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Yuan L.,University of California at Davis | Liu Z.,University of California at Davis | Liu Z.,Zhejiang University | Liu Z.,Laboratory of Horticultural Plant Growth and Quality Regulation | And 5 more authors.
Developmental Cell

Since the discovery of double fertilization, it has been recognized that flowering plants produce two highly dimorphic female gametes, the egg cell and central cell. These give rise, respectively, to the embryo and the endosperm, a nourishing tissue unique to flowering plants. Here we show that in Arabidopsis, endosperm formation requires the CYTOKININ INDEPENDENT 1 (CKI1) histidine kinase, an activator of the cytokinin signaling pathway, which specifies central cells and restricts egg cell fate. Dimorphism of the two adjacent gametes is mechanistically established in the syncytial embryo sac by spatially restricted CKI1 expression, followed by translocation of ER-localized CKI1 protein via nuclear migration. Cell specification by CKI1 likely involves activation of the cytokinin signaling pathway mediated by histidine phosphotransferases. Ectopic CKI1 expression generates non-propagating seeds with dual fertilized endosperms and no embryos. We conclude that CKI1-directed specification of the endosperm precursor central cell results in seeds containing an embryo and an endosperm. © 2016 Elsevier Inc. Source

Yu X.,Zhejiang University | Yu X.,Laboratory of Horticultural Plant Growth and Quality Regulation | Liu Y.,Zhejiang University | Liu Y.,Laboratory of Horticultural Plant Growth and Quality Regulation | And 10 more authors.
Molecular Biology Reports

To establish a simple and rapid method for isolating mitochondrial DNA (mtDNA) from Brassica vegetables, the effects of different factors on mtDNA extraction were investigated firstly. A new protocol includes five steps: organelle isolation, deoxyribonuclease treatment, lysis, RNase treatment, and deproteinization. Results indicate that a 15 min-lysis time can achieve higher mtDNA yields from etiolated seedlings. Moreover, it is found that the inflorescence of the cytoplasmic male sterile (CMS) line is unfit for the isolation of mtDNA. The mtDNA isolated using this method is intact and pure, and can be used for further molecular analysis. Subsequently, the genomic and transcriptional differences of atps and coxs genes on the mitochondria between the petaloid-type CMS line and its maintainer line have been identified. RFLP analysis revealed that out of the five atps and three coxs genes, except of atp4 and cox3 , the others mtDNA protein coding genes exhibited polymorphisms, respectively. This results suggest that atps and coxs genes are located in a long mtDNA fragment, and the mtDNA evolves rapidly in structure between the CMS line and its maintainer line in tuber muster. Northern blot analysis showed that the expression level of these genes in flower bud is higher than that of leaf and flower, and that, alternative splicing have been found among the atp6, atp8 and cox3 genes, respectively. Our results modified a efficient protocol for isolating the mtDNA, and provided some novel molecular markers indicating the CMS trait in tuber mustard. The comparative analysis presented in this study allows a more comprehensive understanding of the molecular mechanism on CMS in Brassica crops. © Springer Science+Business Media 2014. Source

Liu Z.,Zhejiang University | Liu Z.,Laboratory of Horticultural Plant Growth and Quality Regulation | Zhang M.,Zhejiang University | Zhang M.,Laboratory of Horticultural Plant Growth and Quality Regulation | And 12 more authors.
DNA Research

In plants, a two component system (TCS) composed of sensor histidine kinases (HKs), histidine phosphotransfer proteins (HPs), and response regulators (RRs) has been employed in cytokinin signal transduction. ATCS exhibits important functions in diverse biological processes, including plant growth, development, and response to environmental stimuli. Conducting an exhaustive search of the Chinese cabbage genome, a total of 20 HK(L) (11 HKs and 9 HKLs), 8 HP (7 authentic and 1 pseudo), and 57 RR (21 Type-A, 17 Type-B, 4 Type-C, and 15 pseudo) proteins were identified. The structures, conserved domains, and phylogenetic relationships of these protein-coding geneswere analysed in detail. The duplications, evolutionary patterns, and divergence of the TCS genes were investigated. The transcription levels of TCS genes in various tissues, organs, and developmental stages were further analysed to obtain information of the functions of these genes. Cytokinin-related binding elements were found in the putative promoter regions of Type-A BrRR genes. Furthermore, gene expression patterns to adverse environmental stresses (drought and high salinity) and exogenous phytohormones (tZ and ABA)were investigated. Numerous stress-responsive candidate genes were obtained. Our systematic analyses provided insights into the characterization of the TCS genes in Chinese cabbage and basis for further functional studies of such genes. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. Source

Liu Z.,Zhejiang University | Liu Z.,Laboratory of Horticultural Plant Growth and Quality Regulation | Kong L.,Zhejiang University | Kong L.,Laboratory of Horticultural Plant Growth and Quality Regulation | And 14 more authors.

The AP2/ERF transcription factor family is one of the largest families involved in growth and development, hormone responses, and biotic or abiotic stress responses in plants. In this study, 281 AP2/ERF transcription factor unigenes were identified in Chinese cabbage. These superfamily members were classified into three families (AP2, ERF, and RAV). The ERF family was subdivided into the DREB subfamily and the ERF subfamily with 13 groups (I- XI) based on sequence similarity. Duplication, evolution and divergence of the AP2/ERF genes in B. rapa and Arabidopsis thaliana were investigated and estimated. Cytokinin response factors (CRFs), as a subclade of the AP2/ERF family, are important transcription factors that define a branch point in the cytokinin two-component signal (TCS) transduction pathway. Up to 21 CRFs with a conserved CRF domain were retrieved and designated as BrCRFs. The amino acid sequences, conserved regions and motifs, phylogenetic relationships, and promoter regions of the 21 BrCRFs were analyzed in detail. The BrCRFs broadly expressed in various tissues and organs. The transcripts of BrCRFs were regulated by factors such as drought, high salinity, and exogenous 6-BA, NAA, and ABA, suggesting their involvement in abiotic stress conditions and regulatory mechanisms of plant hormone homeostasis. These results provide new insight into the divergence, variation, and evolution of AP2/ERF genes at the genome-level in Chinese cabbage. © 2013 Liu et al. Source

Liu Z.,Zhejiang University | Liu Z.,Laboratory of Horticultural Plant Growth and Quality Regulation | Lv Y.,Zhejiang University | Lv Y.,Laboratory of Horticultural Plant Growth and Quality Regulation | And 14 more authors.
BMC Genomics

Background: Cytokinins (CKs) have significant roles in various aspects of plant growth and development, and they are also involved in plant stress adaptations. The fine-tuning of the controlled CK levels in individual tissues, cells, and organelles is properly maintained by isopentenyl transferases (IPTs) and cytokinin oxidase/dehydrogenases (CKXs). Chinese cabbage is one of the most economically important vegetable crops worldwide. The whole genome sequencing of Brassica rapa enables us to perform the genome-wide identification and functional analysis of the IPT and CKX gene families.Results: In this study, a total of 13 BrIPT genes and 12 BrCKX genes were identified. The gene structures, conserved domains and phylogenetic relationships were analyzed. The isoelectric point, subcellular localization and glycosylation sites of the proteins were predicted. Segmental duplicates were found in both BrIPT and BrCKX gene families. We also analyzed evolutionary patterns and divergence of the IPT and CKX genes in the Cruciferae family. The transcription levels of BrIPT and BrCKX genes were analyzed to obtain an initial picture of the functions of these genes. Abiotic stress elements related to adverse environmental stimuli were found in the promoter regions of BrIPT and BrCKX genes and they were confirmed to respond to drought and high salinity conditions. The effects of 6-BA and ABA on the expressions of BrIPT and BrCKX genes were also investigated.Conclusions: The expansion of BrIPT and BrCKX genes after speciation from Arabidopsis thaliana is mainly attributed to segmental duplication events during the whole genome triplication (WGT) and substantial duplicated genes are lost during the long evolutionary history. Genes produced by segmental duplication events have changed their expression patterns or may adopted new functions and thus are obtained. BrIPT and BrCKX genes respond well to drought and high salinity stresses, and their transcripts are affected by exogenous hormones, such as 6-BA and ABA, suggesting their potential roles in abiotic stress conditions and regulatory mechanisms of plant hormone homeostasis. The appropriate modulation of endogenous CKs levels by IPT and CKX genes is a promising approach for developing economically important high-yielding and high-quality stress-tolerant crops in agriculture. © 2013 Liu et al.; licensee BioMed Central Ltd. Source

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