Agrobacterium rhizogenes: paving the road to research and breeding for woody plants.

Woody plants play a vital role in global ecosystems and serve as valuable resources for various industries and human needs. While many woody plant genomes have been fully sequenced, gene function research and biotechnological breeding advances have lagged behind. As a result, only a limited number of genes have been elucidated, making it difficult to use newer tools such as CRISPR-Cas9 for biotechnological breeding purposes. The use of Agrobacterium rhizogenes as a transformative tool in plant biotechnology has received considerable attention in recent years, particularly in the research field on woody plants. Over the past three decades, numerous woody plants have been effectively transformed using A. rhizogenes-mediated techniques. Some of these transformed plants have successfully regenerated. Recent research on A. rhizogenes-mediated transformation of woody plants has demonstrated its potential for various applications, including gene function analysis, gene expression profiling, gene interaction studies, and gene regulation analysis. The introduction of the Ri plasmid has resulted in the emergence of several Ri phenotypes, such as compact plant types, which can be exploited for Ri breeding purposes. This review paper presents recent advances in A. rhizogenes-mediated basic research and Ri breeding in woody plants. This study highlights various aspects of A. rhizogenes-mediated transformation, its multiple applications in gene function analysis, and the potential of Ri lines as valuable breeding materials.

Overexpression of the Ginkgo biloba dihydroflavonol 4-reductase gene GbDFR6 results in the self-incompatibility-like phenotypes in transgenic tobacco.

Although flavonoids play multiple roles in plant growth and development, the involvement in plant self-incompatibility (SI) have not been reported. In this research, the fertility of transgenic tobacco plants overexpressing the Ginkgo biloba dihydroflavonol 4-reductase gene, GbDFR6, were investigated. To explore the possible physiological defects leading to the failure of embryo development in transgenic tobacco plants, functions of pistils and pollen grains were examined. Transgenic pistils pollinated with pollen grains from another tobacco plants (either transgenic or wild-type), developed full of well-developed seeds. In contrast, in self-pollinated transgenic tobacco plants, pollen-tube growth was arrested in the upper part of the style, and small abnormal seeds developed without fertilization. Although the mechanism remains unclear, our research may provide a valuable method to create SI tobacco plants for breeding.

The absorption of water from humid air by grass embryos during germination.

Grass embryos possess structures that do not occur in any other flowering plants. Due to the specific embryo structure and position, grass embryo surfaces may be exposed to surrounding air under partial caryopsis-soil contact conditions, but whether caryopses of the grass family (Poaceae) can sense soil air humidity to initiate successful germination under partial caryopsis-soil contact conditions remain unknown. Here, we found that grass embryos have the unique ability to absorb water from atmospheric water vapor under partial caryopsis-soil contact conditions. To absorb atmospheric moisture, grass embryos developed profuse and highly elongated hairs on the embryo surface. These hairs, classically known as coleorhiza hairs, developed only on the embryo surface exposed to humid air, and submergence of the embryo surface inhibited their development. In addition to humid air-dependent development, almost all other developmental features of coleorhiza hairs were substantially different from root hairs. However, coleorhiza hair development was regulated by ROOTHAIRLESS 1. Besides the genetic control of coleorhiza hair development, we also identified how caryopses manage to keep the hairs turgid in natural open environments as the hairs were highly sensitive to dry air exposure. Moreover, we video-documented the regulation of developmental processes. The unique humid air-dependent coleorhiza hair development and their ability to absorb water from water vapor present in microsites or soil air give grasses advantages in germination and seedling establishment. Ultimately, coleorhiza hairs may have contributed to the ecological success of the grass family.

Transgenic tobacco plant overexpressing ginkgo dihydroflavonol 4-reductase gene GbDFR6 exhibits multiple developmental defects.

Dihydroflavonol Q 4-reductase (DFR), a key enzyme in the flavonoid biosynthetic pathway in plants, significantly influences plant survival. However, the roles of DFR in the regulation of plant development are largely unknown. In the present study, phenotypes of transgenic tobacco plants overexpressing the Ginkgo biloba DFR gene, GbDFR6, were investigated. Transgenic tobacco seedlings exhibited relatively low fresh weights, long primary roots, decreased lateral root numbers, and impaired root gravitropic responses when compared to wild-type tobacco plants. Adult transgenic tobacco plants exhibited a considerably high percentage of wrinkled leaves when compared to the wild-type tobacco plants. In addition to the auxin-related phenotypic changes, transgenic tobacco plants exhibited delayed flowering phenotypes under short-day conditions. Gene expression analysis revealed that the delayed flowering in transgenic tobacco plants was caused by the low expression levels of NtFT4. Finally, variations in anthocyanin and flavonoid contents in transgenic tobacco plants were evaluated. The results revealed that the levels of most anthocyanins identified in transgenic tobacco leaves increased. Specifically, cyanidin-3,5-O-diglucoside content increased by 9.8-fold in transgenic tobacco plants when compared to the wild-type tobacco plants. Pelargonidin-3-O-(coumaryl)-glucoside was only detected in transgenic tobacco plants. Regarding flavonoid compounds, one flavonoid compound (epicatechin gallate) was upregulated, whereas seven flavonoid compounds (Tamarixetin-3-O-rutinoside; Sexangularetin-3-O-glucoside-7-O-rhamnoside; Kaempferol-3-O-neohesperidoside; Engeletin; 2'-Hydoxy,5-methoxyGenistein-O-rhamnosyl-glucoside; Diosmetin; Hispidulin) were downregulated in both transgenic tobacco leaves and roots. The results indicate novel and multiple roles of GbDFR6 in ginkgo and provide a valuable method to produce a late flowering tobacco variety in tobacco industry.

A nucleoside diphosphate kinase gene OsNDPK4 is involved in root development and defense responses in rice (Oryza sativa L.).

MAIN CONCLUSION: Dysfunctional mutation of OsNDPK4 resulted in severe defects in root development of rice. However, the resistance of Osndpk4 against bacterial blight was significantly enhanced. Nucleoside diphosphate kinases (NDPKs) are an evolutionarily conserved family of important enzymes balancing the energy currency nucleoside triphosphates by catalyzing the transfer of their phosphate groups. The aim of this study was to elucidate the function of OsNDPK4 in rice. A dysfunctional rice mutant was employed to characterize the function of OsNDPK4. Its expression and subcellular localization were examined. The transcriptomic change in roots of Osndpk4 was analyzed by RNA-seq. The rice mutant Osndpk4 showed severe defects in root development from the early seedling stage. Further analysis revealed that meristematic activity and cell elongation were significantly inhibited in primary roots of Osndpk4, together with reduced accumulation of reactive oxygen species (ROS). Map-based cloning identified that the mutation occurred in the OsNDPK4 gene. OsNDPK4 was found to be expressed in a variety of tissues throughout the plant and OsNDPK4 was located in the cytosol. Osndpk4 showed enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) and up-regulation of pathogenesis-related marker genes. In addition, transcriptomic analysis showed that OsNDPK4 was significantly associated with a number of biological processes, including translation, protein modification, metabolism, biotic stress response, etc. Detailed analysis revealed that the dysfunction of OsNDPK4 might reorchestrate energy homeostasis and hormone metabolism and signalling, resulting in repression of translation, DNA replication and cell cycle progression, and priming of biotic stress defense. Our results demonstrate that OsNDPK4 plays important roles in energy homeostasis, development process, and defense responses in rice.

A Pelota-like gene regulates root development and defence responses in rice.

Background and Aims: Pelota (Pelo) are evolutionarily conserved genes reported to be involved in ribosome rescue, cell cycle control and meiotic cell division. However, there is little known about their function in plants. The aim of this study was to elucidate the function of an ethylmethane sulphonate (EMS)-derived mutation of a Pelo-like gene in rice (named Ospelo). Methods: A dysfunctional mutant was used to characterize the function of OsPelo. Analyses of its expression and sub-cellular localization were performed. The whole-genome transcriptomic change in leaves of Ospelo was also investigated by RNA sequencing. Key Results: The Ospelo mutant showed defects in root system development and spotted leaves at early seedling stages. Map-based cloning revealed that the mutation occurred in the putative Pelo gene. OsPelo was found to be expressed in various tissues throughout the plant, and the protein was located in mitochondria. Defence responses were induced in the Ospelo mutant, as shown by enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae, coupled with upregulation of three pathogenesis-related marker genes. In addition, whole-genome transcriptome analysis showed that OsPelo was significantly associated with a number of biological processes, including translation, metabolism and biotic stress response. Detailed analysis showed that activation of a number of innate immunity-related genes might be responsible for the enhanced disease resistance in the Ospelo mutant. Conclusions: These results demonstrate that OsPelo positively regulates root development while its loss of function enhances pathogen resistance by pre-activation of defence responses in rice.

Root Cell-Specific Regulators of Phosphate-Dependent Growth.

Cellular specialization in abiotic stress responses is an important regulatory feature driving plant acclimation. Our in silico approach of iterative coexpression, interaction, and enrichment analyses predicted root cell-specific regulators of phosphate starvation response networks in Arabidopsis (Arabidopsis thaliana). This included three uncharacterized genes termed Phosphate starvation-induced gene interacting Root Cell Enriched (PRCE1, PRCE2, and PRCE3). Root cell-specific enrichment of 12 candidates was confirmed in promoter-GFP lines. T-DNA insertion lines of 11 genes showed changes in phosphate status and growth responses to phosphate availability compared with the wild type. Some mutants (cbl1, cipk2, prce3, and wdd1) displayed strong biomass gain irrespective of phosphate supply, while others (cipk14, mfs1, prce1, prce2, and s6k2) were able to sustain growth under low phosphate supply better than the wild type. Notably, root or shoot phosphate accumulation did not strictly correlate with organ growth. Mutant response patterns markedly differed from those of master regulators of phosphate homeostasis, PHOSPHATE STARVATION RESPONSE1 (PHR1) and PHOSPHATE2 (PHO2), demonstrating that negative growth responses in the latter can be overcome when cell-specific regulators are targeted. RNA sequencing analysis highlighted the transcriptomic plasticity in these mutants and revealed PHR1-dependent and -independent regulatory circuits with gene coexpression profiles that were highly correlated to the quantified physiological traits. The results demonstrate how in silico prediction of cell-specific, stress-responsive genes uncovers key regulators and how their manipulation can have positive impacts on plant growth under abiotic stress.

Isolation, Characterization and Transcriptome Analysis of a Cytokinin Receptor Mutant Osckt1 in Rice.

Cytokinins play important roles in regulating plant development, including shoot and root meristems, leaf longevity, and grain yield. However, the in planta functions of rice cytokinin receptors have not been genetically characterized yet. Here we isolated a rice mutant, Osckt1, with enhanced tolerance to cytokinin treatment. Further analysis showed that Osckt1 was insensitive to aromatic cytokinins but responded normally to isoprenoid and phenylurea-type cytokinins. Map-based cloning revealed that the mutation occurred in a putative cytokinin receptor gene, histidine kinase 6 (OsHK6). OsCKT1 was found to be expressed in various tissues throughout the plant and the protein was located in the endoplasmic reticulum. In addition, whole-genome gene expression profiling analysis showed that OsCKT1 was involved in cytokinin regulation of a number of biological processes, including secondary metabolism, sucrose and starch metabolism, chlorophyll synthesis, and photosynthesis. Our results demonstrate that OsCKT1 plays important roles in cytokinin perception and control of root development in rice.

OsKASI, a ß-ketoacyl-[acyl carrier protein] synthase I, is involved in root development in rice (Oryza sativa L.).

MAIN CONCLUSION: The involvement of OsKASI in FA synthesis is found to play a critical role in root development of rice. The root system plays important roles in plant nutrient and water acquisition. However, mechanisms of root development and molecular regulation in rice are still poorly understood. Here, we characterized a rice (Oryza sativa L.) mutant with shortened roots due to a defect in cell elongation. Map-based cloning revealed that the mutation occurred in a putative 3-oxoacyl-synthase, an ortholog of ß-ketoacyl-[acyl carrier protein] synthase I (KASI) in Arabidopsis, thus designated as OsKASI. OsKASI was found to be ubiquitously expressed in various tissues throughout the plant and OsKASI protein was localized in the plastid. In addition, OsKASI deficiency resulted in reduced fertility and a remarkable change in fatty acid (FA) composition and contents in roots and seeds. Our results demonstrate that involvement of OsKASI in FA synthesis is required for root development in rice.

OsGLU3, a putative membrane-bound endo-1,4-beta-glucanase, is required for root cell elongation and division in rice (Oryza sativa L.).

Plant roots move through the soil by elongation. This is vital to their ability to anchor the plant and acquire water and minerals from the soil. In order to identify new genes involved in root elongation in rice, we screened an ethyl methane sulfonate (EMS)-mutagenized rice library, and isolated a short root mutant, Osglu3-1. The map-based cloning results showed that the mutant was due to a point mutation in OsGLU3, which encodes a putative membrane-bound endo-1,4-ß-glucanase. Osglu3-1 displayed less crystalline cellulose content in its root cell wall, shorter root cell length, and a slightly smaller root meristem as visualized by restricted expression of OsCYCB1,1:GUS. Exogenous application of glucose can suppress both the lower root cell wall cellulose content and short root phenotypes of Osglu3-1. Consistently, OsGLU3 is ubiquitously expressed in various tissues with strong expression in root tip, lateral root, and crown root primodia. The fully functional OsGLU3-GFP was detected in plasma membrane, and FM4-64-labeled compartments in the root meristem and elongation zones. We also found that phosphate starvation, an environmental stress, altered cell wall cellulose content to modulate root elongation in a OsGLU3-dependant way.

Overexpression of a NAC-domain protein promotes shoot branching in rice.

For a better understanding of shoot branching in rice (Oryza sativa), a rice activation-tagging library was screened for mutations in tiller development. Here, an activation-tagging mutant Ostil1 (Oryza sativa tillering1) was characterized, which showed increased tillers, enlarged tiller angle and semidwarf phenotype. Flanking sequence was obtained by plasmid rescue. RNA-interfering and overexpression transgenic rice plants were produced using Agrobacterium-mediated transformation. The mutant phenotype was cosegregated with the reallocation of Ds element, and the flanking region of the reallocated Ds element was identified as part of the OsNAC2 gene. Northern analysis showed that expression of OsNAC2 was greatly induced in the mutant plants. Transgenic rice overexpressing the OsNAC2 resulted in recapture of the mutant phenotype, while downregulation of OsNAC2 in the Ostil1 mutant through RNA interfering (RNAi) complemented the mutant phenotype, confirming that the Ostil1 was caused by overexpression of OsNAC2. Overexpression of OsNAC2 regulates shoot branching in rice. Overexpression of OsNAC2 contributes tiller bud outgrowth, but does not affect tiller bud initiation. This suggests that OsNAC2 has potential utility for improving plant structure for higher light-use efficiency and higher yield potential in rice.

Morphogenesis and molecular basis on naked seed rice, a novel homeotic mutation of OsMADS1 regulating transcript level of AP3 homologue in rice.

The floral organs are formed from floral meristem with a regular initiation pattern in angiosperm species. Flowers of naked seed rice (nsr) were characterized by the overdeveloped lemma and palea, the transformation of lodicules to palea-/lemma-like organs, the decreased number of stamens and occasionally extra pistils. Some nsr spikelets contained additional floral organs of four whorls and/or abnormal internal florets. The floral primordium of nsr spikelet is differentiated under an irregular pattern and an incomplete determination. And molecular analysis indicated that nsr was a novel homeotic mutation in OsMADS1, suggesting that OsMADS1 played a distinct role in regulating the differentiation pattern of floral primordium and in conferring the determination of flower meristem. The gain-of-function of OsMADS1 transgenic lines presented the transformation of outer glumes to lemma-/palea-like organs and no changes in length of lemma and palea, but loss-of-function of OsMADS1 transgenic lines displayed the overdeveloped lemma and palea. Both findings revealed that OsMADS1 played a role in specifying lemma and palea and acted as a repressor of overdevelopment of lemma and palea. Moreover, it was indicated that OsMADS1 upregulated the transcript level of AP3 homologue OsMADS16, using real-time PCR analysis on gain- and loss-of-function of OsMADS1 transgenic lines.