Drought and low temperature-induced NF-YA1 activates FT expression to promote citrus flowering.

Flower induction in adult citrus is mainly regulated by drought and low temperatures. However, the mechanism of FLOWERING LOCUS T regulation of citrus flowering (CiFT) under two flower-inductive stimuli remains largely unclear. In this study, a citrus transcription factor, nuclear factor YA (CiNF-YA1), was found to specifically bind to the CiFT promoter by forming a complex with CiNF-YB2 and CiNF-YC2 to activate CiFT expression. CiNF-YA1 was induced in juvenile citrus by low temperature and drought treatments. Overexpression of CiNF-YA1 increased drought susceptibility in transgenic citrus, whereas suppression of CiNF-YA1 enhanced drought tolerance in silenced citrus plants. Furthermore, a GOLDEN2 - LIKE protein (CiFE) that interacts with CiFT protein was also isolated. Further experimental evidence showed that CiFE binds to the citrus LEAFY (CiLFY) promoter and activates its expression. In addition, the expressions of CiNF-YA1 and CiFE showed a seasonal increase during the floral induction period and were induced by artificial drought and low-temperature treatments at which floral induction occurred. These results indicate that CiNF-YA1 may activate CiFT expression in response to drought and low temperatures by binding to the CiFT promoter. CiFT then forms a complex with CiFE to activate CiLFY, thereby promoting the flowering of adult citrus.

Two citrus KNAT-like genes, CsKN1 and CsKN2, are involved in the regulation of spring shoot development in sweet orange.

Shoot-tip abortion is a very common phenomenon in some perennial woody plants and it affects the height, architecture, and branch orientation of trees; however, little is currently known about the underlying mechanisms. In this study, we identified a gene in sweet orange (Citrus sinensis) encoding a KNAT-like protein (CsKN1) and found high expression in the shoot apical meristem (SAM). Overexpression of CsKN1 in transgenic plants prolonged the vegetative growth of SAMs, whilst silencing resulted in either the loss or inhibition of SAMs. Yeast two-hybrid analysis revealed that CsKN1 interacted with another citrus KNAT-like protein (CsKN2), and overexpression of CsKN2 in lemon and tobacco caused an extreme multiple-meristem phenotype. Overexpression of CsKN1 and CsKN2 in transgenic plants resulted in the differential expression of numerous genes related to hormone biosynthesis and signaling. Yeast one-hybrid analysis revealed that the CsKN1-CsKN2 complex can bind to the promoter of citrus floral meristem gene LEAFY (CsLFY) and inhibit its expression. These results indicated that CsKN1 might prolong the vegetative growth period of SAMs by delaying flowering. In addition, an ethylene-responsive factor (CsERF) was found to bind to the CsKN1 promoter and suppresses its transcription. Overexpression of CsERF in Arabidopsis increased the contents of ethylene and reactive oxygen species, which might induce the occurrence of shoot-tip abscission. On the basis of our results, we conclude that CsKN1 and CsKN2 might work cooperatively to regulate the shoot-tip abscission process in spring shoots of sweet orange.

Metabolomics Insights into Oleate-Induced Disorders of Phospholipid Metabolism in Macrophages.

BACKGROUND: Diet-induced disordered phospholipid metabolism and disturbed macrophage metabolism contribute to the pathogenesis of metabolic diseases. However, the effects of oleate, a main dietary fatty acid, on macrophage phospholipid metabolism are unclear. OBJECTIVES: We aimed to discover oleate-induced disorders of macrophage phospholipid metabolism and potential therapeutic targets for treating diet-related metabolic diseases. METHODS: RAW 264.7 cells were exposed to 65 µg oleate/mL, within the blood concentration range of humans and mice, to trigger disorders of phospholipid metabolism. Meanwhile, WY-14643 and pioglitazone, 2 drugs widely used for treating metabolic diseases, were employed to prevent oleate-induced disorders of macrophage phospholipid metabolism. Subsequently, an untargeted metabolomics approach based on liquid chromatography-mass spectrometry was used to discover relevant metabolic disorders and potential therapeutic targets. RESULTS: We showed that 196 metabolites involved in phospholipid metabolism were altered upon oleate treatment and interventions of WY-14643 and pioglitazone (P < 0.05, 2-tailed Mann-Whitney U test). Notably, most lysophospholipids were decreased, whereas most phospholipids were increased in oleate-treated macrophages. Phosphatidylethanolamines accumulated most among phospholipids, and their acyl chain polyunsaturation increased in oleate-treated macrophages. Additionally, saturated fatty acids were decreased, whereas polyunsaturated fatty acids were increased in oleate-treated macrophages. Furthermore, changes in phosphatidylglycerols, phosphatidylinositols, cardiolipins, phosphatidates, lysophosphatidylglycerols, and acylcarnitines in oleate-treated macrophages could be attenuated or even abolished by WY-14643 and/or pioglitazone treatment. CONCLUSIONS: Oleate induced accumulation of various phospholipids, increased acyl chain polyunsaturation of phosphatidylethanolamines, and decreased lysophospholipids in RAW 264.7 macrophages. This study suggests macrophage phospholipid and fatty acid metabolism as potential therapeutic targets for intervening diet-related metabolic diseases.

Comparative analysis of the transcriptome, methylome, and metabolome during pollen abortion of a seedless citrus mutant.

KEY MESSAGE: Pollen abortion could be mainly attributed to abnormal meiosis in the mutant. Multiomics analysis uncovered significant epigenetic variations between the mutant and its wild type during the pollen abortion process. Male sterility caused by aborted pollen can result in seedless fruit. A seedless Ponkan mandarin mutant (bud sport) was used to compare the transcriptome, methylome, and metabolome with its progenitor to understand the mechanism of citrus pollen abortion. Cytological observations showed that the anther of the mutant could form microspore mother cells, although the microspores failed to develop fertile pollen at the anther dehiscence stage. Based on pollen phenotypic analysis, pollen abortion could be mainly attributed to abnormal meiosis in the mutant. A transcriptome analysis uncovered the molecular mechanisms underlying pollen abortion between the mutant and its wild type. A total of 5421 differentially expressed genes were identified, and some of these genes were involved in the meiosis, hormone biosynthesis and signaling, carbohydrate, and flavonoid pathways. A total of 50,845 differentially methylated regions corresponding to 15,426 differentially methylated genes in the genic region were found between the mutant and its wild type by the methylome analysis. The expression level of these genes was negatively correlated with their methylation level, especially in the promoter regions. In addition, 197 differential metabolites were identified between the mutant and its wild type based on the metabolome analysis. The transcription and metabolome analysis further indicated that the expression of genes in the flavonoid, carbohydrate, and hormone metabolic pathways was significantly modulated in the pollen of the mutant. These results indicated that demethylation may alleviate the silencing of carbohydrate genes in the mutant, resulting in excessive starch and sugar hydrolysis and thereby causing pollen abortion in the mutant.

Involvements of PCD and changes in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis).

BACKGROUND: Citrus shoot tips abscise at an anatomically distinct abscission zone (AZ) that separates the top part of the shoots into basal and apical portions (citrus self-pruning). Cell separation occurs only at the AZ, which suggests its cells have distinctive molecular regulation. Although several studies have looked into the morphological aspects of self-pruning process, the underlying molecular mechanisms remain unknown. RESULTS: In this study, the hallmarks of programmed cell death (PCD) were identified by TUNEL experiments, transmission electron microscopy (TEM) and histochemical staining for reactive oxygen species (ROS) during self-pruning of the spring shoots in sweet orange. Our results indicated that PCD occurred systematically and progressively and may play an important role in the control of self-pruning of citrus. Microarray analysis was used to examine transcriptome changes at three stages of self-pruning, and 1,378 differentially expressed genes were identified. Some genes were related to PCD, while others were associated with cell wall biosynthesis or metabolism. These results strongly suggest that abscission layers activate both catabolic and anabolic wall modification pathways during the self-pruning process. In addition, a strong correlation was observed between self-pruning and the expression of hormone-related genes. Self-pruning plays an important role in citrus floral bud initiation. Therefore, several key flowering homologs of Arabidopsis and tomato shoot apical meristem (SAM) activity genes were investigated in sweet orange by real-time PCR and in situ hybridization, and the results indicated that these genes were preferentially expressed in SAM as well as axillary meristem. CONCLUSION: Based on these findings, a model for sweet orange spring shoot self-pruning is proposed, which will enable us to better understand the mechanism of self-pruning and abscission.

Effects of biochar additions on the mechanical stability of soil aggregates and their role in the dynamic renewal of aggregates in slope ecological restoration.

Mechanical stability of soil aggregates is important for resisting external disturbances in slope soils. Biochar (BC) is widely used in slope remediation. However, biochar application may not be conducive to the formation of mechanical-stable soil aggregates, and the effects of biochar additions on the mechanical stability of soil aggregates in slope restoration remain largely unclear. In this context, an incubation experiment was conducted in this study with four biochar levels added to artificial soil, namely 0 % (BC0), 1.5 % (BC1), 3 % (BC2), and 4.5 % (BC3), corresponding approximately to 0, 0.77, 1.53 and 2.30 M ha-1, respectively. The contributions of different soil aggregate fractions to maintaining the mechanical stability of aggregates, as well as the main influencing factors and pathways of biochar additions on soil aggregate stability in a dynamic renewal process of aggregates, were investigated in this study. The results showed a decreasing trend in the mean weight diameter (MWD) with increasing biochar levels and BC1 has no significant difference with BC0, showing MWD values of 2.74 and 2.75, respectively. In contrast, BC3 is significantly lower MWD value of 2.18. The BC3 exhibited negative impact on the mechanical stability of the aggregates. Redundancy analysis (RDA) showed that large macroaggregates (>5 mm) exhibited a stronger contribution on the aggregate mechanical stability between all soil aggregate fractions. The random forest (RF) algorithm and structural equation modeling (SEM) indicated that microaggregate-associated soil organic carbon (SOC) contents and soil pH values were the main factors driving the changes in the aggregate mechanical stability caused by biochar applications. Indeed, the biochar level of 1.5 % maintained the stability of macroaggregates and increased the microaggregate-associated SOC content by 35.7 %, which was conducive to the formation of microaggregates within macroaggregates. Our study suggests that the application of biochar at a level of 1.5 % is more beneficial for maintaining the mechanical stability of artificial soil aggregates.

Functional analysis of a PISTILLATA-like gene CcMADS20 involved in floral organs specification in citrus.

PISTILLATA (PI), as a member of MADS-box transcription factor, plays an important role in petal and stamen specification in Arabidopsis. However, little is known about PI-like genes in citrus. To understand the molecular mechanism of PI during the developmental process of citrus flower, a PI-like gene CcMADS20 was isolated from Citrus Clemantina. Sequence alignment and phylogenetic analysis revealed that CcMADS20 had relatively high similarity with PI-like homolog and was classified in the core dicotyledonous group. The temporal and spatial expression analyses showed that CcMADS20 was specifically expressed in petal and stamen of citrus flower, which was consistent with PI expression pattern in Arabidopsis. Protein interaction revealed that CcMADS20 could form heterodimer with AP3-like proteins. Furthermore, ectopic overexpression of CcMADS20 in Arabidopsis resulted in transformation of sepals into petal-like structure, as observed in other plants overexpressing a functional PI-like homolog. Additionally, promoter fragments of CcMADS20 were also cloned in the representative 21 citrus varieties. Interestingly, four types of promoters were discovered in these citrus varieties, resulting from two stable insert/deletion fragments (Locus1 and Locus2). The homo/hetero-zygosity of promoter alleles in each variety was strongly related to the evolutionary origin of citrus. Four promoters activity analysis indicated that Locus1 presence inhibited CcMADS20 transcriptional activity and Locus2 presence promoted its transcriptional activity. These findings suggested that CcMADS20 determines petal and stamen development during the evolutionary process of citrus and four promoters discovered, as effective genetic markers, are valuable for citrus breeding practices.

Transcriptome profile analysis of flowering molecular processes of early flowering trifoliate orange mutant and the wild-type [Poncirus trifoliata (L.) Raf.] by massively parallel signature sequencing.

BACKGROUND: After several years in the juvenile phase, trees undergo flowering transition to become mature (florally competent) trees. This transition depends on the balanced expression of a complex network of genes that is regulated by both endogenous and environmental factors. However, relatively little is known about the molecular processes regulating flowering transition in woody plants compared with herbaceous plants. RESULTS: Comparative transcript profiling of spring shoots after self-pruning was performed on a spontaneously early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata) with a short juvenile phase and the wild-type (WT) tree by using massively parallel signature sequencing (MPSS). A total of 16,564,500 and 16,235,952 high quality reads were obtained for the WT and the mutant (MT), respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed genes in the MT (31,468) was larger than in the WT (29,864), suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts revealed that 2735 genes had more than twofold expression difference in the MT compared with the WT. In addition, we identified 110 citrus flowering-time genes homologous with known elements of flowering-time pathways through sequencing and bioinformatics analysis. These genes are highly conserved in citrus and other species, suggesting that the functions of the related proteins in controlling reproductive development may be conserved as well. CONCLUSION: Our results provide a foundation for comparative gene expression studies between WT and precocious trifoliate orange. Additionally, a number of candidate genes required for the early flowering process of precocious trifoliate orange were identified. These results provide new insight into the molecular processes regulating flowering time in citrus.

Molecular cloning and functional characterization of genes associated with flowering in citrus using an early-flowering trifoliate orange (Poncirus trifoliata L. Raf.) mutant.

To isolate differentially expressed genes during the juvenile-to-adult phase transition of an early-flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata), suppression subtractive hybridization was performed. In total, 463 cDNA clones chosen by differential screening of 1,920 clones were sequenced and 178 differentially expressed genes were identified, among which 41 sequences did not match any known nucleotide sequence. Analysis of expression profiles of the differentially expressed genes through hybridization on customized chips revealed their expression change was associated with the phase transition from juvenile to adult in the mutant. Open reading frames of nine selected genes were successfully determined by rapid amplification of cDNA ends. Expression analysis of these genes by real-time RT-PCR showed that transcript levels of several genes were associated with floral induction and inflorescence development. Among these genes, HM596718, a sequence sharing a high degree of similarity with Arabidopsis EARLY FLOWERING 5 (AtELF5) was discovered. Real-time PCR and in situ hybridization indicated its expression pattern was closely correlated with floral induction and flowering of the mutant. Ectopic expression of the gene in Arabidopsis caused early flowering; however, its functional characterization is different than the role of AtELF5 observed in Arabidopsis. A yeast two-hybrid assay indicated that PtELF5 significantly interacted with DUF1336 domain of a hypothetical protein, which has not yet been functionally characterized in woody plants. These findings suggest that PtELF5 may be a novel gene that plays an important role during the early flowering of precocious trifoliate orange.

HD-ZIP I Transcription Factor (PtHB13) Negatively Regulates Citrus Flowering through Binding to FLOWERING LOCUS C Promoter.

For floral induction in adult citrus, low temperature is one of the most important environmental factors. FLOWERING LOCUS C (FLC) plays a very important role in low-temperature-induced Arabidopsis flowering by repressed FLC expression under exposure to prolonged low-temperature conditions. However, little is known about the FLC regulation mechanism in perennial woody plants such as citrus. In this study, the functions of citrus FLC homolog (PtFLC) were investigated by ectopic expression in Arabidopsis. Transcription factor of homeodomain leucine zipper I (HD-ZIP I) as an upstream regulator of PtFLC was identified by yeast one-hybrid screen to regulate its transcription. The HD-ZIP I transcription factor was highly homologous to Arabidopsis ATHB13 and thus was named PtHB13. Ectopically expressed PtHB13 inhibited flowering in transgenic Arabidopsis. Furthermore, the expression of PtFLC and PtHB13 showed a seasonal change during the floral induction period and was also affected by low temperature. Thus, we propose that PtHB13 binds to PtFLC promoter to regulate its activity during the citrus floral induction process.

Genetic regulation of flowering time in annual and perennial plants.

Flowering time plays a significant role in the reproductive success of plants. So far, five major pathways to flowering have been characterized in Arabidopsis, including environmental induction through photoperiod, vernalization, and gibberellins and autonomous floral iation, and aging by sequentially operating miRNAs (typically miR156 and miR172) responding to endogenous cues. The balance of signals from these pathways is integrated by a common set of genes (FLOWERING LOCUS C, FLOWERING LOCUS T, LEAFY, and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) that determine the flowering time. Recent studies have indicated that epigenetic modification, alternative splicing, antisense RNA and chromatin silencing regulatory mechanisms play an important role in this process by regulating related flowering gene expression. In this review, we discuss the current understanding in genetic regulation of the phase transition from vegetative to reproductive growth by using Arabidopsis as a model. We also describe how this knowledge has been successfully applied for identifying homologous genes from perennial crops. Furthermore, detailed analysis of the similarities and differences between annual and perennial plants flowering will help elucidate the mechanisms of perennial plant maturation and regulation of floral initiation.

Identification of miRNAs and their target genes using deep sequencing and degradome analysis in trifoliate orange [Poncirus trifoliata L. Raf] [corrected].

To identify novel as well as conserved miRNAs in citrus, deep sequencing of small RNA library combined with microarray was performed in precocious trifoliate orange (an early flowering mutant of trifoliate orange, Poncirus trifoliata L. Raf.), resulting in the obtainment of a total of 114 conserved miRNAs belonging to 38 families and 155 novel miRNAs. The miRNA star sequences of 39 conserved miRNAs and 27 novel miRNAs were also discovered among newly identified miRNAs, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 172 and 149 genes were identified as targets of conserved miRNAs and novel miRNAs, respectively. GO and KEGG annotation revealed that high ranked miRNA-target genes were those implicated in biological and metabolic processes. To characterize those miRNAs expressed at the juvenile and adult development stages of citrus, further analysis on the expression profiles of these miRNAs through hybridizing the commercial microarray and real-time PCR was performed. The results revealed that some miRNAs were down-regulated at adult stage compared with juvenile stage. Detailed comparison of the expression patterns of some miRNAs and corresponding target genes revealed the negative correlation between them, while few of them are positively correlated.

A global view of gene activity at the flowering transition phase in precocious trifoliate orange and its wild-type [Poncirus trifoliata (L.) Raf.] by transcriptome and proteome analysis.

Most of what we know about the molecular genetics of flowering time regulation comes from studies in the model plants. However, little is known about the regulation of flowering transition in perennial species or in species with particular growth habits compared with model plants. Here comparative transcriptome and proteome profiling of spring shoots was performed on an early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata) and its wild-type. A total of 19,215 read-mapped genes were observed in two genotypes by RNA-Seq. Of these, 1450 and 1159 genes were specifically observed in the mutant and wild-type libraries, respectively. There were 355 genes that were expressed differently in the two genotypes. A total of 1664 proteins were identified by the iTRAQ technique, and transcript and protein profiles were parallel across the time course for 50% of the comparisons made, but divergent patterns were also observed, indicative of post-transcriptional events. In addition, a global survey of messenger RNA splicing events identified 16,343 splice junctions among 12,688 genes and showed that alternative 3' splice is the most prevalent form of alternative splicing. We further identify 5698 novel transcribed regions that are not overlapping with annotated citrus transcriptome in two genotypes. Understanding of the regulation of flowering transition in citrus can help in the development of new genetic or management strategies to improve fruit production.

Identification and comparative profiling of miRNAs in an early flowering mutant of trifoliate orange and its wild type by genome-wide deep sequencing.

MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes.