Identification and characterization of the WOX Gene Family revealed two WUS Clade Members associated with embryo development in Cunninghamia lanceolata.

The WUSCHEL-related homeobox (WOX) gene family is vital for plant development and stress response. In this study, we conducted a comprehensive analysis of WOX genes in Cunninghamia lanceolata (C. lanceolata) and subsequently explored the potential roles of two ClWOX genes within the WUS clade. In total, six ClWOX genes were identified through a full-length transcriptome analysis. These genes, exhibiting conserved structural and functional motifs, were assigned to the ancient clade and Modern/WUS clade, respectively, through a phylogenetic analysis. Our expression analysis indicated that these ClWOX genes were highly expressed in the middle and late developmental stages of zygotic embryos in C. lanceolata. Moreover, only ClWOX5 and ClWOX6 within the Modern/WUS clade exhibited transcriptional activity, and their expressions were also induced in response to auxin and wounding. Overexpression of ClWOX5 and ClWOX6 in Arabidopsis caused a partially sterile phenotype, resulting in a very low seed setting rate. Transcriptomic analysis revealed that expressions of many embryo-defective (EMB) genes, phytohormone-related genes, and transcription factors (TFs) were dramatically altered in ClWOX5 and ClWOX6 transgenic plants, which suggested that ClWOX5 and ClWOX6 may play specific important roles in embryo development via complex gene networks. In addition, overexpression of ClWOX5 and ClWOX6 in leaf segments promoted shoot regeneration in tobacco, indicating that ClWOX5 and ClWOX6 can promote plant regeneration and could be used to improve genetic transformation. In conclusion, these results help to elucidate the function of the WOX gene and provide a valuable basis for future studies of the developmental regulation and applications of WOX genes in C. lanceolata.

AC-UNet: an improved UNet-based method for stem and leaf segmentation in Betula luminifera.

Plant phenotypic traits play an important role in understanding plant growth dynamics and complex genetic traits. In phenotyping, the segmentation of plant organs, such as leaves and stems, helps in automatically monitoring growth and improving screening efficiency for large-scale genetic breeding. In this paper, we propose an AC-UNet stem and leaf segmentation algorithm based on an improved UNet. This algorithm aims to address the issues of feature edge information loss and sample breakage in the segmentation of plant organs, specifically in Betula luminifera. The method replaces the backbone feature extraction network of UNet with VGG16 to reduce the redundancy of network information. It adds a multi-scale mechanism in the splicing part, an optimized hollow space pyramid pooling module, and a cross-attention mechanism in the expanding network part at the output end to obtain deeper feature information. Additionally, Dice_Boundary is introduced as a loss function in the back-end of the algorithm to circumvent the sample distribution imbalance problem. The PSPNet model achieves mIoU of 58.76%, mPA of 73.24%, and Precision of 66.90%, the DeepLabV3 model achieves mIoU of 82.13%, mPA of 91.47%, and Precision of 87.73%, on the data set. The traditional UNet model achieves mIoU of 84.45%, mPA of 91.11%, and Precision of 90.63%, and the Swin-UNet model achieves . The mIoU is 79.02%, mPA is 85.99%, and Precision is 88.73%. The AC-UNet proposed in this article achieved excellent performance on the Swin-UNet dataset, with mIoU, mPA, and Precision of 87.50%, 92.71%, and 93.69% respectively, which are better than the selected PSPNet, DeepLabV3, traditional UNet, and Swin-UNet. Commonly used semantic segmentation algorithms. Experiments show that the algorithm in this paper can not only achieve efficient segmentation of the stem and leaves of Betula luminifera but also outperforms the existing state-of-the-art algorithms in terms of both speed. This can provide more accurate auxiliary support for the subsequent acquisition of plant phenotypic traits.

A CNN-LSTM-att hybrid model for classification and evaluation of growth status under drought and heat stress in chinese fir (Cunninghamia lanceolata).

BACKGROUND: Cunninghamia lanceolata (Chinese fir), is one of the most important timber trees in China. With the global warming, to develop new resistant varieties to drought or heat stress has become an essential task for breeders of Chinese fir. However, classification and evaluation of growth status of Chinese fir under drought or heat stress are still labor-intensive and time-consuming. RESULTS: In this study, we proposed a CNN-LSTM-att hybrid model for classification of growth status of Chinese fir seedlings under drought and heat stress, respectively. Two RGB image datasets of Chinese fir seedling under drought and heat stress were generated for the first time, and utilized in this study. By comparing four base CNN models with LSTM, the Resnet50-LSTM was identified as the best model in classification of growth status, and LSTM would dramatically improve the classification performance. Moreover, attention mechanism further enhanced performance of Resnet50-LSTM, which was verified by Grad-CAM. By applying the established Resnet50-LSTM-att model, the accuracy rate and recall rate of classification was up to 96.91% and 96.79% for dataset of heat stress, and 96.05% and 95.88% for dataset of drought, respectively. Accordingly, the R2 value and RMSE value for evaluation on growth status under heat stress were 0.957 and 0.067, respectively. And, the R2 value and RMSE value for evaluation on growth status under drought were 0.944 and 0.076, respectively. CONCLUSION: In summary, our proposed model provides an important tool for stress phenotyping in Chinese fir, which will be a great help for selection and breeding new resistant varieties in future.

The complete chloroplast genome of Rhododendron huadingense (Ericaceae).

Rhododendron huadingense is an important horticultural plant that belongs to the Ericaceae family. In this study, the chloroplast genome sequence of R. huadingense is reported. The chloroplast genome of R. huadingense was 198,952 bp in length and had an angiosperm-typical quadripartite structure with a large single-copy (LSC) region of 108,557 bp, a small single-copy (SSC) region of 53 bp, and two inverted repeat regions (IRs) of 45,171 bp. One hundred and thirteen unique genes including 79 protein-coding genes, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes were identified in the chloroplast genome. Further phylogenetic analysis revealed a close relationship between R. huadingense and R. molle. The complete chloroplast genome of R. huadingense provides valuable genetic information for the phylogeny, varieties breeding and sustainable utilization of this species.

Genome-wide identification and characterization of OVATE family proteins in Betula luminifera reveals involvement of BlOFP3 and BlOFP5 genes in leaf development.

Ovate family proteins (OFP) are plant-specific transcription factors involved in regulating morphologies of the lateral organs, plant growth and development. However, the functional roles of OFP genes in Betula luminifera, an important timber tree species, are not well studied. In this study, we identified 20 BlOFP genes and analyzed their phylogenetic relationship, gene structure, conserved motifs, and cis-elements. Further, expression analysis indicates that BlOFP genes were up-regulated in leaves on the one-year-old branch compared to leaves on the current-year branch and bract, except BlOFP7, BlOFP11, BlOFP14 and BlOFP12. The overexpression of BlOFP3 and BlOFP5 in Arabidopsis thaliana not only resulted in a slower growth rate but also produced sawtooth shape, flatter and darker green rosette leaves. Further investigation showed that the leaf thickness of the transgenic plants was more than double that of the wild type, which was caused by the increasement in the number and size of palisade tissue cells. Furthermore, the expression analysis also indicated that the expressions of several genes related to leaf development were significantly changed in the transgene plants. These results suggested the significant roles of BlOFP3 and BlOFP5 in leaf development. Moreover, protein-protein interaction studies showed that BlOFP3 interacts with BlKNAT5, and BlOFP5 interacts with BlKNAT5, BlBLH6 and BlBLH7. In conclusion, our study demonstrates that BlOFP3 and BlOFP5 were involved in leaf shape and thickness regulation by forming a complex with BlKNAT5, BlBLH6 and BlBLH7. In addition, our study serves as a guide for future functional genomic studies of OFP genes of the B. luminifera.

Comparative physiological analyses and the genetic basis reveal heat stress responses mechanism among different Betula luminifera populations.

Betula luminifera is a subtropical fast-growing timber species with high economic value. However, along with global warming, heat stress become one of the main environmental variables that limit the productivity of B. luminifera, and the response of diverse geographic populations to high temperatures is still unclear. In order to offer a comprehensive understanding of the behavior of B. luminifera under heat stress, the physiological responses of six B. luminifera populations (across the core distribution area) were described in this work in an integrated viewpoint. The results showed that a multi-level physiological regulatory network may exist in B. luminifera, the first response was the activity of resistant enzymes [e.g., peroxidase (POD)] at a preliminary stage of 2 h heat stress, and then the proline (osmoregulation substance) content began to increase after 24 h of continuous high-temperature treatment. In addition, photosynthesis was stronlgly affected by heat stress, and the net photosynthetic rate (Pn ) showed a downward trend under heat treatment in all six B. luminifera populations. Interestingly, although the physiological change patterns of the six B. luminifera populations were relatively consistent for the same parameter, there were obvious differences among different populations. Comprehensive analysis revealed that the physiological response of Rongshui (RS) was the most stable, and this was the representative B. luminifera population. Illumina RNA-seq analysis was applied to reveal the specific biological process of B. luminifera under heat stress using the RS population, and a total of 116,484 unigenes were obtained. The differentially expressed genes (DEGs) between different time periods under heat stress were enriched in 34 KEGG pathways, and the limonene and pinene degradation pathway was commonly enriched in all pairwise comparisons. Moreover, transcription factors including bHLH (basic helix-loop-helix), MYB, WRKY, and NAC (NAM, ATAF1/2, and CUC2) were identified. In this study, the physiological response and tolerance mechanisms of B. luminifera under high temperature stress were revealed, which can conducive to the basis of B. luminifera selection and resistance assessment for cultivation and breeding.

The chromosome-scale genome of Phoebe bournei reveals contrasting fates of terpene synthase (TPS)-a and TPS-b subfamilies.

Terpenoids, including aromatic volatile monoterpenoids and sesquiterpenoids, function in defense against pathogens and herbivores. Phoebe trees are remarkable for their scented wood and decay resistance. Unlike other Lauraceae species investigated to date, Phoebe species predominantly accumulate sesquiterpenoids instead of monoterpenoids. Limited genomic data restrict the elucidation of terpenoid variation and functions. Here, we present a chromosome-scale genome assembly of a Lauraceae tree, Phoebe bournei, and identify 72 full-length terpene synthase (TPS) genes. Genome-level comparison shows pervasive lineage-specific duplication and contraction of TPS subfamilies, which have contributed to the extreme terpenoid variation within Lauraceae species. Although the TPS-a and TPS-b subfamilies were both expanded via tandem duplication in P. bournei, more TPS-a copies were retained and constitutively expressed, whereas more TPS-b copies were lost. The TPS-a genes on chromosome 8 functionally diverged to synthesize eight highly accumulated sesquiterpenes in P. bournei. The essential oil of P. bournei and its main component, ß-caryophyllene, exhibited antifungal activities against the three most widespread canker pathogens of trees. The TPS-a and TPS-b subfamilies have experienced contrasting fates over the evolution of P. bournei. The abundant sesquiterpenoids produced by TPS-a proteins contribute to the excellent pathogen resistance of P. bournei trees. Overall, this study sheds light on the evolution and adaptation of terpenoids in Lauraceae and provides valuable resources for boosting plant immunity against pathogens in various trees and crops.

Full-Length Transcriptome Sequencing and Comparative Transcriptomic Analyses Provide Comprehensive Insight Into Molecular Mechanisms of Cellulose and Lignin Biosynthesis in Cunninghamia lanceolata.

Cunninghamia lanceolata is an essential timber species that provide 20%-30% raw materials for China's timber industry. Although a few transcriptomes have been published in C. lanceolata, full-length mRNA transcripts and regulatory mechanisms behind the cellulose and lignin biosynthesis have not been thoroughly investigated. Here, PacBio Iso-seq and RNA-seq analyses were adapted to identify the full-length and differentially expressed transcripts along a developmental gradient from apex to base of C. lanceolata shoots. A total of 48,846 high-quality full-length transcripts were obtained, of which 88.0% are completed transcriptome based on benchmarking universal single-copy orthologs (BUSCO) assessment. Along stem developmental gradient, 18,714 differentially expressed genes (DEGs) were detected. Further, 28 and 125 DEGs were identified as enzyme-coding genes of cellulose and lignin biosynthesis, respectively. Moreover, 57 transcription factors (TFs), including MYB and NAC, were identified to be involved in the regulatory network of cellulose and lignin biosynthesis through weighted gene co-expression network analysis (WGCNA). These TFs are composed of a comparable regulatory network of secondary cell wall formation in angiosperms, revealing a similar mechanism may exist in gymnosperms. Further, through qRT-PCR, we also investigated eight specific TFs involved in compression wood formation. Our findings provide a comprehensive and valuable source for molecular genetics breeding of C. lanceolata and will be beneficial for molecular-assisted selection.

Full-length transcriptomic identification of R2R3-MYB family genes related to secondary cell wall development in Cunninghamia lanceolata (Chinese fir).

BACKGROUND: R2R3-MYB is a class of transcription factor crucial in regulating secondary cell wall development during wood formation. The regulation of wood formation in gymnosperm has been understudied due to its large genome size. Using Single-Molecule Real-Time sequencing, we obtained full-length transcriptomic libraries from the developmental stem of Cunninghamia lanceolata, a perennial conifer known as Chinese fir. The R2R3-MYB of C. lanceolata (hereafter named as ClMYB) associated with secondary wall development were identified based on phylogenetic analysis, expression studies and functional study on transgenic line. RESULTS: The evolutionary relationship of 52 ClMYBs with those from Arabidopsis thaliana, Eucalyptus grandis, Populus trichocarpa, Oryza sativa, two gymnosperm species, Pinus taeda, and Picea glauca were established by neighbour-joining phylogenetic analysis. A large number of ClMYBs resided in the woody-expanded subgroups that predominated with the members from woody dicots. In contrast, the woody-preferential subgroup strictly carrying the members of woody dicots contained only one candidate. The results suggest that the woody-expanded subgroup emerges before the gymnosperm/angiosperm split, while most of the woody-preferential subgroups are likely lineage-specific to woody dicots. Nine candidates shared the same subgroups with the A. thaliana orthologs, with known function in regulating secondary wall development. Gene expression analysis inferred that ClMYB1/2/3/4/5/26/27/49/51 might participate in secondary wall development, among which ClMYB1/2/5/26/27/49 were significantly upregulated in the highly lignified compression wood region, reinforcing their regulatory role associated with secondary wall development. ClMYB1 was experimentally proven a transcriptional activator that localised in the nucleus. The overexpression of ClMYB1 in Nicotiana benthamiana resulted in an increased lignin deposition in the stems. The members of subgroup S4, ClMYB3/4/5 shared the ERF-associated amphiphilic repression motif with AtMYB4, which is known to repress the metabolism of phenylpropanoid derived compounds. They also carried a core motif specific to gymnosperm lineage, suggesting divergence of the regulatory process compared to the angiosperms. CONCLUSIONS: This work will enrich the collection of full-length gymnosperm-specific R2R3-MYBs related to stem development and contribute to understanding their evolutionary relationship with angiosperm species.

Colchicine-Induced Polyploidy in Rhododendron fortunei Lindl.

Polyploidy in Rhododendron fortunei has great potential to improve its horticultural and commercial value, and to also meet market demands. In this study, a feasible method for polyploid induction in R. fortunei via colchicine treatment was established, and the obtained polyploid plants were identified and characterized. As a result, the stem bases of tissue-cultured plantlets treated with 0.1% colchicine for 24 h showed the highest polyploid induction with a rate of 36.67%. By flow cytometric analysis, 69 tetraploids and 29 octoploids were identified in the regenerated plants that were examined. Phenotypic analysis indicated that the leaves of tetraploid and octoploid plants were smaller, rounder and thicker with more abundant and longer epidermal hairs than those of diploids. Furthermore, the stomata of polyploids were larger and sparser than those of diploids. An increase in chlorophyll content was also detected in polyploids, which resulted in darker green leaves. In conclusion, our study established an effective method to induce polyploidy in R. fortunei, which could be used to develop new genetic resources for breeding R. fortunei and other Rhododendron species in the future.

Genome survey of Chinese fir (Cunninghamia lanceolata): Identification of genomic SSRs and demonstration of their utility in genetic diversity analysis.

Chinese fir (Cunninghamia lanceolata) is an important coniferous species that accounts for 20-30% of the total commercial timber production in China. Though traditional breeding of Chinese fir has achieved remarkable success, molecular-assisted breeding has made little progress due to limited availability of genomic information. In this study, a survey of Chinese fir genome was performed using the Illumina HiSeq Xten sequencing platform. K-mer analysis indicated that Chinese fir has a large genome of approximately 11.6 Gb with 74.89% repetitive elements and is highly heterozygous. Meanwhile, its genome size was estimated to be 13.2 Gb using flow cytometry. A total of 778.02 Gb clean reads were assembled into 10,982,272 scaffolds with an N50 of 1.57 kb. In total, 362,193 SSR loci were detected with a frequency of 13.18 kb. Dinucleotide repeats were the most abundant (up to 73.6% of the total SSRs), followed by trinucleotide and tetranucleotide repeats. Forty-six polymorphic pairs were developed, and 298 alleles were successfully amplified from 199 Chinese fir clones. The average PIC value was 0.53, indicating that the identified genomic SSR (gSSR) markers have a high degree of polymorphism. In addition, these breeding resources were divided into three groups, and a limited gene flow existed among these inferred groups.

RNA-Seq analysis of differential gene expression in Betula luminifera xylem during the early stages of tension wood formation.

BACKGROUND: Betula luminifera H. Winkler, which is widely distributed in southern China, is an economically important broadleaf tree species. However, little genomic information of B. luminifera is available, and little is known about the molecular mechanisms of wood formation in this species. Meanwhile, few efforts have focused on investigating the early transcriptional changes during tension wood formation in woody plants. RESULTS: A reference transcriptome dataset was first generated containing 45,700 Unigenes, and 35,135 (76.9%) Unigenes were annotated by a BLAST similarity search against four public databases. Then, based on an anatomical investigation, the global gene expression changes during the early stages of tension wood formation were analyzed. Gene expression profiling showed that a total of 13,273 Unigenes were differentially regulated during the early stages of tension wood formation. Most genes involved in cellulose and lignin biosynthesis were highlighted to reveal their biological importance in tension wood formation. In addition, the transcription levels of many genes involved in the auxin response pathway were significantly changed during the early stages of tension wood formation. Furthermore, 18 TFs co-expressed with key enzymes of cellulose synthesis were identified. CONCLUSIONS: Our results revealed the transcriptional changes associated with TW formation and identified potential key genes in the regulation of this process. These results will help to dissect the molecular mechanism of wood formation and provide key candidate genes for marker-assisted selection in B. luminifera.

Molecular Characterization of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) Gene Family in Betula luminifera.

As a major family of plant-specific transcription factors, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes play vital regulatory roles in plant growth, development and stress responses. In this study, 18 SPL genes were identified and cloned from Betula luminifera. Two zinc finger-like structures and a nuclear location signal (NLS) segments were existed in the SBP domains of all BlSPLs. Phylogenetic analysis showed that these genes were clustered into nine groups (group I-IX). The intron/exon structure and motif composition were highly conserved within the same group. 12 of the 18 BlSPLs were experimentally verified as the targets of miR156, and two cleavage sites were detected in these miR156-targeted BlSPL genes. Many putative cis-elements, associated with light, stresses and phytohormones response, were identified in the promoter regions of BlSPLs, suggesting that BlSPL genes are probably involved in important physiological processes and developmental events. Tissue-specific expression analysis showed that miR156-targeted BlSPLs exhibited a more differential expression pattern, while most miR156-nontargeted BlSPLs tended to be constitutively expressed, suggesting the distinct roles of miR156-targeted and nontargeted BlSPLs in development and growth of B. luminifera. Further expression analysis revealed that miR156-targeted BlSPLs were dramatically up-regulated with age, whereas mature BlmiR156 level was apparently declined with age, indicating that miR156/SPL module plays important roles in vegetative phase change of B. luminifera. Moreover, yeast two-hybrid assay indicated that several miR156-targeted and nontargeted BlSPLs could interact with two DELLA proteins (BlRGA and BlRGL), which suggests that certain BlSPLs take part in the GA regulated processes through protein interaction with DELLA proteins. All these results provide an important basis for further exploring the biological functions of BlSPLs in B. luminifera.

De novo characterization of the Chinese fir (Cunninghamia lanceolata) transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis.

BACKGROUND: Chinese fir (Cunninghamia lanceolata) is an important timber species that accounts for 20-30% of the total commercial timber production in China. However, the available genomic information of Chinese fir is limited, and this severely encumbers functional genomic analysis and molecular breeding in Chinese fir. Recently, major advances in transcriptome sequencing have provided fast and cost-effective approaches to generate large expression datasets that have proven to be powerful tools to profile the transcriptomes of non-model organisms with undetermined genomes. RESULTS: In this study, the transcriptomes of nine tissues from Chinese fir were analyzed using the Illumina HiSeq™ 2000 sequencing platform. Approximately 40 million paired-end reads were obtained, generating 3.62 gigabase pairs of sequencing data. These reads were assembled into 83,248 unique sequences (i.e. Unigenes) with an average length of 449 bp, amounting to 37.40 Mb. A total of 73,779 Unigenes were supported by more than 5 reads, 42,663 (57.83%) had homologs in the NCBI non-redundant and Swiss-Prot protein databases, corresponding to 27,224 unique protein entries. Of these Unigenes, 16,750 were assigned to Gene Ontology classes, and 14,877 were clustered into orthologous groups. A total of 21,689 (29.40%) were mapped to 119 pathways by BLAST comparison against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The majority of the genes encoding the enzymes in the biosynthetic pathways of cellulose and lignin were identified in the Unigene dataset by targeted searches of their annotations. And a number of candidate Chinese fir genes in the two metabolic pathways were discovered firstly. Eighteen genes related to cellulose and lignin biosynthesis were cloned for experimental validating of transcriptome data. Overall 49 Unigenes, covering different regions of these selected genes, were found by alignment. Their expression patterns in different tissues were analyzed by qRT-PCR to explore their putative functions. CONCLUSIONS: A substantial fraction of transcript sequences was obtained from the deep sequencing of Chinese fir. The assembled Unigene dataset was used to discover candidate genes of cellulose and lignin biosynthesis. This transcriptome dataset will provide a comprehensive sequence resource for molecular genetics research of C. lanceolata.

Hypocellular acute myeloid leukemia in adults: analysis of the clinical outcome of 123 patients.

BACKGROUND: The hypocellular variant of acute myeloid leukemia accounts for less than 10% of all cases of adult acute myeloid leukemia. It is defined by having less than 20 percent of cellular bone marrow in a biopsy at presentation. It is unclear in the literature whether the outcome of hypocellular acute myeloid leukemia differs from that of non-hypocellular acute myeloid leukemia. DESIGN AND METHODS: We retrospectively analyzed all the cases reported to be hypocellular acute myeloid leukemia between 2000 and 2009. A second pathology review was conducted and the diagnosis was confirmed in all cases. RESULTS: One hundred twenty-three (9%) patients were identified: patients with hypocellular acute myeloid leukemia were older than those with non-hypocellular acute myeloid leukemia (P=0.009) and more frequently presented with cytopenias (P<0.001). Forty-one patients with hypocellular acute myeloid leukemia had an antecedent hematologic disorder and 11 patients had received prior chemo-radiotherapy for non-hematopoietic neoplasms. On multivariate analysis, overall survival, remission duration and event-free survival were comparable to those of other patients with acute myeloid leukemia. CONCLUSIONS: The outcome of hypocellular acute myeloid leukemia does not differ from that of non-hypocellular acute myeloid leukemia.

Bcl-2 suppresses hydrogen peroxide-induced programmed cell death via OsVPE2 and OsVPE3, but not via OsVPE1 and OsVPE4, in rice.

Hydrogen peroxide (H(2)O(2)) is known to be a key player in apoptosis in animals. The components and pathways regulating H(2)O(2)-induced programmed cell death in plants, however, remain largely unknown. In the present study, rice transgenic lines overexpressing Bcl-2, a human apoptotic suppressor, were obtained. These transgenic lines showed increased tolerance to high levels of H(2)O(2), resulting in increased seed germination rates, root elongation, root tip cell viability and chlorophyll retention compared to control lines. In the control lines, treatment with H(2)O(2) resulted in DNA laddering and a clear terminal transferase dUTP nick end labeling signal, which are the hallmarks of programmed cell death. However, this effect was not detected in the Bcl-2-overexpressing transgenic lines. Further investigations indicated that Bcl-2 suppressed H(2)O(2)-induced programmed cell death but did not inhibit stress-elicited reactive oxygen species production in rice. RT-PCR revealed that the expression of the two vacuolar processing enzyme genes (i.e. OsVPE2 and OsVPE3) was dramatically induced by H(2)O(2) in the wild-type line but not in the Bcl-2-overexpressing line. Moreover, treatment with H(2)O(2) resulted in the disruption of the vacuolar membrane in the wild-type line. The expression levels of OsVPE1 and OsVPE4 did not significantly differ between the wild-type line and the transgenic line that was treated or untreated with H(2)O(2). The similar roles of Bcl-2 and OsVPEs during endogenous reactive oxygen species-triggered programmed cell death were also confirmed by NaCl stress in rice. To our knowledge, the present study is the first to demonsatrate that Bcl-2 overexpression inhibits H(2)O(2)-induced programmed cell death and enhances H(2)O(2) tolerance. We propose that Bcl-2 overexpression in rice suppresses the transcriptional activation of OsVPE2 and OsVPE3, but not of OsVPE1 or OsVPE4.

Identification of genes related to the development of bamboo rhizome bud.

Bamboo (Phyllostachys praecox) is one of the largest members of the grass family Poaceae, and is one of the most economically important crops in Asia. However, complete knowledge of bamboo development and its molecular mechanisms is still lacking. In the present study, the differences in anatomical structure among rhizome buds, rhizome shoots, and bamboo shoots were compared, and several genes related to the development of the bamboo rhizome bud were identified. The rice cross-species microarray hybridization showed a total of 318 up-regulated and 339 down-regulated genes, including those involved in regulation and signalling, metabolism, and stress, and also cell wall-related genes, in the bamboo rhizome buds versus the leaves. By referring to the functional dissection of the homologous genes from Arabidopsis and rice, the putative functions of the 52 up-regulated genes in the bamboo rhizome bud were described. Six genes related to the development of the bamboo rhizome bud were further cloned and sequenced. These show 66-90% nucleotide identity and 68-98% amino acid identity with the homologous rice genes. The expression patterns of these genes revealed significant differences in rhizome shoots, rhizome buds, bamboo shoots, leaves, and young florets. Furthermore, in situ hybridization showed that the PpRLK1 gene is expressed in the procambium and is closely related to meristem development of bamboo shoots. The PpHB1 gene is expressed at the tips of bamboo shoots and procambium, and is closely related to rhizome bud formation and procambial development. To our knowledge, this is the first report that uses rice cross-species hybridization to identify genes related to bamboo rhizome bud development, and thereby contributes to the further understanding of the molecular mechanism involved in bamboo rhizome bud development.

Identification and characterization of two bamboo (Phyllostachys praecox) AP1/SQUA-like MADS-box genes during floral transition.

Bamboo (Bambusoideae) is by far the largest member of the grass family Poaceae, which is vital to the economy of many countries in the tropics and subtropics. However, the mechanism of flowering of bamboo (Phyllostachys praecox) is still unknown. In this study, we isolated two novel genes from P. praecox and evaluated their functional characteristics. The sequence and phylogenetic analysis indicated that these two genes, named PpMADS1 and PpMADS2, belong to FUL3 and FUL1 clade of Poaceae AP1/SQUA-like genes, respectively. The PpMADS2 possesses a truncated C terminus lacking the highly conserved paleoAP1 motif. It was further confirmed that the truncated C-terminal region was produced by natural sequence deletion in exons, but not by alternative splicing. Ectopic expression of PpMADS1 and PpMADS2 significantly promoted early flowering through upregulation of AP1 in Arabidopsis. Yeast two-hybrid experiments demonstrated that AP1 protein can interact with PpMADS1 but not PpMADS2, suggesting that these two genes may act differently in signaling early flowering of bamboo plants. RT-qPCR and in situ hybridization analysis revealed distinct expression patterns of these two genes in vegetative and reproductive tissues of bamboo. Taken together, our results suggest that both PpMADS1 and PpMADS2 are involved in floral transition, and PpMADS2 might play more important roles than PpMADS1 in floral development of Phyllostachys praecox.

Molecular cloning, expression analyses and primary evolution studies of REV- and TB1-like genes in bamboo.

Most cultured bamboos are perennial woody evergreens that reproduce from rhizomes. It is unclear why some rhizome buds develop into aerial bamboo shoots instead of new rhizomes. REVOLUTA (REV)-like Class III homeodomain leucine-zipper (HD-Zip) proteins and TEOSINTE BRANCHED1 (TB1)-like transcription factors have been shown to play regulatory roles in meristem initiation and outgrowth. We cloned and analyzed the bamboo (Phyllostachys praecox C.D. Chu & C.S. Chao.) REV- (PpHB1) and TB1-like (PpTB1) gene. Gene expression was mainly detected by in situ hybridization. PpHB1 expression was detected in the tips of lateral buds, on the adaxial portion of the leaf and within the developing procambium, indicating its close correlation to rhizome bud formation and procambial development. PpTB1 expression was mainly detected on the top of buds at later developmental stages, suggesting it was more likely involved in bud outgrowth. Meristem genes might therefore serve as specific molecular markers of rhizome bud development and could be useful in studies designed to elucidate the mechanisms underlying bamboo shoot development. In addition, meristem genes such as TB1-like sequences may be useful in phylogenetic analyses of bamboo species.