ADP-ribosylation factor D1 modulates Golgi morphology, cell plate formation, and plant growth in Arabidopsis.

ADP-ribosylation factor (ARF) family proteins, one type of small guanine-nucleotide-binding (G) proteins, play a central role in regulating vesicular traffic and organelle structures in eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains more than 21 ARF proteins, but relatively little is known about the functional heterogeneity of ARF homologs in plants. Here, we characterized the function of a unique ARF protein, ARFD1B, in Arabidopsis. ARFD1B exhibited both cytosol and punctate localization patterns, colocalizing with a Golgi marker in protoplasts and transgenic plants. Distinct from other ARF1 homologs, overexpression of a dominant-negative mutant form of ARFD1B did not alter the localization of the Golgi marker mannosidase I (ManI)-RFP in Arabidopsis cells. Interestingly, the ARFD1 artificial microRNA knockdown mutant arfd1 displayed a deleterious growth phenotype, while this phenotype was restored in complemented plants. Further, confocal imaging and transmission electron microscopy analyses of the arfd1 mutant revealed defective cell plate formation and abnormal Golgi morphology. Pull-down and liquid chromatography-tandem mass spectrometry analyses identified Coat Protein I (COPI) components as interacting partners of ARFD1B, and subsequent bimolecular fluorescence complementation, yeast (Saccharomyces cerevisiae) two-hybrid, and co-immunoprecipitation assays further confirmed these interactions. These results demonstrate that ARFD1 is required for cell plate formation, maintenance of Golgi morphology, and plant growth in Arabidopsis.

The γ-tubulin complex protein GCP6 is crucial for spindle morphogenesis but not essential for microtubule reorganization in Arabidopsis.

γ-Tubulin typically forms a ring-shaped complex with 5 related γ-tubulin complex proteins (GCP2 to GCP6), and this γ-tubulin ring complex (γTuRC) serves as a template for microtubule (MT) nucleation in plants and animals. While the γTuRC takes part in MT nucleation in most eukaryotes, in fungi such events take place robustly with just the γ-tubulin small complex (γTuSC) assembled by γ-tubulin plus GCP2 and GCP3. To explore whether the γTuRC is the sole functional γ-tubulin complex in plants, we generated 2 mutants of the GCP6 gene encoding the largest subunit of the γTuRC in Arabidopsis thaliana. Both mutants showed similar phenotypes of dwarfed vegetative growth and reduced fertility. The gcp6 mutant assembled the γTuSC, while the wild-type cells had GCP6 join other GCPs to produce the γTuRC. Although the gcp6 cells had greatly diminished γ-tubulin localization on spindle MTs, the protein was still detected there. The gcp6 cells formed spindles that lacked MT convergence and discernable poles; however, they managed to cope with the challenge of MT disorganization and were able to complete mitosis and cytokinesis. Our results reveal that the γTuRC is not the only functional form of the γ-tubulin complex for MT nucleation in plant cells, and that γ-tubulin-dependent, but γTuRC-independent, mechanisms meet the basal need of MT nucleation. Moreover, we show that the γTuRC function is more critical for the assembly of spindle MT array than for the phragmoplast. Thus, our findings provide insight into acentrosomal MT nucleation and organization.

Comparative transcriptomic analyses of glucosinolate metabolic genes during the formation of Chinese kale seeds.

BACKGROUND: To understand the mechanism of glucosinolates (GSs) accumulation in the specific organs, combined analysis of physiological change and transcriptome sequencing were applied in the current study. Taking Chinese kale as material, seeds and silique walls were divided into different stages based on the development of the embryo in seeds and then subjected to GS analysis and transcriptome sequencing. RESULTS: The main GS in seeds of Chinese kale were glucoiberin and gluconapin and their content changed with the development of the seed. During the transition of the embryo from torpedo- to the early cotyledonary-embryo stage, the accumulation of GS in the seed was accompanied by the salient decline of GS in the corresponding silique wall. Thus, the seed and corresponding silique wall at these two stages were subjected to transcriptomic sequencing analysis. 135 genes related to GS metabolism were identified, of which 24 genes were transcription factors, 81 genes were related to biosynthetic pathway, 25 genes encoded catabolic enzymes, and 5 genes matched with transporters. The expression of GS biosynthetic genes was detected both in seeds and silique walls. The high expression of FMOGS-OX and AOP2, which is related to the production of gluconapin by side modification, was noted in seeds at both stages. Interestingly, the expression of GS biosynthetic genes was higher in the silique wall compared with that in the seed albeit lower content of GS existed in the silique wall than in the seed. Combined with the higher expression of transporter genes GTRs in silique walls than in seeds, it was proposed that the transportation of GS from the silique wall to the seed is an important source for seed GS accumulation. In addition, genes related to GS degradation expressed abundantly in the seed at the early cotyledonary-embryo stage indicating its potential role in balancing seed GS content. CONCLUSIONS: Two stages including the torpedo-embryo and the early cotyledonary-embryo stage were identified as crucial in GS accumulation during seed development. Moreover, we confirmed the transportation of GS from the silique wall to the seed and proposed possible sidechain modification of GS biosynthesis may exist during seed formation.

AtNBR1 Is a Selective Autophagic Receptor for AtExo70E2 in Arabidopsis.

Selective autophagy is a subcellular process whereby cytoplasmic materials are selectively sequestered into autophagosomes for subsequent delivery to the vacuole for degradation and recycling. Arabidopsis (Arabidopsis thaliana) NBR1 (next to BRCA1 gene 1 protein; AtNBR1) has been proposed to function as a selective autophagy receptor in plants, whereby AtNBR1 anchors the ubiquitinated targets to autophagosomes for degradation. However, the specific cargos of AtNBR1 remain elusive. We previously showed that Arabidopsis exocyst subunit EXO70 family protein E2 (AtExo70E2), a marker for exocyst-positive organelle (EXPO), colocalized with the autophagosome marker Arabidopsis autophagy-related protein8 (AtATG8) and was delivered to the vacuole for degradation upon autophagic induction. Here, through multiple analyses, we demonstrate that AtNBR1 is a selective receptor for AtExo70E2 during autophagy in Arabidopsis. First, two novel loss-of-function nbr1 CRISPR mutants (nbr1-c1 and nbr1-c2) showed an early-senescence phenotype under short-day growth conditions. Second, during autophagic induction, the vacuolar delivery of AtExo70E2 or EXPO was significantly reduced in nbr1 mutants compared to wild-type plants. Third, biochemical and recruitment assays demonstrated that AtNBR1 specifically interacted and recruited AtExo70E2 or its EXPO to AtATG8-positive autophagosomes in a ubiquitin-associated (UBA)-independent manner during autophagy. Taken together, our data indicate that AtNBR1 functions as a selective receptor in mediating vacuolar delivery of AtExo70E2 or EXPO in a UBA-independent manner in plant autophagy.

Use of Three-Dimensional Arterial Spin Labeling to Evaluate Renal Perfusion in Patients With Chronic Kidney Disease.

BACKGROUND: A noninvasive method for evaluating renal blood flow (RBF) in patients with chronic kidney disease (CKD) may have clinical value in disease staging, management, and prognostication. PURPOSE: To evaluate effectiveness of three-dimensional pseudocontinuous arterial spin labeling (pCASL) and pulsed arterial spin labeling (PASL) in assessment of cortex and outer medulla (cortex/OM) RBF in CKD patients and healthy volunteers (HVs). STUDY TYPE: Prospective, in a single institution. SUBJECTS: A total of 48 CKD patients (stage 1, 2, 3, and 4-5: N = 11, 12, 13, and 12, respectively) and 18 HVs FIELD STRENGTH/SEQUENCE: 3 T, pCASL, and PASL with a three-dimensional hybrid gradient echo/spin echo sequence. ASSESSMENT: Quality of RBF images derived from pCASL and PASL were evaluated and RBF in cortex/OM measured. Clinical and laboratory data were recorded. STATISTICAL TESTS: Image quality differences between pCASL and PASL were evaluated with Wilcoxon signed-rank test. For both methods, analysis of variance, followed by Fisher's LSD-t test, was used to determine whether RBF differed between CKD stages and HVs. Pearson correlation coefficients were calculated to assess strength of relationships between cortex/OM RBF and data from clinical and laboratory tests. RESULTS: Image quality differences were significantly higher in pCASL than PASL in both patients and HVs (both P < 0.05). For pCASL, cortex/OM RBF of patients were significantly lower than those of HVs (P < 0.05). Cortex/OM RBF were higher in S1 and S2 patients than those in S3 and S4-5 (P < 0.05). For PASL, only RBF in cortex of S1 and S2 patients were significantly higher than those of S4-5 (P < 0.05). Good correlations between pCASL RBF and estimated glomerular filtration (eGFR) were found in cortex/OM of patients (rho = 0.796 and 0.798, respectively, both P < 0.05), higher than those between PASL RBF and eGFR (rho = 0.430 and 0.374, respectively, both P < 0.05). DATA CONCLUSION: Three-dimensional pCASL may potentially be a noninvasive technique to assess renal perfusion in CKD patients in different stages. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 2.

Physiological and transcriptomic responses of water spinach (Ipomoea aquatica) to prolonged heat stress.

BACKGROUND: Water spinach (Ipomoea aquatica) is an important heat-resistant leafy vegetable that can survive under long-time heat stress condition. However, the physiological characteristics and molecular changes in its response to heat stress are poorly understood. RESULTS: In this study the selected water spinach cultivars with different thermo resistance and their physiological response to heat stress were examined. Under prolonged heat stress, plant growth was inhibited in all tested cultivars. This inhibition was accompanied by the reduction of photosynthetic performance. The reactive oxygen species system in terms of superoxide and hydrogen peroxide contents, as well as antioxidant polyphenols, were evaluated. The results showed that prolonged heat stress caused reduced antioxidant capacity, but the role of antioxidant capacity in a prolonged thermotolerance was not predominant. Transcriptomic analysis of the water spinach subjected to heat stress revealed that 4145 transcripts were specifically expressed with 2420 up-regulated and 1725 down-regulated in heat-sensitive and heat-tolerant cultivars treated with 42 °C for 15 days. Enrichment analysis of these differentially expressed genes showed that the main metabolic differences between heat-sensitive and heat-tolerant cultivars were the carbohydrate metabolism and phenylpropanoid biosynthesis. The results of carbohydrate profiles and RT-qPCR also suggested that heat stress altered carbohydrate metabolism and associated changes in transcriptional level of genes involved in sugar transport and metabolic transition. CONCLUSIONS: The prolonged heat stress resulted in a reduced antioxidant capacity while the role of antioxidant capacity in a prolonged thermotolerance of water spinach was not predominant. Transcriptome analysis and the measurement of carbohydrates as well as the gene expression evaluation indicated that the response of the metabolic pathway such as carbohydrate and phenylpropanoid biosynthesis to heat stress may be a key player in thermo resistance.

Patterns of Expansion and Expression Divergence of the Polygalacturonase Gene Family in Brassica oleracea.

Plant polygalacturonases (PGs) are closely related to cell-separation events during plant growth and development by degrading pectin. Identifying and investigating their diversification of evolution and expression could shed light on research on their function. We conducted sequence, molecular evolution, and gene expression analyses of PG genes in Brassica oleracea. Ninety-nine B. oleracea PGs (BoPGs) were identified and divided into seven clades through phylogenetic analysis. The exon/intron structures and motifs were conserved within, but divergent between, clades. The second conserved domain (GDDC) may be more closely related to the identification of PGs. There were at least 79 common ancestor PGs between Arabidopsis thaliana and B. oleracea. The event of whole genome triplication and tandem duplication played important roles in the rapid expansion of the BoPG gene family, and gene loss may be an important mechanism in the generation of the diversity of BoPGs. By evaluating the expression in five tissues, we found that most of the expressed BoPGs in clades A, B, and E showed ubiquitous expression characteristics, and the expressed BoPGs in clades C, D, and F were mainly responsible for reproduction development. Most of the paralogous gene pairs (76.2%) exhibited divergent expression patterns, indicating that they may have experienced neofunctionalization or subfunctionalization. The cis-elements analysis showed that up to 96 BoPGs contained the hormone response elements in their promoters. In conclusion, our comparative analysis may provide a valuable data foundation for the further functional analysis of BoPGs during the development of B. oleracea.

Comparative transcriptome analyses revealed different heat stress responses in high- and low-GS Brassica alboglabra sprouts.

BACKGROUND: Chinese kale (Brassica alboglabra) contains high nutritional elements and functional molecules, especially anticarcinogenic and antioxidant glucosinolates (GS), which was highly affected by environment temperature. To investigate the link of GS biosynthesis with heat stress response in Chinese kale, global transcription profiles of high-GS line (HG), low-GS line (LG), high-GS line under heat stress (HGT) and low-GS line under heat stress (LGT) were analyzed. RESULTS: Based on three biological replicates of each RNA sequencing data, 3901, 4062 and 2396 differentially expressed genes in HG vs HGT, LG vs LGT and HGT vs LGT were obtained, respectively. GO annotation, KEGG pathway analysis and a comprehensive analysis of DEGs showed a strong correlation between the GS biosynthesis and heat stress response. It was noticed that 11 differentially expressed genes tied to the GS biosynthesis were down-regulated, 23 heat shock transcription factors and 61 heat shock proteins were up-regulated upon the heat treatment. Another two Chinese kale varieties Cuibao and Shunbao with high- and low- GS content respectively, were used to validate the relationship of GS content and heat-response, and the results showed that high-GS content variety were more thermotolerant than the low-GS content one although GS significantly decreased in both varieties under heat stress. In addition, HSP100/ClpB, HSP90, HSP70 and sHSPs were differentially expressed in high- and low-GS varieties. Notably, HSP90 and sHSPs showed an obviously early response to heat stress than other related genes. CONCLUSION: The higher heat resistance of high-GS Chinese kale and the sharp decrease of glucosinolate content under heat stress indicated a strong relationship of GS accumulation and heat stress response. Combined with the previous report on the low expression of HSP90 at elevated temperatures in GS-deficient mutant TU8 of Arabidopsis, the differential expression pattern of HSP90 in high- and low- GS varieties and its early heat response implied it might be a key regulator in GS metabolism and heat-resistance in Chinese kale.

Classic myrosinase-dependent degradation of indole glucosinolate attenuates fumonisin B1-induced programmed cell death in Arabidopsis.

The mycotoxin fumonisin B1 (FB1) causes the accumulation of reactive oxygen species (ROS) which then leads to programmed cell death (PCD) in Arabidopsis. In the process of studying FB1-induced biosynthesis of glucosinolates, we found that indole glucosinolate (IGS) is involved in attenuating FB1-induced PCD. Treatment with FB1 elevates the expression of genes related to the biosynthesis of camalexin and IGS. Mutants deficient in aliphatic glucosinolate (AGS) or camalexin biosynthesis display similar lesions to Col-0 upon FB1 infiltration; however, the cyp79B2 cyp79B3 double mutant, which lacks induction of both IGS and camalexin, displays more severe lesions. Based on the fact that the classic myrosinase ß-thioglucoside glucohydrolase (TGG)-deficient double mutant tgg1 tgg2, rather than atypical myrosinase-deficient mutant pen2-2, is more sensitive to FB1 than Col-0, and the elevated expression of TGG1, but not of PEN2, correlates with the decrease in IGS, we conclude that TGG-dependent IGS hydrolysis is involved in FB1-induced PCD. Indole-3-acetonitrile (IAN) and indole-3-carbinol (I3C), the common derivatives of IGS, were used in feeding experiments, and this rescued the severe cell death phenotype, which is associated with reduced accumulation of ROS as well as increased activity of antioxidant enzymes and ROS-scavenging ability. Despite the involvement of indole-3-acetic acid (IAA) in restricting FB1-induced PCD, feeding of IAN and I3C attenuated FB1-induced PCD in the IAA receptor mutant tir1-1 just as in Col-0. Taken together, our results indicate that TGG-catalyzed breakdown products of IGS decrease the accumulation of ROS by their antioxidant behavior, and attenuate FB1 induced PCD in an IAA-independent way.

Cloning and expression analysis of betalain biosynthesis genes in Amaranthus tricolor.

OBJECTIVES: To clone genes involved in the betalain biosynthesis pathway and to assess the effects of phytohormones on betalain biosynthesis in Amaranthus tricolor. RESULTS: Five betalain biosynthesis genes were cloned by reverse transcription PCR and rapid amplification of cDNA ends. Betacyanin analyses revealed that pigments accumulated differently in various tissues and under different phytohormone treatments. Quantitative RT-PCR analysis indicated that gene expression levels did not correlate with pigment accumulation. Notably, gene expression and pigment accumulation were negatively regulated by 2,4-dichlorophenoxyacetic acid. The expression of AmaDOPA5-GT, AmaDODA, and AmaB5-GT was induced by pigmentation-promoting 6-benzyl aminopurine (6-BA). and pigmentation-inhibiting gibberellin A3 while AmaTyDC expression was suppressed. AmaTyDC expression was also suppressed by pigmentation-promoting kinetin. Additionally, the expression of AmaB6-GT was suppressed by 6-BA. CONCLUSIONS: The changes in betacyanin levels among various tissues and following phytohormone treatments were related to the differences in betalain biosynthesis gene expression levels.

Ectopic expression of a BZR1-1D transcription factor in brassinosteroid signalling enhances carotenoid accumulation and fruit quality attributes in tomato.

The brassinosteroid (BR) response transcription factor Brassinazole resistant 1 (BZR1)-mediated BR signalling regulates many specific developmental processes including fruit ripening. Here, we report the effect of 2,4-epibrassinolide (EBR) and BZR1-1D overexpression on carotenoid accumulation and quality attributes of tomato (Solanum lycopersicum) fruit. EBR-treated pericarp discs of ethylene-insensitive mutant, Never ripe, accumulated significantly more carotenoid than those of the control. The results suggest that BR seems to be involved in modulating pigments accumulation. When three independent transgenic lines overexpressing the Arabidopsis BZR1-1D were used to evaluate the role of BZR1 in regulating tomato fruit carotenoid accumulation and quality attributes, fruits of all three transgenic lines exhibited enhanced carotenoid accumulation and increased soluble solid, soluble sugar and ascorbic acid contents during fruit ripening. In addition, the fruits of two transgenic lines showed dark green shoulder at mature green stage, in accordance with the up-regulated expression level of SlGLK2, which is involved in chloroplast development. Our results indicate the importance of BZR1-centred BR signalling in regulating carotenoid accumulation and quality attributes of tomato fruit and the potential application of the BZR1-like(s) for improvement of nutritional quality and flavour of tomato through genetic engineering.

Genome-Wide Identification and Expression Analysis of Bx Involved in Benzoxazinoids Biosynthesis Revealed the Roles of DIMBOA during Early Somatic Embryogenesis in Dimocarpus longan Lour.

Benzoxazinoids (BXs) are tryptophan-derived indole metabolites and play a role in various physiological processes, such as auxin metabolism. Auxin is essential in the process of somatic embryogenesis (SE) in plants. In this study, we used bioinformatics, transcriptome data, exogenous treatment experiments, and qPCR analysis to study the evolutionary pattern of Bx genes in green plants, the regulatory mechanism of DlBx genes during early SE, and the effect of 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3-one (DIMBOA) on the early SE in Dimocarpus longan Lour. The results showed that 27 putative DlBxs were identified in the longan genome; the Bx genes evolved independently in monocots and dicots, and the main way of gene duplication for the DlBx was tandem duplication (TD) and the DlBx were strongly constrained by purification selection during evolution. The transcriptome data indicated varying expression levels of DlBx during longan early SE, and most DlBxs responded to light, temperature, drought stress, and 2,4-dichlorophenoxyacetic acid (2,4-D) treatment; qRT-PCR results showed DlBx1, DlBx6g and DlBx6h were responsive to auxin, and treatment with 0.1mg/L DIMBOA for 9 days significantly upregulated the expression levels of DlBx1, DlBx3g, DlBx6c, DlBx6f, DlB6h, DlBx7d, DlBx8, and DlBx9b. The correlation analysis showed a significantly negative correlation between the expression level of DlBx1 and the endogenous IAA contents; DIMBOA significantly promoted the early SE and significantly changed the endogenous IAA content, and the IAA content increased significantly at the 9th day and decreased significantly at the 13th day. Therefore, the results suggested that DIMBOA indirectly promote the early SE by changing the endogenous IAA content via affecting the expression level of DlBx1 and hydrogen peroxide (H2O2) content in longan.

Jasmonic acid and glucose synergistically modulate the accumulation of glucosinolates in Arabidopsis thaliana.

The interplay of plant hormones and glucose (Glu) in regulating glucosinolate accumulation in Arabidopsis thaliana was investigated in this study. Glucose-induced glucosinolate biosynthesis was enhanced significantly by the addition of jasmonic acid (JA), whereas the synergistic effect of salicylic acid (SA) and Glu was less obvious. The enhanced glucosinolate accumulation is associated with elevated expression of genes in glucosinolate biosynthetic pathway, as well as the transcription factors involved in their regulation, such as MYB28, MYB29, MYB34, and MYB122. The induction of indolic and aliphatic glucosinolates after treatment with JA and Glu in JA-insensitive mutants, coi1, jar1, and jin1, was compromised. Moreover, the effect of JA and Glu on glucosinolate contents was dramatically reduced in Glu-insensitive mutants, rgs1-2 and abi5-7. These results indicate a crosstalk between JA and Glu signalling in the regulation of glucosinolate biosynthesis. JA signalling, RGS1 (the putative membrane receptor of Glu signalling), and ABI5, are involved in the synergistic effect of JA and Glu on glucosinolate accumulation.

BZR1 and BES1 participate in regulation of glucosinolate biosynthesis by brassinosteroids in Arabidopsis.

The effect of 24-epibrassinolide (EBR) on glucosinolate biosynthesis in Arabidopsis thaliana was investigated in the present study by using mutants and transgenic plants involved in brassinosteroid (BR) biosynthesis and signal transduction, as well as glucosinolate biosynthesis. The results showed that EBR significantly decreased the contents of major aliphatic glucosinolates including glucoiberin (S3), glucoraphanin (S4), and glucoerucin (T4), as well as the indolic glucosinolates glucobrassicin (IM) and neoglucobrassicin (1IM). In addition, a significantly higher level of glucosinolates accumulated in the BR-deficient mutant cpd and a dramatically lower glucosinolate content in the transgenic plant DWF4-ox overexpressing the BR biosynthetic gene DWF4 compared with their related wild-types, confirmed the repressing effect of BR on glucosinolate biosynthesis. BRI1, the receptor of BR signal transduction, was involved in regulation of glucosinolate biosynthesis by BR. Furthermore, the observation of reduced content of glucosinolates and lower expression levels of glucosinolate biosynthetic genes in 35S-BZR1/bzr1-1D and bes1-D plants compared with the corresponding wild-types suggested that BZR1 and BES1, two important components in BR signal transduction, are responsible for the inhibiting role of BR in glucosinolate biosynthesis. The disappearance of the repressing effect of BR on glucosinolate content in the myb28, myb34, and myb122 mutants indicated that these three MYB factors are important for the regulation of BR in glucosinolate biosynthesis.

The function of BoTCP25 in the regulation of leaf development of Chinese kale.

XG Chinese kale (Brassica oleracea cv. 'XiangGu') is a variety of Chinese kale and has metamorphic leaves attached to the true leaves. Metamorphic leaves are secondary leaves emerging from the veins of true leaves. However, it remains unknown how the formation of metamorphic leaves is regulated and whether it differs from normal leaves. BoTCP25 is differentially expressed in different parts of XG leaves and respond to auxin signals. To clarify the function of BoTCP25 in XG Chinese kale leaves, we overexpressed BoTCP25 in XG and Arabidopsis, and interestingly, its overexpression caused Chinese kale leaves to curl and changed the location of metamorphic leaves, whereas heterologous expression of BoTCP25 in Arabidopsis did not show metamorphic leaves, but only an increase in leaf number and leaf area. Further analysis of the expression of related genes in Chinese kale and Arabidopsis overexpressing BoTCP25 revealed that BoTCP25 could directly bind the promoter of BoNGA3, a transcription factor related to leaf development, and induce a significant expression of BoNGA3 in transgenic Chinese kale plants, whereas this induction of NGA3 did not occur in transgenic Arabidopsis. This suggests that the regulation of Chinese kale metamorphic leaves by BoTCP25 is dependent on a regulatory pathway or elements specific to XG and that this regulatory element may be repressed or absent from Arabidopsis. In addition, the expression of miR319's precursor, a negative regulator of BoTCP25, also differed in transgenic Chinese kale and Arabidopsis. miR319's transcrips were significantly up-regulated in transgenic Chinese kale mature leaves, while in transgenic Arabidopsis, the expression of miR319 in mature leaves was kept low. In conclusion, the differential expression of BoNGA3 and miR319 in the two species may be related to the exertion of BoTCP25 function, thus partially contributing to the differences in leaf phenotypes between overexpressed BoTCP25 in Arabidopsis and Chinese kale.

Revealing spatial multimodal heterogeneity in tissues with SpaTrio.

Capturing and depicting the multimodal tissue information of tissues at the spatial scale remains a significant challenge owing to technical limitations in single-cell multi-omics and spatial transcriptomics sequencing. Here, we developed a computational method called SpaTrio that can build spatial multi-omics data by integrating these two datasets through probabilistic alignment and enabling further analysis of gene regulation and cellular interactions. We benchmarked SpaTrio using simulation datasets and demonstrated its accuracy and robustness. Next, we evaluated SpaTrio on biological datasets and showed that it could detect topological patterns of cells and modalities. SpaTrio has also been applied to multiple sets of actual data to uncover spatially multimodal heterogeneity, understand the spatiotemporal regulation of gene expression, and resolve multimodal communication among cells. Our data demonstrated that SpaTrio could accurately map single cells and reconstruct the spatial distribution of various biomolecules, providing valuable multimodal insights into spatial biology.

Revealing the transcriptional heterogeneity of organ-specific metastasis in human gastric cancer using single-cell RNA Sequencing.

BACKGROUND: Deciphering intra- and inter-tumoural heterogeneity is essential for understanding the biology of gastric cancer (GC) and its metastasis and identifying effective therapeutic targets. However, the characteristics of different organ-tropism metastases of GC are largely unknown. METHODS: Ten fresh human tissue samples from six patients, including primary tumour and adjacent non-tumoural samples and six metastases from different organs or tissues (liver, peritoneum, ovary, lymph node) were evaluated using single-cell RNA sequencing. Validation experiments were performed using histological assays and bulk transcriptomic datasets. RESULTS: Malignant epithelial subclusters associated with invasion features, intraperitoneal metastasis propensity, epithelial-mesenchymal transition-induced tumour stem cell phenotypes, or dormancy-like characteristics were discovered. High expression of the first three subcluster-associated genes displayed worse overall survival than those with low expression in a GC cohort containing 407 samples. Immune and stromal cells exhibited cellular heterogeneity and created a pro-tumoural and immunosuppressive microenvironment. Furthermore, a 20-gene signature of lymph node-derived exhausted CD8+ T cells was acquired to forecast lymph node metastasis and validated in GC cohorts. Additionally, although anti-NKG2A (KLRC1) antibody have not been used to treat GC patients even in clinical trials, we uncovered not only malignant tumour cells but one endothelial subcluster, mucosal-associated invariant T cells, T cell-like B cells, plasmacytoid dendritic cells, macrophages, monocytes, and neutrophils may contribute to HLA-E-KLRC1/KLRC2 interaction with cytotoxic/exhausted CD8+ T cells and/or natural killer (NK) cells, suggesting novel clinical therapeutic opportunities in GC. Additionally, our findings suggested that PD-1 expression in CD8+ T cells might predict clinical responses to PD-1 blockade therapy in GC. CONCLUSIONS: This study provided insights into heterogeneous microenvironment of GC primary tumours and organ-specific metastases and provide support for precise diagnosis and treatment.

Single-Cell RNA Sequencing Reveals the Temporal Diversity and Dynamics of Cardiac Immunity after Myocardial Infarction.

Myocardial infarction (MI) is strongly associated with the temporal regulation of cardiac immunity. However, a variety of current clinical trials have failed because of the lack of post-MI immunomodulating/anti-inflammatory targets. Single-cell RNA sequencing analysis of the cardiac Cd45+ immune cell at 0, 3, 7, and 14 d after injury in a mouse left anterior descending coronary artery ligation model is performed. Major immune cell populations, distinct subsets, and dynamic changes are identified. Macrophages (Mø) are most abundant, peaking at 3 d after infarction. Mø-5 and Mø-6 are the predominant infiltrated subsets at this time point, with strong expression of inflammatory factors. Further analysis demonstrates that suppressing these sets attenuated pathological MI progression by preventing subsequent leukocyte extravasation and adverse remodeling. Abundant apoptotic neutrophils and a profibrotic macrophage subset on days 7 and 14, respectively, are also detected. These results provide a basis for developing cell type- and time-specific interventions in MI.

Sucrose enhances the accumulation of anthocyanins and glucosinolates in broccoli sprouts.

The germination rate, fresh weight, as well as the contents of anthocyanins and glucosinolates in broccoli sprouts treated with different kinds of sugars, including sucrose, glucose, fructose, mannitol, and fructose/glucose (1:1), were investigated. The results showed that all the sugars induced the accumulation of anthocyanins and glucosinolates with sucrose being the most effective one. In accordance with the accumulation of anthocyanins and glucosinolates, the antioxidant level increased while treated with sucrose. The effect of sucrose on expression of genes related to anthocyanin and glucosinolate biosynthesis were also investigated. The genes involved in the biosynthesis and transcriptional regulation of anthocyanin as well as glucosinolate biosynthetic gene Bo-Elong were all up-regulated by sucrose within 12h after the sucrose treatment. The application of sucrose improved the nutrition value of broccoli sprouts by enhancing the biosynthesis of anthocyanin and glucosinolate at the level of transcription.

New insights into AtNBR1 as a selective autophagy cargo receptor in Arabidopsis.

Selective autophagy, mediated by cargo receptors and recruiting specific targets to autophagosomes for degradation and recycling, plays an important role in quality control and cellular homeostasis in eukaryotes. The Arabidopsis AtNBR1 shares a similar domain organization with the mammalian autophagic receptors p62 and NBR1. We recently demonstrated that AtNBR1 functions as a selective autophagy receptor for the exocyst component AtExo70E2, a marker for the Exocyst-positive organelle (EXPO), which was achieved via a specific ATG8-AtNBR1-AtExo70E2 interaction in Arabidopsis. Here we further showed that nbr1 CRISPR mutants exhibit an early senescence phenotype under short-day growth conditions, which can be restored by complementation with expression of AtNBR1pro::AtNBR1-GFP in the mutant. Interestingly, in addition to the typical cytosolic and punctate patterns, YFP-AtNBR1 also exhibited a microtubule pattern particularly in the cortical layer. Treatments with the microtubule depolymerizer oryzalin but not the microfilament depolymerizer latrunculin B abolished the microtubule pattern and affected the vacuolar delivery of YFP-AtNBR1 upon autophagy induction. These results indicated that microtubules may be required for AtNBR1 to shuttle its cargos to the vacuole during plant autophagy. The present study thus sheds new light on the recognition and movement pattern of AtNBR1 in selective autophagy in Arabidopsis.