabolic or regulatory pathways including carbohydrate metabolism (especially cell wall synthesis) and protein synthesis, folding and degradation, providing proteomic confirmation of the notion that chalkiness formation involves diverse but delicately regulated pathways. Protein metabolism was the most abundant category, accounting for 27.4% of the total differentially expressed proteins. In addition, down regulation of PDIL 2–3 and BiP was detected in the chalky tissue, indicating the important role of protein metabolism in grain chalkiness formation.
Using this novel comparison system, our comprehensive survey of endosperm proteomics in the notched-belly mutant provides a valuable proteomic resource for the characterization of pathways contributing to chalkiness formation at molecular and biochemical levels.
Rice; Grain chalkiness; iTRAQ; White-belly; Notched-belly mutants
Cultivated rice consists of two important ecotypes, upland and irrigated, that have respectively adapted to either dry land or irrigated cultivation. Upland rice, widely adopted in rainfed upland areas in virtue of its little water requirement, contains abundant untapped genetic resources, such as genes for drought adaptation. With water shortage exacerbated and population expanding, the need for breeding crop varieties with drought adaptation becomes more and more urgent. However, a previous oversight in upland rice research reveals little information regarding its genetic mechanisms for upland adaption, greatly hindering progress in harnessing its genetic resources for breeding and cultivation.
In this study, we selected 84 upland and 82 irrigated accessions from all over the world, phenotyped them under both irrigated and dry land environments, and investigated the phylogenetic relations and population structure of the upland ecotype using whole genome variation data. Further comparative analysis yields a list of differentiated genes that may account for the phenotypic and physiological differences between upland and irrigated rice.
This study represents the first genomic investigation in a large sample of upland rice, providing valuable gene list for understanding upland rice adaptation, especially drought-related adaptation, and its subsequent utilization in modern agriculture.
Upland rice; Upland adaptation; Genetic mechanisms; Phylogenetics; Population structure; Artificial selection
Ca2+, a versatile intracellular second messenger in various signaling pathways, initiates many responses involved in growth, defense and tolerance to biotic and abiotic stress. Endogenous and exogenous signals induce cytoplasmic Ca2+ ([Ca2+]cyt) elevation, which are responsible for the appropriate downstream responses.
Here we report on an ethyl-methane sulfonate-mediated Arabidopsis mutant that fails to induce [Ca2+]cyt elevation in response to exudate preparations from the pathogenic mibrobes Alternaria brassicae, Rhizoctonia solani, Phytophthora parasitica var. nicotianae and Agrobacterium tumefaciens. The
cytoplasmic Ca2+elevation mutant1 (cycam1) is susceptible to infections by A. brassicae, its toxin preparation and sensitive to abiotic stress such as drought and salt. It accumulates high levels of reactive oxygen species and contains elevated salicylic acid, abscisic acid and bioactive jasmonic acid iso-leucine levels. Reactive oxygen species- and phytohormone-related genes are higher in A. brassicae-treated wild-type and mutant seedlings. Depending on the analysed response, the elevated levels of defense-related compounds are either caused by the cycam mutation and are promoted by the pathogen, or they are mainly due to the pathogen infection or application of pathogen-associated molecular patterns. Furthermore, cycam1 shows altered responses to abscisic acid treatments: the hormone inhibits germination and growth of the mutant.
We isolated an Arabidopsis mutant which fails to induce [Ca2+]cyt elevation in response to exudate preparations from various microbes. The higher susceptibility of the mutant to pathogen infections correlates with the higher accumulation of defense-related compounds, such as phytohormones, reactive oxygen-species, defense-related mRNA levels and secondary metabolites. Therefore, CYCAM1 couples [Ca2+]cyt elevation to biotic, abiotic and oxidative stress responses.
Abiotic stress; Biotic stress; A. brassicae; Camalexin; Cytosolic calcium elevation; Glucosinolates; Phytohormones
A member of the TaHKT2;1 multigene family was previously identified as a Na+ transporter with a possible role in root Na+ uptake. In the present study, the existing full-length cDNA of this member was used as a basis to query the International Wheat Genome Survey Sequence to identify all members of the TaHKT2;1 family. Individual TaHKT2;1 genes were subsequently studied for gene and predicted protein structures, promoter variability, tissue expression and their role in Na+ and K+ status of wheat.
Six TaHKT2;1 genes were characterized which included four functional genes (TaHKT2;1 7AL-1, TaHKT2;1 7BL-1, TaHKT2;1 7BL-2 and TaHKT2;1 7DL-1) and two pseudogenes (TaHKT2;1 7AL-2 and TaHKT2;1 7AL-3), on chromosomes 7A, 7B and 7D of hexaploid wheat. Variability in protein domains for cation specificity and in cis-regulatory elements for salt response in gene promoters, were identified amongst the functional TaHKT2;1 members. The functional genes were expressed under low and high NaCl conditions in roots and leaf sheaths, but were down regulated in leaf blades. Alternative splicing events were evident in TaHKT2;1 7AL-1. Aneuploid lines null for each functional gene were grown in high NaCl nutrient solution culture to identify potential role of each TaHKT2;1 member. Aneuploid lines null for TaHKT2;1 7AL-1, TaHKT2;1 7BL-1 and TaHKT2;1 7BL-2 showed no difference in Na+ concentration between Chinese Spring except for higher Na+ in sheaths. The same aneuploid lines had lower K+ in roots, sheath and youngest fully expanded leaf but only under high (200 mM) NaCl in the external solution. There was no difference in Na+ or K+ concentration for any treatment between aneuploid line null for the TaHKT2;1 7DL-1 gene and Chinese Spring.
TaHKT2;1 is a complex family consisting of pseudogenes and functional members. TaHKT2;1 genes do not have an apparent role in controlling root Na+ uptake in bread wheat seedlings under experimental conditions in this study, contrary to existing hypotheses. However, TaHKT2;1 genes or, indeed other genes in the same chromosome region on 7AL, are candidates that may control Na+ transport from root to sheath and regulate K+ levels in different plant tissues.
IWGSS; Cis regulatory elements; Gene expression; Aneuploid lines; Tissue Na+; Tissue K+
The production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b.
TcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae.
To our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively.
Nucleases; Gene expression; ROS production; Molecular modeling
The shape of grass leaves possesses great value in both agronomy and developmental biology research. Leaf rolling is one of the important traits in rice (Oryza sativa L.) breeding. MYB transcription factors are one of the largest gene families and have important roles in plant development, metabolism and stress responses. However, little is known about their functions in rice.
In this study, we report the functional characterization of a rice gene, OsMYB103L, which encodes an R2R3-MYB transcription factor. OsMYB103L was localized in the nucleus with transactivation activity. Overexpression of OsMYB103L in rice resulted in a rolled leaf phenotype. Further analyses showed that expression levels of several cellulose synthase genes (CESAs) were significantly increased, as was the cellulose content in OsMYB103L overexpressing lines. Knockdown of OsMYB103L by RNA interference led to a decreased level of cellulose content and reduced mechanical strength in leaves. Meanwhile, the expression levels of several CESA genes were decreased in these knockdown lines.
These findings suggest that OsMYB103L may target CESA genes for regulation of cellulose synthesis and could potentially be engineered for desirable leaf shape and mechanical strength in rice.
OsMYB103L; Leaf rolling; MYB transcription factor; Cellulose
TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome.
We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast one-hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato.
The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development.
Transcription factors; Tomato; Yeast one-hybrid; Yeast two-hybrid
Reactive oxygen species (ROS) are used by plants as signaling molecules during stress and development. Given the amount of possible challenges a plant face from their environment, plants need to activate and prioritize between potentially conflicting defense signaling pathways. Until recently, most studies on signal interactions have focused on phytohormone interaction, such as the antagonistic relationship between salicylic acid (SA)-jasmonic acid and cytokinin-auxin.
In this study, we report an antagonistic interaction between SA signaling and apoplastic ROS signaling. Treatment with ozone (O3) leads to a ROS burst in the apoplast and induces extensive changes in gene expression and elevation of defense hormones. However, Arabidopsis thaliana dnd1 (defense no death1) exhibited an attenuated response to O3. In addition, the dnd1 mutant displayed constitutive expression of defense genes and spontaneous cell death. To determine the exact process which blocks the apoplastic ROS signaling, double and triple mutants involved in various signaling pathway were generated in dnd1 background. Simultaneous elimination of SA-dependent and SA-independent signaling components from dnd1 restored its responsiveness to O3. Conversely, pre-treatment of plants with SA or using mutants that constitutively activate SA signaling led to an attenuation of changes in gene expression elicited by O3.
Based upon these findings, we conclude that plants are able to prioritize the response between ROS and SA via an antagonistic action of SA and SA signaling on apoplastic ROS signaling.
Cell death; Ethylene; Gene expression; Jasmonic acid; Reactive oxygen species; Salicylic acid
Maize (Zea Mays L.) is one of the most important cereal crops worldwide and provides food for billions of people. Stalk lodging can greatly undermine the standability of maize plants and therefore decrease crop yields. Rind penetrometer resistance is an effective and reliable method for evaluating maize stalk strength, which is highly correlated with stalk lodging resistance. In this study, two recombinant inbred line populations were constructed from crosses between the H127R and Chang7-2 lines, and between the B73 and By804 lines. We genotyped these two populations and their parents using 3,072 single nucleotide polymorphism markers and performed phenotypic assessment of rind penetrometer resistance in multiple environments to dissect the genetic architecture of rind penetrometer resistance in maize.
Based on two linkage maps of 1,397.1 and 1,600.4 cM with average interval of 1.7 and 2.1 cM between adjacent makers, respectively, seven quantitative trait loci (QTL) for rind penetrometer resistance were detected in the two recombinant inbred line populations. These QTL were distributed in seven genomic regions, and each accounted for 4.4–18.9% of the rind penetrometer resistance variation. The QTL with the largest effect on rind penetrometer resistance, qRPR3-1, was located on chromosome 3 with the flanking markers PZE-103123325 and SYN23245. This locus was further narrowed down to a 3.1-Mb interval by haplotype analysis using high-density markers in the target region. Within this interval, four genes associated with the biosynthesis of cell wall components were considered as potential candidate genes for the rind penetrometer resistance effect.
The inheritance of rind penetrometer resistance is rather complex. A few large-effect quantitative trait loci, together with a several minor-effect QTL, contributed to the phenotypic variation in rind penetrometer resistance in the two recombinant inbred line populations that were examined. A potential approach for improving stalk strength and crop yields in commercial maize lines may be to introgress favorable alleles of the locus that was found to have the largest effect on rind penetrometer resistance (qRPR3-1).
Maize; Rind penetrometer resistance; QTL; SNP
Soybean mosaic virus (SMV) is the most prevalent viral disease in many soybean production areas. Due to a large number of SMV resistant loci and alleles, SMV strains and the rapid evolution in avirulence/effector genes, traditional breeding for SMV resistance is complex. Genetic engineering is an effective alternative method for improving SMV resistance in soybean. Potassium (K+) is the most abundant inorganic solute in plant cells, and is involved in plant responses to abiotic and biotic stresses. Studies have shown that altering the level of K+ status can reduce the spread of the viral diseases. Thus K+ transporters are putative candidates to target for soybean virus resistance.
The addition of K+ fertilizer significantly reduced SMV incidence. Analysis of K+ channel gene expression indicated that GmAKT2, the ortholog of Arabidopsis K+ weak channel encoding gene AKT2, was significantly induced by SMV inoculation in the SMV highly-resistant genotype Rsmv1, but not in the susceptible genotype Ssmv1. Transgenic soybean plants overexpressing GmAKT2 were produced and verified by Southern blot and RT-PCR analysis. Analysis of K+ concentrations on different leaves of both the transgenic and the wildtype (Williams 82) plants revealed that overexpression of GmAKT2 significantly increased K+ concentrations in young leaves of plants. In contrast, K+ concentrations in the old leaves of the GmAKT2-Oe plants were significantly lower than those in WT plants. These results indicated that GmAKT2 acted as a K+ transporter and affected the distribution of K+ in soybean plants. Starting from 14 days after inoculation (DAI) of SMV G7, severe mosaic symptoms were observed on the WT leaves. In contrast, the GmAKT2-Oe plants showed no symptom of SMV infection. At 14 and 28 DAI, the amount of SMV RNA in WT plants increased 200- and 260- fold relative to GmAKT2-Oe plants at each time point. Thus, SMV development was significantly retarded in GmAKT2-overexpressing transgenic soybean plants.
Overexpression of GmAKT2 significantly enhanced SMV resistance in transgenic soybean. Thus, alteration of K+ transporter expression is a novel molecular approach for enhancing SMV resistance in soybean.
Soybean mosaic virus; Resistance; Potassium channel; GmAKT2
Recurrent gene duplication and retention played an important role in angiosperm genome evolution. It has been hypothesized that these processes contribute significantly to plant adaptation but so far this hypothesis has not been tested at the genome scale.
We studied available sequenced angiosperm genomes to assess the frequency of positive selection footprints in lineage specific expanded (LSE) gene families compared to single-copy genes using a dN/dS-based test in a phylogenetic framework. We found 5.38% of alignments in LSE genes with codons under positive selection. In contrast, we found no evidence for codons under positive selection in the single-copy reference set. An analysis at the branch level shows that purifying selection acted more strongly on single-copy genes than on LSE gene clusters. Moreover we detect significantly more branches indicating evolution under positive selection and/or relaxed constraint in LSE genes than in single-copy genes.
In this – to our knowledge –first genome-scale study we provide strong empirical support for the hypothesis that LSE genes fuel adaptation in angiosperms. Our conservative approach for detecting selection footprints as well as our results can be of interest for further studies on (plant) gene family evolution.
Lineage specific expansion (LSE); Gene duplication; Gene retention; Ultraparalogs (UP); Superorthologs (SO); Comparative genomics; Positive selection; Adaptation
Arabidopsis AtHB7 and AtHB12 transcription factors (TFs) belong to the homeodomain-leucine zipper subfamily I (HD-Zip I) and present 62% amino acid identity. These TFs have been associated with the control of plant development and abiotic stress responses; however, at present it is not completely understood how AtHB7 and AtHB12 regulate these processes.
By using different expression analysis approaches, we found that AtHB12 is expressed at higher levels during early Arabidopsis thaliana development whereas AtHB7 during later developmental stages. Moreover, by analysing gene expression in single and double Arabidopsis mutants and in transgenic plants ectopically expressing these TFs, we discovered a complex mechanism dependent on the plant developmental stage and in which AtHB7 and AtHB12 affect the expression of each other. Phenotypic analysis of transgenic plants revealed that AtHB12 induces root elongation and leaf development in young plants under standard growth conditions, and seed production in water-stressed plants. In contrast, AtHB7 promotes leaf development, chlorophyll levels and photosynthesis and reduces stomatal conductance in mature plants. Moreover AtHB7 delays senescence processes in standard growth conditions.
We demonstrate that AtHB7 and AtHB12 have overlapping yet specific roles in several processes related to development and water stress responses. The analysis of mutant and transgenic plants indicated that the expression of AtHB7 and AtHB12 is regulated in a coordinated manner, depending on the plant developmental stage and the environmental conditions. The results suggested that AtHB7 and AtHB12 evolved divergently to fine tune processes associated with development and responses to mild water stress.
AtHB7; AtHB12; Homeodomain-leucine zipper (HD-Zip I); Moderate water stress; Yield; Plant growth
Plant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s.
We performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators have the peroxisomal C441-type, suggesting some limitations in the peroxisomal GB synthesis.
Our findings shed light on the evolution of the synthesis of GB in plants, a metabolic trait of most ecological and physiological relevance for their tolerance to drought, hypersaline soils and cold. Together, our results are consistent with smooth evolutionary pathways for the acquisition of the BADH function from ancestral I441-type AMADHs, thus explaining the relatively high occurrence of this event.
Osmoprotection; Osmotic stress; Aminoaldehyde dehydrogenase; Enzyme kinetics; Substrate specificity; Enzyme subcellular location; Protein stability; Protein structure; Protein evolution
Sorghum genotypes used for grain production in temperate regions are photoperiod insensitive and flower early avoiding adverse environments during the reproductive phase. In contrast, energy sorghum hybrids are highly photoperiod sensitive with extended vegetative phases in long days, resulting in enhanced biomass accumulation. SbPRR37 and SbGHD7 contribute to photoperiod sensitivity in sorghum by repressing expression of SbEHD1 and FT-like genes, thereby delaying flowering in long days with minimal influence in short days (PNAS_108:16469-16474, 2011; Plant Genome_in press, 2014). The GIGANTEA (GI)-CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway regulates flowering time in Arabidopsis and the grasses (J Exp Bot_62:2453-2463, 2011). In long day flowering plants, such as Arabidopsis and barley, CONSTANS activates FT expression and flowering in long days. In rice, a short day flowering plant, Hd1, the ortholog of CONSTANS, activates flowering in short days and represses flowering in long days.
Quantitative trait loci (QTL) that modify flowering time in sorghum were identified by screening Recombinant Inbred Lines (RILs) derived from BTx642 and Tx7000 in long days, short days, and under field conditions. Analysis of the flowering time QTL on SBI-10 revealed that BTx642 encodes a recessive CONSTANS allele containing a His106Tyr substitution in B-box 2 known to inactivate CONSTANS in Arabidopsis thaliana. Genetic analysis characterized sorghum CONSTANS as a floral activator that promotes flowering by inducing the expression of EARLY HEADING DATE 1 (SbEHD1) and sorghum orthologs of the maize FT genes ZCN8 (SbCN8) and ZCN12 (SbCN12). The floral repressor PSEUDORESPONSE REGULATOR PROTEIN 37 (PRR37) inhibits sorghum CONSTANS activity and flowering in long days.
Sorghum CONSTANS is an activator of flowering that is repressed post-transcriptionally in long days by the floral inhibitor PRR37, contributing to photoperiod sensitive flowering in Sorghum bicolor, a short day plant.
Photoperiod; Sorghum; Flowering time; QTL; CONSTANS; PRR37