Transcriptional dynamics reveals the asymmetrical events underlying graft union formation in pecan (Carya illinoinensis).

Grafting is a widely used technique for pecan propagation; however, the background molecular events underlying grafting are still poorly understood. In our study, the graft partners during pecan [Carya illinoinensis (Wangenh.) K. Koch] graft union formation were separately sampled for RNA-seq, and the transcriptional dynamics were described via weighted gene co-expression network analysis. To reveal the main events underlying grafting, the correlations between modules and grafting traits were analyzed. Functional annotation showed that during the entire graft process, signal transduction was activated in the scion, while messenger RNA splicing was induced in the rootstock. At 2 days after grafting, the main processes occurring in the scion were associated with protein synthesis and processing, while the primary processes occurring in the rootstock were energy release-related. During the period of 7-14 days after grafting, defense response was a critical process taking place in the scion; however, the main process functioning in the rootstock was photosynthesis. From 22 to 32 days after grafting, the principal processes taking place in the scion were jasmonic acid biosynthesis and defense response, whereas the highly activated processes associated with the rootstock were auxin biosynthesis and plant-type secondary cell wall biogenesis. To further prove that the graft partners responded asymmetrically to stress, hydrogen peroxide contents as well as peroxidase and ß-1,3-glucanase activities were detected, and the results showed that their levels were increased in the scion not the rootstock at certain time points after grafting. Our study reveals that the scion and rootstock might respond asymmetrically to grafting in pecan, and the scion was likely associated with stress response, while the rootstock was probably involved in energy supply and xylem bridge differentiation during graft union formation.

E3 Ubiquitin Ligase PUB23 in Kiwifruit Interacts with Trihelix Transcription Factor GT1 and Negatively Regulates Immune Responses against Pseudomonas syringae pv. actinidiae.

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) is the most serious disease threatening kiwifruit production. Our previous study found genes encoding the U-box containing proteins were significantly regulated by Psa infection. Here, we report a U-box type E3 ubiquitin ligase PUB23 in kiwifruit which acts as a negative regulator of immune responses against Psa. PUB23 was found to physically interact with GT1, a trihelix transcription factor, in vitro and in vivo. The expression of GT1 was up-regulated in PUB23-silenced plants, indicating that interacting with PUB23 may directly or indirectly suppress GT1 expression. The silencing of PUB23 led to enhanced immune responses of PAMP-triggered immunity (PTI), including a higher expression level of defense marker genes PR1 and RIN4, and increased accumulation of hydrogen peroxide and superoxide anion. Our results reveal a negative role PUB23 plays in kiwifruit immune responses against Psa and may regulate gene expression by interacting with GT1.

Comprehensive Genomic Analysis of SnRK in Rosaceae and Expression Analysis of RoSnRK2 in Response to Abiotic Stress in Rubus occidentalis.

The sucrose nonfermenting 1-related protein kinase (SnRK) plays an important role in responding to abiotic stresses by phosphorylating the target protein to regulate various signaling pathways. However, little is known about the characteristics, evolutionary history, and expression patterns of the SnRK family in black raspberry (Rubus occidentalis L.) or other Rosaceae family species. In this study, a total of 209 SnRK genes were identified in 7 Rosaceae species and divided into 3 subfamilies (SnRK1, SnRK2, and SnRK3) based on phylogenetic analysis and specific motifs. Whole-genome duplication (WGD) and dispersed duplication (DSD) were considered to be major contributions to the SnRK family expansion. Purifying selection was the primary driving force in the SnRK family evolution. The spatial expression indicated that the RoSnRK genes may play important roles in different tissues. In addition, the expression models of 5 RoSnRK2 genes in response to abiotic stresses were detected by qRT-PCR. The proteins encoded by RoSnRK2 genes localize to the cytoplasm and nucleus in order to perform their respective functions. Taken together, this study provided an analysis of the SnRK gene family expansion and evolution, and contributed to the current knowledge of the function of 5 RoSnRK2 genes, which in turn expanded understanding of the molecular mechanisms of black raspberry responses to abiotic stress.

Stem rot on Adenia globosa caused by Lasiodiplodia theobromae in Jiangsu.

Adenia globosa, as an excellent indoor ornamental plant, is planted in Tropical Botanical Museum, Nanjing Zhongshan Botanical Garden, Jiangsu Province, China. In September 2022, a new stem basal rot disease was observed on A. globosa seedlings, being planted here. Stem basal rot were observed on approximately 80% of A. globosa seedlings. The basal stem of cutting seedlings appeared decayed, and stem tip eventually turned dry due to water loss (Figure S1A). To isolate the pathogen, three diseased stems were collected from three cuttings planted in different pots of the Tropical Botanical Museum. The stem sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the dark. Pure cultures were obtained by monosporic isolation. Eight isolates were obtained, and all identified as Lasiodiplodia sp.. The colonies morphology of cultures, growing on PDA were cotton-like, the primary mycelia were black gray after 7 days, and the reverse sides of PDA plates were similar to front sides in color (Figure S1B). A representative isolate, QXM1-2 was selected for the further study. Conidia of QXM1-2 were oval or elliptic, with a mean size of 11.6 µm×6.6 µm (n=35). The conidia are colorless and transparent in the early stage, and become dark brown with one-septum in the later stage (Figure S1C). The conidiophores produced conidia after nearly four weeks of cultivation on PDA plate (Figure S1D). The conidiophore was a transparent cylindrical structure, with a size of (6.4-18.2) µm × (2.3-4.5) µm ( n = 35). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The internal transcribed spacer regions (ITS), translation elongation factor 1-alpha (TEF1α) and ß-tubulin (TUB) genes (GenBank Accession No.OP905639, No.OP921005, and No.OP921006, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. They had 99.8-100% homology to the ITS (504/505 bp) of Lasiodiplodia theobromae strain NH-1 (MK696029), TEF1α (316/316 bp) of strain PaP-3 (MN840491), and TUB (459/459 bp) of isolate J4-1 (MN172230). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate QXM1-2 clustered in the L. theobromae clade with 100% bootstrap support (Figure S2). To test pathogenicity, three A. globosa cutting seedlings that previously had been wounded with a sterile needle were inoculated with 20 µL conidia suspension (1×106 conidia/mL) on the stem base. The seedlings inoculated with 20 µL sterile water was used as the control. All plants were covered with clear polyethylene bags to keep moisture in a greenhouse (25℃, 80% relative humidity). The experiment was repeated three times. After 7 days post-inoculation, typical stem rot were found on the treated cutting seedlings and the control seedlings did not have any symptoms (Figure S1E-F). The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased tissues of the inoculated stems to complete Koch's postulates. This pathogen has been reported infecting the branch of castor bean (Tang et al. 2021) and root of Citrus (Al-Sadi et al. 2014). For our knowledge, this is the first report of L. theobromae infecting A. globosa in China. This study provides an important reference for the biology, epidemiology of L. theobromae.

First report of leaf spot on pecan (Carya illinoinensis) caused by Stemphylium eturmiunum in China.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is an important nut tree species, which has been widely planted in Jiangsu and Anhui Provinces of China in recent years (Mo et al. 2018). In May 2022, a new leaf spot disease was observed on both young and old leaves of pecan trees in the Luhe area, Nanjing, Jiangsu Province. Approximately 30% of pecan trees suffered from the disease, which affected the growth of young trees and nut production to cause economic loss. Initially, the leaf spots were grayish black and round. Then, disease spots enlarged and joined together, forming irregular lesions with uneven edges. In the last stage, the leaflets were withered. To isolate the pathogen, three symptomatic leaves were collected from each of three different pecan trees. Leaf sections (3 to 4 mm) were excised from the margin of spots, surface sterilized in 75% alcohol for 30 s, then sterilized in 1.5% NaClO for 90 s. After rinsing three times with sterile distilled water, leaf sections were placed on potato dextrose agar (PDA) medium and incubated at 25 °C in a dark environment for 5 days. Pure cultures were obtained by monosporic isolation. A total of 20 isolates were obtained, and 12 isolates were identified as Stemphylium sp. with the same morphological features and ITS sequences. A representative isolate, named LH3-3, was selected for further study. Colonies on PDA were light yellow with dense mycelium and were brownish yellow on the reverse side. Conidia were 16.3 to 34.4 × 8.1 to 16.3 µm) (n=35), muriform, brown, with transverse and longitudinal septa, lightly deformed at the transverse septa. Ascomata were not observed. The morphological characteristics were consistent with the description of Stemphylium eturmiunum (Simmons 2001). The internal transcribed spacer region (ITS) and portions of genes for calmodulin (cmdA) and glyceraldehyde-3-phosphate dehydrogenase (gpd) were amplified and sequenced with the primers ITS1/ITS4 (White et al. 1990), CALDF1/CALDR2 (Xu et al. 2022) and GPD-F/R (Xie et al. 2019), respectively. Sequences were deposited in GenBank under accessions OP482492 (ITS), OP495734 (cmdA), and OP495735 (gpd). BLAST analysis showed that the sequences had 99.67-100% homology to ITS (525/525 bp) of S. eturmiunum strain ST14 (MH843733), cmdA (694/694 bp) of strain CBS122124 (KU850832), and gpd (299/300 bp) of isolate UMSe0030 (MK336876). MEGA 7.0 was used to construct a phylogenetic analysis based on concatenated sequences of ITS, cmdA, and gpd using the neighbor-joining method. The results showed that LH3-3 clustered on the branch of S. eturmiunum, and the support rate was 100%. A spore suspension in sterile water was made from strain LH3-3 grown on PDA, and adjusted to 1×106 spores/mL with a hemocytometer. To test pathogenicity, 20 µl drops of the spore suspension were placed on the left sides of four healthy detached leaflets of mature pecan trees and leaves of three 3-month-old seedlings. The right side of each leaflet was inoculated with 20 µl drops of sterile distilled water as the control. All inoculated seedlings and detached leaflets were covered with a transparent plastic bag and cultured in a greenhouse at 25 °C, 80% relative humidity, and a 12 h light cycle until symptom appeared. The experiment was repeated three times. After 7 days of inoculation, grayish black lesions appeared on all inoculation sites with the spore suspension but not in the controls. The leaf spot symptoms were similar to those observed in orchards. The same fungus, identified by morphological characteristics and sequencing of ITS, cmdA, and gpd, was re-isolated from the diseased spots of the inoculated leaflets to complete Koch's postulates. S. eturmiunum has been reported to infect garlic (Dumin et al. 2022) and tomato (Prencipee et al. 2021), but this is the first report of S. eturmiunum causing leaf spot of C. illinoinensis. This study provides a basis for further study on the biology, epidemiology, and management of the disease.

Identification of Candidate Genes Associated with Pulp Color by Transcriptomic Analysis of 'Huaxiu' Plum (Prunus salicina Lindl.) during Fruit-Ripening.

The plum (Prunus salicina Lindl.) is one of the traditional and economically important stone fruit trees in China. Anthocyanins are important pigments in plums. However, little is known about the molecular mechanisms underlying anthocyanin accumulation in plum fruits, which has hindered research on the molecular mechanism of its utilization. Our research shows that the chlorophyll content was gradually decreased and the contents of anthocyanin and flavonoid increased during the coloring process of the pulp in 'Huaxiu' plums (P. salicina). Then, the RNA-Seq technique was used to analyze the transcriptome of pulp color changes with three different stages (yellow, orange, and red) in the 'Huaxiu' plum (P. salicina). A total of 57,119 unigenes with a mean length of 953 bp were generated, and 61.6% of them were annotated to public databases. The Gene Ontology (GO) database assigned 21,438 unigenes with biological process, cellular components, and molecular function. In addition, 32,146 unigenes were clustered into 25 categories for functional classification by the COG database, and 7595 unigenes were mapped to 128 KEGG pathways by the KEGG pathway database. Of these, 1095 (YS-versus-OS), 4947 (YS-versus-RS), and 3414 (OS-versus-RS) genes were significantly expressed differentially between two coloration stages. The GO and KEGG pathway enrichment analysis revealed that 20 and 1 differentially expressed genes (DEG) are involved in flavonoid biosynthesis and anthocyanin biosynthesis, respectively. Finally, we mainly identified three structural genes as candidate genes. The transcriptome information in this study provide a basis for further studies of pulp colors in plum and contribute to our understanding of the molecular mechanisms underlying anthocyanin biosynthesis in pulp.

Transcriptome Analysis Reveals a Comprehensive Virus Resistance Response Mechanism in Pecan Infected by a Novel Badnavirus Pecan Virus.

Pecan leaf-variegated plant, which was infected with a novel badnavirus named pecan mosaic virus (PMV) detected by small RNA deep sequencing, is a vital model plant for studying the molecular mechanism of retaining green or chlorosis of virus-infected leaves. In this report, PMV infection in pecan leaves induced PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). PMV infection suppressed the expressions of key genes of fatty acid, oleic acid (C18:1), and very-long-chain fatty acids (VLCFA) biosynthesis, indicating that fatty acids-derived signaling was one of the important defense pathways in response to PMV infection in pecan. PMV infection in pecans enhanced the expressions of pathogenesis-related protein 1 (PR1). However, the transcripts of phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) were downregulated, indicating that salicylic acid (SA) biosynthesis was blocked in pecan infected with PMV. Meanwhile, disruption of auxin signaling affected the activation of the jasmonic acid (JA) pathway. Thus, C18:1 and JA signals are involved in response to PMV infection in pecan. In PMV-infected yellow leaves, damaged chloroplast structure and activation of mitogen-activated protein kinase 3 (MPK3) inhibited photosynthesis. Cytokinin and SA biosynthesis was blocked, leading to plants losing immune responses and systemic acquired resistance (SAR). The repression of photosynthesis and the induction of sink metabolism in the infected tissue led to dramatic changes in carbohydrate partitioning. On the contrary, the green leaves of PMV infection in pecan plants had whole cell tissue structure and chloroplast clustering, establishing a strong antiviral immunity system. Cytokinin biosynthesis and signaling transductions were remarkably strengthened, activating plant immune responses. Meanwhile, cytokinin accumulation in green leaves induced partial SA biosynthesis and gained comparatively higher SAR compared to that of yellow leaves. Disturbance of the ribosome biogenesis might enhance the resistance to PMV infection in pecan and lead to leaves staying green.

First report of leaf spot on Magnolia grandiflora caused by Lasiodiplodia theobromae in Jiangsu.

Magnolia grandiflora linn, with large and fragrant flowers, is widely planted in the south of Yangtze River valley in China. It is an excellent street tree and a beautiful ornamental tree for landscaping. In October 2021, a new leaf spot disease was observed on M. grandiflora seedlings and mature trees growing in Nanjing Botanical Garden, Jiangsu Province, China. According to statistics, about 300 M. grandiflora trees were planted here, and approximately 60% of M. grandiflora trees suffered from the disease. In the beginning, small black spots appeared on the leaf of M. grandiflora, and then the disease spots were connected into coalesced, and eventually lead to a large area of leaf dead (Figure S1A). To isolate the pathogen, ten diseased leaves were collected from ten plants distributed in different five areas of the botanical garden. The leaf sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% alcohol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in the darkness. Pure cultures were obtained by monosporic isolation. Twenty-three isolates were obtained (the isolate rate of 72%), and identified as Lasiodiplodia sp.. A representative isolate, G-H-1 was used for the further study. The colony of G-H-1, growing on PDA was cotton-like. The primary mycelia was gray and white in the early stage of culture. It gradually turned black gray in the later stage, and the reverse was similar in color (Figure S1B). The pycnidia (fruiting body) was black and appeared over PDA plates after 15 days (Figure S1C). The hyphae of G-H-1 were dark brown, and the conidia were monospora, oval or elliptic, with a size of (9.6 ~ 13.3) µm× (5.7 ~ 8.0) µm (mean 11.7×6.6 µm, n=35) (Figure S1D). In the pycnidia, the conidiophores were inside and produced conidia (Figure S1E). In the early stage, the conidia of G-H-1 were colorless transparent, then gradually turned dark brown with a septum in the center (Figure S1F). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The regions of ITS, translation elongation factor 1-alpha (TEF1α) and ß-tubulin (TUB) genes (GenBank Accession No.OM698339, No.OM942757, and No.OM942756, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995). The obtained sequences showed 99.05-99.81% similarity with those from L. theobromae accessions in GenBank. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate G-H-1 clustered in the L. theobromae clade with 96% bootstrap support (Figure S2). To test pathogenicity, three one-year-old M. grandiflora seedlings that previously had been wounded with a sterile needle were inoculated with 20 µL conidia suspension (1×106 spores/mL) on the left sides of leaves. Inoculation with 20 µL sterile water was treated as the control, which were inoculated on the right sides of leaves. All plants were covered with clear polyethylene bags to keep moisture. And inoculated detached leaves were incubated in a greenhouse (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, typical black spots were found on the left sides of all inoculated leaves and the right sides did not have any leaf spot symptoms (Figure S1G-H). After 25 days of inoculation, perforation occurred at the black spots on the leaves of the inoculated plants, resulting in incomplete leaf (Figure S1I), which is identical disease symptoms to those observed in garden. The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased spots of the inoculated leaves to complete Koch,s postulates. The pathogen has a very wide host range. For example, it has been reported to cause dieback and sooty canker on Ficus trees (Abo Rehab et al. 2014), infected trunk of sultana seedless (Tang et al. 2021) and castor bean (Akgul et al. 2015), root of Citrus (Al-Sadi et al. 2014), date palm, and Mango (Al-Sadi et al. 2013) and Cassia fistula (Deng et al. 2015). But, according to nt.ars-grin.gov, there are no other reports of L. theobromae on M. grandiflora in the world. So, this would be the first one. This study provides an important reference for the biology, epidemiology.

First report of leaf spot on Deutzia crenata caused by Neopestalotiopsis ellipsospora in China.

Deutzia crenata Sieb. et Zucc, native to Japan, with white flowers in early summer, is a high quality ornamental shrub widely planted in China. In October 2021, a new leaf spot disease was observed on approximately 70% of the 320 D. crenata trees growing in Nanjing Botanical Garden, Jiangsu Province, China. The disease started as irregular small gray spots on the leaf of D. crenata that coalesced into larger lesions. Infected leaves turned yellow (Figure S1A) and leaves with multiple spots withered. To isolate the pathogen, leaf sections (3 to 4 mm) were excised from the lesion margin, surface sterilized in 75% alcohol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in the darkness. Pure cultures were obtained by monosporic isolation. The colony of a representative isolate (L-1), growing on PDA was circular, white, and cottony, and the surface undulate and pale luteous (Figure S1B). The reverse was similar in color (Figure S1C). The conidial masses were black and appeared over PDA plates after 12 days (Figure S1D). Conidia [18.3 to 28.4×5.4 to 8.5 µm (mean 24.5×6.7 µm)] (n=35) were fusiform to ellipsoid and four-septate (one basal and one apical cell hyaline, and three brown median cells), with two to three apical appendages (Figure S1E). These characteristics were consistent with the description of Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). Three regions of the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1α), and ß-tubulin (TUB) genes (GenBank Accession No. OM663738, No. OM687134 and No. OM687133, respectively) were amplified and sequenced with the primers pairs ITS1/ITS4 (Innis et al. 1990), EF1-526F/EF1-1567R (Maharachchikumbura et al. 2014) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were 95.4-99.8% similar to those from Neopestalotiopsis sp. accessions in GenBank. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate L-1 clustered in the N. ellipsospora clade with 98% bootstrap support (Figure S2). To test pathogenicity, three detached healthy leaves and three one-year-old D. crenata seedlings were inoculated with 20 µL conidia suspension (1×106 spores/mL) on the left sides of leaves. The right side of each leaf was inoculation with 20 µL of sterile water as the experimental control. All plants were covered with clear polyethylene bags and incubated in a greenhouse (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, leaf spots typical of those observed in the orchards were observed on the left sides of all inoculated leaves and the right sides did not have any leaf spot symptoms (Figure S1F-G). The same fungus was isolated from the diseased spots of the inoculated leaves to complete Koch,s postulates (Figure S1H). N. ellipsospora is known to cause leaf spots on Camellia sinensis and sweet potato, infects fruits of Ardisia crenata in China (Maharachchikumbura et al. 2014; Maharachchikumbura et al. 2016; Wang et al. 2019), and causes stem spots on Acanthopanax divaricatus in Korea (Yun et al. 2015). This is the first report of N. ellipsospora causing leaf spot on D. crenata in the world. The occurrence of this disease needs to be monitored, because it can reduce the ornamental value of D. crenata. This finding provides the foundation to further investigate the biology and epidemiology of this disease so that effective strategies can be developed to manage this disease.

Characterization of the complete chloroplast genome of Carya laciniosa (F.Michx.) G.Don (Juglandaceae).

Carya laciniosa (F.Michx.) G.Don is a slow-growing valuable tree in the family Juglandaceae. In this research, we first reported the chloroplast genome sequence of C. laciniosa. The chloroplast genome was a circular form with 160,832 bp in size, comprising four subregions: a large single-copy (LSC) region of 90,065 bp, a small single-copy (SSC) region of 18,791 bp, and two copies of inverted repeats (IRs; IRa and IRb) of 25,988 bp each. A total of 132 genes were annotated in the chloroplast genome of C. laciniosa, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC content of the whole genome was 36.14%. Phylogenetic analysis suggested that C. laciniosa was closely related to C. ovata.

Genome-Wide Bioinformatics Analysis of MAPK Gene Family in Kiwifruit (Actinidia Chinensis).

Mitogen activated protein kinase (MAPK) cascades are universal signal transduction modules that play crucial roles in various biotic and abiotic stresses, hormones, cell division, and developmental processes in plants. Mitogen activated protein kinase (MAPK/MPK), being a part of this cascade, performs an important function for further appropriate cellular responses. Although MAPKs have been investigated in several model plants, no systematic analysis has been conducted in kiwifruit (Actinidia chinensis). In the present study, we identified 18 putative MAPKs in the kiwifruit genome. This gene family was analyzed bioinformatically in terms of their chromosome locations, sequence alignment, gene structures, and phylogenetic and conserved motifs. All members possess fully canonical motif structures of MAPK. Phylogenetic analysis indicated that AcMAPKs could be classified into five subfamilies, and these gene motifs in the same group showed high similarity. Gene structure analysis demonstrated that the number of exons in AcMAPK genes ranged from 2 to 29, suggesting large variation among kiwifruit MAPK genes. The expression profiles of these AcMAPK genes were further investigated using quantitative real-time polymerase chain reaction (qRT-PCR), which demonstrated that AcMAPKs were induced or repressed by various biotic and abiotic stresses and hormone treatments, suggesting their potential roles in the biotic and abiotic stress response and various hormone signal transduction pathways in kiwifruit. The results of this study provide valuable insight into the putative physiological and biochemical functions of MAPK genes in kiwifruit.

Overexpression of Actinidia deliciosa pyruvate decarboxylase 1 gene enhances waterlogging stress in transgenic Arabidopsis thaliana.

Ethanolic fermentation is classically associated with waterlogging tolerance when plant cells switch from respiration to anaerobic fermentation. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we cloned a full-length PDC cDNA sequence from kiwifruit, named AdPDC1. We determined the expression of the AdPDC1 gene in kiwifruit under different environmental stresses using qRT-PCR, and the results showed that the increase of AdPDC1 expression during waterlogging stress was much higher than that during salt, cold, heat and drought stresses. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis enhanced the resistance to waterlogging stress but could not enhance resistance to cold stress at five weeks old seedlings. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis could not enhance resistance to NaCl and mannitol stresses at the stage of seed germination and in early seedlings. These results suggested that the kiwifruit AdPDC1 gene is required during waterlogging but might not be required during other environmental stresses. Expression of the AdPDC1 gene was down-regulated by abscisic acid (ABA) in kiwifruit, and overexpression of the AdPDC1 gene in Arabidopsis inhibited seed germination and root length under ABA treatment, indicating that ABA might negatively regulate the AdPDC1 gene under waterlogging stress.

De novo transcriptome sequencing and gene expression profiling of spinach (Spinacia oleracea L.) leaves under heat stress.

Spinach (Spinacia oleracea) has cold tolerant but heat sensitive characteristics. The spinach variety 'Island,' is suitable for summer periods. There is lack molecular information available for spinach in response to heat stress. In this study, high throughput de novo transcriptome sequencing and gene expression analyses were carried out at different spinach variety 'Island' leaves (grown at 24 °C (control), exposed to 35 °C for 30 min (S1), and 5 h (S2)). A total of 133,200,898 clean reads were assembled into 59,413 unigenes (average size 1259.55 bp). 33,573 unigenes could match to public databases. The DEG of controls vs S1 was 986, the DEG of control vs S2 was 1741 and the DEG of S1 vs S2 was 1587. Gene Ontology (GO) and pathway enrichment analysis indicated that a great deal of heat-responsive genes and other stress-responsive genes were identified in these DEGs, suggesting that the heat stress may have induced an extensive abiotic stress effect. Comparative transcriptome analysis found 896 unique genes in spinach heat response transcript. The expression patterns of 13 selected genes were verified by RT-qPCR (quantitative real-time PCR). Our study found a series of candidate genes and pathways that may be related to heat resistance in spinach.

[Study on Salt Tolerance of Echinacea purpurea].

OBJECTIVE: To explore the salt tolerance of Echiancea purpurea and its mechanism. METHODS: Echiancea purpurea was used as test material in this study and six salinity levels (0, 30, 60, 90, 120 and 150 mmol/L NaCl) were set. Effects on seed germination and salt tolerance relevant physiological and biochemical indexes of Echiancea purpurea were studied. RESULTS: Salt stress suppressed the germination of Echiancea purpurea seeds, induced osmotic adjustment substances proline, soluble sugar and K+ to increase, and activities of POD and SOD to rise, and meanwhile resulted in accumulation of Na+ and decrease of K+/Na+. CONCLUSION: Echiancea purpurea can tolerant salt stress to a certain degree, but in case of high salt concentrations, severe salt injury would remarkably suppress the growth of Echinacea purpurea.

Comparative proteomic analysis of the stolon cold stress response between the C4 perennial grass species Zoysia japonica and Zoysia metrella.

Zoysiagrass, the most cold-tolerant grass among the warm-season turfgrasses, is often used as a model species for isolating cellular components related to cold stress. To understand the proteomic responses to cold stress in zoysiagrass stolons, we extracted stolon proteins from Zoysiajaponica, cv. Meyer (cold-tolerant) and Z. metrella, cv. Diamond (cold-sensitive), which were grown with or without cold treatment. Approximately 700 proteins were resolved on 2-DE gels, and 70 protein spots were differentially accumulated. We further observed that 45 of the identified proteins participate in 10 metabolic pathways and cellular processes. A significantly greater number of proteins accumulated in the Meyer than in the Diamond and 15 increased proteins were detected only in the Meyer cultivar under cold stress. Furthermore, we propose a cold stress-responsive protein network composed of several different functional components that exhibits a balance between reactive oxygen species (ROS) production and scavenging, accelerated protein biosynthesis and proteolysis, reduced protein folding, enhanced photosynthesis, abundant energy supply and enhanced biosynthesis of carbohydrates and nucleotides. Generally, the cold-tolerant Meyer cultivar showed a greater ROS scavenging ability, more abundant energy supply and increased photosynthesis and protein synthesis than did the cold-sensitive Diamond cultivar, which may partly explain why Meyer is more cold tolerant.

Association of molecular markers with cold tolerance and green period in zoysiagrass (Zoysia Willd.).

Cold tolerance and the green period are key traits in the breeding of zoysiagrass (Zoysia Willd.). Identification of molecular markers associated with cold tolerance and the green period of zoysiagrass will contribute to efficient selection of elite cultivars. These two traits were measured in 96 zoysiagrass accessions in 2004 and 2005-2006, respectively. The mapping population was screened with 29 pairs of simple sequence repeat (SSR) primers and 54 pairs of sequence-related amplified polymorphism (SRAP) primers. A multi-loci in silico mapping approach implemented with an empirical Bayes method was applied for association mapping of cold tolerance and green period. We detected 254 SSR polymorphic loci and 338 SRAP polymorphic loci, among which three SSR loci (Xgwm131-3B-187, Xgwm469-6D-194 and Xgwm234-5B-244) and one SRAP locus (Me11Em7-406) were significantly associated with cold tolerance with effect values of 57.83%, 38.05%, 36.92% and 37%, respectively. Three SSR loci (Xgwm132-6B-225, Xgwm111-7D-34 and Xgwm102-2D-97) and two SRAP loci (Me19Em5-359 and Me16Em8-483) were significantly associated with the green period with effect values of 79.54%, 62.59%, 99.04%, 49.01% and 82.57%. These markers will be useful for genetic improvement of the cold tolerance and green period of zoysiagrass by marker-assisted breeding.