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Response regulator (RR) is the core component of cytokinin (CK) signaling, and it regulates the expression of numerous downstream CK-responsive genes. However, the knowledge regarding the pecan RR (CiRR) gene family is still limited. In this study, we first monitored trans-zeatin riboside (tZR) content in the graft union 0, 7, 14, and 32 days after grafting and then conducted genome-wide analysis and expression profiling of the CiRR gene family using an available genome sequence and RNA-seq dataset, aiming to better understand the roles of CK during pecan grafting. The dynamic contents of tZR showed an increased trend during the specific period for both the scion and rootstock. There were 20 CiRRs in the pecan genome, including 12 type A CiRRs, 5 type B members, and 3 type C genes. All members contained a receiver domain and type B CiRRs possessed an additional Myb-like DNA-binding domain. Promoter analysis showed that the CiRR gene family contained cis-elements associated with growth and development, hormones, and stress. A total of 10 genes, including CiRR18/9/4a/14a/12c/5/12b/14b/2b/2a, were abundantly expressed in the samples of different tissues, drought stress, and kernel development. There were 12 genes (CiRR5/18/4a/12b/2b/12c/14b/2a/14a/4b/9/11a) showing active expressions during grafting, and weighted gene co-expression network analysis (WGCNA) grouped them into six modules. Among them, CiRR14a and CiRR12b were the hub genes for the turquoise and brown modules, respectively. Functional annotation indicated that the turquoise module was associated with gene transcription and translation, while the brown module was related to cell proliferation. Our results suggest that the CiRR gene family central to CK signaling is probably involved in callus formation during pecan grafting.

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.

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.

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.

Patch budding (bud grafting) is a commonly used method for pecan reproduction; however, the grafting survival rate varies with cultivars. Clarifying the underlying mechanisms of successful grafting is pivotal for graft technique improvement. Here, weighted gene co-expression network analysis (WGCNA) was conducted to dissect the key pathways and genes related to the successful grafting of pecan. Based on the transcriptome data of two contrasting cultivars (an easy-to-survive cultivar ‘Pawnee’ and a difficult-to-survive cultivar ‘Jinhua’) in response to budding, all the genes with variable transcripts were grouped into 18 modules. There were two modules that were significantly correlated with the trait of different cultivars. Enrichment analysis showed that several enriched gene ontology (GO) terms were related to oxidative detoxification and genes associated with hormone signaling pathway occupied a high ratio for the two modules. A total of 52 hub genes were identified, and 48 showed promoter polymorphisms between the two cultivars. Our study suggested that oxidative detoxification and hormone signaling were probably the key pathways for the successful grafting of pecan. The 48 hub genes identified here might be the key genes that led to the divergence of graft survival rates among different pecan cultivars. Our results will lay a foundation for future graft technique improvement in pecan.

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.

Research Highlights: For the first time, the complete chloroplast (cp) genome of Carya illinoinensis cv. ‘Pawnee’ was de novo assembled. Comprehensive analysis the cp genome of C. illinoinensis revealed potential cpDNA markers for intraspecies identification, genes involved in adaptation, and its phylogenetic position. Background and Objectives: C. illinoinensis is an economically important nut tree in the family Juglandaceae. Cp-derived markers are helpful for genetic research, but they still need to be developed in C. illinoinensis. Additionally, the adaptation and phylogenetic relationships of C. illinoinensis have not been revealed based on the complete cp genome. Materials and Methods: Chloroplast genomic DNA of C. illinoinensis cv. ‘Pawnee’ was extracted and subjected to Illumina sequencing. Results: The cp genome is 160,819 bp in size, exhibiting a typical quadripartite structure with a large single copy (LSC) of 90,022 bp, a small single copy (SSC) of 18,791 bp, and a pair of inverted repeats (IRA and IRB) regions of 26,003 bp each. The genome was predicted to encode 112 unique genes, including 79 protein-coding genes, 29 tRNAs, and four rRNAs, with 19 duplicates in the IR regions. In total, 213 SSRs and 44 long repeats were identified in the cp genome. A comparison of two different C. illinoinensis genotypes, ‘Pawnee’ and 87MX3-2.11, obtained 143 SNPs and 74 indels. The highly variable regions such as atpF, clpP, and ndhA genes, and matK-rps16, trnS-trnG, and trnT-psbD intergenic spacers might be helpful for future intraspecific identification. Positive selection was acting on the ccsA and rps12 cp genes based on the Ka/Ks ratios. Phylogenetic analysis indicated that C. illinoinensis forms a sister clade to Asian Carya species, represented by C. kweichowensis and Annamocarya sinensis. Conclusions: The genome information in our study will have significance for further research on the intraspecies identification and genetic improvement of C. illinoinensis.

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.

Dehydration-responsive element binding (DREB) proteins that interact specifically with the DRE cis-acting element, have been identified as a group of important transcription factors that regulate the expression of many stress-inducible genes in plants. In this work, a novel DREB gene ortholog, designated ZjDREB1, was isolated from a cold-tolerant zoysiagrass (Zoysia japonica) cultivar 'Meyer' using RT-PCR and RACE methods. It contained an open reading frame of 774 bp encoding 257 amino acid residues. The deduced protein sequence featured a conserved AP2/ERF DNA-binding domain, a nuclear localisation signal, and an acidic activation region, thereby showing the typical characteristics of the DREB gene family. This is the first report on the isolation of a DREB-like gene in zoysiagrass. Expression profiles of the ZjDREB1 gene in response to cold in three cultivars, 'Meyer', 'Palisades', and 'Lanyin 3' was analysed quantitatively by real-time RT-PCR. The results revealed that ZjDREB1 transcript levels started to rise within 1 h at 4°C, indicating rapid induction by the chilling treatment, and reached a maximum level after 6 h of exposure to low temperature. The level of expression of ZjDREB1 in a cold-tolerant cultivar ('Meyer') was higher than that in a cold-susceptible cultivar ('Lanyin 3'). The level in 'Palisades' was intermediate. A decreasing positive temperature resulted in higher levels of accumulation of ZjDREB1 transcripts, with a major increase below 8°C, peaking at 0°C. It was concluded that ZjDREB1 may play a role in the response of zoysiagrass to cold stress. [ABSTRACT FROM AUTHOR]