Reactive oxygen species act as the key signaling molecules mediating light-induced anthocyanin biosynthesis in Eucalyptus.

Light plays a pivotal role in regulating anthocyanin biosynthesis in plants, and the early light-responsive signals that initiate anthocyanin biosynthesis remain to be elucidated. In this study, we showed that the anthocyanin biosynthesis in Eucalyptus is hypersensitive to increased light intensity. The combined transcriptomic and metabolomic analyses were conducted on Eucalyptus leaves after moderate (ML; 100 µmol m-2 s-1) and high (HL; 300 µmol m-2 s-1) light intensity treatments. The results identified 1940, 1096, 1173, and 2756 differentially expressed genes at 6, 12, 24, and 36 h after HL treatment, respectively. The metabolomic results revealed the primary anthocyanin types, and other differentially accumulated flavonoids and phenylpropane intermediates that were produced in response to HL, which well aligned with the transcriptome results. Moreover, biochemical analysis showed that HL inhibited peroxidase activity and increased the ROS level in Eucalyptus leaves. ROS depletion through co-application of the antioxidants rutin, uric acid, and melatonin significantly reduced, and even abolished, anthocyanin biosynthesis induced by HL treatment. Additionally, exogenous application of hydrogen peroxide efficiently induced anthocyanin biosynthesis within 24 h, even under ML conditions, suggesting that ROS played a major role in activating anthocyanin biosynthesis. A HL-responsive MYB transcription factor EgrMYB113 was identified to play an important role in regulating anthocyanin biosynthesis by targeting multiple anthocyanin biosynthesis genes. Additionally, the results demonstrated that gibberellic acid and sugar signaling contributed to HL-induced anthocyanin biosynthesis. Conclusively, these results suggested that HL triggers multiple signaling pathways to induce anthocyanin biosynthesis, with ROS acting as indispensable mediators in Eucalyptus.

Inhibition of flowering by gibberellins in the woody plant Jatropha curcas is restored by overexpression of JcFT.

Jatropha curcas (J. curcas) is a perennial oil-seed plant with vigorous vegetative growth but relatively poor reproductive growth and low seed yield. Gibberellins (GAs) promotes flowering in most annual plants but inhibits flowering in many woody plants, including J. curcas. However, the underlying mechanisms of GA inhibits flowering in perennial woody plants remain unclear. Here, we found that overexpression of the GA biosynthesis gene JcGA20ox1 inhibits flowering in J. curcas and in J. curcas × J. integerrima hybrids. Consistent with this finding, overexpression of the GA catabolic gene JcGA2ox6 promotes flowering in J. curcas. qRTPCR revealed that inhibits floral transition by overexpressing JcGA20ox1 resulted from a decrease in the expression of JcFT and other flowering-related genes, which was restored by overexpressing JcFT in J. curcas. Overexpression of JcGA20ox1 or JcGA2ox6 reduced seed yield, but overexpression of JcFT significantly increased seed yield. Furthermore, hybridization experiments showed that the reduction in seed yield caused by overexpression of JcGA20ox1 or JcGA2ox6 was partially restored by the overexpression of JcFT. In addition, JcGA20ox1, JcGA2ox6 and JcFT were also found to be involved in the regulation of seed oil content and endosperm development. In conclusion, our study revealed that the inhibitory effect of GA on flowering is mediated through JcFT and demonstrated the effects of JcGA20ox1, JcGA2ox6 and JcFT on agronomic traits in J. curcas. This study also indicates the potential value of GA metabolism genes and JcFT in the breeding of new varieties of woody oil-seed plants.

Cytokinin promotes anthocyanin biosynthesis via regulating sugar accumulation and MYB113 expression in Eucalyptus.

Anthocyanins are flavonoid-like substances that play important roles in plants' adaptation to various environmental stresses. In this research, we discovered that cytokinin (CK) alone could effectively induce the anthocyanin biosynthesis in Eucalyptus and many other perennial woody plant species, but not in tobacco and Arabidopsis, suggesting a diverse role of CK in regulating anthocyanin biosynthesis in different species. Transcriptomic and metabolomic strategies were used to further clarify the specific role of CK in regulating anthocyanin biosynthesis in Eucalyptus. The results showed that 801 and 2241 genes were differentially regulated at 6 and 24 h, respectively, after CK treatment. Pathway analysis showed that most of the differentially expressed genes were categorized into pathways related to cellular metabolism or transport of metabolites, including amino acids and sugars. The metabolomic results well supported the transcriptome data, which showed that most of the differentially regulated metabolites were related to the metabolism of sugar, amino acids and flavonoids. Moreover, CK treatment significantly induced the accumulation of sucrose in the CK-treated leaves, while sugar starvation mimicked by either defoliation or shading treatment of the basal leaves significantly reduced the sugar increase of the CK-treated leaves and thus inhibited CK-induced anthocyanin biosynthesis. The results of in vitro experiment also suggested that CK-induced anthocyanin in Eucalyptus was sugar-dependent. Furthermore, we identified an early CK-responsive transcription factor MYB113 in Eucalyptus, the expression of which was significantly upregulated by CK treatment in Eucalyptus, but was inhibited in Arabidopsis. Importantly, the overexpression of EgrMYB113 in the Eucalyptus hairy roots was associated with significant anthocyanin accumulation and upregulation of most of the anthocyanin biosynthetic genes. In conclusion, our study demonstrates a key role of CK in the regulation of anthocyanin biosynthesis in Eucalyptus, providing a molecular basis for further understanding the regulatory mechanism and diversity of hormone-regulated anthocyanin biosynthesis in different plant species.

Identification of flowering genes in Camellia perpetua by comparative transcriptome analysis.

Camellia perpetua has the excellent characteristic of flowering multiple times throughout the year, which is of great importance to solve the problem of "short flowering period" and "low fresh flower yield" in the yellow Camellia industry at present. Observations of flowering phenology have demonstrated that most floral buds of C. perpetua were formed by the differentiation of axillary buds in the scales at the base of the terminal buds of annual branches. However, the molecular mechanism of flowering in C. perpetua is still unclear. In this study, we conducted a comparative transcriptomic study of the terminal buds and their basal flower buds in March (spring) and September (autumn) using RNA-seq and found that a total of 11,067 genes were significantly differentially expressed in these two periods. We identified 27 genes related to gibberellin acid (GA) synthesis, catabolism, and signal transduction during floral bud differentiation. However, treatment of the terminal buds and axillary buds of C. perpetua on annual branch with GA3 did not induce floral buds at the reproductive growth season (in August) but promoted shoot sprouting. Moreover, 203 flowering genes were identified from the C. perpetua transcriptome library through homology alignment, including flowering integrators LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO), as well as MADS-box, SQUAMOSA PROMOTER BINDING PROTEIN-box (SBP-box), and TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) genes, which were specifically upregulated in floral buds and were likely involved in flowering in C. perpetua. The floral inhibitor CperTFL1b was identified and cloned from C. perpetua, and its expression level was specifically regulated in terminal buds in autumn. Ectopic overexpression of CperTFL1b delayed flowering time and produced abnormal inflorescence and floral organs in Arabidopsis, suggesting that CperTFL1b inhibits flowering. In conclusion, this study deepens our understanding of the molecular mechanism of blooms throughout the year in C. perpetua and provides a helpful reference for cultivating new varieties of yellow Camellia with improved flowering traits.

JcSEUSS1 negatively regulates reproductive organ development in perennial woody Jatropha curcas.

MAIN CONCLUSION: Overexpression of JcSEUSS1 resulted in late flowering, reduced flower number, wrinkled kernels, and decreased seed yield in Jatopha curcas, while downregulation of JcSEUSS1 increased flower number and seed production. The seed oil of Jatropha curcas is suitable as an ideal alternative for diesel fuel, yet the seed yield of Jatropha is restricted by its small number of female flowers and low seed setting rate. Therefore, it is crucial to identify genes that regulate flowering and seed set, and hence improve seed yield. In this study, overexpression of JcSEUSS1 resulted in late flowering, fewer flowers and fruits, and smaller fruits and seeds, causing reduced seed production and oil content. In contrast, the downregulation of JcSEUSS1 by RNA interference (RNAi) technology caused an increase in the flower number and seed yield. However, the flowering time, seed number per fruit, seed weight, and size exhibited no obvious changes in JcSEUSS1-RNAi plants. Moreover, the fatty acid composition also changed in JcSEUSS1 overexpression and RNAi plants, the percentage of unsaturated fatty acids (FAs) was increased in overexpression plants, and the saturated FAs were increased in RNAi plants. These results indicate that JcSEUSS1 played a negative role in regulating reproductive growth and worked redundantly with other genes in the regulation of flowering time, seed number per fruit, seed weight, and size.

Transcriptome-Based Construction of the Gibberellin Metabolism and Signaling Pathways in Eucalyptus grandis × E. urophylla, and Functional Characterization of GA20ox and GA2ox in Regulating Plant Development and Abiotic Stress Adaptations.

Gibberellins (GAs) are the key regulators controlling plant growth, wood production and the stress responses in perennial woody plants. The role of GA in regulating the above-mentioned processes in Eucalyptus remain largely unclear. There is still a lack of systematic identification and functional characterization of GA-related genes in Eucalyptus. In this study, a total of 59,948 expressed genes were identified from the major vegetative tissues of the E. grandis × E. urophylla using transcriptome sequencing. Then, the key gene families in each step of GA biosynthesis, degradation and signaling were investigated and compared with those of Arabidopsis, rice, and Populus. The expression profile generated using Real-time quantitative PCR showed that most of these genes exhibited diverse expression patterns in different vegetative organs and in response to abiotic stresses. Furthermore, we selectively overexpressed EguGA20ox1, EguGA20ox2 and EguGA2ox1 in both Arabidopsis and Eucalyptus via Agrobacterium tumefaciens or A. rhizogenes-mediated transformation. Though both Arabidopsis EguGA20ox1- and EguGA20ox2-overexpressing (OE) lines exhibited better vegetative growth performance, they were more sensitive to abiotic stress, unlike EguGA2ox1-OE plants, which exhibited enhanced stress resistance. Moreover, overexpression of EguGA20ox in Eucalyptus roots caused significantly accelerated hairy root initiation and elongation and improved root xylem differentiation. Our study provided a comprehensive and systematic study of the genes of the GA metabolism and signaling and identified the role of GA20ox and GA2ox in regulating plant growth, stress tolerance, and xylem development in Eucalyptus; this could benefit molecular breeding for obtaining high-yield and stress-resistant Eucalyptus cultivars.

Macadamia germplasm and genomic database (MacadamiaGGD): A comprehensive platform for germplasm innovation and functional genomics in Macadamia.

As an important nut crop species, macadamia continues to gain increased amounts of attention worldwide. Nevertheless, with the vast increase in macadamia omic data, it is becoming difficult for researchers to effectively process and utilize the information. In this work, we developed the first integrated germplasm and genomic database for macadamia (MacadamiaGGD), which includes five genomes of four species; three chloroplast and mitochondrial genomes; genome annotations; transcriptomic data for three macadamia varieties, germplasm data for four species and 262 main varieties; nine genetic linkage maps; and 35 single-nucleotide polymorphisms (SNPs). The database serves as a valuable collection of simple sequence repeat (SSR) markers, including both markers that are based on macadamia genomic sequences and developed in this study and markers developed previously. MacadamiaGGD is also integrated with multiple bioinformatic tools, such as search, JBrowse, BLAST, primer designer, sequence fetch, enrichment analysis, multiple sequence alignment, genome alignment, and gene homology annotation, which allows users to conveniently analyze their data of interest. MacadamiaGGD is freely available online (http://MacadamiaGGD.net). We believe that the database and additional information of the SSR markers can help scientists better understand the genomic sequence information of macadamia and further facilitate molecular breeding efforts of this species.

Genome-Wide Identification and Expression Analysis of Cytokinin Response Regulator (RR) Genes in the Woody Plant Jatropha curcas and Functional Analysis of JcRR12 in Arabidopsis.

The cytokinin (CK) response regulator (RR) gene family plays a pivotal role in regulating the developmental and environmental responses of plants. Axillary bud outgrowth in the perennial woody plant Jatropha curcas is regulated by the crosstalk between CK and gibberellins (GA). In this study, we first analyzed the effects of gibberellin A3 (GA3), lovastatin (a CK synthesis inhibitor), decapitation, and their interaction, on the outgrowth of axillary buds. The results indicate that lovastatin completely inhibited GA-promoted axillary bud outgrowth and partially weakened the decapitation-promoted axillary bud outgrowth. To further characterize and understand the role of CK signaling in promoting the development of female flowers and branches, we performed bioinformatics and expression analyses to characterize the CK RR gene (JcRR) family in J. curcas. A total of 14 members of the JcRR family were identified; these genes were distributed on 10 chromosomes. Phylogenetic analysis indicated that the corresponding RR proteins are evolutionarily conserved across different plant species, and the Myb-like DNA-binding domain divides the 14 members of the JcRR family into type-A and type-B proteins. Further analysis of cis-acting elements in the promoter regions of JcRRs suggests that JcRRs are expressed in response to phytohormones, light, and abiotic stress factors; thus, JcRRs may be involved in some plant development processes. Genomic sequence comparison revealed that segmental duplication may have played crucial roles in the expansion of the JcRR gene family, and five pairs of duplicated genes were all subjected to purifying selection. By analyzing RNA sequencing (RNA-seq) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) data, we characterized that the temporospatial expression patterns of JcRRs during the development of various tissues and the response of these genes to phytohormones and abiotic stress. The JcRRs were mainly expressed in the roots, while they also exhibited differential expression patterns in other tissues. The expression levels of all six type-A and one type-B JcRRs increased in response to 6-benzylaminopurine (6-BA), while the four type-B JcRRs levels decreased. The expression levels of two type-B JcRRs increased in response to exogenous GA3 treatment, while those of three type-A and three type-B JcRRs decreased. We found that type-A JcRRs may play a positive role in the continuous growth of axillary buds, while the role of type-B JcRRs might be the opposite. In response to abiotic stress, the expression levels of two type-A and three type-B JcRRs strongly increased. The overexpression of JcRR12 in Arabidopsis thaliana slightly increased the numbers of rosette branches after decapitation, but not under normal conditions. In conclusion, our results provide detailed knowledge of JcRRs for further analysis of CK signaling and JcRR functions in J. curcas.

An ortholog of the MADS-box gene SEPALLATA3 regulates stamen development in the woody plant Jatropha curcas.

MAIN CONCLUSION: Overexpression of JcSEP3 causes defective stamen development in Jatropha curcas, in which brassinosteroid and gibberellin signaling pathways may be involved. SEPALLATAs (SEPs), the class E genes of the ABCE model, are required for floral organ determination. In this study, we investigated the role of the JcSEP3 gene in floral organ development in the woody plant Jatropha curcas. Transgenic Jatropha plants overexpressing JcSEP3 displayed abnormal phenotypes such as deficient anthers and pollen, as well as free stamen filaments, whereas JcSEP3-RNA interference (RNAi) transgenic plants had no obvious phenotypic changes, suggesting that JcSEP3 is redundant with other JcSEP genes in Jatropha. Moreover, we compared the transcriptomes of wild-type plants, JcSEP3-overexpressing, and JcSEP3-RNAi transgenic plants. In the JcSEP3-overexpressing transgenic plants, we discovered 25 upregulated genes involved in anther and pollen development, as well as 12 induced genes in brassinosteroid (BR) and gibberellin (GA) signaling pathways. These results suggest that JcSEP3 directly or indirectly regulates stamen development, concomitant with the regulation of BR and GA signaling pathways. Our findings help to understand the roles of SEP genes in stamen development in perennial woody plants.

Characterization of the bark storage protein gene (JcBSP) family in the perennial woody plant Jatropha curcas and the function of JcBSP1 in Arabidopsis thaliana.

BACKGROUND: Bark storage protein (BSP) plays an important role in seasonal nitrogen cycling in perennial deciduous trees. However, there is no report on the function of BSP in the perennial woody oil plant Jatropha curcas. METHODS: In this study, we identified six members of JcBSP gene family in J. curcas genome. The patterns, seasonal changes, and responses to nitrogen treatment in gene expression of JcBSPs were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Overexpression of JcBSP1 in transgenic Arabidopsis thaliana was driven by a constitutive cauliflower mosaic virus (CaMV) 35S RNA promoter. RESULTS: JcBSP members were found to be expressed in various tissues, except seeds. The seasonal changes in the total protein concentration and JcBSP1 expression in the stems of J. curcas were positively correlated, as both increased in autumn and winter and decreased in spring and summer. In addition, the JcBSP1 expression in J. curcas seedlings treated with different concentrations of an NH4NO3 solution was positively correlated with the NH4NO3 concentration and application duration. Furthermore, JcBSP1 overexpression in Arabidopsis resulted in a phenotype of enlarged rosette leaves, flowers, and seeds, and significantly increased the seed weight and yield in transgenic plants.

Efficiency of graft-transmitted JcFT for floral induction in woody perennial species of the Jatropha genus depends on transport distance.

FLOWERING LOCUS T (FT) promotes flowering by integrating six genetic pathways. In Arabidopsis, the FT protein is transported from leaves to shoot apices and induces flowering. However, contradictory conclusions about floral induction via graft-transmitted FT in trees were reported in previous studies. We obtained extremely early-flowering transgenic woody Jatropha curcas L. by overexpression of J. curcas FT using Arabidopsis thaliana SUCROSE TRANSPORTER 2 (SUC2) promoter (SUC2:JcFT) and non-flowering transgenic J. curcas by RNA interference (RNAi), which were used to investigate the function of graft-transmitted JcFT in floral induction in woody perennials. Scions from five wild-type species of the Jatropha genus and from JcFT-RNAi transgenic J. curcas were grafted onto SUC2:JcFT rootstocks. Most grafted plants produced flowers in 1-2 months, and the flowering percentage and frequency of various grafted plants decreased with increasing scion length. Consistently, FT protein abundance in scions also decreased with increasing distance from graft junctions to the buds. These findings suggest that FT proteins can be transmitted by grafting and can induce the floral transition in woody perennials, and the efficiency of graft-transmitted JcFT for floral induction depends on the scion length, which may help explain previous seemingly contradictory observations regarding floral induction via graft-transmitted FT in trees.

Extended mining of the oil biosynthesis pathway in biofuel plant Jatropha curcas by combined analysis of transcriptome and gene interactome data.

BACKGROUND: Jatropha curcas L. is an important non-edible oilseed crop with a promising future in biodiesel production. However, little is known about the molecular biology of oil biosynthesis in this plant when compared with other established oilseed crops, resulting in the absence of agronomically improved varieties of Jatropha. To extensively discover the potentially novel genes and pathways associated with the oil biosynthesis in J. curcas, new strategy other than homology alignment is on the demand. RESULTS: In this study, we proposed a multi-step computational framework that integrates transcriptome and gene interactome data to predict functional pathways in non-model organisms in an extended process, and applied it to study oil biosynthesis pathway in J. curcas. Using homologous mapping against Arabidopsis and transcriptome profile analysis, we first constructed protein-protein interaction (PPI) and co-expression networks in J. curcas. Then, using the homologs of Arabidopsis oil-biosynthesis-related genes as seeds, we respectively applied two algorithm models, random walk with restart (RWR) in PPI network and negative binomial distribution (NBD) in co-expression network, to further extend oil-biosynthesis-related pathways and genes in J. curcas. At last, using k-nearest neighbors (KNN) algorithm, the predicted genes were further classified into different sub-pathways according to their possible functional roles. CONCLUSIONS: Our method exhibited a highly efficient way of mining the extended oil biosynthesis pathway of J. curcas. Overall, 27 novel oil-biosynthesis-related gene candidates were predicted and further assigned to 5 sub-pathways. These findings can help better understanding of the oil biosynthesis pathway of J. curcas, as well as paving the way for the following J. curcas breeding application.

First Report of Collar Rot in Purple Passion Fruit (Passiflora edulis) Caused by Neocosmospora solani in Yunnan province, China.

Purple passion fruit (Passiflora edulis Sims) is a perennial climbing vine native to South America that is grown worldwide as an edible tropical fruit with excellent nutritional value and high economic value (Zibadi et al. 2007). With the increasing expansion of the plantation area in China, considerable economic loss caused by collar rot has attracted wide attention. From 2018-2020, collar rot resulted in the death of many plants of P. edulis 'Mantianxing', a commercial cultivar in China, in southwest China's Yunnan province. The disease spread quickly, and field incidence reached more than 50%. Stem rot symptoms were observed at the base of the stem, about 5-10 cm from the ground, resulting in wilting, defoliation, and death of plants. Representative symptomatic samples were collected from the base of five plants, surface disinfested for 30 seconds with 75% ethanol and 15 min with 10% hypochlorite, washed three times with sterile distilled water, then transferred to potato dextrose agar (PDA) dishes. After 2 days in the dark at 28℃, emerging fungal colonies were purified on new PDA dishes cultured at 28℃ for 7 days. The mycelia were flocculent. The color of the surface and the reverse colony was white and cream, respectively. On synthetic nutrient agar (SNA) medium, microconidia were oval, ellipsoidal or reniform, 0- or 1-septate, and 6.7-23.1 µm in length (n>30); macroconidia were straight to slightly curved, 3- or 5-septate, and 30.8-53.9 µm in length (n>30). Genomic DNA, extracted from six isolates, was amplified with three pairs of primers, ITS1 and ITS4 (White et al. 1990) , EF1-728F and EF1-986R (Carbone and Kohn 1999), and fRPB2-5F and fRPB2-7cR (Liu et al. 1999). The amplicons from all six isolates were sequenced and identical sequences obtained. The sequence of one representative isolate was uploaded to NCBI (National Center for Biotechnology Information) and analyzed with BLASTn in the Fusarium MLST database (https://fusarium.mycobank.org). The sequence of the internal transcribed spacer 1 (ITS1) region (GenBank MN944550) showed 99.1% (449/453 bp) identity to Fusarium solani strain NRRL 53667 (syn: Neocosmospora solani, GenBank MH582405). The sequence of the translation elongation factor-1 (EF-1) gene (GenBank MN938933) showed 97.8% identity (263/269 bp) to F. solani strain NRRL 32828 (GenBank DQ247135). The sequence of the second largest subunit of RNA polymerase Ⅱ (RPB2) gene (GenBank MW002686) showed 98.7% identity (810/821 bp) to F. solani strain NRRL 43441 (GenBank MH582407). Based on a multilocus phylogenetic analysis of the ITS1, EF-1 and RPB2 sequences, coupled with the morphological characteristics, the isolate (designated as NsPed1) was considered to be Neocosmospora solani (syn: Fusarium solani) (Crespo et al. 2019). Subsequently, three-month-old healthy seedlings and 45-day-old cuttings of P. edulis 'Mantianxing' plants were inoculated with the isolate NsPed1 to test its pathogenicity. Stems were wounded, approximately 1-2 mm deep, in the collar region of plants at 2 cm above the soil. A disk (9 mm in diameter) of NsPed1-colonized PDA was placed on the wound. Sterile PDA served as controls. All plants were kept in a growth chamber with 28-30°C, 60% relative humidity, and 16/8-h light/dark photoperiod. Fifteen plants were used for each treatment and replicated three times. Two weeks after inoculation, the stems of the inoculated plants turned brown with a lesion, 2-5 cm in length, and the leaves wilted. These symptoms were similar to those of the diseased plants in the field. The control plants were asymptomatic. N. solani NsPed1 was re-isolated from the infected plants, satisfying Koch's postulates. Taken together, N. solani NsPed1 was identified as the causal pathogen of collar rot in P. edulis 'Mantianxing'. Knowledge of the causal organism of collar rot in purple passion fruit will lead to improved measures to prevent and control the disease in China and other countries.

Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data.

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

Overexpression of Type 1 and 2 Diacylglycerol Acyltransferase Genes (JcDGAT1 and JcDGAT2) Enhances Oil Production in the Woody Perennial Biofuel Plant Jatropha curcas.

Diacylglycerol acyltransferase (DGAT) is the only enzyme that catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol (DAG) to form triacylglycerol (TAG). The two main types of DGAT enzymes in the woody perennial biofuel plant Jatropha curcas, JcDGAT1 and JcDGAT2, were previously characterized only in heterologous systems. In this study, we investigated the functions of JcDGAT1 and JcDGAT2 in J. curcas.JcDGAT1 and JcDGAT2 were found to be predominantly expressed during the late stages of J. curcas seed development, in which large amounts of oil accumulated. As expected, overexpression of JcDGAT1 or JcDGAT2 under the control of the CaMV35S promoter gave rise to an increase in seed kernel oil production, reaching a content of 53.7% and 55.7% of the seed kernel dry weight, respectively, which were respectively 25% and 29.6% higher than that of control plants. The increase in seed oil content was accompanied by decreases in the contents of protein and soluble sugars in the seeds. Simultaneously, there was a two- to four-fold higher leaf TAG content in transgenic plants than in control plants. Moreover, by analysis of the fatty acid (FA) profiles, we found that JcDGAT1 and JcDGAT2 had the same substrate specificity with preferences for C18:2 in seed TAGs, and C16:0, C18:0, and C18:1 in leaf TAGs. Therefore, our study confirms the important role of JcDGAT1 and JcDGAT2 in regulating oil production in J. curcas.

Silencing of the Ortholog of DEFECTIVE IN ANTHER DEHISCENCE 1 Gene in the Woody Perennial Jatropha curcas Alters Flower and Fruit Development.

DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1), a phospholipase A1, utilizes galactolipids (18:3) to generate α-linolenic acid (ALA) in the initial step of jasmonic acid (JA) biosynthesis in Arabidopsis thaliana. In this study, we isolated the JcDAD1 gene, an ortholog of Arabidopsis DAD1 in Jatropha curcas, and found that it is mainly expressed in the stems, roots, and male flowers of Jatropha. JcDAD1-RNAi transgenic plants with low endogenous jasmonate levels in inflorescences exhibited more and larger flowers, as well as a few abortive female flowers, although anther and pollen development were normal. In addition, fruit number was increased and the seed size, weight, and oil contents were reduced in the transgenic Jatropha plants. These results indicate that JcDAD1 regulates the development of flowers and fruits through the JA biosynthesis pathway, but does not alter androecium development in Jatropha. These findings strengthen our understanding of the roles of JA and DAD1 in the regulation of floral development in woody perennial plants.

Jatropha curcas ortholog of tomato MADS-box gene 6 (JcTM6) promoter exhibits floral-specific activity in Arabidopsis thaliana.

BACKGROUND: Jatropha curcas L., a perennial oilseed plant, is considered as a promising feedstock for biodiesel production. Genetic modification of flowering characteristics is critical for Jatropha breeding. However, analysis of floral-specific promoters in Jatropha is limited. METHODS: In this study, we isolated the Jatropha ortholog of TM6 (JcTM6) gene from Jatropha flower cDNA library and detected the expression pattern of JcTM6 gene by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). We isolated a 1.8-kb fragment from the 5' region of the JcTM6 gene and evaluated its spatiotemporal expression pattern in Arabidopsis using the ß-glucuronidase (GUS) reporter gene and Arabidopsis ATP/ADP isopentenyltransferase 4 (AtIPT4) gene, respectively. RESULTS: JcTM6 was identified as a flower-specific gene in Jatropha. As expected, JcTM6 promoter was only active in transgenic Arabidopsis flowers with the strongest activity in stamens. Moreover, JcTM6:AtIPT4 transgenic Arabidopsis showed a phenotype of large flowers without any alterations in other organs. Furthermore, deletion of the region from -1,717 to -876 bp resulted in the disappearance of promoter activity in stamens but an increase in promoter activity in young leaves, sepals, and petals. Deletion analysis suggests that the -1,717- to -876-bp promoter fragment contains regulatory elements that confer promoter activity in stamens and inhibit activity in young leaves, sepals, and petals.

Comparative transcriptome analysis of gynoecious and monoecious inflorescences reveals regulators involved in male flower development in the woody perennial plant Jatropha curcas.

KEY MESSAGE: ABCE model genes along with genes related to GA biosynthesis and auxin signalling may play significant roles in male flower development in Jatropha curcas. Flowering plants exhibit extreme reproductive diversity. Jatropha curcas, a woody plant that is promising for biofuel production, is monoecious. Here, two gynoecious Jatropha mutants (bearing only female flowers) were used to identify key genes involved in male flower development. Using comparative transcriptome analysis, we identified 17 differentially expressed genes (DEGs) involved in floral organ development between monoecious plants and the two gynoecious mutants. Among these DEGs, five floral organ identity genes, Jatropha AGAMOUS, PISTILLATA, SEPALLATA 2-1 (JcSEP2-1), JcSEP2-2, and JcSEP3, were downregulated in ch mutant inflorescences; two gibberellin (GA) biosynthesis genes, Jatropha GA REQUIRING 1 and GIBBERELLIN 3-OXIDASE 1, were downregulated in both the ch and g mutants; and two genes involved in the auxin signalling pathway, Jatropha NGATHA1 and STYLISH1, were downregulated in the ch mutant. Furthermore, four hub genes involved in male flower development, namely Jatropha SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1, CRYPTOCHROME 2, SUPPRESSOR OF OVEREXPRESSION OF CO 1 and JAGGED, were identified using weighted gene correlation network analysis. These results suggest that floral organ identity genes and genes involved in GA biosynthesis and auxin signalling may participate in male flower development in Jatropha. This study will contribute to understanding sex differentiation in woody perennial plants.

De novo genome assembly and Hi-C analysis reveal an association between chromatin architecture alterations and sex differentiation in the woody plant Jatropha curcas.

BACKGROUND: Chromatin architecture is an essential factor regulating gene transcription in different cell types and developmental phases. However, studies on chromatin architecture in perennial woody plants and on the function of chromatin organization in sex determination have not been reported. RESULTS: Here, we produced a chromosome-scale de novo genome assembly of the woody plant Jatropha curcas with a total length of 379.5 Mb and a scaffold N50 of 30.7 Mb using Pacific Biosciences long reads combined with genome-wide chromosome conformation capture (Hi-C) technology. Based on this high-quality reference genome, we detected chromatin architecture differences between monoecious and gynoecious inflorescence buds of Jatropha. Differentially expressed genes were significantly enriched in the changed A/B compartments and topologically associated domain regions and occurred preferentially in differential contact regions between monoecious and gynoecious inflorescence buds. Twelve differentially expressed genes related to flower development or hormone synthesis displayed significantly different genomic interaction patterns in monoecious and gynoecious inflorescence buds. These results demonstrate that chromatin organization participates in the regulation of gene transcription during the process of sex differentiation in Jatropha. CONCLUSIONS: We have revealed the features of chromatin architecture in perennial woody plants and investigated the possible function of chromatin organization in Jatropha sex differentiation. These findings will facilitate understanding of the regulatory mechanisms of sex determination in higher plants.