Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis.

Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somatic embryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somatic embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somatic embryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species.

The Arabidopsis mutant, fy-1, has an ABA-insensitive germination phenotype.

Arabidopsis FY, a homologue of the yeast RNA 3' processing factor Pfs2p, regulates the autonomous floral transition pathway through its interaction with FCA, an RNA binding protein. It is demonstrated here that FY also influences seed dormancy. Freshly-harvested seed of the Arabidopsis fy-1 mutant germinated readily in the absence of stratification or after-ripening. Furthermore, the fy-1 mutant showed less ABA sensitivity compared with the wild type, Ler, under identical conditions. Freshly-harvested seed of fy-1 had significantly higher ABA levels than Ler, even though Ler was dormant and fy-1 germinated readily. The PPLPP domains of FY, which are required for flowering control, were not essential for the ABA-influenced repression of germination. FLC expression analysis in seeds of different genotypes suggested that the effect of FY on dormancy may not be elicited through FLC. No significant differences in CYP707A1, CYP707A2, NCED9, ABI3, and ABI4 were observed between freshly-harvested Ler and fy-1 imbibed for 48 h. GA3ox1 and GA3ox2 rapidly increased over the 48 h imbibition period for fy-1, with no significant increases in these transcripts for Ler. ABI5 levels were significantly lower in fy-1 over the 48 h imbibition period. The results suggest that FY is involved in the development of dormancy and ABA sensitivity in Arabidopsis seed.

Identification and characterization of annexin gene family in rice.

Plant annexins are Ca(2+)-dependent phospholipid-binding proteins and are encoded by multigene families. They are implicated in the regulation of plant development as well as protection from drought and other stresses. They are well characterized in Arabidopsis, however no such characterization of rice annexin gene family has been reported thus far. With the availability of the rice genome sequence information, we have identified ten members of the rice annexin gene family. At the protein level, they share 16-64% identity with predicted molecular masses ranging from 32 to 40 kDa. Phylogenetic analysis of rice annexins together with annexins from other monocots led to their classification into five different orthologous groups and share similar motif patterns in their protein sequences. Expression analysis by real-time RT-PCR revealed differential temporal and spatial regulation of these genes. The rice annexin genes are also found to be regulated in seedling stage by various abiotic stressors including salinity, drought, heat and cold. Additionally, in silico analysis of the putative upstream sequences was analyzed for the presence of stress-responsive cis-elements. These results provide a basis for further functional characterization of specific rice annexin genes at the tissue/developmental level and in response to abiotic stresses.

Expression of chitinase genes of Metarhizium anisopliae isolates in lepidopteran pests and on synthetic media.

Pathogenecity of the well characterized entomopathogenic fungus Metarhizium anisopliae used for biocontrol of a wide range of insect pests secretes hydrolytic enzymes that degrade the host cuticle. The chitinolytic activity of high and low virulent isolates of M. anisopliae was assayed on minimal medium (MM) + colloidal chitin and MM supplemented with insect cuticles. Ex- pression pattern of four chitinase genes (chitinase (chi), chi 1, chi 2, chi 3) was profiled during pathogenic stages of the entomopathogen under in vitro and in vivo conditions. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed that chitinase cDNAs were expressed during the germination of fungus under nutrient-deprived conditions. RT-PCR analysis performed for the four chitinase genes on the two insect hosts Spodoptera litura and Helicoverpa armigera at six developmental stages of the pathogen displayed up-regulation in S. litura at mycosed and conidiated condition while with H. armigera there was expression only after 48 h of incubation. Differential expression of chi, chi 1 and chi 2 genes in vitro (nitrogen rich and nitrogen limiting media) and in vivo (live insect hosts S. litura and H. armigera) implicate the role of substrate differences in pathogenesis.

Ectopic expression of an annexin from Brassica juncea confers tolerance to abiotic and biotic stress treatments in transgenic tobacco.

Plant annexins belong to a multigene family and are suggested to play a role in stress responses. A full-length cDNA for a gene encoding an annexin protein was isolated and characterized from Brassica juncea (AnnBj1). AnnBj1 message levels were regulated by abscisic acid, ethephon, salicylic acid, and methyl jasmonate as well as chemicals that induce osmotic stress (NaCl, Mannitol or PEG), heavy metal stress (CdCl(2)) and oxidative stress (methyl viologen or H(2)O(2)). In order to determine if AnnBj1 functions in protection against stress, we generated transgenic tobacco plants ectopically expressing AnnBj1 under the control of constitutive CaMV 35S promoter. The transgenic tobacco plants showed significant tolerance to dehydration (mannitol), salt (NaCl), heavy metal (CdCl(2)) and oxidative stress (H(2)O(2)) at the seedling stage and retained higher chlorophyll levels in response to the above stresses as determined in detached leaf senescence assays. The transgenic plants also showed decreased accumulation of thiobarbituric acid-reactive substances (TBARS) compared to wild-type plants in response to mannitol treatments in leaf disc assays. AnnBj1 recombinant protein exhibited low levels of peroxidase activity in vitro and transgenic plants showed increased total peroxidase activity. Additionally, the transgenic plants showed enhanced resistance to the oomycete pathogen, Phytophthora parasitica var. nicotianae, and increased message levels for several pathogenesis-related proteins. Our results demonstrate that ectopic expression of AnnBj1 in tobacco provides tolerance to a variety of abiotic and biotic stresses.

Molecular, biochemical and structural characterization of osmotin-like protein from black nightshade (Solanum nigrum).

A full-length 910bp cDNA encoding osmotin-like protein with an open reading frame of 744bp encoding a protein of 247 amino acids with a calculated molecular mass of 26.8kDa was cloned from Solanum nigrum (SniOLP). Phylogenetic analysis revealed the evolutionary conservation of this protein among diverse taxa. The genomic DNA gel blot showed that SniOLP belongs to a small multigene family and it showed organ-specific expression. Time-course studies revealed that the expression of SniOLP was upregulated by treatment with various signaling molecules, osmotic and oxidative stress inducers. Recombinant protein purified from overexpressed Escherichia coli cells showed hyphal growth inhibition in Rhizoctonia batiticola and Sclerotinia sclerotiorum but without any endo-beta-1,3-glucanase activity. Model built by homology modeling showed that the protein consists of an acidic cleft region that is capable of interacting with the carbohydrate components of the fungal cell walls. Analysis of the structure and functional relationship was carried out by docking of the beta-(1,3)-glucan onto the acidic cleft region on the surface of the protein (SniOLP).

Genome-wide comparative analysis of annexin superfamily in plants.

Most annexins are calcium-dependent, phospholipid-binding proteins with suggested functions in response to environmental stresses and signaling during plant growth and development. They have previously been identified and characterized in Arabidopsis and rice, and constitute a multigene family in plants. In this study, we performed a comparative analysis of annexin gene families in the sequenced genomes of Viridiplantae ranging from unicellular green algae to multicellular plants, and identified 149 genes. Phylogenetic studies of these deduced annexins classified them into nine different arbitrary groups. The occurrence and distribution of bona fide type II calcium binding sites within the four annexin domains were found to be different in each of these groups. Analysis of chromosomal distribution of annexin genes in rice, Arabidopsis and poplar revealed their localization on various chromosomes with some members also found on duplicated chromosomal segments leading to gene family expansion. Analysis of gene structure suggests sequential or differential loss of introns during the evolution of land plant annexin genes. Intron positions and phases are well conserved in annexin genes from representative genomes ranging from Physcomitrella to higher plants. The occurrence of alternative motifs such as K/R/HGD was found to be overlapping or at the mutated regions of the type II calcium binding sites indicating potential functional divergence in certain plant annexins. This study provides a basis for further functional analysis and characterization of annexin multigene families in the plant lineage.

Transcriptional regulation of annexins in Indian Mustard, Brassica juncea and detoxification of ROS in transgenic tobacco plants constitutively expressing AnnBj1.

Annexins in plants constitute a multigene family and there is growing evidence for their involvement in response to various stress treatments. We have cloned and characterized six different annexin genes from mustard. The transcript regulation of these genes was studied in treatments with various signaling molecules, osmotic stress, oxidative stress conditions and wounding. All these annexins were found to be reponsive to Abscisic acid (ABA). Two genes (AnnBj1 and AnnBj3) were found to respond to most of the stress conditions, which suggest their possible role in cross-talk in multiple signaling pathways. Wound response signal methyl jasmonate (mJA) caused rapid and high expression of AnnBj4. We extended our previous study and showed that transgenic tobacco plants heterologously expressing AnnBj1 evidenced mannitol induced ROS detoxification. These results suggest Brassica annexins may have potential role in alleviating abiotic stress, which should be characterized by in vivo function based studies through silencing by RNAi or overexpression in transgenic plants.

Molecular cloning and characterization of five annexin genes from Indian mustard (Brassica juncea L. Czern and Coss).

Plant annexins constitute a multigene family having suggested roles in a variety of cellular processes including stress responses. We have isolated and characterized five different cDNAs of mustard, Brassica juncea (AnnBj1, AnnBj2, AnnBj3, AnnBj6 and AnnBj7) encoding annexin proteins using a RT-PCR/RACE-PCR based strategy. The predicted molecular masses of these annexins are approximately 36.0 kDa with acidic pIs. At the amino acid level, they share high sequence similarity with each other and with annexins from higher plants. Phylogenetic analysis revealed their evolutionary relationship with corresponding orthologous sequences in Arabidopsis and deduced proteins in various plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. The expression patterns of these genes also showed regulation by various stress conditions such as exposure to signaling molecules, salinity and oxidative stress and wounding. Additionally, the in silico promoter analysis (of AnnBj1, AnnBj2 and AnnBj3) showed the presence of different cis-responsive elements that could respond to various stress conditions. These results indicate that AnnBj genes may play important roles in adaptation of plants to various environmental stresses.