Yingdian Wang

Subcellular Distribution of NTL Transcription Factors in Arabidopsis thaliana.

NAC with a transmembrane (TM) motif1‐like (NTL) transcription factors, containing three regions: the N‐terminal NAC domain (ND), the middle regulation region (RR), and the C‐terminal TM domain, belong to the tail‐anchored proteins. Although these NTLs play numerous essential roles in plants, their subcellular distribution and the mechanism of translocation into the nucleus (NU) remain unclear. In this study, we found that most of the full‐length NTLs were localized in the endoplasmic reticulum (ER), with the exception of NTL11 and NTL5, which were restricted to the NU. Furthermore, we found that NTL11 contains a TM domain, whereas NTL5 does not. The ND of all of the NTLs was responsible for nuclear localization in plants. After truncation of the TM domain, NTL8_NR, NTL10_NR and NTL13_NR localized in the cytoplasm (CT) and NU, and other NTL_NRs were only localized in the NU, suggesting that the RR of NTL8, NTL10 and NTL13 contains some inhibitory region to mask the nuclear localization signal sequence in the ND domain and permit their diffusion between CT and NU. Furthermore, the N‐terminus of NTL11 was translocated to the NU, but the C‐terminus was degraded in Arabidopsis mesophyll protoplasts. The chimeric construct of NTL11_ND with NTL10_RR and TM domain (11ND‐10RT) was localized exclusively in the ER, and not in the NU. However, 10ND‐11RT was found mainly in the NU. Our results indicated that the TM domain is essential for NTL targeting the ER and the N‐terminal fragment, including ND and RR, is translocated into the NU after activation through proteolytic cleavage events upon stimulation by internal and external environmental signals.

Four AUXIN RESPONSE FACTOR genes downregulated by microRNA167 are associated with growth and development in Oryza sativa

MicroRNA167 (miR167), as a conserved miRNA, has been implicated in auxin signalling by regulating the expression of certain auxin response factor (ARF) genes to determine the plant developmental process. Among the 10 MIR167 genes of rice, the precursor structures derived from MIR167a, MIR167b and MIR167c produce miR167 with high efficiency. To explore the biological function of miR167 in rice, four of its predicted target genes, OsARF6, OsARF12, OsARF17 and OsARF25, were identified in vivo. Although the expression levels of miR167 and its target OsARFs did not show an obvious negative correlation, the enhanced miR167 level in transgenic rice overexpressing miR167 resulted in a substantial decrease in mRNA levels of the four OsARF genes. Moreover, the transgenic rice plants were small in stature with remarkably reduced tiller number. These results suggest that miR167 is important for the appropriate expression of at least four OsARFs, which mediate the auxin response, to contribute to the normal growth and development of rice.

Gene Structure Analysis of Rice ADP-ribosylation Factors (OsARFs) and Their mRNA Expression in Developing Rice Plants

ADP-ribosylation factors (ARFs) have been widely identified as being involved in regulation of cell differentiation, intracellular vesicle transport, and cytoskeleton organization in animals and plants. To systematically clarify the ARF gene family in rice, eight putative rice ARF genes (OsARFs) were detected by searching the rice genome database. The OsARFs can be classified into class I (OsARFA1a-OsARFA1e) and class II (OsARFB1a-OsARFB1c) by comparing their deduced amino acid sequences with other known ARFs. Most OsARFs are composed of six exons and five introns, though OsARFB1b has three exons and two introns. The OsARFs are usually expressed in various developing organs, whereas OsARFA1a is predominantly expressed in young roots and in caryopses at early development stages. The histochemical localization of OsARFs in various developing organs was confirmed in young leaves, the pericycle of young roots, and the aleurone layer of developing caryopses. Furthermore, the expression of OsARFA1a was confirmed specifically in the aleurone layer and immature embryos of developing caryopses. The present work provides a foundation for further clarifying the physiological functions of OsARFs in rice growth and development.

A Bi-Functional Xyloglucan Galactosyltransferase Is an Indispensable Salt Stress Tolerance Determinant in Arabidopsis

Salinity is an abiotic stress that substantially limits crop production worldwide. To identify salt stress tolerance determinants, we screened for Arabidopsis mutants that are hypersensitive to salt stress and designated these mutants as short root in salt medium (rsa). One of these mutants, rsa3-1, is hypersensitive to NaCl and LiCl but not to CsCl or to general osmotic stress. Reactive oxygen species (ROS) over-accumulate in rsa3-1 plants under salt stress. Gene expression profiling with Affymetrix microarray analysis revealed that RSA3 controls expression of many genes including genes encoding proteins for ROS detoxification under salt stress. Map-based cloning showed that RSA3 encodes a xyloglucan galactosyltransferase, which is allelic to a gene previously named MUR3/KAM1. The RSA3/MUR3/KAM1-encoded xylogluscan galactosyltransferase regulates actin microfilament organization (and thereby contributes to endomembrane distribution) and is also involved in cell wall biosynthesis. In rsa3-1, actin cannot assemble and form bundles as it does in the wild-type but instead aggregates in the cytoplasm. Furthermore, addition of phalloidin, which prevents actin depolymerization, can rescue salt hypersensitivity of rsa3-1. Together, these results suggest that RSA3/MUR3/KAM1 along with other cell wall-associated proteins plays a critical role in salt stress tolerance by maintaining the proper organization of actin microfilaments in order to minimize damage caused by excessive ROS.

Subcellular localization of rice hexokinase (OsHXK) family members in the mesophyll protoplasts of tobacco

Hexokinase (HXK, EC 2.7.1.1) plays an important role in the metabolism and glucose signalling. To examine the characteristics of HXK gene family in rice, the subcellular localizations of ten hexokinases (OsHXK1 — OsHXK10) were determined using OsHXK::GFP fusion proteins in tobacco mesophyll protoplasts. As was previously demonstrated, OsHXK4 was detected in the chloroplast stroma, OsHXK5 and OsHXK6 in the mitochondria, and OsHXK7 and OsHXK10 in the cytoplasm. In the present study, OsHXKs were clearly divided into three types (A, B, C) based on their N-terminal sequences. The new type-C HXKs in plants, OsHXK1, OsHXK7 and OsHXK8, which lack the plastidic transit peptide and the membrane anchor domain, were detected not only in the cytoplasm but also in the nucleus. The type-B HXKs, OsHXK2, OsHXK3, OsHXK9 and OsHXK10, which contained a membrane anchor domain, were distinctly localized in the mitochondria. These results suggest that OsHXKs localized in different cell compartments may be involved in the glucose signalling-related gene expression during growth and development of rice.

Arabidopsis Synaptotagmin 2 Participates in Pollen Germination and Tube Growth and Is Delivered to Plasma Membrane via Conventional Secretion

Arabidopsis synaptotagmin 2 (SYT2) has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen of Arabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmMan1-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.

Cytosolic and Nucleosolic Calcium Signaling in Response to Osmotic and Salt Stresses Are Independent of Each Other in Roots of Arabidopsis Seedlings

Calcium acts as a universal second messenger in both developmental processes and responses to environmental stresses. Previous research has shown that a number of stimuli can induce [Ca2+] increases in both the cytoplasm and nucleus in plants. However, the relationship between cytosolic and nucleosolic calcium signaling remains obscure. Here, we generated transgenic plants containing a fusion protein, comprising rat parvalbumin (PV) with either a nuclear export sequence (PV-NES) or a nuclear localization sequence (NLS-PV), to selectively buffer the cytosolic or nucleosolic calcium. Firstly, we found that the osmotic stress-induced cytosolic [Ca2+] increase (OICIcyt) and the salt stress-induced cytosolic [Ca2+] increase (SICIcyt) were impaired in the PV-NES lines compared with the Arabidopsis wildtype (WT). Similarly, the osmotic stress-induced nucleosolic [Ca2+] increase (OICInuc) and salt stress-induced nucleosolic [Ca2+] increase (SICInuc) were also disrupted in the NLS-PV lines. These results indicate that PV can effectively buffer the increase of [Ca2+] in response to various stimuli in Arabidopsis. However, the OICIcyt and SICIcyt in the NLS-PV plants were similar to those in the WT, and the OICInuc and SICInuc in the PV-NES plants were also same as those in the WT, suggesting that the cytosolic and nucleosolic calcium dynamics are mutually independent. Furthermore, we found that osmotic stress- and salt stress-inhibited root growth was reduced dramatically in the PV-NES and NLS-PV lines, while the osmotic stress-induced increase of the lateral root primordia was higher in the PV-NES plants than either the WT or NLS-PV plants. In addition, several stress-responsive genes, namely CML37, DREB2A, MYB2, RD29A, and RD29B, displayed diverse expression patterns in response to osmotic and salt stress in the PV-NES and NLS-PV lines when compared with the WT. Together, these results imply that the cytosolic and nucleosolic calcium signaling coexist to play the pivotal roles in the growth and development of plants and their responses to environment stresses.

GpDSR7, a Novel E3 Ubiquitin Ligase Gene in Grimmia pilifera Is Involved in Tolerance to Drought Stress in Arabidopsis

The growth and development of plants under drought stress depends mainly on the expression levels of various genes and modification of proteins. To clarify the molecular mechanism of drought-tolerance of plants, suppression subtractive hybridisation cDNA libraries were screened to identify drought-stress-responsive unigenes in Grimmia pilifera, and a novel E3 ubiquitin ligase gene, GpDSR7, was identified among the 240 responsive unigenes. GpDSR7 expression was induced by various abiotic stresses, particularly by drought. GpDSR7 displayed E3 ubiquitin ligase activity in vitro and was exclusively localised on the ER membrane in Arabidopsis mesophyll protoplasts. GpDSR7-overexpressing transgenic Arabidopsis plants showed a high water content and survival ratio under drought stress. Moreover, the expression levels of some marker genes involved in drought stress were higher in the transgenic plants than in wild-type plants. These results suggest that GpDSR7, an E3 ubiquitin ligase, is involved in tolerance to drought stress at the protein modification level.

Microarray-based screening of the microRNAs associated with caryopsis development in Oryza sativa

Plant microRNAs modulate diverse developmental processes by regulating expression of their target genes. To explore potential miRNA-guided gene regulation in developing rice (Oryza sativa L.) caryopses, a miRNA microarray was used to identify miRNAs present at the different developmental stages. We found that 27 miRNAs, of which 16 were conserved miRNAs, were present in developing caryopses. High expression levels were detected for miR159, miR167, and miR530 at the morphogenesis stage and for miR169, miR435, and miR528 at the stage of accumulation of metabolites. Next, 26 target genes were predicted for seven of the detected miRNAs and the expression profiles of these miRNAs and their corresponding target genes were examined in developing caryopses. Our results suggest that the miRNAs and their target genes examined at the two distinct stages could contribute to the developmental progress of rice caryopses in concert with phytohormone signalling.

Transcriptome Profiling Identified Multiple Jasmonate ZIM-Domain Proteins Involved in the Regulation of Alkaloid Biosynthesis in Tobacco BY-2 Cells

Jasmonate (JA) zinc-finger expressed in inflorescence meristem (ZIM)-domain (JAZ) proteins are key regulators of the JA response in plants. Transcriptome profiling of tobacco BY-2 cells was used to identify 17 members of the NtJAZ family, which were divided into 12 distinct groups based on their predicted amino acid sequences and conserved domains. Transcript levels of eight of the NtJAZ groups increased rapidly upon JA treatment, whereas the remaining members did not show a significant response. The majority of JA-induced NtJAZs formed homo- and heteromers and interacted with NtMYC2a (but not NtERF189) in yeast two-hybrid assays. NtJAZ1, NtJAZ3b, NtJAZ7 and NtJAZ10 were localised in the nucleus and degraded rapidly via the 26S proteasome pathway following the treatment of BY-2 with MeJA. RNAi-induced silencing of NtJAZ1, NtJAZ3, NtJAZ7a and NtJAZ10 greatly reduced the levels of NtPMT transcripts and specifically decreased the nicotine content in the four RNAi transgenic BY-2 lines. The levels of transcripts encoding other nicotine biosynthesis enzymes, NtERF189 and NtMYC2a, and other NtJAZs exhibited different expression patterns in RNAi lines with or without MeJA treatment. Our results indicate that cross-talk occurs among different NtJAZs and forms a complex transcription regulatory scheme for JA-induced nicotine biosynthesis in tobacco.