Diverse geotropic responses in the orchid family.

In epiphytes, aerial roots are important to combat water-deficient, nutrient-poor, and high-irradiance microhabitats. However, whether aerial roots can respond to gravity and whether auxin plays a role in regulating aerial root development remain open-ended questions. Here, we investigated the gravitropic response of the epiphytic orchid Phalaenopsis aphrodite. Our data showed that aerial roots of P. aphrodite failed to respond to gravity, and this was correlated with a lack of starch granules/statolith sedimentation in the roots and the absence of the auxin efflux carrier PIN2 gene. Using an established auxin reporter, we discovered that auxin maximum was absent in the quiescent center of aerial roots of P. aphrodite. Also, gravity failed to trigger auxin redistribution in the root caps. Hence, loss of gravity sensing and gravity-dependent auxin redistribution may be the genetic factors contributing to aerial root development. Moreover, the architectural and functional innovations that achieve fast gravitropism in the flowering plants appear to be lost in both terrestrial and epiphytic orchids, but are present in the early diverged orchid subfamilies. Taken together, our findings provide physiological and molecular evidence to support the notion that epiphytic orchids lack gravitropism and suggest diverse geotropic responses in the orchid family.

Protocorm-like-body extract of Phalaenopsis aphrodite combats watermelon fruit blotch disease.

Bacterial fruit blotch, caused by the seedborne gram-negative bacterium Acidovorax citrulli, is one of the most destructive bacterial diseases of cucurbits (gourds) worldwide. Despite its prevalence, effective and reliable means to control bacterial fruit blotch remain limited. Transcriptomic analyses of tissue culture-based regeneration processes have revealed that organogenesis-associated cellular reprogramming is often associated with upregulation of stress- and defense-responsive genes. Yet, there is limited evidence supporting the notion that the reprogrammed cellular metabolism of the regenerated tissued confers bona fide antimicrobial activity. Here, we explored the anti-bacterial activity of protocorm-like-bodies (PLBs) of Phalaenopsis aphrodite. Encouragingly, we found that the PLB extract was potent in slowing growth of A. citrulli, reducing the number of bacteria attached to watermelon seeds, and alleviating disease symptoms of watermelon seedlings caused by A. citrulli. Because the anti-bacterial activity can be fractionated chemically, we predict that reprogrammed cellular activity during the PLB regeneration process produces metabolites with antibacterial activity. In conclusion, our data demonstrated the antibacterial activity in developing PLBs and revealed the potential of using orchid PLBs to discover chemicals to control bacterial fruit blotch disease.

Co-option of the SHOOT MERISTEMLESS network regulates protocorm-like body development in Phalaenopsis aphrodite.

The protocorm is a structure that is formed upon germination of an orchid seed. It lacks cotyledons and is ovoid in shape. The protocorm-like body (PLB), on the other hand, is a protocorm-like organ induced from somatic tissues. PLBs have been widely used for orchid micropropagation. Because of its unique structure and its application in the orchid industry, PLB development has drawn considerable interest from orchid and developmental biologists. Our previous genome-wide comparative transcriptome study demonstrated that protocorms and PLBs share similar molecular signatures and suggested that SHOOT MERISTEMLESS (STM)-dependent organogenesis is important for PLB development. Here, we show that overexpression of Phalaenopsis aphrodite STM (PaSTM) greatly enhances PLB regeneration from vegetative tissue-based explants of Phalaenopsis orchids, confirming its regulatory role in PLB development. Expression of PaSTM restored shoot meristem function of the Arabidopsis (Arabidopsis thaliana) stm-2 mutant. Moreover, we identified class S11 MYB transcription factors (TFs) as targets downstream of PaSTM. A cis-acting element, TTGACT, identified in the promoters of S11 MYB TFs was found to be important for PaSTM binding and activation. Overexpression of PaSTM or its downstream targets, PaMYB13, PaMYB14, and PaMYB17, enhanced de novo shoot regeneration in Arabidopsis, indicating the active role of the PaSTM-S11 PaMYB module in organogenesis. In summary, our data demonstrate that PaSTM is important for PLB development. The STM-S11 MYB regulatory module is evolutionarily conserved and may regulate shoot or shoot-related organ development in plants.

Revised annotation and extended characterizations of components of the Chlamydomonas reinhardtii SUMOylation system.

Small ubiquitin-like modifier (SUMO) conjugation, or SUMOylation, is a reversible post-translational modification that is important for regulation of many cellular processes including cell division cycle in the eukaryotic kingdom. However, only a portion of the components of the Chlamydomonas SUMOylation system are known and their functions and regulation investigated. The present studies are aimed at extending discovery and characterization of new components and improving the annotation and nomenclature of all known proteins and genes involved in the system. Even though only one copy of the heterodimerized SUMO-activating enzyme, SAE1 and SAE2, was identified, the number of SUMO-conjugating enzymes (SCEs) and SUMO proteases/isopeptidase was expanded in Chlamydomonas. Using the reconstituted SUMOylation system, we showed that SCE1, SCE2, and SCE3 have SUMO-conjugating activity. In addition to SUMOylation, components required for other post-translational modifications such as NEDDylation, URMylation, and UFMylation, were confirmed to be present in Chlamydomonas. Our data also showed that besides isopeptidase activity, the SUMO protease domain of SUPPRESSOR OF MAT3 7/SENTRIN-SPECIFIC PROTEASE 1 (SMT7/SENP1) has endopeptidase activity that is capable of processing SUMO precursors. Moreover, the key cell cycle regulators of Chlamydomonas E2F1, DP1, CDKG1, CYCD2, and CYCD3 were SUMOylated in vitro, suggesting SUMOylation may be part of regulatory pathway modulating cell cycle regulators.

SUMO Protease SMT7 Modulates Ribosomal Protein L30 and Regulates Cell-Size Checkpoint Function.

Proliferating cells actively coordinate growth and cell division to ensure cell-size homeostasis; however, the underlying mechanism through which size is controlled is poorly understood. Defect in a SUMO protease protein, suppressor of mat3 7 (SMT7), has been shown to reduce cell division number and increase cell size of the small-size mutant mating type locus 3-4 (mat3-4), which contains a defective retinoblastoma tumor suppressor-related protein of Chlamydomonas (Chlamydomonas reinhardtii). Here we describe development of an in vitro SUMOylation system using Chlamydomonas components and use it to provide evidence that SMT7 is a bona fide SUMO protease. We further demonstrate that the SUMO protease activity is required for supernumerous mitotic divisions of the mat3-4 cells. In addition, we identified RIBOSOMAL PROTEIN L30 (RPL30) as a prime SMT7 target and demonstrated that its SUMOylation is an important modulator of cell division in mat3-4 cells. Loss of SMT7 caused elevated SUMOylated RPL30 levels. Importantly, overexpression of the translational fusion version of RPL30-SUMO4, which mimics elevation of the SUMOylated RPL30 protein in mat3-4, caused a decrease in mitotic division and recapitulated the size-increasing phenotype of the smt7-1 mat3-4 cells. In summary, our study reveals a novel mechanism through which a SUMO protease regulates cell division in the mat3-4 mutant of Chlamydomonas and provides yet another important example of the role that protein SUMOylation can play in regulating key cellular processes, including cell division.

Phalaenopsis LEAFY COTYLEDON1-Induced Somatic Embryonic Structures Are Morphologically Distinct From Protocorm-Like Bodies.

Somatic embryogenesis is commonly used for clonal propagation of a wide variety of plant species. Induction of protocorm-like-bodies (PLBs), which are capable of developing into individual plants, is a routine tissue culture-based practice for micropropagation of orchid plants. Even though PLBs are often regarded as somatic embryos, our recent study provides molecular evidence to argue that PLBs are not derived from somatic embryogenesis. Here, we report and characterize the somatic embryonic tissues induced by Phalaenopsis aphrodite LEAFY COTYLEDON1 (PaLEC1) in Phalaenopsis equestris. We found that PaLEC1-induced somatic tissues are morphologically different from PLBs, supporting our molecular study that PLBs are not of somatic embryonic origin. The embryonic identity of PaLEC1-induced embryonic tissues was confirmed by expression of the embryonic-specific transcription factors FUSCA3 (FUS3) and ABSCISIC ACID INSENSITIVE3 (ABI3), and seed storage proteins 7S GLOBULIN and OLEOSIN. Moreover, PaLEC1-GFP protein was found to be associated with the Pa7S-1 and PaFUS3 promoters containing the CCAAT element, supporting that PaLEC1 directly regulates embryo-specific processes to activate the somatic embryonic program in P. equestris. Despite diverse embryonic structures, PaLEC1-GFP-induced embryonic structures are pluripotent and capable of generating new shoots. Our study resolves the long-term debate on the developmental identity of PLB and suggests that somatic embryogenesis may be a useful approach to clonally propagate orchid seedlings.

A Protoplast Transient Expression System to Enable Molecular, Cellular, and Functional Studies in Phalaenopsis orchids.

The enigmatic nature of the specialized developmental programs of orchids has fascinated plant biologists for centuries. The recent releases of orchid genomes indicate that orchids possess new gene families and family expansions and contractions to regulate a diverse suite of developmental processes. However, the extremely long orchid life cycle and lack of molecular toolkit have hampered the advancement of orchid biology research. To overcome the technical difficulties and establish a platform for rapid gene regulation studies, in this study, we developed an efficient protoplast isolation and transient expression system for Phalaenopsis aphrodite. This protocol was successfully applied to protein subcellular localization and protein-protein interaction studies. Moreover, it was confirmed to be useful in delineating the PaE2F/PaDP-dependent cell cycle pathway and studying auxin response. In summary, the established orchid protoplast transient expression system provides a means to functionally characterize orchid genes at the molecular level allowing assessment of transcriptome responses to transgene expression and widening the scope of molecular studies in orchids.

Adhesion Regulating Molecule 1 Mediates HAP40 Overexpression-Induced Mitochondrial Defects.

Striatal neuron death in Huntington's disease is associated with abnormal mitochondrial dynamics and functions. However, the mechanisms for this mitochondrial dysregulation remain elusive. Increased accumulation of Huntingtin-associated protein 40 (HAP40) has been shown to be associated with Huntington's disease. However, the link between increased HAP40 and Huntington's disease remains largely unknown. Here we show that HAP40 overexpression causes mitochondrial dysfunction and reduces cell viability in the immortalized mouse striatal neurons. HAP40-associated mitochondrial dysfunction is associated with reduction of adhesion regulating molecule 1 (ADRM1) protein. Consistently, depletion of ADRM1 by shRNAs impaired mitochondrial functions and increased mitochondrial fragmentation in mouse striatal cells. Moreover, reducing ADRM1 levels enhanced activity of fission factor dynamin-related GTPase protein 1 (Drp1) via increased phosphorylation at serine 616 of Drp1 (Drp1Ser616). Restoring ADRM1 protein levels was able to reduce HAP40-induced ROS levels and mitochondrial fragmentation and improved mitochondrial functions and cell viability. Moreover, reducing Drp1 activity by Drp1 inhibitor, Mdivi-1, ameliorates both HAP40 overexpression- and ADRM1 depletion-induced mitochondrial dysfunction. Taken together, our studies suggest that HAP40-mediated reduction of ADRM1 alters the mitochondrial fission activity and results in mitochondrial fragmentation and mitochondrial dysfunction.

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples.

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning. This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 °C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

Expression analysis of fertilization/early embryogenesis-associated genes in Phalaenopsis orchids.

One of the distinct reproductive programs in orchid species is pollination-triggered ovule development and megasporogenesis. During sexual reproduction, fertilization occurs days to months after pollination. The molecular mechanisms evolved to carry out this strategic reproductive program remain unclear. In the August issue of Plant Physiology 1 , we report comprehensive studies of comparative genome-wide gene expression in various reproductive tissues and the molecular events associated with developmental transitions unique to sexual reproduction of Phalaenopsis aphrodite. Transcriptional factors and signaling components whose expression is specifically enriched in interior ovary tissues when fertilization occurs and embryos start to develop have been identified. Here, we report verification of additional fertilization-associated genes, DOMAINS REARRANGED METHYLTRANSFERASE 1 (PaDRM1), CHROMOMETHYLTRANSFERASE 1 (PaCMT1), SU(VAR)3-9 RELATED PROTEIN 1 (PaSUVR1), INDOLE-3-ACETIC ACID inducible 30-like 1 (PaIAA30L1), and ETHYLENE INSENSITIVE 3-like 1 (PaEIN3L1), and discuss their potential roles in gametophyte development, epigenetic reprogramming, and hormone regulation during fertilization and establishment of embryo development in Phalaenopsis orchids.

Protocorms and Protocorm-Like Bodies Are Molecularly Distinct from Zygotic Embryonic Tissues in Phalaenopsis aphrodite.

The distinct reproductive program of orchids provides a unique evolutionary model with pollination-triggered ovule development and megasporogenesis, a modified embryogenesis program resulting in seeds with immature embryos, and mycorrhiza-induced seed germination. However, the molecular mechanisms that have evolved to establish these unparalleled developmental programs are largely unclear. Here, we conducted comparative studies of genome-wide gene expression of various reproductive tissues and captured the molecular events associated with distinct reproductive programs in Phalaenopsis aphrodite Importantly, our data provide evidence to demonstrate that protocorm-like body (PLB) regeneration (the clonal regeneration practice used in the orchid industry) does not follow the embryogenesis program. Instead, we propose that SHOOT MERISTEMLESS, a class I KNOTTED-LIKE HOMEOBOX gene, is likely to play a role in PLB regeneration. Our studies challenge the current understanding of the embryonic identity of PLBs. Taken together, the data obtained establish a fundamental framework for orchid reproductive development and provide a valuable new resource to enable the prediction of gene regulatory networks that is required for specialized developmental programs of orchid species.

The long pollen tube journey and in vitro pollen germination of Phalaenopsis orchids.

KEY MESSAGE: Pollen biology in P. aphrodite. Orchids have a distinct reproductive program. Pollination triggers ovule development and differentiation within flowers, and fertilization occurs days to months after pollination. It is unclear how pollen tubes travel through the developing ovaries during ovule development and when pollen tubes arrive at the mature embryo sac to achieve fertilization. Here, we report a robust staining protocol to image and record the timing of pollen germination, progressive growth of pollen tubes in ovaries, and arrival of pollen tubes at embryo sacs in Phalaenopsis aphrodite. The pollen germinated and pollen tubes entered the ovary 3 days after pollination. Pollen tubes continued to grow and filled the entire cavity of the ovary as the ovary elongated and ovules developed. Pollen tubes were found to enter the matured embryo sacs at approximately 60-65 days after pollination in an acropetal manner. Moreover, these temporal changes in developmental events such as growth of pollen tubes and fertilization were associated with expression of molecular markers. In addition, we developed an in vitro pollen germination protocol, which is valuable to enable studies on pollen tube guidance and tip growth regulation in Phalaenopsis orchids and possibly in other orchid species.

Defects in a new class of sulfate/anion transporter link sulfur acclimation responses to intracellular glutathione levels and cell cycle control.

We previously identified a mutation, suppressor of mating type locus3 15-1 (smt15-1), that partially suppresses the cell cycle defects caused by loss of the retinoblastoma tumor suppressor-related protein encoded by the MAT3 gene in Chlamydomonas reinhardtii. smt15-1 single mutants were also found to have a cell cycle defect leading to a small-cell phenotype. SMT15 belongs to a previously uncharacterized subfamily of putative membrane-localized sulfate/anion transporters that contain a sulfate transporter domain and are found in a widely distributed subset of eukaryotes and bacteria. Although we observed that smt15-1 has a defect in acclimation to sulfur-limited growth conditions, sulfur acclimation (sac) mutants, which are more severely defective for acclimation to sulfur limitation, do not have cell cycle defects and cannot suppress mat3. Moreover, we found that smt15-1, but not sac mutants, overaccumulates glutathione. In wild-type cells, glutathione fluctuated during the cell cycle, with highest levels in mid G1 phase and lower levels during S and M phases, while in smt15-1, glutathione levels remained elevated during S and M. In addition to increased total glutathione levels, smt15-1 cells had an increased reduced-to-oxidized glutathione redox ratio throughout the cell cycle. These data suggest a role for SMT15 in maintaining glutathione homeostasis that impacts the cell cycle and sulfur acclimation responses.

Genome-wide annotation, expression profiling, and protein interaction studies of the core cell-cycle genes in Phalaenopsis aphrodite.

Orchidaceae is one of the most abundant and diverse families in the plant kingdom and its unique developmental patterns have drawn the attention of many evolutionary biologists. Particular areas of interest have included the co-evolution of pollinators and distinct floral structures, and symbiotic relationships with mycorrhizal flora. However, comprehensive studies to decipher the molecular basis of growth and development in orchids remain scarce. Cell proliferation governed by cell-cycle regulation is fundamental to growth and development of the plant body. We took advantage of recently released transcriptome information to systematically isolate and annotate the core cell-cycle regulators in the moth orchid Phalaenopsis aphrodite. Our data verified that Phalaenopsis cyclin-dependent kinase A (CDKA) is an evolutionarily conserved CDK. Expression profiling studies suggested that core cell-cycle genes functioning during the G1/S, S, and G2/M stages were preferentially enriched in the meristematic tissues that have high proliferation activity. In addition, subcellular localization and pairwise interaction analyses of various combinations of CDKs and cyclins, and of E2 promoter-binding factors and dimerization partners confirmed interactions of the functional units. Furthermore, our data showed that expression of the core cell-cycle genes was coordinately regulated during pollination-induced reproductive development. The data obtained establish a fundamental framework for study of the cell-cycle machinery in Phalaenopsis orchids.

Regulation of the Chlamydomonas cell cycle by a stable, chromatin-associated retinoblastoma tumor suppressor complex.

We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit-RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle-regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.

A suppressor screen in chlamydomonas identifies novel components of the retinoblastoma tumor suppressor pathway.

The retinoblastoma (RB) protein is a eukaryotic tumor suppressor and negative cell-cycle regulator. Chlamydomonas reinhardtii cells that lack the RB homolog MAT3 show loss of size checkpoint control and deregulated cell-cycle progression leading to the production of tiny cells. We carried out an insertional mutagenesis screen to isolate bypass suppressors of mat3 (smt mutants) that reverted the mat3 cell-size defect. Previously we reported that the loci encoding Chlamydomonas homologs of E2F and DP were frequently disrupted in this screen, indicating that the architecture of the canonical RB pathway is conserved in Chlamydomonas with MAT3/RB acting as a negative regulator upstream of E2F/DP. Here, we describe four novel smt mutants that moderately suppressed the cell-size checkpoint and cell-cycle phenotypes of mat3. As single mutants, three of the smt strains displayed no obvious phenotypes, and one had a slightly small phenotype. Strikingly, several smt double-mutant combinations synergized to cause enhanced suppression of mat3 and even to cause a large-cell phenotype that is comparable to that caused by loss of DP1. Molecular characterization of one smt mutant revealed that suppression is due to a defect in a gene encoding a putative small ubiquitin-like modifier (SUMO) peptidase. Our results reveal a complex genetic network that lies downstream of MAT3/RB and implicate protein sumoylation as an important step for cell-cycle progression in cells that are missing MAT3/RB.

Cell size checkpoint control by the retinoblastoma tumor suppressor pathway.

Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription.

Bacillus thuringiensis crystal proteins that target nematodes.

Bacillus thuringiensis (Bt) crystal proteins are pore-forming toxins used as insecticides around the world. Previously, the extent to which these proteins might also target the invertebrate phylum Nematoda has been mostly ignored. We have expressed seven different crystal toxin proteins from two largely unstudied Bt crystal protein subfamilies. By assaying their toxicity on diverse free-living nematode species, we demonstrate that four of these crystal proteins are active against multiple nematode species and that each nematode species tested is susceptible to at least one toxin. We also demonstrate that a rat intestinal nematode is susceptible to some of the nematicidal crystal proteins, indicating these may hold promise in controlling vertebrate-parasitic nematodes. Toxicity in nematodes correlates with damage to the intestine, consistent with the mechanism of crystal toxin action in insects. Structure-function analyses indicate that one novel nematicidal crystal protein can be engineered to a small 43-kDa active core. These data demonstrate that at least two Bt crystal protein subfamilies contain nematicidal toxins.

Effect of regulated overexpression of the MADS domain factor AGL15 on flower senescence and fruit maturation.

We have examined the effect of regulated overexpression of AGL15, a member of the MADS domain family of regulatory factors, on reproductive tissues. Using molecular and physiological markers, we show that constitutive overexpression of AGL15 in Arabidopsis leads to delay and down-regulation of senescence programs in perianth organs and developing fruits and alters the process of seed desiccation. Through genetic crosses, we show that the rate of water loss in the maturing seeds is dictated by the genetic composition and physiological state of the maternal tissue, rather than the embryo. To define the developmental time and/or place when senescence programs are most affected by elevated AGL15 levels, we expressed AGL15 under the control of various promoters. Expression during senescence or in abscission zone cells did not produce delays in floral organ senescence or abscission. Using a glucocorticoid-inducible expression system, we show that an increase in AGL15 levels around the time of flower opening is necessary to delay senescence and increase floral organ longevity.