Identification and expression analysis of histone modification gene (HM) family during somatic embryogenesis of oil palm.

BACKGROUND: Oil palm (Elaeis guineensis, Jacq.) is an important vegetable oil-yielding plant. Somatic embryogenesis is a promising method to produce large-scale elite clones to meet the demand for palm oil. The epigenetic mechanisms such as histone modifications have emerged as critical factors during somatic embryogenesis. These histone modifications are associated with the regulation of various genes controlling somatic embryogenesis. To date, none of the information is available on the histone modification gene (HM) family in oil palm. RESULTS: We reported the identification of 109 HM gene family members including 48 HMTs, 27 HDMs, 13 HATs, and 21 HDACs in the oil palm genome. Gene structural and motif analysis of EgHMs showed varied exon-intron organization and with conserved motifs among them. The identified 109 EgHMs were distributed unevenly across 16 chromosomes and displayed tandem duplication in oil palm genome. Furthermore, relative expression analysis showed the differential expressional pattern of 99 candidate EgHM genes at different stages (non-embryogenic, embryogenic, somatic embryo) of somatic embryogenesis process in oil palm, suggesting the EgHMs play vital roles in somatic embryogenesis. Our study laid a foundation to understand the regulatory roles of several EgHM genes during somatic embryogenesis. CONCLUSIONS: A total of 109 histone modification gene family members were identified in the oil palm genome via genome-wide analysis. The present study provides insightful information regarding HM gene's structure, their distribution, duplication in oil palm genome, and also their evolutionary relationship with other HM gene family members in Arabidopsis and rice. Finally, our study provided an essential role of oil palm HM genes during somatic embryogenesis process.

Transcriptome analysis of oil palm pistil during pollination and fertilization to unravel the role of phytohormone biosynthesis and signaling genes.

Phytohormones play an important role in the pollination and fertilization of crops, but the regulatory mechanisms of oil palm pollination and fertilization are unclear. The purpose of this study is to explore the hormonal changes of oil palm pistils during flowering. We used RNA sequencing to evaluate differentially expressed genes (DEGs) in oil palm pistils at the pollination and non-pollination stages. In this study, we found that the hormone contents of oil palm pistil changed drastically after pollination. The transcriptome of the oil palm pistil without pollination and at 2 h, 4 h, 12 h, 24 h, and 48 h after pollination was comprehensively analyzed, and a large number of differential genes and metabolic pathways were explored. Based on the transcriptome data, it could be recognized that the changes of indoleacetic acid (IAA), zeatin riboside (ZR), and abscisic acid (ABA) during pollination were consistent with the changes in the corresponding gene transcripts. Differentially expressed genes during pollination and fertilization of oil palm were mainly related to energy metabolism and hormone signal transduction. It provides new insights to elucidate the interaction and regulation mechanisms of plant hormones before and after oil palm pollination, providing a theoretical basis and reference for the research on sexual reproduction of oil palm.

Transcriptome analysis reveals key developmental and metabolic regulatory aspects of oil palm (Elaeis guineensis Jacq.) during zygotic embryo development.

BACKGROUND: Oil palm is the most efficient oil-producing crop in the world, and the yield of palm oil is associated with embryonic development. However, a comprehensive understanding of zygotic embryo development at the molecular level remains elusive. In order to address this issue, we report the transcriptomic analysis of zygotic embryo development in oil palm, specifically focusing on regulatory genes involved in important biological pathways. RESULTS: In this study, three cDNA libraries were prepared from embryos at S1 (early-stage), S2 (middle-stage), and S3 (late-stage). There were 16,367, 16,500, and 18,012 genes characterized at the S1, S2, and S3 stages of embryonic development, respectively. A total of 1522, 2698, and 142 genes were differentially expressed in S1 vs S2, S1 vs S3, and S2 vs S3, respectively. Using Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify key genes and pathways. In the hormone signaling pathway, genes related to auxin antagonize the output of cytokinin which regulates the development of embryo meristem. The genes related to abscisic acid negatively regulating the synthesis of gibberellin were strongly up-regulated in the mid-late stage of embryonic development. The results were reported the early synthesis and mid-late degradation of sucrose, as well as the activation of the continuous degradation pathway of temporary starch, providing the nutrients needed for differentiation of the embryonic cell. Moreover, the transcripts of genes involved in fatty acid synthesis were also abundantly accumulated in the zygotic embryos. CONCLUSION: Taken together, our research provides a new perspective on the developmental and metabolic regulation of zygotic embryo development at the transcriptional level in oil palm.

Progress in Tissue Culture and Genetic Transformation of Oil Palm: An Overview.

Oil palm (Elaeis guineensis, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, Agrobacterium tumefaciens mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.

Identification and transcript profiles of citrus growth-regulating factor genes involved in the regulation of leaf and fruit development.

Growth-regulating factor (GRF) is an important protein in GA-mediated response, with key roles in plant growth and development. However, it is not known whether or how the GRF proteins in citrus to regulate organ size. In this study, nine citrus GRF genes (CsGRF1-9) were validated from the 'Anliu' sweet orange (AL, Citrus sinensis cv. Anliu) by PCR amplification. They all contain two conserved motifs (QLQ and WRC) and have 3-4 exons. The transcript levels of genes were detected by qRT-PCR. Transcript analysis showed that (1) CsGRF 1, 2, 5, 6, 7, and 9 expressed predominantly in young leaf, CsGRF 3 and 4 expressed predominantly in fruit immature juice sacs and CsGRF 8 expressed predominantly in root; (2) all citrus GRF genes had significantly higher expression in young leaves than mature leaf; (3) in juice sacs, the transcript levels of CsGRF1, 4, 5, 6, and 8 increased significantly while the transcript levels of CsGRF2, 3, 7, and 9 had no significant change from 80 DAF to 100 DAF. Besides, GA3 treatment did not affect the transcript levels of CsGRF5 and CsGRF6 but significantly increased the transcript levels of the other seven CsGRF genes in young leaves. These results suggested that all CsGRF genes involve in the leaf development, CsGRF1, 4, 5, 6, and 8 act developmentally whilst CsGRF2, 3, 7, and 9 play fundamental roles in fruit cell enlargement, which may be through GA pathway or GA-independent pathway.

Genome-wide identification of citrus ATP-citrate lyase genes and their transcript analysis in fruits reveals their possible role in citrate utilization.

ATP-citrate lyase (ACL, EC4.1.3.8) catalyzes citrate to oxaloacetate and acetyl-CoA in the cell cytosol, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. We identified three ACL genes, CitACLα1, CitACLα2, and CitACLß1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLß1 encodes a putative ACL ß subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLß1 had 16 exons and 15 introns. CitACLα1 and CitACLß1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLß1, and/or CitACLα2 in cultivars 'Niuher' and 'Owari' increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in 'Egan No. 1' and 'Iyokan', although citrate content also decreased. In 'HB pummelo', in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of CitACLα2, CitACLß1, and/or CitACLα1 increased. Moreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only CitACLß1 was significantly reduced by ABA treatment. Taken together, these data indicate that the effects of ACL on citrate use during fruit ripening depends on the cultivar, and the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment.

Identification and transcript analysis of two glutamate decarboxylase genes, CsGAD1 and CsGAD2, reveal the strong relationship between CsGAD1 and citrate utilization in citrus fruit.

Glutamate decarboxylase (GAD, EC 4.1.1.15) has been suggested to be a key, regulatory point in the biosynthesis of γ-aminobutyrate (GABA) and in the utilization of citric acid through GABA shunt pathway. In this study we discovered two GAD genes, named as CsGAD1 and CsGAD2, in citrus genome database and then successfully cloned. Both CsGAD1 and CsGAD2 have a putative pyridoxal 5-phosphate binding domain in the middle region and a putative calmodulin-binding domain at the carboxyl terminus. Gene structure analysis showed that much difference exists in the size of exons and introns or in cis-regulatory elements in promoter region between the two GAD genes. Gene expression indicated that CsGAD1 transcript was predominantly expressed in flower and CsGAD2 transcript was predominantly expressed in fruit juice sacs; in the ripening fruit, CsGAD1 transcript level was at least 2-time higher than CsGAD2 transcript level. Moreover, CsGAD1 transcript level was increased significantly along with the increase of GAD activity and accompanied by a significant decrease of titratable acid (TA), suggesting that it is CsGAD1 rather than CsGAD2 plays a role in the citric acid utilization during fruit ripening. In addition, injection of abscisic acid and foliar spray of K2SO4 significantly increased the TA content of Satsuma mandarin, and significantly decreased GAD activity as well as CsGAD1 transcript, further suggesting the important role of CsGAD1 in the citrate utilization of citrus fruit.

Crystal structure of (Z)-2-hy-droxy-N'-(4-oxo-1,3-thia-zolidin-2-yl-idene)benzohydrazide.

In the title compound, C10H9N3O3S, the five-membered ring adopts a slightly twisted conformation about the Cm-S (m = methyl-ene) bond. The dihedral angle between this ring and the benzene ring is 7.99 (9)°. A bifurcated intra-molecular N-H⋯(O,S) hydrogen bond helps to establish the near planar conformation of the mol-ecule. In the crystal, mol-ecules are linked by N-H⋯O and O-H⋯O hydrogen bonds to generate (001) sheets.

Crystal structure of (Z)-2-hy-droxy-4-methyl-N'-(4-oxo-1,3-thia-zolidin-2-yl-idene)benzohydrazide trihydrate.

In the title compound, C11H11N3O3S·3H2O, the non-H atoms of the main mol-ecule are approximately planar, with an r.m.s. deviation of 0.030 Å. There is a bifurcated intra-molecular N-H⋯(O,S) hydrogen bond present forming S(6) and S(5) ring motifs. In the crystal, O-H⋯O and N-H⋯O hydrogen bonds link the molecules into a three-dimensional network.

2-Hy-droxy-N'-methyl-5-nitro-benzohydrazide.

In the title compound, C8H9N3O4, there are two mol-ecules in the asymmetric unit, one of which is in the zwitterionic form. The zwitterion contains an intra-molecular N-H⋯O hydrogen bond and the other mol-ecule contains both an intra-molecular N-H⋯O and an intra-molecular O-H⋯O hydrogen bond. In the crystal, N-H⋯O and N-H⋯N hydrogen bonds link the mol-ecules, formimg a two-dimensional network parallel to (10-1).

Methylome and transcriptome analysis of flowering branches building of Citrus plants induced by drought stress.

Citrus plants exhibit positive floral response under water stress conditions, however, the mechanistic understanding of floral induction remains largely unexplored in water deficit. In this study, DNA methylomic and transcriptomic analyses were integrated to explore the flowering bud formation as well as branches building after light drought stress. While comparing with the conventional watering group (CK), the light drought group treated with five months (LD) showed a significant increase in the flowering branches, whereas an apparent decrease in vegetative branches. Global DNA methylation analysis showed that the LD Group acquired DNA methylation in more than 70,090 genomic regions and lost DNA methylation in about 18,421 genomic regions compared with normal watering group, this indicates that water deficiency leads to a global increase in the expression of DNA methylation in citrus. In the same time, we verified that the increase of DNA methylation level in LD group was correlated with the decrease of DNA demethylase related gene expression. Interestingly, in transcription analysis, it was found that the promoting flower genes of the LD group did not increase but decreased similarly with repressing genes, which is contrary to the intended result. Thus, we thought the lower expression of suppressors FLC and BFT were the key influencing factor to stimulate the flowering branches formation after LD treatment. Moreover, there was a strong negative correlation between the genes expression level and methylation level of the flowering induction/flower development genes. In general, we thought high global DNA methylation level induced by water deficit regulate the flowering branches building by reducing FLC and BFT genes expression.

Effects of Interaction of Protein Hydrolysate and Arbuscular Mycorrhizal Fungi Effects on Citrus Growth and Expressions of Stress-Responsive Genes (Aquaporins and SOSs) under Salt Stress.

Protein hydrolysates (PHs) and arbuscular mycorrhizal fungi (AMF) are environmentally friendly biostimulants that effectively promote crop growth and alleviate the damage from abiotic stress. However, the physiological and molecular regulatory mechanisms are still unclear. This study aimed to explore the effects of PHs and AMF on growth, mineral nutrient absorption, and expression of Aquaporins and SOSs in Goutoucheng (Citrus aurantium) under salt stress. Results showed that PH application and AMF inoculation significantly promoted plant growth and enhanced mineral element absorption and sodium effluxion in citrus under salt stress. The biomass, root activity, leaves mineral nutrition contents in PHs, AMF, and combined (PHs and AMF) treatments were significantly higher than those of control. Leaves sodium content in three treatments was significantly lower than in the control. AMF and combined treatments showed dominant effects than PHs alone. Besides, PHs interacted with AMF on growth, nutrient absorption, and sodium effluxion. Importantly, AMF and PHs induced stress-responsive genes. PIP1, PIP3, SOS1, and SOS3 expression in PHs and AMF treatments was significantly higher than control. Thus, it was concluded that AMF and PHs enhanced the salt tolerance of citrus by promoting nutrient absorption and sodium effluxion via up-regulating the expression of PIPs and SOSs. The mixed application of PHs and AMF had a better effect.

[Effects of different fertilizer applications on citrus rhizosphere soil quality and arbuscular mycorrhizae colonization]. / ä¸åŒæ–½è‚¥å¤„ç†å¯¹æŸ‘æ©˜æ ¹å›´åœŸå£¤è´¨é‡åŠä¸›æžèŒæ ¹å®šæ®–çš„å½±å“.

To understand the effects of different fertilizer applications on soil quality and arbuscular mycorrhizal colonization, we examined the changes in soil physical and chemical properties, mycorrhizal colonization and propagules, and their relationships in citrus under inorganic fertilization (IF), organic fertilization (OF), combined organic and inorganic fertilization (CF), and no fertilization (CK) treatments. Results showed that all fertilization treatments improved the content of rhizospheric soil organic carbon (SOC), nutrient contents, and electrical conductivity (EC). Both CF and OF significantly increased soil pH, soil aggregate stability, activities of urease, catalase, and sucrase, and the colonization and reproduction of arbuscular mycorrhizae fungi (AMF) in citrus rhizosphere. However, IF treatment significantly decreased soil pH and the colonization and reproduction of AMF in citrus rhizosphere. The number of mycorrhizal colonization and propagation was positively correlated with soil aggregate stability, SOC content, total nitrogen content, total phosphorus content, urease activity, and soil pH. Combined with the principal component analysis, we concluded that application of inorganic fertilizer alone could cause soil acidification and inhibit AMF colonization in citrus orchards. In contrast, organic fertilizer combined with inorganic fertilizer in citrus could improve the soil quality and AMF colonization.

Benzene-1,3,5-tricarbonyl trichloride.

In the title molecule, C(9)H(3)Cl(3)O(3), there are three short interactions involving the benzene H atoms and the chloro-formyl Cl atoms. In the crystal, mol-ecules stack along the a axis with no significant non-bonded inter-actions.

The auxin response factor (ARF) gene family in Oil palm (Elaeis guineensis Jacq.): Genome-wide identification and their expression profiling under abiotic stresses.

Auxin response factors (ARFs) play vital role in controlling growth and developmental processes of plants via regulating the auxin signaling pathways. However, the identification and functional roles of ARFs in oil palm plants remain elusive. Here, we identified a total of 23 ARF (EgARF) genes in oil palm through a genome-wide identification approach. The EgARF gene structure analysis revealed the presence of intron-rich ARF gene family in genome of oil palm. Further analysis demonstrated the uneven distribution of 23EgARFs on 16 chromosomes of oil palm. Phylogenetic analysis clustered all the EgARFs into four groups. Twenty-one EgARFs contained BDD, ARF, and CTD domains, whereas EgARF5 and EgARF7 lacked the CTD domain. The evolution of ARF genes in oil palm genome has been expanded by segmental duplication events. The cis-acting regulatory elements of EgARF gene family were predominantly associated with the stress and hormone responses. Expression profiling data demonstrated the constitutive and tissue-specific expression of EgARF genes in various tissues of oil palm. Real-time PCR analysis of 19 EgARF genes expression levels under cold, drought, and salt stress conditions proved their prominent role under abiotic stress responses. Altogether, our study provides a basis for studying the molecular and functional roles of ARF genes in oil palm.

Identification and function prediction of iron-deficiency-responsive microRNAs in citrus leaves.

Iron is a critical micronutrient for growth and development of plants and its deficiency limiting the crop productivity. MicroRNAs (miRNAs) play vital roles in adaptation of plants to various nutrient deficiencies. However, the role of miRNAs and their target genes related to Fe-deficiency is limited. In this study, we identified Fe-deficiency-responsive miRNAs from citrus. In Fe-deficiency conditions, about 50 and 31 miRNAs were up-regulated and down-regulated, respectively. The differently expressed miRNAs might play critical roles in contributing the Fe-deficiency tolerance in citrus plants. The miRNAs-mediated Fe-deficiency tolerance in citrus plants might related to the enhanced stress tolerance by decreased expression of miR172; regulation of S homeostasis by decreased expression of miR395; inhibition of plant growth by increased expression of miR319 and miR477; regulation of Cu homeostasis as well as activation of Cu/Zn superoxide dismutase activity due to decreased expression of miR398 and miR408 and regulation of lignin accumulation by decreased expression of miR397 and miR408. The identified miRNAs in present study laid a foundation to understand the Fe-deficiency adaptive mechanisms in citrus plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02669-z.

Characteristics of boron distribution in the 'Newhall' navel orange plant with two root systems.

Grafting is a technique that provides a substantial way to enhance nutrient utilization thereby improves plant growth and yield quality. Although it is commonly practised in horticultural crops, the impact of scion-rootstock interaction on nutrient distribution is still unclear. Here, 'Newhall' navel orange plants grafted on Trifoliate orange (T) as the original rootstock were inarched with trifoliate orange (N/Tt combination) or carrizo citrange (N/Tc combination) rootstock seedlings. The experimental plants were treated with isotope 10B solution for 7 weeks to investigate the effect of different inarched rootstocks on B distribution and translocation by using a two-root system. From this study, the original rootstock played a more dominant role in B distribution to scion tissues than the inarching rootstock either in N/Tt or N/Tc combination. From inarched combinations, the carrizo citrange in the N/Tc combination had a higher ability to distribute B to new leaves, new twigs and old twigs than trifoliate orange in the N/Tt combination. However, the original rootstock of N/Tt distributed more B to scion tissues than N/Tc and the B concentration in old leaves and new leaves of N/Tt plants was significantly higher than that of N/Tc plants. These results suggest that scion B status is influenced by the B distribution of two inarching rootstocks in an inarching plant, as well as the affinity between the inarching rootstock and grafted plant. In addition, by either adding 10B to the inarching rootstock or original rootstock, we could detect 10B in the other rootstock root in both N/Tt and N/Tc combinations. The results further suggest that B can translocate from rootstock to leaves and then, re-translocate from scion to rootstock through the cycling of B transportation.

miRNAs as key regulators via targeting the phytohormone signaling pathways during somatic embryogenesis of plants.

Somatic embryogenesis is the regeneration of embryos from the somatic cell via dedifferentiation and redifferentiation without the occurrence of fertilization. A complex network of genes regulates the somatic embryogenesis process. Especially, microRNAs (miRNAs) have emerged as key regulators by affecting phytohormone biosynthesis, transport and signal transduction pathways. miRNAs are small, non-coding small RNA regulatory molecules involved in various developmental processes including somatic embryogenesis. Several types of miRNAs such as miR156, miR157, miR 159, miR 160, miR165, miR166, miR167, miR390, miR393 and miR396 have been reported to intricate in regulating somatic embryogenesis via targeting the phytohormone signaling pathways. Here we review current research progress on the miRNA-mediated regulation involved in somatic embryogenesis via regulating auxin, ethylene, abscisic acid and cytokinin signaling pathways. Further, we also discussed the possible role of other phytohormone signaling pathways such as gibberellins, jasmonates, nitric oxide, polyamines and brassinosteroids. Finally, we conclude by discussing the expression of miRNAs and their targets involved in somatic embryogenesis and possible regulatory mechanisms cross talk with phytohormones during somatic embryogenesis.

CRISPR/Cas mediated base editing: a practical approach for genome editing in oil palm.

The improvement of the yield and quality of oil palm via precise genome editing has been indispensable goal for oil palm breeders. Genome editing via the CRISPR/Cas9 (CRISPR-associated protein 9) system, ZFN (zinc finger nucleases) and TALEN (transcription activator-like effector nucleases) has flourished as an efficient technology for precise target modifications in the genomes of various crops. Among the genome editing technologies, base editing approach has emerged as novel technology that could generate single base changes i.e. irreversible conversion of one target base in to other in a programmable manner. A base editor (adenine or cytosine) is a fusion of catalytically inactive CRISPR-Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations. However, till date no such genetic modifications have ever been developed in oil palm via base editing technology. Precise genome editing via base editing approach can be a challenging task in oil palm due to its complex genome as well as difficulties in tissue culture and genetic transformation methods. However, availability of whole genome sequencing data in oil palm provides a platform for developing the base editing technology. Here, we briefly review the potential application and future implications of base editing technology for the genetic improvement of oil palm.

Development of SSR markers based on transcriptome data and association mapping analysis for fruit shell thickness associated traits in oil palm (Elaeis guineensis Jacq.).

Present study mainly aimed to ascertain the distribution characteristics of gene-based microsatellite loci and to develop polymorphic SSR markers from the already available transcriptome data of Elaeis guineensis Jacq, an important oil crop. From this study, we identified the sum of 5791 SSRs across 51,425 unigenes from the transcripts of oil palm. We were able to evaluate 331primer pairs and characterized 183 polymorphic gene-based SSR markers. We identified a total of 506 alleles from the 183 polymorphic SSR loci, with an average of 2.77 alleles per locus. The characterized gene-based SSR markers from the transcriptome data of oil palm exhibited moderate levels of polymorphism with a significant level of heterozygosity ranges from 0.096 to 0.594 (mean = 0.336 ± 0.11). Among the identified SSR markers, sixty polymorphic markers were used to analyze genotypes of 55 oil palm accessions selected from three different provinces of China. Association mapping analysis provided the information of four markers that are associated with fruit shell thickness trait of oil palm. Among the four markers identified from association analysis, one SSR marker obtained from Unigene17150 is strictly associated with the oil palm fruit shell thickness trait.