Mohamed Elhiti

Modulation of embryo-forming capacity in culture through the expression of Brassica genes involved in the regulation of the shoot apical meristem.

Somatic embryogenesis in Arabidopsis is achieved by culturing bending-cotyledon embryos on a 2,4-D-containing induction medium for 14 d followed by a transfer on to a hormone-free development medium. Several genes orthologous to Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL) were isolated from Brassica oleracea (Bo), B. rapa (Br), and B. napus (Bn), and ectopically expressed in Arabidopsis to assess their effects on somatic embryogenesis. Ectopic expression of BoSTM, BrSTM, and BnSTM increased the number of somatic embryos, whereas a different effect was observed in lines overexpressing BnCLV1 in which somatic embryo formation was severely repressed. The introduction of BnZLL did not have any effects on Arabidopsis somatic embryogenesis. The increased embryo-forming capacity observed in lines overexpressing Brassica STM was associated with a lower requirement for the inductive signal 2,4-D, and a higher expression of WUSCHEL (WUS) which demarcates the formation of embryogenic cells. This was in contrast to the 35S::BnCLV1 lines which showed the highest requirement for exogenous 2,4-D and a reduced WUS expression. Microarray studies were conducted to monitor global changes in transcript levels during Arabidopsis somatic embryogenesis between the wild-type (WT) line and a BoSTM-overexpressing line, which showed the most pronounced enhancement of somatic embryo yield. The introduction of BoSTM affected the expression of many genes involved in hormone perception and signalling, as well as genes encoding DNA methyltransferases and enzymes of glutathione metabolism. Pharmacological experiments performed to confirm some of the microarray results showed that Arabidopsis somatic embryogenesis is encouraged by a global hypomethylation of the DNA during the induction phase and by a switch of the glutathione pool towards an oxidized state during the subsequent development phase. Both events occurred in the 35S::BoSTM line, but not in the WT line. Altered expression of Brassica STM also had profound effects on B. napus microspore-derived embryogenesis. The yield of microspore-derived embryos increased in lines overexpressing BnSTM and significantly decreased in antisense lines down-regulating BnSTM.

Structure and function of homodomain-leucine zipper (HD-Zip) proteins

Homeodomain-leucine zipper (HD-Zip) proteins are transcription factors unique to plants and are encoded by more than 25 genes in Arabidopsis thaliana. Based on sequence analyses these proteins have been classified into four distinct groups: HD-Zip Ι-ІV. HD-Zip proteins are characterized by the presence of two functional domains; a homeodomain (HD) responsible for DNA binding and a leucine zipper domain (Zip) located immediately C-terminal to the homeodomain and involved in protein-protein interaction. Despite sequence similarities HD-ZIP proteins participate in a variety of processes during plant growth and development. HD-Zip Ι proteins are generally involved in responses related to abiotic stress, abscisic acid (ABA), blue light, de-etiolation, and embryogenesis. HD-Zip ΙΙ proteins participate in light response, shade avoidance, and auxin signalling. Members of the third group (HD-ZipIII) control embryogenesis, leaf polarity, lateral organ initiation, and meristem function. HD-Zip ΙV proteins play significant roles during anthocyanin accumulation, differentiation of epidermal cells, trichome formation, and root development.

Molecular regulation of plant somatic embryogenesis

In vitro embryogenesis is an asexual reproduction process by which embryos are produced from either gametophytic (androecium/gynoecium) or sporophytic (somatic) tissues. Regardless of the type of explant used, the hallmark of this process is that the explant cells undergo dedifferentiation and acquire meristematic identity. The developmental program of such meristematic cells can then be redirected to form somatic embryos, depending on the imposed culture environment. Analysis of proteomes and transcriptomes has led to the molecular identification and functional characterization of many genes involved in the initiation and development of somatic embryos. These genes can be classified into three categories: embryonic induction, embryonic, and maturation. So far, few genes involved in early somatic embryogenesis have been characterized because isolation of early pure embryonic tissue is very difficult. This review focuses on genes regulating the induction process. Furthermore, we employed bioinformatic tools and pathway databases to identify genes that may play roles in regulating early somatic embryogenesis. A total of 51 proteins were identified that may function in early somatic embryogenesis. These proteins are predicted to be involved in hormone signal transduction, chromatin remodeling, cell cycle regulation, cellulose biosynthetic and metabolic activity, GTPase signal transduction, transcription regulation, meristem formation and maintenance, and/or apoptosis and microtubule organization. This review will help advance knowledge and promote research on molecular regulation of early somatic embryogenesis.

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation

Applications of buthionine sulfoximine (BSO), an inhibitor of GSH (reduced glutathione), which switches the cellular glutathione pool towards the oxidized form GSSG, positively influences embryo quality by improving the structure of the shoot apical meristem and promoting embryo maturation, both of which improve the post-embryonic performance of the embryos. To investigate the mechanisms underlying BSO-mediated improvement in embryo quality the transcript profiles of developing Brassica napus microspore-derived embryos cultured in the absence (control) or presence of BSO were analyzed using a 15,000-element B. napus oligo microarray. BSO applications induced major changes in transcript accumulation patterns, especially during the late phases of embryogenesis. BSO affected the transcription and activities of key enzymes involved in ascorbate metabolism, which resulted in major fluctuations in cellular ascorbate levels. These changes were related to morphological characteristics of the embryos and their post-embryonic performance. BSO applications also activated many genes controlling meristem formation and function, including ZWILLE, SHOOTMERISTEMLESS, and ARGONAUTE 1. Increased expression of these genes may contribute to the improved structural quality of the shoot poles observed in the presence of BSO. Compared to their control counterparts, middle- and late-stage BSO-treated embryos also showed increased accumulation of transcripts associated with the maturation phase of zygotic embryo development, including genes encoding ABA-responsive proteins and storage- and late-embryogenic abundant (LEA) proteins. Overall these transcriptional changes support the observation that the BSO-induced oxidized glutathione redox state allows cultured embryos to reach both morphological and physiological maturity, which in turn guarantees successful regeneration and enhanced post-embryonic growth.

Function of type–2 Arabidopsis hemoglobin in the auxin-mediated formation of embryogenic cells during morphogenesis

Suppression of Arabidopsis GLB2, a type-2 nonsymbiotic hemoglobin, enhances somatic embryogenesis by increasing auxin production. In the glb2 knock-out line (GLB2-/-), polarization of PIN1 proteins and auxin maxima occurred at the base of the cotyledons of the zygotic explants, which are the sites of embryogenic tissue formation. These changes were also accompanied by a transcriptional upregulation of WUSCHEL (WUS) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK1), which are markers of embryogenic competence. The increased auxin levels in the GLB2-/- line were ascribed to the induction of several key enzymes of the tryptophan and IAA biosynthetic pathways, including ANTHRANILATE SYNTHASE (α subunit; ASA1), CYTOCHROME P79B2 (CYP79B2) and AMIDASE1 (AMI1). The effects of GLB2 suppression on somatic embryogenesis and IAA synthesis are mediated by increasing levels of nitric oxide (NO) within the embryogenic cells, which repress the expression of the transcription factor MYC2, a well-characterized repressor of the auxin biosynthetic pathway. A model is proposed in which the suppression of GLB2 reduces the degree of NO scavenging by oxyhemoglobin, thereby increasing the cellular NO concentration. The increased levels of NO repress the expression of MYC2, relieving the inhibition of IAA synthesis and increasing cellular IAA, which is the inductive signal promoting embryogenic competence. Besides providing a model for the induction phase of embryogenesis in vitro, these studies propose previously undescribed functions for plant hemoglobins.

The use of zygotic embryos as explants for in vitro propagation: an overview.

Plant propagation in vitro via somatic embryogenesis or organogenesis is a complicated process requiring the proper execution of several steps, which are affected by culture conditions and environment. A key element for a successful outcome is the choice of the explants. Several studies have shown that factors such as age, ontogenic and physiological conditions, and degree of differentiation affect the response of the explants to culture conditions. As a general rule, younger tissues, such as zygotic embryos, are the preferred choice for tissue culturists as they have better potential and competence to produce embryos and organs compared to more differentiated and mature tissues. This chapter focuses on how competence and commitment to regenerate embryos and organs in cultures are acquired by somatic cells and why zygotic embryos are so often utilized for propagation practices.

Altered seed oil and glucosinolate levels in transgenic plants overexpressing the Brassica napus SHOOTMERISTEMLESS gene.

SHOOTMERISTEMLESS (STM) is a homeobox gene conserved among plant species which is required for the formation and maintenance of the shoot meristem by suppressing differentiation and maintaining an undetermined cell fate within the apical pole. To assess further the role of this gene during seed storage accumulation, transgenic Brassica napus (Bn) plants overexpressing or down-regulating BnSTM under the control of the 35S promoter were generated. Overexpression of BnSTM increased seed oil content without affecting the protein and sucrose level. These changes were accompanied by the induction of genes encoding several transcription factors promoting fatty acid (FA) synthesis: LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and WRINKLE1 (BnWRI1). In addition, expression of key representative enzymes involved in sucrose metabolism, glycolysis, and FA biosynthesis was up-regulated in developing seeds ectopically expressing BnSTM. These distinctive expression patterns support the view of an increased carbon flux to the FA biosynthetic pathway in developing transformed seeds. The overexpression of BnSTM also resulted in a desirable reduction of seed glucosinolate (GLS) levels ascribed to a transcriptional repression of key enzymes participating in the GLS biosynthetic pathway, and possibly to the differential utilization of common precursors for GLS and indole-3-acetic acid synthesis. No changes in oil and GLS levels were observed in lines down-regulating BnSTM. Taken together, these findings provide evidence for a novel function for BnSTM in promoting desirable changes in seed oil and GLS levels when overexpressed in B. napus plants, and demonstrate that this gene can be used as a target for genetic improvement of oilseed species.

Depletion of cellular brassinolide decreases embryo production and disrupts the architecture of the apical meristems in Brassica napus microspore-derived embryos

Exogenous applications of brassinolide (BL) increased the number and quality of microspore-derived embryos (MDEs) whereas treatments with brassinazole (BrZ), a BL biosynthetic inhibitor, had the opposite effect. At the optimal concentration (4×10−6 M) BrZ decreased both embryo yield and conversion to less than half the value of control embryos. Metabolic studies revealed that BL levels had profound effects on glutathione and ascorbate metabolism by altering the amounts of their reduced forms (ASC and GSH) and oxidized forms [dehydroascorbate (DHA), ascorbate free radicals (AFRs), and GSSG]. Applications of BL switched the glutathione and ascorbate pools towards the oxidized forms, thereby lowering the ASC/ASC+DHA+AFR and GSH/GSH+GSSG ratios. These changes were ascribed to the ability of BL to increase the activity of ascorbate peroxidase (APX) and decrease that of glutathione reductase (GR). This trend was reversed in a BL-depleted environment, effected by BrZ applications. These metabolic alterations were associated with changes in embryo structure and performance. BL-treated MDEs developed zygotic-like shoot apical meristems (SAMs) whereas embryos treated with BrZ developed abnormal meristems. In the presence of BrZ, embryos either lacked a visible SAM, or formed SAMs in which the meristematic cells showed signs of differentiation, such as vacuolation and storage product accumulation. These abnormalities were accompanied by the lack or misexpression of three meristem marker genes isolated from Brassica napus (denoted as BnSTM, BnCLV1, and BnZLL-1) homologous to the Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL). The expression of BnSTM and BnCLV1 increased after a few days in cultures in embryos treated with BL whereas an opposite tendency was observed with applications of BrZ. Compared with control embryos where these two genes exhibited abnormal localization patterns, BnSTM and BnCLV1 always localized throughout the subapical domains of BL-treated embryos in a zygotic-like fashion. Expression of both genes was often lost in the SAM of BrZ-treated embryos. The results suggest that maintenance of cellular BL levels is required to modulate the ascorbate and glutathione redox status during embryogenesis to ensure proper development of the embryos and formation of functional apical meristems.

Jasmonic acid is a downstream component in the modulation of somatic embryogenesis by Arabidopsis Class 2 phytoglobin

Highlight Suppression of the phytoglobin GLB2 enhances jasmonic acid which promotes the accumulation of auxin and the formation of somatic embryos in Arabidopsis

Identification and characterization of PgHZ1, a novel homeodomain leucine-zipper gene isolated from white spruce (Picea glauca) tissue.

A member of the homeodomain-leucine zipper (HD-Zip) family was isolated from white spruce (Picea glauca) and designated as PgHZ1 (Gene Bank Accession No. DQ201170). The gene has an open reading frame of 1268bp and encodes a protein of 309 amino acid residues. PgHZ1 has all the features of a HD-ZIP protein: a homeodomain composed by three alpha-helices involved in DNA binding and an adjacent leucine zipper motif for protein-protein interaction. Phylogenetic analyses and sequence allignments with several Arabidopsis HD-ZIP members reveal that PgHZ1 belongs to the same monophyletic group of ATHB3, 13, 20, and 23 with which it shares a respective amino acid similarities of 74%, 71%, 68%, and 61%. Expression studies during spruce somatic embryogenesis reveal that the transcript levels of PgHZ1 increase during the late phases of proliferation and remain high during the subsequent embryo growth on the ABA-containing maturation medium. Such an increase does not occur in a non-embryogenic line characterized by a developmental block. Arabidopsis plants with ectopic PgHZ1 expression show an increase sensitivity to ABA, as estimated in seed germination and root growth tests. Compared to wild type plants, plants over-expressing PgHZ1 driven by the CAMV 35S promoter show a variety of phenotypic deviations, including a reduced inflorescence growth, increased branching, small rosette leaves and a delay in flowering. Somatic embryos produced from 35S:PgHZ1 Arabidopsis plants display a heavy accumulation of storage products and remain in a developmental program even if subjected to prolonged culture. This is in contrast to wild type somatic embryos in which storage products are quickly mobilized and the germination program is initiated after only 15 days in maturation. Overall these data support the notion that PgHZ1 confers hypersensitivity to ABA and that proper expression of this gene may be required for proper embryonic growth.