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Eukaryotic genes can be classified into intronless (no introns), intron-poor (three or fewer introns per gene) or intron-rich. Early eukaryotic genes were mostly intron-rich, and their alternative splicing into multiple transcripts, giving rise to different proteins, might have played pivotal roles in adaptation and evolution. Interestingly, extant plant genomes contain many gene families with one or sometimes few sub-families with genes that are intron-poor or intronless, and it remains unknown when and how these intron-poor or intronless genes have originated and evolved, and what their possible functions are. In this study, we identified 33 such gene families that contained intronless and intron-poor sub-families. Intronless genes seemed to have first emerged in early land plant evolution, while intron-poor sub-families seemed first to have appeared in green algae. In contrast to intron-rich genes, intronless genes in intron-poor sub-families occurred later, and were subject to stronger functional constraints. Based on RNA-seq analyses in Arabidopsis and rice, intronless or intron-poor genes in AP2, EF-hand_7, bZIP, FAD_binding_4, STE_STE11, CAMK_CAMKL-CHK1 and C2 gene families were more likely to play a role in response to drought and salt stress, compared with intron-rich genes in the same gene families, whereas intronless genes in the B_lectin and S_locus_glycop gene family were more likely to participate in epigenetic processes and plant development. Understanding the origin and evolutionary trajectory, as well as the potential functions, of intronless and intron-poor sub-families provides further insight into plant genome evolution and the functional divergence of genes.

Background The mitogen-activated protein kinases (MAPKs), as a part of the MAPKKK-MAPKK-MAPK cascade, play crucial roles in plant development as an intracellular signal transduction pathway to respond various environmental signals. However, few MAPKK have been functionally characterized in grapevine. Results In the study, five MAPKK (MKK) members were identified in grapevine (cultivar ‘Pinot Noir’), cloned and designated as VvMKK1-VvMKK5. A phylogenetic analysis grouped them into four sub-families based on the similarity of their conserved motifs and gene structure to Arabidopsis MAPKK members. qRT-PCR results indicated that the expression of VvMKK1, VvMKK2, VvMKK4, and VvMKK5 were up-regulated in mature leaf and young blades, and roots, but exhibited low expression in leaf petioles. VvMKK2, VvMKK3, and VvMKK5 genes were differentially up-regulated when grapevine leaves were inoculated with spores of Erisyphe necator, or treated with salicylic acid (SA), ethylene (ETH), H2O2, or exposed to drought, indicating that these genes may be involved in a variety of signaling pathways. Over expression of VvMKK2 and VvMKK4 genes in transgenic Arabidopsis plants resulted in the production of seeds with a significantly higher germination and survival rate, and better seedling growth under stress conditions than wild-type plants. Overexpression of VvMKK2 in Arabidopsis improved salt and drought stress tolerance while overexpression of VvMKK4 only improved salt stress tolerance. Conclusions Results of the present investigation provide a better understanding of the interaction and function of MAPKKK-MAPKK-MAPK genes at the transcriptional level in grapevine and led to the identification of candidate genes for drought and salt stress in grapes.

Pathogen invasion leads to fast, local-to-systemic signal transduction that initiates plant defense responses. Despite tremendous progress in past decades, aspects of this process remain unknown, such as which cell types respond first and how signals are transferred among cell types. Here, we used single-cell RNA-seq of >50 000 single cells to document the gene expression landscape in leaves of woodland strawberry during infection by Botrytis cinerea and identify major cell types. We constructed a single-cell atlas and characterized the distinct gene expression patterns of hydathode, epidermal, and mesophyll cells during the incubation period of B. cinerea infection. Pseudotime trajectory analysis revealed signals of the transition from normal functioning to defense response in epidermal and mesophyll cells upon B. cinerea infection. Genes related to disease resistance showed different expression patterns among cell types: disease resistance-related genes and genes encoding transcription factors were highly expressed in individual cell types and interacted to trigger plant systemic immunity to B. cinerea. This is the first report to document the single-cell transcriptional landscape of the plant pathogenic invasion process; it provides new insights into the holistic dynamics of host–pathogen interactions and can guide the identification of genes and the formulation of strategies for resistant cultivar development.

Arbuscular mycorrhizal symbiosis (AMS) is widespread mutualistic association between plants and fungi, which plays an essential role in nutrient exchange, enhancement in plant stress resistance, development of host, and ecosystem sustainability. Previous studies have shown that plant small secreted proteins (SSPs) are involved in beneficial symbiotic interactions. However, the role of SSPs in the evolution of AMS has not been well studied yet. In this study, we performed computational analysis of SSPs in 60 plant species and identified three AMS-specific ortholog groups containing SSPs only from at least 30% of the AMS species in this study and three AMS-preferential ortholog groups containing SSPs from both AMS and non-AMS species, with AMS species containing significantly more SSPs than non-AMS species. We found that independent lineages of monocot and eudicot plants contained genes in the AMS-specific ortholog groups and had significant expansion in the AMS-preferential ortholog groups. Also, two AMS-preferential ortholog groups showed convergent changes, between monocot and eudicot species, in gene expression in response to arbuscular mycorrhizal fungus Rhizophagus irregularis. Furthermore, conserved cis-elements were identified in the promoter regions of the genes showing convergent gene expression. We found that the SSPs, and their closely related homologs, in each of three AMS-preferential ortholog groups, had some local variations in the protein structural alignment. We also identified genes co-expressed with the Populus trichocarpa SSP genes in the AMS-preferential ortholog groups. This first plant kingdom-wide analysis on SSP provides insights on plant-AMS convergent evolution with specific SSP gene expression and local diversification of protein structures.

Small secreted proteins (SSPs) are less than 250 amino acids in length and are actively transported out of cells through conventional protein secretion pathways or unconventional protein secretion pathways. In plants, SSPs have been found to play important roles in various processes, including plant growth and development, plant response to abiotic and biotic stresses, and beneficial plant–microbe interactions. Over the past 10 years, substantial progress has been made in the identification and functional characterization of SSPs in several plant species relevant to agriculture, bioenergy, and horticulture. Yet, there are potentially a lot of SSPs that have not been discovered in plant genomes, which is largely due to limitations of existing computational algorithms. Recent advances in genomics, transcriptomics, and proteomics research, as well as the development of new computational algorithms based on machine learning, provide unprecedented capabilities for genome-wide discovery of novel SSPs in plants. In this review, we summarize known SSPs and their functions in various plant species. Then we provide an update on the computational and experimental approaches that can be used to discover new SSPs. Finally, we discuss strategies for elucidating the biological functions of SSPs in plants.

Rosa rugosa, commonly known as rugged rose, is a perennial ornamental shrub. It produces beautiful flowers with a mild fragrance and colorful seed pods. Unlike many other cultivated roses, R. rugosa adapts to a wide range of habitat types and harsh environmental conditions such as salinity, alkaline, shade, drought, high humidity, and frigid temperatures. Here, we produced and analyzed a high-quality genome sequence for R. rugosa to understand its ecology, floral characteristics and evolution. PacBio HiFi reads were initially used to construct the draft genome of R. rugosa, and then Hi-C sequencing was applied to assemble the contigs into 7 chromosomes. We obtained a 382.6 Mb genome encoding 39,704 protein-coding genes. The genome of R. rugosa appears to be conserved with no additional whole-genome duplication after the gamma whole-genome triplication (WGT), which occurred ~100 million years ago in the ancestor of core eudicots. Based on a comparative analysis of the high-quality genome assembly of R. rugosa and other high-quality Rosaceae genomes, we found a unique large inverted segment in the Chinese rose R. chinensis and a retroposition in strawberry caused by post-WGT events. We also found that floral development- and stress response signaling-related gene modules were retained after the WGT. Two MADS-box genes involved in floral development and the stress-related transcription factors DREB2A-INTERACTING PROTEIN 2 (DRIP2) and PEPTIDE TRANSPORTER 3 (PTR3) were found to be positively selected in evolution, which may have contributed to the unique ability of this plant to adapt to harsh environments. In summary, the high-quality genome sequence of R. rugosa provides a map for genetic studies and molecular breeding of this plant and enables comparative genomic studies of Rosa in the near future.

1-Methylcyclopropene (1-MCP) is an inhibitor of ethylene perception that is widely used to maintain the quality of several climacteric fruits during storage. A large body of literature now exists on the effects of 1-MCP on climacteric fruit ripening for different species and environmental conditions, presenting an opportunity to use meta-analysis to systematically dissect these effects. We classified 44 ripening indicators of climacteric fruits into five categories: physiology and biochemistry, quality, enzyme activity, color, and volatiles. Meta-analysis showed that 1-MCP treatment reduced 20 of the 44 indicators by a minimum of 22% and increased 6 indicators by at least 20%. These effects were associated with positive effects on delaying ripening and maintaining quality. Of the seven moderating variables, species, 1-MCP concentration, storage temperature and time had substantial impacts on the responses of fruit to 1-MCP treatment. Fruits from different species varied in their responses to 1-MCP, with the most pronounced responses observed in rosaceous fruits, especially apple, European pear fruits, and tropical fruits. The effect of gaseous 1-MCP was optimal at 1 μl/l, with a treatment time of 12–24 h, when the storage temperature was 0 °C for temperate fruits or 20 °C for tropical fruits, and when the shelf temperature was 20 °C, reflecting the majority of experimental approaches. These findings will help improve the efficacy of 1-MCP application during the storage of climacteric fruits, reduce fruit quality losses and increase commercial value.

Background Crassulacean acid metabolism (CAM) plants use water 20–80% more efficiently by shifting stomata opening and primary CO2 uptake and fixation to the nighttime. Protein kinases (PKs) play pivotal roles in this biological process. However, few PKs have been functionally analyzed precisely due to their abundance and potential functional redundancy (caused by numerous gene duplications). Results In this study, we systematically identified a total of 758 predicted PK genes in the genome of a CAM plant, pineapple (Ananas comosus). The pineapple kinome was classified into 20 groups and 116 families based on the kinase domain sequences. The RLK was the largest group, containing 480 members, and over half of them were predicted to locate at the plasma membrane. Both segmental and tandem duplications make important contributions to the expansion of pineapple kinome based on the synteny analysis. Ka/Ks ratios showed all of the duplication events were under purifying selection. The global expression analysis revealed that pineapple PKs exhibit different tissue-specific and diurnal expression patterns. Forty PK genes in a cluster performed higher expression levels in green leaf tip than in white leaf base, and fourteen of them had strong differential expression patterns between the photosynthetic green leaf tip and the non-photosynthetic white leaf base tissues. Conclusions Our findings provide insights into the evolution and biological function of pineapple PKs and a foundation for further functional analysis of PKs in CAM plants. The gene duplication, expression, and coexpression analysis helped us to rapidly identify the key candidates in pineapple kinome, which may play roles in the carbon fixation process in pineapple and help engineering CAM pathway into C3 crops for improved drought tolerance.

Protein kinases (PKs) have evolved as the largest family of molecular switches that regulate protein activities associated with almost all essential cellular functions. Only a fraction of plant PKs, however, have been functionally characterized even in model plant species. In the present study, the entire grapevine kinome was identified and annotated using the most recent version of the grapevine genome. A total of 1168 PK-encoding genes were identified and classified into 20 groups and 121 families, with the RLK-Pelle group being the largest, with 872 members. The 1168 kinase genes were unevenly distributed over all 19 chromosomes, and both tandem and segmental duplications contributed to the expansion of the grapevine kinome, especially of the RLK-Pelle group. Ka/Ks values indicated that most of the tandem and segmental duplication events were under purifying selection. The grapevine kinome families exhibited different expression patterns during plant development and in response to various stress treatments, with many being coexpressed. The comprehensive annotation of grapevine kinase genes, their patterns of expression and coexpression, and the related information facilitate a more complete understanding of the roles of various grapevine kinases in growth and development, responses to abiotic stress, and evolutionary history., Genetics: Mapping molecular switches in grape A comprehensive map of all the protein kinases (PKs) in grape is now available. PKs switch other proteins on or off by adding or removing a phosphate group. They regulate many cellular processes, coordinate plant growth, and trigger responses to various stresses. However, the number, relationships, and functions of PKs remain uncharacterized in most plants. Zong-Ming Cheng at Nanjing Agricultural University and the University of Tennessee mapped all the PKs in grape. Using the most up-to-date version of the genome, Cheng and coworkers identified over 1000 PKs, traced their evolutionary relationships, and identified which PKs are active in key growth phases. They also identified many PKs involved in response to stresses such as heat and drought. These results may help to improve stress tolerance in one of the world’s most economically important fruits.

Background Protein kinases (PKs) play an important role in signaling cascades and are one of the largest and most conserved protein super families in plants. Despite their importance, the woodland strawberry (Fragaria vesca) kinome and expression patterns of PK genes remain to be characterized. Results Here, we report on the identification and classification of 954 Fragaria vesca PK genes, which were classified into nine groups and 124 gene families. These genes were distributed unevenly among the seven chromosomes, and the number of introns per gene varied from 0 to 47. Almost half of the putative PKs were predicted to localize to the nucleus and 24.6% were predicted to localize to the cell membrane. The expansion of the woodland strawberry PK gene family occurred via different duplication mechanisms and tandem duplicates occurred relatively late as compared to other duplication types. Moreover, we found that tandem and transposed duplicated PK gene pairs had undergone stronger diversifying selection and evolved relatively faster than WGD genes. The GO enrichment and transcriptome analysis implicates the involvement of strawberry PK genes in multiple biological processes and molecular functions in differential tissues, especially in pollens. Finally, 109 PKs, mostly the receptor-like kinases (RLKs), were found transcriptionally responsive to Botrytis cinerea infection. Conclusions The findings of this research expand the understanding of the evolutionary dynamics of PK genes in plant species and provide a potential link between cell signaling pathways and pathogen attack.

Additional files 5 : Table S2. The number of MAPKK gene family members in different plant species.

Additional file 19: Figure S12. A heatmap of the expression data of the strawberry kinase genes in cluster 2 response to gray mold.

Additional files 6 : Table S3. The primer sequences used to clone the MAPKK genes in grapevine by PCR.

Additional files 7 : Table S4. The primer sequences used for 3′ RACE of the MAPKK genes in grapevine.

Additional file 20: Figure S13. A heatmap of the expression data of the strawberry kinase genes in cluster 3 response to gray mold.

Additional files 9 : Table S6. The primer sequences used to insert VvMKK2 and VvMKK4 genes into transformation vectors.

Additional file 1 : Table S1. The nucleic acid sequence of cloned VvMKK genes.

Additional files 8 : Table S5. The primer sequences of the MAPKK genes in grapevine for quantitative RT-PCR.

Additional file 9: Figure S2. A heatmap of the expression data of strawberry kinase genes in cluster 1 in 16 different strawberry tissues and developmental stages.

Background Adventitious root (AR) formation is a critical developmental process in cutting propagation for the horticultural industry. While auxin has been shown to regulate this process, the exact mechanism and details preceding AR formation remain unclear. Even though AR and lateral root (LR) formation share common developmental processes, there are exist some differences that need to be closely examined at the cytological level. Tomato stem cuttings, which readily form adventitious roots, represent the perfect system to study the influence of auxin on AR formation and to compare AR and LR organogenesis. Results Here we show the progression by which AR form from founder cells in the basal pericycle cell layers in tomato stem cuttings. The first disordered clumps of cells assumed a dome shape that later differentiated into functional AR cell layers. Further growth resulted in emergence of mature AR through the epidermis following programmed cell death of epidermal cells. Auxin and ethylene levels increased in the basal stem cutting within 1 h. Tomato lines expressing the auxin response element DR5pro:YFP showed an increase in auxin distribution during the AR initiation phase, and was mainly concentrated in the meristematic cells of the developing AR. Treatment of stem cuttings with auxin, increased the number of AR primordia and the length of AR, while stem cuttings treated with the pre-emergent herbicide/auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) occasionally developed thick, agravitropic AR. Hormone profile analyses showed that auxin positively regulated AR formation, whereas perturbations to zeatin, salicylic acid, and abscisic acid homeostasis suggested minor roles during tomato stem rooting. The gene expression of specific auxin transporters increased during specific developmental phases of AR formation. Conclusion These data show that AR formation in tomato stems is a complex process. Upon perception of a wounding stimulus, expression of auxin transporter genes and accumulation of auxin at founder cell initiation sites in pericycle cell layers and later in the meristematic cells of the AR primordia were observed. A clear understanding and documentation of these events in tomato is critical to resolve AR formation in recalcitrant species like hardwoods and improve stem cutting propagation efficiency and effectiveness.

Background: The mitogen-activated protein kinase (MAPK) cascade plays an important role in plant development and stress response. In particular, MAPK kinases (MAPKKs/MKKs) have been reported to play a crucial role in MAPK cascades that mediate a variety of stress responses in plants. Few MKKs in grapevine (Vitis vinifera), however, have been functionally characterized.Results: In the present study, five MKK members in grapevine (‘Pinot Noir’) were identified, cloned and designated as VvMKK1-VvMKK5. A phylogenetic analysis grouped these into four sub-families based on the similarity of their conserved motifs and gene structure to other Arabidopsis MKK members. RT-qPCR results indicated that the expression of VvMKK1, VvMKK2, VvMKK4, and VvMKK5 was up-regulated in mature leaves, young leaves, and roots, but exhibited low expression levels in leaf petioles. VvMKK2, VvMKK3, and VvMKK5 genes were differentially up-regulated when grapevine leaves were inoculated with spores of Erisyphe necator, or treated with SA, ETH, H2O2, or exposed to drought, indicating that these genes may be involved in a variety of signaling pathways. Over expression of VvMKK2 and VvMKK4 genes in transgenic Arabidopsis plants resulted in the production of seeds with a significantly higher germination and survival rate, and better seedling growth under stress conditions than wild-type plants. Overexpression of VvMKK2 in Arabidopsis resulted in improved salt and drought stress tolerance while overexpression of VvMKK4 improved salt stress tolerance.Conclusions: Results of the present investigation provide a better understanding of the interaction and function of MAPKKK-MAPKK-MAPK genes at the transcriptional level in grapevine and led to the identification of candidate genes for improved drought and salt stress in grapes.

Summary Cation/proton antiporter 1 (CPA1) genes encode cellular Na+/H+ exchanger proteins, which act to adjust ionic balance. Overexpression of CPA1s can improve plant performance under salt stress. However, the diversified roles of the CPA1 family and the various parameters used in evaluating transgenic plants over‐expressing CPA1s make it challenging to assess the complex functions of CPA1s and their physiological mechanisms in salt tolerance. Using meta‐analysis, we determined how overexpression of CPA1s has influenced several plant characteristics involved in response and resilience to NaCl stress. We also evaluated experimental variables that favour or reduce CPA1 effects in transgenic plants. Viewed across studies, overexpression of CPA1s has increased the magnitude of 10 of the 19 plant characteristics examined, by 25% or more. Among the ten moderating variables, several had substantial impacts on the extent of CPA1 influence: type of culture media, donor and recipient type and genus, and gene family. Genes from monocotyledonous plants stimulated root K+, root K+/Na+, total chlorophyll, total dry weight and root length much more than genes from dicotyledonous species. Genes transformed to or from Arabidopsis have led to smaller CPA1‐induced increases in plant characteristics than genes transferred to or from other genera. Heterogeneous expression of CPA1s led to greater increases in leaf chlorophyll and root length than homologous expression. These findings should help guide future investigations into the function of CPA1s in plant salt tolerance and the use of genetic engineering for breeding of resistance.

Background and Aims Protein kinases are widely involved in the response in plants to external environment stress signals. Sucrose non-fermenting 1-related protein kinase 2 (SnRK2) is an important class of plant-specific protein kinases that plays a crucial role in abscisic acid responses and abiotic stresses across some model plant species. Genome-wide annotation, however, and specific expression patterns of SnRK2s in grapevine remain unclear. Methods and Results From a genome-wide survey of grapevine, we identified eight VviSnRK2 genes through phylogenetic and local BLAST methods and various bioinformatics analyses. Phylogenetic analysis indicated that these VviSnRK2s could be classified into three subgroups as found in Arabidopsis. Comparison of the structure of the genes with those of Arabidopsis suggests that intron length may be a special evolutionary imprint of the SnRK2 gene family in grapevine. Sequence alignment and structural annotation provided an overview of variations that may contribute to functional divergence from Arabidopsis SnRK2s. Expressional analyses based on public microarray data and quantitative real-time polymerase chain reaction and co-expression network analyses suggest both conserved and variant biological functions exist in SnRK2s across different species. Conclusions Our thorough analysis of the SnRK2 gene family in the grapevine genome revealed functional conservation and diversity synchronously existed in grapevine SnRK2 gene family. Significance of the Study Our research provides a comprehensive overview of both the expression pattern and functional diversity of the grapevine SnRK2 gene family, which will facilitate the future functional characterisation of individual VviSnRK2 genes.

DNA methylation plays important roles in genome protection and the regulation of gene expression and it is associated with plants’ responses to environments. DNA demethylases are very important proteins in DNA methylation regulation. In this study, we performed genome-wide and deep analysis of putative demethylases (DMEs) in pear. Seven DME genes were found in the pear genome and were defined as PbDME1–7 based on their domain organization. Results were supported by the gene structural characteristics and phylogenetic analysis. The gene structure of the DME genes were relatively complex and the DME7 proteins didn’t contain the Perm_CXXC domain. The DME genes experienced a whole genome duplication event (WGD) that occurred in the ancestor genome of pear and apple before their divergence based on the Ks values. Expression results showed that high salinity stress could influence the expression level of DMEs and salt-responsive genes in Pyrus betulaefolia. Furthermore, the methylation levels of salt-responsive genes changed under salt stress treatment. Results suggested important roles of PbDME genes in response to salt stress and are useful for better understanding the complex functions of this DME genes, which will facilitate epigenetic studies in pear trees salt tolerance.

Eukaryotes utilize Ca2+ as a universal second messenger to convert and multiply environmental and developmental signals to downstream protein phosphorylation responses. However, the phylogenetic relationships of the genes that convert Ca2+ signal (CS) to protein phosphorylation responses (PPRs) remain highly controversial, and their origin and evolutionary trajectory are unclear, which greatly hinders functional studies. Here we examined the deep phylogeny of eukaryotic CS converter gene families and identified a phylogenetically and structurally distinctive monophyly in Archaeplastida. This monophyly can be divided into four subfamilies, and each can be traced to ancestral members that contain a kinase domain and a calmodulin-like domain. This strongly indicates that the ancestor of this monophyly originated by a de novo fusion of a kinase gene and a calmodulin gene. This gene family, with a proposed new name, Calmodulin Fused Kinase (CFK), had expanded and diverged significantly both in sizes and in structures for efficient and accurate Ca2+ signalling, and was shown to play pivotal roles in all the six major plant adaptation events in evolution. Our findings elucidated the common origin of all CS-PPR converter genes except CBL-CIPK converter genes, and revealed that CFKs act as the main CS conversion system in plants.

Calcium plays a central role in regulating signal transduction pathways. Calcineurin B-like proteins (CBLs), which harbor a crucial region consisting of EF hands that capture Ca2+, interact in a specific manner with CBL-interacting protein kinases (CIPKs). This two gene families or their interacting-complex widely respond to various environment stimuli and development processes. The genome-wide annotation and specific expression patterns of CBLs and CIPKs, however, in grapevine remain unclear. In the present study, 8 CBL and 20 CIPK genes were identified in grapevine genome, and divided into four and five subfamilies, respectively, based on phylogenetic analysis, and validated by gene structure and the distribution of conserved protein motifs. Four (50%) out of eight VvCBLs and eight (40%) out of twenty VvCIPKs were found to be derived from tandem duplication, and five (25%) out of twenty VvCIPKs were derived from segmental duplication, indicating that the expansion of grapevine CBL and CIPK gene families were mainly contributed by gene duplication, and all duplication events between VvCIPK genes only detected in intron poor clade. Estimating of synonymous and non-synonymous substitution rates of both gene families suggested that VvCBL genes seems more conserved than VvCIPK genes, and were derived by positive selection pressure, whereas VvCIPK genes were mainly derived by purifying selection pressure. Expressional analyses of VvCBL and VvCIPK genes based on microarray and qRT-PCR data performed diverse expression patterns of VvCBLs and VvCIPKs in response to both various abiotic stimuli and at different development stages. Furthermore, the co-expression analysis of grapevine CBLs and CIPKs suggested that CBL-CIPK complex seems to be more responsive to abiotic stimuli than during different development stages. VvCBLs may play an important and special role in regulating low temperature stress. The protein interaction analysis suggested divergent mechanisms might exist between Arabidopsis and grapevine. Our results will facilitate the future functional characterization of individual VvCBLs and VvCIPKs.

Background and Aims Mitogen-activated protein kinases have been found to play essential roles in mediating biotic and abiotic stress responses in plants. Investigation of their possible involvement in grapevine resistance to biotic or abiotic stresses and of their development will be possible only through future functional genomics experiments of gain/loss of function in the grapevine. Methods and Results We identified and re-annotated all 12 mitogen-activated protein kinases genes from the 12X V1 sequenced grapevine genome and re-nominated them according to international standards as VvMPK. All were validated by cloning their cDNA sequences through polymerase chain reaction amplification. Expression analysis of VvMPK genes using microarray analysis and quantitative real-time polymerase chain reaction demonstrated that all VvMPK genes are expressed during grapevine growth and development. Based on expression analysis of grapevine tissues and organs at several developmental stages, and of leaf tissues treated with Erysiphe necator (powdery mildew), salicylic acid, ethylene, hydrogen peroxide and drought, we identified for further functional characterisation several VvMPK candidate genes which might be involved in grapevine growth and development and in biotic and abiotic responses. Conclusions We identified several grapevine MPK genes modulated at the transcriptional level in several stages of grapevine growth and development and during the response to development and environmental stresses. Significance of the Study This is the first comprehensive experimental survey of the grapevine MPK gene family, which provides insights into their potential roles in regulating responses to biotic and abiotic stresses. Ongoing functional characterisation of important candidate VvMPK genes will assist unravelling their biological roles in grapevine.

Calcium plays a central role in regulating signal transduction pathways. Calcineurin B-like proteins (CBLs), which harbor a crucial region consisting of EF hands that capture Ca2+, interact in a specific manner with CBL-interacting protein kinases (CIPKs). This two gene families or their interacting-complex widely respond to various environment stimuli and development processes. The genome-wide annotation and specific expression patterns of CBLs and CIPKs, however, in grapevine remain unclear. In the present study, eight CBL and 20 CIPK genes were identified in grapevine genome, and divided into four and five subfamilies, respectively, based on phylogenetic analysis, and validated by gene structure and the distribution of conserved protein motifs. Four (50%) out of eight VvCBLs and eight (40%) out of 20 VvCIPKs were found to be derived from tandem duplication, and five (25%) out of 20 VvCIPKs were derived from segmental duplication, indicating that the expansion of grapevine CBL and CIPK gene families were mainly contributed by gene duplication, and all duplication events between VvCIPK genes only detected in intron poor clade. Estimating of synonymous and non-synonymous substitution rates of both gene families suggested that VvCBL genes seems more conserved than VvCIPK genes, and were derived by positive selection pressure, whereas VvCIPK genes were mainly derived by purifying selection pressure. Expressional analyses of VvCBL and VvCIPK genes based on microarray and qRT-PCR data performed diverse expression patterns of VvCBLs and VvCIPKs in response to both various abiotic stimuli and at different development stages. Furthermore, the co-expression analysis of grapevine CBLs and CIPKs suggested that CBL-CIPK complex seems to be more responsive to abiotic stimuli than during different development stages. VvCBLs may play an important and special role in regulating low temperature stress. The protein interaction analysis suggested divergent mechanisms might exist between Arabidopsis and grapevine. Our results will facilitate the future functional characterization of individual VvCBLs and VvCIPKs.

A genome-wide identification and cloning of CaM genes in pear was conducted and in compared withArabidopsisthat indicated a conserved expansion of CaM genes in pear, and PbCaMs and AtCaMs had a similar distribution of cis-elements and expressions in response to salt and osmotic stress. In particular, PbCaM1 and PbCaM3 were both significantly upregulated in response to salt and osmotic stress in pear.

Eukaryotes utilize Ca2+ as a universal second messenger to convert and multiply environmental and developmental signals to downstream protein phosphorylation responses. However, the phylogenetic relationships of the genes that convert Ca2+ signal (CS) to protein phosphorylation responses (PPRs) remain highly controversial, and their origin and evolutionary trajectory are unclear, which greatly hinders functional studies. Here we examined the deep phylogeny of eukaryotic CS converter gene families and identified a phylogenetically and structurally distinctive monophyly in Archaeplastida. This monophyly can be divided into four subfamilies, and each can be traced to ancestral members that contain a kinase domain and a calmodulin-like domain. This strongly indicates that the ancestor of this monophyly originated by a de novo fusion of a kinase gene and a calmodulin gene. This gene family, with a proposed new name, Calmodulin Fused Kinase (CFK), had expanded and diverged significantly both in sizes and in structures for efficient and accurate Ca2+ signalling, and was shown to play pivotal roles in all the six major plant adaptation events in evolution. Our findings elucidated the common origin of all CS-PPR converter genes except CBL-CIPK converter genes, and revealed that CFKs act as the main CS conversion system in plants.

A genome-wide identification and cloning of CaM genes in pear was conducted and in compared with Arabidopsis that indicated a conserved expansion of CaM genes in pear, and PbCaMs and AtCaMs had a similar distribution of cis-elements and expressions in response to salt and osmotic stress. In particular, PbCaM1 and PbCaM3 were both significantly upregulated in response to salt and osmotic stress in pear.

Duplication, lateral gene transfer, domain fusion/fission and de novo domain creation play a key role in formation of initial common ancestral protein. Abundant protein diversities are produced by domain rearrangements, including fusions, fissions, duplications, and terminal domain losses. In this report, we explored the origin of the RPW8 domain and examined the domain rearrangements that have driven the evolution of RPW8-encoding genes in land plants. The RPW8 domain first emerged in the early land plant, Physcomitrella patens, and it likely originated de novo from a non-coding sequence or domain divergence after duplication. It was then incorporated into the NBS-LRR protein to create a main sub-class of RPW8-encoding genes, the RPW8-NBS-encoding genes. They evolved by a series of genetic events of domain fissions, fusions, and duplications. Many species-specific duplication events and tandemly duplicated clusters clearly demonstrated that species-specific and tandem duplications played important roles in expansion of RPW8-encoding genes, especially in gymnosperms and species of the Rosaceae. RPW8 domains with greater Ka/Ks values than those of the NBS domains indicated that they evolved faster than the NBS domains in RPW8-NBSs.

Calcium ion is an intracellular messenger that plays a central role in signal transduction pathways. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) signal network have shown different functions in the Ca2+ signaling process. In this work, we identified the entire soybean (Glycine max) CIPK gene family, which comprised 52 genes and divided into four subgroups (I to IV) based on phylogeny. The gene structural analysis separated these 52 genes into an intron-rich clade and an intron-poor clade. Chromosomal location analysis resulted in the identification of 22 duplicated blocks and six tandem duplication events. Phylogenetic classification of 193 CIPK proteins from representative plant species suggested that the intron-poor clade of CIPKs originated in seed plants. Analysis of global gene expression patterns of soybean CIPK family revealed that most intron-poor soybean CIPK genes are drought-inducible; a finding that was further confirmed using qRT-PCR. Our study provides a foundation for further functional analysis to reveal the roles that CIPKs and more specifically the intron-poor clade play in drought tolerance in soybean.

I wrote the inaugural Editorial when Horticulture Research was launched in January 2014. This second Editorial was trigged by the manuscript that was submitted to Horticulture Research (HORTRES.2015.49, www.nature.com/articles/hortres201549), a summary of 17 years of traditional genetics on a woody ornamental tree called redbud. I have always been fascinated by the wealth of unique ornamental trees, mostly caused by genetic mutations. Since the redbud and dogwood are the two signature ornamental trees in eastern Tennessee, where University of Tennessee locates, I called Dr Dennis Werner and asked him to write a story behind this research and how he could continue and sustain this research for 17 years. In his story (see below), I see several important questions that are in need of open discussions. Over the past 25 years as a faculty in land-grant universities in the United States, I have seen eliminations or switching from the traditional breeding and genetics research programs to biotechnology, molecular biology and genomics, and alike. University administrators are shifting funds and changing evaluation matrixes, and have been placing more emphasis on publications, grants, and high impact factor journal articles. During the six years of my joint appointment at Nanjing Agricultural University, China, I have been struck by the “typhoon” of publishing high impact factor journal articles and changing evaluation matrix that swept through the Chinese research institutions. I have contacted many faculty members in the United States and China, and asked the following questions: “Will the traditional breeding and genetics (or similar programs) be fairly valued in academia, especially in land grant, or similar mission-oriented universities where horticultural research is primarily conducted?” “In the current academic environment, how can young scientists set long-term research goals without worrying about short-term pressures of publishing?” “How do we evaluate high impact research and does it equal to high impact factor journal articles?” As the Editor-in-Chief, I accepted Dr Werner’s article, which has taken 17 years to collect and aggregate the data, in Horticulture Research, to offer my view and support on this solid, traditional genetics research. I invite and encourage faculty and administrators to openly discuss these questions which may greatly impact research and service we perform to the horticultural science, horticultural industry, and ultimately our life in general.

Crassulacean acid metabolism (CAM) is a CO2 fixation pathway that maximizes water-use efficiency (WUE), compared with the C3/C4 CO2 pathway, which permits CAM plants to adapt to arid environments. The CAM pathway provides excellent opportunities to genetically design plants, especially bioenergy crops, with a high WUE and better photosynthetic performance than C3/C4 in arid environments. The information available on the origin and evolution of CAM is scant, however. Here, we analyzed transcriptomes from 13 orchid species and two existing orchid genomes, covering CAM and C3 plants, with an emphasis on comparing 13 gene families involved in the complete carbon fixation pathway. The dosage of the core photosynthesis-related genes plays no substantial role in the evolution of CAM in orchids; however, CAM may have evolved primarily by changes at the transcription level of key carbon fixation pathway genes. We proposed that in both dark and light, CO2 is primarily fixed and then released through two metabolic pathways via known genes, such as PPC1, PPDK and PPCK. This study reports a comprehensive comparison of carbon fixation pathway genes across different photosynthetic plants, and reveals the importance of the level of expression of key genes in the origin and evolution of CAM.

WRKY proteins are a class of transcription factors (TFs) involved in the regulation of various physiological processes, including the plant response to biotic and abiotic stresses. Recent studies in Arabidopsis have revealed that some WRKY TFs interact with a class of proteins designed as VQ proteins because of their typical conserved motif (FxxxVQxLTG). So far, no information is available about the genomic organization and the function of VQ motif-containing protein in grapevine (Vitis vinifera L). In the current study, we analyzed the 12X V1 prediction of the nearly homozygous PN40024 genotype identifying up to 18 predicted VQ genes (VvVQ). VvVQs phylogenetic and bioinformatic analyses indicated that the intron-exon structures and motif distribution are highly divergent between different members of the grapevine VQ family. Moreover, the analysis of the V. vinifera cv. Corvina expression atlas revealed a tissue- and stage-specific expression of several members of the family which also showed a significant correlation with WRKY TFs. Grapevine VQ genes also exhibited altered expression in response to drought, powdery mildew infection, salicylic acid (SA) and ethylene (ETH) treatments. The present study represents the first characterization of VQ genes in a grapevine genotype and it is a pivotal foundation for further studies aimed at functionally characterizing this mostly unknown grapevine multigenic family., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Disease resistance (R) genes from different Rosaceae species have been identified by map-based cloning for resistance breeding. However, there are few reports describing the pattern of R-gene evolution in Rosaceae species because several Rosaceae genome sequences have only recently become available. Results Since most disease resistance genes encode NBS-LRR proteins, we performed a systematic genome-wide survey of NBS-LRR genes between five Rosaceae species, namely Fragaria vesca (strawberry), Malus × domestica (apple), Pyrus bretschneideri (pear), Prunus persica (peach) and Prunus mume (mei) which contained 144, 748, 469, 354 and 352 NBS-LRR genes, respectively. A high proportion of multi-genes and similar Ks peaks (Ks = 0.1- 0.2) of gene families in the four woody genomes were detected. A total of 385 species-specific duplicate clades were observed in the phylogenetic tree constructed using all 2067 NBS-LRR genes. High percentages of NBS-LRR genes derived from species-specific duplication were found among the five genomes (61.81% in strawberry, 66.04% in apple, 48.61% in pear, 37.01% in peach and 40.05% in mei). Furthermore, the Ks and Ka/Ks values of TIR-NBS-LRR genes (TNLs) were significantly greater than those of non-TIR-NBS-LRR genes (non-TNLs), and most of the NBS-LRRs had Ka/Ks ratios less than 1, suggesting that they were evolving under a subfunctionalization model driven by purifying selection. Conclusions Our results indicate that recent duplications played an important role in the evolution of NBS-LRR genes in the four woody perennial Rosaceae species. Based on the phylogenetic tree produced, it could be inferred that species-specific duplication has mainly contributed to the expansion of NBS-LRR genes in the five Rosaceae species. In addition, the Ks and Ka/Ks ratios suggest that the rapidly evolved TNLs have different evolutionary patterns to adapt to different pathogens compared with non-TNL resistant genes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1291-0) contains supplementary material, which is available to authorized users.

Highlight Identification and functional classification of soybean kinase gene family revealed wide expansion and extensive divergence in gene structure, subcellular localizations, and tissue and stress gene expression patterns., The protein kinase (PK) gene family is one of the largest and most highly conserved gene families in plants and plays a role in nearly all biological functions. While a large number of genes have been predicted to encode PKs in soybean, a comprehensive functional classification and global analysis of expression patterns of this large gene family is lacking. In this study, we identified the entire soybean PK repertoire or kinome, which comprised 2166 putative PK genes, representing 4.67% of all soybean protein-coding genes. The soybean kinome was classified into 19 groups, 81 families, and 122 subfamilies. The receptor-like kinase (RLK) group was remarkably large, containing 1418 genes. Collinearity analysis indicated that whole-genome segmental duplication events may have played a key role in the expansion of the soybean kinome, whereas tandem duplications might have contributed to the expansion of specific subfamilies. Gene structure, subcellular localization prediction, and gene expression patterns indicated extensive functional divergence of PK subfamilies. Global gene expression analysis of soybean PK subfamilies revealed tissue- and stress-specific expression patterns, implying regulatory functions over a wide range of developmental and physiological processes. In addition, tissue and stress co-expression network analysis uncovered specific subfamilies with narrow or wide interconnected relationships, indicative of their association with particular or broad signalling pathways, respectively. Taken together, our analyses provide a foundation for further functional studies to reveal the biological and molecular functions of PKs in soybean.

Background Basic leucine zipper (bZIP) transcription factor gene family is one of the largest and most diverse families in plants. Current studies have shown that the bZIP proteins regulate numerous growth and developmental processes and biotic and abiotic stress responses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of plant bZIP family members remains very limited. Results We identified 55 bZIP transcription factor-encoding genes in the grapevine (Vitis vinifera) genome, and divided them into 10 groups according to the phylogenetic relationship with those in Arabidopsis. The chromosome distribution and the collinearity analyses suggest that expansion of the grapevine bZIP (VvbZIP) transcription factor family was greatly contributed by the segment/chromosomal duplications, which may be associated with the grapevine genome fusion events. Nine intron/exon structural patterns within the bZIP domain and the additional conserved motifs were identified among all VvbZIP proteins, and showed a high group-specificity. The predicted specificities on DNA-binding domains indicated that some highly conserved amino acid residues exist across each major group in the tree of land plant life. The expression patterns of VvbZIP genes across the grapevine gene expression atlas, based on microarray technology, suggest that VvbZIP genes are involved in grapevine organ development, especially seed development. Expression analysis based on qRT-PCR indicated that VvbZIP genes are extensively involved in drought- and heat-responses, with possibly different mechanisms. Conclusions The genome-wide identification, chromosome organization, gene structures, evolutionary and expression analyses of grapevine bZIP genes provide an overall insight of this gene family and their potential involvement in growth, development and stress responses. This will facilitate further research on the bZIP gene family regarding their evolutionary history and biological functions.

The plant WRKY gene family represents an ancient and complex class of zinc-finger transcription factors (TFs) that are involved in the regulation of various physiological processes, such as development and senescence, and in plant response to many biotic and abiotic stresses. Despite the growing number of studies on the genomic organisation of WRKY gene family in different species, little information is available about this family in grapevine (Vitis vinifera L.). In the present study, a total number of 59 putative grapevine WRKY transcription factors (VvWRKYs) were identified based on the analysis of various genomic and proteomic grapevine databases. According to their structural and phylogentic features, the identified grapevine WRKY transcription factors were classified into three main groups. In order to shed light into their regulatory roles in growth and development as well as in response to biotic and abiotic stress in grapevine, the VvWRKYs expression profiles were examined in publicly available microarray data. Bioinformatics analysis of these data revealed distinct temporal and spatial expression patterns of VvWRKYs in various tissues, organs and developmental stages, as well as in response to biotic and abiotic stresses. To also extend our analysis to situations not covered by the arrays and to validate our results, the expression profiles of selected VvWRKYs in response to drought stress, Erysiphe necator (powdery mildew) infection, and hormone treatments (salicilic acid and ethylene), were investigated by quantitative real-time reverse transcription PCR (qRT-PCR). The present study provides a foundation for further comparative genomics and functional studies of this important class of transcriptional regulators in grapevine.

Cation/proton antiporter 1 (CPA1) proteins function as regulators of monovalent ions, pH homeostasis, and other developmental processes in plants. Better understanding of the expression and regulation of CPA1 in plant responses to salinity would help the development of scientific practices in crops worldwide. In this report, we characterized all seven CPA1 family genes in grapevine (Vitis vinifera) in response to short-term osmotic and NaCl stresses. We found that two of the seven genes have subfunctionalized to be differentially expressed in response to NaCl stress in the early stage in different organs, whereas the other five members seem to play little or no role in this response. Specifically, VIT_19s0090g01480 may control Na(+) compartmentalization in grapevine roots; and VIT_05s0020g01960 may influence Na(+) transfer in stems. Based on the dynamics of ion concentrations, electrolyte leakage rates, and CPA1 gene expression in root, stem, and leaf tissues under osmotic and NaCl stresses, we suggest how grapevine responds physiologically and molecularly to the osmotic and ion toxicity of NaCl stress in the short term. This work lays a foundation for future research on the CPA1 gene family regarding its evolutionary history and biological functions for modulating salt responses in grapevine.

Gene co-expression analysis has been widely used for predicting gene functions because genes within modules of a co-expression network may be involved in similar biological processes and exhibit similar biological functions. To detect gene relationships in the grapevine genome, we constructed a grapevine gene co-expression network (GGCN) by compiling a total of 374 publically available grapevine microarray datasets. The GGCN consisted of 557 modules containing a total of 3834 nodes with 13 479 edges. The functions of the subnetwork modules were inferred by Gene ontology (GO) enrichment analysis. In 127 of the 557 modules containing two or more GO terms, 38 modules exhibited the most significantly enriched GO terms, including 'protein catabolism process', 'photosynthesis', 'cell biosynthesis process', 'biosynthesis of plant cell wall', 'stress response' and other important biological processes. The 'response to heat' GO term was highly represented in module 17, which is composed of many heat shock proteins. To further determine the potential functions of genes in module 17, we performed a Pearson correlation coefficient test, analyzed orthologous relationships with Arabidopsis genes and established gene expression correlations with real-time quantitative reverse transcriptase PCR (qRT-PCR). Our results indicated that many genes in module 17 were upregulated during the heat shock and recovery processes and downregulated in response to low temperature. Furthermore, two putative genes, Vit_07s0185g00040 and Vit_02s0025g04060, were highly expressed in response to heat shock and recovery. This study provides insight into GGCN gene modules and offers important references for gene functions and the discovery of new genes at the module level.