No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth
Abstract
The molecular mechanisms underlying the initiation and maintenance of the embryonic pathway in plants are largely unknown. To obtain more insight into these processes, we used subtractive hybridization to identify genes that are upregulated during the in vitro induction of embryo development from immature pollen grains of Brassica napus (microspore embryogenesis). One of the genes identified, BABY BOOM (BBM), shows similarity to the AP2/ERF family of transcription factors and is expressed preferentially in developing embryos and seeds. Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of explants, and alterations in leaf and flower morphology. The expression pattern of BBM in developing seeds combined with the BBM overexpression phenotype suggests a role for this gene in promoting cell proliferation and morphogenesis during embryogenesis.
... Boutilier et al. discovered the gene that regulates pollen microspore embryogenesis in Brassica napus immature pollen and named it the BABY BOOM (BBM) gene [40]. Studies have shown that the BBM activates downstream genes, such as LEAFY COTYLEDON1 (LEC1), ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), AGAMOUS-LIKE15 (AGL15), and LEAFY COTYLEDON1 (LEC2), to initiate somatic embryogenesis in plant cells [41]. ...
... Promote cell proliferation and somatic cell embryo formation [40] AtBBM Arabidopsis thaliana Promote somatic embryogenesis [41] AtBBM Arabidopsis thaliana Promote cell proliferation and somatic embryogenesis [45] AtBBM ...
... Promote asexual embryo development [46] AtBBM:GR Arabidopsis thaliana Activation of complex developmental pathways for cell proliferation and cell growth [44] BnBBM1/BnBBM2 Brassica napus Promote cell proliferation and morphogenesis during embryogenesis [40] BnBBM Capsicum annuum Promote somatic embryogenesis and improve regeneration efficiency [47] CaBBM Coffea arabica Involved in somatic embryogenesis [48] GmBBM Glycine max Promote somatic embryogenesis and embryo development [49] LdBBM Larix decidua Involved in somatic embryogenesis [50] MaBBM1/MaBBM2 Musa acuminata Promote somatic embryogenesis [51] PsASGR-BBML Pennisetum glaucum Induction of apomixis [52] RcBBM1 RcBBM2 Rosa canina Promote somatic embryogenesis and improve shoot regeneration efficiency [53] TaBBM Triticum aestivum Expressed during somatic embryogenesis [54] TcBBM Theobroma cacao Increase efficiency of somatic embryogenesis [55] ZmBBM Zea mays (B73)/Sorghum bicolor (P898012) Improve transformation efficiency [56] ZmBBM Zea mays Sorghum bicolor Saccharum officinarum (v. CP01-1372) Oryza sativa ssp. ...
Establishing plant regeneration systems and efficient genetic transformation techniques plays a crucial role in plant functional genomics research and the development of new crop varieties. The inefficient methods of transformation and regeneration of recalcitrant species and the genetic dependence of the transformation process remain major obstacles. With the advancement of plant meristematic tissues and somatic embryogenesis research, several key regulatory genes, collectively known as developmental regulators, have been identified. In the field of plant genetic transformation, the application of developmental regulators has recently garnered significant interest. These regulators play important roles in plant growth and development, and when applied in plant genetic transformation, they can effectively enhance the induction and regeneration capabilities of plant meristematic tissues, thus providing important opportunities for improving genetic transformation efficiency. This review focuses on the introduction of several commonly used developmental regulators. By gaining an in-depth understanding of and applying these developmental regulators, it is possible to further enhance the efficiency and success rate of plant genetic transformation, providing strong support for plant breeding and genetic engineering research.
... These gain-of-function mutant lines generated embryogenic callus and somatic embryos at much higher frequencies than the wild-type, and somatic embryogenesis and embryogenic callus could be induced from a variety of plant tissues without the use of external growth regulators (Zuo et al., 2002). The same year, Boutilier et al. (2002) identified another transcription factor in Arabidopsis, Baby Boom (Bbm), which triggered somatic embryogenesis when ectopically expressed. Mean-while, Gordon-Kamm et al. (2002) found expression of a protein isolated from wheat dwarf virus, RepA, promoted cell division in maize and subsequently improved transformation efficiency in amenable genotypes such as Hi-II. ...
... In the model plant A. thaliana, many genes associated with somatic embryogenesis and embryogenic culture response have been identified (Gulzar et al., 2020). In some instances, these genes have induced somatic embryogenesis when ectopically expressed (Boutilier et al., 2002;Jia et al., 2014;Stone et al., 2001;Zuo et al., 2002) or knocked out (Ikeuchi et al., 2015;Rider et al., 2003). The precursor to somatic embryogenesis in many culture systems is treatment of the target tissue with a synthetic herbicide such as 2,4-D or dicamba, which induce a stress response and partially mimic the activity of auxin (Eyer et al., 2016;Parry et al., 2009). ...
... Bbm orthologues in maize have been found to induce somatic embryogenesis when ectopically expressed (Du et al., 2019;K. Lowe et al., 2016), similar to results obtained with Arabidopsis and Brassica napus (Boutilier et al., 2002). Wus, a core regulator of meristem homeostasis (Daum et al., 2014;Laux et al., 1996), has been demonstrated to induce somatic embryogenesis when ectopically expressed in diverse species, including maize and other monocot crops (K. ...
The production of embryogenic callus and somatic embryos is integral to the genetic improvement of crops via genetic transformation and gene editing. Regenerable embryogenic cultures also form the backbone of many micro‐propagation processes for crop species. In many species, including maize, the ability to produce embryogenic cultures is highly genotype dependent. While some modern transformation and genome editing methods reduce genotype dependence, these efforts ultimately fall short of producing truly genotype‐independent tissue culture methods. Recalcitrant genotypes are still identified in these genotype‐flexible processes, and their presence is magnified by the stark contrast with more amenable lines, which may respond more efficiently by orders of magnitude. This review aims to describe the history of research into somatic embryogenesis, embryogenic tissue cultures, and plant transformation, with particular attention paid to maize. Contemporary research into genotype‐flexible morphogenic gene‐based transformation and genome engineering is also covered in this review. The rapid evolution of plant biotechnology from nascent technologies in the latter half of the 20th century to well‐established, work‐horse production processes has, and will continue to, fundamentally changed agriculture and plant genetics research.
... As one of the largest transcription factor families in plants, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors are widely present in plants and play important functions in response to adversity and stress and in growth and development [19]. The BABY BOOM (BBM) gene belongs to the AP2 subfamily of the AP2/ERF transcription factor family and is proven to be one of the key genes for promoting plant regeneration [20,21]. BBM encodes an AINTEGUMENTA-LIKE (AIL) APETALA2/ ethylene-responsive element-binding factor. ...
... as the reference [35], the full-length coding sequence of (Figure 1(A)). GmBBM7 (Glyma.09G248200), the target gene of this study, was located in cluster I; ZmBBM (Zm00001d042492) and AtBBM (AT5G17430), which are reported to regulate the embryonic development of maize and Arabidopsis, were located in the same cluster [20,33]. This indicated that the members of BBM gene families located in this cluster may play important functions in plant growth and development. ...
... In summary, we can predict that during somatic embryogenesis of soybean, GmBBM7 may first respond to external hormonal signals, thereby inducing somatic embryogenesis [20]. When GmBBM7 was overexpressed, the callus formation rate and density increased significantly ( Figure 6(C,D)), indicating that GmBBM7 positively regulates somatic embryogenesis and accelerates the proliferation of callus cells in soybean. ...
The BABY BOOM (BBM) subfamily of the AP2/ERF transcription factor family is the main regulator of totipotency in plant cells and plays an important role in regulating cell proliferation and plant growth and development. Previously, we identified GmBBM7, a key gene of the soybean BBM gene subfamily (GmBBM family). In this study, we aimed to analyze its molecular characteristics and role in somatic embryogenesis in soybean. First, we identified 17 members of GmBBM family. Phylogenetic and collinear analyses revealed that the GmBBM family genes mainly exhibited a strong genetic relationship with the those of PvBBM family, the BBM family of common bean (Phaseolus vulgaris). Analysis of the promoters revealed that GmBBM family genes were mainly regulated by plant hormones. The expression of GmBBM7 varied in various tissues of soybean. Particularly, it was higher in the roots, immature embryos, grains and callus. Subcellular localization analysis indicated that GmBBM7 encodes a nuclear protein. Furthermore, overexpressed GmBBM7 could significantly enhance callus formation by regulating the levels of gibberellin A1, A3 and A7; abscisic acid; and salicylic acid and could promote root growth and development. In summary, GmBBM7 is an important regulatory gene for somatic embryogenesis and root elongation in soybean. GmBBM7 overexpression in soybean could increase the callus formation rate and density through hormonal pathways and could increase root growth. This study provided theoretical basis for efficient breeding of soybean via gene editing, transgenic and other techniques.
... However, the specific conditions that activate their differentiation into different cell types are still largely unknown [6]. To study this process, the standard models in plants are embryos [7], protoplasts [8], calli [9], and meristems [10]. These models are valuable as they contain dedifferentiated cells, which can develop into differentiated cells, ultimately giving rise to different plant tissues. ...
... However, there are many other extinct orders [72]. Lycophytes display considerable variation in the organization of their meristems, ranging 7 from the monoplex meristem with one or two apical cells found in Selaginellales [73,74] to the simplex meristems found in Lycopodiales and Isoëtales [70]. Monilophytes (ferns) usually have a meristem composed of an apical initial cell and its surrounding cells, this multicellular structure has also been proposed for Selaginella [42,75]. ...
The shoot apical meristem (SAM) gives rise to the aerial structure of plants by producing lateral organs and secondary meristems. The SAM is responsible for plant developmental patterns, thus determining plant morphology and, consequently, many agronomic traits such as the number and size of fruits and flowers and kernel yield. Our current understanding of SAM morphology and regulation is based on studies conducted mainly on some angiosperms, including economically important crops such as maize (Zea mays) and rice (Oryza sativa), and the model species Arabidopsis (Arabidopsis thaliana). However, studies in other plant species from the gymnosperm class are scant, making difficult comparative analyses that help us understand SAM regulation in diverse plant species. This limitation prevents deciphering the mechanisms by which evolution gave rise to the multiple plant structures within the plant kingdom, and determined the conserved mechanisms involved in SAM maintenance and operation. This review aims to integrate and analyze the current knowledge of SAM evolution by combining the morphological and molecular information recently reported from the plant kingdom.
... Overexpression of developmental regulators in Arabidopsis, maize, rice, sorghum, and citrus can effectively improve plant SE, genetic transformation, and gene editing efficiency [68]. For example, Leafy cotyledon 1 (LEC1), LEC2, and BABY BOOM (BBM) are specifically expressed during zygotic embryogenesis and development [69][70][71][72]; WUSCHEL (WUS) is specifically expressed in pluripotent stem cells [73]; and Growth-Regulating Factor (GRF) and its cofactor GRF-interacting factor (GIF) regulate plant growth and development [68]. Therefore, due to species specificity, it is necessary to understand the molecular mechanism and identify the key genes associated with SE in H. brasiliensis, which can be used for targeted breeding to improve SE. ...
... The ectopic expression of these regulators is associated with improving the developmental reprogramming of somatic cells to a pluripotent state and the reacquisition of an embryonic or meristematic development fate, and the loss-offunction mutations in these regulators significantly impair regeneration [75]. Recently, several developmental regulators have been used to improve the SE or organogenesis ability in monocots and dicots [68,69,71,[81][82][83]. For example, overexpression of the maize BBM and WUS2 genes can significantly improve SE and transformation frequencies in several recalcitrant maize inbred lines, sorghum, sugarcane, and rice [81,84]. ...
The rubber tree (Hevea brasiliensis) is the most important commercial plant for producing natural rubber. Immature seed inner integument and anther-derived somatic embryogenesis techniques play a crucial role in the in vitro large-scale propagation and genetic transformation of the rubber tree. However, somatic embryogenesis is highly genotype-dependent, that is, only a limited number of H. brasiliensis genotypes, such as CATAS73397, CATAS917, and PB260, can be efficiently induced by somatic embryogenesis and used for large-scale propagation or transformation. The genotype dependence of the somatic embryogenesis technique is a conundrum for the application of Hevea biotechnology in most commercially important cultivars, such as Reken628 and CATAS879. Previous studies have shown that several somatic embryogenesis regulators can overcome genotype dependence and enhance the transformation and regeneration efficiency of recalcitrant plants and cultivars. In this review, we first describe the relevant successful applications of somatic embryogenesis technology in seedling production and genetic modification of H. brasiliensis. Second, we discuss the genotype dependence of somatic embryogenesis as the major challenge currently. Third, we summarize the recent significant advances in the understanding of the molecular mechanisms underlying somatic embryogenesis in other plants. Finally, we suggest a roadmap for using somatic embryogenesis regulatory genes to facilitate genotype-dependent somatic embryogenesis technology in H. brasiliensis.
... Morrone (sub Pennisetum squamulatum), is the APOSPORY SPECIFIC GENOMIC REGION-BABY BOOM-LIKE (PsASGR-BBML) gene [32]. This gene is an ortholog of the BBM gene in the clade APETALA 2 of highly conserved transcription factors whose ectopic expression induces embryo formation in Brassica and Arabidopsis [141]. The regulatory element in PsASGR-BBML is still to be proven, although it triggers parthenogenesis at a low frequency in tetraploid tobacco, but not in diploid Arabidopsis [142,143]. ...
Potato is the most important non-cereal crop worldwide, and, yet, genetic gains in potato have been traditionally delayed by the crop’s biology, mostly the genetic heterozygosity of autotetraploid cultivars and the intricacies of the reproductive system. Novel site-directed genetic modification techniques provide opportunities for designing climate-smart cultivars, but they also pose new possibilities (and challenges) for breeding potato. As potato species show a remarkable reproductive diversity, and their ovules have a propensity to develop apomixis-like phenotypes, tinkering with reproductive genes in potato is opening new frontiers in potato breeding. Developing diploid varieties instead of tetraploid ones has been proposed as an alternative way to fill the gap in genetic gain, that is being achieved by using gene-edited self-compatible genotypes and inbred lines to exploit hybrid seed technology. In a similar way, modulating the formation of unreduced gametes and synthesizing apomixis in diploid or tetraploid potatoes may help to reinforce the transition to a diploid hybrid crop or enhance introgression schemes and fix highly heterozygous genotypes in tetraploid varieties. In any case, the induction of apomixis-like phenotypes will shorten the time and costs of developing new varieties by allowing the multi-generational propagation through true seeds. In this review, we summarize the current knowledge on potato reproductive phenotypes and underlying genes, discuss the advantages and disadvantages of using potato’s natural variability to modulate reproductive steps during seed formation, and consider strategies to synthesize apomixis. However, before we can fully modulate the reproductive phenotypes, we need to understand the genetic basis of such diversity. Finally, we visualize an active, central role for genebanks in this endeavor by phenotyping properly genotyped genebank accessions and new introductions to provide scientists and breeders with reliable data and resources for developing innovations to exploit market opportunities.
... Other proteins associated with apomixis were transcription factors, such as BABY BOOM (BBM), that induces parthenogenesis and somatic embryogenesis in Brassica napus and A. thaliana (Boutilier et al., 2002;Schmidt, 2020), and apogamy in C. richardii (Bui et al., 2017); and the AGAMOUS-LIKE proteins, belonging to the MADS-box family, widely represented in our transcriptome. We remark here two members: AGAMOUS-LIKE 62 (AGL62) and 6 (AGL6), the primer promoting cellularization in early endosperm development in A. thaliana (Kang et al., 2008), while AGL6 is involved in megasporogenesis in the apomictic and sexual angiosperm Brachiaria brizantha (Guimaraes et al., 2013). ...
The gametophyte of the fern Dryopteris affinis ssp. affinis represents a good model to explore the molecular basis of vegetative and reproductive development, as well as stress responses. Specifically, this fern reproduces asexually by apogamy, a peculiar case of apomixis whereby a sporophyte forms directly from a gametophytic cell without fertilization. Using an RNA-sequencing approach, we have previously annotated more than six thousand transcripts. Here, we selected one hundred of the inferred proteins that seemed particularly interesting for a detailed study of their potential functions, protein-protein interactions, and molecular phylogenies. As expected, a plethora of proteins associated with gametogenesis and embryogenesis in angiosperms, such as FERONIA (FER) and CHROMATING REMODELING 11 (CHR11) were identified, and more than a dozen candidates potentially involved in apomixis, such as ARGONAUTE4 (AGO4), AGO9, and AGO10, BABY BOOM (BBM), FASCIATED STEM4 (FAS4), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), and MATERNAL EFFECT EMBRYO ARREST29 (MEE29). In addition, proteins involved in the response to biotic and abiotic stresses were widely represented, as shown by the enrichment of heat-shock proteins. Using the String platform, studying interactomes revealed that most of the protein-protein interactions were predicted based on experimental, database, and text mining datasets, with MULTICOPY SUPPRESSOR OF IRA4 (MSI4) showing the highest number of 16 interactions. Lastly, some proteins were studied from a phylogenetic point of view, comparing the alignments with respect to more distantly or closely related plant groups, identifying AGO1 as the evolutionarily most similar to that other ferns and the most distant to the predicted common ancestor. This work sets the stage for future functional characterizations in relation to gametophyte development including apomictic reproduction.
... Recent empirical data illustrated the repeated AP2 domain regulates during the developmental processes in crop variety (i.e. flower, meristem, leaf, and embryo development) [2][3][4][5][6]. On the other hand, the ethylene-responsive factor (ERF) family divides into two main subfamilies: C-repeat/dehydration-responsive element binding factors family (CBF/ DREB family) [7][8][9]. ...
Background
The Apetala 2/ethylene-responsive factor family has diverse functions that enhance development and torment resistance in the plant genome. In variation, the ethylene-responsive factor (ERF) family of TF’s genes is extensive in the crop genome. Generally, the plant-specific ethylene-responsive factor family may divided by the dehydration-responsive element-binding (DREB) subfamily. So, the AP2/ERF super-family demonstrated the repeated AP2 domain during growth. The sole AP2 domain function represents abiotic stress resistance. Also, the AP2 with B3 domain enhances during the replication of brassinosteroid.
Objective
The study objective is to investigate the Apetala 2/ethylene-responsive factor family in a model organism of the Arabidopsis thaliana for comparative analysis towards Solanum lycopersicum (Tomato), Brassica juncea (Indian and Chinese mustard), Zea mays L. (Maize) and Oryza sativa (Indian and Japanese Rice). So, examinations of the large AP2/ERF super-family are mandatory to explore the Apetala 2 (AP2) family, ERF family, DREB subfamily, and RAV family involved during growth and abiotic stress stimuli in crops.
Methods
Therefore, perform bioinformatics and computational methods to the current knowledge of the Apetala 2/ethylene-responsive factor family and their subfamilies in the crop genome. This method may be valuable for functional analysis of particular genes and their families in the plant genome.
Results
Observation data provided evidence of the Apetala 2/ethylene-responsive factor (AP2/ERF) super-family and their sub-family present in Arabidopsis thaliana (Dicots) and compared with Solanum lycopersicum (Dicots), Brassica juncea (Dicots), Zea mays L. (Monocots) and Oryza sativa (Monocots). Also, remarks genes in Oryza sativa. This report upgraded the Apetala 2/ethylene-responsive factor (AP2/ERF) family in the crop genome. So, the analysis documented the conserved domain, motifs, and phylogenetic tree towards Dicots and Monocots species. Those outcomes will be valuable for future studies of the defensive Apetala 2/ethylene-responsive factor family in crops.
Conclusion
Therefore, the study concluded that the several species-specific TF genes in the Apetala 2/ethylene-responsive factor (AP2/ERF) family in Arabidopsis thaliana and compared with crop-species of Solanum lycopersicum, Brassica juncea, Zea mays L. and Oryza sativa. Those plant-specific genes regulate during growth and abiotic stress control in plants.
... They then control the secondary metabolism and other life activities of plants and play a key role in plant growth and development Hao et al., 2020). Among these factors, gene members in the AP2 family have been proved to be involved in the regulation of plant development, such as flower development, embryo development, and initiation of leaf epidermal cell differentiation (Boutilier et al., 2002;Elliott et al.,1996;Moose & Sisko, 1996). The APETALA2/ethylene-responsive factor (AP2/ERF) family is a large family of transcription factors in plants and members of this family usually contain one to two highly conserved AP2 domains. ...
Terpenoids are the main active components in the Zanthoxylum armatum leaves, which have extensive medicinal value. The Z. armatum leaf is the main by‐product in the Z. armatum industry. However, the transcription factors involved in the biosynthesis of terpenoids are rarely reported. This study was performed to identify and classify the APETALA2/ethylene‐responsive factor (AP2/ERF) gene family of Z. armatum. The chromosome distribution, gene structure, conserved motifs, and cis‐acting elements of the promoter of the species were also comprehensively analyzed. A total of 214 ZaAP2/ERFs were identified. From the obtained transcriptome and terpenoid content data, four candidate ZaAP2/ERFs involved in the biosynthesis of terpenoids were selected via correlation and weighted gene co‐expression network analysis. A phylogenetic tree was constructed using 13 AP2/ERFs related to the biosynthesis of terpenoids in other plants. ZaERF063 and ZaERF166 showed close evolutionary relationships with the ERFs in other plant species and shared a high AP2‐domain sequence similarity with the two closest AP2/ERF proteins, namelySmERF8 from Salvia miltiorrhiza and AaERF4 from Artemisia annua. Further investigation into the effects of methyl jasmonate (MeJA) treatment on the content of terpenoids in Z. armatum leaves revealed that MeJA significantly induced the upregulation of ZaERF166 and led to a significant increase in the terpenoids content in Z. armatum leaves, indicating that ZaERF166 might be involved in the accumulation of terpenoids of Z. armatum. Results will be beneficial for the functional characterization of AP2/ERFs in Z. armatum and establishment of the theoretical foundation to increase the production of terpenoids via the manipulation of the regulatory elements and strengthen the development and utilization of Z. armatum leaves.
... The WUS clade genes have been shown to be involved in regulating plant regeneration [58,[60][61][62]. The application of regeneration-related genes could potentially enhance plant transformation efficiency during the in vitro culture media process [57,[63][64][65][66][67][68][69]. Carbohydrate metabolism is essential for plant callus induction and shoot regeneration [70]. ...
Background
The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide.
Results
A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis.
Conclusions
Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.
... e which codes for a transcription factor of the family AP2/ERF. The members of this family are involved in the control of events as the determination of the identity of the floral organs that are expressed preferentially in the development of embryos and seeds, and the formation of somatic embryos in A. thaliana by stimulating their overexpression (Boutilier et. al., 2002;El Ouakfaoui et al 2010;Chen et al., 2022). Genetic analysis indicates that WUS protein interacts with many other regulators of meristematic outbreaks and floral meristems suggesting that WUS integrates positional and temporal information of these genes as is the case of CLAVATA (CLV) genes and SHOOT MERISTEM LESS (STM) genes (Klaus et. ...
p> Background: During the last few years the home box transcription factor WUSCHEL (WUS) has been shown to cause dedifferentiation when expressed on somatic cells followed by a production of new stem cells that can lead to somatic embryogenesis or organogenesis. WUS has been shown to promote the transition from a vegetative to an embryogenic state when overexpressed. Objective/Hypothesis . The genetic in vitro transformation of meristematic tissue of Musa acuminata L. AAA, cv. “Grand Nain” was carried out using a heterologous gene WUSCHEL from Arabidopsis thaliana , via vacuum infiltration with Agrobacterium tumefaciens , to establish and ascertain if its expression modifies the progression of the explants to the embryogenesis process and or reduces the time needed for in vitro embryogenic induction phase . Methodology : Explants of proliferating shoot meristems , named “scalps”, of Musa acuminata L. AAA, cv. “Grand Nain” was transformed with WUS gene under the control of promoter GAL4, inducible by 17 b -estradiol. The effect of steroid 17 b -estradiol ( b-E st) and homobrassinolide (HomoBra) on the in vitro somatic embryogenesis induction phase of non-transformed banana scalps was investigated. Results : The successful transformation of the explants was confirmed by PCR, for the transferred neomycin phosphotransferase II (NPTII) and the WUS gene. In addition, the expression of the red fluorescent protein (RFP) for the corresponding transferred reporter gene was verified by fluorescence microscopy in proliferated transformed tissues. Besides, the transformed tissue response to the induction of embryogenesis with either b-E st and/or HomoBra, inducers of the transgene were investigated. Implications: The improvement of the process of somatic embryogenesis in this way, generates a more effective and productive study model in a short time. Conclusions: WUS can promote the meristematic tissue-to-embryonic transition, and eventually somatic embryo formation, suggesting that the homeodomain protein can play a critical role during embryogenesis. </p
... Recent research indicated that the key genes regulate the SEs process. These genes mainly include FUSCA (FUC3), LEAFY COTYLEDON genes (LEC), WUSCHEL (WUS), BABY BOOM (BBM), AGAMOUSLIKE-15 (AGL15), and SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE1 (SERK1), etc. (Tsuwamoto et al., 2010;Lotan et al., 1998;Zuo et al., 2002;Boutilier et al., 2002;Horstman et al., 2017;Harding et al., 2003;Wang et al., 2020;Fambrini et al., 2022). The transcription control, miR156-SPL, chromatin accessibility, and DNA methylation control the process of somatic embryogenesis (Pais et al., 2019;Ji et al., 2019;Grzybkowska et al., 2020;Markulin et al., 2021;Zang et al., 2021). ...
... Recent empirical data illustrated the repeated AP2 domain regulates during the developmental processes in crop variety (i.e. ower, meristem, leaf, and embryo development) [2] [3][4][5][6]. On the other hand, the ethylene-responsive factor (ERF) family divides into two main subfamilies: Crepeat/dehydration-responsive element binding factors family (CBF/DREB family) [7][8][9]. ...
Background: The Apetala 2/ethylene-responsive factor family has diverse functions that enhance development and torment resistance in the plant genome. In variation, the ethylene-responsive factor (ERF) family of TF’s genes is extensive in the crop genome. Generally, the plant-specific ethylene-responsive factor family may divided by the dehydration-responsive element-binding (DREB) subfamily. So, the AP2/ERF super-family demonstrated the repeated AP2 domain during growth. The sole AP2 domain function represents abiotic stress resistance. Also, the AP2 with B3 domain enhances during the replication of brassinosteroid.
Objective: The study objective is to investigate the Apetala 2/ethylene-responsive factor family in a model organism of the Arabidopsis thaliana for comparative analysis towards Solanum lycopersicum (Tomato), Brassica juncea (Indian and Chinese mustard), Zea mays L. (Maize) and Oryza sativa (Indian and Japanese Rice). So, examinations of the large AP2/ERF superfamily are mandatory to explore the Apetala 2 (AP2) family, ERF family, DREB subfamily, and RAV family involved during growth and abiotic stress stimuli in crops.
Methods: Therefore, perform bioinformatics and computational methods to the current knowledge of the Apetala 2/ethylene-responsive factor family and their subfamily in crop species. This method may be valuable for functional analysis of particular genes and their families in the plant genome.
Results: Observation data provided evidence of the Apetala 2/ethylene-responsive factor (AP2/ERF) superfamily and their subfamily present in Arabidopsis thaliana (Dicots) and compared with Solanum lycopersicum (Dicots), Brassica juncea (Dicots), Zea mays L. (Monocots) and Oryza sativa(Monocots). Also, remarks genes in Oryza sativa. This report upgraded the Apetala 2/ethylene-responsive factor (AP2/ERF) family in crop genomes. So, the analysis documented the conserved domain, motifs, and phylogenetic tree towards Dicots and Monocots species. Those outcomes will be valuable for future studies of the defensive Apetala 2/ethylene-responsive factor family in crops.
Conclusion: Therefore, the study concluded that the several species-specific TF genes in the Apetala 2/ethylene-responsive factor (AP2/ERF) in Arabidopsis thaliana and compared with crop-species of Solanum lycopersicum, Brassica juncea, Zea mays L. and Oryza sativa. Those plant-specific genes regulate during growth and abiotic stress control in plants. So, the study will provide extensive knowledge of the agronomic, economic, and ecological traits and possibly other benefits of crops.
... In this study, we found that Lus10031831 and Lus10031260 are significantly more highly expressed in roots compared to other tissues. In previous studies, BBM was discovered by in vitro induction of immature pollen grains in rapeseed, showing similarity to the AP2/ERF family of transcription factors, which are preferentially expressed in developing embryos and seeds and promote cell proliferation, differentiation and morphogenesis [46,47]. In our study, we found that Lus10015055, Lus10026477, Lus10019905, Lus10004990, and Lus10041595 show significantly increased expression in pistils, stamens, leaves, and roots, but the expression level in fruits remains stable, which differs from previous research. ...
Flax (Linum usitatissimum L.) is an important oilseed crop widely cultivated for its oil and fiber. This study conducted transcriptome analysis to analyze the gene expression profiles of roots, leaves, stamens, pistils, and fruits in the flax cultivar Longya10. A total of 43,471 genes were detected in the RNA-seq data, with 34,497 genes showing differential expression levels between different organs. Gene expression patterns varied across different organs, with differences observed in expression-regulating genes within specific organs. However, 23,448 genes were found to be commonly expressed across all organs. Further analysis revealed organ-specific gene expressions, with 236, 690, 544, 909, and 1212 genes identified in pistils, fruits, leaves, roots, and stamens, respectively. Gene Ontology (GO) enrichment analysis was performed on these organ-specific genes, and significant enrichment was observed in various biological processes, cellular components, and molecular functions, providing new insights for the specific growth patterns of flax organs. Furthermore, we investigated the expression differences of AP2 transcription factors in various tissues and organs of Longya10. We identified 96 AP2 genes that were differentially expressed in different organs and annotated them into various biological pathways. Our results suggest that AP2 transcription factors may play important roles in regulating the growth and development of flax organs including stress response. In summary, our study provides a comprehensive analysis of gene expression patterns in different organs and tissues of flax plant and identifies potential critical regulators of flax organ growth and development. These findings contribute to a better understanding of the molecular mechanisms underlying flax organ development and may have important implications for the genetic improvement of flax crops.
... Baby boom (Bbm) and Wuschel2 (Wus2) are key regulatory factors in the development of plant stem cells [134]. Bbm encodes an AP2/ERF transcription factor, which plays an important role in maintaining stem cells in an undifferentiated state. ...
Genetic transformation is an important strategy for enhancing plant biomass or resistance in response to adverse environments and population growth by imparting desirable genetic characteristics. Research on plant genetic transformation technology can promote the functional analysis of plant genes, the utilization of excellent traits, and precise breeding. Various technologies of genetic transformation have been continuously discovered and developed for convenient manipulation and high efficiency, mainly involving the delivery of exogenous genes and regeneration of transformed plants. Here, currently developed genetic transformation technologies were expounded and compared. Agrobacterium-mediated gene delivery methods are commonly used as direct genetic transformation, as well as external force-mediated ways such as particle bombardment, electroporation, silicon carbide whiskers, and pollen tubes as indirect ones. The regeneration of transformed plants usually involves the de novo organogenesis or somatic embryogenesis pathway of the explants. Ectopic expression of morphogenetic transcription factors (Bbm, Wus2, and GRF-GIF) can significantly improve plant regeneration efficiency and enable the transformation of some hard-to-transform plant genotypes. Meanwhile, some limitations in these gene transfer methods were compared including genotype dependence, low transformation efficiency, and plant tissue damage, and recently developed flexible approaches for plant genotype transformation are discussed regarding how gene delivery and regeneration strategies can be optimized to overcome species and genotype dependence. This review summarizes the principles of various techniques for plant genetic transformation and discusses their application scope and limiting factors, which can provide a reference for plant transgenic breeding.
The generation of complex plant architectures depends on the interactions among different molecular regulatory networks that control the growth of cells within tissues, ultimately shaping the final morphological features of each structure. The regulatory networks underlying tissue growth and overall plant shapes are composed of intricate webs of transcriptional regulators which synergize or compete to regulate the expression of downstream targets. Transcriptional regulation is intimately linked to phytohormone networks as transcription factors (TFs) might act as effectors or regulators of hormone signaling pathways, further enhancing the capacity and flexibility of molecular networks in shaping plant architectures. Here, we focus on homeodomain-leucine zipper (HD-ZIP) proteins, a class of plant-specific transcriptional regulators, and review their molecular connections with hormonal networks in different developmental contexts. We discuss how HD-ZIP proteins emerge as key regulators of hormone action in plants and further highlight the fundamental role that HD-ZIP/hormone networks play in the control of the body plan and plant growth.
Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.
Innovations and novel technologies are essential to address the needs of the future food system. Enhancing the genetics of important oil crops and millets, as well as domesticating new high-yielding varieties, is crucial to address the growing need for food security. Genome editing in plants is accomplished by changing the genome of cultured cells, which is followed by the regeneration of entire plants. Emerging as a ground-breaking breeding tool, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) may provide a framework for accommodating increasing demand and potentially reducing regulatory burden. Although the CRISPR/Cas system is highly efficient for genome editing, several barriers and challenges impede its further improvement and adoption. Successful implementation of editing technologies requires an effective plant regeneration system. This review gives a comprehensive insight into many standard tissue culture-based regeneration and non-tissue culture-based genetic transformation techniques, making gene editing suitable for application in much less explored recalcitrant crops like oilseed and millets. This review also outlines the existing obstacles and introduces the techniques and emerging platforms that are being devised to address these limitations.
Wide variation in amenability to transformation and regeneration (TR) among many plant species and genotypes presents a challenge to the use of genetic engineering in research and breeding. To help understand the causes of this variation, we performed association mapping and network analysis using a population of 1204 wild trees of Populus trichocarpa (black cottonwood).
To enable precise and high‐throughput phenotyping of callus and shoot TR, we developed a computer vision system that cross‐referenced complementary red, green, and blue (RGB) and fluorescent‐hyperspectral images. We performed association mapping using single‐marker and combined variant methods, followed by statistical tests for epistasis and integration of published multi‐omic datasets to identify likely regulatory hubs.
We report 409 candidate genes implicated by associations within 5 kb of coding sequences, and epistasis tests implicated 81 of these candidate genes as regulators of one another. Gene ontology terms related to protein–protein interactions and transcriptional regulation are overrepresented, among others.
In addition to auxin and cytokinin pathways long established as critical to TR, our results highlight the importance of stress and wounding pathways. Potential regulatory hubs of signaling within and across these pathways include GROWTH REGULATORY FACTOR 1 (GRF1), PHOSPHATIDYLINOSITOL 4‐KINASE β1 (PI‐4Kβ1), and OBF‐BINDING PROTEIN 1 (OBP1).
Genome-editing technology represents a potent tool for functional genomics to improve the quality and commercial value of oil palm, one of the world’s most important commercial oil crops. The availability of the oil palm genetic transformation system and the accessibility of the entire oil palm genome sequence data will revolutionise the technology to improve agricultural traits and create new plant varieties. This chapter highlights the application and recent progress of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9, commonly known as CRISPR/Cas9 genome editing in oil palm. Their associated strategies and challenges to developing an improved crop with the addition of valuable traits with high precision and future utilisation potential are also discussed. CRISPR/Cas9 is the most recently developed and rapidly adopted targeted genome modification system. Comparing it to other genome-editing technologies such as transcriptional activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs), it has been shown to be more effective, specific, adaptable, straightforward, and multiplexed. The CRISPR/Cas9 technology enables the editing single to multiple genes to produce oil palm varieties with high-value-added fatty acids and novel agronomic traits. Such traits have always been of interest to the oil palm industry for ease of fruit harvesting, improve labour productivity, extend economic lifespan, and increase nutritional value in response to growing demand.
Hybrids with high yield and stress tolerance have been developed by researchers and cultivated by farmers over the years. However, their genetic constitution will change in the segregating generations and the elite traits of the hybrids will not be retained completely in the subsequent generations. These limitations can be overcome by exploiting the asexual mode of reproduction that exists in flowering plants known as apomixis. This phenomenon has the potential to produce clonal or genetically identical seeds as the mother genotype and it can also preserve the hybrid vigour for several generations. In angiosperms, it is essential to understand the mechanism and molecular basis of apomixis to transfer or convert non-apomictic plants to apomictic plants. Different genes/alleles or genetic mechanisms controlling various steps of apomixis have already been reported. Recently, few studies were reported on how non-apomictic plants can be converted into apomictic lines by using two approaches viz., hybridization and CRISPR/Cas gene editing method, to develop synthetic apomictic lines. This synthetic apomixis brought an initial breakthrough by producing clonal hybrid seeds (> 90 per cent) over multiple generations. This review highlights the mechanisms, molecular basis of apomixis and recent developments, achievements and challenges in apomixis, as a tool for clonal seed production, preservation of heterosis and crop improvement.
The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.
The conventional concept of plant breeding as art and science is shifting toward less art and more science due to the rapid development in genomics, transcriptomics, and the availability of advanced molecular tools. Conventional plant breeding methods were made rapid and more precise with the help of molecular markers. However, these methods lack ways to reduce or avoid unwanted variation incorporated during the transfer of any desirable trait. Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9), a genome-editing tool, has revolutionized plant sciences and made genetic studies more accessible and easier. This tool can supplement breeding methods in terms of creating precise and targeted variation for a trait and that too with negligible background variation. It can also be used to create new variations through chromosome engineering and targeted recombination, aiding in breaking undesirable linkages that otherwise cannot be broken by conventional plant breeding methods. Besides this, genome editing is also assisting in rapid de novo domestication of crop wild relatives, generating haploid-inducer lines, and developing apomictic hybrids. Genome editing is poised to remove many major bottlenecks of plant breeding.
Understanding somatic cell totipotency remains a challenge facing scientific inquiry today. Plants display remarkable cell totipotency expression, illustrated by single-cell differentiation during somatic embryogenesis (SE) for plant regeneration. Determining cell identity and exploring gene regulation in such complex heterogeneous somatic cell differentiation have been major challenges. Here, we performed high-throughput single-cell sequencing assays to define the precise cellular landscape and revealed the modulation mode of marker genes during embryogenic differentiation in cotton (Gossypium hirsutum L.) as the crop for biotechnology application. We demonstrated that nonembryogenic calli (NEC) and primary embryogenic calli (PEC) tissues were composed of heterogeneous cells that could be partitioned into 4 broad populations with 6 distinct cell clusters. Enriched cell clusters and cell states were identified in NEC and PEC samples, respectively. Moreover, a broad repertoire of new cluster-specific genes and associated expression modules were identified. The energy metabolism, signal transduction, environmental adaptation, membrane transport pathways, and a series of transcription factors were preferentially enriched in cell embryogenic totipotency expression. Notably, the SE-ASSOCIATED LIPID TRANSFER PROTEIN (SELTP) gene dose-dependently marked cell types with distinct embryogenic states and exhibited a parabolic curve pattern along the somatic cell embryogenic differentiation trajectory, suggesting that SELTP could serve as a favorable quantitative cellular marker for detecting embryogenic expression at the single-cell level. In addition, RNA velocity and Scissor analysis confirmed the pseudo-temporal model and validated the accuracy of the scRNA-seq data, respectively. This work provides valuable marker-genes resources and defines precise cellular taxonomy and trajectory atlases for somatic cell embryogenic differentiation in plant regeneration.
Yam is an important staple in sub-Saharan Africa, but the availability of quality seed yam is majorly constrained by the low propagation ratio. This is because the propagating explant is limited to the tuber and nodal parts as yam rarely flowers. There are several reports of the use of somatic embryogenesis (SE) in the rapid propagation of different crop species and as a regenerative pathway in plant genetic engineering. However, SE deployment in yam is still at the protocol development stage. This review thus exploits the status of SE application in improving the yam propagation rate. This article reviews the potential of the various yam propagation techniques in rapidly multiplying disease-free yam with their propagating explants. The advantages SE offers are rapidly propagating yam, the factors to consider in the protocol optimization of SE application in rapidly multiplying different yam varieties, and as a platform for full utilization of genetic engineering in yam. The findings so far show that SE potentially offers a faster rate of propagating yam varieties. However, due to the differences in varietal endogenous hormonal and gene products, response to SE in yam is constrained by varietal specificity. Hence, the applicability of SE in yam is still at the protocol development state. This review, thus, presents the need for more research efforts to elucidate the molecular and phytochemical controlling mechanisms of SE in yam to improve the yam multiplication rate and lay an efficient platform for the exploitation of other biotechnological advancements in improving yam species.
The shoot apical meristem (SAM) gives rise to the aerial structure of plants by producing lateral organs and other meristems. The SAM is responsible for plant developmental patterns, thus determining plant morphology and, consequently, many agronomic traits such as the number and size of fruits and flowers and kernel yield. Our current understanding of SAM morphology and regulation is based on studies conducted mainly on some angiosperms, including economically important crops such as maize (Zea mays) and rice (Oryza sativa), and the model species Arabidopsis (Arabidopsis thaliana). However, studies in other plant species from the gymnosperms are scant, making difficult comparative analyses that help us understand SAM regulation in diverse plant species. This limitation prevents deciphering the mechanisms by which evolution gave rise to the multiple plant structures within the plant kingdom and determines the conserved mechanisms involved in SAM maintenance and operation. This review aims to integrate and analyze the current knowledge of SAM evolution by combining the morphological and molecular information recently reported from the plant kingdom.
Transcription factors (TFs) are diverse groups of regulatory proteins. Through their specific binding domains, TFs bind to their target genes and regulate their expression, therefore TFs play important roles in various growth and developmental processes. Plant embryogenesis is a highly regulated and intricate process during which embryos arise from various sources and undergo development; it can be further divided into zygotic embryogenesis (ZE) and somatic embryogenesis (SE). TFs play a crucial role in the process of plant embryogenesis with a number of them acting as master regulators in both ZE and SE. In this review, we focus on the master TFs involved in embryogenesis such as BABY BOOM (BBM) from the APETALA2/Ethylene-Responsive Factor (AP2/ERF) family, WUSCHEL and WUSCHEL-related homeobox (WOX) from the homeobox family, LEAFY COTYLEDON 2 (LEC2) from the B3 family, AGAMOUS-Like 15 (AGL15) from the MADS family and LEAFY COTYLEDON 1 (LEC1) from the Nuclear Factor Y (NF-Y) family. We aim to present the recent progress pertaining to the diverse roles these master TFs play in both ZE and SE in Arabidopsis, as well as other plant species including crops. We also discuss future perspectives in this context.
Transferring an asexual mode of reproduction by seeds (apomixis) to cultivated plants would enable clonal reproduction of heterozygous genotypes such as F1 hybrids with hybrid vigor (heterosis), facilitating their access and multiplication by small-scale growers. Although sources of apomixis and the genetic loci controlling its constituent elements have been identified in wild species, their transfer by crossing to cultivated species has so far been unsuccessful. Here, we have introduced synthetic apomixis in hybrid rice to produce a high (95-100%) frequency of clonal seeds, via the inactivation of three meiotic genes-resulting in unreduced, non-recombined gametes-and the addition of an egg cell parthenogenesis trigger. The genotype and phenotype, including grain quality, of the F1 hybrid are reproduced identically in the clonal apomictic progenies. These results make synthetic apomixis compatible with future use in agriculture.
BABY BOOM (BBM) is one of the members of the plant-specific APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor superfamily. It acts as a key regulator of plant cell pluripotency, playing a significant role in promoting somatic embryogenesis. In this study, a BBM gene named PoBBM was screened, cloned, and identified from the third-generation full-length transcriptome data of Paeonia ostii. Its open reading frame was 2136 bp, encoding 711 amino acids. Sequence feature analysis revealed that it possessed two AP2 conserved domains and eight motifs, including bbm-1. The phylogenetic tree indicated that PoBBM clusters with AtBBM in the euANT group of the Arabidopsis AP2 family, which is most closely related to grape VvBBM and may have the same ancestry as grape. Subcellular localization demonstrated that the PoBBM protein was localized in the nucleus. Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess the PoBBM transcript levels during ten developmental stages of somatic embryos and in five tissue types of peonies. The results indicate that PoBBM was highly expressed in the early stages of peony somatic embryo development. The expression on 0–15 d was the highest and decreased gradually with somatic embryogenesis. The gene is almost not expressed after 40 d since somatic embryo formation. PoBBM was expressed in roots, stems, leaves, seeds, and calli, with the highest levels in seeds, followed by leaves and calli. The PoBBM protein displayed transcriptional self-activation activity, which may facilitate further research on its relationships with other proteins. The above results provide a key gene PoBBM for somatic embryogenesis in peonies, which is significant for advancing the establishment of a stable and efficient regeneration and genetic transformation system for peonies.
This chapter summarizes the status of the genome editing efforts in Hordeum vulgare L. and provides an overview of the technical advances and obstacles of applying genome editing in barley. It also highlights the potential of genome editing in barley breeding with the focus on breeding for high yielding, disease resistant and stable varieties. The CRISPR/Cas technology is a breakthrough in genome editing due to its robustness and easy to use programming, especially for generating targeted mutations to switch off genes that have a negative impact on food quality, increase susceptibility to pathogens, or divert metabolic flux away from useful end products. Genome editing studies are expected to advance barley breeding by accelerating the breeding process and enabling easier multiplexing of traits. The chapter offers an outlook on the future of barley genome editing techniques based on CRISPR/Cas system.
CRISPR technology is revolutionizing genomic engineering by enabling scientists to precisely modify plant DNA, thus representing a powerful tool for plant breeding.
This chapter provides a summary of the approaches and constraints of CRISPR-mediated genome editing in plants, with a focus on the critical prerequisite of efficient CRISPR reagents for successful gene editing in plants.
While computational tools have tremendously improved our ability to design specific guides, their limitations make guide effectiveness prediction unreliable, especially for plants. Therefore, it is strongly recommended to validate CRISPR reagents before investing time and resources in the challenging process of plant transformation.
A number of in vitro and in planta assays coupled with analytical methods have been proposed to assess the editing performances. Each approach has its own strengths and weaknesses, so the choice of the most suitable system depends on the specific plant species and the type and depth of the genotypic data required.
In many cases, the hairy root assay can provide a good compromise between rapidity, reliability and cost-effectiveness for assessing editing performance in numerous plant species.
Sexual reproduction in plants is the main pathway for creating new genetic combinations in modern agriculture. In heterozygous plants, after the identification of a plant with desired traits, vegetative propagation (cloning) is the primary path to create genetically uniform plants. Another natural plant mechanism that creates genetically uniform plants (clones) is apomixis. In fruit crops like citrus and mango, sporophytic apomixis results in polyembryony, where seeds contain multiple embryos, one of which is sexually originated and the others are vegetative clones of the parent mother tree. Utilizing the mango genome and genetic analysis of a diverse germplasm collection, we identified MiRWP as the gene that causes polyembryony in mango. There is a strong correlation between a specific insertion in the gene’s promoter region and altered expression in flowers and developing fruitlets, inducing multiple embryos. The MiRWP gene is an ortholog of CitRWP that causes polyembryony in citrus. Based on the data, we speculate that promoter insertion events, which occurred independently in citrus and mango, induced nucellar embryogenesis. The results suggest convergent evolution of polyembryony in the two species. Further work is required to demonstrate the utility of these genes (mango and citrus) in other biological systems as a tool for the clonal production of other crops.
Callus browning during tissue culture of indica rice is genotype dependent, thus limiting the application of genetic transformation for editing-assisted breeding and elucidation of gene function. Here, using 124 introgression lines (HCLs) derived from a cross between the indica rice 9311 and Chaling common wild rice and 2059 SNPs for single-point and interval analysis, we identified two major QTLs, qCBT7 on chromosome 7 and qCBT10 on chromosome 10, related to callus browning, explaining 8–13% of callus browning. Moreover, we performed RNA-seq of two introgression lines with low callus browning, HCL183 and HCL232, with Oryza. rufipogon introgression fragments on chromosomes 10 and 7, respectively. Three candidate genes (Os07g0620700, Os10g0361000, and Os10g0456800) with upregulation were identified by combining interval mapping and weighted gene coexpression network analysis using the DEGs. The qRT-PCR results of the three candidate genes were consistent with those of RNA-seq. The differentiation of indica and japonica subspecies Oryza. sativa and Oryza. rufipogon suggests that these candidate genes are possibly unique in Oryza. rufipogon. GO analyses of hub genes revealed that callus browning may be mainly associated with ethylene and hormone signaling pathways. The results lay a foundation for future cloning of qCBT7 or qCBT10 and will improve genetic transformation efficiency in rice.
Somatic embryogenesis (SE) is a key regeneration process in plant. AcSERK1 is a gene specifically expressed in the early stage of SE in pineapple (Ananas comosus), suggesting that the promoter of SERK1 might contain specific cis-acting element regulating SE. To identify embryonic cell-specific element in the SERK1 promoter, a series of binary plant transformation vectors with GUS (β-glucuronidase) reporter gene were systematically analyzed by transient gene expression system in wild-type and transgenic pineapple embryogenic callus. Histochemical and quantitative GUS assays demonstrated that the activity of the AcSERK1 upstream regulatory sequence lacking − 921 to -911 or -910 to -880 was significantly reduced in the embryonic callus of the pineapple, and these two regions were needed for the embryonic cell-specific. Besides, a promoter lacking − 943 to -922 was shown to significantly increase GUS activity in embryogenic callus, suggesting repressive elements exist in this region. Our data of stable transformation assays confirmed again the 5’ upstream regulatory sequence (-921 to -880) of the AcSERK1 gene is an essential functional region. Our findings lay the basis for better understanding of the molecular mechanisms of AcSERK1 gene in the regulation in early stage of SE.
In the realm of genetically transformed crops, the process of plant regeneration holds utmost significance. However, the low regeneration efficiency of several wheat varieties currently restricts the use of genetic transformation for gene functional analysis and improved crop production. This research explores overexpression of TaLAX PANICLE1 (TaLAX1), which markedly enhances regeneration efficiency, thereby boosting genetic transformation and genome editing in wheat. Particularly noteworthy is the substantial increase in regeneration efficiency of common wheat varieties previously regarded as recalcitrant to genetic transformation. Our study shows that increased expression of TaGROWTH-REGULATING FACTOR (TaGRF) genes, alongside that of their co-factor, TaGRF-INTERACTING FACTOR 1 (TaGIF1), enhances cytokinin accumulation and auxin response, which may play pivotal roles in the improved regeneration and transformation of TaLAX1-overexpressing wheat plants. Overexpression of TaLAX1 homologs also significantly increases the regeneration efficiency of maize and soybean, suggesting that both monocot and dicot crops can benefit from this enhancement. Our findings shed light on a gene that enhances wheat genetic transformation and elucidate molecular mechanisms that potentially underlie wheat regeneration.
Adventitious shoot (AS) regeneration is a significant factor in the genetic transformation of horticultural plants. It is also a noteworthy approach for their vegetative propagation. AS regeneration remains highly dependent on the genotype or maturity of explants. We here found that the AS regeneration abilities of apple leaves were positively correlated with MdAIL5 expression. MdAIL5 overexpression dramatically increased the AS regeneration efficiency. Notably, MdAIL5 overexpression could restore the AS formation ability of explants to a certain extent, which was lost with an increase in maturity. Endogenous hormone detection revealed that MdAIL5 overexpression changed the contents of auxin, cytokinin (CK), and other hormones in apple leaves. Transcriptome analysis revealed that many genes related to auxin, CK, and brassinolide signaling pathways were significantly and differentially expressed between MdAIL5-overexpressing transgenic apple and wild-type apple plants. Yeast one-hybrid assays, electrophoretic mobility shift assay, and dual-luciferase reporter assay revealed that MdAIL5 directly binds to MdARF9 and MdHB14 promoters and positively affects their expression. We here established a model of MdAIL5 regulating AS formation, which acts as a theoretical basis for facilitating genotype- or explant maturity-independent AS regeneration in the future.
Prevention of autonomous division of the egg apparatus and central cell in a female gametophyte before fertilization ensures successful reproduction in flowering plants. Here we show that rice ovules of Polycomb repressive complex 2 (PRC2) Osfie1 and Osfie2 double mutants exhibit asexual embryo and autonomous endosperm formation at a high frequency, while ovules of single Osfie2 mutants display asexual pre-embryo-like structures at a lower frequency without fertilization. Earlier onset, higher penetrance and better development of asexual embryos in the double mutants compared with those in Osfie2 suggest that the autonomous endosperm facilitated asexual embryo development. Transcriptomic analysis showed that male genome-expressed OsBBM1 and OsWOX8/9 were activated in the asexual embryos. Similarly, the maternal alleles of the paternally expressed imprinted genes were activated in the autonomous endosperm, suggesting that the egg apparatus and central cell convergently adopt PRC2 to maintain the non-dividing state before fertilization, possibly through silencing of the maternal alleles of male genome-expressed genes.
Key message
This review discusses the epigenetic changes during somatic embryo (SE) development, highlights the genes and miRNAs involved in the transition of somatic cells into SEs as a result of epigenetic changes, and draws insights on biotechnological opportunities to study SE development.
Abstract
Somatic embryogenesis from somatic cells occurs in a series of steps. The transition of somatic cells into somatic embryos (SEs) is the most critical step under genetic and epigenetic regulations. Major regulatory genes such as SERK, WUS, BBM, FUS3/FUSA3, AGL15, and PKL, control SE steps and development by turning on and off other regulatory genes. Gene transcription profiles of somatic cells during SE development is the result of epigenetic changes, such as DNA and histone protein modifications, that control and decide the fate of SE formation. Depending on the type of somatic cells and the treatment with plant growth regulators, epigenetic changes take place dynamically. Either hypermethylation or hypomethylation of SE-related genes promotes the transition of somatic cells. For example, the reduced levels of DNA methylation of SERK and WUS promotes SE initiation. Histone modifications also promote SE induction by regulating SE-related genes in somatic cells. In addition, miRNAs contribute to the various stages of SE by regulating the expression of auxin signaling pathway genes (TIR1, AFB2, ARF6, and ARF8), transcription factors (CUC1 and CUC2), and growth-regulating factors (GRFs) involved in SE formation. These epigenetic and miRNA functions are unique and have the potential to regenerate bipolar structures from somatic cells when a pluripotent state is induced. However, an integrated overview of the key regulators involved in SE development and downstream processes is lacking. Therefore, this review discusses epigenetic modifications involved in SE development, SE-related genes and miRNAs associated with epigenetics, and common cis-regulatory elements in the promoters of SE-related genes. Finally, we highlight future biotechnological opportunities to alter epigenetic pathways using the genome editing tool and to study the transition mechanism of somatic cells.
Efficient regeneration of transgenic plants from explants after transformation is one of the crucial steps in developing genetically modified plants with desirable traits. Identification of novel plant growth regulators and developmental regulators will assist to enhance organogenesis in culture. In this study, we observed enhanced shoot regeneration from tomato cotyledon explants in culture media containing timentin, an antibiotic frequently used to prevent Agrobacterium overgrowth after transformation. Comparative transcriptome analysis of explants grown in the presence and absence of timentin revealed several genes previously reported to play important roles in plant growth and development, including Auxin Response Factors (ARFs), GRF Interacting Factors (GIFs), Flowering Locus T (SP5G), Small auxin up-regulated RNAs (SAUR) etc. Some of the differentially expressed genes were validated by quantitative real-time PCR. We showed that ticarcillin, the main component of timentin, degrades into thiophene acetic acid (TAA) over time. TAA was detected in plant tissue grown in media containing timentin. Our results showed that TAA is indeed a plant growth regulator that promotes root organogenesis from tomato cotyledons in a manner similar to the well-known auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). In combination with the cytokinin 6-benzylaminopurine (BAP), TAA was shown to promote shoot organogenesis from tomato cotyledon in a concentration-dependent manner. To the best of our knowledge, the present study reports for the first time demonstrating the function of TAA as a growth regulator in a plant species. Our work will pave the way for future studies involving different combinations of TAA with other plant hormones which may play an important role in in vitro organogenesis of recalcitrant species. Moreover, the differentially expressed genes and long noncoding RNAs identified in our transcriptome studies may serve as contender genes for studying molecular mechanisms of shoot organogenesis.
The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium‐mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9) genome editing in a perennial, stress‐tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single‐guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co‐expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass.
We encountered unexpected transgene silencing in Arabidopsis thaliana sperm cells; transgenes encoding proteins with no specific intracellular localization (cytoplasmic proteins) were silenced transcriptionally or post-transcriptionally. The mRNA of cytoplasmic protein transgenes tagged with a fluorescent protein gene was significantly reduced, resulting in undetectable fluorescent protein signals in the sperm cell. Silencing of the cytoplasmic protein transgenes in the sperm cell did not affect the expression of either its endogenous homologous genes or co-transformed transgenes encoding a protein with targeted intracellular localization. This transgene silencing in the sperm cell persisted in mutants of the major gene silencing machinery including DNA methylation. The incomprehensible, yet real, transgene silencing phenotypes occurring in the sperm cell could mislead the interpretation of experimental results in plant reproduction, and this Commentary calls attention to that risk and highlights details of this novel cytoplasmic protein transgene silencing.
A new term — the “blastozone” — is proposed to designate regions of the shoot competent for organogenesis. It is argued that the notion of “marginal meristems” is based on the cell theory and thus may not be appropriate to elucidate the process of organ formation. For instance, with respect to the occurrence of initials and of an elevated cell division rate marginal meristems have been shown to be doubtful structures. Furthermore, organogenetic competent regions form only parts of the meristems of the shoot. The study of blastozones from an organismic perspective reveals primary morphogenetic events such as initiation, incorporation, and fusion processes. Loss of morphogenetic competence is associated with histogenetic events, e.g., trichome outgrowth, and indicates the onset of processes leading to maturation. The marginal blastozone of the leaf is then used up although meristem features continue to be expressed. A series of SEM studies in several genera exemplifies the proposed viewpoint, demonstrating some of the morphogenetic potentialities of angiosperm leaf marginal blastozones.
The orderly production of meristems with specific fates is crucial for the proper elaboration of plant architecture. The maize inflorescence meristem branches several times to produce lateral meristems with determinate fates. The first meristem formed, the spikelet pair meristem, produces two spikelet meristems, each of which produces two floral meristems. We have identified a gene called indeterminate spikelet1 (ids1) that specifies a determinate spikelet meristem fate and thereby limits the number of floral meristems produced. In the absence of ids1 gene function, the spikelet meristem becomes indeterminate and produces additional florets. Members of the grass family vary in the number of florets within their spikelets, suggesting that ids1 may play a role in inflorescence architecture in other grass species. ids1 is a member of the APETALA2 (AP2) gene family of transcription factors that has been implicated in a wide range of plant development roles. Expression of ids1 was detected in many types of lateral organ primordia as well as spikelet meristems. Our analysis of the ids1 mutant phenotype and expression pattern indicates that ids1 specifies determinate fates by suppressing indeterminate growth within the spikelet meristem.
cDNA clones encoding proteins of approximately 18 kDa in which 83% of the amino acids are conserved relative to the published sequences of mammalian cyclophilin/rotamase (CyP) have been isolated from tomato, maize, and Brassica napus. In correspondence with the mammalian genes, but in contrast with the Neurospora gene and one yeast CyP gene, the plant CyP genes encode only mature proteins lacking transit peptides. RNA blot analyses demonstrate that CyP genes are expressed in all plant organs tested. Southern blots of genomic DNA indicate that there are small families (two to eight members) of CyP-related genes in maize and B. napus. A vector was constructed for expression of the tomato cDNA in E. coli. SDS/polyacrylamide gels show that extracts of appropriately induced cells harboring this vector contain nearly 40% of the protein as a single approximately 18-kDa band. While the majority of this protein is sequestered in insoluble inclusion bodies, the soluble extracts have higher levels of peptidyl-prolyl cis-trans isomerase (rotamase) activity than extracts of wild-type cells. This additional activity is sensitive to inhibition by the cyclic undecapeptide cyclosporin A.
We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number in E. coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes.
APETALA2 (AP2) plays a central role in the establishment of the floral meristem, the specification of floral organ identity, and the regulation of floral homeotic gene expression in Arabidopsis. We show here that in addition to its functions during flower development, AP2 activity is also required during seed development. We isolated the AP2 gene and found that it encodes a putative nuclear protein that is distinguished by an essential 68-amino acid repeated motif, the AP2 domain. Consistent with its genetic functions, we determined that AP2 is expressed at the RNA level in all four types of floral organs--sepals, petals, stamens, and carpels--and in developing ovules. Thus, AP2 gene transcription does not appear to be spatially restricted by the floral homeotic gene AGAMOUS as predicted by previous studies. We also found that AP2 is expressed at the RNA level in the inflorescence meristem and in nonfloral organs, including leaf and stem. Taken together, our results suggest that AP2 represents a new class of plant regulatory proteins that may play a general role in the control of Arabidopsis development.
The EMB-1 mRNA of carrot (Daucus carota) was isolated as an embryo abundant cDNA clone (T.H. Ulrich, E.S. Wurtele, B.J. Nikolau [1990] Nucleic Acids Res 18: 2826). Northern analyses of RNA isolated from embryos, cultured cells, and a variety of vegetative organs indicate that the EMB-1 mRNA specifically accumulates in embryos, beginning at the early stages of embryo development. In situ hybridization with both zygotic and somatic embryos show that the EMB-1 mRNA begins to accumulate at low levels throughout globular embryos. Accumulation of EMB-1 mRNA increases and becomes more localized as embryos mature; in torpedo embryos, EMB-1 mRNA preferentially accumulates in the meristematic regions, particularly the procambium. The similarity in distribution of EMB-1 mRNA in both zygotic and somatic embryos indicates that much of the spatial pattern of expression of the emb-1 gene is dependent on the developmental program of the carrot embryo and does not require maternal or endosperm factors. The EMB-1 protein (relative molecular weight 9910) is a very hydrophilic protein that is a member of a class of highly conserved proteins (typified also by the Em protein of wheat and the Lea D19 protein of cotton) that may be ubiquitous among angiosperm embryos but whose functions are as yet unknown. The carrot genome appears to contain one or two copies of the emb-1 gene. A 1313-base pair DNA fragment of the carrot genome containing the emb-1 gene was isolated and sequenced. The gene is interrupted by a single intron of 99 base pairs. Primer extension experiments identify two EMB-1 mRNAs, differing by 6 bases at their 5' ends that are transcribed from this gene.
A novel transposon-tagging strategy designed to recover dominant gain-of-function alleles was performed with Arabidopsis by using a Dissociation element with a cauliflower mosaic virus 35S promoter transcribing outward over one terminus. Lines containing transposed copies of this transposon were screened for mutants, and a semidominant mutation affecting plant height, hypocotyl elongation, and fertility was recovered. The pleiotropic effects of this mutation appear to result from a general reduction in cell expansion, and some of the effects are similar to those caused by supplying exogenous ethylene or cytokinin to wild-type seedlings. In addition, the arrangement of cells in some organs such as the etiolated hypocotyl, is disorganized. The mutation was called tiny, and the affected gene was cloned by first using transposon sequences to isolate the mutant allele. The predicted protein product of the TINY gene shows strong homology with the DNA binding domain of a recently identified class of plant transcription factors. This domain, called the APETALA2 domain, was initially identified as a duplicated region within the APETALA2 gene of Arabidopsis and then as a conserved region between APETALA2 and the ethylene responsive element binding proteins of tobacco. In the mutant allele, the Dissociation element is inserted in the untranslated leader of the TINY gene, 36 bp from the ATG, and the mutant contains a novel transcript that initiates from the cauliflower mosaic virus 35S promoter within the transposon. This transcript is present in greater abundance than the wild-type TINY transcript; therefore, the semidominant tiny mutation most likely results from increased, or ectopic, expression of the gene.
To understand better the role of genes in controlling ovule development, a female-sterile mutant, aintegumenta (ant), was isolated from Arabidopsis. In ovules of this mutant, integuments do not develop and megasporogenesis is blocked at the tetrad stage. As a pleiotropic effect, narrower floral organs arise in reduced numbers. More complete loss of floral organs occurs when the ant mutant is combined with the floral homeotic mutant apetala2, suggesting that the two genes share functions in initiating floral organ development. The ANT gene was cloned by transposon tagging, and sequence analysis showed that it is a member of the APETALA2-like family of transcription factor genes. The expression pattern of ANT in floral and vegetative tissues indicates that it is involved not only in the initiation of integuments but also in the initiation and early growth of all primorida except roots.
Vegetative development in many plants progresses through distinct juvenile and adult phases. In maize, the transition from juvenile to adult shoot development affects a variety of leaf epidermal cell traits. These include epicuticular waxes, leaf hairs, and cell wall characteristics. Previous genetic and phenotypic analyses have shown that the maize Glossy15 (Gl15) gene is required for the expression of juvenile epidermal traits after leaf 2. We report here the molecular cloning of the Gl15 gene using a defective Suppressor-Mutator (dSpm) element insertion as a transposon-tag. Consistent with the gl15 mutant phenotype, the pattern of Gl15 mRNA expression was correlated with a juvenile leaf epidermal cell identity and was regulated by upstream factors such as Corngrass1. The Gl15 gene encodes a putative transcription factor with significant sequence similarity to the Arabidopsis regulatory genes APETALA2 and AINTEGUMENTA, which act primarily to regulate floral organ identity and ovule development. This finding expands the known functions of APETALA2-related genes to include the control of both vegetative and reproductive lateral organ identity and provides molecular support for the hypothesis that leaves and floral organs are related structures derived from a common growth plan.
We have analyzed the contributions of phytochrome and gibberellin signal transduction to the control of flower meristem identity in the Arabidopsis mutants apetala1 (ap1) and apetala2 (ap2). ap1 flowers are partially defective for the establishment of flower meristem identity and are characterized by the production of ectopic secondary or axillary flowers and by branching. Axillary flower production is also induced in ap2-1 flowers by short-day photoperiod and is suppressed by hy1, a mutation blocking phytochrome activity. The production of axillary flower by ap2-1 is also suppressed by exogenous gibberellins and by spindly (spy), a mutation that activates basal gibberellin signal transduction in hormone-independent manner. Ectopic axillary flower production and floral branching by ap1 flowers are also suppressed by spy. We conclude that gibberellins promote flower meristem identity and that the inflorescence-like traits of ap2-1 and ap1-1 flowers are due in part to SPY gene activity.
The first somatic single cells of carrot hypocotyl explants having the competence to form embryos in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) were identified using semi-automatic cell tracking. These competent cells are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the provascular elements of the hypocotyl. A search for marker genes to monitor the transition of somatic into competent and embryogenic cells in established suspension cell cultures resulted in the identification of a gene transiently expressed in a small subpopulation of the same enlarged single cells that are formed during the initiation of the embryogenic cultures from hypocotyl explants. The predicted amino acid sequence and in vitro kinase assays show that this gene encodes a leucine-rich repeat containing receptor-like kinase protein, designated Somatic Embryogenesis Receptor-like Kinase (SERK). Somatic embryos formed from cells expressing a SERK promoter-luciferase reporter gene. During somatic embryogenesis, SERK expression ceased after the globular stage. In plants, SERK mRNA could only be detected transiently in the zygotic embryo up to the early globular stage but not in unpollinated flowers nor in any other plant tissue. These results suggest that somatic cells competent to form embryos and early globular somatic embryos share a highly specific signal transduction chain with the zygotic embryo from shortly after fertilization to the early globular embryo.
In tomato, the Pto kinase confers resistance to bacterial speck disease by recognizing the expression of a corresponding avirulence gene, avrPto, in the pathogen Pseudomonas syringae pv. tomato. Using the yeast two-hybrid system, we have identified three genes, Pti4, Pti5 and Pti6, that encode proteins that physically interact with the Pto kinase. Pti4/5/6 each encode a protein with characteristics that are typical of transcription factors and are similar to the tobacco ethylene-responsive element-binding proteins (EREBPs). Using a gel mobility-shift assay, we demonstrate that, similarly to EREBPs, Pti4/5/6 specifically recognize and bind to a DNA sequence that is present in the promoter region of a large number of genes encoding 'pathogenesis-related' (PR) proteins. Expression of several PR genes and a tobacco EREBP gene is specifically enhanced upon Pto-avrPto recognition in tobacco. These observations establish a direct connection between a disease resistance gene and the specific activation of plant defense genes.
Arabidopsis abscisic acid (ABA)-insensitive abi4 mutants have pleiotropic defects in seed development, including decreased sensitivity to ABA inhibition of germination and altered seed-specific gene expression. This phenotype is consistent with a role for ABI4 in regulating seed responses to ABA and/or seed-specific signals. We isolated the ABI4 gene by positional cloning and confirmed its identity by complementation analysis. The predicted protein product shows homology to a plant-specific family of transcriptional regulators characterized by a conserved DNA binding domain, the APETALA 2 domain. The single mutant allele identified has a single base pair deletion, resulting in a frameshift that should disrupt the C-terminal half of the protein but leave the presumed DNA binding domain intact. Expression analyses showed that despite the seed-specific nature of the mutant phenotype, ABI4 expression is not seed specific.
The Arabidopsis LEAFY COTYLEDON1 (LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box-binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.
Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved
in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some
flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box.
Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pm range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that
only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses
demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the
first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments
studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that
these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible
for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity.
Jasmonate (JA) is an important plant stress hormone that induces various plant defense responses, including the biosynthesis of protective secondary metabolites. The induction of the secondary metabolite biosynthetic gene Strictosidine synthase (Str) in Catharanthus roseus (periwinkle) cells by elicitor requires JA as a second messenger. A 42 bp region in the Str promoter is both necessary and sufficient for JA- and elicitor-responsive expression. This region is unlike other previously identified JA-responsive regions, and contains a GCC-box-like element. Yeast one-hybrid screening identified cDNAs encoding two AP2-domain proteins. These octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) proteins bind in a sequence-specific manner the JA- and elicitor-responsive element. ORCA2 trans-activates the Str promoter and its expression is rapidly inducible with JA and elicitor, whereas Orca1 is expressed constitutively. The results indicate that a GCC-box-like element and ORCA2 play key roles in JA- and elicitor-responsive expression of the terpenoid indole alkaloid biosynthetic gene Str.
Somatic embryogenesis can be synchronized by enriching carrot (Daucus carota L.) suspension cultures for small, dense clusters of cells termed proembryogenic masses (PEMs). Gene-expression programs of PEMs were compared with those of embryonic and mature tissues by in-vitro translation of representative mRNA populations and by nucleic-acid hybridization. Analysis of invitro-translated polypeptides by two-dimensional polyacrylamide gel electrophoresis revealed striking similarities between the mRNA populations of PEM and torpedo-stage embryos; substantial differences, however, were observed when in-vitro translation products of PEMs and torpedo embryos were compared with those of hypocotyls and leaves. Northern blots of RNA isolated from PEMs, staged embryos, and mature carrot tissues were hybridized with cDNA probes for Dc3, Dc5 and Dc13; these cDNA recombinants represent mRNAs that are regulated during carrot somatic embryogenesis. The pattern of expression of these embryo-regulated transcripts was similar in PEMs and somatic embryos but differed in other carrot tissues. These results indicate that many of the molecular processes of embryogenesis are already established in PEMs in the presence of auxin. Additional experiments indicate the utility of Dc3 as a molecular marker for the acquisition of embryogenic potential.
We report the identification, via their cDNAs, of genes which are temporarily transcribed during the initiation of somatic embryogenesis in carrot (Daucus carota L.) cells cultured in an auxin-free medium. Their expression is roughly associated with the first morphogenetic, or globular, stage. A cDNA library (λ gt 10) was established using poly(A)(+) -rich RNAs from cells deprived of auxin for 8 d. By differential screening a number of clones corresponding to early-induced embryogenic genes were identified. For several a temporary accumulation of the specific mRNA between 6 and 16 d after induction was observed. With regard to the nucleotide sequence and the respective deduced amino-acid sequence, two glycine-rich proteins and a polypeptide with a proline-rich domain were among the products of genes activated at the onset of somatic embryogenesis.
Temperature controls the developmental fate of isolated Brassica napus microspores in vitro. Culture at 32.5°C leads to sporophytic development and the formation of embryos. Here we show that culture at 17.5°C leads to gametophytic development, and the formation of pollen-like structures at high frequencies (up to 80% after 7 days in culture). Early stages of both developmental pathways are observed in culture at 25.0°C, and embryos are produced at low frequencies (0.7%) at that temperature. Culturing B. napus microspores at 32.5°C versus 17.5°C brings the switch from gametophytic to sporophytic development under simple experimental control and provides a convenient tool for investigating the cellular and molecular mechanisms controlling this developmental switch.
Brassica napus L. microspores at the late uninucleate to early binucleate stage of development can be induced in vitro to alter their development from pollen to embryo formation. High temperatures or other stress treatments are required to initiate this redirection process. The critical period for induction of microspore embryogenesis is within the first 8 h of temperature-stress imposition. During this period, which precedes the first embryogenic nuclear division, the process regulating the induction and sustainment of microspore embryogenesis is activated. A number of mRNAs and proteins, some of them possibly heat-shock proteins, appear in microspores during the commitment phase of the induction process.
We have recently isolated two sunflower polyubiquitin genes (UbB1 and UbB2) which are induced by heat-stress and show a highly conserved region of about 250 bp upstream of the transcription initiation site. The promoter activity of this region has been analyzed by transient expression using the β-glucoronidase (GUS) gene as reporter in tobacco protoplasts. The GUS expression in absence of any external inducer is five times higher than that observed under the control of the CaMV 35S promoter. Analysis of different deletions within the 250-bp region showed that a 65-bp sequence is responsible for most of the promoter activity in tobacco protoplasts. This activity is intron-independent and is not enhanced when protoplasts are heat-stressed. Sequence homologies strongly suggest that UbB1 plays a major role in the stress response in sunflower.
Brassica napus L. microspores at the late uninucleate to early binucleate stage of development can be induced in vitro to alter their development from pollen to embryo formation. High temperatures or other stress treatments are required to initiate this redirection process. The critical period for induction of microspore embryogenesis is within the first 8 h of temperature-stress imposition. During this period, which precedes the first embryogenic nuclear division, the process regulating the induction and sustainment of microspore embryogenesis is activated. A number of mRNAs and proteins, some of them possibly heat-shock proteins, appear in microspores during the commitment phase of the induction process.
ABSTRACTA tobacco (Nicotiana tabacum L.) cDNA clone, Nthsp 18P, encoding a class I low-molecular-weight heat shock protein (LMW HSP), has been isolated from a mid-biccllular pollen cDNA library by hcterologous hybridization with a low-molecular-weight heat shock cDNA clone of alfalfa. Northern analysis showed that Nthsp 18P expression is activated at normal temperature during the dehydration phase of in situ pollen development, just before anthesis. Induction of pollen embryogenesis by an in vitro starvation treatment of mid-bicellular tobacco pollen was accompanied by a dramatic increase in the levels of the Nthsp 18P transcript, which accumulated in embryogenic pollen grains at 25°C; lower levels of this transcript were also detected in early pollen-derived embryos. A similar pattern of expression was observed in transgcnic tobacco plants containing the promoter of Gmhsp 17.3-B, a soybean homologue of the Nthsp 18P gene, fused to the GUS reporter gene. This is the first characterized gene shown to be transcriptionally activated during the induction of pollen embryogenesis.
A synthetic transcribed, untranslated leader sequence from alfalfa mosaic virus RNA4 (AMV leader) has been assessed for its in vivo properties as a cis-active ‘translational activator’ in transient expression assays in protoplasts of Nicotiana tabacum and Picea glauca, as well as in stable expression in transformed Nicotiana tabacum. Levels of GUS enzyme activity produced by chimeric genes with or without the AMV leader sequence, in combination with either a CaMV 35S promoter or a duplicated-enhancer CaMV 35S promoter construct were assessed. In transient assay systems, the presence of a synthetic 40-base leader sequence lead to a 20-fold elevation in GUS activity when the constructs contained a native cauliflower mosaic virus (CaMV) 35S promoter, whereas a 4-fold elevated expression lebel was observed in constructs containing a duplicated-enhancer 35S promoter. Furthermore, elevated expression in chimeric constructs was influenced by the sequence context for translation initiation of the marker gene. In transgenic tobacco plants the mean values for steady-state expression of GUS-containing 35S/AMV constructs were elevated about 8-fold relative to plasmids containing the native 35S promoter alone. A quantitative PCR approach was used to assess relative transcript levels in plants expressing GUS from AMV-containing chimeric constructs. The results showed that elevated expression attributable to the AMV leader sequence was independent of abundance of the corresponding AMV-gus transcript, suggesting a post-transcriptional mechanism of action in vivo. Further, we describe the construction of general-purpose constitutive high expression plant promoter cassettes which incorporate the AMV translational enhancer sequence, as well a duplicated-enhancer 35S promoter in an optimized translational context.
To gain insight into the molecular events occurring in the very early stages of barley microspore embryogenesis, cDNA clones corresponding to genes differentially expressed during the early stages of microspore culture were isolated and characterized. A cDNA library established from barley microspores cultured for three days was differentially screened against probes generated from freshly isolated microspores. Three cDNAs representing genes not previously identified in barley were isolated. ECA1 (early culture abundant 1) lacked significant homology to known genes or proteins, and the transcript was only expressed during the early stages of culture. Expression was also reduced in low-density control cultures, therefore this gene may play a role in the early stages of barley microspore embryogenesis. ECGST (early culture glutathione S-transferase) had homology to parA-like genes, which are members of a newly discovered group of glutathione S-transferases (GSTs). The protein corresponding to ECGST may be important in protecting cells from oxidative stress during the culture process. ECLTP (early culture lipid transfer protein) had homology to lipid transfer proteins (LTPs), and had an expression pattern similar to that of an LTP known to be a marker of the early stages of embryogenesis in the carrot somatic embryogenesis system. The identification and characterization of the clones isolated in this study provides new information on the events involved in barley microspore embryogenesis.
The subject of this review is the development of the plant embryo. Plant embryo-genesis is a unique process in the sense that it can be started not only from the fertilized egg but can also be initiated from other cells of the reproductive apparatus and even from somatic cells. One of the challenges of this field is therefore to unravel the molecular mechanisms that lead to the formation of a cell destined to form an embryo. A second important area of research is to determine the molecular basis of pattern formation in the embryo, a process that results in a stereotyped organization of a seedling. On the one hand, the pattern formation process has to establish precisely arranged tissue organization, but on the other hand sufficient flexibility during plant development has to be maintained to allow continuous formation of new organs from meristems.
Brassica species possess the most complex acetohydroxyacid synthase (AHAS) multigene family reported for plants. The AHAS genes code for an essential enzyme in branched-chain amino acid biosynthesis. In the allotetraploid species, B. napus, four (AHAS1-4) of the five AHAS genes have been cloned and sequenced. The transcripts were examined by RNase protection assays using gene-specific, antisense RNA probes. Only AHAS1, AHAS2 and AHAS3 were shown to be expressed in B. napus and one of the diploid progenitor species B. campestris or B. oleracea. AHAS1 and AHAS3 are highly conserved genes that presumably code for the essential AHAS housekeeping functions. They were expressed as low abundance mRNA in all somatic and reproductive tissues examined. AHAS2, which is structurally distinct from all other plant AHAS genes, was only expressed in mature ovules and extraembryonic tissues of immature seeds. This study provides direct evidence for multiple AHAS isoforms in plants and for an AHAS gene which is developmentally regulated in a tissue-specific manner. The discovery raises questions concerning the functional significance of AHAS in seed development.
Napins are a family of small, basic storage proteins synthesized in Brassica napus (rapeseed) embryos during seed maturation. Cultured embryos also synthesize napins but require exogenous abscisic acid (ABA) to maintain high accumulation rates. We synthesized cDNA from total RNA of embryos cultured on a medium containing ABA, and cloned it into the Pst1 site of pBR322. Two clones containing napin cDNA sequences selected by differential colony hybridization using [32P]cDNA probes from embryos grown with or without ABA were analyzed. These clones, pN1 (insert size = 583 bp) and pN2 (insert size = 739 bp), contained cDNA from two different napin mRNAs. The mRNAs to which they hybridized were found to encode a 21,000-dalton polypeptide that was immunoprecipitated by antibodies to mature napin (subunits of 9,000 and 4,000 daltons). The cDNA clones hybridized to an 850-base mRNA. Nucleotide sequencing demonstrated 95% homology between pN1 and pN2 cDNA inserts and predicted a precursor polypeptide of 178 amino acids, consistent with the 21,000 dalton in vitro translation product. Comparison of the deduced amino acid sequence with published amino acid compositions of mature napin subunits suggests that both the large and the small subunits are present in one precursor polypeptide, and that other regions of the precursor are removed during processing.
We demonstrated that the GCC box, which is an 11-bp sequence (TAAGAGCCGCC) conserved in the 5' upstream region of ethylene-inducible pathogenesis-related protein genes in Nicotiana spp and in some other plants, is the sequence that is essential for ethylene responsiveness when incorporated into a heterologous promoter. Competitive gel retardation assays showed DNA binding activities to be specific to the GCC box sequence in tobacco nuclear extracts. Four different cDNAs encoding DNA binding proteins specific for the GCC box sequence were isolated, and their products were designated ethylene-responsive element binding proteins (EREBPs). The deduced amino acid sequences of EREBPs exhibited no homology with those of known DNA binding proteins or transcription factors; neither did the deduced proteins contain a basic leucine zipper or zinc finger motif. The DNA binding domain was identified within a region of 59 amino acid residues that was common to all four deduced EREBPs. Regions highly homologous to the DNA binding domain of EREBPs were found in proteins deduced from the cDNAs of various plants, suggesting that this domain is evolutionarily conserved in plants. RNA gel blot analysis revealed that accumulation of mRNAs for EREBPs was induced by ethylene, but individual EREBPs exhibited different patterns of expression.
Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.
Ovules play a central role in plant reproduction, generating the female gametophyte within sporophytic integuments. When fertilized, the integuments differentiate into the seed coat and support the development of the embryo and endosperm. Mutations in the AINTEGUMENTA (ANT) locus of Arabidopsis have a profound effect on ovule development. Strong ant mutants have ovules that fail to form integuments or a female gametophyte. Flower development is also altered, with a random reduction of organs in the outer three whorls. In addition, organs present in the outer three floral whorls often have abnormal morphology. Ovules from a weak ant mutant contain both inner and outer integuments but generally fail to produce a functional female gametophyte. We isolated the ANT gene by using a mutation derived by T-DNA insertional mutagenesis. ANT is a member of a gene family that includes the floral homeotic gene APETALA2 (AP2). Like AP2, ANT contains two AP2 domains homologous with the DNA binding domain of ethylene response element binding proteins. ANT is expressed most highly in developing flowers but is also expressed in vegetative tissue. Taken together, these results suggest that ANT is a transcription factor that plays a critical role in regulating ovule and female gametophyte development.
A clone for an embryoid-abundant, early cysteine-labeled metallothionein (EcMt) gene has been isolated from a wheat pollen embryoid cDNA library. The transcript of this gene was only expressed in embryogenic microspores, pollen embryoids, and developing zygotic embryos of wheat. Accumulation of the EcMt mRNA showed a direct and positive correlation with an increase of the plant hormone, abscisic acid (ABA) in developing pollen embryoids. Treating cultures with an inhibitor of ABA biosynthesis, fluridone, suppressed not only ABA accumulation but also the appearance of the EcMt gene transcript and the ability of microspores to form embryoids. These results suggest that the EcMt gene may act as a molecular marker for pollen embryogenesis because ABA biosynthesis is accompanied by the increased expression of the EcMt transcript that coincides with the differentiation of pollen embryoids in wheat anther cultures.
Ocs elements are a group of promoter sequences required for the expression of both pathogen genes in infected plants and plant defense genes. Genes for ocs element binding factors (OBFs), belonging to a specific class of basic-region leucine zipper (bZIP) transcription factors, have been isolated in a number of plants. Using protein-protein interaction screening with OBF4 we have isolated AtEBP, an Arabidopsis protein that contains a novel DNA-binding domain, the AP2/EREBP domain. One class of proteins that contain this domain are the tobacco ethylene-responsive element binding proteins (EREBPs). The EREBPs bind the GCC box that confers ethylene responsiveness to a number of pathogenesis related (PR) gene promoters. AtEBP expression is inducible by exogenous ethylene in wild-type plants and AtEBP transcripts are increased in the ctr1-1 mutant, where ethylene-regulated pathways are constitutively active. Electrophoretic mobility-shift assay and DNase I footprint analysis revealed that AtEBP can specifically bind to the GCC box. Interestingly, the highest level of AtEBP expression was detected in callus tissue, where ocs elements are very active. Synergistic effects of the GCC box with ocs elements or the related G-box sequence have been previously observed, for example, in the ethylene-induced expression of a PR gene promoter. Our results suggest that cross-coupling between EREBP and bZIP transcription factors occurs and may therefore be important in regulating gene expression during the plant defense response.
The plant growth regulator gibberellin (GA) has a profound effect on shoot development and promotes developmental transitions
such as flowering. Little is known about any analogous effect GA might have on root development. In a screen for mutants,
Arabidopsisplants carrying a mutation designated pickle(pkl) were isolated in which the primary root meristem retained characteristics of embryonic tissue. Expression of this aberrant
differentiation state was suppressed by GA. Root tissue from plants carrying the pkl mutation spontaneously regenerated new embryos and plants.
Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2, 4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.
Genetic transformation and clonal propagation are techniques that play an important role in the identification and characterization
of plant genes and their products. The Joint efforts to develop Arabidopszs thaliana as a model for genetic and molecular
analysis of higher plants have produced methods for in vitro propagation (1), regeneration and transformation (2)–(15) using either Agrobacterium or direct gene transfer.
AP2 (APETALA2) and EREBPs (ethylene-responsive element binding proteins) are the prototypic members of a family of transcription factors unique to plants, whose distinguishing characteristic is that they contain the so-called AP2 DNA-binding domain. AP2/ REBP genes form a large multigene family, and they play a variety of roles throughout the plant life cycle: from being key regulators of several developmental processes, like floral organ identity determination or control of leaf epidermal cell identity, to forming part of the mechanisms used by plants to respond to various types of biotic and environmental stress. The molecular and biochemical characteristics of the AP2/EREBP transcription factors and their diverse functions are reviewed here, and this multigene family is analyzed within the context of the Arabidopsis thaliana genome sequence project.
Plant growth is greatly affected by drought and low temperature. Expression of a number of genes is induced by both drought and low temperature, although these stresses are quite different. Previous experiments have established that a cis-acting element named DRE (for dehydration-responsive element) plays an important role in both dehydration- and low-temperature-induced gene expression in Arabidopsis. Two cDNA clones that encode DRE binding proteins, DREB1A and DREB2A, were isolated by using the yeast one-hybrid screening technique. The two cDNA libraries were prepared from dehydrated and cold-treated rosette plants, respectively. The deduced amino acid sequences of DREB1A and DREB2A showed no significant sequence similarity, except in the conserved DNA binding domains found in the EREBP and APETALA2 proteins that function in ethylene-responsive expression and floral morphogenesis, respectively. Both the DREB1A and DREB2A proteins specifically bound to the DRE sequence in vitro and activated the transcription of the b-glucuronidase reporter gene driven by the DRE sequence in Arabidopsis leaf protoplasts. Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration. Overexpression of the DREB1A cDNA in transgenic Arabidopsis plants not only induced strong expression of the target genes under unstressed conditions but also caused dwarfed phenotypes in the transgenic plants. These transgenic plants also revealed freezing and dehydration tolerance. In contrast, overexpression of the DREB2A cDNA induced weak expression of the target genes under unstressed conditions and caused growth retardation of the transgenic plants. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.
We have cloned and characterized two novel DNA binding proteins designated RAV1 and RAV2 from Arabidopsis thaliana. RAV1 and RAV2 contain two distinct amino acid sequence domains found only in higher plant species. The N-terminal regions
of RAV1 and RAV2 are homologous to the AP2 DNA-binding domain present in a family of transcription factors represented by
the Arabidopsis APETALA2 and tobacco EREBP proteins, while the C-terminal region exhibits homology to the highly conserved C-terminal domain,
designated B3, of VP1/ABI3 transcription factors. Binding site selection assays using a recombinant glutathione S-transferase
fusion protein have revealed that RAV1 binds specifically to bipartite recognition sequences composed of two unrelated motifs,
5′-CAACA-3′ and 5′-CACCTG-3′, separated by various spacings in two different relative orientations. Analyses using various
deletion derivatives of the RAV1 fusion protein show that the AP2 and B3-like domains of RAV1 bind autonomously to the CAACA
and CACCTG motifs, respectively, and together achieve a high affinity and specificity of binding. From these results, we suggest
that the AP2 and B3-like domains of RAV1 are connected by a highly flexible structure enabling the two domains to bind to
the CAACA and CACCTG motifs in various spacings and orientations.
The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
AINTEGUMENTA (ANT) was previously shown to be involved in floral organ initiation and growth in Arabidopsis. ant flowers have fewer and smaller floral organs and possess ovules that lack integuments and a functional embryo sac. The present work shows that young floral meristems of ant plants are smaller than those in wild type. Failure to initiate the full number of organ primordia in ant flowers may result from insufficient numbers of meristematic cells. The decreased size of ant floral organs appears to be a consequence of decreased cell division within organ primordia. Ectopic expression of ANT under the control of the constitutive 35S promoter results in the development of larger floral organs. The number and shape of these organs is not altered and the size of vegetative organs is normal. Microscopic and molecular analyses indicate that the increased size of 35S::ANT sepals is the result of increased cell division, whereas the increased sizes of 35S::ANT petals, stamens, and carpels are primarily attributable to increased cell expansion. In addition, 35S::ANT ovules often exhibit increased growth of the nucellus and the funiculus. These results suggest that ANT stimulates cell growth in floral organs.
The life cycle of angiosperms is punctuated by a dormant phase that separates embryonic and postembryonic development of the sporophyte. In the pickle (pkl) mutant of Arabidopsis, embryonic traits are expressed after germination. The penetrance of the pkl phenotype is strongly enhanced by inhibitors of gibberellin biosynthesis. Map-based cloning of the PKL locus revealed that it encodes a CHD3 protein. CHD3 proteins have been implicated as chromatin-remodeling factors involved in repression of transcription. PKL is necessary for repression of LEC1, a gene implicated as a critical activator of embryo development. We propose that PKL is a component of a gibberellin-modulated developmental switch that functions during germination to prevent reexpression of the embryonic developmental state.
The control of cell proliferation during organogenesis plays an important role in initiation, growth, and acquisition of the intrinsic size of organs in higher plants. To understand the developmental mechanism that controls intrinsic organ size by regulating the number and extent of cell division during organogenesis, we examined the function of the Arabidopsis regulatory gene AINTEGUMENATA (ANT). Previous observations revealed that ANT regulates cell division in integuments during ovule development and is necessary for floral organ growth. Here we show that ANT controls plant organ cell number and organ size throughout shoot development. Loss of ANT function reduces the size of all lateral shoot organs by decreasing cell number. Conversely, gain of ANT function, via ectopic expression of a 35S::ANT transgene, enlarges embryonic and all shoot organs without altering superficial morphology by increasing cell number in both Arabidopsis and tobacco plants. This hyperplasia results from an extended period of cell proliferation and organ growth. Furthermore, cells ectopically expressing ANT in fully differentiated organs exhibit neoplastic activity by producing calli and adventitious roots and shoots. Based on these results, we propose that ANT regulates cell proliferation and organ growth by maintaining the meristematic competence of cells during organogenesis.
In higher plants, the correct patterning of the floral meristem in terms of organ type, number and form is the result of a concerted expression of a network of genes. We describe phenotypes of flower patterning, resulting from a reduction of transcript levels of the Arabidopsis SHAGGY-related protein kinase genes AtSK11(ASKalpha) and AtSK12(ASKgamma). The AtSK genes are plant homologues of the Drosophila shaggy (SGG) gene and the mammalian Glycogen-Synthase Kinase-3 (GSK-3). The SGG protein kinase is a key component of the wingless signalling pathway and is required for the establishment of tissue patterning and cell fate determination. The expression patterns of the AtSK11(ASKalpha) and AtSK12(ASKgamma) genes during wild-type Arabidopsis inflorescence development, detected by in situ hybridisation, have been shown to be consistent with a possible role in floral meristem patterning. AtSK11(ASKalpha) and AtSK12(ASKgamma) transcripts were detected at the periphery of the inflorescence meristem and in the floral meristem. At later stages the expression of the AtSK genes became localised in specific regions of developing flower organ primordia. Furthermore, we have obtained and analysed transgenic plants containing AtSK11(ASKalpha) and AtSK12(ASKgamma) gene specific antisense constructs. These plants developed flowers showing a higher number of perianth organs and an alteration of the apical-basal patterning of the gynoecium.