Comparative genomics and transcriptomic analyses were performed on two agronomically important groups of genes from oil palm versus other major crop species and the model organism, Arabidopsis thaliana. The first analysis was of two gene families with key roles in regulation of oil quality and in particular the accumulation of oleic acid, namely stearoyl ACP desaturases (SAD) and acyl-acyl carrier protein (ACP) thioesterases (FAT). In both cases, these were found to be large gene families with complex expression profiles across a wide range of tissue types and developmental stages. The detailed classification of the oil palm SAD and FAT genes has enabled the updating of the latest version of the oil palm gene model. The second analysis focused on disease resistance (R) genes in order to elucidate possible candidates for breeding of pathogen tolerance/resistance. Ortholog analysis showed that 141 out of the 210 putative oil palm R genes had homologs in banana and rice. These genes formed 37 clusters with 634 orthologous genes. Classification of the 141 oil palm R genes showed that the genes belong to the Kinase (7), CNL (95), MLO-like (8), RLK (3) and Others (28) categories. The CNL R genes formed eight clusters. Expression data for selected R genes also identified potential candidates for breeding of disease resistance traits. Furthermore, these findings can provide information about the species evolution as well as the identification of agronomically important genes in oil palm and other major crops.
A cross between IR64 (high-yielding but drought-susceptible) and Aday Sel (drought-tolerant) rice cultivars yielded a stable line with enhanced grain yield under drought screening field trials at International Rice Research Institute. The major effect qDTY4.1 drought tolerance and yield QTL was detected in the IR77298-14-1-2-10 Backcrossed Inbred Line (BIL) and its IR87705-7-15-B Near Isogenic Line (NIL) with 93.9% genetic similarity to IR64. Although rice yield is extremely susceptible to water stress at reproductive stage, currently, there is only one report on the detection of drought-responsive microRNAs in inflorescence tissue of a Japonica rice line. In this study, more drought-responsive microRNAs were identified in the inflorescence tissues of IR64, IR77298-14-1-2-10 and IR87705-7-15-B via next-generation sequencing. Among the 32 families of inflorescence-specific non-conserved microRNAs that were identified, 22 families were up-regulated in IR87705-7-15-B. Overall 9 conserved and 34 non-conserved microRNA families were found as drought-responsive in rice inflorescence with 5 conserved and 30 non-conserved families induced in the IR87705-7-15-B. The observation of more drought-responsive non-conserved microRNAs may imply their prominence over conserved microRNAs in drought response mechanisms of rice inflorescence. Gene Ontology annotation analysis on the target genes of drought-responsive microRNAs identified in IR87705-7-15-B revealed over-representation of biological processes including development, signalling and response to stimulus. Particularly, four inflorescence-specific microRNAs viz. osa-miR5485, osa-miR5487, osa-miR5492 and osa-miR5517, and two non-inflorescence specific microRNAs viz. osa-miR169d and osa-miR169f.2 target genes that are involved in flower or embryonic development. Among them, osa-miR169d, osa-miR5492 and osa-miR5517 are related to flowering time control. It is also worth mentioning that osa-miR2118 and osa-miR2275, which are implicated in the biosynthesis of rice inflorescence-specific small interfering RNAs, were induced in IR87705-7-15-B but repressed in IR77298-14-1-2-10. Further, gene search within qDTY4.1 QTL region had identified multiple copies of NBS-LRR resistance genes (potential target of osa-miR2118), subtilisins and genes implicated in stomatal movement, ABA metabolism and cuticular wax biosynthesis.
Climate change-induced abiotic stress results in crop yield and production losses. These stresses result in changes at the physiological and molecular level that affect the development and growth of the plant. Reactive oxygen species (ROS) is formed at high levels due to abiotic stress within different organelles, leading to cellular damage. Plants have evolved mechanisms to control the production and scavenging of ROS through enzymatic and non-enzymatic antioxidative processes. However, ROS has a dual function in abiotic stresses where, at high levels, they are toxic to cells while the same molecule can function as a signal transducer that activates a local and systemic plant defense response against stress. The effects, perception, signaling, and activation of ROS and their antioxidative responses are elaborated in this review. This review aims to provide a purview of processes involved in ROS homeostasis in plants and to identify genes that are triggered in response to abiotic-induced oxidative stress. This review articulates the importance of these genes and pathways in understanding the mechanism of resistance in plants and the importance of this information in breeding and genetically developing crops for resistance against abiotic stress in plants.
Fruit ripening is a complex and highly coordinated developmental process involving the expression of many ripening-related genes under the control of a network of signalling pathways. The hormonal control of climacteric fruit ripening, especially ethylene perception and signalling transduction in tomato has been well characterized. Additionally, great strides have been made in understanding some of the major regulatory switches (transcription factors such as RIPENING-INHIBITOR and other transcriptional regulators such as COLOURLESS NON-RIPENING, TOMATO AGAMOUS-LIKE1 and ETHYLENE RESPONSE FACTORs), that are involved in tomato fruit ripening. In contrast, the regulatory network related to non-climacteric fruit ripening remains poorly understood. However, some of the most recent breakthrough research data have provided several lines of evidences for abscisic acid- and sucrose-mediated ripening of strawberry, a non-climacteric fruit model. In this review, we discuss the most recent research findings concerning the hormonal regulation of fleshy fruit ripening and their cross-talk and the future challenges taking tomato as a climacteric fruit model and strawberry as a non-climacteric fruit model. We also highlight the possible contribution of epigenetic changes including the role of plant microRNAs, which is opening new avenues and great possibilities in the fields of fruit-ripening research and postharvest biology.
CBF/DREB1 is a group of transcription factors that are mainly involved in abiotic stress tolerance in plants. They belong to the AP2/ERF superfamily of plant-specific transcription factors. A gene encoding a new member of this group was isolated from ripening oil palm fruit and designated as EgCBF3. The oil palm fruit demonstrates the characteristics of a climacteric fruit like tomato, in which ethylene has a major impact on the ripening process. A transgenic approach was used for functional characterization of the EgCBF3, using tomato as the model plant. The effects of ectopic expression of EgCBF3 were analyzed based on expression profiling of the ethylene biosynthesis-related genes, anti-freeze proteins (AFPs), abiotic stress tolerance and plant growth and development. The EgCBF3 tomatoes demonstrated altered phenotypes compared to the wild type tomatoes. Delayed leaf senescence and flowering, increased chlorophyll content and abnormal flowering were the consequences of overexpression of EgCBF3 in the transgenic tomatoes. The EgCBF3 tomatoes demonstrated enhanced abiotic stress tolerance under in vitro conditions. Further, transcript levels of ethylene biosynthesis-related genes, including three SlACSs and two SlACOs, were altered in the transgenic plants' leaves and roots compared to that in the wild type tomato plant. Among the eight AFPs studied in the wounded leaves of the EgCBF3 tomato plants, transcript levels of SlOSM-L, SlNP24, SlPR5L and SlTSRF1 decreased, while expression of the other four, SlCHI3, SlPR1, SlPR-P2 and SlLAP2, were up-regulated. These findings indicate the possible functions of EgCBF3 in plant growth and development as a regulator of ethylene biosynthesis-related and AFP genes, and as a stimulator of abiotic stress tolerance.
Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-alpha,beta-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin.
An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.
Amphidiploid species in the Brassicaceae family, such as Brassica napus, are more tolerant to environmental stress than their diploid ancestors.A relatively salt tolerant B. napus line, N119, identified in our previous study, was used. N119 maintained lower Na(+) content, and Na(+)/K(+) and Na(+)/Ca(2+) ratios in the leaves than a susceptible line. The transcriptome profiles of both the leaves and the roots 1 h and 12 h after stress were investigated. De novo assembly of individual transcriptome followed by sequence clustering yielded 161,537 nonredundant sequences. A total of 14,719 transcripts were differentially expressed in either organs at either time points. GO and KO enrichment analyses indicated that the same 49 GO terms and seven KO terms were, respectively, overrepresented in upregulated transcripts in both organs at 1 h after stress. Certain overrepresented GO term of genes upregulated at 1 h after stress in the leaves became overrepresented in genes downregulated at 12 h. A total of 582 transcription factors and 438 transporter genes were differentially regulated in both organs in response to salt shock. The transcriptome depicting gene network in the leaves and the roots regulated by salt shock provides valuable information on salt resistance genes for future application to crop improvement.
Citrus maxima "seedless" is originally from Malaysia, and now is widely cultivated in Hainan province, China. The essential features of this cultivar are thin skin, green epicarp and seedless at the ripening stage. Here, using C. maxima "seedless" as experimental material, we investigated the physical and inclusion indicators, and found the accumulation of storage compounds during 120-210 DAF leading to inconsistent increase between volume and weight. Component analysis of soluble sugar indicated that arabinose and xylose have a high content in early development of pummelo juice sacs (PJS), whereas fructose, glucose and sucrose show a significant increase during PJS maturation. To clarify a global overview of the gene expressing profiles, the PJSs from four periods (60, 120, 180 and 240 DAF) were selected for comparative transcriptome analysis. The resulting 8275 unigenes showed differential expression during PJS development. Also, the stability of 11 housekeeping genes were evaluated by geNorm method, resulting in a set of five genes (UBC, ACT, OR23, DWA2 and CYP21D) used as control for normalization of gene expression. Based on transcriptome data, 5 sucrose synthases (SUSs) and 10 invertases (INVs) were identified to be involved in sucrose degradation. Importantly, SUS4 may be responsible for arabinose and xylose biosynthesis to form the cell wall in early development, while SUS3 and VIN2 may be important in the accumulation of soluble hexose leading to cell expansion through an osmotic-independent pathway in late development. The information provides valuable metabolite and genetic resources in C. maxima "seedless", and is important for achieving high fruit yield and quality.
Plants maintain extensive growth flexibility under different environmental conditions, allowing them to continuously and rapidly adapt to alterations in their environment. A large portion of many plant genomes consists of transposable elements (TEs) that create new genetic variations within plant species. Different types of mutations may be created by TEs in plants. Many TEs can avoid the host's defense mechanisms and survive alterations in transposition activity, internal sequence and target site. Thus, plant genomes are expected to utilize a variety of mechanisms to tolerate TEs that are near or within genes. TEs affect the expression of not only nearby genes but also unlinked inserted genes. TEs can create new promoters, leading to novel expression patterns or alternative coding regions to generate alternate transcripts in plant species. TEs can also provide novel cis-acting regulatory elements that act as enhancers or inserts within original enhancers that are required for transcription. Thus, the regulation of plant gene expression is strongly managed by the insertion of TEs into nearby genes. TEs can also lead to chromatin modifications and thereby affect gene expression in plants. TEs are able to generate new genes and modify existing gene structures by duplicating, mobilizing and recombining gene fragments. They can also facilitate cellular functions by sharing their transposase-coding regions. Hence, TE insertions can not only act as simple mutagens but can also alter the elementary functions of the plant genome. Here, we review recent discoveries concerning the contribution of TEs to gene expression in plant genomes and discuss the different mechanisms by which TEs can affect plant gene expression and reduce host defense mechanisms.
The cell wall determines morphology and the environmental responses of plant cells. The primary cell wall (PCW) is produced during cell division and expansion, determining the cell shape and volume. After cell expansion, specific types of plant cells produce a lignified wall, known as a secondary cell wall (SCW). We functionally analyzed Group IIId Arabidopsis AP2/EREBP genes, namely ERF34, ERF35, ERF38, and ERF39, which are homologs of a rice ERF gene previously proposed to be related to SCW biosynthesis. Expression analysis revealed that these four genes are expressed in regions related to cell division and/or cell differentiation in seedlings (i.e., shoot apical meristems, the primordia of leaves and lateral roots, trichomes, and central cylinder of primary roots) and flowers (i.e., vascular tissues of floral organs and replums and/or valve margins of pistils). Overexpression of ERF genes significantly upregulated PCW-type, but not SCW-type, CESA genes encoding cellulose synthase catalytic subunits in Arabidopsis seedlings. Transient co-expression reporter analysis indicated that ERF35, ERF38, and ERF39 possess transcriptional activator activity, and that ERF34, ERF35, ERF38, and ERF39 upregulated the promoter activity of CESA1, a PCW-type CESA gene, through the DRECRTCOREAT elements, the core cis-acting elements known to be recognized by AP2/ERF proteins. Together, our findings show that Group IIId ERF genes are positive transcriptional regulators of PCW-type CESA genes in Arabidopsis and are possibly involved in modulating cellulose biosynthesis in response to developmental requirements and environmental stimuli.
KEY MESSAGE: Several hypomethylated sites within the Karma region of EgDEF1 and hotspot regions in chromosomes 1, 2, 3, and 5 may be associated with mantling. One of the main challenges faced by the oil palm industry is fruit abnormalities, such as the "mantled" phenotype that can lead to reduced yields. This clonal abnormality is an epigenetic phenomenon and has been linked to the hypomethylation of a transposable element within the EgDEF1 gene. To understand the epigenome changes in clones, methylomes of clonal oil palms were compared to methylomes of seedling-derived oil palms. Whole-genome bisulfite sequencing data from seedlings, normal, and mantled clones were analyzed to determine and compare the context-specific DNA methylomes. In seedlings, coding and regulatory regions are generally hypomethylated while introns and repeats are extensively methylated. Genes with a low number of guanines and cytosines in the third position of codons (GC3-poor genes) were increasingly methylated towards their 3' region, while GC3-rich genes remain demethylated, similar to patterns in other eukaryotic species. Predicted promoter regions were generally hypomethylated in seedlings. In clones, CG, CHG, and CHH methylation levels generally decreased in functionally important regions, such as promoters, 5' UTRs, and coding regions. Although random regions were found to be hypomethylated in clonal genomes, hypomethylation of certain hotspot regions may be associated with the clonal mantling phenotype. Our findings, therefore, suggest other hypomethylated CHG sites within the Karma of EgDEF1 and hypomethylated hotspot regions in chromosomes 1, 2, 3 and 5, are associated with mantling.
KEY MESSAGE: Potentially embryogenic oil palms can be identified through leaf transcriptomic signatures. Differential expression of genes involved in flowering time, and stress and light responses may associate with somatic embryogenesis potential. Clonal propagation is an attractive approach for the mass propagation of high yielding oil palms. A major issue hampering the effectiveness of oil palm tissue culture is the low somatic embryogenesis rate. Previous studies have identified numerous genes involved in oil palm somatic embryogenesis, but their association with embryogenic potential has not been determined. In this study, differential expression analysis of leaf transcriptomes from embryogenic and non-embryogenic mother palms revealed that transcriptome profiles from non- and poor embryogenic mother palms were more similar than highly embryogenic palms. A total of 171 genes exhibiting differential expression in non- and low embryogenesis groups could also discriminate high from poor embryogenesis groups of another tissue culture agency. Genes related to flowering time or transition such as FTIP, FRIGIDA-LIKE, and NF-YA were up-regulated in embryogenic ortets, suggesting that reproduction timing of the plant may associate with somatic embryogenesis potential. Several light response or photosynthesis-related genes were down-regulated in embryogenic ortets, suggesting a link between photosynthesis activity and embryogenic potential. As expression profiles of the differentially expressed genes are very similar between non- and low embryogenic groups, machine learning approaches with several candidate genes may generate a more sensitive model to better discriminate non-embryogenic from embryogenic ortets.
KEY MESSAGE: Transcriptomes generated by laser capture microdissected abnormal staminodes revealed adoption of carpel programming during organ initiation with decreased expression of numerousHSPs,EgDEF1, EgGLO1but increasedLEAFYexpression. The abnormal mantled phenotype in oil palm involves a feminization of the male staminodes into pseudocarpels in pistillate inflorescences. Previous studies on oil palm flowering utilized entire inflorescences or spikelets, which comprised not only the male and female floral organs, but the surrounding tissues as well. Laser capture microdissection coupled with RNA sequencing was conducted to investigate the specific transcriptomes of male and female floral organs from normal and mantled female inflorescences. A higher number of differentially expressed genes (DEGs) were identified in abnormal versus normal male organs compared with abnormal versus normal female organs. In addition, the abnormal male organ transcriptome closely mimics the transcriptome of abnormal female organ. While the transcriptome of abnormal female organ was relatively similar to the normal female organ, a substantial amount of female DEGs encode HEAT SHOCK PROTEIN genes (HSPs). A similar high amount (20%) of male DEGs encode HSPs as well. As these genes exhibited decreased expression in abnormal floral organs, mantled floral organ development may be associated with lower stress indicators. Stamen identity genes EgDEF1 and EgGLO1 were the main floral regulatory genes with decreased expression in abnormal male organs or pseudocarpel initials. Expression of several floral transcription factors was elevated in pseudocarpel initials, notably LEAFY, FIL and DL orthologs, substantiating the carpel specification programming of abnormal staminodes. Specific transcriptomes thus obtained through this approach revealed a host of differentially regulated genes in pseudocarpel initials compared to normal male staminodes.
Panduratin A extracted from Boesenbergia rotunda is a flavonoid reported to possess a range of medicinal indications which include anti-dengue, anti-HIV, anti-cancer, antioxidant and anti-inflammatory properties. Boesenbergia rotunda is a plant from the Zingiberaceae family commonly used as a food ingredient and traditional medicine in Southeast Asia and China. Reports on the health benefits of secondary metabolites extracted from Boesenbergia rotunda over the last few years has resulted in rising demands for panduratin A. However large scale extraction has been hindered by the naturally low abundance of the compound and limited knowledge of its biosynthetic pathway.
Phosphatidylinositol 3-kinase (PI3K) is a key enzyme that phosphorylates phosphatidylinositol at 3'-hydroxyl position of the inositol head group initiating the generation of several phosphorylated phosphatidylinositols, collectively referred to as phosphoinositides. The function of PI3K in plant senescence and ethylene signal transduction process was studied by expression ofSolanum lycopersicumPI3K in transgenicNicotiana tabacum, and delineating its effect on flower senescence. Detached flowers of transgenic tobacco plants with overexpressedSl-PI3K(OX) displayed accelerated senescence and reduced longevity, when compared to the flowers of wild type plants. Flowers from PI3K-overexpressing plants showed enhanced ethylene production and upregulated expression of 1-aminocyclopropane-1-carboxylic acid oxidase 1 (ACO1). Real time polymerase chain reaction (PCR) analysis showed thatPI3Kwas expressed at a higher level in OX flowers than in the control. Seedlings of OX-lines also demonstrated a triple response phenotype with characteristic exaggerated apical hook, shorter hypocotyls and increased sensitivity to 1-aminocyclopropane-1-carboxylate than the control wild type seedlings. In floral tissue from OX-lines,Solanum lycopersicumphosphatidylinositol 3-kinase green fluorescent protein (PI3K-GFP) chimera protein was localized primarily in stomata, potentially in cytoplasm and membrane adjacent to stomatal pores in the guard cells. Immunoblot analysis of PI3K expression in OX lines demonstrated increased protein level compared to the control. Results of the present study suggest that PI3K plays a crucial role in senescence by enhancing ethylene biosynthesis and signaling.
A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens.
The mantled floral phenotype of oil palm (Elaeis guineensis) affects somatic embryogenesis-derived individuals and is morphologically similar to mutants defective in the B-class MADS-box genes. This somaclonal variation has been previously demonstrated to be associated to a significant deficit in genome-wide DNA methylation. In order to elucidate the possible role of DNA methylation in the transcriptional regulation of EgDEF1, the APETALA3 ortholog of oil palm, we studied this epigenetic mark within the gene in parallel with transcript accumulation in both normal and mantled developing inflorescences. We also examined the methylation and expression of two neighboring retrotransposons that might interfere with EgDEF1 regulation. We show that the EgDEF1 gene is essentially unmethylated and that its methylation pattern does not change with the floral phenotype whereas expression is dramatically different, ruling out a direct implication of DNA methylation in the regulation of this gene. Also, we find that both the gypsy element inserted within an intron of the EgDEF1 gene and the copia element located upstream from the promoter are heavily methylated and show little or no expression. Interestingly, we identify a shorter, alternative transcript produced by EgDEF1 and characterize its accumulation with respect to its full-length counterpart. We demonstrate that, depending on the floral phenotype, the respective proportions of these two transcripts change differently during inflorescence development. We discuss the possible phenotypical consequences of this alternative splicing and the new questions it raises in the search for the molecular mechanisms underlying the mantled phenotype in the oil palm.
SAG21/LEA5 is an unusual late embryogenesis abundant protein in Arabidopsis thaliana, that is primarily mitochondrially located and may be important in regulating translation in both chloroplasts and mitochondria. SAG21 expression is regulated by a plethora of abiotic and biotic stresses and plant growth regulators indicating a complex regulatory network. To identify key transcription factors regulating SAG21 expression, yeast-1-hybrid screens were used to identify transcription factors that bind the 1685 bp upstream of the SAG21 translational start site. Thirty-three transcription factors from nine different families bound to the SAG21 promoter, including members of the ERF, WRKY and NAC families. Key binding sites for both NAC and WRKY transcription factors were tested through site directed mutagenesis indicating the presence of cryptic binding sites for both these transcription factor families. Co-expression in protoplasts confirmed the activation of SAG21 by WRKY63/ABO3, and SAG21 upregulation elicited by oligogalacturonide elicitors was partially dependent on WRKY63, indicating its role in SAG21 pathogen responses. SAG21 upregulation by ethylene was abolished in the erf1 mutant, while wound-induced SAG21 expression was abolished in anac71 mutants, indicating SAG21 expression can be regulated by several distinct transcription factors depending on the stress condition.
Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia.