Swee-Suak Ko

Arabidopsis Hsa32, a Novel Heat Shock Protein, Is Essential for Acquired Thermotolerance during Long Recovery after Acclimation

Plants and animals share similar mechanisms in the heat shock (HS) response, such as synthesis of the conserved HS proteins (Hsps). However, because plants are confined to a growing environment, in general they require unique features to cope with heat stress. Here, we report on the analysis of the function of a novel Hsp, heat-stress-associated 32-kD protein (Hsa32), which is highly conserved in land plants but absent in most other organisms. The gene responds to HS at the transcriptional level in moss (Physcomitrella patens), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). Like other Hsps, Hsa32 protein accumulates greatly in Arabidopsis seedlings after HS treatment. Disruption of Hsa32 by T-DNA insertion does not affect growth and development under normal conditions. However, the acquired thermotolerance in the knockout line was compromised following a long recovery period (>24 h) after acclimation HS treatment, when a severe HS challenge killed the mutant but not the wild-type plants, but no significant difference was observed if they were challenged within a short recovery period. Quantitative hypocotyl elongation assay also revealed that thermotolerance decayed faster in the absence of Hsa32 after a long recovery. Similar results were obtained in Arabidopsis transgenic plants with Hsa32 expression suppressed by RNA interference. Microarray analysis of the knockout mutant indicates that only the expression of Hsa32 was significantly altered in HS response. Taken together, our results suggest that Hsa32 is required not for induction but rather maintenance of acquired thermotolerance, a feature that could be important to plants.

Complex Regulation of Two Target Genes Encoding SPX-MFS Proteins by Rice miR827 in Response to Phosphate Starvation

Here we report on the characterization of rice osa-miR827 and its two target genes, OsSPX-MFS1 and OsSPX-MFS2, which encode SPX-MFS proteins predicted to be implicated in phosphate (Pi) sensing or transport. We first show by Northern blot analysis that osa-miR827 is strongly induced by Pi starvation in both shoots and roots. Hybridization of osa-miR827 in situ confirms its strong induction by Pi starvation, with signals concentrated in mesophyll, epidermis and ground tissues of roots. In parallel, we analyzed the responses of the two OsSPX-MFS1 and OsSPX-MFS2 gene targets to Pi starvation. OsSPX-MFS1 mRNA is mainly expressed in shoots under sufficient Pi supply while its expression is reduced on Pi starvation, revealing a direct relationship between induction of osa-miR827 and down-regulation of OsSPX-MFS1. In contrast, OsSPX-MFS2 responds in a diametrically opposed manner to Pi starvation. The accumulation of OsSPX-MFS2 mRNA is dramatically enhanced under Pi starvation, suggesting the involvement of complex regulation of osa-miR827 and its two target genes. We further produced transgenic rice lines overexpressing osa-miR827 and T-DNA knockout mutant lines in which the expression of osa-miR827 is abolished. Compared with wild-type controls, both target mRNAs exhibit similar changes, their expression being reduced and increased in overexpressing and knockout lines, respectively. This suggests that OsSPX-MFS1 and OsSPX-MFS2 are both negatively regulated by osa-miR827 abundance although they respond differently to external Pi conditions. We propose that this is a complex mechanism comprising fine tuning of spatial or temporal regulation of both targets by osa-miR827.

A Positive Feedback Loop between HEAT SHOCK PROTEIN101 and HEAT STRESS-ASSOCIATED 32-KD PROTEIN Modulates Long-Term Acquired Thermotolerance Illustrating Diverse Heat Stress Responses in Rice Varieties

Rice heat-shock proteins modulate long-term acquired thermotolerance, which can be decoupled from basal thermotolerance in different rice cultivars. Heat stress is an important factor that has a negative impact on rice (Oryza sativa) production. To alleviate this problem, it is necessary to extensively understand the genetic basis of heat tolerance and adaptability to heat stress in rice. Here, we report the molecular mechanism underlying heat acclimation memory that confers long-term acquired thermotolerance (LAT) in this monocot plant. Our results showed that a positive feedback loop formed by two heat-inducible genes, HEAT SHOCK PROTEIN101 (HSP101) and HEAT STRESS-ASSOCIATED 32-KD PROTEIN (HSA32), at the posttranscriptional level prolongs the effect of heat acclimation in rice seedlings. The interplay between HSP101 and HSA32 also affects basal thermotolerance of rice seeds. These findings are similar to those reported for the dicot plant Arabidopsis (Arabidopsis thaliana), suggesting a conserved function in plant heat stress response. Comparison between two rice cultivars, japonica Nipponbare and indica N22 showed opposite performance in basal thermotolerance and LAT assays. ‘N22’ seedlings have a higher basal thermotolerance level than cv Nipponbare and vice versa at the LAT level, indicating that these two types of thermotolerance can be decoupled. The HSP101 and HSA32 protein levels were substantially higher in cv Nipponbare than in cv N22 after a long recovery following heat acclimation treatment, at least partly explaining the difference in the LAT phenotype. Our results point out the complexity of thermotolerance diversity in rice cultivars, which may need to be taken into consideration when breeding for heat tolerance for different climate scenarios.

The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice

This study shows that rice bHLH142 functions in tapetal programmed cell death and pollen development, finding that bHLH142 and TDR1 form a heterodimer that binds to the EAT1 promoter to activate its expression. Protein modeling also suggests that EAT1 can form a heterodimer with TDR1, presumably serving as a feedback regulator. Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.

Overexpression of Arabidopsis thaliana tryptophan synthase beta 1 (AtTSB1) in Arabidopsis and tomato confers tolerance to cadmium stress

Tryptophan (Trp) is an essential amino acid in humans, and in plants, it plays a major role in the regulation of plant development and defence responses. However, little is known about Trp-mediated cadmium (Cd) tolerance. Gene expression analysis showed that Arabidopsis thaliana tryptophan synthase beta 1 (AtTSB1) is up-regulated in plants treated with Cd; hence, we investigated whether this gene is involved in Cd tolerance. Exogenous application of Trp to wild-type Arabidopsis enhances Cd tolerance. Cd tolerance in the Trp-overproducing mutant trp5-1 was associated with high chlorophyll levels and low lipid peroxidation, as indicated by malondialdehyde 4-hydroxyalkenal level, whereas the wild-type developed symptoms of severe chlorosis. Moreover, the Trp-auxotroph mutant trp2-1 was sensitive to Cd. CaMV 35S promoter-driven AtTSB1 enhanced Trp accumulation and improved Cd tolerance in transgenic Arabidopsis and tomato plants without increasing the level of Cd. Moreover, reverse transcription-polymerase chain reaction confirmed that enhanced level of Trp in AtTSB1 transgenic Arabidopsis plants affected the expression of AtZIP4 and AtZIP9 metal transporters, which interfered with Cd ion trafficking, a mechanism of transcriptional regulation that does not exist in wild-type plants. Overexpression of AtTSB1 in transgenic tomato also produced higher Trp synthase-beta enzyme activity than that in wild-type plants. These results implicate that Trp could be involved in Cd defence.

BeMADS1 is a key to delivery MADSs into nucleus in reproductive tissues-De novo characterization of Bambusa edulis transcriptome and study of MADS genes in bamboo floral development

BackgroundThe bamboo Bambusa edulis has a long juvenile phase in situ, but can be induced to flower during in vitro tissue culture, providing a readily available source of material for studies on reproductive biology and flowering. In this report, in vitro-derived reproductive and vegetative materials of B. edulis were harvested and used to generate transcriptome databases by use of two sequencing platforms: Illumina and 454. Combination of the two datasets resulted in high transcriptome quality and increased length of the sequence reads. In plants, many MADS genes control flower development, and the ABCDE model has been developed to explain how the genes function together to create the different whorls within a flower.ResultsAs a case study, published floral development-related OsMADS proteins from rice were used to search the B. edulis transcriptome datasets, identifying 16 B. edulis MADS (BeMADS). The BeMADS gene expression levels were determined qRT-PCR and in situ hybridization. Most BeMADS genes were highly expressed in flowers, with the exception of BeMADS34. The expression patterns of these genes were most similar to the rice homologs, except BeMADS18 and BeMADS34, and were highly similar to the floral development ABCDE model in rice. Transient expression of MADS-GFP proteins showed that only BeMADS1 entered leaf nucleus. BeMADS18, BeMADS4, and BeMADS1 were located in the lemma nucleus. When co-transformed with BeMADS1, BeMADS15, 16, 13, 21, 6, and 7 translocated to nucleus in lemmas, indicating that BeMADS1 is a key factor for subcellular localization of other BeMADS.ConclusionOur study provides abundant B. edulis transcriptome data and offers comprehensive sequence resources. The results, molecular materials and overall strategy reported here can be used for future gene identification and for further reproductive studies in the economically important crop of bamboo.

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

Abstract 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.

Transcriptome‐wide analysis of the MADS‐box gene family in the orchid Erycina pusilla

Orchids exhibit a range of unique flower shapes and are a valuable ornamental crop. MADS-box transcription factors are key regulatory components in flower initiation and development. Changing the flower shape and flowering time can increase the value of the orchid in the ornamental horticulture industry. In this study, 28 MADS-box genes were identified from the transcriptome database of the model orchid Erycina pusilla. The full-length genomic sequences of these MADS-box genes were obtained from BAC clones. Of these, 27 were MIKC-type EpMADS (two truncated forms) and one was a type I EpMADS. Eleven EpMADS genes contained introns longer than 10 kb. Phylogenetic analysis classified the 24 MIKC(c) genes into nine subfamilies. Three specific protein motifs, AG, FUL and SVP, were identified and used to classify three subfamilies. The expression profile of each EpMADS gene correlated with its putative function. The phylogenetic analysis was highly correlated with the protein domain identification and gene expression results. Spatial expression of EpMADS6, EpMADS12 and EpMADS15 was strongly detected in the inflorescence meristem, floral bud and seed via in situ hybridization. The subcellular localization of the 28 EpMADS proteins was also investigated. Although EpMADS27 lacks a complete MADS-box domain, EpMADS27-YFP was localized in the nucleus. This characterization of the orchid MADS-box family genes provides useful information for both orchid breeding and studies of flowering and evolution.

Tightly Controlled Expression of bHLH142 Is Essential for Timely Tapetal Programmed Cell Death and Pollen Development in Rice

Male sterility is important for hybrid seed production. Pollen development is regulated by a complex network. We previously showed that knockout of bHLH142 in rice (Oryza sativa) causes pollen sterility by interrupting tapetal programmed cell death (PCD) and bHLH142 coordinates with TDR to modulate the expression of EAT1. In this study, we demonstrated that overexpression of bHLH142 (OE142) under the control of the ubiquitin promoter also leads to male sterility in rice by triggering the premature onset of PCD. Protein of bHLH142 was found to accumulate specifically in the OE142 anthers. Overexpression of bHLH142 induced early expression of several key regulatory transcription factors in pollen development. In particular, the upregulation of EAT1 at the early stage of pollen development promoted premature PCD in the OE142 anthers, while its downregulation at the late stage impaired pollen development by suppressing genes involved in pollen wall biosynthesis, ROS scavenging and PCD. Collectively, these events led to male sterility in OE142. Analyses of related mutants further revealed the hierarchy of the pollen development regulatory gene network. Thus, the findings of this study advance our understanding of the central role played by bHLH142 in the regulatory network leading to pollen development in rice and how overexpression of its expression affects pollen development. Exploitation of this novel functionality of bHLH142 may confer a big advantage to hybrid seed production.

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.